FEMS Microiology Ecology 49 (24) 26 268 www.fems-microiology.org Direct inhiition of methnogenesis y ferric iron Peter M. vn Bodegom,, *, Johnnes C.M. Scholten c, Alfons J.M. Stms Lortory of Microiology, Wgeningen University, H.v. Suchtelenweg 4, 67 CT Wgeningen, The Netherlnds Lortory of Theoreticl Production Ecology, Wgeningen University, P.O. Box 4, 67 AK Wgeningen, The Netherlnds c Deprtment of Biologicl Sciences, Pcific Northwest Ntionl Lortory, P.O. Box 999, MS P7-5, Richlnd, WA 9952, USA Received 6 Ferury 24; received in revised form 26 Mrch 24; ccepted 26 Mrch 24 First pulished online 22 April 24 Astrct Oserved inhiition of methnogenesis under Fe(III)-reducing conditions is usully explined y competition of methnogens nd Fe(III)-reducing cteri for the common sustrtes cette nd hydrogen. However, sustrte competition lone cnnot explin the strong inhiition of methnogenesis during Fe(III)-reduction. We demonstrte direct inhiition of methnogenesis y morphous Fe(OH) t concentrtions etween nd mm in experiments with pure cultures of methnogens. The sensitivity towrd Fe(III) ws higher for Methnospirillum hungtei nd Methnosrcin rkeri grown with H 2 /CO 2 thn for Methnoset concilii nd Methnosrcin rkeri grown with cette. Cultures of Methnosrcin rkeri grown with H 2 /CO 2 nd methnol demonstrted cpcity for Fe(III) reduction, which suggests tht Fe(III)-reduction y methnogens my lso contriute to Fe(III) inhiition of methnogenesis. Our results hve importnt implictions for kinetic modelling of microil redox processes in noxic soils nd sediments. Ó 24 Pulished y Elsevier B.V. on ehlf of the Federtion of Europen Microiologicl Societies. Keywords: Methne production; Ferric iron reduction; Iron toxicity; Sulphide; Aceticlstic methnogens; Hydrogenotrophic methnogens. Introduction * Corresponding uthor. Present ddress: Deprtment of Systems Ecology, Institute of Ecologicl Science, de Boeleln 85, 8 HV Amsterdm, The Netherlnds. Tel.: +-2-4446964; fx: +-2-44472. E-mil ddress: peter.vn.odegom@ecology.flw.vu.nl (P.M. vn Bodegom). In noxic soils nd sediments, the pthwy of orgnic mtter degrdtion depends on the types nd quntities of electron cceptors present. Ferric iron (Fe(III)) reduction nd methnogenesis re dominnt processes in most freshwter environments [,2]. Understnding the iogeochemicl controls on methne production is importnt to predict sptil nd temporl ptterns of the emission to the tmosphere of this importnt greenhouse gs. Severl studies hve shown tht methne production is severely inhiited under iron reducing conditions [2,]. This phenomenon is often explined y competition etween Fe(III)-reducing nd methnogenic microorgnisms for common sustrtes such s cette nd hydrogen. Fe(III)-reducers re le to utilize cette nd H 2 t concentrtions fr elow levels tht cn e metolised y methnogens [4,5]. However, experimentl dt of Rtering nd Conrd [6] nd recently of Lueders nd Friedrich [7] nd Roden [8] indicted tht sustrte competition cnnot completely explin the strong inhiition of methnogenesis during Fe(III)-reduction. In ddition, mechnistic model of microil redox processes showed tht, lthough the higher ffinity for cette nd H 2 gve Fe(III)-reducers some competitive dvntge over methnogens, this process could not explin the temporl dynmics of methne production [9]. Insted, such dynmics could only e explined y the introduction of n empiricl constnt threshold reducile Fe(III) concentrtion ove which no methne production occurred. Inhiition of methne production y Fe(III) ws n importnt cuse for cette ccumultion mesured in noxic sediments [2,6]. Moreover, previous investigtions hve shown tht methnogens my trnsfer electrons to 68-6496/$22. Ó 24 Pulished y Elsevier B.V. on ehlf of the Federtion of Europen Microiologicl Societies. doi:.6/j.femsec.24..7 Downloded from https://cdemic.oup.com/femsec/rticle-strct/49/2/26/482548 y guest on 2 Decemer 27
