Bacillus stamsii sp. nov., a facultatively anaerobic sugar degrader that is numerically dominant in freshwater lake sediment
Nicolai Müller
∗, Frank D. Scherag
1, Michael Pester, Bernhard Schink
DepartmentofBiology,UniversityofKonstanz,78457Konstanz,Germany
Keywords:
Bacillus
Facultativelyanaerobicmetabolism Syntrophicoxidation
Sugarfermentation
a b s t r a c t
Anoveltypeofanaerobicbacteriawaspreviouslyisolatedfromprofundallakesedimentbydirectdilution ofthesedimentinmineralagarmediumcontainingglucoseandabackgroundlawnofMethanospirillum hungateiasasyntrophicpartner.TheisolatedbacteriagroupedwithaerobicBacillusspp.accordingto their16SrRNAgenesequence,andthemostcloselyrelatedspeciesisBacillusthioparans.Fermentative growthofthenovelstrainwithglucosewaspossibleonlyinthepresenceofsyntrophicpartners,and coculturesproducedacetateandmethane,insomecasesalsolactateandtracesofsuccinateasfermenta- tionproducts.Incontrast,thecloselyrelatedstrainsBacillusjeotgaliandBacillussp.strainPeC11areable togrowwithglucoseaxenicallybymixedacidfermentationyieldinglactate,acetate,formate,succinate, andethanolasfermentationproducts.Alternatively,theisolatedstraingrewanaerobicallyinpurecul- tureifpyruvatewasaddedtoglucose-containingmedia,andlactate,acetateandformatewerethemajor fermentationproducts,butthestrainneverproducedethanol.Aerobicgrowthwasfoundwithavariety oforganicsubstratesinthepresenceofpartlyreducedsulfurcompounds.Intheabsenceofsulfideand oxygen,nitrateservedasanelectronacceptor.StrainBoGlc83wascharacterizedasthetypestrainofa newspeciesforwhichthenameBacillusstamsiisp.nov.(DSM19598=JCM30025)isproposed.
Introduction
Inastudyonanaerobicsaccharolyticbacteriaintheprofundal sedimentofLakeConstance,Germany,weisolatedaslow-growing, spore-formingbacteriumwhichgrewwithglucose,fructose,and fewothersugarsonlyin syntrophicassociationwithhydrogen- orformate-oxidizingmethanogenicpartnerorganisms[18].This bacteriumdependedoncooperationwithapartnerorganism;inhi- bition of the methanogenic partner by bromoethane sulfonate preventedgrowthandsubstrateutilizationcompletely.Phyloge- neticanalysisindicatedthatthisnewisolateisrelatedtoaerobic sporeformersofthegenusBacillus,mostcloselyrelatedtoBacil- lus jeotgali [18]. Growth tests indicated that our isolate strain BoGlc83 couldalso grow aerobically, however, aerobic growth wasfoundonlyin complexgrowthmediaand wasnot easyto reproduce.
∗Correspondingauthor.Tel.:+497531883558;fax:+497531884047.
E-mailaddress:Nicolai.Mueller@uni-konstanz.de(N.Müller).
1Present address:Laboratory for Chemistry and Physics of Interfaces CPI, DepartmentofMicrosystemsEngineering–IMTEK,UniversityofFreiburg,79110 Georges-Köhler-Allee103,Freiburg,Germany.
Inthepresentstudy,thephysiologyofthisnewisolateisstudied, togetherwithataxonomicdescription.Further,thephysiologyof closelyrelatedstrainswasinvestigatedincludingBacillusthioparus whichwasdescribedinthemeantimeandisevencloserrelatedto strainBoGlc83[21,56].ThespeciesnameB.thioparuswasrevised laterandBacillusthioparanswasintroducedinstead[7].Moreover, Bacillussp.strainPeC11,anisolatefromgutsofbeetlelarvae,falls withinthesame groupof Bacillusstrains[10].The latterstrain cancoupleoxidationofe.g.glucosetoFe(III)reduction,whileB.
thioparuscangrowlithoautotrophicallyonthiosulfate[10,21].
OurdatashowthatBacillussp.BoGlc83isaversatileorganism abletogrowaerobicallyandanaerobicallywithvariousorganic substrates.Yet,inreducingmediathesubstraterangeisnarrow andthestraindependsonamethanogenicpartner,thusaccentu- atingitspotentialroleasaspecializedsugar-utilizingbacteriumin sulfidicsedimentsofafreshwaterlake.
Materialsandmethods
Originofbacterialstrains
EnrichmentandisolationofstrainBoGlcfromprofundalsed- iments of Lake Constance, Konstanz, Germany, were described earlier [18].Thestrain wasdepositedat theGermanCollection
Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-309355
Erschienen in: Systematic and Applied Microbiology ; 38 (2015), 6. - S. 379-389 https://dx.doi.org/10.1016/j.syapm.2015.06.004
ofMicroorganismsandCell-Cultures(DSM19598)andtheJapan CollectionofMicroorganisms(JCM30025).Methanospirillumhun- gateistrainM1h wasfromourown straincollection.B.jeotgali (DSM18226)andBacillusmegaterium(DSM319)werepurchased fromtheGermanCollectionofMicroorganismsandCell-Cultures (DSMZ).B.thioparans strainBMP-1(CECT7196) waspurchased fromtheSpanishTypeCultureCollection(CECT).Bacillussp.PeC11 waskindlyprovidedbySvenHobbieandDr.AndreasBrune,Mar- burg,Germany.
Cultivationconditions
Foraerobicgrowth,cultureswereincubatedin10mlglasstubes withaluminumcapsorinErlenmeyerflaskswithcottonstoppers onashakerat200rpmand30◦C.AllBacillusstrainsusedinthis studywereroutinelycultivatedin LBmedium containing10g/l Bactotryptone,5g/lyeastextract,10g/lNaCland10mg/lMnSO4 forsporulation(modifiedDSMZ-mediumNo.1).Thismodification helpedtoincreasereproducibilityofaerobicgrowth.
Foraerobiccultivationindefinedmedium,weusedafreshwa- terminimalmediumcontainingHEPES(10mM),NaCl(17.1mM), MgCl2·6H2O(2mM),NH4Cl(4.7mM),andKCl(6.7mM).Thebasal medium was autoclaved at 121◦C and 1bar overpressure for 20min. After cooling toroom temperature, the following sup- plements wereaddedtothemedium fromconcentratedsterile stocksolutions:CaCl2·2H2O(1mM),K–Na–phosphatebuffer,pH 7.0(1mM),7-vitamin-solution(1×,afterWiddelandPfennig[53]), traceelementsolutionSL13(1×,afterWiddeletal.[51]andMüller etal.[18]).Thiosulfatewasaddedassulfursourceatconcentrations between2and10mM.Glucoseorothersubstrateswereaddedat concentrationsbetween2and10mMfromfilter-sterilizedstock solutions.
