van der Maarel, Marc Jos Elise Cornelis

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   University of GroningenAnaerobic microbial degradation of methylated sulfur compoundsvan der Maarel, Marc Jos Elise CornelisIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1996Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):van der Maarel, M. J. E. C. (1996). Anaerobic microbial degradation of methylated sulfur compounds. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 11-10-2022, ÀChapter IDimethylsulfide (DMS) is one of the most abundant organic sulfur compounds in themarine environment and is thought to play an important role in the formation of cloudcondensation uclei through its atmospheric oxidation products methanesulfonic acid andsulfuric acid. DMS also forms a crucial link in the global sulfur cycle because it is themain sulfur species that is transported from marine to terrestrial environments. Theprecursor of DMS in marine environments is the osmolyte dimethylsulfoniopropionate(DMSP), which is produced by a variety of micro- and macroalgae, e.g. Phaeocystis p.and Emiliqnia huxleyi. Processes such as senescence, grazing of zooplankton, or lysis ofthe algae result in the liberation of DMSP. Most of the free DMSP is likely degraded inthe oxic water phase of the oceans and a variety of aerobic DMSP-degrading bacteriahave been isolated in recent years. However, at the start of this thesis project less wasknown about the microorganisms which are involved in the conversion of DMSP inanoxic environments uch as intertidal sediments and the gut of marine animals.The degradation of DMSP in anoxic marine environments had only been studied insediment slurry experiments at the start of this thesis project (Kiene and Visscher 1987;Kiene and Taylor 1988). These experiments howed that DMSP can be degraded via acleavage to DMS and acrylate, via a demethylation to 3-mercaptopropionate (MPA), with3-S-methylmercaptopropionate (MMPA) as a possible intermediate, or via an initialdemethylation coupled to the production of methanethiol. A strain of Clostridiumpropionicum, which had been isolated from river mud, was the only pure culture of aDMSP-cleaving bacterium (Wagner and Stadtman 1962). This thesis describes tudiesthat provide a better insight into the nature and properties of the microorganisms that areinvolved in the degradation of DMSP in anoxic intertidal sediments. In Chapter I somegeneral information on DMS(P) as well as the anaerobic microbial degradation ofDMS(P) is given.A DMSP-demethylating, sulfate-reducing bacterium (strain WN) was isolated froman enrichment culture of 10s times diluted anoxic sediment of the Wadden Sea (Chapter3). Strain WN was tentatively identified as a strain of Desulfobacterium autotrophicum,based on its substrate utilisation and hybridisation with a Desulfubacterium-specific 165rRNA probe. However, phylogenetic analysis of the 165 rRNA gene sequence revealedYZtetdrdteherte;p.ofinria/asinSummary and concluding remarksthat strain WN is more closely related to the genus Desulfubacter. Determination of thecellular fany acid composition and measurement of certain enzyme activities lead to theconclusion that strain WN taxonomically has an intermediate position between the generaDesulfobacterium and Desulfobacter. It was also found that Db. vacuolatum(DsM 33g5).Db. niacini (DSM 2650), and Db. niacini strain PM4 (Chapter 2) are ableto demethylateDMSP to MMPA. Eubacterium limosum strain PM3 1, which is salt-tolerant, also growson the demethylation of DMSp, albeit very slowly and with a very low growth yield.Substrate progress curves in cell suspensions indicated that strain WN and Db. niacinistrain PM4 have a high affinity for DMSp (K. approximately 10 prM).The product of the demethylation of DMSp, MMPA, rurned out to be a goodsubstrate for the growth of Methanosarcina sp. strain MTp4 (DSM 6636), Ms. siciliae(DSM 3028), and Ms. acetivorans (DSM 2g34), which all originare from marineenvrronments and are also able to utilize DMS and methanethiol (Chapter 4). Themethanogenic onversion of MMPA to MPA was also studied in sediment slurries.Addition of 500 pM MMPA resulted in the production of methanethiol, which wassubsequently converted to 411 ;lM methane. In the presence of the antibiotics ampicillin,vancomycin, and kanamycin (20 pglml each),275 pM methane was formed from 3g0 pMMMPA; no methanethiol was formed during these incubations. Only methanethiol wasformed from MMPA when 2-bromoethanesulfonic acid (25 mM) was added to a sedimentsuspension. These results indicate that in anoxic intertidal sediments, DMSP is not onlya precursor of a negative effector of global warming, but can also result in the productionof a potent greenhouse gas.The finding that certain marine Methanosarcina strains are able to metabolizeMMPA raised some interesting biochemical questions, which are discussed in Chapter4. It also raised the question what the possible role of MMPA and other algal metabolitescan be in the foÍmation of methane in open ocean water. In most