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Production of Methane and Acetate from Five Varied Sediments when

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While usinç tatheno! as the aor Substrate.

Dan SmithWoods Rote rcrob1e1 DIversIty ProJect

Summer, 1991

Introduction:In a varied number of inocfle, whet would be the predomInant end product

when incuoted under normal conCftiocs which promoteacetogen/methanogen growth If methanol is tsed as the major substrate?ft is inferred that when using the alternative methoxyleted aromaticcompound TMO as a substrete which promotes the growth of acetogens(methanogens cannot use TMB due to theIr ftabiflty to cleave thephs;lether bond) (8) that methanol Is produced wiIch ft a known substratefor both acetogecs and methenogens (2,5). The question arises, therefore,as to which metabolic rout would be favored if the THU were bypassed endmethanol supplied directly. 8u using BC to measure methane production endHPLC to measure acetate formation the relative competitIve advantages ofmethanogens or ecetogens may be revealed.

Method: —Each sample was fficculated into three methanogen/acetogenmedium vi ci s

1) control forarimgnzousn,ethane/acetate concentrations Head spaceof nitrogen end carbon dioxide, no substrate added.

2) Control formethaneproduction: Heed space with Hydrogen end carbondioxide as the onlu substrate.

3) Expentasotat Contains 4mM Methanol as sole substrate, head spaceof nitrogen and carbon dioxide.* Media recipe from course hand out end fo!low!ng strict anaerobictechnique.1.0 mIlliliter of s)urrj sediment from five locations was Inoculated Intomethanogen vials containing 2Onil medium and subjected to the conditionslisted above. Samples included:—Sippewissit Salt Mars,**—Falmouth Pond—Non—aerated Sludge—Ceder Swamp—bike Path ?ond** NeC was added to a flne concentraticn of 2% to eproxUnate set weter.

A sampes wars then ?‘cetec! at 30D : ft ta dtic. The sempes wicconZe1ted ro;r er ceron dIoxIde In te ee ssa:e were IncueteC ontheir sides In order to provide an extensive surface fcr ges exchance. Attwo day intervals, samples from the head space from each vial were testedfor the presence of methane bu tising gas chromatography. As a control, 5%methane was lnected to ascertaIn the retention tIme of methane as weB asthe area essociatec wIts 5% methane. 1.0 m of inoc.oteC media was 8150removed and stbjected to HPLC analysIs In order to observe acetateproduction at simIlar time intervals. As a control, 10mM Na—acetate stocksolution was run in order to reveal the retention tme and the areaassociated with 10mM acetate. To confer the identification of acetate as apeak, the unknown sample was again run with an acetate spike” andmonitored for an incre9se in the area of the putative acetate peak. (Note:Due to mechanical faults with the Waters HPLC, only two runs during thetime course were cbtained end will be presented In the data section.)

Data: See accompanying data sheets.

Results:Methane Production from Non—aerated Sludoe

The contro Ior endogenous methane which could be present In the sampleshowed that over a thirteen day course, no significant levels of methanewere present and so no subtraction of “background methane wasnecessitated. Initially, the hydrogen/carbon dioxide substrata vial showedthe highest percentage of methane and steadily increased over the thirteenday period until reachIng 7%. The rate of methane production wasapproximatey 0.4%/day and so seems fairly slow. Negative pressure In thevial was noted on day 5 of the experiment end continued there after. Thisindicated the utilization of hydrogen and carbon dioxide from the head spacepresumabig for methane production. The experimental ia with 4mMmethanol as a substrate showed a higher rate of methane production as wellas a higher fine! concentration when the experiment was terminated. Alarge pcsttive pressure was noted on day 9 and continued. This wasindIcative of the production of methane in the head space of the vial with enaccompanying increase in the pressure. The rather sudden decrease inpercentage of methane from day 9 to 11 may be the result of experimentalerror caused by inappropriate use of the GC (the same “dip” is seen from theFelmouth Pond Sample) or coud represent a succession of differentmethenogans. An early methancgen coW have been nibttsd by !ts ownwaste proCucts and a second consortium of methanogens not affected coudhave then te<en over methane production. This is purely speculation on thepart of the investigator and seems unlikely &nce if thIs were the case, onewouid expect to see a leg in methane production. Dn .he ccntrar, en ag 13,the methane production level was epproxmetel at Pt percentage of da 9.