Fortesting thepHtolerance andoptimum,themediumwas bufferedwithamixtureofMES,HEPES,TrisandCHES(10mMeach) tocoverapHbufferingrangefrompH5.0to9.5.ThedesiredpHwas adjustedbyHClorNaOHtothebasalmediumbeforeautoclaving.
Glucosewasaddedataconcentrationof4mMandthiosulfateat 2mMforpH-tests.
Growthunderanoxic,reducingconditionswastestedinoxygen- freefreshwatermediumbufferedwith30mMsodiumbicarbonate plus1mMsodiumsulfideasreducingagentasdescribedbefore [18,51,52].Cultivation wasdonein 15-mlor 25-mlglass tubes sealedwithbutylrubberstoppersthatwererenderedanoxicby flushingtheheadspacewithaN2/CO2mixture(80%/20%).Tubes were filled after autoclaving aseptically with anoxic media by meansofN2-flushedsyringes.
Forcultivationofbacteriaunderanoxic,non-reducingcondi- tionsthesameanoxicmediumwasusedasmentionedabove,but Na2Swasomittedandinstead2mMNa2SO4or2mMNa2S2O3was addedasasulfursource.
Substratesorsupplementswereaddedfromanoxicstocksolu- tions. These stock solutions were made anoxic by repeatedly stirringundervacuumandgassingwith100%nitrogenasreported before[18].
Growthexperiments
Bacterialgrowthwasmonitoredat578nmwavelengthwitha tubespectrophotometer(M107,CamspecAnalyticalinstruments Ltd., Leeds, UK) measuring optical densities directly in culture tubes.Whenopticaldensitieshadtobemeasuredinsamplesfrom largerculturevessels,adouble-beamcuvettespectrophotometer (Uvicon860,Kontron,Zurich,CH)wasused.Opticaldensitiesin anoxic,reducedmediaweremeasuredbyaddingafewgrainsof sodiumdithionitetothecuvettestokeepresazurineinitsreduced andcolorlessstate.
Growthwas definedas an overall changein optical density OD578of≥0.1.TurbiditiesofafinalOD578of0.05–0.09werecat- egorizedas“poorgrowth”.LowervaluesofOD578weredefined as“nogrowth”. Cultureswereinoculated toaninitialOD578 of 0.005–0.05,meaningthat,withthedefinitionofgrowthmentioned above,aOD578 of 0.1correspondstoapproximately2–4dou- blings.
PCRofbacterial16SrRNAgenesandphylogeneticanalysis
DNA extraction, amplification,and analysisof the16S rRNA genesaswellascalculationofthephylogenetictreeweredone asdescribed earlier[18].Primersusedfor amplificationofbac- terial16SrRNAgeneswere27F(5-AGAGTTTGATCCTGGCTC AG-3)[6] and1492R (5-TACGGYTACCTT GTTACG ACTT-3) [50].Primersspecificforthe16SrRNAgeneofBacillussp.BoGlc83 wereconstructedusingtheBioEditsoftwaretool[9].The100most similar gene sequences available on theNCBI-database (www.
ncbi.nlm.nih.gov/) wereused for comparison. Theprimers thus obtainedwereBoGlcfw(5-CCTTGACGGTACCTGCCAGA-3)and BoGlcrev(5-GGCTCCAAGGTTGCCCCTAG-3)andwereusedto amplifya991DNAfragmentatanannealingtemperatureof55◦C asdescribedearlier[18].
Since several newbacterial isolates closely related to Bacil- lussp.BoGlc83weredescribedin themeantime,we createdan updatedphylogenetictreeusingthepreviouslypublishedpartial sequenceofthe16SrRNAgeneofstrainBoGlc83withalengthof 1488bp(accessionnumberAY189804,[18]).Sequencesofinter- est were manuallyselected and aligned with theSINA aligner [24].Thephylogenetictreewascalculatedwith1358unambigu- ouslyalignednucleicacidpositionsusingthemaximum-likelihood methodRAxMLasimplementedintheARB5.5softwarepackage (http://www.arb-home.de,[15,41]).Thenon-redundantSSURef- erencedatasetNr.99fromthearb-silvahomepagewasusedfor phylogenetic analysis (http://www.arb-silva.de, [25]). Bootstrap supportfortheindividualbranchesinthephylogenetictreewas calculated using 1000 bootstraps and the RAxMLalgorithm as implementedinARB(http://www.arb-home.de,[15,41]).
StrainBoGlc83 wasalsoidentified using theEzTaxonserver (http://www.ezbiocloud.net/eztaxon; [13]) on the basis of 16S rRNA gene sequence data.For aligning sequence datanot con- tainedintheEzTaxondatabase,NCBIBLAST(http://blast.ncbi.nlm.
nih.gov/Blast.cgi,[2])wasused.
DNA–DNAhybridization
DNA–DNAhybridizationwascarriedoutbyDr.CathrinSpröer oftheIdentificationServiceoftheDSMZ,Braunschweig.Germany.
Analyticalmethods
Glucose,pyruvate,succinate,lactate,formate,andethanolwere analyzedbyHPLCusinganAminexHPX-87Hion-exchangecolumn (BioRad,Munich,Germany)heatedto60◦Cwith5mMH2SO4as eluentataflowrateof0.6ml/minsuppliedbyaLC-10ATvppump (Shimadzu,Munich,Germany).Sampleswereinjectedintothesys- temwitha234autoinjector(Gilson,Limburg-Offheim,Germany).
Theanalysistimewas25minpersample.Analytesweredetected witharefractionindexdetectorRID-10A(Shimadzu)andthedata analyzedusingtheShimadzuLCsolutionsoftware.Sampleswere preparedasdescribedelsewhere[18].
Nitrate,nitrite,sulfate,andsulfitewereanalyzedwithanion chromatography systemusinganLCA A14anion exchangecol- umn(Sykam,Fürstenfeldbruck,Germany).Thedetectionrangewas between0.05and 1mM,therefore,sampleswerediluted1:100 withwaterbeforemeasurement.
ThiosulfatewasquantifiedbycyanolysisinthepresenceofCu2+
(modifiedafterNorandTabatabai[20]).Samplesweredilutedin distilled water toa final volumeof 900land in a concentra- tionrangeof10–400M.Then,50lofa0.1MKCNsolutionwas addedandthesampleswereincubatedatroomtemperaturefor 5min.Thereafter,thesamplesweremixedwith50lofa50mM CuCl2solutionandincubatedforfurther5minatroomtempera- turetorelease1molofSCN−permoleofS2O32−.Last,50l0.75M Fe(NO3)3in3MHNO3wasaddedandafteranadditionalincuba- tiontimeof2min,theresultingiron(III)cyanatewasquantifiedby recordingtheabsorbanceat460nminaU-1100spectrophotome- ter(Hitachi,Tokyo,Japan)againstacontrolassaymixturewithout thiosulfate.CalibrationcurveswererunusingNaSCNstandardsin arangeof10–400Minafinalvolumeof1mltowhicheach50l Fe(NO3)3in3MHNO3wasadded.