oceans a supersaturationof ntethane is found at certain depths that still do contain oxygen. Since methanogenesisis a strict anaerobic proces, this phenomenon has been called lhe oceanic methaneparadox (Kiene 1991). Active methanogenesis has been reported to occur in theMediteranean Sea (Marty 1993) and several strains of methanogens have been isolatedl i na a,ithtialumr f aliesarermeof'omIterUffi,l6siledChapter Ifrom open ocean water (Cynar and Yayanos 1991; Sieburth 1993). Two possiblehypothesis have been put forwa-rd to explain the oceanic methane paradox: (i) formationof methane in the gut of zooplankton that graze on phytoplankton and subsequent leakagefrom the faecal pellets of this zooplankton (Marty 1993; de Angelis and Lee 1994; Karland Tilbrook 1994); (ii) formation of methane inside reduced microzones of particlesderived from decaying algal matter (Sieburth 1993). Compounds derived from algae, suchas glycine betaine and DMSP, could through their degradation products trimethylamine,DMS, and MMPA indirectly be good substrates for these oceanic methanogens. It is notknown whether the isolates that have been obtained from ocean water are able to srowon DMS or MlvÍPA.A new DMSP-cleaving strain (W218) was isolated from enrichments with DMSPand yeast extract as substrates and anoxic Wadden Sea sediment as inoculum (Chapter5). After the cleavage of DMSP to DMS and acrylate, strain W218 did not ferment theacrylate but used it as an electron acceptor. Besides acrylate it can also reduce sulfate,thiosulfate, and sulfite. Strain W218 was identified as a new species of the genusDesulfovibrio, derived from its phylogenetic position based on 165 rRNA gene sequenceanalysis and physiological characteristics, and was named Desulfovibrio acrylicus.Thereduction of the acrylate from DMSP by Dv. acrylicus was a complete surprise. Weexpected to find a fermentation of acrylate, as had been described for the DMSP-cleavingstrain of C. propionicum (Wagner and Stadtman 1962). The reduction of acrylate isenergetically more favourable than the reduction of sulfate, as already pointed out inChapter 5. At limiting electron donor concentrations this thermodynamic advantage maybe turned into a competitive advantage for Dv. qcmlicus and may explain why a sulfate-reducing bacterium is also able to reduce acrylate in an environment where sulfate ispresent in high concentrations (up to 28 mM). The ability to reduce acrylate was notfound among a variety of other sulfate-reducing bacteria.The enzyme which is responsible for the cleavage of DMSP to DMS and acrylatehas received considerable attention in the last three years and has been purified andcharacterized from the red alga Polysiphonia panictilata (Nishiguchi and Goff 1995), thefacultative anaerobe Alcaligenes p. strain M3A (de Souza and Yoch 1995a), and the sul-941I- l,Shf,tw;P.erhete,USrceheVeSummary and concluding remarksfate-reducing bacterium Dv. acrylicus strain W218 (Chapter 6). Also the generalcharacteristics of the DMSP lyase of Phaeocystis sp. have been studied in cell extracts(Stefels and van Boekel 1993). Strain W218 has a very active DMSP-cleaving enzyme(9.8 U/mg protein in cell extracts), which was purified 390-fold and had a K^for DMSPof 0.45 mM and a V,nu" of 2593Ulmg at 30"C (Chapter 6). It is only active with DMSPas a substrate and is most effectively inhibited by dimethylsulfoniobutyrate (K,= 0.25mM).The general picture that is emerging from these data is that the algae have a DMSPlyase with a high affinity, but a low maximum specific activity for DMSP. Bacteria onthe contrary have DMSP lyases with kinetic properties that are opposite to the algalDMSP lyases. However, when considering the turnover numbers, the DMSP lyase fromDv. acrylicus is able to convert more DMSP per unit of time than the P. paniculata andthe Alcaligenes DMSP lyase (À,,,, =I27 |s,25ls, and 20/s, respectively). The V.^/K-ratio, which is indicative for the efficiency of the enzyme, indicates that the Dv.acrylicus DMSP lyase (K,,,/V,,,,, = 0. l7) is as efficient as the P. paniculata DMSP lyase(K^|V,,^ = 0.28), but much more efficient than the Alcalígenes DMSP lyase (K/V^*=3.4). Contparison of such kinetic properties indicates that the three DMSP lyases whichhave been purified and characterized so far are different from each other and are probablynot closely related. Future analysis of their gene sequences will have to show whether thisassumption is true or not.The localization of the DMSP lyase in Dv. acrylicus has not been determined.Because the K. of the purified Dv. acrylicus enzyme for DMSP is in the same order asthe apparent K- of whole cells, it can be speculated that this DMSP lyase is present in theperiplasmic space and not intracellularly. That would be different from the DMSP lyaseot Pseudomonas doudorffii which is believed to be localized intracellularly, because theapparent K- of cell extracts (l.82 mM DMSP) is much higher than the K- value of wholecells (less than 20 pM DMSP). It has been suggested that in P. doudorffil DMSP istransported by a