The average rots of methane prcthction (not incWdirg the “dipi was./oey wmcn s a suostenuai increase wnen comparsa to metonogenes:susing hydrogen end carbon dioxide as the substrate.

Nethans Production from ?eimouth PondExcept for the toto’ percentages of meThane produced end a “dip” at

day 9 recorded for the hydrogen/carbon dioxide sample, these date arecomparable to the s;ud ge data. Again, no background was recorded endmethanol seemed to be the preferred substrate for more production ofmethane. The rates of methane production for hydrogen/carbon dioxideand methanol as substrates (not including “dips”) were 0.7%/day end1.6%/day respectively. Analogous pressures were observed in thesamples as those seen in the non—aerated sludge samples.

Methane Production from Ceder SwemoThe results from these data are interesting in several respects. Prodtctionof methane was expected to be quite high from this sample since methaneproduction is well documented from this site. The greatest percentage ofmethane, however, was only 4.7% in the hydrogen/carbon dioxide vial whichturned out to be the least methane produced over the thirteen days of oH thesamples. interesting too is that there was essentially no methane producedvia the utflizotion of methanol. The only methane produced was that fromhydrogen/carbon dioxide. This could reflect several interesting aspects ofthis sample. One could be that the mathenogens from the swamp ore acompletely different type which cannot utilize methanol. More likely,however, is that environmental conditions such as pH inhibit the use ofmethanol and therefore hydrogen/carbon dioxide is utilized preferentiafly.Since pH was not investigated during this experiment, it would beinteresting to find out the differences between the Ceder Swamp sample endthe other samples tested. The average rate of methenogenesis for thehydrogen/carbon dioxide substrate sample was 0.92%/day. Similar pressureobservation were observed as above, however, no positive pressure was seenin the methanol substrate vie].

Methane Production from the Sippewissit Salt MarshAgain, no background methane was detected in the sample and moreinteresting resuUs were obtained. The total production of methane is fairlylow end initial rates were slow. However, after dog 9, the production rateincreased and methane comparable to that seen in the other samples wasrecorded. (This is an interestng note, since methanogens from brackishsediments are not well studied or documented) (I). The methane productionon the two substrates seem essentially identical making this sample uniquein that it was the only sample to exhibit such a phenomenon. The rate of

nethenogsness frc :c 3 to 9 was O.2%/-c end ircrsessd to rcnC!!/iç thrs e:tr under oorCtios of utflzr5 both substrates. Xegetveressure in ; droen/certor dioxide was noted onij after d&! 9 as ws thepositive pressure from the methanol vial.

Niethene Pr2duction from Elks Path cndHydrogen/carbon dioxide was again utilized well (O.5Vdag)but the profileof methanogenisis under methanol utilization was quite different. Therewas a leo time of nine deus before any methane was detected and thereaftera large increse in mehee production was noted 1 4Vdeg) This suddenincrease ifl tue rae of meLnenogeness wme using metnenol as tnesubstrate is interesting end may reflect the synthesis of new enzymes f orthe metabolism of methanol although this seem unlikely due to the length oftime of the leg. A satisfactory explanation of this phenomenon has not beenfound as of this writing.

Acetate Production From Sediment SamQJflcetete croauction was Getectea In each sample en interesUng patterns ofacetate production evolved. Firstly, the Sludge, Falmouth Pond end CedarSwamp samples all contained relatively equal amounts of acetate where asthe Bike Path Pond samples formed about twice as much acetate (7.7 mM vs.3.2, 3.5 end 2.6 mM respectively). The Sippewissit sample formed only asmell amount of acetate (0.53mM) which could suggest either the lack oflarge numbers of acetogens or ecetogens being out competed for substratealmost to the point of being excluded. The second interesting pattern whichdeveloped 15 that there was acetate formed, without exception, only by thehydrogen/carbon dioxide substrate samples and none detected in themethanol samples. This suggests that the onginel question as to wmch typeof organism would use methono’ more efficiently end hence produce endproducts has been answered and that methanol is easily used formethenogenesis end not easily used by ecetogens for acetogenesis. Theseresults also suggest that using hydrogen/carbon dioxide as a substratewithout any other selective pressures will yield both methanogenesis andecs:ogenes:s at least in the five sample tpes chosen n this experiment.Also without exception, no acetate was detected in samples incubated withno substrate which iflustrates that there was essentially no backgroundacetate to cloud results of this experiment much as that seen in themethane production samples.