Determinationofintra-andextracellularelementalsulfurwas donebymeltingelementalsulfurinsamplesfrombacterialcultures at90◦CinthepresenceofKCNtoyieldSCN−(modifiedafterSchedel andTrüper[30]).Samplesof50–100lcultureweremixedwith 3mlofa0.1MKCNsolutionin15mlplastictubesandincubated for20mininawaterbathheatedto90◦C.Sampleswereallowed tocooltoroomtemperatureand6.95–6.90mldistilledwaterand 500l0.75MFe(NO3)3in3MHNO3wereadded.Theabsorbance wasmeasured inplastic cuvettesat 460nmin a U-1100 spec- trophotometer(Hitachi,Tokyo,Japan)andconcentrationsofSCN− werecalculatedusingcalibrationcurveswithNaSCNasdescribed above.
SulfitewasqualitativelydetectedusingsulfitetestpaperNo.907 63(Macherey-Nagel,Düren,Germany).Sulfatewasassayedusing thebariumchloridemethod[43].
Analyses ofrespiratory quinones,polarlipids, and fattyacid compositionwerecarriedoutbytheIdentificationServiceofthe DSMZandDr.BrianTindall,DSMZ,Braunschweig.Germany.Anal- ysisoftheG+CcontentofgenomicDNAwascarriedoutbythe IdentificationServiceoftheDSMZandDr.PeterSchumann,DSMZ, Braunschweig.Germany.
Oxidasewastestedusingoxidaseteststrips(Fluka)following themanufacturer’sinstructions. Presenceof catalasewastested by dropwise adding a 3% H2O2 solution to a colony on solid mediaortocellsuspensionsdroppedonaglassslide.Theoxidase andcatalase-negativeLactobacillusplantarumandtheoxidaseand catalase-positiveParacoccusdenitrificanswereusedasreference strains.
Chemicals
All chemicals were of analytical or reagent grade quality and purchased from Sigma (Deisenhofen, Germany), Fluka (Neu-Ulm, Germany), Serva (Heidelberg, Germany), Boehringer (Mannheim, Germany), Eastman Kodak (Rochester, NY, USA), Merck(Darmstadt,Germany),andPharmacia(Freiburg,Germany), gases were purchased from Messer-Griesheim (Darmstadt, Germany),andSauerstoffwerkeFriedrichshafen(Friedrichshafen, Germany).
Results
Quantificationofsugar-degradingbacteriainLakeConstance sediments
Sugar-degradingbacteriawerecountedaerobicallyandanaero- bicallybothinthepresenceandabsenceofM.hungateiasapartner.
After incubation for 2–3 months, anaerobic glucose-degrading colonieswerefounduptoatotalof3.8×107cells/cm3sediment.
Aerobiccountsindefinedfreshwatermediumwith2mMglucose
assolesubstrateyieldedcountsof7.4×107cfucm−3.Coloniesin theagarwereoftenfluffyand,inthecaseofanaerobicgrowth,sur- roundedbycoloniesofthemethanogenicpartnerorganism[18].
Bacterialcellsinthecoloniesinbothcaseswereshortrods,often withsubterminal ellipticalspores[18].ControlsbyPCRanalysis confirmedthatinbothcasesthesametypeofbacteriawasculti- vated.
OccurrenceofthistypeofbacteriainLakeConstancesediments wasverifiedalsobyamplificationofthe16SrRNAgeneofBacillus sp.BoGlc83usingprimersspecificforthisstrain.Directamplifica- tionoftheexpectedPCRfragmentfromDNAisolatedfromlake sediment was not successful.In anotherexperiment, DNA was isolatedfromsedimentstakenatdifferentwaterdepths(littoral sedimentandprofundalsedimentsat77mand145mwaterdepth) andusedastemplateforunspecific16SrRNAgeneamplification.
TheresultingPCRproductwasusedastemplateforanotherPCR withtheprimersspecificforBacillussp.BoGlc83.ThisPCRyielded theexpected991bpfragmentoftheBacillussp.BoGlc8316SrRNA genefromallthreesamples(datanotshown).
Chemotaxonomicandphenotypiccharacterizationofstrain BoGlc83
UponaerobiccultivationinmodifiedLBmedium,strainBoGlc83 grewasshort,irregularrodsof5–10mlengthand0.5–0.8m width. Chains of fiveor more cells were oftenobserved, espe- ciallyintheearlyexponentialphase.Thephylogeneticallyrelated strainsB.thioparans,B.jeotgali,andBacillussp.PeC11wereslightly shorterandthinner.StrainBoGlc83belongstothegenusB.accord- ingtoprevious16S-rRNAgeneanalyses[18].Itsclosestdescribed relativesareB.thioparans(similarity98.91%[21]),Bacillussubter- raneus(similarity98.51%[12]),B.jeotgali(similarity98.44%[56]), Bacillusboroniphilus(similarity98.31%[1]),andBacillusselenatarse- natis(similarity98.30% [54])afteranalyzingthe16SrRNAgene sequence of Bacillus strainBoGlc83 using the EzTaxon Identify tool [13]. In addition, the 16S rRNA gene sequence of Bacillus strain BoGlc83 wasaligned withthe 16S rRNA gene sequence of the taxonomically undescribed Bacillus sp.PeC11 [10] using the BLAST bl2seq tool and the similarity was 98.1%. The phy- logenetic distancetreeof BacillusstrainBoGlc83and itsclosely relatedstrainsbasedonthe16SrRNAgenesequenceisshownin Fig.1.Basedonthethresholdvalueof98.7%fordifferentiatingtwo bacterialspeciesbytheir16SrRNAgenesequence[28,39,55]Bacil- lussp.BoGlc83 andB.thioparansseemedtorepresentthesame species.Therefore, DNA–DNAhybridizationof thesetwo strains wasperformedandtheDNA–DNAsimilaritywasdeterminedto be23.8%and25.5%induplicatemeasurements.Consideringthat theDNA–DNAsimilarityoftwostrainsmustbesmallerthan70%
forclassifyingthemasdifferentspecies[49],westatethatBacillus sp.BoGlc83andB.thioparanshavetobegroupedintwodistinct species.
StrainBoGlc83wasoxidasepositivebutwascatalasenegative when tested after aerobic or anaerobic growth. The G+C con- tentwasdeterminedto42.8mol%(Table4).Respiratoryquinones were menaquinone-7 (MK-7, 97%) and menaquinone-6 (MK-6, 3%). Thestrain containsthepolarlipidsdiphosphatidylglycerol, phosphatidylglycerolandphosphatidylethanolamineasjudgedby thin-layerchromatographycarriedoutbytheDSMZidentification service.Analysisofthefattyacidcompositionclassifiesitasamem- beroftheBacilluspumilussubgroupB(DSMZanalysis).Themajor fattyacidwasiso-C15:0with48.8%ofthetotalfattyacidcontent.
