Toluene: biological waste-gas treatment, toxicity and microbial adaptation

Transcriptie

1 Tluene: bilgical waste-gas treatment, txicity and micrbial adaptatin

2 Prmtr: C-prmtr: dr. ir. J.A.M, de Bnt hgleraar in de Industriële Micrbilgie dr. ir.s. Hartmans universitair dcent bij de sectie Industriële Micrbilgie

3 MÔ8EO', zt Frans J. Weber Tluene: bilgical waste-gas treatment txicity and micrbial adaptatin Prefschrift ter verkrijging van de graad van dctr in de landbuw- en milieuwetenschappen p gezag van de rectr magnificus, dr. C.M.Karssen, in het penbaar te verdedigen p wensdag 15 nvember 1995 des namiddags te vier uurin de Aula van de Landbuwuniversiteit te Wageningen. t*m C\l8 \Ojb

4 BIBLJOTHEEK LANDBOUWUNIVERSITEIT WAGENINGEN CIP-DATA KONINKLIJKE BIBLIOTHEEK,DENHAAG Weber, Frans J. Tluene: bilgical waste-gas treatment, txicity and micrbial adaptatin / Frans J. Weber. -[S.I.:s.n.] Thesis Landbuwuniversiteit Wageningen. - Withréf. - With summary in Dutch. ISBN Subject headings: tluene / waste-gas treatment / txiclgy Omslag: Laurens Mrel

5 50MOÖ>2S', ZO'O Stellingen i Enten met een specifieke cultuur kan een grte invled hebben p de werking van een bireactr die nder niet steriele cndities werkt. Dit prefschrift, hfdstuk 3. 2 Ok schimmels zijn in staat m p tlueen als enige klstf- en energiebrn te greien. Dit prefschrift, hfdstuk 4. 3 De HPLC-UV methde zals beschreven dr.a. Rehman (1991) kan niet wrden gebruikt vr een kwantitatieve fsflipide bepaling. Rehman. S.U. (1991). Rapid iscratic methd fr the separatin and quantificatin f majr phsphlipid classes by high-perfrmance liquid chrmatgraphy. J. Chrm. 567: De cnclusie van Lepage ef a/. (1987) en Sajbidr et al. (1992) dat de gevnden veranderingen in de samenstelling van het celmembraan een adaptatie is aan de giftige werking van rganische plsmiddelen, is njuist gezien de waarneming van deze auteurs dat de grei van de gebruikte micr-rganismen sterk wrdt geremd in de aanwezigheid van deze plsmiddelen. Lepage. C, F. Faytle. M. Hermann and J.P. Vandecasteele. (1987). Changes in membrane lipid cmpsitin f Clstridium acetbutylicum during acetne-butanl fermentatin: effects f slvents, grwth temperature and ph. J. Gen. Micrbil. 133: Sajbidr. J., and J. Greg. (1992). Fatty acid alteratins in Saccharmyces cerevisiae expsed t ethanl stress. FEMS Micrbil. Lett. 93: Gezien de tegenvergestelde effecten die benzeen en druk hebben p het celmembraan, lijkt het screenen van diepzeemnsters p benzeentlerante micrrganismen weinig zinvl. Mriya. K. and K. Hrikshi. (1993). Islatin f benzene-tlerant bacterium and its hydrcarbn degradatin. J. Ferm. Bieng. 76:

6 6 Het mduleren van het prces in een bifilmreactr kmt neer p het schatten van 12 parameters m de 13 e te kunnen vrspellen. 7 Juryleden bij verschillende sprten meten ver bvennatuurlijke gaven beschikken m het verschil tussen een 9.95 en een 10.0 te kunnen waarnemen. 8 Om de kansen vr een AIO/OIO p de arbeidsmarkt te verhgen, zu er net als bij bepaalde studies een "numerus fixus" meten wrden ingesteld vr prmvendi. 9 Het ptreden van de milieu-rganisatie "Greenpeace" tegen het in de ceaan dumpen van het lieplatfrm "Brent Spar" is ngelfwaardig nadat deze rganisatie de dr een explsie verweste "Rainbw Warrir" in de ceaan liet afzinken. 10 Het televisiekijkgent is mgekeerd evenredig met het aantal aangebden TV-zenders. 11 De tename in de grtte van cmputerprgrammatuur is geen garantie vr een evenredige verhging van kwaliteit en snelheid. 12 De intrductie van nieuwe elementen in bestaande natuurgebieden behrt niet tt de taken van een natuurbeheerder. Stellingen behrende bij het prefschrift 'Tluene: bilgical waste-gas treatment, txicity and micrbial adaptatin", Frans Weber, Wageningen, 15 nvember 1995.

7 Vrwrd Znder de bijdrage van een grt aantal mensen, die mij de afgelpen jaren hebben gehlpen, had ik mijn prmtienderzek nit kunnen afrnden tt het prefschrift zals het nu vr u ligt. Graag wil ik iedereen hiervr bedanken, een aantal van hen wil ik met name nemen: Allereerst Jan de Bnt en Sybe Hartmans. Dankzij jullie vertruwen en kritische blik heb ik de afgelpen jaren veel geleerd en met veel plezier aan het nderzek gewerkt. Hans Rijs en Ed Waalewijn van Ecsens vr prettige samenwerking en de cntacten met de praktijksituatie. Met name de gebden vrijheid in het nderzek heb ik zeer gewaardeerd. Hierdr was het vr mij mgelijk m naast de bilgische afvalgasreiniging, interessante "zij-spren" te bestuderen. Zals gezegd, veel van de resultaten zijn verkregen dr de inbreng van anderen. Met name nem ik Ruud Schemen,Snja Isken, Lydia Oijkaas, Randall Cffie, Marjn Diender, en K Hage. Jullie hebben als nderdeel van het dcteraal nderzek gewerkt aan vaak weer een nieuw deel van het nderzek. Bedankt vr jullie enthusiaste inbreng en vr de fijne samenwerking. Natuurlijk bedank ik k alle (ex)cllega's en studenten van de sectie Industriële Micrbilgie vr jullie belangstelling, discussies en hulp, maar met name de gede sfeer p het lab en daar buiten. Hermann Heipieper en Jan Sikkema vr verschillende discussies.keesteunis van de vakgrep Organische Chemie, Flkert Hekstra, vakgrep Plantenfysilgie en Jacques Vervrt, vakgrep Bichemie vr de hulp bij respectievelijk, de GC-MS,FIR en NMR analyses. Tensltte wil ik Bernadette bedanken vr de steun en het bendigde "zetje in de rug" dat vral tijdens de afrnding van mijn prefschrift welkm was, en mijn uders vr de mgelijkheden ze mij hebben gebden. Helaas is in februari 1995 tijdens het schrijven van de laatste hfdstukken van dit prefschrift, mijn vader pltseling verleden. Hij had z graag bij mijn prmtie willen zijn. Ik draag dit prefschrift aan hem p.

8 Cntents Chapter 1 General intrductin 9 Chapter 2 Use f activated carbn as a buffer in bifiltratin f waste gases with fluctuating cncentratins f tluene 19 Chapter 3 Remval f tluene frm cntaminated air with a bilgical trickle-bed reactr 31 Chapter 4 Grwth f the fungus Cladsprium sphaerspermum with tluene as the sle carbn and energy surce 47 Chapter 5 Txicity f cntaminants in waste gases fr micrrganisms 61 Chapter 6 Chapter 7 Adaptatin f Pseudmnas putida S12 t high cncentratins f styrene and ther rganic slvents 69 Cis/trans ismerizatin f fatty acids as a defence mechanism f Pseudmnas putida strains t txic cncentratins f tluene 81 Chapter 8 Adaptatin mechanisms f micrrganisms t the txic effects f rganic slvents n membranes 93 Chapter 9 Cncluding remarks 151 Summary 159 Samenvatting 163 Curriculum vitae 167 Bibligraphy 169

9 ? General intrductin Since the industrial revlutin the prductin and use f chemicals has increased immensely. As a cnsequence all kinds f wastes are prduced, which are released in t the envirnment. Many prducts f the petrchemical industry, like rganic slvents, fuels, herbicides r insecticides, will eventually als be released int the envirnment, due t prductin r strage lsses, slvent evapratin, r emissins frm mtr vehicles and aerplanes. Fr example fr tluene it has been estimated that mre than 86% f the *10^ tnnes f tluene prduced in 1981 was ultimately released int the envirnment (Annymus, 1985). Nwadays there is a grwing awareness cncerning the pssible txic r even carcingenic effects f these xenbitics. Therefre the release f these chemicals int the envirnment is restricted by legislatin. In rder t meet these requirements, the industry has t reduce her waste streams. In general this can be achieved by implementing anther r a better cntrl f the prcess. Fr instance, recvery and recirculatin f rganic slvents can significantly reduce the waste streams. Hwever, this will ften require expensive prcess mdificatins and therefre, quite ften an end-f-pipe technique is used t remve cntaminants frm the waste streams such as incineratin r bilgical treatment. Fig. 1 clearly shws that the emissin f rganic hydrcarbns in The Netherlands is mainly t air. In the perid between 1982 and 1992 treatment f waste waters and changes in prductin prcesses have resulted in a reductin f the emissin

10 JO Chapter (0 0) >. Ol X ^~ *, c 0 in (/) F LU m Air Water e Figure 1: Industrial emissin f rganic hydrcarbns t air (-) and water (--) in The Netherlands between 1982 and (Frm van der Plas & Verheve, 1994) f rganic hydrcarbns t water by mre than 50% (Fig. 1). Treatment f waste gases was, hwever, less successful, althugh a gradual decrease in the emissin f rganic hydrcarbns t air is bserved since The grwing envirnmental awareness and mre stringent legislatin is expected t result in a further reductin f the gaseus emissins in the future. In The Netherlands, the release f gaseus cmpunds is cntrlled by the NER (Nederlandse Emissie Richtlijnen; Annymus, 1992). Depending n the nature f the cmpund the NER describes a maximal ff-gas cncentratin abve a certain quantity f cntaminant emitted. Nrmally, the maximal allwable ffgas cncentratin fr an emissin f mre than 3.0 kg/h is 150 mg/m 3, hwever, fr carcingenic cmpunds, e.g. benzene, the limit is 5 mg/m 3 when the emissin exceeds 25 g/h (Annymus, 1992). In rder t meet these limits, varius techniques fr waste-gas treatment are applied and being develped. Either physic-chemical r bilgical techniques are used t treat waste-gas streams. Examples f physic-chemical methds are incineratin, catalytic xidatin, scrubbing r adsrptin n activated carbn (Heck et a/., 1988). Bilgical techniques make use f the capacity f micrrganisms t degrade the txic cntaminants t less txic prducts, ften resulting in cmplete mineralizatin. Bilgical techniques are ften the mst cst

11 General intrductin 11 effective fr waste gases cntaining less than gram f bidegradable rganic cmpunds per m 3 (Lesn &Winer, 1991; Bhn, 1992;Kk, 1992). BIOLOGICALWASTE-GASTREATMENT Bilgical waste-water treatment has becme an established methd t reduce the release f cntaminants int the envirnment. Bilgical treatment f waste gases, hwever, isstill nly applied n a minr scale, mainly fr the treatment f drus air (Ottengraf, 1987; Annymus, 1989; Lesn & Winer, 1991). These drus waste-gas streams generally cntain lw cncentratins f cmplex mixtures f cmpunds. Due t the increasing stringent legislatin cncerning the emissin f vlatile rganic cmpunds, there is nwadays a grwing interest t als apply bilgical waste-gas treatment techniques fr the remval f higher cncentratins f specific cntaminants. Fr the bilgical remval f pllutants frm waste gases several types f bireactrs have been develped (Ottengraf, 1987; Grenestijn & Hesselink, 1993) (Fig. 2). Biftlters Bifilters were the first bireactrs used fr the bilgical treatment f waste gases. Already since 1920 bifilters have been used n a limited scale t remve drus cmpunds frm waste gases f fr instance waste-water treatment plants (Lesn & Winer, 1991). A bifilter is essentially a packed-bed reactr thrugh which the waste gas is frced (Fig. 2). Micrrganisms grwing n the packing material, usually cmpst, will degrade the cntaminants present in the waste-gas stream. Besides the simple and cst effective peratin f bifilters, anther advantage is the absence f a distinct water layer in the bifilter. Therefre, the mass transfer f prly water sluble cmpunds t the bifilm is relatively gd inthis type f reactr. The first bifilters were actually sil beds. Nwadays generally cmpst r peat, mixed with bark chips r plystyrene particles are used as packing material.the additin f wd bark r plystyrene results in a hmgeneus structure f the packing with a relatively lw pressure drp (Ottengraf, 1986; van Langenhve et al., 1986). Since the intrductin f the first bifilters anther imprtant imprvement has been the develpment f clsed bifilters, allwing a better cntrl f the physical cnditin f the bifilter. In the pen bifilters the packed bed is expsed t fluctuating weather cnditins (temperature, rain). With the clsed bifilters, the micrbial activity within the bifilters can be better maintained, as temperature and humidity which strngly affect the bilgical activity can be better cntrlled (Heslinga, 1992). Usually the in-let gas stream is humidified t almst cmplete saturatin. Generally, additinal humidificatin f

12 12 Chapter 1 A) B) Cmpst Clean gas r-^-j: Clean gas Packing with bimass Water»- Waste gas Waste gas Humidifier Drain Water Nutrients Titrants C) D) Clean gas Inerl pack ii '"" \ Activated sludge bireactr Water ~f Nutrients Drain Waste _ > gas I Water Nutrients Titreints Dram Gas Water Membrane Bifilm Figure 2: Varius reactrs fr bilgical waste-gas treatment. (A) Bifilter, (B) Trickle-bed bireactr, (C) Biscrubber, (D) Membrane bireactr.

13 General intrductin 13 the filter is als necessary t cmpensate fr water lss due t an increase in temperature f the waste gas caused by the micrbial activity. Besides the stringent cntrl f the humidity ther factrs which are difficult t cntrl are the ph and the supply f additinal (inrganic) nutrients. Althugh several additives, like lime, can be mixed thrugh the packing t buffer the ph, these additives will nt be sufficient t cntinuusly neutralise the HCl frmed due t the bidégradatin f chlrinated cmpunds. Several studies have shwn that nutrient (e.g. nitrgen) limitatins can strngly influence the verall perfrmance f the bifilter (Dn, 1986; Beyreitz et ai, 1989; Weckhuysen et ai, 1993). As a hmgenus supply f these nutrients t the bifilter is difficult, it is expected that the reprted eliminatin rates f mre than 100 g carbn per rr\3 packing material per hur (Ottengraf ef a/., 1986; Windsperger et ai. 1990; Kirchner et ai, 1991) can nt be maintained during a lnger perid f time. Nutrients in the cmpst will becme exhausted, and als ph and humidity cntrl are expected t becme a prblem. Biscrubber In biscrubbers the cntaminants in the waste gas are transferred t a water phase in a scrubber with an inert packing. The cmpunds in the water are subsequently degraded in a separate reactr, which is usually a traditinal activated sludge waste water treatment facility. The water phase is subsequently recirculated thrugh the scrubber cmpartment (Fig. 2). In cntrast t bifilters, a circulating water phase is present, which allws a better cntrl f reactr cnditins (ph, nutrients) and makes it pssible t treat waste gases cntaining cmpunds like ammnia r chlrinated hydrcarbns. Hwever, a drawback cmpared t the bifilter is, that this type f reactr can nly be used t remve cntaminants with a relatively high water slubility. In rder t increase the mass transfer f less water sluble cmpunds, the use f rganic slvents t enhance the mass transfer has been suggested (Schippert, 1994).The intermediate slvent shuld be nn-txic, nn-vlatile and nt bidegradable. By applying such a slvent in a three-phase reactr the transfer f the cntaminants frm the gas phase t the slvent phase can be significantly enhanced. Hwever, the bidégradatin will nly take place in the water phase which requires a transfer f the cntaminant frm the slvent t the water phase. In this situatin the mass transfer frm the slvent t the water phase will prbably becme rate limiting. An verall increase in the mass transferis nly bserved when a very high specific exchange area between the slvent and water phase can be achieved (Cesâri et ai, 1994).

14 M Chapter 1 Trickle-bed bireactr In a trickle-bed reactr sme f the advantages f the bifilter and the biscrubber are cmbined. The circulating water phase allws better cntrl f the reactr cnditins. Due t the cmbinatin f the scrubber and bireactr cmpartments, the cntaminants transferred t the water phase will be immediately degraded resulting in lw cntaminant cncentratins in the water phase and cnsequently a better mass transfer f mderately water sluble cmpunds. Althugh trickle-bed reactrs are used in waste water treatment systems, they are nt yet frequently used fr the treatment f waste gases. Bilgical waste-gas treatment using trickle-bed reactrs has been successfully demnstrated fr the remval f dichlrmethane frm waste gases (Diks & Ottengraf, 1991; Hartmans & Tramper, 1991). Dichlrmethane, which is mderately water sluble was shwn t be eliminated with a sufficient efficiency ver a lnger perid f time at rates f abut 50 g/(nri3 h). Hwever, this specific degradatin rate amunts t nly 7 grams f carbn per m 3 per h. Using ther gas cmbinatins, resulting in higher rganic lads, clgging f the reactr due t excessive bimass frmatin was bserved (Diks,1992). In this thesis results are presented which describe pssible methds t prevent clgging f the reactr. Subsequently the applicatin f trickle-bed reactrs fr the degradatin f a waste-gas stream cntaining a high tluene lad, during a lnger perid f time with a high degradatin rate, will be demnstrated. Membrane bireactr In a membrane bireactr the gas phase and the liquid phase, cntaining the micrrganisms, are separated by a prus hydrphbic membrane. As the membrane makes it pssible t prevent the release f micrrganisms int the envirnment, this reactr type is being evaluated fr use in space-cabins t maintain a clean atmsphere during lng-term manned space-flights. The pres f the hydrphbic membrane are filled with air resulting in a lw mass transfer resistance (Hartmans et a/., 1992). As the micrrganisms frm a bifilm directly n the membrane, the mass transfer resistance fr cntaminants frm the gas phase t the bifilm is expected t be lw. Byusing a hllw-fibre reactr a large specific gas/bifilm surface can be created, resulting in a reactr with a lw mass transfer resistance and a high specific surface area, and the ability t cntrl the envirnment f the bifilm by the circulating water phase like in a trickle-bed reactr. It has been shwn that with this type f reactr, a stable bidégradatin f prpene culd be btained during prlnged peratin (Reij et a/., 1995).

15 General intrductin 15 MICROBIOLOGICAL ASPECTS OFWASTE-GASTREATMENT The first criterin fr a successful bidégradatin prcess is f curse the availability f micrrganisms capable f degrading the cntaminant. Fr the bidégradatin f xenbitics which were previusly thught t be nnbidegradable nwadays mre and mre micrrganisms are being selected frm nature, which are capable f degrading these cmpunds. The metablic pathways fr the degradatin f a great number f cmpunds have been elucidated in many different micrrganisms. Furthermre, genetic engineering has ffered pssibilities t cmbine and cnstruct new pathways fr the degradatin f xenbitics that were nt degraded previusly. Althugh many (xenbitic) cmpunds are intrinsically bidegradable, actual degradatin rates depend n a number f parameters. One f the mst imprtant parameters is the cntaminant (substrate) cncentratin. The ff-gas cncentratins required by legislatin, especially fr carcingenic cmpunds, are extremely lw and in sme cases t lw fr bidégradatin at an acceptable rate (Hartmans, 1994). Applicatin f bilgical waste-gas treatment techniques fr the remval f higher cncentratins f rganic slvents seems t be prmising. Hwever, a prblem culd be the txicity f the pllutants in the waste gas fr the micrrganisms in the bireactr. Althugh many xenbitic cmpunds are tlerated and degraded by micrrganisms at lw cncentratins, they can already becme txic at slightly higher cncentratins (Vlsky et a/., 1990; Blum & Speece, 1991). Especially cmpunds with a gd slubility in water can be expected t accumulate in the water phase f the reactr during the start-up perid. This accumulatin can result in the inactivatin f the bimass in the reactr as the cntaminant cncentratins reach txic levels. Als fluctuatins in the cntaminant cncentratins in the waste gas culd result in levels which are txic fr the micrrganisms in the reactr. Interestingly, several micrbial strains have been described that can adapt t these txic levels, resulting in the capacity t tlerate and grw in the presence f these txic slvents (Inue & Hrikshi, 1989; Cruden et a/., 1992). OUTLINE OFTHISTHESIS In this thesis several aspects f the bidégradatin f xenbitics, especially tluene, and the remval f tluene frm waste gases with trickle-bed reactrs are discussed. In waste gases, the cncentratin f the cntaminants generally

16 16 Chapter 1 fluctuate strngly. In Chapter 2 the selectin and use f adsrbents t buffer these fluctuating cncentratins is discussed. The applicatin f trickle-bed reactrs fr the remval f xenbitics frm waste gases als depends n the ability t cntrl the bimass cntent f these reactrs (i.e. t prevent clgging). In Chapter 3 tw pssible methds are addressed with which the frmatin and remval f bimass frm these reactrs culd be cntrlled. In ne f the tricklebed reactrs used fr the remval f tluene, a fungal culture develped. This rganism was islated and a tentative pathway fr the bidégradatin f tluene by this fungus, Cladsprium sphaerspermum, is presented in Chapter 4. Ultimately, mst pllutants and hazardus substances are f cncern because f their ptential txic effects n man. Hwever, these xenbitics can als be txic fr micrrganisms (Chapter 5) and this might limit the applicatin f these rganisms fr bidégradatin prcesses. Generally, the txicity f lipphilic slvents is due t interactins f these slvents with the cell membrane. Cnsequently the functining f the membrane as a permeability barrier is affected. These effects f varius rganic slvents n the functining f the cell membrane, and the adaptatin mechanisms f micrrganisms t cmbat these effects are reviewed and discussed in Chapter 8. Pseudmnas putida SI2 is a strain which can grw in the presence f supersaturating slvent cncentratins which are nrmally extremely txic t micrrganisms. In Chapter 6 the grwth f P. putida SI2 n supersaturating cncentratins f styrene is presented. Aspects f the adaptatin f this strain and ther slvent tlerant P. putida strains t allw grwth in the presence f supersaturating amunts f slvents are described in Chapters 7 and 8. Finally, in Chapter 9 the ptential f trickle-bed reactrs and slvent-tlerant bacteria fr bitechnlgical applicatins are discussed. REFERENCES Annymus. (1985). Tluene. Envirnmental Health Criteria 52, Wrld Health Organizatin, Geneva. Annymus. (1989). Bilgische Abgas-/ Abluftreinigung: Bifilter. VDI-Handbuch Reinhaltung der Luft. Band 6. VDI 3477 (entwurf), Verein Deutscher Ingenieure, Düsseldrf. Annymus. (1992). Nederlandse emissie richtlijnen lucht. Cmmissie emissies lucht, Stafbur NER, Bilthven. Beyreitz, G., R. Hübner and M. Saake. (1989). Bitechnlgische Behandelung lösemittelhaltige Abluft. Wasser Luft und Bden 9: Blum, D.J.W, and R.E. Speece. (1991). A database f chemical txicity t envirnmental bacteria and itsuse in interspecies cmparisns and crrelatins.res. J. WPCF 63:

17 General intrductin 17 Bhn,H. (1992). Cnsider bifiltratin fr decntaminating gases. Chem. Eng. Prg. (April) Cesâri, M.T., H.H. Beeftink and J. Tramper. (1994). Remval f prly water-sluble cmpunds frm waste gases using water-immiscible rganic slvents. In: L Alberghina, L. Frntali and P. Sensi (eds.). Prceedings f the 6th Eurpean cngress n bitechnlgy, pp ,Elsevier, Amsterdam. Cruden, D.L, J.H.Wlfram, R.D.Rgers and D.T. Gibsn. (1992). Physilgical prperties f a Pseudmnas strain which grws with p-xylene in a tw-phase (rganic-aqueus) medium. Appl. Envirn. Micrbil. 58: Diks, R.M.M, and S.P.P.OHengraf. (1991). Verificatin studies f a simplified mdel fr the remval f dichlrmethane frm waste gases using a bilgical trickling filter. (Part II). Biprc. Eng. 6: Diks, R.M.M. (1992). The remval f dichlrmethane frm waste gases in a bilgical trickling filter. PhD-Thesis, Technical University, Eindhven. Dn, J.A. (1986). The rapid develpment f bifiltratin fr the purificatin f diversified waste gas streams. In:VDI Berichte 561, Geruchstffe, pp , VDI Verlag, Düsseldrf. Grenestijn, J.W. and P.G.M. Hesselink. (1993). Bitechniques fr air pllutin cntrl. Bidegradatin 4: Hartmans, S. and J. Tramper. (1991). Dichlrmethane remval frm waste gases with a trickle-bed bireactr. Biprc. Eng.6: Hartmans, S., E.J.T.M. Leenen and G.T.H. Vskuilen. (1992). Membrane bireactr with hydrphbic membranes fr waste-gas treatment. In: A.J. Dragt, and J. van Ham (eds.), Bi techniques fr air pllutin abatement and dur cntrl plicies, pp Elsevier, Amsterdam. Hartmans, S. (1994). Micrbilgical aspects f bilgical waste gas cleaning. In: VDI Berichte 1104, Bilgische Abgasreinigung, pp. 1-12, VDI Verlag, Düsseldrf. Heck, G., G. Müller and M. Ulrich. (1988). Reinigung lösungsmittelhaltiger Abluft: alternative Möglichkeiten. Chem. Ing. Tech. 4: Heslinga, D.C. (1992). Bifiltratin technlgy. In: A.J. Dragt, and J. van Harn (eds.). Bitechniques fr air pllutin abatement and dur cntrl plicies, pp , Elsevier, Amsterdam. Inue, A. and K. Hrikshi. (1989). A Pseudmnas thrives in high cncentratins f tluene. Nature 338: Kirchner, K., CA. Gssen and H.-J. Rehm. (1991). Purificatin f exhaust air cntaining rganic pllutants in a trickle-bed bireactr. Appl. Micrbil. Bitechnl. 35: Kk, H.J.G. (1992). Biscrubbing f air cntaminated with high cncentratins f hydrcarbns. In: A.J. Dragt, and J. van Ham (eds.). Bitechniques fr air pllutin abatement and dur cntrl plicies, pp ,Elsevier, Amsterdam. van Langenhve, H., E. Wuyts and N. Schamp. (1986). Eliminatin f hydrgen sulphide frm drus air by a wd bark bifilter. Waf. Res. 20:

18 18 Chapter 1 Lesn, G. and A.M. Winer. (1991). Bifiltratin: An innvative air pllutin cntrl technlgy fr VOC emissins. J. Air Waste Manage. Assc. 41: Ottengraf, S.P.P. (1986). Exhaust gas purificatin. In: H.-J. Rehm, and G. Reed (eds.). Bitechnlgy, Vl. 8, pp , VCH Verlagsgesellschaft, Weinheim. Ottengraf, S.P.P., J.J.P. Meesters, A.H.C. van den Oever and H.R. Rzema. (1986). Bilgical eliminatin f vlatile xenbitic cmpunds in bifilters. B/prc. Eng. 1: Ottengraf, S.P.P. (1987). Bilgical systems fr waste gas eliminatin. Trends in B/bfechn/. 5: van der Plas, A. and P. Verheve. (1994). Trends in industriële emissies van priritaire stffen. Peride 1981 t/m Publikatiereeks Emissieregistratie, Nr 18, juli 1994, Ministerie van VROM, "s-gravenhage. Rei],M.W., K.D. de Gijer, J.A.M. de Bnt and S. Hartmans. (1995). Membrane bireactr with a prus hydrphbic membrane as a gas-liquid cntactr fr waste gas treatment. B/techn/. Bieng. AS: Schippert, E. (1994). Biwäschertechnlgie. In: VDI Berichte 1104, Bilgische Abgasreinigung, pp.39-56, VDI Verlag, Düsseldrf. Vlsky Jr.. V.T., C.P.L Grady Jr. and H.H. Tabak. (1990). Effect f selected RCRA cmpunds n activated sludge activity. Res. J. WPCF 62: Weckhuysen,B., L Vriens and H. Verachtert. (1993). The effect f nutrient supplementatin n the bifiltratin remval f butanal in cntaminated air. Appl. Micrbil. Bitechnl. 39: Windsperger, A., R. Büchner and K. Stefan. (1990). Reinigung lösungsmittelhaltiger Abluft mit Bifiltern. Teil I: Labruntersuchungen und Ergebnisse aus verschiedenen Industriezweigen. Staub-Reinhaltung der Luft 50:

19 2 Use f activated carbn as a buffer in bifiltratin f waste gases with fluctuating cncentratins f tluene Frans J. Weber and Sybe Hartmans Summary: Fluctuatins in cntaminant cncentratins ften adversely influence the effectiveness f bireactrs fr waste-gas treatment. Applicatin f an adsrbent t minimize such fluctuatins culd imprve the verall prcess. Therefre the buffer capacity f a number f activated carbns and ther adsrbents was tested. The buffer capacity f the adsrbents depends n the desired cncentratin range f the cntaminants entering the bireactr and n the time available fr desrptin. When fluctuatins between 0 and 1000 mg tluene per m 3 were supplied t a bifilter this resulted in significant cncentratins f tluene leaving the bifilter. Using ne selected type f activated carbn it was demnstrated that these fluctuatins culd be decreased t a value f abut 300 mg/m 3, which was subsequently cmpletely degraded in the bifilter. Applied Micrbilgy and Bitechnlgy (1995) 49:

20 20 Chapter 2 INTRODUCTION It has been shwn that bilgical waste-gas treatment is pssible with high efficiencies and degradatin velcities using bifilters r trickle-bed reactrs (Ottengraf, 1986; Diks & Ottengraf, 1991; Hartmans & Tramper, 1991). Hwever, fluctuating cncentratins f the cntaminants in the waste gas can have a negative influence n these perfrmances. Temprary high cncentratins can be txic fr the micrrganisms in the reactr, resulting in inactivatin f the system. Furthermre, fr a reliable peratin the design f the reactr shuld be based n the peak cncentratins in the waste gas, which is nt an ecnmically favurable situatin. Therefre, it wuld be desirable t buffer the fluctuatins in the cncentratin f cntaminants in waste gases by means f an adsrbent, s that a cnstant supply f cntaminants t the bireactr can be achieved. MATERIALS AND METHODS Equilibrium istherm The equilibrium istherm f the amunt f tluene adsrbed versus the gas-phase cncentratin was determined by placing 10 g f adsrbent in 250-ml flasks. These flasks were clsed with a Tefln valve. Knwn amunts f tluene were added t these flasks which were placed in a shaking water-bath at 30 C. After 24 hurs equilibrium was reached and the tluene cncentratin in the gas phase was determined. Desrptin prfile T determine the desrptin rate the adsrbent was first laded with tluene in a 1-1 flask clsed with a Tefln valve (24 h at 30 C). Enugh tluene was added t achieve a gas phase equilibrium cncentratin f abut 5000 mg/m 3. The adsrbent laded with tluene was subsequently placed in a thermstated clumn (15 cm x 28 mm, 30 C). Thrugh this clumn air was blwn at a flw rate f 1.5 l/min (1000 h ), and the tluene cncentratin in the effluent gas was mnitred cntinuusly with an n-line gas-analyzer. Experimental set-up f bifilter The remval efficiency f fluctuating cncentratins f tluene was determined in a bifilter (40 cm x 98 mm) packed with cmpst and plystyrene (20% v/v). Air was saturated with tluene by passing it thrugh a bubble clumn with tluene at 30 C and was diluted with fresh air t the desired cncentratin. The ttal air flw was subsequently saturated with water and blwn thrugh the bifilter at a flwrate f 0.65 m 3 /h (210 hr 1 ). When activated carbn (450 ml) was mixed thrugh

21 Activated carbn as a buffer in bifiltratin 21 the cmpst, n plystyrene was added. Fr sme experiments a separate activated-carbn clumn (25 cm x 50 mm) was placed in series with the bifilter. The air was humidified befre entering the bifilter after it had passed thrugh the activated-carbn clumn. The tluene cncentratins in the air entering and leaving the activated-carbn clumn and/r the bifilter were analyzed with an n-line gas-analyzer. Analytical methds The tluene cncentratin in the gas phase was either determined by analyzing head-space samples n a gas-chrmatgraph r with an n-line gas-analyzer. The head-space samples (100 ul) were analyzed n a Packard 430 gaschrmatgraph with a Prapak R clumn ( mesh, 110 cm x 1/8 inch i.d.). Fr the n-line gas analysis a Chrmpack CP2001 gas-analyzer with a OV-1 clumn (4 m) was used. Carbn dixide was measured using the n-line gasanalyzer with a Hayesep A clumn (25 cm). Materials The activated carbns were a gift f Nrit N.V., Amersfrt, The Netherlands. Silica gel was btained frm Merck, and Al frm Aldrich. RESULTS Equilibrium istherm Different amunts f tluene were added t activated carbn in clsed flasks. After equilibrium was reached between the tluene which adsrbed t the adsrbent and the amunt left in the gas phase the tluene cncentratin in the gas phase was determined. In Fig. 1 such an equilibrium istherm is shwn fr Nrit R3 Extra at 30 C. Using these istherms the amunt f tluene adsrbed n the activated carbn at gas-phase cncentratins f 100 mg/m 3 and 1000 mg/m 3 was determined. Frm these data the buffer capacity was calculated (Table 1). The equilibrium istherm was als used t calculate the cncentratin f tluene in air that was passed thrugh a clumn f Nrit R3 Extra laded with 400 mg tluene/g at a flw-rate f 1000 rr' (Fig. 2).