DMSP binding protein into the cell (de Souza and Yoch 1995b).It had been reported in the literature that sulfate-reducing bacteria are not able toreduce dimethylsulfoxide (DMSO) to DMS (Kiene and Capone 1988). This conclusiontatelndthesul-95Chapter Iwas based on sediment slurry experiments to which DMSO and molybdate, a specificinhibitor of sulfate reduction, had been added. Because inhibitor studies have to beinterpreted with caution, pure cultures of sulfate-reducing bacteria were tested tor theirability to reduce DMSO. Db. niacini and several different strain of the genusDesulfovibrio, all from marine or saline environments, were found to reduce DMSO toDMS (Chapter 7). In Dv. desulfuricans strain PA 2805 DMSO was reducedsimultaneously with sultate and the reduction of DMSO was not inhibited by molybdate.It is not yet clear whether the microorganisrns which have been studied in this thesisor the metabolic pathways these microorganisms use, such as acrylate reduction, areabundantly present in nature. Novel molecular ecological techniques can be usefull to getmore information on such questions, Íbr instance the use of species- or group-specific 165rRNA probes either for slorblot hybridization (Lee and Fuhrman 1990; Raskin et al.1994) or for in-situ hybridization using fluorescent dyes (Amann et al. 1992). Once genesequences for the different bacterial DMSP lyases or the acrylate-reducing enzyme areknown, more specific probes can be developed for use in denaturing gradient gelelectrophoresis (Wawer and Muyzer 1995) or reversed sample genome probing(Voordouw et al. 1991). These techniques have been applied successfully to study e.g. thediversity of Desulfoviàrio species in microbial mats (Wawer and Muyzer 1995) or in oilfield samples (Voordouw et al. 1991). The use of such molecular ecological techniquesin future studies hould also reveal whether the strains that have been studied in this thesisare ecologically relevant or not.The results presented in this thesis underline the physiological potential of sulfate-reducing bacteria as a group in total. At the moment more than 100 different substrateshave been identified for sulfate-reducing bacteria (Hansen 1993). It has recently beenshown that certain sulfate-reducing bacteria are able to reductively degrade halogenatedcompounds (DeWeerd et al. 1990) or trinitrotoluene (Boopathy et al. 1993), to reduceuranium VI to uranium IV (Lovley and Phillips 1992), and are also involved in thedegradation of polycyclíc aromatic hydrocarbons (Coates et al. 1996). In the course of thisthesis project it became clear that the conversion of DMSP to MMPA by sultate-reducingbacteria may find biotechnological applications in the production of natural aromatic96ecificto be' their3enusSO tolucedrdate.thesisn, areto getc  165et al.) genele arert gelobing,g. thein oildquesthesisrlfate-;trates'been:natededucein theof thislucingaticSummary and concluding remarkscompounds. A patent application on the MMPA production by sulfate-reducing bacteriahas been filed in collaboration with a Dutch biotechnology company (Hansen and van derMaarel 1994).ReferencesAmann, R.I., J. stromley, R. Devereux, R. Key, and D. A. staht. 1992. Molecular andmicroscopic identification of sulfate-reducing bacteria in multispecies biofilms. Appl.Environ. Microbiol. 582614-623.Boopathy, R., c. F. Kulpa, and M. wilson. 1993. Metabolism of 2,4,6-trinitrotoluene(TNT) by Desulfovibrio sp. (B strain). Appl. Microbiol. Biotechnol.3g:210-275.coates, J. D., R. T. Anderson, and D. R. Lovley. 1996. oxidation of polycyclicaromatic hydrocarbons under sulfate-reducing conditions. Appl. Environ. Microbiol.6 2 : 1 0 9 9 - 1 1 0 1 .Cynar, F. J., and A. Yayanos. 199 I . Enrichment and characterization of a methanogenicbacterium from the oxic upper layer of the ocean. current Microbiol. 23:g9-96.de Angelis, M. A., and c. Lee, 1994. Methane production during zooplankton grazingon marine phytoplankton. Limnol. Oceanogr. 39: l29g_130g.de souza, M. P., and D. c. yoch. r995a. purification and characterization ofdimethylsulfoniopropionate lyase from an Alcaligenes-like dimethyl sulfide-producingmarine isolate. Appl. Environ. Microbiol .61:21-26.de souza, M. P., and D. c. yoch. 1995b. compararive physiology of dimethyl sulfideproduction by dimethylsulfoniopropionate lyase in pseudomonas doudorffii andAlcaligenes p. strain M3A. Appl. Environ. Microbiol. 6l:39g6-3991.DeWeerd, K. A., L. Mandelco, R. S. Tanner, C. R. Woese, and J. M. Suflita. 1990.Desulfomonile tiedje gen. nov. and sp. nov., a novel anaerobic, dehalogenating, sulfate-reducing bacterium. Arch. Microbiol. 154:23-30.Hansen, T. A. 1993. carbon metabolism of sulfate-reducing bacteri a, p. 2l-40. In: J. M.Odom' and R. Singleton (ed.), The sulfate-reducing bacteria: contemporary perspecrrves.Springer Verlag, New York.97

Anaerobic microbial degradation of methylated sulfur compounds

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Anaerobic microbial degradation of methylated sulfur compounds

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