Dscsson/Conc!usiunThe results of this experiment answer the original question; namely: WIUmethanogenesis a— acetogeness be the prevalent metabcllc route whenmethanol is used as the substrate or will there be a mixture thereof? LThder

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the conditions used end in the y’9 sedjmer;ts studied methonogers outoompee eno uuizs rre:’ano: wrre as no acewte was oeteztsc sugest:ngtot ecetcgens were out competed for this substrate. One could rcua thatsince no ecetogenec control was used (T13 as en e;I.ernetive substrate) thatthe results ore not conclusive. However, since, without exception, acetatewas detEcted in the h.yCrccsn/ceion dioxide vials (3—7mM) ecetogens wereindeed present end meteboizing. (it should be ncted, however, that it iscommon to see 30—50mM acetate production in pure ocetogen cultures (5)Snce no acetate was seen under methanol utilizing conritions these resultsseem reasonable. Such results hove also been reported else where 0,8).When methanol was used, methenogens were invariably isolated. Due to thisobservation and considering that bath methenogens and ecetogens grow wellon methanol the demethylation of en alternative substrate such as TMO bçecetocens to methanol must be a sequestered event whereby methanol ismode but remains internalized end utilized solely by the ccstcgen. (Theactual demethylation to methanol ma be similar to initial events inmethanol utilization bg methenogens) (7). Otherwise, by the resultsreported here, if methanol was reeesed into the media it would beefficiently utilized by methanogens which would thus exclude acetogenesis.The results reported here, therefore, coincide with previous notions thatalternative substrate demethylation to methanol by acetogens is an internalevent.

Several interesting aspects of methane production in sediments utilizingmethanol were also seen. Methanol utilizing methanogens seemed moreefficient in 3 out of 5 cases than hydrogen/carbon dioxide utilizingmethenogens (Sludge, Fel mouth, Dike Path). This suggests that highermethers production from a given sediment may be expected using methanolrather than hydrogen/carbon dioxide. This is unexpected since the energyyield of methanol is chemically less (5). Interesting too ore the resullsfrom ceder swamp suggesting that methanol was rot utilized what so ever.The question as to what the differences are between this sample end theothers remains unanswered. There are some methenogens such as&yontllend #ethonoèectsr/um thermaoutotrophicum(9) which utilizehydrogen/carbon dioxide exclusively and such related methanogens may be inabundance in this sample due to a oH optimum, temperature or consortiumconsiderations. t would be interesting to physically characterize thesevarious sediments and to tru and discover a difference which could accountfor the lack of methanol utilization by methenogens. A lest interestingobservation was the unexpected amount of methane produced from the saltmers sempe since this enyrcnmert corta!ns a high concentration ofsulfate recuction (2,3). Sulfate reduction is a kncwn inhibitor ofmethenogeresis since sulfate reducers can out compete methenogens icrhydrogen (3). Also supportina this finding is that methane production

increases when s.1fete retuoticn is inhibited using mchdete (2). :nretrzs2ect, this ProJect answered the oigina1 uesticn and spc•rtedseyer& other ftvastigatcrs observetions regarding the possible ivolvemsnt

Q of methanol in the biochemistr of ecetogens when utflizing methoxletedaromatic acids such as Th8. there is methanol in an environment, whetheradded or as a b product of m•etobol]sm, it will inveriab be utilized bmethanogans for methane production to the excusion of aceto9enesis.

er9nces:1)Drelmend, (1982) App thy /YicroIz43:4522) Love1 at. l, (1983) $pa Er,y /‘/icralz 45:107-1923) Ward etel.j1985) Ad Aquet flicroh3l4I—1794) JHoc/er(1963) 155: 1206-12165) App Lw M&o(1953)45: 1310-13156)Jfloct(1983) 153: 1415-14237) Arch MfcriW(1904) 136: 360-3548) Uache, . (1961) Arch tllcrotz 130: 255-2619) Zehnder, C 988) 3iolou of Anaerobic Microorganisms. Ch. 13fiiochemistnjaffletheneProoucticnYooels at. 81, John wileu an Sons nc.

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