22 22 Chapter Weight Adsrbed (mg/g) Figure 1: Adsrptin istherm f Nrit R3Extra - tluene at 30 C E c m ( CD U r U Figure 2: Theretical desrptin f tluene frm Nrit R3Extra by an air-flw f 1000 h" 1 at 30 C. The tluene cncentratin in the air leaving a clumn f activated carbn and the amunt f tluene remaining adsrbed are calculated.

23 Activated carbn as a buffer in bifiltratin 23 Table 1: Effective buffer capacities f adsrbents fr tluene. The effective buffer capacity was determined frm the adsrptin istherm r the desrptin prfiles in the cncentratin range mg tluene per m 3 air. The time required fr a decrease in gas-phase cncentratin f 1000 t 100 mg/m 3 at a flw rate f 1000 tr' and the amunt desrbed (starting frm an equilibrium gas-phase cncentratin f 1000 mg/m 3 ) in 12 h at this flw rate were determined frm the desrptin prfiles. Adsrbent NritRB3 NritROX0.8 Nrit R3Extra Nrit R03 Nrit ROW0.8 Supra Nrit Car 115' Effective buffering capacity (mg/g) Equilibrium istherm Desrptin prfile Time (d) Desrbed in 12 hurs (mg/g) Silica gel Al < Water Car 115is an experimental type f activated carbn. - Desrptin prfile All the adsrbents were laded with tluene and placed in a clumn. Thrugh this clumn a cnstant air flw was blwn and the tluene cncentratin in the ff-gas was determined. Such a desrptin prfile is shwn in Fig. 3. Using these prfiles again the buffer capacities between 100 and 1000 mg/m 3 were calculated (Table 1). Als shwn in this table is the time required t reduce the gas-phase cncentratin leaving the carbn clumn frm 1000 t 100 mg/m 3 and the amunt f tluene which is desrbed in 12 hurs. Bifilter Nrit R3 Extra was chsen t study the remval f fluctuating cncentratins f tluene frm waste gases with a bifilter. Three different cnfiguratins were tested. In the first cnfiguratin a bifilter was used withut activated carbn. In the secnd set-up activated carbn was mixed with the cmpst in the bifilter. In the third system a separate activated-carbn clumn in series with the bifilter was used. Air cntaining 900 mg tluene per m 3 was supplied t these three reactrs fr 8 h a day. The bifilter withut activated carbn had a remval

24 24 Chapter 2 Figure 3: Theretical desrptin ( ), calculated with the adsrptin istherm and assuming equilibrium, and measured ( ) desrptin f tluene frm Nrit R3 Extra.T: 30 C. Flw-rate: 1000 lv'. efficiency f abut 50% (Fig. 4A). A similar remval efficiency was bserved fr the reactr in which activated carbn was mixed with the cmpst (Fig. 4B). When a separate clumn with activated carbn was placed befre the bifilter the fluctuating tluene cncentratins were buffered t a cncentratin f abut 300 mg/m 3, which was cmpletely degraded in the bifilter (Fig. 4C). The degradatin velcities bserved fr the three cnfiguratins are shwn in Table 2. Als shwn are the bserved degradatin velcities when tluene was supplied cntinuusly t these reactrs. DISCUSSION Bilgical waste-gas treatment is especially useful t remve lw cncentratins f cntaminants frm waste gases. Higher cncentratins f cntaminants can be treated mre effectively and cheaper using ther systems (as fr instance incineratin) (Dragt, 1992). Therefre a cncentratin f 1000 mg/m 3 was chsen as the maximum cncentratin which shuld be allwed t enter a reactr fr bilgical waste-gas treatment. In practise it is expected that cntaminant cncentratins in waste gases will fluctuate strngly, depending n the surce. Fr a reliable system it wuld therefre be desirable t be able t buffer temprarily high (txic) cncentratins f cntaminants by using an adsrbent

25 Activated carbn as a buffer in bifiltratin S\ n KAI r\ O) Q) +-* (0 \_ +^ c (1) u c 0) c dl D I A_ L_ s\ B A, k h 1 L \ l r\ [\ c rhul Time (d) Figure 4A-C: Effect f Nrit R3 Extra n the remval f fluctuating cncentratins f tluene in a bifilter. (A) Bifilter withut activated carbn. (B) Bifilter with activated carbn mixed with cmpst. (C) Bifilter with a separate activated carbn filter placed befre the bifilter. ( ) Tluene cncentratin f inlet air, ( ) gas-phase cncentratin leaving activatedcarbn clumn, ( ) cncentratin in air leaving the bifilter. The results shwn were btained 20 t 30 days after start-up f the bifilters.

26 26 Chapter 2 c 0) c "c ö C u O).<y ö. a D C O A b "O JU > u 3 JZ ~0 c 2 'n c.0 V ö) eu û «Si 0) «.>a a V» a> c (U _2 u u es CM > n - vo > c* c <N CN -* O CN es T O * G O CN tv O CO > 00 uï -f tr> C._ 0 <0 8 i= 0 0 V 3 l + c O 0 < 0 1 XI 0) L_ 0 0 c 00 c 0.Q b JU 0 > 0 a X) a CQ CN CO c 0.a 0 > x> a.c c 0

27 Activated carbn as a buffer in bifiltratin 27 During perids with lw cntaminant cncentratins in the waste gas, desrptin shuld take place. Inthis manner there will be less fluctuatins in the cntaminant cncentratin supplied t the bireactr. A useful adsrbent shuld therefre have a high buffer capacity belw an equilibrium gas-phase cncentratin f 1000 mg/m 3. At a cncentratin f 1000 mg tluene/m 3 in the gas phase Nrit R3 Extra can adsrb abut 300 mg/g (Fig. 1). Hwever, the effective buffer capacity will be much lwer than thisasit will take mnths fr the tluene t desrb ttally again. Thisis shwn mre clearly when a desrptin prfile fr Nrit R3Extra laded with 400 mg tluene/g is calculated (Fig. 2). Fr this calculatin it was assumed that there was equilibrium between the gas phase and the activated carbn. At gasphase cncentratins belw 100 mg/m 3 a significant amunt f tluene is still adsrbed n the carbn, which is nly very slwly remved. Thus a significant amunt f tluene adsrbed t the carbn will nt be remved by desrptin. This capacity will therefre nt be available t buffer fluctuatins in the cntaminant cncentratin. Therefre we have defined the effective buffer capacity as the amunt which can be adsrbed at equilibrium gas-phase cncentratins between 100 and 1000 mg/m 3. Desrptin resulting in equilibrium gas-phase cncentratins belw 100 mg/m 3 are nt expected in practise as it can take mre than a week t reach this equilibrium cncentratin (Fig. 2).These effective buffer capacities between 100 and 1000 mg/m 3 vary frm 24 t 225 mg/g depending n the type f adsrbent (Table 1 ). The actual amunt f tluene desrbed frm the adsrbent isless than predicted by the adsrptin istherm (Fig. 3). This difference between the calculated and measured desrptin was prbably due t the shrt residence time f the air in the clumn. An empty-bed cntact time f 4 s was apparently t shrt fr equilibrium between air and activated carbn t be reached.thisresults in lwer tluene cncentratins in the gas phase than calculated with the equilibrium istherm. Owing t this effect the effective buffer capacity (between 100 and 1000 mg/m 3 ) is lwer than calculated frm the equilibrium istherm. If a flw rate f 1000 h" 1 is used it still takes mre than 4 days befre enugh tluene is desrbed t reduce the utlet gas-phase cncentratin frm 1000 t 100 mg/m 3 (Table 1).These lng desrptin times are nt very realistic in view f the fluctuatins in cntaminant cncentratins bserved under practical cnditins, which are ften the result f the 8-h wrking day. The effective buffer capacity therefre strngly depends n the time available fr desrptin. In 12 h nly 23 mg tluene/g is desrbed frm Nrit R3 Extra laded with 300 mg/g (equilibrium gas-phase cncentratin: 1000 mg/m 3 ) at a flw rate f 1000 tv'.

28 28 Chapter 2 Althugh the buffer capacity f the tested adsrbents fr tluene is particularly strngly influenced by the desrptin time, they can prbably still be used as a buffering agent in waste-gas treatment. Of all tested adsrbents Nrit Car 115 and Nrit R3 Extra had the highest buffer capacity fr tluene in the cncentratin range between 100 and 1000 mg/m 3. Nrit R3Extra was chsen fr further study as a buffer in bilgical waste-gas treatment. Mixing the activated carbn with the cmpst in the bifilter resulted in remval efficiencies cmparable t thse in a bifilter withut activated carbn. During the 16 hurs when n tluene was supplied t the reactr, almst n carbn dixide prductin (signifying degradatin) was bserved, indicating that tluene was nt buffered by the activated carbn in the bifilter.thisis prbably caused by the water present in the bifilter. The cntaminant first has t diffuse thrugh a water layer t reach the carbn surface. As liquid-phase diffusin is much slwer than gas-phase diffusin almst n tluene will be adsrbed at these shrt cntact times. Fr this reasn much lnger residence times (several hurs) are generally used in waste water treatment t buffer shck lads f cntaminants (Najm ef a/., 1991; Chatzpulus et al ). Ottengraf (1986) als cncluded that water present in a bifilter diminished the buffer capacity f activated carbn. By using a separate activated-carbn clumn placed befre the bifilter, the presence f water in the carbn filter can be prevented. Under these cnditins fluctuating tluene cncentratin culd indeed be effectively buffered (Fig. 4C). Tluene (900 mg/m 3 ) supplied t this system during 8 h a day was buffered t a stable cncentratin f abut 300 mg/m 3, which was cmpletely degraded in the bifilter resulting in a specific remval rate f 1240 g-c/(m 3 day). The degradatin rate in the bifilter withut activated carbn during the 8 h that tluene was supplied t the reactr was abut 85 g-c/(m 3 h). When tluene was supplied cntinuusly, the bserved degradatin rates were halved. This effect has als been bserved fr the degradatin f ethyl acetate in a bifilter (Nlte, 1992). These differences in maximal specific remval rates are remarkable but can be explained when the degradatin rc'e is assumed t be limited by mineralizatin prcesses in the bifilter. Release f inrganic nutrients frm inactive bimass (mineralizatin) will take place cntinuusly, and this can explain the higher degradatin rates bserved when tluene is supplied discntinuusly t the bifilter. The mineralizatin rate is apparently sufficient t allw a cntinuus remval rate f abut 40 g-c/(m 3 h) r a tw- t three-times higher activity when cntaminant remval nly takes place during 8 h per 24 h.

29 Activated carbn as a buffer in bif 'titratin 29 Several experiments have indeed shwn that the degradatin rate in bifilters is limited by the available amunt f nutrients (Dn, 1986; Beyreitz et al ; Weckhuysenef a/., 1993). In cnclusin, we have demnstrated that applicatin f an activated-carbn filter befre treatment f waste gases with a fluctuating cntaminant cntent can result in a better verall perfrmance f a bifilter. As a cnsequence smaller bifilters can smetimes be applied fr the treatment f a specific waste gas. It shuld, hwever, be emphasized that the waste gas entering the carbn filter has t be relatively dry. Fr the treatment f waste gases with a high water cntent the prcess seems less suitable. ACKNOWLEDGEMENTS We like t thank Ecsens bv (Merkapelle, The Netherlands) fr financial and technical supprt and Nrit nv (Amersfrt, The Netherlands) fr the dnatin f the activated carbns. REFERENCES Beyreitz, G., R. Hübner and M. Saake. (1989). Bitechnlgische Behandelung lösemittelhaltige Abluft. Wasser Luft und Bden 9: Chatzpulus, D., A. Varma and R.L. Irvine. (1993). Activated carbn adsrptin and desrptin f tluene in the aqueus phase. Am. Inst. Chem. Eng. J. 39: Diks, R.M.M, and S.P.P. Ottengraf. (1991). Verificatin studies f a simplified mdel fr the remval f dichlrmethane frm waste gases using a bilgical trickling filter. (Part II). Biprc. Eng. 6: Dn, J.A. (1986). The rapid develpment f bifiltratin fr the purificatin f diversified waste gas streams. In:VDI Berichte 561: Geruchstffe, pp , VDI Verlag, Düsseldrf. Dragt, A.J. (1992). Opening address. In: A.J. Dragt, and J. van Ham (eds.). Bitechniques fr air pllutin abatement and dur cntrl plicies, pp. 3-9,Elsevier, Amsterdam. Hartmans, S. and J. Tramper. (1991). Dichlrmethane remval frm waste gases with a trickle-bed bireactr. Biprc. Eng. 6: Najm, I.M., V.L. Sneyink, B.W. Lykins Jr. and J.Q. Adams. (1991). Using pwdered activated carbn: a critical review. J. Am. Wat. Wrks Ass. 83: Ntte, F.J. (1992). Ermittlung der Eliminatinsleistung eines Bifilters bei der Behundlung vn Ethylacetatablüften - Halbtechnische Piltanlage. In: A.J. Dragt, and J. van Harn (eds.), Bitechniques fr air pllutin abatement and dur cntrl plicies, pp , Elsevier, Amsterdam.

30 30 Chapter 2 Ottengraf, S.P.P. (1986). Exhaust gas purificatin. In: H.-J. Rehm, and G. Reed (eds.). Bitechnlgy, vl. 8, pp , VCH Verlagsgesellschaft, Weinheim. Weckhuysen, B.,L. Vriens and H.Verächter». (1993).The effect f nutrient supplementatin n the bifiltratin remval f butanal in cntaminated air. Appl. Micrbil. Bitechnl. 39:

31 3 Remval f tluene frm cntaminated air with a bilgical trickle-bed reactr Frans J. Weber and Sybe Hartmans Summary: Generally remval f relatively high cncentratins f rganic cmpunds like tluene frm waste gases in a trickle-bed reactr results in clgging f the reactr due t the frmatin f an excessive amunt f bimass. We therefre limited the amunt f nutrients available fr grwth, t prevent clgging f the reactr. As a cnsequence f this nutrient limitatin a lw remval rate was bserved. Hwever, when a fungal culture was used t inculate the reactr, the tluene remval rate under nutrient-limited cnditins was higher. Over a perid f 375 days an average remval rate f 27 g-c/(m 3 h) was btained with this fungal culture. We als studied the applicatin f a NaOHwash t remve excess bimass, as a methd t prevent clgging. Under these cnditins an average tluene remval rate f 35 g-c/(m 3 h) was btained. After abut 50 days there was n net increase in the bimass cntent f the reactr. The amunt f bimass which was frmed in the reactr equalled the amunt remved by the NaOH-wash. Submitted fr publicatin

32 32 Chapter 3 INTRODUCTION Bilgical treatment can be an effective and relatively cheap technique fr the remval f relatively lw cncentratins (<1 g/m 3 ) f cntaminants frm waste gases (Dragt, 1992; Ottengraf & Diks, 1992; Grenestijn & Hesselink, 1993). In Germany and The Netherlands bifilters have becme an accepted technique, especially fr the treatment f drus waste-gas streams (Annymus, 1989; Mildenberger, 1992). In bifilters the waste gas is blwn thrugh a packed bed f cmpst, peat, bark r ther rganic material, in which the natural micrbial flra will degrade the cntaminants present in the waste gas. A limitatin f bifilters is that nutrient levels (e.g. nitrgen) and the ph are difficult t cntrl. Humidificatin f the waste gas and maintaining a cnstant misture cntent f the filter can als be prblematic. Treatment f waste-gas streams cntaining chlrinated hydrcarbns is nt pssible in bifilters, due t the frmatin f hydrchlric acid. In a trickle-bed reactr these parameters can easily be cntrlled due t the presence f a circulating water phase. Previusly, efficient remval f dichlrmethane and dimethylacetamide using a trickle-bed reactr has been demnstrated (Diks & Ottengraf, 1991; Hartmans & Tramper, 1991; Waalewijn et a/., 1994). Althugh trickle-bed reactrs have certain advantages cmpared t bifilters, a majr disadvantage can be a reductin f reactr perfrmance due t the frmatin f excessive amunts f bimass. A stable remval f dichlrmethane frm waste gases ver a perid f mre than 600 days has been reprted (Diks & Ottengraf, 1991). Hwever, the simultaneus remval f dichlrmethane and acetne resulted in serius clgging f the reactr within 50 days (Diks, 1992). A reduce reactr perfrmance due t clgging was als bserved with several ther cmpunds (Kirchner et ai, 1991; Diks, 1992; van Lith et ai, 1994; Weber & Hartmans, 1994). Previusly, we have shwn that these clgging prblems nt nly ccurred due t the amunt f bimass frmed but als because f the pr bifilm frmatin n the packing material. Mst f the bimass was present in lumps between the packing material (Weber & Hartmans, 1994). As mst bacteria and packing materials are charged negatively an electrstatic repulsin exists between bacteria and between bacteria and the packing which prevents the frmatin f a stable bifilm. A higher inic strength f the medium results in decreased repulsin and thus better bifilm frmatin (Oliveira, 1992; Rijnaarts et ai, 1993; Zita & Hermanssn, 1994). Althugh an increase f the inic strength f the water phase resulted in a better bifilm frmatin (Weber & Hartmans, 1994), excessive

33 Tluene degradatin in a trickle bed reactr 33 bimass frmatin still can result in clgging and reduced reactr perfrmance and shuld thus be prevented. Especially, the remval efficiency f tluene in a trickle-bed reactr was shwn t be sensitive t changes in the cnditins in the reactr. The tluene remval rate sharply decreased when sme clgging f the reactr ccurred (Weber & Hartmans, 1994). The degradatin f ther cmpunds like butanne and butylacetate was nt significantly affected under these cnditins. The mderate water slubility f tluene prbably causes this high sensitivity t changes in the reactr cnditins. Therefre, tluene was chsen as a mdel cntaminant t further study and ptimize the perfrmance f trickle-bed reactrs. In this reprt we describe tw appraches t prevent clgging f the bireactr. MATERIALS AND METHODS Reactr All experiments were perfrmed in a semi-pilt scale bireactr, cnsisting f a clumn with a diameter f 0.3 m and a height f 1.5 m. The reactr was filled t 1 m with a randm-packing f Pall 50 mm rings (Vereinigte Füllkörper Fabrieken, Ramsbach-Baumbach, Germany; Specific surface area: 110 m 2 /m 3 ). The gas stream was intrduced at the bttm f the reactr, after being heated t 30 C by a heat-exchanger. The air flw was cntrlled at a rate f 7 m 3 /h (100 m 3 / (m 2 h)) by a swirl-meter (Fischer & Prter, Göttingen, Germany) and an autmatic valve. Air saturated with tluene was added t this gas stream t give the desired inlet cncentratin. Tluene-saturated air was btained by bubbling air thrugh a thermstated bubble clumn (10 cm x 50 cm) with tluene, the air flw thrugh this clumn was cntrlled by a mass-flw cntrller (Brks Instrument, Veenendaal, The Netherlands). The gas-phase pressure drp ver the reactr was cntinuusly mnitred by a differential pressure transducer. A mineral salts medium was circulated cunter-currently ver the reactr at a flw rate f 0.5 m 3 /h. The liquid was distributed n tp f the packing by means f a nzzle (BETE Fg Nzzle, Spraybest, Zwanenburg, The Netherlands) and was cllected at the bttm in a 70-dm 3 vessel. The temperature f the liquid in the vessel was cntrlled at 30 C and the ph was maintained at 7.0 by the additin f 0.5 N NaOH. Water was added t this vessel t cmpensate fr the water lst by evapratin. Liquid and bimass were nly remved frm this vessel as indicated in the varius experiments.

34 34 Chapter 3 Micrrganisms A bimass suspensin rich in fungi (>50% as examined by micrscpy) was btained by washing a bifilter, which has been used t remve tluene, with a 0.8% NaCI slutin. This suspensin is further designated as a fungal culture. An enrichment culture f especially bacteria was islated frm a water sample f the river Rhine near Wageningen, The Netherlands, using a phsphate-buffered (ph 7.0) mineral salts medium (Hartmans et al., 1989) supplemented with 200 mg tluene/liter. Medium In the trickle-bed reactr tap water supplemented with varius inrganic salts was used as the mineral salts medium.t a vessel f 70 I were added: 200 g NH 4 CI,20 g (NH 4 )2S0 4, 31 g KH 2 P0 4, 17 g NaH 2 P0 4 -H 2 0, 14 g MgCI 2-7H 2 0, 2.0 g FeS0 4, and 2.0 g CaCI 2. Fr the experiments where the bifilm grwth was cntrlled by a limitatin f the nutrients, half the amunt f nutrients were added. In all cases 820 g f NaCI was added t increase the inic strength t 0.2 M. Experimental cnditins The bireactrs were cnnected with an n-line gas-analyzer which cntinuusly mnitred the tluene and C0 2 cncentratins f the inlet and ff-gas streams. Rutinely abut 15 measurements were made frm each gas flw every day. Frm these measurements average cncentratins per day were calculated, which are used in the figures shwn here. Fr the nutrient limited trickle-bed reactr experiments (Fig. 1, 2 & 3), half the amunt f inrganic nutrients was added n day 1. One trickle-bed reactr was inculated with a bacterial culture, and a secnd reactr with a fungal culture. Bth reactrs were perated simultaneus under identical cnditins. On day 23 the tluene remval rate at varius rganic lads was measured in bth reactrs. Varius tluene cncentratins were applied t the reactrs and during ne hur the inlet and utlet cncentratins were measured every 10 minutes, after which the tluene lad was changed. In anther experiment the circulatin medium f the trickle-bed reactr was discharged and replaced with fresh medium every 14 days (Fig. 4). In a secnd reactr a NaOH wash was applied befre the medium was replaced. Fr the NaOH-wash 280 g f NaOH was added t the reactr medium which circulated thrugh the reactr, and after 3 hurs the medium was discharged and replaced with fresh medium. As the ph f the circulatin medium was still high after the medium replacement, the ph was restred t ph7.0 by the additin f 2N HCl.

35 Tluene degradatin in a trickle bed reactr 35 Analytical methds Bimass cncentratins were determined by weighing dried (24 h, 105 C) 10-ml samples f the liquid phase. The dry-weights were crrected fr the salts present in the medium. Tluene, 1,2-dichlrethane and C0 2 were analyzed with a CP 2001 gas-analyzer (Chrmpack, Middelburg, The Netherlands) equipped with an OV-1 clumn (4 m) and a Hayesep A clumn (25 cm). RESULTS Nutrient limitatin. A majr disadvantage, limiting the use f trickle-bed reactrs fr bilgical wastegas treatment, can be clgging caused by the frmatin f excessive amunts f bimass. This excessive bimass frmatin shuld be prevented and it is expected that this can be cntrlled by limiting the amunt f inrganic nutrients available fr grwth. T test this we perated a 70-1 trickle-bed reactr with a high lading rate f tluene (71 g/(m 3 h)) fr 145 days with a single dse f inrganic nutrients cntaining 100 g NH 4 CI. This amunt f nitrgen is expected t be sufficient t frm a bifilm f 400 urn n the packing, assuming a bimass nitrgen-cntent f 9% (w/w) (CH l8 O 05 N 016 S P ) (Rels, 1983) and a bifilm density f 100 kg dry-weight/m 3. u I Figure 1: Tluene degradatin rate ( ) and C0 2 frmatin rate ( ) in a trickle-bed reactr with an rganic lad f 65 g-c/(m 3 h). Additinal nutrients were supplied n day 145.

36 36 Chapter 3 In Fig. 1 the bserved tluene remval rate is shwn. On day 145 a secnd dse f inrganic nutrients was added t the reactr. Initially abut 45% f the tluene present in the inlet gas-flw was degraded. After a number f weeks the degradatin efficiency decreased t a stable level f abut 20% (13 g-c/(m 3 h)). When additinal nutrients were supplied n day 145, the degradatin efficiency almst immediately increased t mre than 75%, clearly illustrating that the bimass was nutrient limited. Als shwn in Fig. 1is the C0 2 frmatin rate (n carbn basis). The difference in C-remval and C0 2 frmatin indicates that, during the whle perid, a significant part f the degraded tluene is nt cnverted int C0 2. Effect f inculum The data shwn in Fig. 1 are fr a reactr that was inculated with an enrichment culture frm river water grwn n tluene. This culture mainly cntained bacteria. A secnd reactr was perated under the same cnditins, but this time a fungal culture was used t inculate the reactr. Interestingly, much higher tluene remval rates were bserved with this reactr (Fig. 2). During the first 120 days an average tluene remval f 48% (31 g-c/ (m 3 h)) was bserved in the trickle-bed reactr with the fungal culture. During the 375 days that the reactr was perated additinal nutrients were added nly 4 times. The average remval rate during these 375 days was 27 g-c/(m 3 h). During this perid there was n significant change in the pressure drp ver the reactr (<100 Pa/m). The difference in the remval rate between the reactrs inculated with respectively the bacterial and fungal culture are mre clearly shwn in Fig. 3. Here the bserved degradatin velcities are pltted against the tluene lad fr bth reactrs as determined n day 23. The bserved differences between the degradatin rates culd be caused by differences in the packing f the bifilm in bth reactrs resulting in differences in the specific surface area available fr exchange f tluene between the gas and liquid phase. T test the mass-transfer characteristics f the tw reactrs we used 1,2-dichlrethane t determine the transfer rate f this cmpund in bth reactrs. Dichlrethane was chsen as it is nt degraded by the bimass present in the reactrs. Fresh water at 30 C was pumped thrugh the reactrs nce (0.5 m 3 /h), and it was determined hw much dichlrethane was transferred t the water phase. At a gas-phase cncentratin f 950 mg/m 3 and a flw rate f 7 m 3 /h, 60% f the dichlrethane was transferred t the water phase in a reactr withut a bifilm. In the reactr with the bacterial and fungal cultures this was 64% and 66% respectively.

37 Tluene degradatin in a trickle bed reactr Time (d) Figure 2: Tluene degradatin rate ( ) in a trickle-bed reactr inculated with a fungal culture and an rganic lad f 64 g-c/(m 3 h). ( ) C0 2 frmatin rate. Additinal nutrients were nly supplied n days 145, 177, 275 and 316(A). u I 01 > c r "O (C b> <D Q Organic lad (g-c/(m 3 h)) Figure3: Observed tluene remval rates at varius rganic lads in a tricklebed reactr inculated with a bacterial culture (D) r a fungal culture (O). Dtted line crrespnds t 100% remval.

38 38 Chapter 3 Sdium hydrxide wash By limiting the amunt f nutrients available fr grwth, it was pssible t prevent clgging f the trickle-bed reactr, but the remval rate decreased due t the nutrient limitatin. We have als perated the trickle-bed reactr with regular additins f mineral salts medium, cntaining 200 g NH 4 CI (Fig. 4).This clearly results in higher average tluene remval rates than under nutrient limiting cnditins. The reactr was inculated with a mixture f bth the bacterial and fungal culture. The remval rate fluctuates between 15 and 45 g-c/(m 3 h) as a result f the tw-weekly nutrients additins, but a gradual decrease in the remval efficiency is als 60 O I Time (d) Figure 4 A-B: Tluene degradatin rate ( ) and C0 2 prductin rate ( ) in tw trickle-bed reactrs.the circulatin medium f bth reactrs is refreshed every 14 days ( ), in reactr Bthis is preceded by a NaOH-wash (0.1 M) f 3 h.the tluene lad f bth reactrs is65 g-c/(m 3 h).

39 Tluene degradatin in a trickle bed reactr 39 bserved. This is prbably due t the increase in the bimass cntent f the reactr resulting in clgging and a less efficient mass-transfer. As a result f the increase in bimass in the reactr the pressure drp ver the reactr fluctuated. Pressure drps as high as 1000 Pa/m were bserved a number f times during a few days. Usually the pressure drp was between 100 and 200 Pa/m. Anther methd t prevent clgging f the reactr can be the regular remval f bimass. Under nrmal cnditins mst f the bimass frmed in the reactr remains there. Only a small percentage is remved frm the packing by the liquid flwing thrugh the packing material. We have tried t remve mre bimass by intrducing a sdium hydrxide wash. Every tw weeks NaOH (endcncentratin 0.1 M) was added t the circulatin medium in the reactr. As a result excessive faming was bserved, indicating the release f prtein due t the lysis f bimass. After 3 hurs the medium was discharged and fresh medium was added t the reactr. The bserved remval rates in the reactr perated in this manner is shwn in Fig. 4B. After the NaOH-wash, the tluene remval rate was fully restred within ne day (Fig. 5). During the last 50 days, the average rganic lad f bth trickle-bed reactrs was 65 g-c/(m 3 h). In the reactr which was treated with the NaOH-wash tluene was remved at an average rate f 36 g-c/(m 3 h). In the reactr withut the NaOHwash this was 24 g-c/(m 3 h). The amunt f bimass which was remved by the NaOH-wash was estimated by dry-weight determinatins. An average f 3.3 g/l ( ) bimass was remved, crrespnding t 230 g dry-weight f bimass. u i Figure 5: Tluene remval rate befre and after an NaOH-wash (\P) f 3 hurs.