Similarresultswereshownearlierforthecloselyrelatedstrains B.thioparans(77.3%iso-C15:0,[21])andB.jeotgali(49.3%iso-C15:0
[56]).Detailedresultsofthefattyacidanalysisaresummarizedin Table1.
Fig.1. PhylogenetictreeshowingthepositionofBacillussp.strainBoGlc83andcloselyrelatedstrainsbasedona1488bplongfragmentofthe16SrRNAgene.Thetreewas createdusing1358unambiguouslyalignednucleotidepositionsandthemaximum-likelihoodmethodRAxMLasimplementedinARB(http://www.arb-home.de,[15,41]).
Fornodeswithbootstrapvalueshigherthan70%,therespectivepercentagesareshown.Barequals5%estimatedsequencedivergence.
Table1
AnalysisoffattyacidcompositionofBacillussp.strainBoGlc83.
BacillusstrainBoGlc83–compositionoffattyacids%
Fattyacid Percentage
C10:0 0.03
i-C13:0 0.26
ai-C13:0 0.05
i-C14:0 1.32
C14:0 0.84
iF-C15:1 2.06
ai-C15:1 0.05
i-C15:0 48.81
ai-C15:0 10.93
C15:0 0.08
C16:17calcohol 0.99
i-C16:0 1.33
C16:111c 1.42
C16:0 4.52
i-C17:110c 7.32
ai-C17:19c 0.18
i-C17:0 12.78
ai-C17:0 3.73
C18:19c 0.12
C18:17c 0.07
C18:0 0.19
i-C19:0 0.11
Summedfeature1 0.06
Summedfeature3 0.47
Summedfeature4 2.29
AnalysiswasdonebytheDSMZidentificationservice.Chromatographicpeaksthat couldnotbeseparatedcontained:Summedfeature1:i-C15:1H/i-C15:1I/C13:03OH;
Summedfeature3:C16:17c/i-C15:02OH;Summedfeature4:i-C17:1I/ai-C17:1B.
AerobicgrowthofstrainBoGlc83
If strain BoGlc83 was cultivated aerobically in freshwater medium with sulfate as sulfur source, growthwas not always reproducibleaftertransferring stationarycells tofreshmedium andwasfoundpreferentiallywithcomplexgrowthmediacontain- ingyeastextractorotherundefinedconstituents.Aerobicgrowth wasobservedalwaysforonly3–4generations,i.e.,onetransfer afteranaerobiccultivation;furtheraerobiccultivationyieldedno furthergrowth.Aerobiccultivationovermorethan3–4cellgenera- tionswaspossibleinthepresenceofyeastextract(0.05%w/v).This growth-stimulatingeffectwasfurtherelucidatedusingfreshwater mediumwithsingleaminoacidsorcombinationsofaminoacids.
Sulfur-containingaminoacidscausedsubstantiallyhighergrowth stimulation(OD578=0.44±0.11with1mMl-methionine+4mM
Fig.2.AerobicgrowthofstrainBoGlc83withdifferentsulfursources.Inoculawere obtainedbycentrifugationof500lofanaerobiccoculturewithMethanospirillum hungatei.Pelletswerewashedthreetimesinoxicminimalmediumwithoutsul- fursource(filleddiamonds)orinsulfide-reducedanoxicmedium(filledsquares).
Afterthelastwash,inoculaweretransferredtooxicminimalmediumwithout sulfursource.Anon-washedcontrolwasruninparallel(emptycircles).Intwo additionalexperiments,sulfur-freewashedinoculaweretransferredtomediacon- tainingeither1.8mMthiosulfate(opensquares)or0.1%w/velementalsulfur(filled triangles).Shownaremeanvaluesofn=3ofOD578±standarddeviation.
glucose)thanotheraminoacidsdid(l-glutamine,OD578=0.25;
glycine, leucine, alanine, asparagine OD578=0.15–0.19). Also glutathionestimulatedgrowthsubstantially(OD578=0.51with 2mM glutathione). Obviously, the cells depended on a partly reducedsulfursourcebeyondthesodiumsulfatethatwaspresent in the standard medium. As documented in Fig. 2, also partly reduced inorganicsulfurcompounds such asthiosulfate orsul- furflowerstimulatedgrowthwithsucroseinasimilarmanneras methioninedid.AsalsoshowninFig.2,smallcarry-oversofsul- fidefromreducedanaerobicpreculturescouldstimulategrowthof aerobiccultures.
Fig.3. AerobicgrowthofstrainBoGlc83with5mMglucoseplus10mMthiosul- fate.Shownareopticaldensitiesat578nm(filledsquares),glucoseconcentrations (emptydiamonds),acetateconcentrations(emptytriangles),andthiosulfatecon- centrations(emptysquares).N=3,±standarddeviation.
Growthstimulationbythiosulfatewithglucoseasmainenergy sourcerequiredatleast2mMthiosulfatetoyieldsignificantyield increases.Inthepresenceof4mMglucose,thethiosulfate-specific growthstimulationwashigher thanwith2mMglucose;higher thiosulfate additions (4mM,10mM)didnotstimulate glucose- dependentgrowthanyfurther.Additionofthiosulfatestimulated glucose consumption alsoin densesuspensions of resting cells (resultsnotshown).
Thiosulfatewasconsumedtogetherwithglucoseinaerobiccell suspensionsatabouta1:1stoichiometry.Thesecellsuspensions werecontinuouslyspargedwithairandtheemittedgaswaspassed throughavesselcontainingcupricchloridesolution.Formationof abrownprecipitateinthisvesselindicatedtheformationofhydro- gensulfide.Sulfateorsulfitecouldnotbedetected.
Ingrowingbatchcultures,thiosulfate wasconsumedincom- pletely during theexponential growthphase (Fig.3).From an averageof11.3mMthiosulfateaddedtotriplicatecultures,9.4mM wasstillpresentattheendofgrowth(Fig.3).Elementalsulfurcould bedetectedintheculturesatanaverageconcentrationof4.9mM.
Assumingthat1molofthiosulfatecouldbeconvertedto2molof elementalsulfur,thissulfurconcentrationapproximatelyaccounts forthe1.9mMthiosulfateconsumed.