262 P.M. vn Bodegom et l. / FEMS Microiology Ecology 49 (24) 26 268 pthwys other thn methnogenesis, e.g. to reduce moleculr sulphur [], humic sustnces [] nd Fe(III) [2]. Collectively, these studies suggest tht some fctor(s) other thn sustrte competition plys significnt role in inhiition of methnogenesis during Fe(III)-reduction. We conducted experiments with pure cultures of methnogens to evlute two nonmutully-exclusive hypotheses relted to potentil mechnisms for inhiition of methne production y Fe(III): (i) methnogens re directly inhiited y the presence of Fe(III) (lso in sence of Fe(III)-reducers); nd (ii) methnogens use the electron flow generted y cette or H 2 preferentilly to reduce Fe(III), s long s Fe(III) is ville. Concomitntly with these conditions, no methne is produced. 2. Mterils nd methods Three methnogenic pure cultures were used for the experiments: Methnoset concilii (DSM 67) n oligte ceticlstic methnogen, Methnospirillum hungtei (DSM 864) methnogen tht uses H 2 /CO 2 nd formte nd Methnosrcin rkeri (DSM 8) tht cn use H 2 /CO 2, methnol nd cette. These model cultures cover the most importnt methnogenic conversions in freshwter sediments. All cultures were pregrown without Fe(III) nd were trnsferred when the cells were growing in the exponentil phse. Methnogenesis resumed immeditely fter trnsfer in sence of Fe(III). All experiments were crried out t C. 2.. Experiment Sterile 2-ml ottles were filled with 5 ml fresh icronte-phosphte-uffered, mm sodium sulphide reduced noxic medium [] tht did not contin (prt from sulphide) cysteine or other components tht my ct s electron crrier tht my contriute to iotic Fe(III) reduction [4] nd were closed with utyl stoppers. The ottles were inoculted with 5 ml of culture of exponentilly growing methnogens. M. hungtei nd M. rkeri were grown with mm cette nd 4 kp 2% CO 2 /8% H 2 nd M. concilii ws grown with 2 mm cette nd % N 2 t finl pressure of 4 kp. Freshly inoculted cultures were mended with,, 5 nd mm Fe(III). Fe(III) ws dded s neutrlized FeCl. Added FeCl hydrolysed rpidly to produce colloidl suspension of morphous Fe(OH). At ech iron concentrtion, the experiment ws crried out in triplicte for ll species nd control of uninoculted mended medium. Totl ionic concentrtion ws kept the sme in ll tretments y NCl ddition, thus dding, 27, 5 nd mm NCl to the tretments with,, 5 nd mm Fe(III), respectively. The ottles were incuted in the drk t C while shken continuously t 25 rpm. At t ¼,, 2, 5, 9, 2 nd 6 dys,. ml liquid smples were tken quickly using.8-mm thick needles fter shking vigorously to ensure n even, representtive, distriution of the colloids. Smples were extrcted with wter nd.5 N HCl, respectively nd nlysed for Fe(II) nd Fe(III). In ddition,. ml gs smples were tken for nlysis of CH 4 nd CO 2. All syringes were flushed in ottles with % N 2 tmosphere nd fresh sodium dithionite solution efore smpling. 