40 40 Chapter 3 DISCUSSION Althugh, bilgical waste-gas treatment using trickle-bed reactrs has been successfully demnstrated fr the remval f dichlrmethane frm waste gases (Hartmans & Tramper, 1991; Diks & Ottengraf, 1991), the degradatin f several ther cmpunds resulted in the inactivatin f the reactr due t clgging. T prevent this inactivatin f the reactr, excessive bimass accumulatin shuld be prevented. It is expected that by limiting the amunt f nutrients available fr grwth the bimass frmatin can be cntrlled. Using a bacterial culture as inculum an initial tluene remval rate f 30 g-c/ (m 3 h) was bserved, under nutrient-limited cnditins. Hwever, several weeks after depletin f the nutrients present in the reactr, the tluene remval rate decreased t an average (stable) level f 13 g-c/(m 3 h). Over a perid f 120 days an average tluene remval efficiency f 26% was btained. This is bviusly t lw fr a practical applicatin. Hwever, this lw remval efficiency was expected because f the high tluene lad applied t the reactr (65 g-c/ (m 3 h)). Bifilters, which are used t treat industrial waste gases are generally perated under significantly lwer lading cnditins (< 15 g-c/(m 3 h)) (Annymus, 1989; Mildenberger, 1992). At a reduced tluene lad f abut 10 g-c/ (m 3 h) a remval efficiency f abut 80% is btained in the trickle-bed reactr (Fig. 3). Differences in the perfrmance f a bireactr, hwever, can nt be effectively studied at lw lading cnditins (resulting in a high remval efficiency). Interestingly, when a fungal culture was used instead f a bacterial culture t inculate the reactr much higher remval rates were btained. Especially during the perid f nutrient depletin this difference was bvius. Over a perid f 375 days an average degradatin rate f 27 g-c/(m 3 h) is btained. In literature ften much higher activities have been reprted fr the remval f rganic cmpunds in bireactrs. Hwever, it is unclear if these high activities can be maintained during lnger perids f time. Cmpared t bifilters, which are used t treat waste gases frm industry the activity f bth the bacterial and fungal reactr is gd. In these industrial bifilters the remval rate is ften less than 15 g-c/(m 3 h) (Mildenberger, 1992). Als cmpared t the dichlrmethane remval in a trickle-bed reactr the bserved tluene remval rate is gd. Over a perid f 600 days an average dichlrmethane eliminatin capacity f 7 g- C/(m 3 h) (50 g dichlrmethane/(m 3 h)) has been btained in a trickle-bed reactr. (Diks& Ottengraf, 1991). The bserved differences in degradatin rates between the reactrs inculated with a bacterial r fungal culture are surprising. These results clearly shw that,

41 Tluene degradatin in a trickle bed reactr 41 Table 1: Averages f the rganic lad in the different experiments, and the btained remval efficiencies and the fractin f degraded tluene which is cnverted int C0 2. Reactr cnditins Nutrient-limitatin, "Bacteria" "Fungi" Average between (day) Lad (g Tluene-C/ (m 3 h)) Remval (g Tluene-C/ (m 3 h)) C0 2 frmed (g C0 2 -C/ (m 3 h)) Nutrient-additins nrmal NaOH-wash even under nn-sterile cnditins, inculatin f the reactr with a specific starter culture can influence the reactr perfrmance ver a prlnged perid f time. As the gas-liquid transfer rate f dichlrethane (which is nt degraded by the bimass in the reactr) was almst identical in bth the bacterial and fungal reactr, it is nt expected that differences in the bifilm structure explain the remarkable differences in degradatin rate. Apparently, the fungal culture is capable t degrade mre tluene under nutrient-limitated cnditins than the bacterial culture. It is expected that under nutrient-limited cnditins there will be n increase in the ttal amunt f bimass nce the nutrients have been incrprated int bimass. Besides fr the frmatin f bimass rganic carbn is als required as an energy surce fr maintenance requirements. A maintenance cefficient f 0.02 g glucse-c/(g bimass-c h) is a average value reprted in literature (Andersn & Dmsch, 1985;Tijhuis et ai, 1993). With this maintenance cefficient and assuming that 155 g bimass-c is frmed frm the nutrients added t the reactr a maintenance activity f 44 g-c/(m 3 h) can be calculated. Assuming that all the bimass is still active, this activity is in the range f the activities that were bserved in the reactrs. When, the reactr is running n the maintenance requirements f the bimass, a cmplete cnversin f tluene int C0 2 is expected. Hwever, less C0 2 is frmed, indicating that even when the nutrients are depleted a substantial amunt f the tluene is assimilated by the bimass resulting in the accumulatin f reserve materials (e.g. glycgen). Under nutrient-limited cnditins.

42 42 Chapter 3 accumulatin f these carbhydrate plymers up t 50% f the cell dry-weight has been bserved (Herbert, 1961; Dawes & Senir, 1973). Extracellular plysaccharides (EPS) can als be an imprtant carbn-sink during nutrientlimited grwth. These plysaccharides are prduced by the micrrganisms and frm a plymer which traps the micrbial cells. The extracellular plymer matrix can accunt fr 50 t 90% f the bifilm rganic carbn (Bakke et a/., 1984). Althugh the limitatin f nutrients will restrict the grwth f cells, a large amunt f the tluene is apparently cnverted int biplymers like glycgen and EPS. In the reactr with the bacterial culture, abut 8 g-c/(m 3 h) is nt cnverted int C0 2. Over the perid f 120 days this crrespnds t 1.6 kg-c f cells and plymers which is frmed in the reactr. Frm the 28 g-n f nitrgen-cntaining cmpunds, which were added t the reactr, nly abut 310 g f bimass was expected t be frmed. This wuld indicate that under these nutrient-limited cnditins an enrmus amunt f EPS and ther plymers are frmed. Althugh this seems t be an unrealistic amunt, we have bserved that in a trickle-bed reactr t which nly 17 g f nitrgen was added, mre then 15 kg f wet weight bimass was frmed within ne mnth (unpublished results). When mre than 1.6 kg-c bimass is present in the reactr, this will crrespnd t a bifilm thickness f abut 3.5 mm (Assuming a bifilm density f 100 kg/m 3 with an average carbn cntent f 60% fr the bifilm, including EPS and ther plymers). Visual examinatin f the bifilm in the reactr did indeed shw a thick bifilm. Althugh the nutrient limitatin did nt result in a thin bifilm, a high and stable tluene remval rate was btained with the fungal culture. As fungi are prminent decmpsers f nitrgen-pr plant plymers, they may have develped special mechanisms that enable them t grw in these nitrgendeficient envirnments. Several studies have suggested that hyphal utgrwth f fungi is pssible withut a net increase in cell cytplasm. It is assumed that the cytplasm is translcated t the grwing hyphae (Cwling & Merill, 1966; Levi & Cwling, 1969; Watkinsn et a/., 1981; Littke ef a/., 1984; Paustian & Schnürer, 1987). As the cytplasm cntains mst f the nitrgen and phsphate an internal recycle f the cytplasm seems t be an effective mechanism t grw under these nutrient-limited cnditins. Sme ther fungi may be able t further extend N resurces by lysing degenerated hyphae and reassimilating the N released. (Levi et al., 1968). Especially fungi are knwn fr there capacity t grw n dead bimass (Fermr & Wd, 1981; Grant et a/., 1986). It is expected that this capacity f fungi t mbilize nutrients fr grwth is a reasn fr the higher degradatin rate that is bserved in the reactr with the fungal culture.

43 Tluene degradatin in a trickle bed reactr 43 As als under nutrient-limited cnditins the bimass cntent (including EPS and ther plymers) f the reactr increases, this can eventually result in clgging f the reactr. The fact that n clgging was bserved under the nutrient-limited cnditins in ur experiments is presumably due the relatively lw specific surface area f the packing material we used (110 m 2 /m 3 ). Hwever, t be sure that clgging will nt ccur eventually bimass has t be remved frm the reactr. With a lw rganic lad, the amunt f bimass frmed in the reactr can be remved frm the reactr by the liquid drain as was the case after abut 200 days fr a trickle-bed reactr remving dichlrmethane (Diks er a/., 1994). The bimass yield n tluene, hwever, is much higher cmpared t dichlrmethane and the amunt f bimass that is remved by the liquid drain is nt sufficient t cunterbalance the bimass that is frmed. Fr a higher and stable tluene remval under nn-limiting cnditins, bimass remval by a NaOH-wash seems t be an effective methd t prevent clgging. By washing the reactr with 0.1 M NaOH during 3 hurs an average f 230 g f dry-weight bimass culd be remved frm the reactr every tw weeks. Based n the nitrgen cntent f the added nutrients, 560 g f bimass (CH, 8O 05 N 0]6 ) can be frmed. Based n the amunt f tluene degraded and the carbn dixide frmed 235 g bimass is frmed in the perid prceeding the NaOHwash. Especially the value calculated frm the carbn balance ver the reactr crrespnds very well with the amunt which is actually remved frm the reactr by the NaOH-wash. Once an equilibrium is reached between the amunt f bimass which is remved by the NaOH-wash and the amunt f bimass which is frmed in the reactr, it shuld be pssible t maintain a high tluene remval rate withut clgging f the reactr taking place. ACKNOWLEDGEMENTS We wuld like t thank K Hage fr perfrming sme f the experiments and Ecsens bv (Merkapelle, The Netherlands) fr their financial and technical supprt. REFERENCES Andersn, T.-H. and K.H. Dmsch. (1985). Maintenance carbn requirements f activelymetablizing micrbial ppulatins under in situ cnditins. S/7 Bil. Bichem. 17: Annymus. (1989). Bilgische Abgas-/Abluftreinigung: Bifilter. VDI-Handbuch Reinhaltung der Luft. Band 6. VDI 3477 (entwurf), Verein Deutscher Ingenieure, Düsseldrf.

44 44 Chapter 3 Bakke, R., M.G. Trulear, J.A. Rbinsn and W.G. Characklis. (1984). Activity f Pseudmnas aeruginsa in bifilms: Steady State. Bitechnl. Bieng. 26: Cwling, E.B. and W. Merill. (1966). Nitrgen in wd and its rle in wd deteriratin. Can. J. Btany 44: Dawes, E.A. and P.J.Senir. (1973).The rle and regulatin f energy reserve plymers in micr-rganisms. Adv. Micrbial. Physil.10: Diks, R.M.M, and S.P.P. OHengraf. (1991). Verificatin studies f a simplified mdel fr the remval f dichlrmethane frm waste gases using a bilgical trickling filter. (Part II). Biprc. Eng. 6: Diks, R.M.M. (1992). The remval f dichlrmethane frm waste gases in a bilgical trickling filter. PhD-Thesis, Technical University, Eindhven. Diks, R.M.M., S.P.P. Ottengraf and S. Vrijland. (1994). The existence f a bilgical equilibrium in a trickling filter fr waste gas purificatin. Bitechnl. Bieng. 44: Dragt, A.J. (1992). Opening address. In: A.J. Dragt, and J. van Ham (eds.). Bitechniques fr air pllutin abatement and dur cntrl plicies, pp.3-9, Elsevier, Amsterdam. Fermr, T.R. and A.D. Wd. (1981). Degradatin f bacteria by Agaricus bisprus and ther fungi. J. Gen. Micrbil. 126: Grant, W.D., L.L. Rhdes, B.A. Prsser and R.A. Asher. (1986). Prductin f bacterilytic enzymes and degradatin f bacteria by filamentus fungi. J. Gen. Micrbil. 132: Grenestijn, J.W. and P.G.M. Hesselink. (1993). Bitechniques fr air pllutin cntrl. Bidegradatin 4: Hartmans, S., J.P. Smits, M.J. van der Werf, F. Vlkering and J.A.M. de Bnt. (1989). Metablism f styrène xide and 2-phenylethanl in the styrene-degrading Xanfhbacfer strain 124X. Appl. Envirn. Micrbil. 55: Hartmans, S. and J.Tramper. (1991). Dichlrmethane remval frm waste gases with a trickle-bed bireactr. Biprc. Eng.6: Herbert, D. (1961). The chemical cmpsitin f micr-rganisms as a functin f their envirnment. In: G.G. Meynell, and H. Gds (eds.). Micrbial reactin t envirnment, pp , Sciety fr General Micrbilgy, Cambridge. Kirchner, K., CA. Gssen and H.-J. Rehm. (1991). Purificatin f exhaust air cntaining rganic pllutants in a trickle-bed bireactr. Appl. Micrbil. Bitechnl. 35: Levi, M.P., W. Merill and E.B. Cwling. (1968). Rle f nitrgen in wd deteriratin. VI. Mycelial fractins and mdel nitrgen cmpunds as substrates fr grwth f Plyprus versiclr and ther wd-destrying and wd-inhabiting fungi. Phyfpafh/gy 58: Levi, M.P. and E.B. Cwling. (1969). Rle f nitrgen in wd-deteriratin. VII. Physilgical adaptatin f wd-destrying and ther fungi t substrates deficient in nitrgen. Phyfpafh/gy 59:

45 Tluene degradatin in a trickle bed reactr 45 van LIth,C.P.M.,S.P.P.Ottengraf and R.M.M. Diks. (1994).The cntrl f a bitrickling filter. In:VDI Berichte 1104, Bilgische Abgasreinigung, pp , VDI Verlag, Düsseldrf. Littke,W.R., CS. Bledse and R.L Edmnds. (1984). Nitrgen uptake and grwth in vitr by Hebelma crutulinifrme and ther Pacific Nrthwest mycrrhizal fungi. Can. J. Btany 62: Mildenberger, H.-J. (1992). Bifiltersysteme zur Geruchsbeseitigung und zur Reduzierung vn Organika-Emissinen auf Kläranlagen und in der chemischen Industrie. In: A.J. Dragt, and J. van Harn (eds.), Bitechniques fr air pllutin abatement and dur cntrl plicies, pp ,Elsevier, Amsterdam. Oliveira, D.R. (1992). Physic-chemical aspects f adhesin. In: L.F. Mel, T.R. Bth, M. Fletcher, and B. Capdeville (eds.), Bifilms - Science and technlgy, NATO ASISeries E, Vl. 223, pp ,Kluwer Academic Publishers, Drdrecht. Ottengraf, S.P.P. and R.M.M. Diks. (1992). Prcess technlgy f bitechniques. In: A.J. Dragt, and J. van Dam (eds.). Bitechniques fr air pllutin abatement and dur cntrl plicies, pp , Elsevier, Amsterdam. Paustian, K. and J. Schnürer. (1987). Fungal grwth respnse t carbn and nitrgen limitatin: Applicatin f a mdel t labratry and field data. S/7 Bil. Bichem. 19: Rijnaarts, H.H.M., W. Nrde, E.J. Buwer, J. Lyklema and A.J.B. Zehnder. (1993). Bacterial adhesin under static and dynamic cnditins. Appl. Envirn. Micrbil. 59: Rels, J.A. (1983). Energetics and kinetics in bitechnlgy. Elsevier Bimedical Press, Amsterdam. Tijhuis, I., M.C.M. van Lsdrecht and J.J. Heijnen. (1993). A thermdynamically based crrelatin fr maintenance Gibbs energy requirements in aerbic and anaerbic grwth. Bitechnl. Bieng. 42: Waalewijn, E., CD. Meijer, F.J. Weber, U.C. Duursma and P. van Rijs. (1994). Practice runs f a bi-trickling filter in the exhaust gases frm a fibre factry. In: VDI Berichte 1104, Bilgische Abgasreinigung, pp ,VDI Verlag, Düsseldrf. Watkinsn,S.C., E.M. Davidsn and J.Bramah. (1981). The effect f nitrgen availability n grwth and cellullysis by Serpula lacrimans. New Phytlgist 89: Weber, F.J. and S. Hartmans. (1994). Tluene degradatin in a trickle bed reactr. - Preventin f clgging. In: VDI Berichte 1104, Bilgische Abgasreinigung, pp , VDI Verlag, Düsseldrf. ZHa, A. and M. Hermanssn. (1994). Effects f inic strength n bacterial adhesin and stability f fles in a wastewater activated sludge system. Appl. Envirn. Micrbil. 60:

46 46

47 4 Grwth f the fungus Cladsprium sphaerspermum with tluene as the sle carbn and energy surce Frans J. Weber K C. Hage and Jan A.M. de Bnt Summary: The fungus Cladsprium sphaerspermum was islated frm a bifilter used fr the remval f tluene frm waste gases.thisis the first reprt describing grwth f a eukarytic rganism with tluene as the sle surce f carbn and energy. The xygen cnsumptin rates, as well as the measured enzyme activities, f tluene-grwn C. sphaerspermum indicate that tluene is degraded by an initial attack n the methyl grup. Applied and Envirnmental Micrbilgy (1995) 61: inpress

48 48 Chapter 4 INTRODUCTION The cmplete aerbic bidégradatin f tluene t carbn dixide, water, and bimass by bacteria has been studied extensively. Different initial xidative reactins have been identified fr a variety f bacteria, and the bidégradatin f the resulting xygenated cmpunds is well dcumented (Kitagawa, 1956; Claus & Walker, 1964; Gibsn et al., 1970; Wrsey & Williams, 1975; Shields et a/., 1989; Kaphammer et a/., 1990; Shields et al., 1991; Whited & Gibsn, 1991). The presently knwn five different pathways fr the utilizatin f tluene in bacteria are summarized in Fig. 1. In humans and animals, the initial step in the metablic transfrmatin f tluene appears t be hydrxylatin f tluene t benzyl alchl (Fig. 1) by a mnxygenase. Benzyl alchl is further cnverted t benzic acid and excreted in urine, either unchanged r as its glycine cnjugate, hippuric acid (Annymus, 1985). In cntrast, utilisatin f tluene by fungi as the sle carbn and energy surce has nt been reprted. Partial degradatin f tluene by fungi was bserved by Hlland et al. (1988). Cultures f Mrtierella isabeilina and Helminthsprium species were pregrwn n a rich medium with glucse and when the mycelium was subsequently incubated in water fr 72 hurs, it cnverted tluene int benzyl alchl. Degradatin f tluene by the white rt fungus Phanerchaete chryssprium has been demnstrated by using ring-labeled [ 14 C]tluene and by measuring the prductin f 14 C0 2 (Yadav & Reddy, 1993). During these experiments, the fungus was grwn fr 3 weeks in media cntaining glucse (1%) and tluene was added at 5 mg/l. The quantity f tluene degraded was very small n the rder f 1 mg f tluene degraded per g f mycelium per day. Interestingly, the degradatin tk place under nnligninlytic culture cnditins, indicating that lignin perxidases r ther perxidases were nt invlved. It is knwn frm many ther studies that these enzymes play a key rle in the partial xidatin f anthracene t anthraquinne (Field et al., 1992) and in the partial xidatin f many ther xenbitic armatic cmpunds (Field et a/., 1993). A number f nnligninlytic fungi have intracellular mechanisms fr the partial degradatin f cmplex armatic structures, as dcumented by Cerniglia (1984), and yeasts are als able t partially xidize plycyclic armatic hydrcarbns (Hfmann, 1986; MacGillivray & Shiaris, 1993). Fungal grwth with armatic hydrcarbns ther than tluene has been tested fr n-alkylbenzenes and fr styrene. Fungi islated n n-alkanes were grwn in the presence f n-alkylbenzenes t determine the effect f the side chain length (C, t C 12 ). The rganisms did nt grw in the presence f tluene, maybe as a result f the txicity f this cmpund, but they grew n ther cmpunds, fr instance

49 Grwth f C. sphaerspermum n tluene 49 CH-OH CHO COOH [HOOC A. ~] HOOC OH Fissin (A) / Benzylalchl Benzate Benzaldehyde C/s-Benzatedil Catechl CH, C/s-2,3-Dihydrxy- 2,3 Dihydrtluene Fissin 3-Methylcatechl L-- OH OH OH OH OH p-cresl 4-Hydrxy- 4-Hydrxy- 4-Hydrxy- Prtcatechuate benzylalchl benzaldehyde benzate CH,OH CHO COOH C-NH-CHj-COOH ^... ^... ^ tîf fy Benzyl alchl Benzaldehyde Benzate Hppuricacid Fissin Excretin Figure 1: Tluene bidégradatin rutes in bacteria ( ), and mammals ( J. Cmpsed after (A) Wrsey & Williams (1975), (B) Gibsn et al. (1970). (C) Shields et al. (1989). (D) Kaphammer et al. (1990) (E) Whited & Gibsn (1991), and (F) Epstein & Braunstein (1931)

50 50 Chapter 4 n ddecylbenzene, frm which the rganism accumulated benzic and phenylacetic acids (Fedrak & Westlake, 1986). Tw styrene-degrading fungi have been islated (Hartmans et ai, 1990), and ne strain identified as Exph/ala jeanselmei was subsequently studied in mre detail (Cx et ai, 1993a).Theresults btained with E. jeanselmei (Cx et ai, 1993a) indicate that the styrene degradatin pathway in the fungus was similar t a pathway bserved fr a styrene-degrading bacterium (Hartmans et ai, 1990). The metablism by fungi f xygenated armatic cmpunds has been amply studied, and reviews f this field are available (Cain et ai, 1968; Middelhven et ai, 1992; Wright, 1993). During ur investigatins f the remval f tluene frm cntaminated air by use f a cmpst bifilter, we macrscpically bserved abundant fungal grwth. Micrscpic examinatin cnfirmed that fungi were predminant in this system, remving tluene frm waste gases. On the basis f current knwledge f tluene bidégradatin, it might be expected that the fungi nly partly metablize tluene. Alternatively, they might be invlved in the degradatin f partly xidized prducts derived by bacteria frm tluene, since it is knwn that fungi are able t metablize xygenated armatic cmpunds, as, fr instance, benzate. Hwever, in view f the abundance f the fungi in the bifiltratin system, it seemed mre reasnable t expect that the fungi were invlved in the cmplete bidégradatin f tluene. T test if a eukarytic tluene-degrading rganism was indeed present, we decided t islate fungi frm the bifilter and t investigate ne pure culture in mre detail. MATERIALS ANDMETHODS Islatinf fungi Fungi were islated frm a cmpst bifilter (70 liters), which had been used t remve tluene frm cntaminated air («150 mg/m 3, 7 m 3 /h) fr 3 mnths, by washing the bifilter with a 0.8% NaCI slutin. Frm this suspensin dilutins were made, and the resulting suspensins were seeded n agar plates with a mineral salts medium (Hartmans et ai, 1990) and 10 U9 f streptmycin per ml t minimize bacterial grwth.these plates were incubated at 30 C in a desiccatr cntaining tluene vapr, resulting in a cncentratin f abut 200 mg/l in the slidified 'liquid' phase as calculated by using a water/air partitin cefficient f 3.8 (Amre & Hautala, 1983).Pure fungal cultures were btained by subsequent transfers t new agar plates.

51 Grwth f C. sphaerspermum n tluene 51 Grwth with vlatile armatic cmpunds Fr grwth experiments, 250-ml flasks cntaining 10 ml f a phsphate buffered (ph 7) mineral salts medium (Hartmans et a/., 1990) were used t which the varius armatic hydrcarbns were added. The bttles were clsed with Tefln valves (Mininert, Phase Separatins, Waddinxveen, The Netherlands) t prevent evapratin f the varius added armatic hydrcarbns and incubated under statinary cnditins at 30 C. Grwth was assessed by mnitring the prductin f C0 2. Preparatin f washed mycelium suspensins In rder t btain larger amunts f mycelium, Cladsprium sphaerspermum was grwn in a flat-bttmed rund flask (2 liters) with 500 ml f phsphatebuffered mineral salts medium at 30 C. Air cntaining apprximately 3000 mg f tluene per m 3 was bubbled thrugh the medium at a flw rate f 125 ml/min. Mycelium was harvested frm the grwth medium by filtratin ver cheeseclth, washed with 50 mm ptassium phsphate buffer (ph 7.0), and resuspended in the same buffer. Oxygen cnsumptin experiments The xygen cnsumptin f washed mycelium suspensins was determined with a Clark type xygen electrde. The xygen cnsumptin f a 4-ml mycelium suspensin was mnitred fr at least 5 min after the additin f 50 pi f a stck slutin f 20 mm substrate in N,N-dimethylfrmamide. Additin f N,Ndimethylfrmamide resulted in a minimal increase in the endgenus xygen uptake rate. After measuring the xygen cnsumptin rate f the substrate, we checked if the substrate cncentratin used was nt txic by measuring the xygen cnsumptin rate after the additin f 50 ul f the tluene stck slutin. Determinatin f enzyme activities in cell-free extracts Washed mycelium suspensins f tluene-grwn C. sphaerspermum were frzen at -30 C and disrupted by tw passages thrugh a prechilled (-30 C) 5-ml X-press (AB Bix, Götebrg, Sweden). After being slwly thawed the paste was diluted in 50 mm ptassium phsphate buffer (ph 7.0) and centrifuged at 20,000 x g fr 10 min at 4 C. The supernatant btained was used fr the enzyme activity determinatins. The activities f the mnxygenases and dehydrgenases were determined spectrphtmetrically. The reactin mixture (ttal vlume, 1 ml) cntained cell-free extract diluted in the phsphate buffer and 0.2 uml f cfactr (NAD +, NADP +, NADH, r NADPH). The activities f bth 4-hydrxybenzyl alchl dehydrgenase and 4-hydrxybenzaldehyde dehydrgenase were crrected fr the level f high adsrbance f 4-hydrxy-benzaldehyde at

52 52 Chapter nm. p-cresl methylmnxygenase was assayed by mnitring the 0 2 uptake as described previusly (Hpper & Taylr, 1977), except that a 50 mm ptassium phsphate buffer was used. Catechl-1,2-dixygenase was mnitred by measuring the frmatin f c/'s-c/'s-mucnic acid at 260 nm (Gibsn, 1971b). The activity f prtcatechuate-3,4-dixygenase was measured spectrphtmetrically at 290 nm (Gibsn, 1971b). 3-Methylcatechl-l,2- dixygenase was determined by measuring the frmatin f the ring fissin prduct at 390 nm. Analytical methds Carbn dixide cncentratins were determined by injecting 100 \i\ head space samples int a Packard 427 gas Chrmatgraph (Packard/Becker, Delft, The Netherlands) equipped with a Hayesep Q clumn (Chrmpack B.V. Middelburg, The Netherlands). Prtein was quantified by the Bradfrd methd (Bradfrd, 1976) with bvine serum albumin as the standard. Cell dry weight was determined by weighing dried (24 h 105 C) cell suspensins. RESULTS Islatin f fungi frm the bifilter Frm a cmpst bifilter, which had been used t remve tluene frm cntaminated air, a bimass suspensin rich in fungi (>50% as examined by micrscpy) was btained by washing the bifilter with an NaCI slutin. Frm this suspensin several pure fungal cultures were islated, and they grew n mineral agar plates incubatin in the presence f tluene vapr. Grwth f fungi n tluene As these islated fungi culd pssibly grw n impurities frm the agar plates, the capacity f these fungi t grw n tluene was checked by using liquid medium. One f the islated fungi grew in liquid medium with tluene as the sle carbn and energy surce. This fungus was identified by the Centraalbureau vr Schimmelcultures (CBS) (Baarn, The Netherlands) as Cladsprium sphaerspermum Penzig. In Fig. 2, a typical grwth curve fr C. sphaerspermum grwn with an initial amunt f 6 (il (56 Mml) f tluene in the incubatin system (10 ml f liquid medium in a 250-ml culture bttle) is shwn. This initial amunt f tluene in the incubatin system, n the basis f its partitin cefficient, results in a cncentratin f 0.8 mm in the water phase. In abut 10 days all the added tluene was degraded and abut 210 u.ml f C0 2 and abut 6 mg (dry weight)

53 Grwth f C. sphaerspermum n tluene 53 </> m E 200 ~ O) "D r b> <D "O 0) c <D O <1) u T> O O ü Time (h) Figure 2: Grwth f C. sphaerspermum with tluene in 10 ml f mineral salts medium. D, amunt f tluene degraded; O, ttal amunt f C0 2 prduced. f cells was frmed. The grwth rate was maximal at ph 7.0 under statinary cnditins. Grwth f C. sphaerspermum with ther armatic slvents Grwth f C. sphaerspermum with several ther armatic hydrcarbns was als tested. As the armatic hydrcarbns used can be txic even at lw cncentratins, varius amunts f the hydrcarbns were tested (10, 20, and 50 uml). After 10 days f incubatin, grwth was assessed by C0 2 determinatin. Besides tluene, C. sphaerspermum culd als use styrene, ethylbenzene, and prpylbenzene as sle carbn and energy surces fr grwth at all cncentratins tested (>40 uml f C0 2 prduced). N grwth was bserved with -xylene, benzene r phenl at any f the cncentratins tested. Whether C. sphaerspermum culd grw with several intermediates f the knwn bacterial degradatin pathways f tluene was als tested (Fig. 1). Grwth was bserved with benzyl alchl, benzaldehyde, benzate, and catechl. Grwth with benzate and catechl was bserved nly at 10 and 20 ^ml; n grwth was bserved at 50 (jml. N grwth with 3-methylcatechl was bserved at any f the cncentratins tested.

54 54 Chapter 4 Table 1: Rates f xygen cnsumptin by washed cell suspensins f C. sphaerspermum grwn n tluene. Assay substrate Oxygen cnsumptin (nml 0 9 / min mg cell dry-weight) Tluene 164 Benzyl alchl 154 Benzaldehyde 155 Benzate 13 Catechl 77 3-Methylcatechl <5 -Cresl <5 m-cresl <5 p-cresl' 11 4-Hydrxybenzyl alchl <5 4-Hydrxybenzaldehyde Hydrxybenzate 6 4-Hydrxybenzate 7 Prtcatechuate <5 ' Inhibited the tluene induced xygen cnsumptin rate. Table 2: Activities f enzymes pssible invlved in the catablism f tluene in cell-free extracts f C. sphaerspermum. Enzyme Dehydrgenases Benzyl alchl Benzaldehyde 4-Hydrxybenzyl alchl 4-Hydrxybenzaldehyde Mnxygenase 4-Hydrxybenzate Dixygenases (intra dil) Catechl Prtcatechuate 3-Methylcatechl Cfactr NAD + NADP + NADH NADPH NAD + NADP + NADH NADPH NADH +FAD NADPH +FAD Enzyme activity (nml / min mg prtein) 169 < < <25

55 Grwth fc. sphaerspermum n tluene 55 Oxygen cnsumptin experiments Oxygen cnsumptin rates f washed suspensins f C. sphaerspermum grwn with tluene were mnitred in the presence f varius pssible intermediates f the tluene degradatin pathway (Table 1). Additin f tluene after measurement f the p-cresl-induced xygen cnsumptin rate resulted in a reduced tluene-induced xygen cnsumptin rate.this inhibitin f the tluene xygen cnsumptin rate was bserved nly fr p-cresl, even when a ten-fld lwer cncentratin f p-cresl was used.the results presented in Table 1 have been crrected fr the endgenus xygen uptake (96 nml 0 2 / min mg [dry weight] f cells ). Enzymeactivitiesin cell-free extracts Cell-free extracts f tluene-grwn C. sphaerspermum were used t measure the activities f varius enzymes pssibly invlved in the degradatin f tluene (Fig. 1). 4-Hydrxybenzate mnxygenase activity culd be measured nly after the additin f FAD. Additin f FAD and ferrus ammnium sulfate t the reactin mixture did nt enable in vitr mnxygenase activity t be btained with any f the ther substrates tested (tluene, benzate, 3-hydrxybenzate, - cresl, r p-cresl). As many f these mnxygenases are unstable enzymes and have prven difficult t islate (Gibsn, 1971a), the inability t measure these enzyme activities is nt very surprising. The activities f the measured dixygenases and dehydrgenases f tluene-grwn C. sphaerspermum cells are shwn in Table 2. DISCUSSION Frm a bifilter used t remve tluene frm cntaminated air, we have islated a fungus, C. sphaerspermum, which is able t grw with tluene as the sle surce f carbn and energy. T ur knwledge this is the first reprt f tluene catablism by a eukarytic micrrganism. The spectrum f substrates used fr grwth by C. sphaerspermum cells indicates that tluene is degraded by an initial attack n the methyl grup, leading t benzate. C. sphaerspermum culd use benzyl alchl, benzaldehyde and benzate as sle carbn and energy surces. The rute fr tluene degradatin was further investigated by measuring the xygen cnsumptin rates f varius pssible intermediates in tluene-grwn mycelium f C. sphaerspermum. A prerequisite fr measuring a substrateinduced increase in the xygen cnsumptin rate by whle cells is the ability f the cmpund t pass thrugh the cytplasmic membrane. It is expected that

56 56 Chapter 4 substrates like tluene, -cresl, m-cresl, and p-cresl will enter the cell by diffusin thrugh the cytplasmic membrane. The fact that n significant activity was bserved with -cresl, m-cresl, and p-cresl indicates that these cmpunds are nt intermediates f the tluene degradatin pathway. Charged cmpunds like benzate, hwever, may require an active transprt system t enter the cell. The absence f an increase in xygen cnsumptin after the additin f these substrates culd thus very well be caused by the lack f a suitable membrane transprt system. Since an appreciable increase in xygen cnsumptin was bserved after the additin f benzyl alchl, benzaldehyde, and catechl and n activity was btained with -cresl, m-cresl, p-cresl, p- hydrxybenzyl alchl, and 3-methylcatechl, it appears that tluene is degraded by a hydrxylatin f the methyl grup (Fig. 1 ). The results frm bth the grwth and xygen cnsumptin experiments indicated that tluene is degraded by an initial attack n the methyl grup. The high activities measured fr benzyl alchl dehydrgenase and benzaldehyde dehydrgenase cnfirm these results. The activities fr the degradatin f 4- hydrxybenzaldehyde bth in cell suspensins (Table 1) and in extracts (Table 2) are nt in keeping with the prpsed pathway, but these activities are likely due t an aspecific reactin f the benzaldehyde dehydrgenase. It has indeed been shwn that in Pseudmnas cnvexa, 4-hydrxybenzaldehyde and benzaldehyde are xidized by the same dehydrgenase (Bhat & Vaidyanathan, 1976). In mammals, armatic hydrcarbns usually are xidized t an epxide and subsequently a frans-dihydrdil is frmed (Gibsn, 1971a, 1993). Such a pathway has been established, fr instance, fr benzene (Gibsn, 1971a), but tluene was degraded in a different fashin with an initial xidatin at the methyl grup (Gibsn, 1971a; Annymus, 1985). Our results indicate that the mammalian and fungal degradatins f tluene are similar in that the initial reactin in bth cases is with the methyl grup and results in benzate. Benzate in mammals either is an end prduct and is excreted r is cnjugated t hippuric acid. In bacteria, benzate is further metablized via ci's-dihydrdil t catechl as the ring fissin substrate. In all fungi studied, the sle pathway fr benzate metablism is by hydrxylatin f benzate t 4-hydrxybenzate, leading t prtcatechuate as the ring fissin substrate (Wright, 1993). The measured activities fr 4- hydrxybenzate mnxygenase and prtcatechuate-3,4-dixygenase indicate that benzate in C. sphaerspermum is als degraded via 4- hydrxy benzate. In cell-free extracts f C. sphaerspermum a catechl dixygenase activity, besides a prtcatechuate dixygenase activity, was als detected, indicating