AerobicgrowthwaspossiblebetweenpH6.5and8.5,withan optimumatpH7.0–7.5.NogrowthwasfoundatpH5.5and9.5 (Table4).UnderoptimalconditionsatpH7.2,growthwascompa- rablyslowwith=0.16–0.19h−1(td3.7–4h).Theglucose-specific growthyieldincreasedlinearlyintherangeof0–1.5mMglucose;
at higher glucoseconcentrations, the increase wasnot propor- tionaltotheavailablesubstrate(Fig.4).Themolargrowthyield was57.8±1.3gdrycellmasspermolglucosedissimilated.Sev- eralothersugars(fructose,galactose,mannose,lactose,maltose, sucrose)wereutilizedwithsimilargrowthefficiencies.Besidessug- ars,alsoacetate,citrate,fumarate,gluconate,lactate,pyruvate,or succinatewereusedassubstratesforaerobicgrowth.Therewas nogrowthwithsingleamino acidsor combinationsofdifferent aminoacidsnorwithmonovalentalcohols.Alistofsubstratesuti- lizedornotutilizedisfoundinthespeciesdescriptionattheendof thispaper.SeveralcharacteristicsofstrainBoGlc83anditsclosest relativesaresummarizedinTable4.
Fig.4.Influenceofglucoseconcentrationonmaximalopticaldensitiesreached byculturesofstrainBoGlc83growninoxicminimalmedium.Averagemaximal changesofOD578ofthreeindependentcultures±standarddeviationarepresented.
Someerrorbarsaresmallerthansymbolsize.
Fig.5. Highestmaximalchangesofopticaldensitiesat578nmreachedbycultures ofstrainBoGlc83duringanaerobicgrowthinnon-reducingmediumwithdifferent combinationsofglucose,nitrate,casaminoacids(CA),andyeastextract(YE).Mean valuesofthreeindependentcultures±standarddeviationafter5daysofincubation.
Anaerobicgrowthinnon-reducingmedium
Inanoxic,non-reducedmedium,strainBoGlc83grewwithglu- coseplusnitrateinthepresenceof0.05%casaminoacidsand0.05%
yeastextractassupplements(Fig.5).Growthwaspossiblealsowith casaminoacids,yeastextract,andtryptoneassolesourcesofcarbon andenergyrespectivelyandwithnitrateasanelectronacceptor.
However,growthonglucoseplusnitratewaspoorifnocomplex supplementswerepresent(Fig.5).Therefore,growthwastested bothinthepresenceandabsenceofglucose.AsshowninFig.5,glu- coseinadditiontocasaminoacids,yeastextract,andnitrateyielded muchhigheropticaldensitiesthancontrolswithoutglucoseaddi- tion.Thus,strainBoGlc83canoxidizeglucoseanaerobicallywith nitrateaselectronacceptorinthepresenceofcomplexmedium additions.Forfurthercharacterizationofnitratereductionbystrain BoGlc83,sulfate(2mM)wasaddedasasulfursource.Of20mM nitrateinitiallyaddedtothemedium,anaverageof10.2mMnitrite wasproduced,and3.9mMnitrateand3.9mMglucosewerestill presentattheendofgrowth.Oxidationofglucosewasincomplete eventhoughnitratewasstillpresent,indicatingthattheaccumu- latednitriteinhibitedfurthergrowth.Acetate(5.9mM)andlactate (2.8mM)wereformedasfurtherproducts. Similarresultswere
Fig.6.Highestmaximalchangesofopticaldensitiesat578nmreachedbycultures ofstrainBoGlc83duringanaerobicgrowthinnon-reducingmediumwithdifferent combinationsofthiosulfate,nitrate,casaminoacids(CA),andyeastextract(YE).
Meanvaluesofthreeindependentcultures±standarddeviationafter22daysof incubation.
obtainedincultureswith2mMthiosulfateassulfursource(not shown).However,asmentionedabove,undertheseconditionssul- furwasmostlikelyassimilatedfromorganicsourcesinthecomplex supplements.Inacontrolexperiment, glucosewasomitted and 1%casaminoacidsascarbonandenergysourcewereaddedinthe presenceof20mMnitrateand2mMsulfateassulfursource.After growthendedinthreeindependentcultures,cultureswithsulfate assulfursourcecontained9.4mMnitrite,while17.2mMnitrite wasstillpresent incultureswith2mM thiosulfate.Nitratewas completelyconsumedinbothcases.
We also tested for possible use of thiosulfate as an energy source. Strain BoGlc83 was cultivated with 50mM thiosulfate, 50mMnitrate,0.05%casaminoacids,and0.05%yeastextract(Fig.6) Controlcultures contained either nocomplex supplements,no thiosulfate,ornonitrate(Fig.6).Cultureswithoutcasaminoacids andyeastextract didnot grow,whereas allculturescontaining thesecomplexsupplements andnitrategrew wellandreached stationaryphaseafter76h(averagemaxOD578=0.31,Fig.6).Opti- caldensitiesincultureswiththiosulfateinadditiontocasamino acids,yeastextract,andnitrateincreasedfurther,andthecultures reachedstationaryphaseafter22days(averagemaxOD578=0.59, Fig.6).Formation ofa white precipitatewasobserved inthese cultures suggestive ofproduction of elemental sulfur. Thecon- centration of elemental sulfur in these cultures was 10.1mM (±2.4mM),while4.1mMthiosulfatewasconsumedasjudgedby colorimetricassays.
Anaerobicgrowthinreducingmedium
StrainBoGlc83didnotgrowinreducingmediuminpurecul- ture(OD578below0.1),neitherwithglucosenorwithpyruvate addedseparatelytothemedium.However,culturesweremetabol- icallyactiveundertheseconditions,asglucoseorpyruvatewas slowlyconvertedtolactateandacetate(Table2).Ifbothglucose andpyruvateweresuppliedinthepresenceof0.05%casaminoacids and0.05%yeastextract,culturesgrewtoanaveragemaxOD578
of0.19 (Fig.7).Weakgrowthwasfoundalsointheabsenceof casamino acids and yeast extract, yet to a much lower extent thanincultureswiththesesupplements(datanotshown).Fer- mentationproductswerelactate,succinate,acetate,andformate (Tables2and3).EthanolwasneverproducedbystrainBoGlc83.
However,thecloselyrelatedB.jeotgaliandBacillussp.PeC11pro-
ducedsignificantamounts ofethanolduringgrowthonglucose Table2 FermentationbalancesofstrainBoGlc83andotherBacillusstrainstestedforanaerobicgrowthinreducingmediumwith5mMglucose.Shownareaveragevalues±standarddeviationofn=3. Glucose consumed (mM) Glucose assimilated (mM)a
Glucose dissimilated (mM) Fermentationproducts(mM)Electronrecovery SuccinateLactateAcetateFormateEthanolFromtotal glucose consumptionb
Fromglucose dissimilatedb Bacillussp.BoGlc830.08±0.240.01±0.020.07±0.020.07±0.010.53±0.120.28±0.090.24±0.040±0NANA Bacillusjeotgali5.05±0.010.58±0.024.47±0.020.57±0.025.09±0.241.98±0.163.72±0.512.24±0.3695.5±3.4%108±4.5% Bacillussp.PeC115.17±0.180.64±0.004.53±0.000.12±0.021.08±0.064.02±0.167.97±0.364.11±0.1890±5.5%102.5±3.8% aCalculatedassuminganOD/drymasscorrelationof263.5mg/lperOD578=1asin[18]fromthehighestobservedaverageofn=3correctedforinitialODvaluesatthebeginningoftheexperiment.Fromtheassimilation equation,acorrelationof6.877molglucoseper1mgcelldrymasswasderivedasdescribedin[18]. bElectronrecoverieswerecalculatedforbothtotalglucoseconsumptionanddissimilatedglucoseasthegrowthmediumcontained0.05%casaminoacidsplus0.05%yeastextractrequiredforgrowth.Thissupplementaddition ledtoanelectronrecoveryhigherthan100%,aspartofthecellmasswasproducedfromthesupplements.