2.2. Experiments 2 nd Experiments 2 nd differed in few spects from the first experiment: The tretment of mm Fe(III) ws omitted. M. rkeri ws grown oth with mm cette nd 4 kp 2% CO 2 /8% H 2 nd t 2 mm cette nd % N 2 t 4 kp. For oth conditions control of uninoculted mended medium ws dded. Experiment contined dditionl tretments of M. rkeri t 5 mm Fe(III), mm cette nd 4 kp 2% CO 2 /8% H 2 with the ddition of mm romoethne sulfonte (BrES), n nlogue of methylcoenzyme M nd inhiitor of methnogenesis, of M. rkeri grown with 25 mm methnol nd % N 2 t 4 kp nd of M. rkeri grown with 5 mm Fe(III), 25 mm methnol nd % N 2 t 4 kp. Finlly, n uninoculted tretment with 5 mm Fe(III) with the filter-sterile ddition of 5 ml cell free extrct from ctively growing M. rkeri cultures ws included. Smples were tken t t ¼,, 2, 5, 7,, 4 nd 9 dys,. ml liquid smples were tken for cid voltile sulphur (AVS), Fe(II) nd Fe(III) nd.2 ml liquid smples were tken for nlysis of SO 2,SO2 4 nd S 2 O 2. Iron ws extrcted y wter (to quntify dissolved iron),.5 N HCl, M mmonium cette nd 5 g/l sodium dithionite in.5 M cetic cid nd.2 M sodium citrte..5 N HCl is supposed to extrct lone nd only reduced Fe [4]. Ammonium cette is commonly used extrction method in geochemistry to extrct morphous iron oxides [5], which is presumly the predominnt source for iron reduction [6]. Dithionite extrctle iron, on the other hnd, represents oth morphous nd crystlline iron oxides nd morphous FeS [7] nd would in this cse represent ll iron present. In ddition,.2 ml gs smples were tken for nlysis of CH 4, H 2 S nd CO 2.Att¼5,, 4 nd 9 dys, ml liquid smples were tken for S nlysis. At t ¼ nd t ¼ 9 dys, 2 ml liquid smples were tken for nlysis of chromium reducile sulphur (CRS). Microscopic oservtions on the cultures were performed y phse contrst microscopy. Downloded from https://cdemic.oup.com/femsec/rticle-strct/49/2/26/482548 y guest on 2 Decemer 27
P.M. vn Bodegom et l. / FEMS Microiology Ecology 49 (24) 26 268 26 2.. Chemicl nlyses All liquid smples were centrifuged (fter extrction, if ny) for 5 min t 6,g nd superntnt ws nlysed. Fe(II) ws nlysed colorimetriclly with ferrozine s regents [4]. After mesurement,.25 M hydroxylmine in.25 M HCl ws dded nd fter min sornce ws nlysed gin (for the determintion of Fe(totl) in solution). SO 2,SO2 4 nd S 2 O 2 were determined s descried previously [2]. AVS ws determined s descried y Tr uper nd Schlegel [8]. S ws nlysed indirectly y conversion of S with SO 2 to S 2 O 2. A 7.5% N 2SO -solution in mm mnnitol ws prepred noxiclly. In rection tue,.5 ml of this solution ws dded with.5 ml M NOH to. ml liquid smple. The tues were incuted for 48 h t 65 C, while shking. Converted S ws mesured s S 2 O 2 s descried ove. CRS ws determined ccording to the single step titrtion descried y Fossing nd J orgensen [9]. CH 4 ws nlysed t 7 C ygcon moleculr sieve column, coupled to FID. H 2 S nd CO 2 were nlysed on Porpk Q column coupled to TCD. 2.4. Sttisticl nlysis Effects of Fe(III) concentrtions on methne production nd differences in iron dynmics nd dynmics of sulphurous compounds mong species or etween species nd uninoculted medi were nlysed y pired student s t-test. Correltions etween methne production nd.5 N HCl extrctle Fe(III) were clculted using the Person s correltion coefficient. Bckwrd multiple regression nlysis ws crried out for ech species nd sustrte with methne production s dependent vrile nd.5 N HCl extrctle Fe(II) nd Fe(III) concentrtions s independent vriles. For this purpose, Fe(II) nd Fe(III) concentrtions were log-trnsformed to pproch norml distriution. Proilities of difference in mens seprte for ech point in time, s depicted in the figures, re sed on Tukey s post-hoc tests following ANOVA nlyses using sqrt-trnsformed methne dt nd log-trnsformed.5 N HCl extrctle Fe(II) concentrtions to pproch norml distriution of the