57 Grwth f C. sphaerspermum n tluene 57 that catechl is a ring fissin substrate. As the prtcatechuate dixygenase activity is measured by disappearance f prtcatechuate, this activity culd als be caused by the cnversin f prtcatechuate int catechl. It has been shwn in several fungi that 4-hydrxybenzate is degraded via prtcatechuate, leading t catechl as the ring fissin substrate (Cain er ai, 1968). Whether prtcatechuate is the actual ring fissin substrate r is cnverted int catechl is uncertain and will require further investigatin. The discvery f tluene-degrading fungi is f imprtance fr bifiltratin. A disadvantage f bifilters used fr waste-gas treatment is that the ph cannt be cntrlled, and humidificatin f the filter can be prblematic (Ottengraf, 1986). Bth these parameters shuld be carefully cntrlled t allw an extended peratin f the filter. The applicatin f armatic hydrcarbn-degrading fungi in these bifilters might have tw advantages, as fungi generally require less stringent cntrl f bth the ph and the water activity. A reductin f the water activity in the bifilter might als enhance the mass transfer f substrates prly sluble in water (Cx et a/., 1993b). REFERENCES Amre, J.E. and E. Hautala. (1983). Odr as an aid t chemical safety: dr threshlds cmpared with threshld limit values and vlatilities fr 214 industrial chemicals in air and water dilutin. J. Appl. Txicl. 3: Annymus. (1985). Tluene. Envirnmental Health Criteria 52, Wrld Health Organizatin, Geneva. Bhat, S.G. and CS. Vaidyanathan. (1976). Invlvement f 4-hydrxymandelic acid in the degradatin f mandelic acid by Pseudmnas cnvexa. J. Bacteril. 127: Bradfrd, M.M. (1976). A rapid and sensitive methd fr the quantitatin f micrgram quantities f prtein utilizing the principle f prtein-dye binding. Anal. Bichem. 72: Cain, R.B.,R.F. Biltn and J.A. Darrah. (1968).The metablism f armatic acids by micrrganisms. Bichem. J. 108: Cerniglia, CE. (1984). Micrbial metablism f plycyclic armatic hydrcarbns. Adv. Appl. Micrbil. 30: Claus, D. and N. Walker. (1964). The decmpsitin f tluene by sil bacteria. J. Gen. Micrbil. 36: Cx, H.H.J., J.H.M. Hutman, H.J. Dddema and W. Harder. (1993a). Grwth f the black yeast Exphiala jeanselmei n styrene and styrene-related cmpunds. Appl. Micrbil. Bitechnl. 39:

58 58 Chapter 4 Cx, H.H.J., J.H.M. Hutman. H.J. Dddema and W. Harder. (1993b). Enrichment f fungi and degradatin f styrène in bifilters. Bitechnl. Lett. 15: Epstein,S. and A.E. Braunstein. (1931). Über den Einfluß der sgen, sauren und basischen Nahrung auf das Schicksal armatischer Substanzen im Organismus. II. Oxydatin vn Tlul. Bi'chem. Z. 235: Fedrak, P.M. and D.W. Westlake. (1986). Fungal metablism f n-alkylbenzenes. Appl. Envirn. Micrbil. 51: Field, J.A., E. de Jng, G. Feij-Csta and J.A.M. de Bnt. (1992). Bidegradatin f plycyclic armatic hydrcarbns by new islates f white rt fungi. Appl. Envirn. Micrbil. 58: Field, J.A., E. de Jng, G. Feij-Csta and J.A.M. de Bnt. (1993). Screening fr ligninlytic fungi applicable fr the bidégradatin f xenbitics. Trends Bitechnl. 11: Gibsn, D.T., M. Henley, H. Yshika and T.J. Mabry. (1970). Frmatin f (+)-cfe-2,3- dihydrxy-l-methyl-cyclhexa-4,6-diene frm tluene by Pseudmnas putida. Bichem. 9: Gibsn, D.T. (1971a). The micrbial xidatin f armatic hydrcarbns. CRC Crit. Rev. Micrbil. 1: Gibsn, D.T. (1971b). Assay f enzymes f armatic metablism. In: J.R. Nrris, and D.W. Ribbns (eds.), Methds in micrbilgy, vl 6a, pp , Academic Press, Lndn. Gibsn, D.T. (1993). Bidegradatin, bitransfrmatin and the Belmnt.J. Ind. Micrbil. 12: Hartmans, S., M.J. van der Wert and J.A.M. de Bnt. (1990). Bacterial degradatin f styrene invlving a nvel flavin adenine dinucletide-dependent styrene mnxygenase. Appl. Envirn. Micrbil. 56: Hfmann, K.H. (1986). Oxidatin f naphthalene by Saccharmyces cerevisiae and Candida utilis. J. Basic Micrbil. 26: Hlland, H.L., F.M. Brwn, B. Munz and R.W. Ninniss. (1988). Side chain hydrxylatin f armatic hydrcarbns by fungi. Part 2: Istpe effects and mechanism. J. Chem. Sc. Perkin Trans. I11988: Hpper, D.J. and D.G. Taylr. (1977). The purificatin and prperties f p-cresl- (acceptr) xidreductase (hydrxylating), a flavcytchrme frm Pseudmnas putida. Bichem. J. 167: Kaphammer, B.,J.J.Kukr and R.H.Olsen. (1990). Clning and characterizatin f a nvel tluene degradative pathway frm Pseudmnas pickettii PK01. In: Abstracts f the 90th Annual Meeting f the American Sciety fr Micrbilgy, abstr. K-145, pp. 243, American Sciety fr Micrbilgy, Washingtn D.C. Kitagawa, M. (1956). Studies n the xidatin mechanisms f methyl grups. J. Bichem (Tky) 43:

59 Grwth f C. sphaerspermum n tluene 59 MacGillivray, A.R. and M.P. Shiaris. (1993). Bitransfrmatin f plycyclic armatic hydrcarbns by yeast islated frm castal sediments. Appl. Envirn. Micrbil. 59: Middelhven, W.J., M. Krevaar and G.W. Schuur. (1992). Degradatin f benzene cmpunds by yeast in acidic sils. Plant and Sil145: Ottengraf, S.P.P. (1986). Exhaust gas purificatin. In: H.-J. Rehm, and G. Reed (eds.), Bitechnlgy, Vl.8, pp , VCH Verlagsgesellschaft, Weinheim. Shields, M.S., S.O. Mntgmery, P.J. Chapman, S.M. Cuskey and P.H. Pritchard. (1989). Nvel pathway f tluene catablism in the trichlrethylene-degrading bacterium GA.Appl. Envirn. Micrbil. 55: Shields, M.S., S.O. Mntgmery, M. Cuskey, S, P.J. Chapman and P.H. Pritchard. (1991). Mutants f Pseudmnas cepacia G4 defective in catablism f armatic cmpunds and trichlrethylene. Appl. Envirn. Micrbil. 57: Whited, G.M. and D.T. Gibsn. (1991). Separatin and partial characterizatin f the enzymes f the tluene-4-mnxygenase catablic pathway in Pseudmnas mendcina KR1. J. Bacteril. 173: Wrsey, M.J. and P.A. Williams. (1975). Metablism f tluene and xylenes by Pseudmnas putida [arvilla] mt-2: Evidence fr a new functin f the TOL plasmid. J. Bacteril. 124:7-13. Wright,J.D. (1993). Fungal degradatin f benzic acid and related cmpunds. Wrld J. Micrbil. Bitechl. 9: Yadav, J.S. and C.A. Reddy. (1993). Degradatin f benzene, tluene, ethylbenzene, and xylenes (BTEX) by the lignin-degrading basidimycete Phanerchaete chryssprium. Appl. Envirn. Micrbil. 59:

60 60

61 5 Txicity f cntaminants in waste gases fr micrrganisms Frans J. Weber Ruud M.W. Schemen and Sybe Hartmans Summary: The txicity f varius vlatile rganic cmpunds frequently present as cntaminants in waste gases has been determined. Fr bth the Gram-psitive Rhdcccus S5 and the Gram-negative Pseudmnas SI2 the txicity was assessed as the cncentratin which reduced the grwth rate f the bacterium with 50%. N significant differences were bserved between the IC50% values fr these tw bacteria. A relatinship between the txicity and hydrphbicity f varius substituted benzene cmpunds was bserved. Txicity prblems in a bireactr fr waste-gas treatment are nly expected fr cmpunds with a high affinity t disslve in water. Surprisingly, ne f the selected bacteria, Pseudmnas strain SI2 was able t adapt t the txic effects caused by the cntaminants.this adaptatin resulted in the capacity f this strain t grw in the presence f supersaturating cncentratins f tluene.

62 62 Chapter 5 INTRODUCTION Already fr abut 20years bilgical waste-gas treatment has been used fr the reductin f emissins frm different industrial prcesses. Especially bifilters are used in Germany and The Netherlands fr treatment f drus waste-gas streams (Lesn & Winer, 1991). Nwadays bifilters are als used t treat wastegas streams cntaminated with vlatile rganic slvents. Anther type f reactr, the trickle-bed reactr, has especially been studied t treat waste-gas streams with higher cncentratins f these rganic cntaminants. The applicatin f trickle-bed reactrs fr the treatment f gas cntaminated with dichlrmethane has been demnstrated t be applicable with high efficiencies and degradatin velcities (Diks & Ottengraf, 1991; Hartmans & Tramper, 1991). The use f bilgical waste-gas treatment fr the remval f higher cncentratins f rganic slvents seems t be prmising. Hwever, a serius prblem culd be the txicity f the pllutants in the waste gas t the micrrganisms in the reactrs. Especially cmpunds with a gd slubility in water can be expected t accumulate in the water phase f the reactr during the start-up perid.this accumulatin can result in the inactivatin f the bimass in the reactr as the cntaminant cncentratins reach txic levels. Als fluctuatins in the cntaminant cncentratins in the waste gas culd result in levels which are txic fr the micrrganisms in the reactr. In this reprt we will describe the txicity f varius vlatile cmpunds t micrrganisms in relatin t bilgical waste-gas treatment. MATERIALS AND METHODS Micrrganisms The Gram-negative Pseudmnas strain SI2 and the Gram-psitive Rhdcccus strain S5 have been used fr the txicity tests. These micrrganisms have previusly been islated n lw cncentratins f styrene as sle carbn and energy surce (Hartmans et ai, 1990). The micrrganisms were rutinely maintained n yeast extract - glucse agar slants. Txicitytest The bacteria were grwn n mineral salts medium (Hartmans ef a/., 1989) with 40 mm sdium acetate as the carbn and energy surce. Frm an expnentially grwing preculture 1 ml f cell suspensin was transferred t varius 250-ml flasks with 15 ml acetate medium.t these incubatin flasks varius cncentratins f

63 Txicity f cntaminants in waste gases 63 the cntaminant t be studied were added. The flasks were clsed with a Tefln Mininert valve t prevent evapratin f the cntaminants and were incubated at 30 C in a water-bath with intensive shaking. The influence f the cntaminants n the grwth rate f the bacteria was determined by measuring carbn dixide frmatin in time. The measured carbn dixide cncentratins were crrected fr (1) the amunt f carbn dixide present in the bttle befre inculatin, and (2) the amunt f carbn dixide that was frmed by the inculum, which is nt transferred with the inculum. Analytical methds Carbn dixide was measured by analysing 100 fil-samples f the gas phase n a Packard mdel 427 gas Chrmatgraph (Packard, Delft, The Netherlands) with a Hayesep Q clumn (Chrmpack, Middelburg, The Netherlands). RESULTS Txicity The txicity f a cntaminant is measured as an inhibitin f the grwth rate f a bacterium grwing n acetate. In Fig. 1 the carbn dixide prductin f Rhdcccus S5 in the presence f varius tluene cncentratins is shwn. The liquid-phase cncentratins are calculated using the Henry cefficient f the E 73 O ü 3 ~ O a O ü 10" O Time (h) Figure 1: C0 2 prductin by Rhdcccus S5 grwn n acetate medium in the presence f 0 (A), 100 (O), 200 (V), 300 (0), and 400 (D) mg/l tluene. The lgarithm f the C0 2 cncentratin in the gas phase is shwn. Inset: Grwth rate f Rhdcccus S5 n acetate in the presence f varius cncentratins f tluene.

64 64 Chapter 5 Table 1: IC50% values f varius cntaminants fr Rhdcccus S5 and Pseudmnas S12. Cntaminant Lg P 0 w Water-Air Maximum IC50% Prpylbenzene Ethylbenzene Styrene Tluene Benzene rati b 2.5 C slubility b (mg/l) 55 c RhdcccusS5 water (mg/l) >500 Acrylnitril Frmaldehyde a Calculated accrding t the methd f Rekker and de Krt (1979) b frm Amre & Hautala (1983) c frm Mackay & Shiu (1981) gas (g/m 3 ) > Pseudmnas SI2 water (mg/l) gas (g/m 3 ) cntaminants. Frm these lgarithmic plts f the carbn dixide prductin in time, the grwth rate was calculated. In Fig. 1 b these grwth rates are shwn as a functin f the tluene cncentratin in the liquid phase. Using this plt the tluene cncentratin that inhibited the grwth rate t half f the maximum grwth rate was determined (IC50%). Similarly the IC50% values have been determined fr the ther cntaminants. These results are summarized in Table 1. Als shwn are the cntaminant cncentratins in the gas phase which are in equilibrium with the determined IC50% values in the liquid phase. Fr the substituted benzene cmpunds a linear relatin between the txicity and the hydrphbicity was bserved (Fig. 2). Adaptatin Surprisingly, ne f the bacteria used fr the txicity tests (Pseudmnas strain S12) started t grw in the presence f txic cncentratins f the armatic cmpunds after abut 20 hurs. This adaptatin was studied in mre detail fr grwth f Pseudmnas SI 2 n acetate in the presence f varius cncentratins f tluene. After an adaptatin perid grwth was bserved in the presence f 400 mg tluene/1 and als in the presence f a supersaturating amunt f tluene (1500 mg/l). When the cells were pre-cultured in the presence f 400 mg tluene/1 an adaptatin perid was nly bserved when the cells were transferred t medium cntaining 1500 mg tluene/1. N adaptatin time was bserved when the cells were pre-grwn in the presence f 1500 mg tluene/1.

65 Txicity f cntaminants in waste gases 65 uu 01 E 400 \ \ m U Lg P w Figure 2: IC50% f the tested substituted benzene cmpunds versus their Lg P w value. Data fr (O) RhdcccusS5 and (D) Pseudmnas SI2. DISCUSSION Numerus studies have shwn that xenbitics present in the envirnment can be txic fr micrrganisms (Bringmann & Kühn, 1977; Davidsn & Branden, 1981 ; Blum & Speece, 1991; Sierra-Alvarez & Lettinga, 1991). This txicity can seriusly hamper the applicatin f micrrganisms t remve pllutants frm waste streams. The cntaminant cncentratins in waste gases are expected t fluctuate, with temprarily high cncentratins being txic, pssibly resulting in an inactivatin f the system. Als during the start-up perid f a bireactr, cntaminants can accumulate in the water phase f the reactr reaching txic levels. In rder t estimate the txicity levels f varius vlatile cmpunds we have determined the IC50% value (cncentratins which inhibits the grwth rate by 50%) f these cmpunds fr tw types f micrrganisms (the Gram-psitive Rhdcccus S5 and the Gram-negative Pseudmnas S12). The bserved IC50% values where similar fr bth types f micrrganisms. This in spite f the difference in cell envelpe structure f Gram-psitive and Gram-negative bacteria. Fr the substituted benzene cmpunds a relatinship was bserved between the txicity and the hydrphbicity f the cmpund. The hydrphbicity is

66 66 Chapter 5 expressed as the lgarithm f partitining f this cmpund between ctanl and water (Lg P w ). A similar relatinship has als been bserved fr ther micrrganisms and ther grups f chemicals (Sierra-Alvarez & Lettinga, 1991; Vermuë et ai, 1993) These results indicate that the primary site f txicity is the cell membrane. An increase in hydrphbicity crrelates with in increase in txicity, and an increase in affinity f the cmpund t accumulate in the cell membrane (Sikkema era/., 1994). Of the tested xenbitics, especially the cmpunds with a gd water slubility were already txic at lw cncentratins in the gas phase, (e.g. frmaldehyde, at 100 mg/m 3 ). Under nrmal peratin f a bireactr frmaldehyde will be degraded and such a txic level in the water phase will nt be reached. Hwever, during the start-up perid f the bireactr r when temprarily high cncentratins are present in the waste gas these txic levels can be reached, resulting in inactivatin f the micrrganisms. Fr a reliable peratin f waste gas purificatin systems these situatins shuld be prevented. Therefre, during the start-up perid it is advisable t reduce the cntaminant cncentratin r flw-rate until enugh bimass is frmed t degrade all f the cntaminants present in the waste gas. Temprarily high cncentratins, due t fluctuatins, can als be buffered by using activated carbn, thereby preventing inactivatin f the system (Weber & Hartmans, 1995). Surprisingly ne f the micrrganisms used in the txicity test is capable t adapt t the presence f txic cmpunds. This adaptatin results in the capacity f this bacterium t grw in the presence f supersaturating cncentratins f tluene. Until nw nly a few micrrganisms have been described which are able t grw in the presence f such high cncentratins f tluene. Applicatin f this type f micrrganism in waste-gas treatment culd result in a rbust system which is nt sensitive t fluctuatins in cntaminant cncentratins. REFERENCES Amre, J.E. and E. Hautala. (1983). Odr as an aid t chemical safety: dr threshlds cmpared with threshld limit values and vlatilities fr 214 industrial chemicals in air and water dilutin. J. Appl. Txicl. 3: Blum, D.J.W. and R.E. Speece. (1991). A database f chemical txicity t envirnmental bacteria and itsuse in interspecies cmparisns and crrelatins.res. J. WPCF 63:

67 Txicity f cntaminants in waste gases 67 Bringmann, G. and R. Kühn. (1977). Grenzwerte der Schadwirkung wassergefährender Stffe gegen Bakterien (Pseudmnas putida) und Grünalgen {Scenedes quadricauda) im Zellvermehrungshemmtest. 1. f. Wasser- und Abwasser-Frschung 10: Davidsn, P.M. and A.L. Branden. (1981). Antimicrbial activity f nn-halgenated phenlic cmpunds. J. Fd Prtectin 44: Diks, R.M.M, and S.P.P. OHengraf. (1991). Verificatin studies f a simplified mdel fr the remval f dichlrmethane frm waste gases using a bilgical trickling filter. (Part li). Biprc. Eng. 6: Hartmans, S., J.P. Smits, M.J. van der Werf, F. Vlkering and J.A.M. de Bnt. (1989). Metablism f styrène xide and 2-phenylethanl in the styrene-degrading Xanthbacter strain 124X. Appl. Envirn. Micrbil. 55: Hartmans, S., M.J. van der Werf and J.A.M, de Bnt. (1990). Bacterial degradatin f styrène invlving a nvel flavin adenine dinucletide-dependent styrene mnxygenase. Appl. Envirn. Micrbil. 56: Hartmans, S. and J. Tramper. (1991). Dichlrmethane remval frm waste gases with a trickle-bed bireactr. Biprc. Eng. 6: Lesn, G. and A.M. Winer. (1991). Bifiltratin: An innvative air pllutin cntrl technlgy fr VOC emissins. J. Air Waste Manage. Assc. 41: Mackay, D. and Y.W.Shiu. (1981). A critical review f Henry's law cnstants fr chemicals f envirnmental interest. J. Phys. Chem. Ref. Data 10: Rekker, R.F. and H.M. de Krt. (1979).The hydrphbic fragmental cnstant; an extensin t a 1000 data pint set. Eur. J. Med. Chem. 14: Sierra-Alvarez, R. and G. Lettinga. (1991). The effect f the armatic structure n the inhibitin f acetclastic methangenesis in granular sludge. Appl. Micrbil. Bitechnl. 34: Sikkema, J., J.A.M. de Bnt and B. Plman. (1994). Interactin f cyclic hydrcarbns with bilgical membranes. J. Bil. Chem. 269: Vermuë, M., J. Sikkema, A. Verheul, R. Bakker and J. Tramper. (1993). Txicity f hmlgus series f rganic slvents fr the gram-psitive bacteria Arthrbacter and Ncardia sp. and the gram-negative bacteria Acinetbacter and Pseudmnas sp. Bitechnl. Bieng. 42: Weber, F.J. and S. Hartmans. (1995). Use f activated carbn as a buffer in bifiltratin f waste gases with fluctuating cncentratins f tluene. Appl. Micrbil. Bitechnl. 43:

68 68

69 ó Adaptatin f Pseudmnas putida SI2 t high cncentratins f styrene and ther rganic slvents Frans J. Weber Lydia P. Oijkaas Ruud M.W. Schemen Sybe Hartmans and Jan A.M. de Bnt Summary: Pseudmnas putida S12, previusly selected fr its capacity t grw n lw cncentratins f styrene, was shwn t be able t grw n supersaturating amunts f styrene after a lng lag-phase. Similarly, grwth was btained n supersaturating cncentratins f ctanl and heptanl. Using acetate r prpinate as a carbn and energy surce, grwth was btained in the presence f slvents with a Lg P w f 2.3 and higher. Hwever, in the presence f supersaturating cncentratins f tluene n grwth was bserved using ther carbn surces. Acetate was txic fr P. putida SI 2 but cells were able t adapt t higher acetate cncentratins. Only using these acetateadapted cells adaptatin t and grwth in the presence f supersaturating cncentratins f tluene was bserved. Applied and Envirnmental Micrbilgy (1993) 59:

70 70 Chapter 6 INTRODUCTION By using styrene as the sle carbn and energy surce in subsaturating cncentratins we have previusly islated 14 bacteria which were thught nt t grw at styrene cncentratins exceeding the slubility f styrene in water (Hartmans et ai, 1990). This substrate inhibitin was nt surprising since it has been established repeatedly that armatic slvents with a lg P w belw 4.0, as fr instance tluene and tetralin, are txic fr micrrganisms even at lw cncentratins (de Smet et a/., 1978; Sikkema et ai, 1992). The lgarithm f the partitining cefficient f a slvent in a defined ctanl-water mixture (Lg P w ) is cmmnly used as a measure fr the lipphilicity f a slvent (Rekker & de Krt, 1979). Armatic slvents with a Lg P w belw 4.0 can accumulate in the cytplasmic membrane f bacteria causing impairment f membrane functins and expansin f the cell membrane, resulting in leakage f cellular metablic prducts (Sikkema ef a/., 1992). Nevertheless, tw Pseudmnas species which grew n yeast peptne glucse medium in the presence f 50% tluene (Lg P w = 2.5) have been islated (Inue & Hrikshi, 1989; An ef a/., 1992) Recently, it has been shwn that P. putida Idah is nt nly resistant t tluene in a tw-phase system, but can even use tluene at these high cncentratins as the carbn and energy surce (Cruden ef a/., 1992). Several studies demnstrate that bacteria are able t adapt t hydrphbic cmpunds in their envirnment. These adaptatins may be due t the inductin f degradative enzymes r alteratins in the structure and dynamics f the cells (Heipieper et ai, 1991; Mishra & Kaur, 1991; Leying ef a/., 1992). Pssibly, such phentypic adaptatins eventually may als lead t a new gentype which wuld be irreversibly resistant t slvents. Thus, the cnstitutively tluene-resistant Pseudmnas species islated by the grup f Hrikshi and by Cruden et al (Inue & Hrikshi, 1989; An et ai, 1992; Cruden ef a/., 1992) may have evlved by an adaptatin mechanism. In this reprt we describe the adaptatin f a culture f the styrene-sensitive Pseudmnas strain SI2 t high styrene cncentratins. In additin the capacity f the strain t grw in a tw-phase styrene-water medium and the resistance t ther txic slvents was investigated.

71 Adaptatin f P. putida S12 t styrene 71 MATERIALS AND METHODS Organism and grwth Pseudmnas strain S12 has previusly been islated n styrene (Hartmans et al., 1990). Cells were grwn n phsphate buffered (ph 7.0) mineral salts medium (Hartmans et a/., 1989) with sdium acetate (60 mm) r ther carbn surces (3.0 g/l). Cultures were incubated at 30 C in a hrizntally shaking water bath (160 scillatins per minute, amplitude: 2.2 cm). Grwth in the presence f slvents An inculum f 5% (v/v) frm an vernight culture f P. putida SI2 was transferred t fresh medium. When these cells had reached the expnential grwth phase (OD 660 «0.4) slvents were added. The culture bttles (250 ml) cntaining 25 ml f medium were clsed with Mininert valves (Phase Separatins, Waddinxveen, the Netherlands) t prevent slvent evapratin. Bacterial survival The number f viable cells befre and after slvents were added t expnentially grwing cultures (OD 660 «0.4) was determined by plating 0.1 ml f suitable dilutins in 0.8% (w/v) saline n agar plates with the same grwth substrate (e.g. acetate r glucse) as the liquid culture. The adapted inculum was btained by plating a culture grwing n acetate in the presence f tluene (1% v/v) n an acetate-plate. Analytical methds Dry weight was determined by drying washed cell suspensins at 105 C fr 24 h. C0 2 cncentratins were determined using a Packard mdel 427 gas Chrmatgraph (Packard, Delft, The Netherlands) with a Hayesep Q clumn (Chrmpack, Middelburg, The Netherlands). 100 ul headspace samples were analyzed. A partitin cefficient f C0 2 between medium (ph 7.0) and air f 5.1 was measured and used t calculate the ttal amunt f C0 2 prduced. RESULTS Identificatin Strain SI2 was previusly islated n styrene and was tentatively identified as a Pseudmnas species (Hartmans et ai, 1990). The rganism was further identified by its grwth and bichemical characteristics as a Pseudmnas putida species (Pallerni, 1984). The results f the tests used are summarized in Table 1. The identificatin was cnfirmed by the fatty acid analysis f the strain using the

72 72 Chapter 6 Table 1: Mrphlgical, bichemical and grwth characteristics f P. putida S12. Frm Gram stain Mtility Reactin t xygen Flurescent pigment Grwth at 41 C Arginine dehydrlase Oxidase reactin Catalase Denitrificatin Rds Negative Mtile Aerbic Carbn surces fr grwth: Glucse mes-lnsitl Geranil L-Valine ß-Alanine DL-Arginine Micrbial Identificatin System f MIDI (Newark, DE, USA) (Janse & Smits, 1990). Using the aerbe TSBA database f the MIDI system, strain SI2 was identified as a Pseudmnas putida with 95% cnfidence. Adaptatin Pseudmnas putida SI2 was precultured n styrene and grwth was assessed by determining C0 2 -evlutin. Expnential grwth ( J = 0.6 h -1 ) was bserved withut an appreciable lag-phase (Fig. 1 ) at an initial amunt f 2 ul styrene in the incubatin system (25 ml liquid medium in a ttal vlume f 250 ml). This initial amunt f styrene in the incubatin system, based n its partitin cefficient, results in a cncentratin f 0.3 mm in the water phase, which is well belw the water-saturating level f 1.5 mm (Banerjee et a/., 1980). Grwth f the rganism was cmpletely inhibited by raising the initial amunt f styrene t supersaturating amunts (0.25 ml/25 ml). But surprisingly, the culture started t evlve C0 2 after abut 20 h (Fig. 1 ). Subsequently, cells grwn n this high cncentratin f styrene were used t inculate fresh medium (25 ml) with either 2 pi r 0.25 ml styrene. N significant lag-time was bserved fr bth cnditins and the grwth rates (0.6 h' 1 ) n basis f the C0 2 prfiles were identical. Adapted cells culd als be btained fllwing the abve prcedure but by starting frm a single clny f P. putida S12 grwn n acetate plates. P. putida SI2 als grew n supersaturating cncentratins f ctanl r heptanl as the sle carbn and energy surce. Using these slvents at supersaturating amunts als resulted in lag-times f apprximately 20 h.

73 j Adaptatin f P. putida SJ2 t styrene 73 ^ E E TS m "Ö 0 CL CM O 1 aß> ó à ó 1 / A ' 1- -t& A^ 0.01 ' / / / / j f X jf P,^ Time (h) Figure 1: Grwth f P. putida SI2 n styrene at tw different cncentratins. Cells were precuitured with 0.008% (v/v) styrene and C0 2 prductin was measured at lw (O) and high (A) initial styrene cncentratins (0.008% (v/v) and 1% (v/v) respectively). Grwth f cells adapted t 1% (v/v) f styrene was studied in mre detail by determining dry weight and viable cunts as well as C0 2 -evlutin. Fig. 2 shws a typical grwth-curve f P. putida SI2 grwing at 1% (v/v) styrene. After a shrt lag-time, expnential grwth was bserved. In the statinary phase a rapid decline in the viable cell cunt was bserved. Slvent tlerance It was als tested if a culture f the rganism wuld develp tlerance t a slvent if it was grwing n a nn-txic substrate in the presence f a nnmetablizable slvent. Unadpted cells f P. putida SI2 were grwn n several carbn surces and t the grwing cultures tluene (1% v/v) was added. Only cultures grwing n either acetate r prpinate eventually cntinued t grw in the presence f tluene. N grwth in the presence f tluene was bserved using either glucse, fructse, glycerl, ethanl, arginine, alanine, succinate, lactate r pyruvate as carbn surce. Using acetate as carbn surce it was subsequently tested if unadpted cells culd grw in the presence f several ther slvents (Table 2). Grwth was bserved in the presence f slvents with a Lg P w f 2.3 r higher. Lag times f mre than 20 h were bserved, especially fr slvents with a lw Lg P w value.

74 74 Chapter 6 3 O 0) Figure2: Grwth f P. putida S12 n 1% (v/v) styrene in 25 ml mineral salts medium. (O) Ttal amunt f C0 2 prduced (mmles), (D) dry weight (g/l), (A) viable cunt (cells per milliliter). Survival after the additin f tluene The effect f a slvent n unadpted cells was further studied by expsing either acetate-grwn r glucse-grwn cells t tluene. Numbers f viable cells in expnentially grwing cultures were determined just befre tluene additins (1% v/v) as well as 1 hur after additins (Table 3). Only 0.002% f the initial number f cells was still viable ne hur after the additin f 1% (v/v) tluene t cells grwing n acetate (60 mm). Grwth f the culture eventually resumed as was cnfirmed by measuring C0 2 cncentratins after 48 h. Hwever, when glucse medium was used, n viable cells were detected ne hur after the additin f tluene and n grwth within 48 h was bserved. It was subsequently tested if cells riginating frm a culture grwn n acetate in the presence f tluene wuld retain their resistance t tluene when grwn in the absence f a slvent. If such cells were grwn n acetate fr abut 10 generatins in the absence f tluene, abut 10% f the cells survived the additin f tluene (Table 3). Hwever, when cells frm the slvent-adapted acetate culture were transferred t and grwn in glucse medium fr 10 generatins, n survivrs were bserved ne hur after the additin f tluene.