Table3 FermentationbalancesofstrainBoGlc83andotherBacillusstrainsgrownanaerobicallyinreducingmediumwith5mMglucoseplus10mMpyruvate.Shownareaveragevalues±standarddeviationofn=3. Glucose consumed (mM) Glucose assimilated (mM)a
Glucose dissimilated (mM) Pyruvate consumed (mM)
Fermentation products(mM)Electronrecovery SuccinateLactateAcetateFormateEthanolFromtotal Glucoseand Pyruvate consumptionb
FromGlucose dissimilatedb Bacillussp.BoGlc835.47±0.240.34±0.025.13±0.022.5±1.030.16±0.0310.15±1.092.93±0.681.54±0.680±096.6±13.2%101±10.7% Bacillusjeotgali3.7±1.210.36±0.133.34±0.131.4±0.770.53±0.104.53±1.312.51±0.682.61±0.751.2±0.2099.1±18.8%104±23.9% Bacillussp.PeC115.16±0.200.33±0.044.83±0.041.7±0.320.22±0.081.54±0.145.9±0.189.19±0.584.21±0.1397±6.7%103±5.11% acalculatedassuminganOD/drymasscorrelationof263.5mg/lperOD578=1asin[18]fromthehighestobservedaverageofn=3correctedforinitialODvaluesatthebeginningoftheexperiment.Fromtheassimilation equation,acorrelationof6.877molglucoseper1mgcelldrymasswasderivedasdescribedin[18]. bElectronrecoverieswerecalculatedforbothtotalglucoseconsumptionanddissimilatedglucoseasthegrowthmediumcontained0.05%casaminoacidsplus0.05%yeastextractrequiredforgrowth.Thissupplementaddition ledtoanelectronrecoveryhigherthan100%,aspartofthecellmasswasproducedfromthesupplements. Table4 CharacteristicsofstrainBoGlc83andcloselyrelatedstrains. CharacteristicBacillusstrain BoGlc83Bacillus thioparans CECT7196
Bacillusjeotgali DSM18226Bacillusstrain PeC11Bacillus subterra-neusBacillus boroni-philusBacillusselenat- arsenatis Cellsize(m)5–10×0.5–0.81–1.7×0.5–0.7a4–6×0.8–1.1cND2–25×0.5–0.8d1.8–5.5×0.5–0.9e3–6×1f pHoptimum7.0–7.57.0a7.0–8.0cND7.0–9.0d7.5–8.5e8.0f pHrange6.5–8.5ND5.0–8.0cND6.0–9.5d6.5–9.0e7.5–9.0f Temperatureoptimum30◦C30◦C–35◦Ca30◦C–35◦CcND37◦C–40◦Cd30◦Ce40◦Cf Temperaturerange20◦C–40◦Cb30◦C–45◦Ca10◦C–45◦CcND25◦C–45◦Cd16◦C–37◦Ce25◦C–40◦Cf Catalase−+a+cND+d+e+f Oxidase+−a−cND−d+e−f GCcontent42.8%43.8%a41%cND43%d42.2%e42.8%f Sporeformation+++ND−d+e+f Nitratereduction++a+cND+d−e+f Axenic,fermentativegrowthwithglucose−−+++dNDND Axenic,fermentativegrowthwithglucoseandpyruvate+ND++NDNDND Syntrophic,fermentativegrowthwithglucose++NANANDNDND Autotrophicgrowthwiththiosulfate−+a−aNDNDNDND Ethanolproductionduringfermentativegrowthwithglucose−ND++NDNDND ND=notdetermined. aDataextractedfrom[21]. bDataextractedfrom[18]. cDataextractedfrom[56]. dDataextractedfrom[12]. eDataextractedfrom[1]. fDataextractedfrom[54].
Fig.7.AnaerobicgrowthofstrainBoGlc83,Bacillusjeotgali,andBacillussp.PeC11 inreducingmedium.Mediacontainedboth0.05%casaminoacidsand0.05%yeast extractandeither5mMglucoseplus10mMpyruvate,5mMglucose,or10mM pyruvate.Shownaremeanvaluesofthehighestchangesofopticaldensitiesat 578nmofthreeindependentcultures±standarddeviationafter135hofincubation.
(Tables2–4),andwerealsoabletogrowwithoutaddedpyruvate (Fig.7).Thepatternoffermentationproductsduringgrowthwith glucosepluspyruvatedifferedgreatlybetweenthethreestrains (Table3).ThemajorfermentationproductofstrainBoGlc83was lactate,withminoramountsofacetateandformate.AlsoB.jeotgali producedmainlylactate,butalsoformate,acetate,ethanol, and succinate(Table3).Incontrast,Bacillussp.strainPeC11produced mainlyformate,acetate,andethanol,togetherwithminoramounts oflactateandsuccinate(Table3).Noneofthesestrainstestedgrew with10mMpyruvatealone.
Syntrophicgrowth
Anaerobicgrowthunderreducingconditionswithglucoseinthe presenceofM.hungateiwasshownearlierforBacillussp.BoGlc83 [18].WealsotestedothercloselyrelatedBacillusstrainsforsyn- trophicgrowth.OfthetestedB.megaterium,B.thioparans,B.jeotgali, andBacillussp.Pec11,onlyB.thioparanswasabletogrowinthis mediumwithglucoseinthepresenceofM.hungateiafteranincu- bationtimeofapproximately1month,whileaxenicgrowthofB.
thioparanswithglucosewasnotpossible(Table4).B.megaterium didnotgrowatallundertheseconditionsandB.jeotgaliandBacillus sp.PeC11reachedstationaryphasealreadyafter7daysasbothare abletogrowwithglucosewithoutamethanogenicpartner.There wasnoindicationofafermentationproductshiftbythepresence ofthemethanogenicpartner.
Discussion
Thebacterialstraindescribedinthisstudy,strainBoGlc83,was originallyisolated asanumerically predominantutilizerofglu- coseandothersugarsinthesedimentofLakeConstance,Germany.