dt.. Results Tretment specific results did not significntly depend upon the experiment (P > :) for ny vrile mesured. The three experiments could thus sttisticlly considered to e replictes nd were thus comined, resulting in 9 replictes depending on the tretment. In the sence of Fe(III), methnogens grown on H 2 / CO 2 produced more methne thn those grown on cette (Fig. ). In most cses, methne production ws inhiited y the presence of Fe(III) (Fig. ). Sensitivity to Fe(III) vried with methnogenic sustrte nd species (Fig. ). M. hungtei ws highly sensitive to Fe(III) nd ws lmost completely inhiited y mm Fe(III). M. rkeri, when grown on H 2 /CO 2, showed significntly (P < :5) decresed methne production t 5 nd mm Fe(III). When M. rkeri ws grown on cette, methne production ws lso significntly (P < :5) reduced t 5 nd mm Fe(III), lthough the effects were less thn when grown on H 2 /CO 2. Effects of Fe(III) on M. concilii, which lso grew on cette, were minor nd not sttisticlly significnt (P > :5). In controls without methnogens, methne ws never produced. Rtes of methne production (in mol CH 4 l medium dy )ym. rkeri nd M. hungtei grown on H 2 /CO 2 showed strong negtive correltion with.5 N HCl extrctle Fe(III) concentrtions (P ¼ :8 nd P < :, respectively). Such correltions were insignificnt for M. rkeri nd M. concilii grown n cette (P ¼ :48 nd P ¼ :2, respectively). The potentil iron reducing ctivity of methnogens ws determined from the dynmics of different iron species. The totl iron lnce, determined from dithionite iron extrcts, did not chnge in time in ny tretment, indicting tht ll iron ws retrieved. During the incution, chemicl chnges in iron specition occurred s ws shown y significnt decrese (P < :5) in time for mmonium cette extrctle totl iron nd for H 2 O extrctle totl iron. These chnges were not significntly (P > :) different for methnogenic cultures nd uninoculted medi, indicting some chemicl crystllistion of iron phses. In most methnogenic cultures, the chnge in Fe(II) in the.5 N HCl extrcts, which represents Fe(III) reduction est, ws not significntly (P > :) different from uninoculted medi. However, for M. rkeri cultures grown with H 2 /CO 2, the increse in Fe(II) ws significntly higher (P < :) thn in ny of the other cultures (Fig. 2). Also distinct colour chnge ws detected in the H 2 /CO 2 -grown M. rkeri culture grown t 5 mm Fe(III)-which hd the highest methne production rtes of ll cultures t 5 mm Fe(III). Initilly ll cultures mended with 5 mm Fe(III) hd reddish colour, cused y colloidl Fe(OH). After 7 dys of incution, the colloidl Fe(OH) ws converted into lck precipitte. None of the other cultures or controls showed such ehviour (Fig. ). Also M. rkeri grown with methnol nd 5 mm Fe(III) showed significntly higher (P < :) increses in Fe(II) in time thn ny of the other cultures (Fig. 4()). Fe(II) production with methnol ws similr to the one with H 2 /CO 2, lthough methne production ws slightly higher (Fig. 4()). No significnt increses in Fe(II) were found with the ddition of BrES or if cell Downloded from https://cdemic.oup.com/femsec/rticle-strct/49/2/26/482548 y guest on 2 Decemer 27