75 Adaptatin f P. putida S12 t styrene 75 Table 2: Grwth f P. putida SI2 n slvents (1% v/v) as sle carbn surce, r in presence f slvent (1% v/v) with acetate as carbn surce. Slvent Decane Prpylbenzene Hexane Cyclhexane Ethylbenzene p-xylene Styrene Octanl Tluene Heptanl Dimethylphthalate Flurbenzene Benzene a Lg P 0 w a Acetate in the presence f slvent (1%,v/v) /- - - Grwth b n: Slvent (1%, v/v) Lg P w values were calculated accrding t the methd f Rekker and de Krt (1979) b Symbls: +, grwth (>0.5 mml C0 2 prduced after 48 h); +/-, grwth (>0.5 mml C0 2 prduced after 120 h); - n grwth (<0.1 mml C0 2 prduced after 120 h) Table 3: Effect f the additin f 1% (v/v) tluene n the viable cunt f expnentially grwing P. putida SI2 n acetate r glucse media. Adapted cells were previusly grwn in the presence f 1% (v/v) tluene. Strain Substrate Viable cells (ml" Survival Befre 1 h After Wild Type Acetate (60mM) 3.8* * 10 2 Wild Type Glucse 3.7* 10 7 <10 Adapted Acetate (60mM) 1.T *10 7 Adapted Glucse 3.2*10 7 < % 10% Grwth after 48 h yes n yes n

76 76 Chapter 6 m E E "Ö ü D "O O rp O Om / D ^~-~ f ^ v Û ü / /& [A UT vt Time (h) Figure3: Grwth f P. putida SI2 at varius acetate cncentratins at ph 7.0. O:20 mm; D:40 mm;a:60 mm;v:80 mm acetate. Txicity f acetate In view f the resistance f cells t tluene when grwn n acetate r prpinate it was tested if acetate itself is als txic fr the cells. Grwth f glucseprecultured cells at different cncentratins f acetate was determined and it was bserved that grwth f the cells was inhibited at acetate cncentratins higher than 40 mm (Fig. 3). When cells had reached the statinary phase and were transferred t fresh medium with the same respective acetate cncentratin, n grwth inhibitin was bserved. When tluene (1% v/v) was added t such cultures in the beginning f their expnential grwth phase, grwth after 48 h was nly bserved at initial acetate cncentratins f 40 mm r higher. DISCUSSION Previusly, we have islated the styrene-degrader P. putida SI2 by using subsaturating cncentratins f styrene (Hartmans et al., 1990). It was bserved that grwth f the rganism was inhibited at higher cncentratins f styrene. Surprisingly, we have nw fund that this rganism is able t adapt t higher cncentratins f styrene. Grwth n styrene in a tw-phase styrene-water system

77 Adaptatin f P. putida S12 t styrène 77 eventually ccurred and similarly grwth was als bserved in the presence f a secnd phase f heptanl r ctanl.this resistance t high cncentratins f a slvent, which is als used as carbn and energy surce, is similar t the resistance fr p-xylene bserved in P. putida Idah (Cruden et al., 1992). The rganism als grew in the presence f supersaturating cncentratins f nngrwth slvents with a Lg P w value f 2.3 r higher using acetate as carbn surce. Similar slvent-resistance has been bserved in P. putida IH-2000 and P. aeruginsa ST-001 (Inue & Hrikshi, 1989; Anef a/., 1992). Althugh P. putida S12 eventually grew n acetate in the presence f tluene, mre than 99.99% f the cells were killed when tluene was added t cells grwing n acetate (60 mm).thesurvivrs eventually grew t a high cell density. Hwever, 10% f the cells in a culture previusly expsed t tluene and grwn in acetate medium survived a tluene shck. Even when the cells had been transferred fr 10 successive times in an acetate medium lacking slvents a similar high percentage f survivrs was bserved. When several ther carbn surces were used instead f acetate r prpinate n grwth was bserved after the additin f 1% tluene t a expnentially grwing culture. Neither was grwth in the presence f tluene bserved when cells previusly grwn n acetate and adapted t tluene were subsequently grwn n glucse as carbn surce. The respnse f P. putida SI2 t the presence f a txic slvent is quite remarkable. Cells either precultured at subsaturating cncentratins f the txic styrene r at elevated acetate cncentratin were able t acquire slvent resistance. The undissciated acids f acetate and prpinate are txic fr micrrganisms and are generally used as fd preservatives (Chichester & Tanner, 1975). Sheu and Freese (1972) have reprted quite similar inhibitin prfiles fr Bacillus subtilis. B. subtilis grws at half its maximum grwth velcity at 80 mm acetate (ph 6.5) whereas fr P. putida SI2 this pint was reached at 60 mm acetate (ph 7.0). At 60 mm acetate (ph7.0) and 80 mm acetate (ph 6.5) the cncentratins f the undissciated acetic acids are 0.3 mm and 1.4 mm respectively. The results f Sheu and Freese (1972) suggest that micrrganisms grwn in the presence f shrt-chain fatty acids have a lwer membrane fluidity. Due t this reduced membrane fluidity f acetate-grwn cells a small number f P. putida SI2 cells apparently can survive and adapt t tluene. A similar situatin is expected fr cells grwing n subsaturating cncentratins f styrene. Whereas glucse-grwn cells, pssessing a nrmal membrane fluidity, are all killed by the additin f tluene. Several studies have demnstrated that bacteria can change their lipid cmpsitin when grwn in the presence f

78 78 Chapter 6 rganic slvents (Ingram, 1977; Heipieper et ai, 1991). Currently we are studying whether such changes are imprtant in the adaptatin t P. putida SI2 t high cncentratins f styrene as the grwth substrate r t ther slvents when grwing n acetate. ACKNOWLEDGEMENTS We like t thank Mariët J. van der Werf (Industrial Micrbilgy, Wageningen Agricultural University, Wageningen, The Netherlands) fr perfrming sme f the identificatin experiments. J.D. Janse (Plant Prtectin Service, Wageningen, The Netherlands) is acknwledged fr the identificatin f strain SI2 using the MIDI Micrbial Identificatin system. REFERENCES An, R., M. It, A. Inue, K. Hrikshi. (1992). Islatin f nvel tluene-tlerant strain f Pseudmnas aeruginsa. Bisci. Bitechnl. Bichem. 1: Banerjee, S.,S.H.Yalkwsky, and S.C. Valvani. (1980). Water slubility and ctanl/water partitin cefficients f rganics. Limitatins f the slubility-partitin cefficient crrelatin. Envirn. Sei. Technl. 14: Chichester, D.F., and F.W.TannerJr. (1975). Antimicrbial fd additives. In:T.E. Furia (ed.). Handbk f fd additives, pp ,CRC Press, Cleveland. Cruden, D.L., J.H.Wlfram, R.D. Rgers, and D.T. Gibsn. (1992). Physilgical prperties f a Pseudmnas strain which grws with p-xylene in a tw-phase (rganic-aqueus) medium. Appl. Envirn. Micrbil. 58: Hartmans, S., J.P. Smits, M.J. van der Werf, F. Vlkering, and J.A.M, de Bnt. (1989). Metablism f styrene xide and 2-phenylethanl in the styrene-degrading Xanfhbacter strain 124X. Appl. Envirn. Micrbil. 55: Hartmans, S., M.J. van der Werf, and J.A.M. de Bnt. (1990). Bacterial degradatin f styrene invlving a nvel flavin adenine dinucletide-dependent styrene mnxygenase. Appl. Envirn. Micrbil. 56: Heipieper, HJ., H. Kewelh, and H.J. Rehm. (1991). influence f phenls n grwth and membrane permeability f free and immbilized Escherichia cli. Appl. Envirn. Micrbil. 58: Ingram, LO. (1977). Changes in lipid cmpsitin f Escherichia cli resulting frm grwth with rganic slvents and with fd additives. Appl. Envirn. Micrbil. 33: Inue, A., and K. Hrikshi. (1989). A Pseudmnas thrives in high cncentratins f tluene. Nature 338: Janse, J.D., and P.H.Smits. (1990). Whle cell fatty acid patterns f Xenrhabdus species. Lett. Appl. Micrbil. 10:

79 Adaptatin f P. putida SJ2 t styrene 79 Leylng, H.J., K.H. Büscher, W. Cullmann, and R.L. Then. (1992). Lipplysaccharide alteratins respnsible fr cmbined quinlne and ß-lactam resistance in Pseudmnas aeruginsa. Chemtherapy 38: Mishra, P., and S. Kaur. (1991). Lipids as mdulatr f ethanl tlerance in yeast. Appi. Micrbil. Bitechnl. 34: Pallerni, N.J. (1984). Genus I. Pseudmnas. In: N.R. Krieg, and J.G. Hlt (eds.). Bergey's manual f systematic bacterilgy. Vl. 1, Williams & Wilkins C., Baltimre. Rekker, R.F. and H.M. de Krt. (1979). The hydrphbic fragmental cnstant; an extensin t a 1000 data pint set. Eur. J. Med. Chem. - Chimica Therapeutica 14: Sheu, C.W., and E. Freese. (1972). Effects f fatty acids n grwth and envelpe prteins f Bacillus subtilis. J. Bacteril. Ill: Sikkema, J., B. Plman, W.N. Knings, J.A.M. de Bnt. (1992). Effects f the membrane actin f tetralin n the functinal and structural prperties f artificial and bacterial membranes. J. Bacteril. 174: de Smet,M.J., J.Kingma, and B.Withlt. (1978).The effect f tluene n the structure and permeability f the uter and cytplasmic membranes f Escherichia cll. Bichim. Biphys. Acta 506:64-80.

80 80

81 7 Cis/trans ismerizatin f fatty acids as a defence mechanism f Pseudmnas putida strains t txic cncentratins f tluene Frans J. Weber Snja Isken and Jan A.M. de Bnt Summary: Defence mechanisms f three Pseudmnas putida strains grwing in the presence f tluene up t 50% (v/v) were investigated. The three strains reacted t txic cncentratins f tluene by accumulating trans unsaturated fatty acids in the membrane instead f the cis ismers. The membranes f the tluene-adapted cells pssessed a higher trans/cis rati and had a higher lipidrdering since the transitin temperature was abut 7 C higher cmpared t the nn-adapted cells. Micrbilgy (1994) 140:

82 82 Chapter 7 INTRODUCTION Armatic slvents like tluene, styrene and tetralin are txic fr micrrganisms even at subsaturating cncentratins in water (de Smet et al., 1978; Sikkema et al., 1992). These slvents partitin preferentially in the cell membrane and this excessive accumulatin causes expansin f the membrane and impairment f membrane functins (Sikkema et ai, 1994). Ntwithstanding the clear txicity f varius armatic slvents t mst micrrganisms, bacteria d exist that are able t tlerate high cncentratins f such cmpunds in their envirnment. Clear examples are Pseudmnas putida IH-2000 (Inue & Hrikshi, 1989) and P. putida PpGl (Shima et al., 1991) which grew in the presence f 50% (v/v) tluene. These strains, hwever, were nt able t metablize tluene. Recently, tw ther strains have been islated which can utilize txic slvents as carbn and energy surce. P. putida Idah grew n tluene and p-xylene in cncentratins f mre than 50% (Cruden et ai, 1992) and P. putida SI2 grew n supersaturating cncentratins f styrene (Weber et al., 1993). The mechanism f this tlerance f these P. putida strains is presently unknwn, but it is reasnable t expect adaptatins at the level f the membrane cmpsitin. Changes in the fatty acid cmpsitin f the membrane may alter the partitining f slvents in the membrane. An increase f the acyl chain length f the phsphlipids frm tetradecanic (CI4:0) t ctadecanic acid (CI8:0) reduced the partitining f lindane int lipsmes 50-fld (Antunes-Madeira & Madeira, 1989). Incubatins f Escherichia cli with aplar slvents such as benzene and ctanl resulted in an increased synthesis f saturated fatty acids (Ingram, 1976, 1977). Anther adaptatin mechanism has been bserved in a Pseudmnas putida species grwing n phenl at high cncentratins (Heipieper et al., 1992). These cells adapted by cnverting the cis unsaturated fatty acids int the trans ismers which have a higher transitin temperature and a decreased fluidity f the membrane. In this reprt we describe changes in the fatty acid cmpsitin f the phsphlipids f the three slvent-tlerant strains P. putida PpGl, P. putida Idah and P. putida SI2, in respnse t the presence f supersaturating cncentratins f rganic slvents in the grwth medium.

83 Cis/trans ismerizatin f fatty acids in P. putida strains 83 MATERIALS AND METHODS Organism and grwth Pseudmnas putida S12 had been islated n styrene (Hartmans et ai, 1990). P. putida Idah (NRRL B-18435) and P. putida PpGl (ATCC 17453) were btained frm culture cllectins. Cells were grwn in phsphate buffered (ph 7.0) mineral salts medium (Hartmans et al., 1989) with sdium acetate (60 mm) r glucse (15 mm) as carbn and energy surce. Cultures were incubated at 30 C with hrizntal shaking in a water bath (160 scillatins per minute, amplitude: 2.2 cm). Grwth in the presence f slvents An inculum f 5% (v/v) frm an vernight culture was transferred t fresh medium. When the culture reached the expnential grwth phase (OD 660 «0.4) rganic slvents were added. The culture bttles (250 ml) cntaining 25 ml f medium were clsed with Mininert valves (Phase Separatins, Waddinxveen, The Netherlands) t prevent evapratin. A partitin cefficient f tluene between medium and air f 3.8 (Amre & Hautala, 1983) was used t calculate the tluene cncentratins in the water phase. Chemstat Cells were grwn n mineral salts medium with 15 mm glucse in a fermentr with 700 ml wrking vlume at 30 C, 350 rpm and at a dilutin rate f 0.2 h _1. Varius tluene cncentratins were supplied t the fermentr via the gas phase by passing an adjustable part f the air flw thrugh a clumn (15 cm) f tluene which was kept at 30 C. The ttal air flw was kept cnstant at 140 ml/min. Bacterial survival The numbers f viable cells befre and after slvents were added t expnentially grwing cultures (OD 660 «0.4) r t cells harvested frm steady state chemstat cultures were determined by plating 0.1 ml f suitable dilutins in 0.8% (w/v) saline n slidified agar-medium with the same grwth substrate (e.g., acetate r glucse) as the liquid cultivatin medium. Fatty acid analysis The ttal lipids f cells were extracted with chlrfrm/methanl (Bligh & Dyer, 1959). The fatty acids were methylated using brn trifluride/methanl (Mrrisn & Smith, 1964). The fatty acid methyl esters were extracted int hexane and nrmally analyzed n a Chrmpack CP9000 gaschrmatgraph with a CP-Sil 88 WCOT fused-silica clumn. Fatty acids identities were cnfirmed by GC-MS

84 84 Chapter 7 analysis using a 5890A Hewlett/Packard Chrmatgraph with a CP-Sil 19CBWCOT fused-silica capillary clumn; mass spectra were recrded n a 5970series massselective detectr with a 59822B Inizatin Gauge Cntrller (Hewlett/Packard). Phasetransitintemperature The temperature-dependent vibratinal frequency f the CH 2 -stretch in lipids f whle bacterial cells was measured with Furier transfrm infrared spectrscpy (FTIR). FTIR measurements were carried ut using a Perkin-Elmer series 1750FTIR spectrmeter equipped with a 7500 data statin, as described by Crwe et al. (1989). Expnentially grwing cells were harvested and washed twice with water. The pellet was placed between tw CaF 2 windws. The sample was cled t -10 C and slwly heated and spectra were recrded after every stepwise increase (2 t 3 C) in temperature. RESULTS Fatty acid cmpsitin The three slvent resistant strains, P. putida PpGl, P. putida Idah and P. putida SI2 were grwn in mineral medium cntaining either glucse, acetate, r acetate in the presence f tluene (1% v/v). Expnentially grwing cells frm these cultures were harvested, washed and the fatty acid prfiles were determined (Table 1 ). When glucse-grwn P. putida SI2 cells were cmpared t acetate-grwn cells, an increase in the amunt f trans- and a decrease in the c/s-unsaturated fatty acids was seen. A further increase in the trans/cis rati was bserved when P. putida S12 cells were grwn n acetate in the presence f tluene (1% v/v). The acetate-grwn cells als had a higher percentage f cyclprpane fatty acids when cmpared t glucse-grwn cells. Hwever, the presence f tluene had n effect n these cyclprpane fatty acids.similarly, in tw ther slvent tlerant strains P. putida PpGl and P. putida Idah high trans/cis ratis were bserved when grwn in the presence f high tluene cncentratins. Stabilityf the adaptatin Subsequently, we determined whether the bserved changes in fatty acid prfiles,as dependent n grwth cnditins, were reversible.p. putida S12 grwn n acetate in the presence f 1% (v/v) tluene was used t inculate (1% v/v) acetate and glucse medium withut slvents.the fatty acid cmpsitin f the cells grwn n acetate was almst identical t the fatty acid cmpsitin f the

85 Cis/trans ismerizatin f fatty acids in P. putida strains 85 Table 1: Fatty acid cmpsitin (%) f three tluene-tlerant P. putida strains grwn n glucse, acetate, r acetate in the presence f tluene (1% v/v) respectively. P. putida S12 P. putida PpGl P. putida Idah Glucse Acetate Acetate Glucse Acetate Acetate Glucse Acetate Acetate + 1% + 1% + 1% Tluene Tluene Tluene Fatty Acid 16: :1 trans 16:1 as 17 cycl 18:0 18:1 trans 18:1 cis 19 cycl Rati < < < Sat./unsat. Trans/cis :1 18: parent culture grwn in the presence f tluene. Hwever, a decrease in the trans/cis rati was bserved, when grwn n glucse (Table 2). Subsaturating cncentratins f tluene Tluene cncentratins belw the maximum slubility in water (640 mg/l at 30 C) (Bhn & Claussen, 1951) als slwed the grwth f P. putida SI2 n glucse r acetate medium. A tluene cncentratin f abut 350 mg/l reduced the grwth rate t ne-half f the maximum. When these cells were transferred t fresh medium with the same tluene cncentratin, n grwth inhibitin was bserved. The effect f tluene n the fatty acid cmpsitin f P. putida SI2 was als determined by grwing the rganism in a glucse-limited chemstat at a dilutin rate f 0.2 tr'. Varius subsaturating tluene cncentratins were applied t the fermentr via the gas phase. The fatty acid cmpsitin f the cultures were determined after a steady-state was reached in the chemstat. Tluene cncentratins belw 300 mg/l in the water phase did nt result in changes in the fatty acid cmpsitin. But substantial changes in the trans/cis rati f the unsaturated fatty acids were bserved in cells grwn in the presence f higher tluene cncentratins (Fig. 1). The steady-state chemstat cultures were als

86 86 Chapter 7 Table 2: Fatty acid cmpsitin (%) f tluene-adapted P. putida SI2 grwn in the absence f tluene fr abut 100 generatins n glucse r acetate respectively. Fatty Acid 16:0 16:1 trans 16:1 as 17 cycl 18:0 18:1 trans 18:1 cis 19 cycl Rati Sat./Unsaturated 16:1 trans/cis 18:1 trans/cis Glucse < Acetate used t determine the effect n cell viability f a "shck" additin f tluene (1% v/v). Only a small number f survivrs were recrded in cultures grwn in the presence f less than 300 mg tluene per liter. Hwever, when grwn in the presence f mre than 300 mg tluene per liter, a high percentage f cells survived this additin f tluene. Phase transitin temperature Fatty acids underging melting frm gel t liquid phase shw an adsrptin shift f 2 t 4 wavenumbers in the infrared spectrum (Camern & Dluhy, 1987). The symmetric vibratin bands f CH 2 -grups at a frequency arund 2850 cm -1 was measured using FIR spectrscpy. This adsrptin maximum was determined at different temperatures fr P. putida SI2 grwn n glucse, acetate r acetate medium cntaining 400 mg tluene per liter respectively. In Fig. 2 it can be seen that cells grwn in the presence f tluene pssess a lipid melting temperature which is abut 7 C higher than in cells grwn in the absence f tluene. DISCUSSION Previusly we have islated 14 bacteria n subsaturating cncentratins f styrene as sle carbn and energy surce (Hartmans et al., 1990). These bacteria were thught nt t grw n supersaturating cncentratins f styrene, as these high styrene cncentratins were expected t be txic (de Smet et ai, 1978; Sikkema et al., 1992). Hwever, ne f the selected strains, P. putida S12,

87 Cis/trans ismerizatin f fatty acids in P. putida strains Tluene cncentratin (mg/l) Figure 1: Effect f tluene n the fatty acid cmpsitin f P. putida SI2 and n survival f cells given a shck additin f tluene. Cells were grwn in a glucse-limited chemstat in the presence f varius tluene cncentratins. Rati ( ) f saturated/unsaturated fatty acids (O), trans/cis rati f hexadecenic acid (16:1) (A), and ctadecenic acid (18:1) (D), and the percentage survivrs ( ) 1 h after the extra additin f tluene (1% v/v) (V) E 0) A E 3 C > r O Temperature ( C) Figure 2: Phase transitin melting temperature f P. putida SI2 grwn n glucse (O), acetate (A) and acetate medium cntaining 400 mg/l tluene (D) respectively. Wavenumber f the CH 2 symmetric stretch in the lipids were measured using Furier Transfrming Infrared Spectrscpy at varius temperatures.

88 88 Chapter 7 eventually grew n styrene at cncentratins f mre than 50% (v/v) after a lng lag-phase (Weber et al., 1993). In the present investigatin, significant changes in the fatty acid prfiles f slventadapted strains have been bserved. P. putida SI2 and tw ther slventtlerant strains respnded t the presence f tluene (1% v/v) by increasing the amunt f trans fatty acids and by decreasing the crrespnding c/s-ismer. This adaptatin in P. putida S12, ccurred nt nly at supersaturating cncentratins but als at subsaturating cncentratins f tluene in water. N effects were seen belw 300 mg tluene per liter, but abve 300 mg/l the trans/cis ismer rati f the cells increased dramatically. Cells having a high trans/cis ismer rati were well equipped t survive "shck" additins f tluene (1% v/v). The cis/trans ismerizatin as an adaptatin mechanism in P. putida SI2 is quite remarkable as mst bacteria adapt t membrane-active cmpunds by changing the saturatin degree f the lipids (Ingram, 1976, 1977; Ingram & Buttke, 1984; Kewelh et al., 1990). Althugh 15 years ag trans mnunsaturated fatty acids were regarded as nn-natural fatty acids (Lehninger, 1977), ver the last ten years trans fatty acids in bacteria have been reprted in, fr instance Arcbacter cryaerphila (Mss & Daneshvar, 1992), Pseudmnas aeruginsa (de Andres et al., 1991), Pseudmnas atlantica (Guckert et al., 1987), Pseudmnas putida (Heipieper et al., 1992), Vibri chlerae (Guckert et ai, 1986) and ther Vibri species (Okuyama et ai, 1990), a marine islate (Gillan et al., 1981) and in methane-utilizing bacteria (Makula, 1978). Recently, a cis/trans ismerizatin has been bserved as an adaptatin mechanism f a P. putida t txic phenl cncentratins (Heipieper et al., 1992). Frm ur results it nw appears that this defence mechanisms is widespread in P. putida species. The trans ismer has a steric cnfiguratin which is similar t that f saturated fatty acids. The c/s-ismer has a kink in the acyl-chain f the fatty acids, which causes steric hindrance and results in a membrane with a high fluidity. Studies with Achleplasma laidlawii membranes enriched with exgenusly supplied fatty acids have shwn that the phase transitin temperature between membranes cntaining abut 80% cis r trans ctadecenic acid respectively differ abut 45 C (Macdnald et al., 1985). In the tluene-adapted strain pssessing a high trans/cis rati, the transitin temperature f the membrane in viv was apprximately 7 centigrade degrees higher cmpared t the nn-adapted cells. This prvides the cell with a mechanism t cmpensate fr lss f membrane integrity as a result f the accumulatin f lipphilic cmpunds. Furthermre, an

89 Cis/trans ismerizatin f fatty acids in P. putida strains 89 increased lipid rdering als ppses partitining f lipphilic cmpunds in the membrane (Antunes-Madeira & Madeira, 1989). Apart frm an increase in the trans/cis rati f the unsaturated fatty acids, an increase in the amunt f cyclprpane fatty acids was bserved in acetategrwn cells when cmpared t glucse-grwn cells. The lipids f acetate-grwn P. putida SI2 cnsisted f abut 6% cyclprpane fatty acids, whereas glucsegrwn cells did nt cntain these fatty acids. Hwever, adaptatin t tluene either in batch r in chemstat cultures did nt enhance the level f cyclprpane fatty acids. Grwth f P. putida SI2 in batch-cultures in the presence f supersaturating tluene cncentratins was nly bserved when using txic cncentratins f acetate r prpinate as carbn surce (Weber et al., 1993). At 60 mm acetate (ph 7.0), abut 0.3 mm f the undissciated acetic acid will be present in the medium. Results f Sheu & Freese (1972) suggest that these lw cncentratins f acetic acid are membrane active. Acetate-adapted cells had an increased trans/cis rati f the mnunsaturated fatty acids. As a result, cells frm such cultures had a higher degree f lipid rdering in the membrane and cnsequently these cells were able t further adapt and grw in the presence f tluene. Nakajima et al. (1992) and An et al., (1992) have shwn that tlueneresistant bacteria can be islated effectively by first adapting bacteria t the presence f a secnd phase f the less txic slvent xylene. Pre-adaptatin f cells t less txic cmpunds (acetate, xylene) r t subsaturating cncentratins f txic slvents (tluene) seems t be an effective technique t islate slventtlerant micrrganisms. P. putida SI2 cells pssessing a high trans/cis ismer rati after grwth n acetate medium cntaining tluene, did nt revert back t nrmal fatty acid cntent upn several generatins grwth withut tluene. This respnse seems t be quite unusual, but these cells were grwn n a medium with an acetate cncentratin (60 mm) which is nrmally txic fr the cells. Apparently, the mdificatin in the fatty acid cmpsitin induced by tluene, is f benefit fr the cells t grw in the presence f txic acetate cncentratins. Upn remval f acetate, a nrmal fatty acid cmpsitin was bserved, indicating that the trans/cis ismerizatin is an adaptatin mechanism and nt a mutatin. The present investigatin has fcused nly n ne pssible defence mechanisms f P. putida strains t txic slvents. Further investigatins will be necessary t determine whether trans/cis ismerizatin f the fatty is acids is an imprtant

90 90 Chapter 7 factr in gaining resistance r that ther, as yet undiscvered adaptatins are f primary imprtance. In this respect it will be wrthwhile t cnsider the knwn effects f alchls n micrrganisms including changed prtein/lipid ratis r changes in the phsphlipid classes (Ingram & Buttke, 1984). ACKNOWLEDGEMENTS We thank Hermann J. Heipieper fr helpful discussins during the preparatin f the manuscript; C.J. Teunis, Department f Organic Chemistry (Wageningen Agricultural University) fr GC-MS analyses; and Dr. F.A. Hekstra, Department f Plant Physilgy (Wageningen Agricultural University) fr assistance with phase transitin temperature determinatins. REFERENCES Amre, J.E. and E. Hautala. (1983). Odr as an aid t chemical safety: dr threshlds cmpared with threshld limit values and vlatilities fr 214 industrial chemicals in air and water dilutin. J. Appl. Txicl. 3: de Andres, C. M.J. Espuny, M. Rbert, M.E. Mercade, A. Manresa and J. Guinea. (1991). Cellular lipid accumulatin by Pseudmnas aeruginsa 44T1. Appl. Micrbil. Bitechnl. 35: Antunes-Madeira, M.C. and V.M.C. Madeira. (1989). Membrane fluidity as affected by the insecticide lindane. Bichim. Biphys. Acta 982: An,R., M. It, A. Inue and K. Hrikshi. (1992). Islatin f nvel tluene-tlerant strain f Pseudmnas aeruginse. Bisci. Bitechnl. Bichem. 1: Bligh,E.G. and W.J.Dyer. (1959). A rapid methd f ttal lipid extractin and purificatin. Can. J. Bichem. Physil. 37: Bhn, R.L. and W.F.Claussen. (1951).The slubility f armatic hydrcarbns in water.j. Am. Chem. Sc. 73: Camern, D.G. and R.A. Dluhy. (1987). FIR studies f mlecular cnfrmatins in bilgical membranes. In: R.M. Gendreau (ed.). Spectrscpy in the medical sciences, pp , CRC Press, Bca Ratn. Crwe, J.H., F.A. Hekstra, LM. Crwe, T.J. Anchrdguy and E. Drbnis. (1989). Lipid phase transitins measured in intact cells with furier transfrm infrared spectrscpy. Crybilgy 26: Cruden, D.L, J.H.Wlfram, R.D. Rgers and D.T. Gibsn. (1992). Physilgical prperties f a Pseudmnas strain which grws with p-xylene in a tw-phase (rganic-aqueus) medium. Appl. Envirn. Micrbil. 58:

91 Cis/trans ismerizatin f fatty acids in P. putida strains 91 Gillan, F.T., R.B. Jhns, T.V. Verheyen. J.K. Vlkman and H.J. Bavr. (1981). Transmnunsaturated acids in a marine bacterial islate. Appl. Envirn. Micrbil. 41: Guckert, J.B.. M.A. Hd and D.C. White. (1986). Phsphlipid ester-linked fatty acid prfile changes during nutrient deprivatin f Vibri chlerea: increases in the trans/cis rati and prprtins f cyclprpyl fatty acids. Appl. Envirn. Micrbil. 52: Guckert. J.B.,D.B. Ringelberg and D.C. White. (1987). Bisynthesis f trans fatty acids frm acetate in the bacterium Pseudmnas atlantica. Can. J. Micrbil. 33: Hartmans, S., J.P. Smits, M.J. van der Werf, F. Vlkering and J.A.M. de Bnt. (1989). Metablism f styrène xide and 2-phenylethanl in the styrene-degrading Xanthbacter strain 124X. Appl. Envirn. Micrbil. 55: Hartmans, S., M.J. van der Wert and J.A.M. de Bnt. (1990). Bacterial degradatin f styrene invlving a nvel flavin adenine dinucletide-dependent styrene mnxygenase. Appl. Envirn. Micrbil. 56: Heipieper, H.-J., R. Diefenbach and H. Kewelh. (1992). Cnversin f cis unsaturated fatty acids t trans, a pssible mechanism fr the prtectin f phenl-degrading Pseudmnas putida P8 frm substrate txicity. Appl. Envirn. Micrbil. 58: Ingram, L.O. (1976). Adaptin f membrane lipids t alchls. J. Bacteril. 125: Ingram, 1.0. (1977). Changes in lipid cmpsitin f Escherichia cli resulting frm grwth with rganic slvents and with fd additives. Appl. Envirn. Micrbil. 33: Ingram, L.O. and T.M. Buttke. (1984). Effects f alchls n micr-rganisms. Adv. Micrbil. Physil. 25: Inue, A. and K. Hrikshi. (1989). A Pseudmnas thrives in high cncentratins f tluene. Nature 338: Kewelh, H., G. Weyrauch and H.J. Rehm. (1990). Phenl-induced membrane changes in free and immbilized Escherichia cli. Appl. Micrbil. Bitechnl. 33: Lehninger, A.L. (1977). Bichemistry, Wrth Publishers, New Yrk. Macdnald, P.M., B.D. Sykes and R.N. McElhaney. (1985). Flurin-19 nuclear magnetic resnance studies f lipid fatty acyl chain rder and dynamics in Achleplasma laidlawii B membranes. A direct cmparisn f the effects f cis and trans cyclprpane ring and duble-band substituents n rientinal rder. Bichem. 24: Makula, R.A. (1978). Phsplipid cmpsitin f methane-utilizing bacteria. J. Bacteril. 134: 77V777. Mrrisn, W.R. and L.M. Smith. (1964). Preparatin f fatty acid methyl esters and dimethylacetals frm lipids with brn fluride-methanl. J. Lipid Res. 5: Mss, C.W. and M.I. Daneshvar. (1992). Identificatin f sme uncmmn mnunsaturated fatty acids f bacteria. J. Clin. Micrbil. 30:

92 92 Chapter 7 Nakajlma, H., H. Knayashi, R. An and K. Hrikshi. (1992). Effective islatin and identificatin f tluene-tlerant Pseudmnas strains. Bisci. Bitechnl. Bichem. 56: Okuyama, H.,S.Sasaki,S.HIgaski and N. Murata. (1990). A trans-unsaturated fatty acid in a psychrphilic bacterium, Vibri sp. strain ABE-1. J. Bacteril. 172: Sheu, C.W. and E. Freese. (1972). Effects f fatty acids n grwth and envelpe prteins f Bacillus subtilis. J. Bacteril. Ill: Shima, H., T. Kud and K. Hrikshi. (1991). Islatin f tluene-resistant mutants frm Pseudmnas putida PpGl (ATCC 17453). Agric. Bil. Chem. 55: Sikkema, J., B. Plman, W.N. Knings and J.A.M. de Bni. (1992). Effects f the membrane actin f tetralin n the functinal and structural prperties f artificial and bacterial membranes. J. Bacteril. 174: Sikkema, J., J.AM. de Bnt and B. Plman. (1994). Interactin f cyclic hydrcarbns with bilgical membranes. J. Bil. Chem. 269: de Smet, NU.,J.Kingma and B.Withlt. (1978). The effect f tluene n the structure and permeability f the uter and cytplasmic membranes f Escherichia cli. Bichim. Biphys. Acta 506: Weber, F.J., LP. Oijkaas, R.M.W. Schemen, S. Hartmans and J.A.M, de Bnt. (1993). Adaptatin f Pseudmnas putida SI2 t high cncentratins f styrene and ther rganic slvents. Appl. Envirn. Micrbil. 59:

93 8 Adaptatin mechanisms f micrrganisms t the txic effects f rganic slvents n membranes. Frans J. Weber and Jan A.M. de Bnt Summary: Several rganic slvents are txic fr micrrganisms already at relatively lw cncentratins. In general, this txicity is caused by the accumulatin f these lipphilic slvents in the membrane lipid bilayer, affecting the structural and functinal prperties f the membrane. Inthis review effects f alkanls, alkanes and ther hydrcarbns n membranes are summerized. The physic-chemical aspect are cnsidered bth in terms f membrane fluidity and f bilayer stability. Althugh rganic slvents can be highly txic fr micrrganisms, sme micrrganisms are able t adapt and grw in the presence f txic cncentratins f these slvents.the emphasis f this review will be n the ability f micrrganisms t adapt their membrane structure in rder t survive the deleterius effects f slvents. These adaptatin mechanisms are cnsidered in view f the effects f rganic slvents n the prperties and functining f the membrane. Submitted fr publicatin

94 94 Chapter 8 LIST OFABBREVIATIONS DGDG Digalactsyldiglyceride DMPC Dimyristyl (CI4:0) phsphatidylchline DPG Diphsphatidylglycerl DPPC Dipalmityl (CI6:0) phsphatidylchline H, Nrmal hexagnal lipid cnfiguratin H,, Inverted hexagnal lipid cnfiguratin L a Lß Lpl MGDG PC PE PG PI P MB P w PS Lamellar liquid-crystalline phase Lamellar gel phase Interdigitated gel phase Mngalactsyldiglyceride Phsphatidylchline Phsphatidylethanlamine Phsphatidylglycerl Phsphatidylinsitl Partitining f a cmpund ver a membrane/buffer twphase system Partitining f a cmpund ver a ctanl/water tw-phase system Phsphatidylserine T LH Temperature at which the transitin frm a lamellar cnfiguratin t an inverted hexagnal lipid phase is 50% cmplete T M a Temperature at which the transitin frm the lamellar gel t the lamellar liquid-crystalline is 50% cmplete Area f the phsphlipid headgrup at the water interface / Length f the acyl-chain in a phsphlipid v Vlume f the fatty acids in a phsphlipid

95 Adaptatin f micrrganisms t txic slvents 95 CONTENTS 1. Intrductin Partitining f slvents int membranes Psitin f slvent partitining int membranes Effect f slute partitining n the bilayer prperties Effect f alkanls n the bilayer functining Bilayer fluidity Bilayer stability Effects f alkanes and armatic hydrcarbns n bilayer fluidity and stability Effect f slute partitining n membrane functins Membrane permeability Effect n membrane-embedded enzymes Adaptatin Adaptatins at the level f the membrane Lipid cmpsitin a. Adaptatin t alkanls b. Adaptatin t ther rganic slvents Headgrup cmpsitin Membrane prtein cntent Other membrane cmpnents a. Sterls and Hpanids b. Cartenids C Dicarbxylic acids Bilayer fluidity versus bilayer stability Outer membrane Other respnses f micrrganisms t slvents Cmpatible slutes Plasma-membrane ATPase Heat Shck Prteins Methds fr preventing the accumulatin f slvents Decreased affinity f membranes fr slvents Degradatin r mdificatin f the slvent Active efflux mechanisms Cncluding remarks Acknwledgements References 136

96 9ó Chapter 8 1. INTRODUCTION The main functin f the cell membrane f micrrganisms is t frm a permeability barrier, regulating the passage f slutes between the cell and the external envirnment. The membrane keeps essential metablites and macrmlecules inside the cell, it pumps nutrients int the cell against a cncentratin gradient, and it prevents the entry f certain slutes present in the envirnment. In the envirnment, micrrganisms may be cnfrnted with lipphilic cmpunds which preferentially accumulate in membranes. Such cmpunds may be naturally ccurring cmpunds as fr instance alkanls and terpenes, r may be xenbitics such as alkanes and armatic hydrcarbns. This accumulatin f such cmpunds will affect physic-chemical prperties f membranes and cnsequently their functining. The physic-chemical prperties f membranes as affected by slvents and the resulting bilgical functining are usually described by referring t the lamellargel t liquid-crystalline phase transitin. A great number f studies have dealt with the effects f rganic slvents n the transitin frm the lamellar-gel t the liquidcrystalline state. Anther apprach is t cnsider the stability f membranes in lamellar structures as affected by slvents. Many phsphlipids present in membranes prefer nn-lamellar structures and depending n the cmpund this tendency will be affected psitively r negatively by the slvent. Relative t studies n lamellar-gel t liquid-crystalline phase transitins, few articles have appeared dealing with this aspect. Several aspects f the effects f a variety f slvents n the physic-chemical prperties f the cell membrane, bth in terms f phase transitins and lamellar stability, have been reviewed by Lhner (1991). In this review we will nly summarize these physic-chemical effects f alkanls, alkanes and ther hydrcarbns n membranes. The emphasis here will be n the ability f micrrganisms t adapt their membrane structure in rder t survive the deleterius effects f slvents. Such adaptatin mechanisms include changes in the phsphlipid cmpsitin and changes in the cntent f membrane embedded prteins, sterls, hpanids, r cartenids. These adaptatin mechanisms will be cnsidered in view f the effects f rganic slvents n the prperties and functining f the membrane.

97 Adaptatin f micrrganisms t txic slvents PARTITIONINGOFSOLVENTSINTO MEMBRANES Several studies have shwn that a crrelatin exists between the lipphilicity f a cmpund and the partitining f such a cmpund int a lipid bilayer n the ne hand and the txicity t micrrganisms n the ther hand (Vighi & Calamari, 1987; Osbrne et al., 1990; Sierra-Alvarez & Lettinga, 1991; Sikkema et al., 1994a). The lipphilicity f a cmpund usually is expressed in terms f Lg P w, which gives the partitining f a cmpund ver an ctanl/water twphase system. Lipphilic cmpunds, which pssess a high affinity fr the cell membrane, are mre txic than less lipphilic cmpunds. Fr instance ethanl (Lg P w = -0.28) is nly txic fr micrrganisms at high cncentratins (several %), whereas slvents like tluene (Lg P w = 2.5) are already txic in the mm range. (Heipieper et ai, 1994). Hwever, this crrelatin des nt apply fr hydrphbic slvents with a Lg P w > 4-5. These slvents are generally nt txic fr micrrganisms (Inue& Hrikshi, 1991; Vermuë et ai, 1993) and are actually used as a nn-txic secnd phase that functins as a reservir fr txic cmpunds in bitransfrmatin reactins (Laane et ai, 1987). Althugh these slvents with a Lg P w abve 4 have a high affinity t partitin int the lipid bilayer, the pr slubility f these cmpunds in water will prevent them frm reaching high cncentratins in the membrane (Osbrne ef ai, 1990). This may be illustrated by cnsidering the accumulatin f a series f hmlgus alkanls in membranes. As may be seen frm Table 1, the mst hydrphilic alchl included (ddecanl) has a far strnger preference fr partitining in membranes than the lwer alkanls. Nevertheless, the maximum cncentratin which this cmpund can reach in the membrane is lwer than the less hydrphbic alkanls because f its lw water slubility. The maximum membrane cncentratin (Table 1) was calculated frm the membrane/buffer partitin cefficient (P MB ) and the maximum water slubility. The partitin Table 1: Maximum achievable membrane alkanl cncentratin, calculated frm the membrane partitin cefficient (Osbrne ef ai, 1990) and the maximum water slubility (Riddickefa/., 1986) Cmpund Lg P w Lg P MB Max. Aqueus Max. Membrane slubility (mm) cncentratin (mm) Ethanl (C2) Butanl (C4) Hexanl (C6) Octanl (C8) Decanl (CIO) Ddecanl (CI2)

98 98 Chapter 8 cefficient fr the slutes between the membrane and aqueus buffer (P MB ) was calculated using Lg P w values (Rekker & de Krt, 1979) and the experimentally determined relatinship between these tw partitin cefficients (Osbrne et al., 1990). Such calculatins are valid under the assumptin that n direct cntact exists between the slvent phase and the rganism. 3. POSITION OFSOLVENTPARTITIONING INTOMEMBRANES The effect which a lipphilic cmpund has n the integrity f a membrane depends n the psitin in the membrane where it accumulates. A slute near the phsphlipid headgrup area will have anther effect n the membrane than a slute partitining deeply in the lipid acyl chains. Depending n the hydrphbicity f the slute, it will accumulate mre r less deeply int the bilayer. Alkanls like ethanl will interact with the headgrup area and alkanes with the fatty acid acyl chains. The psitin f slvents in membranes has been determined bth fr alkanls and alkanes using a variety f techniques, including X-ray diffractin, and magnetic resnance spectrscpy (NMR and ESR). Westerman et al. (1988) have used phsphlipids, which were deuterium labelled at varius specific places, t study the interactin f n-alkanls (butanl, ctanl, ddecanl and tetradecanl) with bilayers f DMPC. Different patterns f changes in lipid rder induced by the alkanls were bserved by 2 H-NMR (Westerman ef a/., 1988). All alkanls studied prduced a large disrder in the glycerl backbne f the lipid, indicating that the terminal hydrxyl grup f the alkanl is anchred near the aqueus interface f the lipid. Of the fur alkanls studied, butanl prduced the largest disrdering in the glycerl backbne. This prbably reflects the greater fractin f time spent by the hydrxyl grup f butanl in the vicinity f the lipid plar headgrup cmpared with the ther alkanls (Westerman ef a/., 1988). A very hydrphbic cmpund like chlesterl had a minimal effect n the rdering in the glycerl backbne suggesting that this slute is embedded mre deeply in the bilayer. These findings suggest that the amphiphatic alkanls partitin int the bilayer with their hydrxyl miety near the phsphlipid plar headgrup and their aliphatic chains intercalated between the fatty acyl chains f the phsphlipids. Similar results have been btained fr ther amphiphatic cmpunds, like benzyl-alchl (Ppe et al., 1986) r cannabinids (Yang et a/., 1992). Nn-amphiphatic cmpunds like alkanes d nt interact with the headgrup and accumulate mre deeply in the lipid bilayer, aligning themselves parallel t

99 Adaptatin f micrrganisms t txic slvents 99 the lipid hydrcarbn chains (Mcintsh ef a/., 1980). 2 H-NMR studies f Ppe et al. (1989) have shwn that the phsphlipid carbns 2 t 10, which are mre rdered, are entrpically less accessible fr alkanes. They predicted that the mre disrded bilayer centre is the favured lcatin f hydrphbic slutes (Ppe ef a/., 1989). Studies with hexane have shwn that this mlecule is lcated in the centre f the bilayer in a zne f 10 Â (White et a/., 1981). Shrt-chain alkanes are expected t fit easily int this regin (King ef a/., 1985; Ppe ef al., 1989). As the alkane chain-length increases a pint is reached when the entire alkane mlecule can nt be lcated in this disrdered lipid regin, but must expand t the mre rdered regin. Due t the interactin with this mre rdered lipid regin, these lnger chain alkanes tend t be less sluble in the bilayer. The maximum amunt f hexadecane that can be slubilized in the bilayer is abut ne hexadecane mlecule per 6-10 lipid mlecules, which is lwer than fr the shrt-chain alkanes (Mcintsh ef al., 1980; Ppe & Dubr, 1986; Ppe ef al., 1989). Accumulatin f up t 1 ml f hexane per ml f lipid has been reprted (King etal., 1985). The psitin f mlecules ther than alkanls and alkanes in a lipid bilayer is nt very well dcumented. Less hydrphbic, nn-amphiphatic mlecules like tluene are expected t accumulate in between the lipid acyl chains near the headgrup area (Lhner, 1991; Sikkema ef al., 1994b). Only fr benzene several reprts are available describing its psitin in a membrane. Unfrtunately, hwever, there is disagreement in the literature regarding the phsphlipid a (0 c O) U) ^ ^ - ^ O ^-^"^ LL Lg F^w slvent Figure 1: F-NMR signal f p-flurtluene disslved in varius slvents.

100 100 Chapter 8 regins where benzene resides. Studies with surfactant micelles and lipid bilayers indicated that benzene mlecules tend t partitin int the hydrphbic regin (Simn et a/., 1982; Bden et al., 1991), whereas anther study shwed that benzene adsrbs at the headgrup interface (Ward et a/., 1986). In view f the high txicity f these armatic hydrcarbns fr micrrganisms, we are interested in the psitin f these cmpunds in the bacterial membrane. We have used 4-flurtluene as a mdel cmpund, because this flurcmpund gives a F-NMR signal depending n the hydrphbicity f the envirnment. Based n this prperty it is pssible t determine the psitin f tluene in the membrane. First, we have determined the F-NMR signal f F- tluene disslved in varius slvents. In Fig. 1 these F-NMR signals f flurtluene are shwn as a functin f the hydrphbicity f the slvent used t disslve tluene. Subsequently, we have used lipid vesicles t determine the psitin f F-tluene in the membrane. Frm Pseudmnas putida S12, a Gram-negative bacterium which is able t tlerate high cncentratins f tluene (Weber et ai, 1993), the phsphlipids were extracted and further purified. These phsphlipids were used t make multi-laminar vesicles t which F-tluene was added. F-tluene accumulated in these vesicles gave a F-NMR signal at 4.0, which crrespnds t a Lg P w f 0.8 (Fig. 1). Thus F-tluene accumulated in phsphlipids vesicles in an envirnment with a hydrphbicity crrespnding t a Lg P w value f 0.8. In Fig. 2 the hydrphbicity f varius parts f a phsphlipid mlecule is shwn, as calculated using the hydrphbic fragmental set f Rekker and De Krt (1979). The envirnment in the lipid bilayer were tluene accumulated, has a hydrphbicity crrespnding t a Lg P w value f 0.8. The nly place in a phsphlipid were such a hydrphbicity exists is in between the lipid acyl chains., 'O-P-O-CH CH NH 2 T W LgP w = -2.0' LgP w <-4 LgP 0 Figure 2: Hydrphbicity f varius areas in a phsphlipid. The Lg P w values were calculated accrding t the methd f Rekker and De Krt (1979).

101 Adaptatin f micrrganisms t txic slvents 101 near the headgrup area (Fig. 2). Using the same methd it was determined that F-tetradecane accumulated in an area with a hydrphbicity with a Lg P w f abut 9.6, which indicates that this cmpund is lcated deeply embedded in between the lipid acyl chains.this result nicely cnfirms the bservatins f thers n the psitin f lng-chain alkanes in the membrane (Lhner, 1991;Sikkema et al., 1994b). 4. EFFECT OF SOLUTE PARTITIONING ON THE BILAYER PROPERTIES A great number f studies have shwn that the accumulatin f a slute in a lipid bilayer strngly influences the functining f membranes. The Van der Waals interactins between the lipid acyl chains in a membrane will be affected by the partitining f a slute in the membrane and the disturbance f these lipid interactins will have an effect n the fluidity f the membrane (Melchir, 1982; Shinitzky, 1984;Sikkema et a/., 1995).This fluidity usually is taken as a measure t describe the statues f a membrane. Hwever, the cncept f membrane fluidity is cnsidered as an unfrtunate ne by sme authrs (Rilfrs et ai, 1984) and in this review we will therefre als pay attentin t the effects f slvents n the stability f lipid bilayers in bilgical membranes. Such effects f slvents n the transitin f a bilayer t nn-bilayer structures have, hwever, been studied less frequently Effectfalkanls n the bilayerfunctining Bilayer fluidity Numerus studies have shwn that interactins f alkanls with the lipid bilayer affects the fluidity f the membrane (see Shinitzky, 1984; Lhner, 1991;Sikkema et al., 1995 fr reviews). Fr a detailed descriptin cncerning the effects f alkanls n the bilayer fluidity these reviews shuld be cnsulted.as a specific example t illustrate the effects f alkanls, results btained by Eliasz ef a/. (1976) are cnsidered here.these authrs determined the effect f several n-alkanls n the lamellar-gel (Lp) t the liquid-crystalline (L a ) transitin temperature (T M ) f DPPC lipsmes. As shwn in Fig.3, they bserved that decanl had n effect n the T M. Accumulatin f n-alkanls with a chain length f >10 carbn atms decreased the membrane fluidity, whereas thse with a chain length <10 carbn atms had a fluidizing effect n DPPC lipsmes (Fig. 3). These n-alkanls will accumulate in the membrane with their hydrxyl miety near the headgrup area and their hydrcarbn chain aligned in between the phsphlipid acyl chains (Lhner, 1991). The effect f these alkanls n the T M are therefre cmparable t changes in the acyl-chain length f the phsphlipids.

102 »02 Chapter 8 Tetradecanl Ddecanl Decanl Octanl Rati Alkanl/Phsphhpid Figure3: Effect f n-alkanls n the lamellar-gel t liquid-crystalline phase transitin temperature f dipalmityl phsphatidylchline lipsmes. Alkanl cncentratins are shwn as the verall mle rati f alkanl t phsphlipid. (Frm Eliaszef a/., 1976). Table 2: Membrane cncentratin f alkanl which causes a cnstant shift in transitin temperature in DPPC lipid bilayers (75 mm. Membrane cncentratins calculated frm the experimental data f Jain and Wu (1977) using the membrane-buffer partitin cefficient, calculated as described by Osbrn et al. (1990). Methanl Ethanl Prpanl Butanl Pentanl Hexanl Heptanl Octanl Nnanl Decanl HHW' 100 (') (ttal amunt f alkanl (mm) added) Lg PMB (2) Membrane cncentratin (mm) (1 ) Cncentratin at which the half-height width f the btained DSC-prfileis shifted 100% alng the temperature axis. (Jain & Wu,1977) (2) Partitin cefficient f a slute in a membrane/aqueus buffer system. Calculated as described by Osbrn ef a). (1990).

103 Adaptatin f micrrganisms t txic slvents 103 Incrpratin f rigid phsphlipids r rigid lng-chain alkanls will increase the T M, whereas incrpratin f less rigid phsphlipids r shrt-chain alkanls will have an ppsite effect. As seen befre (Table 1 ) the tendency f shrt-chain alkanls t accumulate in a bilgical membrane is nt very high. Therefre, quite high cncentratins f these alchls have t be used t detect an effect n the membrane fluidity. Jain and Wu (1977) determined the verall alkanl cncentratin which was required t cause a decrease in the transitin temperature by a certain magnitude. With the aid f the membrane partitin cefficient (Osbrne et ai, 1990) it is pssible t calculate the actual membrane cncentratin f these alkanls. As can been seen in Table 2 much higher cncentratins f the shrtchain alkanls are required t affect the membrane fluidity than the lng-chain alkanls. This can be explained by cnsidering that alkanls partitin in the bilayer with their hydrxyl miety near the phsphlipid plar headgrup. Shrt-chain alkanls will thus nt interfere strngly with the lipid acyl chains and will therefre have a less prnunced effect n the lipid fluidity than the lnger chain alkanls Bilayer if ability Frm theretical calculatins by Israelachvili et al. (1976, 1977) and thers (Tanfrd, 1973; Mitchell et ai, 1983) it can be cncluded that the effective gemetry f lipid mlecules determines the packing f the lipids int a certain cnfiguratin (e.g. lamellar r nn-lamellar). Frm their theretical mdel a simplified cncept emerged, which visualized each phsphlipid as a building blck with a phsphlipid headgrup water interfacial area 'a', hydrcarbn chain length 7', and a hydrcarbn chain vlume V'. Lipids were simply classed as cnes, cylinders r inverted cnes depending n the relative packing requirements f the tw regins in the phsphlipid: the crss-sectinal area f the hydrcarbn prtin (v//) cmpared t the ptimal surface area required by the plar headgrup (a). Depending n these parameters three phsphlipid cnfiguratins can be distinguished (Fig. 4). Fr instance, an unsaturated phsphatidylethanlamine has a relative small headgrup area (a) cmpared t the hydrcarbn area (v/l) resulting in a packing gemetry >1.This lipid can be visualized as a cne and will prefer t frm an inverted hexagnal (H N ) lipid structure (Fig. 4). In bilgical membranes the situatin bviusly is by far mre cmplex, because they cnsist f a mixture f varius phsphlipids and prteins. Other factrs, like ph, temperature, presence f divalent catins (e.g. Ca 2+ ) r the inic strength f the envirnment will als affect the packing f the phsphlipids int a certain cnfiguratin (Cullis & de Kruijff, 1979; Rilfrs er ai, 1984; Gennis, 1989; Seddn,

104 104 Chapter 8 Mlecular shape Inverted Cne Cylindrical Cne Factrs effecting packing gemetry 1 ) One acyl-chain 1 )Smaller headgrup 2) Increased unsaturatin Packing ()tm\ gemetry \a) < > 1.0 Cnfiguratinf the phsphlipids ft Hexagnal type I r Micellar Bilayer Hexagnal type II Figure 4: Mlecular shape f varius phsphlipids and their crrespnding plymrphic lipid cnfiguratin. (Adapted frm Cullis &. de Kruijff, 1979) 1990). Calcium ins, fr instance, diminish the electrstatic repulsin between the negatively charged plar head-grup f DPG and will reduce the headgrup area 'a'. Thus calcium ins can induce the frmatin f a hexagnal H M lipid phases f DPG mdel membranes (Mandersltefa/., 1980). As far as the effects f n-alkanls are cncerned, studies have been dne with artificial membranes. Egg phsphatidylethanlamine shwed a transitin frm a lamellar t inverted hexagnal (H ) lipid phase when the temperature was raised up t 30 C (Hrnby & Cullis, 1981). The temperature at which this phase transitin ccurs is termed T LH. The effect f alkanls n the T LH f egg PE phsphlipids has been studied by Hrnby and Cullis (1981). They fund that the partitining f lngchain alkanls (C 6) int lipid bilayers decreased the T LH. In ther wrds, the bilayer structure was made less stable by these alkanls. This bservatin may be explained by cnsidering that these alkanls interact with bth the headgrup area and lipid acyl chain. Althugh this accumulatin will increase the

105 Adaptatin f micrrganisms t txic slvents Ethanl Ü I _J h- Decanl \> Octanl Rati Alkanl/Phsphlipid Figure 5: Influence f increasing amunts f n-alkanls n the lamellar t inverted hexagnal (H ( ) phase transitin temperature (T LH ) f egg phsphatidylethanlamine. T LH is estimated as the temperature where 50% f the lipids is in the bilayer rganisatin and 50% is in a hexagnal structure (Frm Hrnby & Cullis, 1981). headgrup area 'a', they particularly have a prnunced effect n the lipid vlume V', thus resulting in an increase f (v//)/a and hence prmte the frmatin f the hexagnal type II lipid phase. Ethanl, which partitins in membranes especially in the headgrup area f the phsphlipids, will increase the headgrup area 'a'. Accrding t the lipid packing thery an increase in headgrup area 'a' will ppse the frmatin f an inverted hexagnal (H N ) lipid phase. This is indeed bserved, additin f ethanl t egg PE lipsmes resulted in an increase f the lamellar-hexagnal transitin temperature (T LH ) (Fig. 5). Shrt-chain alkanls cnsequently have a psitive effect n the stability f bilayers, unless they wuld be able t transfrm a phsphlipid bilayer int a nrmal hexagnal (H,) r micellar lipid structure. Hwever, this H, phase is nt nrmally bserved fr diacyl phsphlipids (Seddn, 1990). Only lys-phsphlipids, cntaining nly ne acyl-chain, will frm this structure. Als very shrt-chain diacyl phsphlipids (six t eight carbns per fatty acid acyl chain) might frm a nrmal hexagnal (H,) structure (Lin et al., 1987). The inductin f a H, phase by methanl r ethanl has nt been bserved, nly tetracaine-hydrchlride has been reprted t be capable f breaking up the lamellar phsphlipid structure f PC int micelles (Fernandez & Calderón, 1980).

106 106 Chapter 8 This effect has been cntributed t the large charged grups f tetracaine which will increase the phsphlipid head-grup area 'a'. Althugh phsphlipids within bilgical membranes are rganized in a bilayer, almst every bilgical membrane cntains substantial amunts f lipids which have a strng preference fr the inverted hexagnal phase (H ) (Cullis & de Kruijff, 1979). In bth A. laidlawii (Wieslander et ai, 1980) and Clstridium butyricum (Gldfine et ai, 1987) a prper balance between bilayer and nn-bilayer frming lipids is maintained by the micrrganisms in respnse t changes in envirnmental cnditins. Als in E. cli the phase preference f the membrane lipids is regulated by adjustments in the rati f bilayer t nn-bilayer lipids (Rietveld et ai, 1993). Rietveld et al. (1993) used a mutant E. cli strain which lacks the ability t synthesize PE, a nn-bilayer frming lipid which nrmally accunts fr 70-80% f the E. cli phsphlipids. Inthis mutant strain the absence f PE is cmpensated by a large increase in the levels f PG and DPG (DeChavigny et ai, 1991; Rietveld et ai, 1993). This strain als has an abslute requirement fr high cncentratins f divalent catins (DeChavigny et ai, 1991). Since divalent catins are knwn t induce H u frmatin in DPG mdel systems, this suggests that DPG in cmbinatin with divalent catins can replace PE in membranes (DeChavigny et ai, 1991). The mutant strain did nt grw in the absence f divalent catins. As bth PG and DPG will nrmally frm a stable bilayer these results strngly indicate that the membrane shuld cntain phsphlipids which prefer t frm an inverted hexagnal (H N ) structure. In all experiments, a bilayer t nn-bilayer transitin was bserved at apprximately 10 C abve the grwth temperature (Rietveld et ai, 1993). By maintaining this tendency f the membrane t nn-bilayer structures, it is pssible that lcally cnditins exists which can result in the frmatin f nn-bilayer structures.thisis prbably imprtant in functinal prcesses such as membrane fusin, cell divisin, and transbilayer mvement f lipids and prteins (de Kruijff et ai, 1985; Lindblm & Rilfrs, 1989; Seddn, 1990). Althugh shrt-chain alkanls like ethanl will stabilize the bilayer structure, this culd pssibly be unfavurable fr cellgrwth, as micrrganisms apparently need t maintain the balance between bilayer and nn-bilayer frming phsphlipids in such a way that a tendency twards an inverted hexagnal (H N ) structureis maintained. When cmparing the effects f alkanls n bth T M and T LH it is interesting t nte that lwer alchls (C 3) decrease the T M (mre fluid membrane) and increase T LH, while lng-chain alkanls (C > 10) result in mre rigid (increased T M ) but less stable bilayers (decreased T LH ). The alkanls with a chain length between C4 and

107 Adaptatin f micrrganisms t txic slvents 107 CIO increase the membrane fluidity and destabilize the bilayer structure (decreased T M and T LH ). The interactin f shrt-chain alkanls with the membrane apparently stabilizes the bilayer structure since the T LH increases. Hwever, it has been demnstrated that these shrt-chain alkanls (C 3) can prmte the frmatin f an unusual phsphlipid cnfiguratin, the interdigitated phase (Lpl) (Simn & Mcintsh, 1984; Slater & Huang, 1988; Vieri et ai, 1994). In the interdigitated cnfiguratin the lipid acyl-chains frm the ppsing mnlayers are fully interpenetrated, thereby expsing the terminal methyl grups. (Fig. 6). These shrt-chain alkanls (C 3) will anchr with their plar miety t the phsphlipid headgrup, and with the nn-plar part between the phsphlipid acyl-chains. Since the nn-plar miety f these mlecules is shrt cmpared t the fatty acid acyl-chains, these mlecules wuld ptentially cause vids between the lipid chains in the bilayer interir. As the energy f frmatin f hles between hydrcarbns is extremely large, the lipids respnd by frming the interdigitated phase (Simn & Mcintsh, 1984). Althugh the interdigitated gel phase has been bserved in varius phsphlipids, this lipid structure is nt frequently bserved in PE systems (Lhner, 1991). As PE is the majr phsphlipid in many micrrganisms, the bilgical significance f the interdigitated gel phase is unclear. PE has, cmpared t ther phsphlipids, a small headgrup area. It is expected that due t this small headgrup, the interactin f the small alkanls (C ^ 3) with the membrane will nt cause the frmatin f an interdigitated phase. Masn and Stephensn (1990) studied the thermtrpic prperties f varius mixed acyl PEs. They used PEs in which the chain-length f ne fatty acid was cnstant (CI8) and the ther was varied between CIO and CI8. Only with the C18-C10-PE a mixed mm Fully interdigitated Figure 6: Schematic drawing, shwing the ethanl induced cnversin f DPPC bilayers frm the nn-interdigitated gel phase (L) t the fully interdigitated gel phase (Lpl).