Anaerobicgrowthwaspossibleonlyinthepresenceofformate- orhydrogen-utilizingmethanogenicpartners.Later,itturnedout thatthisbacteriumgroupedwiththegenusBacillusonthebasis of16SrRNAgenesequencedata,andthatitwasalsoabletogrow aerobically.However,asdocumentedinthepresentstudy,aerobic growthwasnoteasytoreproduceonstandardmediabutrequired partlyreducedsulfurcompounds,eitherorganicorinorganicones, forreproducibleaerobicgrowth.Sulfur,thiosulfate,orsulfitecould serve assulfur sourceunder theseconditions. Nonetheless,the requirementforsuchpartlyreducedsulfurcompoundswascom- parablyhigh,intherangeof2mM,farmorethantheamountof
sulfurrequiredforassimilationonly(whichwouldbeintherange of20–50MSwiththecelldensitiesreached,assumingasulfur contentofcelldrymatterofabout1%).Inaerobiccultures,thio- sulfate wasconverted toelementalsulfur especially duringthe exponentialgrowthphase,indicatingthatthiosulfatecanserveas anadditionalelectronacceptorwhenoxygenbecomeslimitingdue totheincreasingcelldensity.Indensecellsuspensionsofaerobi- callygrowncellsofstrainBoGlc83,consumptionofthiosulfatewas evenmorepronounced,thussupportingthehypothesisthatthe cellsbecomeoxygen-limitedwithincreasingcelldensity,evenif thecellsuspensionsarevigorouslyspargedwithair.Unfortunately, elementalsulfurwasnotmeasuredincellsuspensionexperiments.
Itisknownfromotherthiosulfate-metabolizingbacteriathatele- mentalsulfurandsulfiteareproductsofthiosulfatecleavage[3].In strainBoGlc83,itappearsthat1molofthiosulfateisconvertedto 2molofelementalsulfuraccordingtothemeasuredstoichiometry.
Moreover,sulfiteorsulfatecouldneverbedetected,neitherafter aerobicnorafteranaerobicgrowth.
StrainBoGlc83grewoptimallyaerobicallywithdoublingtimes of3–4h.Instandardenrichmentcultureswithsugarsassubstrates, itwouldalwaysbeoutcompetedbyfasteraerobicbacteria.The sameapplies forcultivationunder anoxicconditions(td>24h):
classicalsugar-fermentingbacteriasuchasClostridiumspp.would outcompeteitveryquicklyalthoughsuchbacteriawerefoundin oursedimentonlyatsubstantiallylowernumbers[18].Isolation ofthisnovelbacteriumwaspossibleonlyindirectdilutionseries inthepresenceofabackgroundlawnofmethanogenicpartners.
Nonetheless,thefactthatthisbacteriumappearstobeadominant sugarutilizer inthelakesedimentstudiedindicates thatunder theconditionsprevailingtherethistypeoforganismhasaclear advantageoverothers,e.g.,byoptimalATPgenerationinsyntrophic cooperation[31].
Strain BoGlc83 can grow auxotrophically in anoxic, non- reducingmediumbynitraterespirationonglucose,withcasamino acids, yeast extract, tryptone, or thiosulfate present. However, nitratereduction wasincompleteinbatchcultures andmostof thenitratepresentintheculturesaccumulatedasnitrite.Nitrate reductionisrathercommoninBacillusstrains,however,oxidation ofthiosulfate asanelectronsourcefornitratereductionwithin thegenusBacillushassofarbeendescribedonlyforB.thioparans [21,35].Yet,thetypeof metabolismis alreadyknownforother bacteria like Thiobacillus denitrificans [11]. In ourhands, strain BoGlc83didnotgrowlithoautotrophicallywiththiosulfateasthis wasshownearlierforB.thioparans[21].Yet,duringauxotrophic growthinthepresenceofyeastextractandcasaminoacids,opti- caldensitiesincreasedandelementalsulfurwasdetectedinthe cultureswhilepartofthethiosulfatewasconsumed.Formation ofawhiteprecipitatewasobservedinthesecultureswhichcould beinsoluble elementalsulfurandtherefore makesit difficultto judgewhethertheobservedincreaseinturbidityisgrowthrelated.
However,cellsareobviouslymetabolicallyactiveunderthesecon- ditions.Therefore,itislikelythatstrainBoGlc83usesthiosulfate asan electronacceptor,e.g. ifnitrite concentrationsin anaero- bic cultures are too highto allowfurther nitratereduction. In cultures withthiosulfate, casamino acids and yeast extract no growthwasobserved whichshows thatnitrateisessential and thiosulfateadditionalonecannotsupportgrowth.Butthefactthat duringbothaerobicandanaerobicgrowth1molofthiosulfateis convertedto2molofelementalsulfurindicatesthat thiosulfate servesaselectronacceptorratherthanaselectrondonorforstrain BoGlc83.ThiosulfateoxidationasinB.thioparansorThiobacillus intermediuswouldyieldsulfiteandsulfateasintermediatesorend products[3,21],whichcouldneverbedetectedinculturesofstrain BoGlc83.
If grown anaerobically under reducing conditions, strain BoGlc83 was unable to grow on glucose without a syntrophic
partneratfirst,eventhoughaccumulationoffermentationprod- uctscouldbeobservedinthemedium.However,ifpyruvateand supplementswereaddedstrainBoGlc83grewbymixed-acidfer- mentation.Limitedgrowthwaspossiblealsoifnosupplementsbut onlyglucoseandpyruvatewereadded.AlsoBacillussubtiliscan growbyfermentationofglucoseinanoxic,non-reducedSpizizen’s mediumamendedwithaminoacidmixtures,however,additionof pyruvatetocultureswithglucoseaugmentedgrowthsignificantly [19].It wasconcludedthatpyruvatemightserve asastimulat- ing agent that triggers expressionof genes required for mixed acidfermentation,astheintracellularpyruvatepoolmightbetoo smallto initiateexpressionof genes requiredfor fermentation.
Also,pyruvatewasdiscussedasaprecursorofcertainaminoacids thatcannotbesynthesizedbyB.subtilis[19].Similarconclusions weredrawnbeforefromobservationsmadewiththemethanogenic archaeonMethanosarcinabarkeri[17].Here, amutantthat lacks ech-hydrogenasecouldnotgrowonmethanol.Uponadditionof pyruvatetothemedium,theabilitytogrowwasrestored,andit wasconcludedthattheech-hydrogenaseprovidesthecellswith reducedferredoxintosecurepyruvatesynthesisfromacetyl-CoA.
Pyruvateinturncouldthenserveasaprecursorforbiosynthesis [17].
InthecaseofstrainBoGlc83,noneofthelatterconclusionsof geneexpressiontriggeredbypyruvateordeficiencyinbiosynthe- sispathwayscanexplainthegrowth-supportingeffectofpyruvate.
First, fermentation pathways are expressedifstrain BoGlc83 is incubatedinpurecultureonglucose,asindicatedbytheproduction offermentationproducts;thestrainjustdoesnotgrowmeasurably.