264 P.M. vn Bodegom et l. / FEMS Microiology Ecology 49 (24) 26 268 CH4 (mmol/l).4.2.8.6.4 Fe(III) Fe(III) 5 Fe(III) Fe(III) CH4 (mmol/l).6.4.2..8.6.4 Fe(III) Fe(III) 5 Fe(III) Fe(III) ().2 5 4 9 time (dys).2 () 5 9 2 6 time (dys) CH4 (mmol/l) (c).6.4.2.8.6.4.2 Fe(III) Fe(III) 5 Fe(III) Fe(III) 5 7 4 time (dys) c c c CH4 (mmol/l) (d).4.2..8.6.4.2 Fe(III) 5 Fe(III) Fe(III) 5 4 9 time (dys) Fig.. Influence of Fe(III) indicted in mm on methne produced y methnogenic cultures for () Methnospirillum hungtei grown with H 2 / CO 2, () Methnoset concilii grown with 2 mm cette, (c) Methnosrcin rkeri grown with H 2 /CO 2 nd (d) Methnosrcin rkeri grown with 2 mm cette. Dt re the comined result of three different incution experiments. Error rs indicte stndrd errors, while letters indicte significnt differences etween tretments for given moment in time (P < :5). Note the different scles on the xes. Fe(II) extrcted y HCl (mm) 2.5 2.5.5 H2/CO2 5 Fe(III) H2/CO2 Fe(III) cette 5 Fe(III) cette Fe(III) no MB 5 Fe(III) no MB Fe(III) c c c c c 2 5 7 4 time (dys) ccc c Fig. 2. Formtion of Fe(II), extrcted y.5 N HCl, in cultures of Methnosrcin rkeri (MB) grown with either H 2 /CO 2 or with 2 mm cette nd in uninoculted medium (no MB) in time. All comintions were incuted with 5 or mm Fe(III). Error rs indicte stndrd errors, while letters indicte significnt differences etween tretments for given moment in time (P < :5). Fig.. Colour chnges in Fe(III)-mended methnogenic cultures grown with H 2 /CO 2 fter dys of incution. free extrcts of M. rkeri were incuted with Fe(III) (Fig. 4()). Given tht oth Fe(II) nd Fe(III) concentrtions vried during the incutions, ckwrd multiple regression nlysis ws crried out for ech species nd sustrte with methne production s dependent vrile nd.5 N HCl extrctle Fe(II) nd Fe(III) concentrtions s independent vriles. This nlysis llows seprting the effects of Fe(II) nd Fe(III) on the inhiition of methnogenesis. Neither Fe(II) nor Fe(III) were significnt (P > :) in cse of M. rkeri nd M. concilii grown n cette, which is in greement with the correltion nlysis. Methne production of M. rkeri Downloded from https://cdemic.oup.com/femsec/rticle-strct/49/2/26/482548 y guest on 2 Decemer 27
P.M. vn Bodegom et l. / FEMS Microiology Ecology 49 (24) 26 268 265 Fe(II) extrcted y HCl (mm) () 2.5 2.5.5 H2/CO2 25 mm methnol H2/CO2 + BrES H2/CO2+MB products 5 7 time (dys) CH 4 (mmol/l) ().2.8.6.4.2 H2/CO2 25 mm methnol H2/CO2 + BrES H2/CO2+MB products 5 7 time (dys) Fig. 4. Product formtion y Methnosrcin rkeri (MB) grown with 5 mm Fe(III) nd with H 2 /CO 2, 25 mm methnol nd with H 2 /CO 2 with the ddition of mm BrES, respectively. An uninoculted control with H 2 /CO 2 nd 5 mm Fe(III) nd cell free extrct from ctively growing Methnosrcin rkeri (MB products) ws dded. Products shown re () Fe(II) formtion nd () methne formtion. Error rs indicte stndrd errors, while letters indicte significnt differences etween tretments for given moment in time (P < :5). nd M. hungtei grown on H 2 /CO 2 ws significntly (P ¼ :2 nd P ¼ :4) decresed in presence of Fe(III), while Fe(II) dropped s n insignificnt vrile out of the regression eqution. Aprt from H 2 /CO 2 or methnol, sulphide ws dded s potentil electron donor in ll medi. However, no Fe(III)-reduction ws detected in M. rkeri cultures grown with cette tht lso included sulphide. In medi with Fe(III), sulphide ws not detected in the gs phse nd ws lwys elow.5 mm in the liquid phse without cler trend in time. Moreover, no significnt differences (P > :) etween M. rkeri cultures nd inoculted medi were found for ny of the potentil oxidtion products of S 2 ;S, sulphte, sulphite nd thiosulphte. CRS ws for ll cultures constnt in time nd round mm, indicting tht ll sulphur ws retrieved. 