108 108 Chapter 8 interdigitated gel phase packing f the phsphlipids was bserved (Masn & Stephensn, 1990). In DMPE vesicles high cncentratins f 1-prpanl resulted in a phase, which had characteristics f the interdigitated gel phase, hwever, n direct evidence that a true interdigitated gel phase had frmed is available (Centen & O'Leary, 1990). Besides shrt-chain alkanls (C 3) als several ther mlecules, such as benzyl alchl, can cause the lipid hydrcarbn chains t interpenetrate and induce the frmatin f an interdigitated gel phase (Mcintsh ef a/., 1983). All these cmpunds interact with the headgrup area f the phsphlipids and d nt partitin deeply in the lipid bilayer. Althugh the interdigitated lipid phase can be induced by varius additives it shuld be nted that this lipid cnfiguratin is nly bserved fr bilayers in the gel-phase (Simn & Mcintsh, 1984; Slater & Huang, 1988; Vieri et al., 1994). In micrrganisms, hwever, mst f the phsphlipids are generally in the liquid-crystalline phase. It is therefre unclear if this interdigitated phase will ccur r can be induced by slvents in micrrganisms Effects f alkanes and armatic hydrcarbns n bilayer fluidity and stability In general n-alkanes have a similar effect n the membrane fluidity as the crrespnding n-alkanls. Lng-chain alkanes (C ^ 12) increased the T M f varius phsphlipids, and shrt-chain alkanes (C < 12) decreased the T M (Lhner, 1991). The alignment f the rigid, lng-chain alkanes parallel t the acyl-chains f the phsphlipids, will have a rigidifying effect n the membrane similar t the lngchain alkanls. The lwer alkanes increase the membrane fluidity, as they will disturb the Van der Waals interactin between the phsphlipid acyl chains. n-alkanes accumulate between the acyl-chains f the phsphlipids, near the centre f the bilayer. The accumulatin f these alkanes can be visualized as a wedge which is placed in between the phsphlipid acyl-chains and will thus destabilize the bilayer structure and prmte the frmatin f a reversed hexagnal (H N ) lipid structure. Indeed incrpratin f C6-C20 n-alkanes prmted the frmatin f such a nn-lamellar structure (Seddn, 1990; Lhner, 1991). In general lnger alkanes are mre effective in reducing the L a - Hu transitin temperature. The C20 saturated alkane eicsane fr instance lwers the T LH transitin by 15 C at a cncentratin f abut 3 mle %. In cntrast, the gel t liquid-crystalline transitin temperature (T M ) is nt significantly altered in the presence f 3% eicsane (Espand, 1985). Cmpared t n-alkants, the crrespnding n-alkanes were mre ptent in destabilizing the bilayer structure. As the alkanls interact with the phsphlipid headgrup area, they will nt nly increase the tail area V/l' but als the

109 Adaptatin f micrrganisms t txic slvents 109 headgrup area 'a' and are thus less effective in prmting an inverted hexagnal lipid phase than the crrespnding alkanes (Hrnby & Cullis, 1981). The effects f armatic hydrcarbns n the membrane fluidity and stability have received nly limited attentin. In an extensive study Jain and Wu (1977) shwed that rganic slvents like benzene, tluene and chlrfrm all reduced the lamellar-gel t liquid-crystalline phase transitin temperature (T M ) f DPPC lipsmes. As these slvents are expected t partitin in between the lipid acylchains f the phsphlipids, they will be able t induce the transfrmatin f a lamellar lipid cnfiguratin int a hexagnal (H N ) cnfiguratin. The frmatin f an H,, phsphlipid cnfiguratin due t benzene additin has been shwn in DPPC lipsmes, at a lw water cntent (McDaniel ef a/., 1982). 5. EFFECT OFSOLUTE PARTITIONING ON MEMBRANEFUNCTIONS 5.1. Membrane permeability The main functin f the cell membrane is a permeability barrier, regulating the passage f slutes between the cell and the external envirnment. The barrier prperties f the cytplasmic membrane is f special imprtance fr the energy transductin f the cell (Nichlls, 1982). An increase in the permeability f the membrane fr prtns r ther ins may lead t a dissipatin f the prtn mtive frce (Ap), resulting in a less effective energy transductin. Furthermre, an increased permeability f the cell might als affect the internal ph cntrl f the cell (Sikkema et al., 1995) r culd result in the lss f essential metablites. As seen in the previus paragraphs, the accumulatin f varius slvents has an effect n the membrane fluidity. Studies with bth lipsmes and intact cell have shwn repeatedly that the permeability is affected by the membrane fluidity. An increase in the fluidity due t the incrpratin f mre unsaturated fatty acids r lipids with a reduced chain length, resulted in an increased permeability f the membrane (de Gier et al., 1968; McElhaney ef al., 1970, 1973; Cullen et ai, 1971; Rmijn et al., 1972; Melchir, 1982). An increase in the permeability has als been bserved in varius membranes, upn accumulatin f membrane-fluidizing slvents like ethanl, narctics, antibitics, and armatics (Bangham et al., 1965; Silver & Wendt, 1967; Jhnsn & Bangham, 1969; Saha et al., 1970; Cartwright ef a/., 1986; Heipieper ef a/., 1991; Szgyief a/., 1993; Sikkema ef a/., 1994a). The effect f alkanls n the membrane permeability can be illustrated by the results btained, already 30 years ag, by Bangham and clleagues. They determined the 42 K + -permeability f lipsmes and shwed that alkanls

110 MO Chapfer8 150 Sutanl CD W (0 100 Pentanl Hexanl Heptanl D Octanl Alkanl cncentratin (C/C 0 > Figure 7: Leakage rate f 42 K + frm phsphatidylchline/phsphatic acid (85:15) vesicles in the presence f varius cncentratins f alkanls. (Cncentratin is expressedas the fractin (C/C 0 ) f an alkanl saturated KCl slutin added). Adapted frm Bangham ef a/., (1965). significantly increased the K + efflux. (Bangham ef a/., 1965) (Fig. 7). Recently, it has been shwn that the txicity f cyclic armatic hydrcarbns fr micrrganisms is caused by an increase in the membrane permeability induced by these hydrcarbns. Accumulatin f varius cyclic hydrcarbns in the lipid bilayer enhanced the prtn permeability f whle cells (Sikkema ef ai, 1992) and f lipsmes derived frm E. cli phsphlipids (Sikkema ef a/., 1994a). As a result f this increase in prtn permeability the prtn-mtive frce f the cell was significantly decreased and in sme instances cmpletely dissipated. Fr all hydrcarbns tested a maximum increase in the permeability f the membrane was bserved at a membrane cncentratin f apprximately 1 slvent mlecule per 2 phsphlipid mlecules (Sikkema et al., 1994a). As pinted ut befre, slvents have an effect n bth the fluidity and the bilayer stability. The accumulatin f a slute in the membrane can eventually result in the frmatin f a nn-bilayer phsphlipid structure r an interdigitated phase. The frmatin f such structures will f curse strngly affect the barrier prperties f the membrane. It is, hwever, unclear if an effect n the membrane permeability will be bserved when the packing gemetry f the phsphlipids is nly slightly changed and n nn-bilayer structures are frmed. Accumulatin f a slute in a phsphlipid mnlayer might result in the spntaneus curvatin

111 Adaptatin f micrrganisms t txic slvents 111 (psitively r negatively) f the lipid mnlayer, due t an imbalance f the frces acrss the layer (Seddn, 1990). In a symmetric bilayer, hwever, the tw phsphlipid mnlayers want t curve the same way, and s cunteract each ther since they are ppsitely riented back-t-back (Grüner ef a/., 1985; Seddn, 1990). This "state f physical frustratin" in the bilayer may mdify many prperties f the bilayer such as thickness and permeability (Seddn, 1990). Ganglisides are nrmal cmpnents f the plasma membrane f vertebrate cells. These lipids have a large headgrup area (140 A 2 ) cmpared t ther phsphlipids (e.g. egg PC: 62 Â 2 ) and will therefre frm a nrmal hexagnal (H,) lipid cnfiguratin (Jain, 1988). Incrpratin f varius ganglisides in membrane vesicles will decrease the fluidity f the membrane (Bertli ef a/., 1981), which is expected t result in a reduced permeability f the membrane. Hwever, an increase in the membrane permeability is bserved upn incrpratin f ganglisides (Sarti ef a/., 1990). It is likely that the "state f physical frustratin" in the bilayer, caused by the large headgrup f the gangliside will have caused this increased permeability. The interactin f very shrt-chain alkanls (C 3) with the membrane is rather cmplex. These alkanls can transfrm the nrmal bilayer structure in an interdigitated phsphlipid cnfiguratin (Lpl). In this cnfiguratin the lipid acylchains are expsed t the phsphlipid-aqueus interface (Fig. 6). As plar headgrups are imprtant fr the barrier prperties f the membrane bilayer, it is expected that the frmatin f such lipid cnfiguratins will result in an increased permeability f the membrane. At 20 C DPPC is in the nn-interdigitated gel phase at ethanl cncentratins belw 1.0 M (Zeng ef a/., 1993). A dramatic increase in the prtn permeability is bserved at 1.2 M ethanl at 20 C and 2.0 M at 14 C.This abrupt increase in permeability ccurs at almst the same ethanl cncentratin which causes the frmatin f the fully interdigitated gel phase at bth temperatures (Zeng ef a/., 1993) Effect n membrane-embedded enzymes The membrane is besides a barrier als a matrix fr varius imprtant enzymes. These include enzymes invlved in slute transprt, and enzymes participating in the electrn transprt chains. Many studies have shwn that the activity f membrane-embedded enzymes can be influenced by the physic-chemical prperties f the membrane (Yeagle, 1989; In 't Veld ef a/., 1991). The interactin f a slvent with the membrane will influence these membrane prperties, and therefre will affect the activity f membrane-embedded enzymes.

112 112 Chapter 8 In E. cli bth the K m and V f the ß-galactside transprt system increased in the presence f n-alkanls (Sullivan et a/., 1974). A reductin f the enzyme activity by the accumulatin f varius cyclic hydrcarbns in the membrane was bserved fr cytchrme c xidase recnstituted in lipsmes (Sikkema er ai, 1994a). Similarly, benzyl alchl affected the activity f varius intrinsic membrane enzymes f the rat liver plasma membrane (Grdn et ai, 1980). A systematic examinatin f hw varius regins f the bilayer affect the catalytic activity f sugar transprt prteins has been carried ut by Carruthers and Melchir (1986). Changes in the lipid cmpsitin can drastically alter the transprter's activity. Hwever, these authrs cncluded that the bilayer fluidity is f minimal imprtance fr the activity f this enzyme. Only the transitin f the membrane frm the gel t the liquid-crystalline phase had a limited influence n the activity in certain bilayers. Especially bilayer thickness and the type f phsphlipid headgrup had an influence n the enzyme activity (Carruthers & Melchir, 1986). In 't Veld et al (1991) pstulated that the activity f transmembrane carrier prteins is strngly affected by the degree f matching between the bilayer thickness and the hydrphbic thickness f the prtein. Interactin f n-alkanes with membranes have been shwn t increase the bilayer thickness (Mcintsh ef a/., 1980; Ppe et a/., 1984) and will cnsequently affect the activity f transmembrane carrier prteins. McElhany (1989) reviewed the influence f the membrane lipid cmpsitin n the activity f membrane-bund enzymes in Achleplasma laidlawii B. NADH xidase and p-nitrphylphsphatase were bth unaffected by the fluidity and phase state f the membrane. Fr the (Na + + Mg 2+ )-ATPase the activity varied with the fatty acid cmpsitin, hwever, abve the phsphlipid phase transitin temperature the ATPase activities were rather independent f the fatty acid cmpsitin. This indicates that alteratins in the fatty acid cmpsitin, and thus membrane fluidity had nly a small effect n the enzyme activity (McElhaney, 1989). The activity f the enzyme was, hwever, strngly influenced by the headgrup cmpsitin. Recnstitutin f the enzyme in DMPC vesicles resulted in a fully active enzyme, but in DMPC lipsmes it was fully inactive (McElhaney, 1989). The activity f the Ca 2+ -ATPase f rabbit muscle is als strngly affected by the phsphlipid headgrup cmpsitin. An increase in the activity was bserved when the enzyme was recnstituted in membranes with either a high PE r MGDG cntent (Navarr et ai, 1984; Yeagle, 1989). A prperty that PE and MGDG have in cmmn is the ability t frm the inverted hexagnal (H M ) phase. Chlesterl, which can induce the H M phase, als stimulated the activity f the Ca 2+ -ATPase recnstituted in vesicles dminant in PE (Cheng et a/., 1986). Althugh the reasn fr these variatins in enzyme activity have nt yet been

113 Adaptatin f micrrganisms t txic slvents 113 discvered, it is clear that many factrs, including the phase preference f the phsphlipids, have an influence n the enzyme activity. Several membrane-embedded enzymes depend fr their activity n a specific bundary phsphlipid (Sandermann, 1978; Carruthers & Melchir, 1986). It is expected that these phsphlipids are necessary fr the crrect cnfrmatin and rientatin f the enzyme. Displacement f these bundary lipids by the interactin f rganic slvents with the membrane might result in a lipid/prtein mismatch and inactivatin f the enzyme (Sandermann, 1993). Furthermre, direct interactins f the slvent with hydrphbic parts f the enzyme might als ccur, which are expected t affect the enzymatic activity. 6. ADAPTATION As has been shwn in the previus paragraphs the accumulatin f slvents in the membrane bilayer can influence bth the membrane fluidity and the bilayer stability. These changes in the physic-chemical prperties f the membrane can affect the permeability f the membrane as well as the activity f membraneembedded enzymes. Cnsequently, the viability f the ceil will be affected. Althugh there are many examples f the txicity f rganic slvents fr micrrganisms, sme micrrganisms are able t adapt and grw in the presence f txic cncentratins f these slvents. Spectacular examples are Lactbacillus heterhichii which is able t grw in the presence f up t 20% ethanl (Uchida, 1975a) and the adaptatin f several Pseudmnas strains allwing them t grw in the presence f a secnd phase f tluene, p-xylene r styrene (Inue& Hrikshi, 1989; Cruden et al., 1992; Weber et ai, 1993) 6.1. Adaptatins at the level f the membrane Several adaptive changes in the membrane cmpsitin have been bserved in reactin t the accumulatin f rganic slvents in the membrane f micrrganisms (Table 3). These changes will, as discussed belw, cunteract the effects induced by the accumulatin f a slute. These mdificatins f the membrane cmpsitin can be explained either as a mechanism t restre the fluidity f the membrane (hmeviscus adaptatin) (Sinensky, 1974) r as a methd t restre the balance between bilayer and nn-bilayer frming phsphlipids. Changes in the membrane cmpsitin can als reduce the slute partitining int the membrane, resulting in lwer cncentratins in the membrane, thus als reducing the txicity (Antunes-Madeira & Madeira, 1985, 1989).

114 'J4 Chapter 8 c > lo 'c > 0 (D c - 0 "3> in 0 r (i S s > t CK 'S <5 0 r (1 S s * t> lu 5> 0 r 0 8 S CK CO CK 0 <1) c O > O Ü ' c x: U O CK "5 _2 ^ S CK a Ol tu (- O) c* n O) <u c XL 1) t XI «O > m n a CO CK U <u tu CO 3 S (U 0 a- - a. tu X! E Q g 'tu x 0 ai * tu tu Q_ TJ a CK - n t) r u n n r u «5 c 0) a -C c a. c ö) ö u g b > _c TJ (D Q) t/1 XI O t <u O) c XL O 0 _> Q. Ö < CO J» SI c 0 0> O O O Û Q Q Ü O Ü Q Q Û Ö O Ü Û Q Q O O O m U a K TI n fc.0 0 IU u TJ U 0 ^- U Tl a> t r 0 cû c a S 01 (1 1/1 TJ O 0 ^K 0 T) tu L. t) r t) CO g -0 3 i - 11 t u c _> û I) 0 E 3 TJ 0 U c 0 c u <-!) U O O E tu i TJ O U <- 0."C <r t m t. «- 0 0 r 3 c.c r U) tu t n.c U 8 IJ A O c c.c ÜJ U LE u u E g a u 3 5

115 Adaptatin f micrrganisms t txic slvents 115 XI Ç ~c ü c 0) _> t/> 'c D) O O 0) c (U (D a a) x: c a c ra b D > ç "Ö ai i. 0) ta _Q O t O) ca c.c 0 _> Q. O XI < «n < -^ - ^ D - (1> 0) a a> X 0) n CO a a> X CS CS "~ O et) "5 Q_ <D U g 0 4 a I CM CK D a> c 0) > 0 0) y 0 U»â c «CO TD O S c CK CS *" n <a 0) fi c 0 < 2 CO CS cs " D ö) c O CS CS 0 Ol 0 0 CS CS D a> O O F a 0> ó >^ 11 cö CS : U <D 0 x r X CÛ CO CS 0 t: U c c CO CS CO Û. ^ V O E a> a - s- 0 cl) O) LU O X 1 0> X <- > > l) ü a c c X X U U c a> x a x 3! V =S O c X Lu uu O c X S 3 a E "O 1) E 3 E c u u 'O c a x O O «- 0 Vï 0 c 3 -> 'S < > c 3 c g <-" ö V? g ie 3 U c 0 t -> D g c 3 <-.2 y C g * -» "5 vi g c 3 C 'a x U *-" Ô ^ 0 c 3 <- X 0) c 1) u 0 > a c 'a x u > c 1 -» 0 «jo 0 i/> C 3 <- OO g c 3 a eu c 0 X u c 3 XI a E S c E E

116 116 Chapter 8 6. h 1. lipid cmpsitin a. Adaptatin t alkanls The changes bserved in lipid cmpsitin f micrrganisms due t the presence f alkanls and especially ethanl, have been reviewed befre (Casey & Ingledew, 1983; Ingram & Buttke, 1984; Ingram, 1986, 1990; Mishra & Kaur, 1991). One f the changes in membrane lipid cmpsitin in respnse t ethanl is an increase in the amunt f unsaturated fatty acids bserved in Lactbacillus heterhichii (Uchida, 1975a), Lactbacillus hmhichii (Uchida, 1975b), Clstridium thermcellum (Herrer et al., 1982), Escherichia cli (Ingram, 1976), and Saccharmyces cerevisiae (Beaven et al., 1982). Hwever, an ppsite effect, in respnse t ethanl, is bserved in several ther micrrganisms. An increased cntent f less fluid lipids is bserved in ßac/7/us subtilis (Rigmier et al., 1980; Bhin & Lubchinsky, 1982), Clstridium acetbutylicum (Vllherbst-Schneck et al., 1984; Baer et al., 1987; Lepage et al., 1987) and tw Micrsprum species (Bansal & Khuller, 1981). The high levels f unusual lng-chain fatty acids fund in L heterhichii are als expected t reduce the fluidity (Uchida, 1975a). Apparently, tw mdes f membrane adaptatin ccur: (1) The increased incrpratin f unsaturated fatty acids which will cunteract the effects f ethanl n the bilayer structure. (2) The increased incrpratin f less fluid lipids which will cmpensate the increase in membrane fluidity caused by ethanl (Hmeviscus adaptatin). As will be discussed belw either f the tw mechanisms emerged. Adaptatins t maintain the bilayer stability. Accumulatin f ethanl in lipid bilayers has a prnunced effect n the headgrup area f the membrane phsphlipids and results in an increase in the apparent phsphlipid headgrup area 'a'. As a result f this increase ethanl can (1) induce the frmatin f the interdigitated gel phase, and (2) will increase the T LH f the membrane. The frmatin f the interdigitated phase, resulting in an increased permeability f the membrane, is expected t affect the viability f the cell. The bserved incrpratin f unsaturated fatty acids in varius micrrganisms in respnse t ethanl, will increase the lipid vlume V' and is therefre expected t prevent the frmatin f the interdigitated lipid phase. As pinted ut earlier, mst f the lipids in micrrganisms will be in the liquid-crystalline phase. The interdigitated phase, hwever, is nly bserved in the gel phase. It is therefre dubtful if this phase will ccur in bilgical membranes under nrmal cnditins. Ethanl, and ther shrt-chain alkanls (C 3) increase the T LH and cnsequently have a psitive effect n the stability f the bilayer, unless they wuld be able t induce the frmatin f a nrmal hexagnal (H,) r micellar lipid structure.

117 Adaptatin f micrrganisms t txic slvents I17 Hwever, the inductin f this lipid cnfiguratin by ethanl has nt been bserved. Apparently, ethanl has a stabilizing effect n the bilayer cnfiguratin. Several studies have shwn that micrrganisms have t maintain a certain balance between bilayer and nn-bilayer phsphlipids in rder t remain viable (Wieslander er ai, 1980; Gldfine et al., 1987; Rietveld et ai, 1993). As discussed befre, grwth f an E cli mutant, defective in the synthesis f PE,is nly bserved when the T LH f the phsphlipids is abut 10 C abve the grwth temperature (Rietveld er al., 1993). Ethanl increased the T LH, and will thus disturb the bilayer t nn-bilayer phsphlipid rati, which is expected t affect the cell viability. The bserved increase in unsaturated fatty acids in varius micrrganisms is expected t cmpensate fr the ethanl induced increase in T LH, as unsaturated fatty acids decrease the T LH. In Lacfbac/7/us heterhichii and Lactbacillus hmhichii, the mst ethanl resistant micrrganisms knwn that are capable f grwing in cncentratins up t 20%, an increase in the amunt f unsaturated fatty acids is bserved in respnse t ethanl (Uchida, 1975a, 1975b). Similar changes have been bserved in Clstridium thermcellum (Herrer et ai, 1982), Escherichia cli (Ingram, 1976), and Saccharmyces cerevisiae (Beaven et ai, 1982). The membranes f Zymmnas mbiles, an bligate fermentative bacterium prducing high cncentratins f ethanl, are particularly rich in 18:1 cis. This fatty acid accunts fr mre than 70% f the ttal fatty acids (Carey & Ingram, 1983; Barrw et ai, 1983; Buchhlz et ai, 1987). Only in cntinuus cultures where a lw glucse feed results in a lw cncentratin f ethanl a reduced cntent f 18:1 eis was bserved (Bringer et ai, 1985). E. cli grwn in the presence f shrt-chain alkanls (C ^ 3) shwed an increased incrpratin f unsaturated fatty acids. Lng-chain alkanls (C5 - C9) caused the ppsite effect: an increase in the amunt f saturated fatty acids. Decanl (CIO) did nt induce changes in the fatty acid cmpsitin (Ingram, 1976; Sullivan et ai, 1979). A very gd agreement exists between the bserved adaptive changes in the membrane cmpsitin and the effects f these alkanls n the T LH. The shrt-chain alkanls (C 3) will increase the headgrup area f the phsphlipids in the membrane. The bserved increase in the amunt f unsaturated fatty acids will decrease '/' and increase V' and cmpensate the increase in 'a' caused by these alkanls. The ppsite is bserved with the lngchain alkanls (C5 - C9). Accumulatin f lng-chain alkanls prmtes the frmatin f an inverted hexagnal (H M ) phase (increase in V). The higher amunt f saturated fatty acids fund (decrease in V', increase in '/') cmpensates this and stabilizes the bilayer structure. Decanl will, just as the ther lng-chain alkanls, increase the lipid-vlume V'. Hwever, n changes in the lipid

118 118 Chapter 8 cmpsitin are bserved in respnse t decanl (Ingram, 1976; Sullivan et ai, 1979). These changes were expected when the adaptatin mechanisms was directed at restring the balance between bilayer and nn-bilayer frming phsphlipids. Experiments with mutants f E. cli, defective in the synthesis f 18:1 cis, have shwn that an increase in 18:1 cis cntent is beneficial fr ethanl tlerance. These mutants, lacking 18:1 cis, are hypersensitive t killing by ethanl (Ingram et al., 1980). Supplementatin f these mutants with 18:1 cis rendered these cells less sensitive t ethanl-killing (Ingram et ai, 1980; Ingram, 1990). The incrpratin f 18:1 cis increases the fluidity f the membrane which apparently is beneficial t acquire ethanl resistance asit cunteracts the increase int LH due t ethanl accumulatin in the membrane. These results suggest that cntrl f membrane fluidity is nt imprtant fr cells as a defence mechanism against txic cncentratins f alkanls. Als fr A. laidlawii, Wieslander and c-wrkers cncluded frm extensive studies that this micrrganism nly changes its membrane cmpsitin t maintain a certain balance between bilayer and nnbilayer prmting phsphlipids (Rilfrs et a/., 1984; Wieslander et ai, 1986).The bserved changes in membrane cmpsitins in respnse t hydrcarbns, alkanls and detergents culd nt be explained as a mechanism t maintain a cnstant membrane fluidity (Wieslander et ai, 1986). Furthermre the capacity f varius micrrganisms t grw at temperatures that are well abve the gel t liquid crystalline phase transitin temperature (T M ) f the membrane lipids, wuld als indicate that regulatin f the membrane fluidity is less imprtant fr cell viability (Steim et ai, 1969; McElhaney, 1974). Similar cnclusins might be drawn frm wrk f Jacksn and Crnan (1978) wh studied the grwth rate f E. cli in relatin t the physical state f the membrane. The membrane lipids f wild-type E. cli were entirely fluid at bth 25 C and 37 C.They shwed that at least sme part f the lipids must be in the fluid state fr E. cli t grw, and that cells can tlerate large variatins in the amunt f fluid lipids withut shwing any majr effects n vital prcesses (Jacksn and Crnan, 1978). Hmeviscus adaptatin. Althugh the results presented in the previus paragraph indicate that a regulatin f membrane fluidity is nt imprtant t acquire slvent tlerance, several ther studies d shw a regulatin f membrane fluidity. An interesting example in this respect is the reactin f E. cli t decanl. This alkanl has a strng effect n the packing f lipids in the membrane and can induce the frmatin f the H N phase (Fig. 5). ButE. cli des nt change its lipid cmpsitin in respnse t decanl (Ingram, 1976; Sullivan ef a/., 1979), which is in keeping with the hmeviscus adaptatin thery, since decanl has n effect

119 Adaptatin f micrrganisms t txic slvents 119 n the membrane fluidity (Fig. 3). Ethanl in the same rganism induces besides an increase in the amunt f unsaturated fatty acids als an increase in chain length f the fatty acids. This increase in chain length reduces the membrane fluidity, while the increased unsaturatin has the ppsite effect. The net result f bth effects apparently is a less fluid membrane. Further evidence that cntrl f membrane fluidity is imprtant cmes frm studies with E. cli mutants, defective in the synthesis f 18:1 cis. Supplementatin f these mutants with trans fatty acids was mre beneficial t acquire ethanl tlerance than supplementatin f these mutants with c/s-fatty acids (Ingram et al., 1980; Ingram, 1990). Trans fatty acids have a higher T M than the crrespnding cis ismer and will thus reduce the membrane fluidity. Further evidence that the regulatin f membrane fluidity is imprtant fr cells t acquire ethanl-tlerance has been btained in mutant-studies f Clstridium thermcellum. Accumulatin f ethanl, similar as in cli, resulted in the synthesis f lipids with a reduced melting pint (Herrer et ai, 1982). Hwever, it was speculated that these changes are maladaptive, as in an ethanl-tlerant mutant strain the changes in fatty acid cmpsitin upn ethanl additin were nt as dramatic as in the wild type (Herrer et al., 1982). Other infrmatin available n fatty acid cmpsitin as affected by alkanls in several ther micrrganisms als indicates that cntrl f membrane fluidity might be imprtant. In the extreme ethanl tlerant L heferh/ch/7 high levels f unusual lng-chain fatty acids (C20-C24) have been fund (Uchida, 1975a) which will reduce the membrane fluidity. Similarly, an ethanl-induced increase in the cntent f less fluid lipids was bserved in Bacillus subtilis (Rigmier et al., 1980; Bhin & Lubchinsky, 1982), Clstridium acetbutylicum (Vllherbst-Schneck et al., 1984; Baer et ai, 1987; Lepage et ai, 1987) and tw Micrsprum species (Bansal & Khuller, 1981). Apparently in these micrrganisms the membrane fluidity is cmpensated by this increase in the amunt f rigid fatty acids. Hwever, these membrane changes will nt cmpensate fr the effect which ethanl has n the T LH. In Schizsaccharmyces pmbe and Mycbacterium smegmatis ppsite adaptatin mechanisms in respnse t ethanl were bserved at varius grwth cnditins. In aerbic-grwn cells f S. pmbe, similar t cli, an increase in the amunt f unsaturated fatty acids was bserved in respnse t ethanl (Kuku et ai, 1990). Hwever, in anaerbically-grwn cells an ppsite effect is bserved. In these cells an increase in saturated fatty acids was fund (Kuku ef ai, 1990). In M. smegmatis ethanl induced ppsite effects n the saturatin index f cells grwn at either 27 and 37 C. At 37 C a decrease in the amunt f saturated fatty acids was bserved, whereas at 27 C the amunt f saturated fatty acids increased (Taneja & Khuller, 1980).

120 120 Chapter l.b. Adaptatin t ther rganic slvents In cntrast t the huge amunt f literature cncerning the adaptatin mechanisms f micrrganisms t alkanls, nly a limited amunt f infrmatin is available cncerning the adaptatin t ther rganic slvents. Ingram (1977) has studied the changes in lipid cmpsitin f E. cli, resulting frm grwth in the presence f several rganic slvents. Frm this study it appears that E. cli dramatically alters its lipid cmpsitin in respnse t the presence f lipphilic cmpunds. Relatively plar slvents, like acetne, dimethyl sulfxide, and tetrahydrfuran all caused an increase in the synthesis f lipids cntaining unsaturated fatty acids, analgus t the effect f shrt-chain alkanls. Incubatin with mre aplar slvents such as benzene, chlrfrm, r aniline caused an ppsite effect, an increase in the amunt f saturated fatty acids, similar as caused by the lng-chain alkanls. In certain Pseudmnas strains a fully different adaptatin mechanism has been bserved. A cnversin f cis fatty acids int the crrespnding frans-ismer was fund in a P. putida strain in reactin t phenl and chlrphenls (Diefenbach et al., 1992; Heipieper et ai, 1992). A similar adaptatin has been bserved in P. putida strains capable f grwing in the presence f supersaturating cncentratins f tluene (Weber et al., 1994). Cells grwn in the presence f 1% (v/v) tluene pssessed a membrane with an increased amunt f transunsaturated fatty acids, and a decreased amunt f the c/'s-ismers (Weber et al., 1994).This adaptatin nt nly ccurred at supersaturating cncentratins but als at subsaturating cncentratins (< 640 mg/l) f tluene in water. N effects were seen in P. putida SI2 belw 300 mg/l tluene, but abve 300 mg/l the trans:cis rati f the cells increased dramatically. Cells having a high transicis rati were well equipped t survive shck additins f tluene (1%(v/v)). The benefit f this ismerizatin reactin lies in the steric differences between cis- and frans-unsaturated fatty acids. Trans fatty acids have a steric cnfiguratin similar t saturated fatty acids. The c/s-ismer, hwever, has a nick in the acyl chain f the fatty acids, which causes steric hindrance and a membrane with a higher fluidity (Fig. 8). The bserved increase in the transicis rati will thus have a rigidifying effect n the membrane. In the tluene-adapted strain, pssessing a high transicis rati, the transitin temperature f the membrane in viv was 7-9 C higher cmpared t the nn-adapted cells (Weber ef a/., 1994).

121 Adaptatin f micrrganisms t txic slvents f 2 / Cis-Unsaturatedfattyacids Trans-Unsaturatedfattyacids Figure8: Cnfiguratin f cis and frans unsaturated fatty acids in a membrane Tluene will, besides increasing the membrane fluidity, als prmte the frmatin f an inverted hexagnal lipid phase. The bserved cnversin f cis unsaturated fatty acids int trans fatty acids in P. putida is expected t cunteract the frmatin f a nn-bilayer structure. This bservatin can be ratinalized because the lipid vlume V' f the trans fatty acids is smaller than the cis ismer. In studies with lipsmes it was shwn that 18:1 trans PE exhibits a lamellar t hexagnal (H N ) transitin between 60 C and 63 C, whereas fr 18:1 cis PE this transitin ccurs between 7 and 12 C (Tilcck & Cullis, 1982). Interestingly, P. putida cells are still able t frm frans-fatty acids when the fatty acid bisynthesis is inhibited by cerulenin (Diefenbach et al., 1992; Heipieper et al., 1992). This cisitrans ismerizatin is an adaptatin mechanism which des nt require de nv synthesis f lipids like the ther adaptatin mechanisms knwn (Diefenbach et al., 1992) and prvide the cell with a very quick mechanism t cmpensate fr the increase in membrane fluidity and the risk f excessive frmatin f nn-bilayer structures, caused by tluene. In summary, it can be cncluded that micrrganisms change their lipid cmpsitin in the membrane in respnse t the accumulatin f rganic slvents. Apparently these changes are a mechanism t maintain a certain rati between bilayer and nn-bilayer phsphlipids which prevent the frmatin f nn-bilayer structures r are mechanisms t restre the fluidity f the membrane (hmeviscus adaptatin). Hwever, especially fr ethanl, ppsite adaptatin mechanisms have been bserved in varius micrrganisms. It is difficult t explain the adaptatin f these varius micrrganisms n the basis f the bserved changes in membrane cmpsitin nly. As will be discussed hereafter ther adaptive changes, which als have an effect n bth the fluidity and the T LH, have t be taken int accunt as well Headgrup cmpsitin Besides the fatty acid cmpsitin, bviusly the headgrups are als f majr imprtance when cnsidering the physic-chemical prperties f lipid bilayers.

122 122 Chapter 8 Cnsiderably less data are, hwever, available n the headgrup cmpsitin than n fatty acid cmpsitin. Headgrups usually determined by TLC techniques include phsphatidylethanlamine (PE), phsphatidylchline (PC), phsphatidylglycerl (PG), diphsphatidylglycerl r cardilipin (DPG), mngalactsyidiglyceride (MGDG) and digalactsyldiglyceride (DGDG) (Fig. 9). Adaptive changes in the headgrup cmpsitin will have an effect n the physic-chemical prperties f the membrane. The differences in T M between phsphlipids with different plar headgrups can be as great as C (Bggs, 1984). At neutral ph, PG has the lwest transitin temperature. Prtnatin increases the T M f the acidic PG. Diphsphatidylglycerl has the highest T M f all phsphlipids at neutral ph (Bggs, 1984; Keugh & Davis, 1984). The difference f C in T M f PG and DPG might be bilgically imprtant as a mechanism t cmpensate fr changes in the membrane fluidity. Hwever, extensive changes in the phsphlipid cmpsitin shuld be necessary t cntrl the fluidity, and such changes are nt generally bserved. Furthermre, changes in the fatty acid cmpsitin have a mre prnunced effect n the membrane fluidity. Fr instance the intrductin f a duble bnd in ne fatty acid chain f a phsphlipid reduces thet M by C (Keugh & Davis, 1984). Besides an effect n the membrane fluidity, adaptive changes in the headgrup cmpsitin will have a strng effect n the transitin frm a bilayer t a hexagnal phase (H n ) (Fig. 4). Fr instance, in A. laidlawii MGDG and DGDG ccur as the tw main phsphlipid species. The rati between these headgrups was affected by slvents. Accumulatin f ethanl in the membrane resulted in an increase in the MGDG : DGDG phsphlipid rati. MGDG has a smaller lipid headgrup area than DGDG, and the changed rati cmpensates fr the increase in headgrup area caused by ethanl (Wieslander et al ). Benzene has an ppsite effect n membranes and prmtes the frmatin f a hexagnal phase, which was cunteracted in A. laidlawii with a decrease in the MGDG : DGDG-rati (Wieslander et al., 1986). We have been studying the adaptatin mechanisms f P. putida S12, a Gramnegative bacterium which can grw in the presence f supersaturating amunts f tluene (Weber et a/., 1993). Apart frm changes in fatty acid cmpsitin (Weber et a/., 1994) als changes in the phsphlipid cmpsitin were bserved when the rganism was grwn in the presence f tluene (Table 4).