Second,eveninthepresenceofcasaminoacidsandyeastextract, nogrowthisobservedonglucosealone,indicatingthataminoacid synthesisdeficienciesarenotasuitableexplanationfortheinabil- ity ofthestraintogrowundertheseconditions. Moreover,the straingrowswellinthepresenceofamethanogenicpartnerwith- outadditionofsupplements.Yet,itispossiblethatstrainBoGlc83 lacksuptakesystemsforcertainaminoacidsbutnotforpyruvate, meaningthattheseaminoacidscouldonlybesynthesizedifhigh amountsofpyruvateareavailable.Suchasituationcouldoccurdur- ingsyntrophicgrowth,i.e.ifpyruvatedoesnotneedtobereduced tolactateorsuccinatetoregenerateelectroncarriers.Instead,dur- ing syntrophic growth,a reversed electron transport systemin strain BoGlc83 was suspected earlier to beresponsible for the regenerationofNADHtoNADwithalow-potentialelectronaccep- tor,suchthatelectronsfinallycouldbetransferredtoprotonsorto protonspluscarbondioxide,toformhydrogenorformate[18].In thepreviouslypublishedarticle,wereportedspecificactivitiesfor formatedehydrogenasewithbenzylviologenaselectronacceptor thatwereabout7foldhigherthanthespecificactivityforhydro- genase.Itwasfurtherconcludedthatformatemightbethemajor electron carrier mediating interspecies hydrogentransfer, even thoughasmallpartofelectronsfromglucoseoxidationmightalso bereleasedashydrogen.Thiswassupportedbythefactthatstrain BoGlc83didnotgrowwellinthepresenceofthehydrogen-only consuming Methanobrevibacterarboriphilus[18].Furtherindica- tionsthatformateactsasanelectroncarriertoitsmethanogenic partnerarepresentedinthispaper.Whileinsyntrophiccocultures ofstrainBoGlc83andthehydrogenandformate-consumingM.
hungateionlytracesofformateweredetected[18],formatewas producedinsubstantialamounts(1.54mM)duringgrowthinpure cultureon5mMglucoseplus10mMpyruvate,indicatingthatthe pathwayofformateproductionisexpressedandcouldserveasa majorelectronsinkduringsyntrophicgrowth.Formateproduction inthetwootherBacillusstrainstestedinthepresentstudywas evenhigher,2.6mMforB.jeotgaliand9.2mMforBacillussp.strain PeC11,whichwasalsoshownearlierforstrainPeC11underanaer- obic,non-reducingconditions[10].Apparently,theBacillusstrains B.jeotgaliand Bacillussp.strainPeC11 donotneedpyruvateto
initiate fermentativegrowth onglucose. However,the fermen- tation patterns differ between the different Bacillus spp., and syntrophicgrowthofthosestrainscouldnotbeobservedyetas thestrainsoutgrowtheslow-growingM.hungateiwhenincubated inmediumwithglucose.Thefactthatallthreestrainscanproduce formateuptoseveralmillimolarconcentrationsalsosuggeststhat theinabilityofstrainBoGlc83togrowwithglucosealonecannot beexplainedbygrowthinhibitionthroughformate.
Duringgrowthonglucoseandpyruvate,lactateis themajor fermentationproductinstrainBoGlc83,incontrasttoB.jeotgali andBacillussp.PeC11whichgrowonglucosewithoutadditionof pyruvate.Thisindicatesthatthepathwayoflactateformationfrom pyruvateis stronglyexpressedinstrainBoGlc83, whichinturn couldimplythatpyruvateisrapidlyconsumedinsidethecellby lactatedehydrogenase.Consequently,thesizeoftheintracellular pyruvatepoolmightbeinsufficienttosynthesizeessentialamino acidswhichpossiblycannotbetakenupbythecellthoughpresent inthemedium.Externallyaddedpyruvatemighthelptocompen- satefortheintracellularpyruvatelossthroughlactateproduction, whichwouldexplain whystrainBoGlc83cangrowunderthese conditions.
According to the 16S rRNA gene sequence analysis, strain BoGlc83andsimilarstrainsisolatedwithit havetobeassigned tothegenusBacilluswhichcontainsmainlystrictlyaerobic,Gram- positivespore-formingbacteria.SeveralBacillusspeciescanalso grow anaerobically by a fermentative metabolism, e.g., B. sub- tilis[19],B.cereus,B.thuringiensis,B.licheniformis,B.coagulans,B.
polymyxa,B.macerans,B.alvei,B.laterosporus,B.larvae,B.popilliae, andB.lentimorbus[35].Nonetheless,syntrophiccooperationwith methanogenicpartnershasnotbeendescribedsofarforanyBacil- lusspecies.Theclosestrelativeaccordingtoourinitialsequence analysis[18],B.jeotgali,wasdescribedasa facultativelyaerobic bacteriumandwasisolatedfromfermentingseafood[56].Accord- ingtoadetailedanalysisof16SrRNAgenesequencesofmorethan 2600Bacillusstrains,ourisolatesfallintocluster9,togetherwith severalsofarnon-describedstrains[23].Mostofthesestrainswere foundasnumericallydominantrepresentativesofthecultivable communityinDutchgrasslandsoils[8],anoxicricepaddysoil[4], pasturesoil[34],farmsoil[47],orcontaminatedgelatineprepara- tions[5].Obviously,representativesofcluster9arewidespreadin natureandrathernumerousinvariousenvironments.
Analysis of the 16S rRNA gene sequence of strain BoGlc83 showedthatthestrainiscloselyrelatedtootherdescribedspecies.
Eventhough,athresholdvalueof97%sequencesimilarityhasbeen commonlyusedsince20yearsforthedescriptionofnewspecies,a lessconservativethresholdvalueof98.7%waspostulatedinrecent years[28,38,39,55].The16SrRNAgenesequencesimilarityofstrain BoGlc83andthesequenceofB.thioparansis98.91%,thesequences of allother closely related and taxonomically described strains havea similaritybelowthethresholdvalueof98.7%.Therefore, DNA–DNAhybridizationexperimentsweredoneonlywithstrain BoGlc83andB.thioparans.ThedeterminedDNA–DNAsimilarityof maximally25.5%taxonomicallydifferentiatesstrainBoGlc83and B.thioparansandthusestablishesBacillusstrainBoGlc83asnew species.
DescriptionofBacillusstamsiisp.nov.
Bacillusstamsiispec.nov.(stam’si.i.N.L.gen.n.,honoringAlfons J.M.Stams,aDutchmicrobiologistwhohascontributedessentially toourunderstandingofsyntrophicmicrobialassociations).
Facultativelyaerobic,Gram-positivespore-formingbacterium.
Catalase-negative,oxidasepositiveafteraerobicgrowth.Cellsrod- shaped, 0.5×5m in size, with subterminal to terminal oval spores. Motile. Aerobic growth requires partly reduced sulfur sourcesforassimilation(sulfurflower,thiosulfate,sulfide,sulfite,