4. Discussion 4.. Inhiition of methnogenesis y Fe(III) Our experiments clerly demonstrte direct inhiiting effect of Fe(III) on methnogenesis. This inhiition my hve een the result of n incresed redox potentil (Eh) of the medium cused y ferrihydrite ddition to the medium. Prt of the Fe(III) will hve rected with the dded mm sulphide, ecuse sulphide concentrtions were strongly decresed in medi to which Fe(III) ws dded. The stoichiometry of the rections indictes tht Fe(III) will lwys hve een left, thus incresing the Eh. The Eh could not e mesured given the risks of oxygen intrusion involved in such mesurement. In our icronte uffered system with ph of 6.8 [] the E for the previling couple, (Fe(OH) +HCO )/Fe2þ, is +2 mv t ph ¼ 7. [2]. From this, in comintion with the mesured dissolved Fe(II) nd Fe(III) concentrtions, the Eh ws clculted to vry in smll rnge etween +5 nd +5 mv. Fetzer nd Conrd [2] found no inhiition effects of Eh on methnogenesis t Eh vlues elow +42 mv. In our experiments, Eh remined fr elow this vlue nd direct Eh-effects thus seem unlikely. Microil modifiction of the dynmics of sulphurous compounds, e.g. through Fe(III), cn lso e ruled out ecuse chnges in ny sulphurous compound compred to uninoculted medi were not significnt. The iotic rections etween Fe(III) nd S 2 decresed S 2 toxicity which would hve stimulted methnogenesis [9], while the sulphte in the medium served s n S-source (unpul. results). Becuse no Fe(III)-reducers were present in the cultures, competition etween species for common sustrtes cnnot explin the results either. Initil Fe(II) concentrtions were very low nd ecuse methnogenesis t nturl conditions occurs fter Fe(III)-reduction thus while potentilly Fe(II) ccumulted, it seems unlikely tht Fe(II) inhiits methnogens (lthough this could not e proven directly y dding Fe(II), ecuse this would hve strongly ffected the Eh). Our multiple regression nlysis, in which Fe(II) dropped out of the equtions s insignificnt vrile, confirmed the non-inhiiting effects of Fe(II) nd suppression effects could e explined y Fe(III) lone. Fe(II) thus does not seem the inhiiting compound. Hence, direct suppression y Fe(III) is the most likely explntion for the oserved suppression of methnogenesis. Sensitivity towrd Fe(III) ws much stronger for methnogens grown with H 2 /CO 2 thn for methnogens grown with cette. Fe(III) ws present in colloidl suspension. Therefore effects through direct Fe(III) uptke into the cell seem unlikely. Sensitivity differences my, however, e explined from the different enzymtic pthwys involved in ceticlstic vs. hydrogenotrophic methnogenesis nd especilly from Fe(III) dsorption to co-fctors nd/or proteins t the outer memrne. Fctor F 42, n importnt electron crrier in methne production from H 2 /CO 2 [22] t the outer memrne Downloded from https://cdemic.oup.com/femsec/rticle-strct/49/2/26/482548 y guest on 2 Decemer 27
266 P.M. vn Bodegom et l. / FEMS Microiology Ecology 49 (24) 26 268 [2], might hve een inctivted t high Fe(III) concentrtions, s suggested previously [2]. Sensitivity of M. rkeri grown on cette ws greter thn tht of M. concilii. The pthwy of cette conversion in these species only differs in the ctivtion of cette to cetyl- CoA, which thus might hve plyed role in the inhiition. However, the precise iochemicl mechnism(s) of either inhiition remins unknown. More reserch will e needed to elucidte these spects. Our findings my explin previously poorly understood phenomenon in noxic sediments; the chnges in predominnce of ceticlstic vs. hydrogenotrophic methnogenesis. After domintion of methne production y the hydrogenotrophic pthwy during the first dy fter flooding [24], hydrogenotrophic methnogenesis quickly dropped to 2% of the totl