123 Adaptatin f micrrganisms t txic slvents CH 2 CH 2 NH 3 Phsphatidylethanlamine R, C O CH 2 R c O C H 2 O ÇH 3 - CH 2 CH 2 N-CH 3 Phsphatidylchline CH 3 OH - CH 2 CH CH 2 OH Phsphatidylglycerl II I I 0 H 2 C O P - R,-C--0-CH 2 R -f 0 O II R 3-C- - c 1 HjC - -O CH, c -H H R 4-f -- HzC p= ÇH 1 2 ÇH p= L 0 CHOH H 2 COH HP J O..OH HÓ OH J 0^ O ÇH 2.OH HC O C R 1 I OH H 2 C O C R 2 Digalactsyldiglyceride HzCOH HO J O^Ç ÇH 2.OH HC O C R, OH H 2 C O C R 2 Diphsphatidylglycerl (Cardilipin) Mngalactsyldiglyceride Figure 9: Structures f varius membrane phsphlipids, illustrating the variety f plar headgrups.

124 124 Chapter 8 Table 4: Phsphlipid cmpsitin f P. putida SI2 grwn in a chemstat n glucse medium in the presence f varius cncentratins f tluene. Phsphlipids were identified by TLC (Skipski ef a/., 1967) and quantified by a phsphate determinatin (Ruse et al ). Tluene cncentratin (mg/l) % (v/v) PE 82.0 ± ± ± ± ± ±4.0 DPG 9.6 ± ± ± ± ±2.6 PG 8.4 ± ± ± ± ± ±0.8 PE/IDPG+PG) Tluene, as seen in Fig. 2, will accumulate in the vicinity f the headgrup area in between the lipid acyl chains. Cnsequently, it is expected t increase (v/l)/a and it will induce the frmatin f a hexagnal H M lipid phase. As shwn in Table 4, P. putida SI2 reacts t the presence f tluene by decreasing the incrpratin f PE in the membrane. PE is a phsphlipid with a relatively small headgrup area 'a' cmpared t PG and DPG. The decreased incrpratin f PE and the increased incrpratin f DPG will thus increase the average phsphlipid headgrup area 'a', which is expected t cmpensate fr the increase in lipid vlume V caused by the accumulatin f tluene and thus stabilizes the bilayer structure f the membrane. As the T M f DPG is abut 10 C higher than PE (Keugh & Davis, 1984), the increased incrpratin f DPG will als have a rigidifying effect n the membrane and is expected t (partially) cmpensate fr the tluene induced increase in membrane fluidity. Regulatin f the phsphlipid headgrup cmpsitin nt nly cntrls the bilayer/nn-bilayer phase preference f the lipids, but there is als evidence that the bilayer surface charge density is regulated. As shwn befre, accumulatin f certain slvents in the lipid bilayer might increase the distance between the phsphlipids in the lipid bilayer. These changes in the crss sectin area f the phsphlipids will affect the verall surface ptential r surface charge density f the membrane. Grwth f A. laidlawii in the presence f different amunts f palmitic acid (16:0) and leic acid (18:1 c/'s) resulted in cell membranes with varius degrees f unsaturatin. A. laidlawii decreased the MGDG : DGDG rati in respnse t an increase in unsaturatin in rder t maintain a prper balance between bilayer and nn-bilayer frming phsphlipids. Hwever, an increasing degree f

125 Adaptatin f micrrganisms t txic slvents 125 unsaturatin als resulted in an increased rati f charged lipids (PG) t uncharged lipids (MGDG plus DGDG). An increase in leic acid and the resulting decrease in the MGDG : DGDG rati f the phsphlipids results in an increase in the mlecular area ccupied by the lipids. The increased incrpratin f aninic PG keeps the surface charge density cnstant. (Cristianssn et a/., 1985; McElhaney, 1989). Similarly, in E cli the rati between the aninic and zwitterinic phsphlipids is kept almst cnstant. Studies with. cli mutants, defective in the synthesis f varius phsphlipids, have shwn that this rati can nt be varied withut disturbing cell grwth (Hawrt & Kennedy, 1975; Raetz, 1976, 1978). Accumulatin f ethanl in a lipid bilayer will increase the surface area f the lipids, and thus influences the surface charge f the lipids. Studies with S. cerevisiae have shwn that PS-enriched cells are mre ethanl resistant cmpared t PC r PE enriched cells. This increased ethanl tlerance has been attributed t the increased anin-zwitterinic phsphlipid rati f the phsphlipids. (Mishra & Prasad, 1988). A similar crrelatin between ethanltlerance and an increased aninic:zwitterinic rati has been bserved in. cli (Clark & Beard, 1979) and Bacillus subtilis (Bhin & Lubchinsky, 1982). The mechanism and reasn fr this regulatin f surface charge density are nt well understd. It is expected that it plays a rle in the bilayer stability. Due t the electrstatic repulsin between charged lipids, these are mre prnunced t frm lamellar phases. (Gennis, 1989). The activity f membrane-bund enzymes will als be influenced by an altered surface charge (Gennis, 1989) Membrane prtein cntent Studies with flurescent prbes have shwn that artificial membranes prepared f phsphlipids extracted frm ethanl-grwn E. cli cells were mre fluid than thse frm cntrl cells. This increase in fluidity was expected frm the bserved changes in lipid cmpsitin. Hwever, the intact plasma membrane f ethanlgrwn E. cli cells is mre rigid, indicating that a nn-lipid cmpnent cmpensated fr the increase in fluidity caused by ethanl (Dmbek & Ingram, 1984). The ethanl-adapted cells had an increased prtein-t-lipid rati, which has an rigidifying effect n the membrane (Dmbek & Ingram, 1984). In general prteins can be envisaged as a bulky rigid dmain in the bilayer. The mtin f the lipids in the vicinity is expected t be markedly hindered, resulting in an verall reductin in membrane fluidity (Shinitzky, 1984). Similar t ethanl, phenl als induced an increase in the prtein cntent f E. cli (Kewelh ef a/., 1990). An ethanl dse-dependent increase in the prtein cntent has als been bserved in Z. mbiles (Carey & Ingram, 1983). A decrease in the ttal amunt f phsphlipids, prbably caused by an increase in the

126 126 Chapter 8 prtein cntent, has been bserved in ethanl-grwn cells f B. subtilis (Rigmier etal., 1980) and Mycbacterium smegmaf/'s (Taneja & Khuller, 1980) Other membrane cmpnents In the previus paragraphs we have shwn that micrrganisms can change their phsphlipid cmpsitin in respnse t the txic actin f rganic slvents. Several ther specific methds t cunterbalance the effects f external factrs n bth the T M and T LH have been bserved. 6.1 A.a. Sterls and Hpanids Sterls are an imprtant cnstituent f the yeast plasma-membrane and can influence bth the fluidity and the bilayer stability. A decrease in ergsterl cntent f the Saccharmyces cerevisiae cell membrane has been directly related t a decrease in cell viability in the presence f ethanl (Larue et al., 1980; Lees et ai, 1980; Del Castill Agud, 1992). Similarly, supplementatin f Saccharmyces sake and K/uyvermyces fragilis with sterls enhanced the ethanl tlerance (Hayashida & Ohta, 1980; Janssens et al., 1983; Nvtny et ai, 1992). The rigidifying effect f sterls n membrane lipids has been suggested t be crucial in enhancing the viability f cells in the presence f ethanl. (Hssack &Rse, 1976). Sterls are nly present in eukarytic rganisms, but sterl-like structures have als been identified in prkarytes. Zymmnas mbilis is a bacterium which can prduce high cncentratins f ethanl. The membranes f this bligate fermentative bacterium are particularly rich in 18:1 cis (>70%) (Carey & Ingram, 1983; Barrw ef a/., 1983), presumably t cunterbalance the frmatin a hexagnal H tl structure. This high amunt f unsaturated fatty acids can nt cmpensate fr the ethanl-effect n the fluidity. Hwever, in this rganism hpanids have been identified as anther imprtant membrane cnstituent. The amunt f hpanids (l,2,3,4-tetrahydrxypentane-29-hptane) synthesized by Z.mb/7/s rises with increasing ethanl cncentratins. Since these hpanids reduce the membrane fluidity, accumulatin f these cmpunds is apparently a mechanism t cmpensate fr the increase in fluidity caused by ethanl (Buchhlz et al., 1987). 6.I A.b. Cartenids Cartenids may be present in sme bacterial membranes and, similar t sterls and hpanids, they have an influence n lipid fluidity. In Staphylcccus aureus, a direct crrelatin has been bserved between cartenid prductin, membrane fluidity, and resistance t leic acid (Chamberlain ef a/., 1991 ). Als A. laidlawii is knwn t prduce cartenids (Smith, 1963). The regulatry rle f cartenids in cntrlling the fluidity f A. laidlawii cell membranes was

127 Adaptatin f micrrganisms t txic slvents 127 suggested by shwing that the cartenids bisynthesis is regulated by membrane fluidity (Rttem & Markwitz, 1979). This finding suggests that the cartenid cntent f the membrane might restre the fluidity f the membrane when affected by slvents. Changes in the cartenid cmpsitin f A. laidlawii in respnse t the presence f tluene in the medium have indeed been bserved (Gvrun et ai, 1989) C Dicarbxylic acids In Sarcina ventriculi very high percentages («45 %) f dicarbxylic acids are frmed in respnse t ethanl r butanl (Jung et a/., 1993). Cntrl cells nly cntained abut 7% f these biplar lng chain fatty acids (C28-C36). These lipids are believed t be imprtant fr membrane integrity under stress cnditins. The tw plar grups n the dicarbxylic acids, which are present at each end suggests that this unusual lipid cmpnent spans the membrane (Jung et al., 1993). Lipsmes cmpsed f these lipids, riginating frm the thermacidphilic Sulflbus acidcaldarius, were cnsiderably mre stable and had a much lwer prtn permeability than lipsmes cmpsed f (nrmal) diester phsphlipids (Elferink ef a/., 1994) Bilayer fluidity versus bilayer stability In the previus paragraphs we have seen that the accumulatin f slutes in the membranes f micrrganisms can be cmpensated fr by several mechanisms. These mechanisms can be explained as either a mechanism t restre the fluidity (hmeviscus adaptatin) r as a mechanisms t maintain a prper balance between bilayer and nn-bilayer prmting phsphlipids. Wieslander and c-wrkers have pstulated frm their studies with A. laidlawii that all the bserved changes in membrane cmpsitin are a mechanism t maintain an ptimal rati between the bilayer and nn-bilayer frming phsphlipids and are nt a mechanism t cntrl the membrane fluidity (hmeviscus adaptatin). The ethanl induced increase in the amunt f unsaturated fatty acids bserved in E. cli and several micrrganisms seems t supprt this thery. The increase can nly be explained as a mechanism t cntrl the bilayer t nn-bilayer frming phsphlipid rati and nt as a mechanism t cmpensate the fluidity. Hwever, it seems unlikely that this hypthesis is valid in all cases: Firstly, E. cli des nt change its lipid cmpsitin in respnse t decanl. Such changes wuld be expected since decanl decreases the T LH (Fig. 5) f the phsphlipids. The absence f membrane changes suggests the cntrl f membrane fluidity is imprtant since decanl has n effect n the membrane fluidity (Fig. 3). Secndly, the amunt f unsaturated fatty acids decreased in respnse t ethanl in several micrrganisms ther than E. cli (Table 3). Thirdly, in A. laidlawii strain B a regulatin f the phsphlipid

128 128 Chapter 8 cmpsitin in respnse t either changes in temperature r chlesterl levels was nt bserved (McElhaney et al., 1970, 1973; McElhaney 1974, 1984). Finally, it is wrth nting that lng-chain alkanes, as fr instance hexadecane strngly decrease the T LH f phsphlipids. Hwever, these slvents are nn-txic fr micrbial cells, althugh small amunts f these alkanes have a large effect n the T LH and destabilize the bilayer structure. Cautin shuld be taken t crrelate, fr instance, changes in fatty acid cmpsitin, with an increase in slvent tlerance when ther imprtant parameters have nt been studied. These ther parameters (e.g. headgrup cmpsitin, prtein r sterl cntent) can have ppsite effects n the membrane integrity. A clear example f this is the adaptatin f E. cli t ethanl. The bserved increase in 18:1 cis cntent will restre the rati between bilayer and nnbilayer prmting phsphlipids. As this increase in 18:1 cis cntent will further fluidize the membrane, it culd be cncluded that cntrl f the membrane fluidity is nt imprtant t acquire slvent tlerance. Hwever, further studies with E. cli have shwn that the membrane fluidity is restred by an increased prtein cntent in the membrane. The pssibility that changes in the prtein cntent f the A. laidlawii membranes play a rle in maintaining a certain membrane fluidity can nt be ruled ut. And althugh mycplasmas as A. laidlawii can nt synthesize sterls, they are knwn t prduce cartenids (Smith, 1963). The regulatry rle f cartenids in cntrlling the fluidity f A. laidlawii cell membranes was suggested by shwing that the cartenid bisynthesis system senses membrane fluidity (Rttem & Markwitz, 1979). Changes in the cartenid cmpsitin f A. laidlawii in respnse t the presence f tluene in the medium have indeed been bserved (Gvrun et al., 1989) Outer membrane We have discussed adaptatin mechanisms s far at the level f the cytplasmic membrane under the assumptin that it is the key cmpnent f the bacterial cell envelpe in cmbating txic rganic slvents. The ther elements f the envelpe are quite different in Gram-psitive and Gram-negative bacteria. The Gram-psitive bacteria have a thick cell wall cnsisting f murein and ther plymers which can nt be expected t have a rle in excluding slvents frm the cell. Gram-negative rganisms have nly a very thin murein layer but have an uter membrane utside this layer. The inner leaflet f this membrane resembles the cytplasmic membrane cmpsitin but the uter leaflet is cmpsed f lipplysaccharides (LPS) (Lugtenberg & van Alphen, 1983). These LPSs have an unusually lw permeability fr hydrphbic cmpunds (Yshimura & Nikaid, 1982; Hiruma et al., 1984; Nikaid & Vaara, 1985; Vaara ef al., 1990).

129 Adaptatin f micrrganisms t txic slvents 129 The questin nw arises whether the uter membrane has a specific functin in the defence f an rganism against slvents. Frm cmparative studies, ne might cnclude that Gram-negative bacteria are in general less sensitive t hydrphbic rganic slvents than Gram-psitive rganisms (Sheu & Freese, 1973; Harrp et al., 1989; Inue & Hrikshi, 1991), but the differences are nt verwhelming. Cnsequently, the cytplasmic membrane indeed is a very critical part f the cell envelpe with respect t slvent txicity. On the ther hand, certain rganisms that are able t acquire resistance against very txic slvents, as, fr instance, tluene are all Gram-negative rganisms. Furthermre, in several cases it has been bserved that the resistance t these hydrphbic slutes is decreased drastically when the structure f the uter membrane is mdified by mutatin r (chemical r enzymatic) remval f parts f the LPS mlecule (Sheu & Freese, 1973; Nikaid, 1976; Hancck, 1984; Okuyama et al ; Tamura et ai, 1993; Vaara, 1993). Deep-rugh mutants (lacking mst f the saccharides) f cli and S. typhimurium had a very high sensitivity t a variety f antibitics, detergents and ther drugs. In additin an enhanced uptake f the hydrphbic dye gentian vilet was shwn in these mutants (Newtn, 1954; Stan-Ltter et ai, 1979). The grwth and xygen cnsumptin f. cli and S. typhimurium is inhibited by shrt-chain fatty acids (C2-C6) but nt by medium r lng chain fatty acids (C10-C18) (Sheu & Freese, 1973). Remval f a prtin f the lipplysaccharide layer by EDTA made the rganisms sensitive t these lnger-chain fatty acids (Sheu& Freese, 1973). Anther imprtant cmpnent f lipplysaccharide, besides the plysaccharide chain are fatty acids. Unlike phsphlipids which have usually nly tw fatty acids linked t the backbne structure, the LPS mlecule has six t seven fatty acids linked t the glucsamine dissaccharide backbne (Lugtenberg & van Alphen, 1983). Anther imprtant difference with fatty acids present in phsphlipids is that the fatty acids present in LPS are all saturated (Nikaid & Vaara, 1985). The absence f unsaturated fatty acids is expected t make the interir f the LPS leaflet much less fluid. Als the large number f fatty acids chains linked t a single backbne will decrease the fluidity (Nikaid, 1994a). Indeed the fluidity f the LPS interir has experimentally been shwn t be very lw (Seydel et al ). Due t the presence f LPS mlecules in the uter membrane, this membrane has a very lw permeability (Yshimura & Nikaid, 1982; Hiruma et al., 1984; Nikaid & Vaara, 1985; Vaara ef ai, 1990). Nevertheless these Gram-negative bacteria must exchange nutrients and waste prducts with the envirnment. Mst f this exchange takes place thrugh prtein channels in the uter membrane (Nikaid, 1994b). These channel-frming prteins include specific channels, which cntain specific binding sites facilitating the transprt f certain mlecules, and

130 J 30 Chapter 8 prins, which allw nnspecific and spntaneus diffusin f small slutes. These prins are water-filled channels that allw the influx f small hydrphilic slutes but exclude large and mre hydrphbic mlecules (Nikaid, 1994a, 1994b). The resistance f Gram-negative bacteria against small, rather hydrphilic antibitics has been cntributed t changes in the prins present in the uter membrane (Nikaid, 1989). Fr instance, spntaneus mutants f E. cli pssessing an increased resistance twards carbenicillin lacked the OmpF prin, which prduces relatively large pres, but retained the OmpC prin which gives a narrw channel (Harder et a/., 1981 ). The specific rle f the uter membrane in cping with rganic slvents has nt been investigated s far. Mst likely, the cmplex structure f the lipplysaccharide has discuraged researchers frm dealing with this aspect f a pssible respnse f cells t txic rganic slvents. Fr lipphilic antibitics, which usually have a relatively high mlecular weight, hwever, several adaptive changes in the uter membrane have been bserved (Diver et a/., 1991; Leying et a/., 1991; Michea-Hamzehpur et ai, 1991). The carbapenem resistance in Enterbacter aergenes, fr instance, has been crrelated with alteratins in the lipplysaccharide cmpsitin (Leying et al., 1991). The islated resistant strains pssessed mre carbhydrates (thus a lnger saccharide chain) than the cntrl cells (Leying et a/., 1991). We have investigated the rle f the uter membrane in the slvent-tlerant P. putida SI2 at tw levels. Bth the effects f divalent catins n the rganizatin f the LPS-layer and changes in the hydrphbicity f the uter membrane will be discussed. Especially Mg 2+ and Ca 2+ ins are imprtant in the rganisatin f the uter membrane (Nikaid & Vaara, 1985). Adjacent plyaninic LPS mlecules are linked electrstatically by divalent catins t each ther frming a stable "tiled rf' n the surface f the uter membrane (Labischinski et ai, 1985; Okuyama et ai, 1991). The uter membrane can be disrganized by remving these divalent catins with chelatrs such as EDTA (Lugtenberg & van Alphen, 1983; Nikaid & Vaara, 1985; Okuyama et ai, 1991). As a cnsequence. Salmnella and ther Gram-negative bacteria were less sensitive t hydrphbic inhibitrs upn the additin Mg 2+ and Ca 2+ (Stan-Ltter et al., 1979). When P. putida S12 was grwn with acetate as carbn surce, a shck lad f tluene resulted in the killing f mre than 99.99% f the cells (Table 5). The survivrs eventually grew t a high cell density in the presence f a secnd-phase f tluene (Weber et ai, 1993). Supplementing the grwth medium f P. putida SI2 with 5 mm MgS0 4 r CaCI 2 resulted in a 70- t 350-times increase in cell

131 Adaptatin f micrrganisms t txic slvents 131 Table5: Effect f the additin f Mg 2+ and Ca 2+ ins n the viable cunt f expnentially grwing P. putida S12 n acetate after the additin f 1% (v/v) tluene. Additin nne 5 mm MgS0 4 5 mm CaCI, Viable cells (ml" 1 ) Befre 3.8* T * h After 7.8* * * 10 5 Survival 0.002% 0.14% 0.7% survival after the additin f tluene (Table 5).This effect is even mre remarkable when the destructive effects f these ins n the phsphlipid bilayer are taken int accunt. Nrmally, these ins will prmte the frmatin f a hexagnal H )( cnfiguratin by aninic phsphlipids (Seddn, 1990). These results suggest that the uter-membrane might cntribute t the resistance f P. putida SI2 t tluene. Similar results were reprted by Inue et al. (1991) fr the tluene resistance f P. putida IH Additin f Mg 2+ and Ca 2+ t the grwth medium resulted in a tw- t three-fld increase in cell yield when the rganisms was grwn in the presence f 30% (v/v) tluene (Inue ef a/., 1991 j. P. putida SI2 apparently als changed the cmpsitin f its uter-membrane in respnse t tluene, resulting in a reduced hydrphbicity f the cell. As a measure f the hydrphbicity the cntact angle f a water drplet n a layer f cells was used (van Lsdrecht, 1987). Glucse-grwn cells are very sensitive t tluene, and ne hur after the additin f a shck lad f tluene n viable cells culd be bserved (Weber ef a/., 1993). These glucse-grwn cells have quite a hydrphbic uter membrane, as indicated by the high water cntact angle (Table 6). Acetate-grwn cells, hwever, are less hydrphbic and are mre tluene resistant. Cells grwn n acetate in the presence f tluene were even less hydrphbic and are mre tluene resistant (Table 6). The bserved reductin in the hydrphbicity f the uter membrane is expected t reduce the permeability f the membrane fr the lipphilic cmpund tluene Other respnses f micrrganisms t slvents Besides changes in the cytplasmic membrane and uter membrane cmpsitin several ther respnses have been bserved in micrrganisms in respnse t the effects f rganic slvents Cmpatible slutes Cmpatible slutes like trehalse, betaine and prline are fund in high cncentratins in a wide variety f micrrganisms that survive smtic stress, severe hydratatin and temperature stress (van Laere, 1989; Wiemken, 1990).

132 132 Chapter 8 Table 6: Hydrphbicity f P. putida SI2 cells and the effect f the additin f 1% (v/v) tluene n the viable cunt f expnentially grwing cells (frm Weber ef a/., 1993). Adapted cells had been grwn in the presence f 1% (v/v) tluene. Strain Wild Type Wild Type Adapted Grwth substrate Glucse Acetate Acetate Viable cells (ml-') Befre 3.7* * T h After <10 7.8* 10 2 l.l'lo 7 Survival 0% 0.002% 10% Water cntact angle These cmpatible slute have a prtective effect n membranes subjected t desiccatin r freezing by affecting bth the lipid packing gemetry and fluidity f the membrane (Crwe et a/., 1984a,1984b; Rudlph ef ai, 1986). As cmpatible slutes are generally fund in micrrganisms itis expected that they can als stabilize membrane structures against slvent stress. Indeed, an accumulatin f intracellular trehalse has been reprted in Saccharmyces strains in respnse t an ethanl shck (Odumera ef a/., 1993).Itis pstulated that this accumulatin f trehalse is a respnse t the decrease in water activity (a w ) caused by the high cncentratins f ethanl (Jnes & Greenfield, 1987).Studies f Mansure ef a/ (1994) have shwn that additin f trehalse t varius PC lipsmes significantly decreased the ethanl-induced leakage f the vesicles. Whether cmpatible slutes als have a prtective effect against the actin f ther rganic slventsstill has t be established Plasma-membrane ATPase Accumulatin f ethanl in the lipid bilayer will increase the permeability f the membrane (Fig. 7). An increase in the permeability f the membrane fr prtns will result in a dissipatin f the prtn mtive frce and will affect the viability f the cell. In5.cerev/'s/ae ethanl was shwn t activate the membrane H + -ATPase activity (Rsa & Sâ-Crreia, 1991; Alexandre ef a/., 1993). It is expected that this increase in H + -ATPase activity is a respnse t the increase in permeability. Similarly, in a Rhizbium sp. cnfrnted with 2,4-dichlrphenxyacetic acid, an increase in the H + -ATPase activity was bserved in respnse t the increased membrane fluidity (Fabra de Peretti ef a/., 1992). Althugh, this increase in activity is merely a respnse t the increase in permeability, differences in the tlerance f the H + -ATPase t slvents are expected t cntribute t the slvent tlerance f the cell. The H + -ATPase f Kluyvermyces marxianus was mre sensitive t ethanl than the H + -ATPase fs. cerevisiae (Rsa & Sa-Crreia, 1992). Apparently, differences in these H + -ATPases have cntribute t the ethanl tlerance f

133 Adaptatin f micrrganisms t txic slvents 133 S. cerevisiae, which has a higher tlerance than K. marxianus (Rsa & Sa-Crreia, 1992) Heat Shck Prteins Expsure f. cli t benzene and ther pllutants led t the inductin f numerus prteins. Sme f these prteins were identified as heat shck r carbn starvatin prteins, but at least 50% were nly induced by a given chemical (Blm et a/., 1992). Ethanl induced heat shck prteins ins. cerevisiae resulting in an increased viability after a heat shck (Plesset et ai, 1982; Curran & Khalawan, 1994). Similarly, a heat shck treatment resulted in a remarkable increased tlerance f the cell t a subsequent ethanl shck (Watsn & Cavichili, 1983; Odumera et ai, 1992). The fact that a chemical stress such as ethanl can prtect against heat shck suggests that these heat shck r ethanl induced prteins functin in a general cellular respnse mechanism t stress (Mager & Ferreira, 1993) Methdsfrpreventing the accumulatin fslvents Txicity f rganic slvents due t their accumulatin in membranes can be cunterbalanced by cells by mdifying the membrane t make it mre suitable t cpe with the slvents. As seen befre, the fatty acid prfile and the headgrup cmpsitin may be altered, specific mlecules may be incrprated in membranes, and the prtein/phsphlipid rati can be shifted. These reactins are meant t stabilize the physical structure f the membrane and t allw it t play its rle as barrier als in the presence f slvents. These reactins all are very defensive in nature in the sense that they try t make the best f an unwanted situatin already in existence. A mre aggressive apprach f cells t cpe with slvents wuld be t prevent r reduce accumulatin f slvents in membranes. When it wuld be pssible t keep the cncentratin f slvent in the membrane lw, then the negative effects f slvents wuld be minimized. In principle, several ptins are pen t an rganism t exclude slvents frm the membrane, but all methds will be difficult due t the intrinsic prperties f lipphilic slvents and their tendency t accumulate in the lipid bilayer Decreased affinity f membranes fr slvents One methd t cunterbalance partitining f slvents in membranes is t make the membrane less attractive fr the disslving mlecule by altering its lipid cmpsitin.the scpe fr such adaptatin has been shwn by wrk f Antunes- Madeira and Madeira (1984, 1986, 1987, 1989) and by De Yung and Dill (1988). These authrs used artificial membranes and shwed that the partitining f hydrphbic cmpunds very strngly depended n the fatty acid cmpsitin

134 134 Chapter 8 f the phsphlipids. Fr instance, the partitining f lindane is 50 times lwer in lipsmes f DSPC (CI8:0) than in lipsmes f DMPC (CI4:0) (Antunes-Madeira & Madeira, 1989). By mdifying the fatty acid cmpsitin micrrganisms can thus nt nly cunteract the effects f slvents n the membrane but can als reduce the partitining f the cmpund in the membrane Degradatin r mdificatin f the slvent Anther methd t prevent the accumulatin f slvents in membranes is t mdify the cmpund and make it less lipphilic r metablize it all tgether. Mdificatin f a cmpund is pssible fr instance in the case f armatic hydrcarbns by partially xidizing the cmpund r by making it mre watersluble by making cnjugates frm it. Cmplete mineralizatin f a cmpund may als be an effective methd t vercme the txic effect prvided its cncentratin is nt t high at any specific mment. Such situatin may ccur, fr instance, in the case f rganisms grwing n armatic hydrcarbns like tluene r styrene Active efflux mechanisms A very interesting methd t reduce the actual cncentratin f slvents in membranes wuld be t transprt slvents mlecules ut f the membrane by an active efflux system. Such a mechanisms wuld be f great help t a cell because any passive defence mechanism based n a lw-permeability barrier r based n a mdified membrane will have its drawback n the cell fr its nrmal functining. With ne pssible exceptin t be discussed later, n such active efflux system fr rganic slvents has been detected in bacteria, but a great deal f infrmatin has becme available n active efflux mechanisms fr lipphilic antibitics. These systems have been reviewed by Levy (1992), Leewis (1994) and Nikaid (1994a). In summary, it can be said that these multi-drug efflux systems are widespread in micrrganisms and the mst salient feature is their being nnspecific: a great variety f lipphilic substrates can be handled by these translcases. The efflux cmplexes are lcated in the cytplasmic membrane f, fr instance, E. cli where it captures drug mlecules that have managed t pass the uter membrane. The drug is transprted via an accessry prtein t a channel in the uter membrane and thus is excluded frm the bacterial cell envelpe. The interesting questin nw arises whether such multi-drug resistance pumps play a rle in slvent resistance in bacteria. Recently An and clleagues (1995) made the very interesting bservatin that indicates that this indeed may be the case. They islated cyclhexane-tlerant mutants f E. cli and subsequently shwed that these mutants were resistant t lw levels f ampicillin, chlramphenicl, naladixic acid and tetracycline. The ther way arund, mutants f E. cli resistant t ampicillin and chlramphenicl had

135 Adaptatin f micrrganisms t txic slvents 135 resistance t cyclhexane. The relatinship between rganic slvent tlerance n the ne hand and resistance t antibitics n the ther hand therefre appears bvius. Frm these results it might be speculated that a multi-drug efflux mechanism f E. cli cntributes t the rganic slvent tlerance. It will be f great interest t see if a crrelatin exists in ther bacteria as well, and particularly in slvent-tlerant strains, between slvent tlerance and multi-drug resistance. 7. CONCLUDING REMARKS In this review we have shwn that accumulatin f rganic slvents in the membrane f micrrganisms affects the physic-chemical prperties f the lipid bilayer and cnsequently the bilgical functining f the membrane. The insight in the varius effects which especially alkanls have n the prperties and functining f the bilgical membrane is grwing. Especially the effects f alkanls n bth the fluidity (T M ) and bilayer stability (T LH ) f the membrane are nw well characterized in mdel membrane systems like lipsmes. Hwever, the cnsequences f these effects n the functining f the cell membrane and viability f micrbial cells are less clear. Althugh the effects f alkanls n (mdel) membranes are quite well characterized, the effects f varius ther rganic slvents (e.g. armatics) are unfrtunately nt yet as extensively studied. In view f the extreme txicity f these armatics fr micrrganisms, further insight in the effects f these slvents wuld be desirable. Althugh rganic slvents can affect the prperties and functining f the cell membrane, varius micrrganisms have develped adaptatin mechanisms t cunteract these effects. In many micrrganisms changes in the lipid cmpsitin have been bserved in respnse t several rganic slvents. These changes can generally be explained as a mechanism t restre the fluidity f the membrane r as a mechanism t cntrl the required balance between bilayer and nn-bilayer prmting phsphlipids. Hwever, especially fr ethanl, apparently cntradicting adaptatin mechanisms have been bserved, which makes interpretatin f the results difficult. Further research int these changes in lipid cmpsitin as well as ther pssible adaptatin mechanisms is therefre required. In view f practical applicatins, it is imprtant t gain a better understanding f the effects f rganic slvents n the functining and prperties f the cell membrane as well as f the adaptatin mechanisms f micrrganisms. Ptentially, knwledge abut slvent txicity and slvent resistant micrrganisms might be useful in bth bitransfrmatins and envirnmental bitechnlgy. Bicatalytical prcesses perfrmed in aqueus phases ften result in lw cncentratins f the prduct, which must be recvered frm the water phase.