methne production for week to month [24 26], mtching exctly the period dominted y iron reduction. After 2 months of flooding, hydrogenotrophic methnogenesis gined gin in importnce, up to % [26], wht theoreticlly should e their mximum contriution to methnogenesis [9]. These chnges cnnot e explined from chnges in the popultion size, ecuse the numers of ceticlstic nd hydrogenotrophic methnogens remined rther constnt during this whole period [27]. Insted, reltive ctivities must hve een chnged. These chnges might e explined from the difference in sensitivity towrd Fe(III) s descried ove, which is more likely thn n explntion sed on H 2 production y ceticlstic methnogens [2]. Sensitivity differences towrd Fe(III) my lso explin why rrna levels of Methnoset incresed t ferrihydrite mendment, while Methnosrcin spp. showed suppressed dynmics [7]. 4.2. Fe(III)-reduction y methnogens No significnt increse in.5 N HCl-extrctle Fe(II) ws oserved in most Fe(III)-mended methnogenic cultures compred to uninoculted controls. However, M. rkeri cultures grown with H 2 /CO 2 nd with 25 mm methnol in the presence of 5 or mm Fe(III) formed significntly more Fe(II) thn ny other culture. No methne or Fe(II) ws formed if BrES ws dded to the culture. Bond nd Lovley [2] found no methne formtion, ut continution of Fe(III) reduction t the ddition of oth BrES for M. rkeri. However, their medium [2] contined cysteine nd AQDS, which my hve led to continued (iotic) electron shuttling leding to Fe(III) reduction s discussed in detil y Doong nd Schink [4]. Bsed on the redox rections, it ws clculted tht on verge 22%, 28% nd 7% of the sum of electrons used within tretment y M. rkeri went to Fe(III)- reduction t 5 mm Fe(III) with methnol, 5 mm Fe(III) with H 2 /CO 2 nd mm Fe(III) with H 2 /CO 2, respectively. The diversion of electrons to Fe(III)-reduction compred to the totl metolic ctivity ws surprisingly constnt with time for given tretment. The totl metolic ctivity, clculted s the sum of electrons used reltive to control tretments without Fe(III) ddition, ws more dynmic (Fig. 5). During the first two dys, Fe(III) reduction led to metolic ctivity on top of continuing methne production (Fig. (c)). After this initil stimultion, totl metolic ctivity decresed compred to tretments without Fe(III) ddition, which mens tht not only quntittively electrons were diverted from methnogenesis to Fe(III) reduction, ut tht lso totl metolism ws inhiited y Fe(III), s discussed ove. Purity of our culture ws confirmed y microscopic oservtions, showing only the typicl pseudosrcin morphology of M. rkeri [28]. In ddition, no growth occurred with glucose or lctte with nd without Fe(III). No methne or Fe(II) ws formed either, if cell free extrcts from ctively growing M. rkeri cultures in 5 mm Fe(III) were trnsferred into fresh medium with 5 mm Fe(III) (Fig. 4). These results indicte tht Fe(III)-reduction is most likely medited directly y living cells nd not indirectly, e.g. through extrcellulr products. Cytochrome-c present in Methnosrcinles ut not in other methnogenic orders [29] nd known to e involved in dissimiltory iron reduction [] of which otherwise little is known [] my ply key role in Fe(III)-reduction y M. rkeri. Fe(III) reduction y M. rkeri ws confirmed y colour chnges in the cultures (Fig. ) nd microscopic oservtion showed lck Fe-precipittes round M. rkeri clusters. The lck precipitte formed in the H 2 / CO 2 -grown M. rkeri cultures ws likely mgnetite nd/or mixed Fe(II) Fe(III) compounds which re known to e produced during dissimiltory morphous Fe(OH) reduction []. These results re in greement metolic ctivity reltive to control without Fe(III) (%) 4 5 25 2 5 5 5 mm Fe(III) with methnol 5 mm Fe(III) with H2/CO2 mm Fe(III) with H2/CO2 5 5 time (dys) Fig. 5. Metolic ctivity of Methnosrcin rkeri mesured s the sum of electrons used for methnogenesis nd Fe(III)-reduction compred to ctivity in control tretment without Fe(III) ddition for the three tretments with Fe(III) reduction s function of time. Downloded from https://cdemic.oup.com/femsec/rticle-strct/49/2/26/482548 y guest on 2 Decemer 27
P.M. vn Bodegom et l. / FEMS Microiology Ecology 49 (24) 26 268 267 with results on reduction of solule Fe(III)-citrte y hyperthermophilic methnogens [2] nd of insolule Fe(III)-oxide with hydrogen y some mesophilic methnogens [2]. The Fe-precipittes my hve impired the efficiency of ferrihydrite reduction, which my explin why only 2 25% of the dded Fe(III) hd een converted into Fe(II) in 4 dys. The ility of M. rkeri to reduce Fe(III) my explin why this orgnism ws less inhiited y Fe(III) thn M. hungtei, ecuse Fe(III) reduction decreses the inhiiting effects of Fe(III). The iochemicl mechnism of Fe(III)-reduction in M. rkeri remins unknown. However, the results for M. rkeri suggest tht reduction of Fe(III) y methnogenic rche my lso e prtly responsile for suppression of methnogenesis in Fe(III)-reducing environments. Bond nd Lovley [2] suggest tht H 2 -oxidtion, rther thn methnogenesis per se, ws linked to Fe(III)-reduction y hydrogenotrophic methnogens. This process represents type of intrcellulr competition for electron cceptor utilistion, which is nlogous to the ility of certin sulphte-reducing cteri to reduce Fe(III) in comintion with incresed efficiency of H 2 scvenging []. The resulting lower H 2 concentrtions y Fe(III)-reducing methnogens such s M. rkeri [2] my contriute directly to Fe(III) suppression of H 2 -dependent non-fe(iii)- reducing methnogens. All together, this shows tht kinetic nd competition experiments (t electron donor nd/or cceptor limiting conditions) etween Fe(III)- reducing nd non-fe(iii)-reducing methnogens, nd Fe(III)-reducing methnogens nd Fe(III)-reducing cteri re needed to understnd more dequtely the interctions etween these importnt physiologicl groups. Our results hve significnt implictions for understnding the sptil nd temporl distriution of redox processes in noxic sediments given tht the pplied reducile Fe(III) contents re not unresonle for nturl conditions. Studies tht found inhiition of methne production t nturl iron reducing conditions hd similr, 4 mm, reducile Fe(III) contents mesured y.5 N HCl extrctions s used in the present study [,9,4]. Our results thus imply tht direct inhiition of methnogenesis y Fe(III) is likely n importnt ecologicl fctor in controlling the redox sequence in freshwter environments which hs thus fr lwys een neglected. In ddition, M. rkeri, when grown with H 2 /CO 2, is le to reduce Fe(III). Even with this dpttion, the sensitivity to Fe(III) is lrger for hydrogenotrophic methnogens thn for ceticlstic methnogens. Tking these phenomen into ccount my sustntilly improve the ility of kinetic models to predict microil redox processes in sediments, therey incresing the utility of such models for vrious environmentl purposes. Acknowledgements This work ws supported finncilly y the Dutch Ntionl Reserch Progrm on Glol Air Pollution nd Climte Chnge. Eric Roden is thnked for the inspiring discussions tht led to this study. 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