Acetate Oxidation Is the Dominant Methanogenic Pathway from Acetate in the Absence of Methanosaetaceae† Dimitar Karakashev, Damien J

Acetate Oxidation Is the Dominant Methanogenic Pathway from Acetate in the Absence of Methanosaetaceae† Dimitar Karakashev, Damien J. Batstone, Eric Trably, and Irini Angelidaki*

Institute of Environment & Resources DTU, Technical University of Denmark, Building 113, DK-2800 Kgs. Lyngby, Denmark Downloaded from

Received 1 March 2006/Accepted 8 May 2006

The oxidation of acetate to hydrogen, and the subsequent conversion of hydrogen and carbon dioxide to methane, has been regarded largely as a niche mechanism occurring at high temperatures or under inhibitory conditions. In this study, 13 anaerobic reactors and sediment from a temperate anaerobic lake were surveyed for their dominant methanogenic population by using ﬂuorescent in situ hybridization and for the degree of acetate oxidation relative to aceticlastic conversion by using radiolabeled [2-14C]acetate in batch incubations.

When Methanosaetaceae were not present, acetate oxidation was the dominant methanogenic pathway. Aceti- http://aem.asm.org/ clastic conversion was observed only in the presence of Methanosaetaceae.

Acetate is the main precursor for methane production dur- carbon dioxide from [2-14C]acetate will be formed only during ing anaerobic digestion of organic matter. Two mechanisms for the oxidation of acetate. methane formation from acetate have been described. The ﬁrst The diversity of environments in which syntrophic acetate one is aceticlastic, being carried out by Methanosarcinaceae or oxidation has been found indicates it may also be important for Methanosaetaceae (2). Methanosarcinaceae generally have a commercial gas production in biogas reactors, digesting waste- higher acetate threshold but a higher growth rate and yield water sludge and manure. Aceticlastic activity has generally been on November 10, 2015 by University of Queensland Library than Methanosaetaceae (2). The second mechanism encom- considered to be the dominant pathway, with either Methano- passes a two-step reaction in which acetate is ﬁrst oxidized to sarcinaceae or Methanosaetaceae dominating (9, 15). If a sec-

H2 and CO2 and, with these products, subsequently converted ond pathway, such as acetate oxidation, dominates, it is nec- to methane (15). This reaction is performed by acetate-oxidiz- essary to re-evaluate reactor operation and optimization, ing bacteria (often Clostridium spp.) in a syntrophic association which are currently based on maintaining Methanosaetaceae with hydrogenotrophic methanogens (often Methanomicrobiales populations. The objective of this work was to assess the de- or Methanobacteriales) (4, 10, 12). gree of acetate oxidation relative to aceticlastic conversion in a Some important environmental factors influencing the rate wide range of industrial anaerobic digesters, fed with either of anaerobic aceticlastic activity are temperature, organic acid manure or wastewater sludge. A low-temperature environmen- concentrations, and ammonia concentration (9). At tempera- tal sample was also evaluated. tures between 50°C and 65°C, acetate oxidation is favored at Sampling. Thirteen Danish full-scale anaerobic digesters low acetate concentrations, while aceticlastic methanogenesis digesting manure together with waste from food industries is favored at high acetate concentrations (15). The dominance were sampled as described in reference 5. An anaerobic of acetate oxidation at lower concentrations increases with sediment sample was collected from a lake situated in Or- increased temperature. Syntrophic acetate oxidation is the holm (Sollerod municipality, Denmark) at a 0.2-m depth main mechanism for acetate degradation in the presence of with a gravity corer (6). inhibitors, particularly ammonium and volatile fatty acids Analysis of the samples. The samples were analyzed for (VFAs) (13). Syntrophic acetate oxidation has been reported VFAs and ammonia by standard methods (1). Microbial ecol- for natural anoxic environments in subtropical lake sediments ogy was evaluated with fluorescent in situ hybridization (FISH) at temperatures as low as 15°C (8). using established probes (see Table S1 in the supplemental It is relatively straightforward to detect acetate oxidation material) and a previously reported method (5). Methanogenic 14 14 activity by measuring the production of CH4 and CO2 from populations not identified by FISH were assessed using PCR- acetate labeled in the methyl group (C-2). When aceticlastic temporal temperature gradient gel electrophoresis (PCR- methanogens degrade acetate, the labeled methyl group will TGGE). form only labeled methane (2). During syntrophic acetate ox- Medium. Basal anaerobic medium was used for acetic oxi- idation, both carbon atoms of acetate are converted to carbon dation batch tests as described previously (5). The medium was dioxide, and some of the carbon dioxide is subsequently re- dispensed anaerobically under a N2/CO2 (80%:20%) head- duced to methane (13). Therefore, significant levels of labeled space in 100-ml incubation bottles, amended with labeled [2-14C]sodium acetate and nonlabeled sodium acetate. The

medium was reduced with Na2S·9H2O and supplemented * Corresponding author. Mailing address: Institute of Environment aseptically with a sterilely ﬁltered anaerobic vitamin solution as & Resources DTU, Technical University of Denmark, Building 113, described previously (5). After inoculation with raw sample, DK-2800 Kgs. Lyngby, Denmark. Phone: (45) 45251429. Fax: (45) 45932850. E-mail: [email protected]. the bottles were closed hermetically and incubated until meth- † Supplemental material for this article may be found at http://aem ane production ceased. This was considered the end of the test, .asm.org/. and analysis followed.

5138 VOL. 72, 2006 ACETATE OXIDATION DOMINATES WITHOUT METHANOSAETACEAE 5139 oeta 0 ftettlnme fmtaoei el acaarsodn oAC1) odmnn ehngn o1%o h oa ubro ehngnccls el n2 ed eecounted. were ﬁelds 20 in Cells cells. methanogenic of number total the of 10% to 1 methanogen, nondominant ARC915); to responding (archaea cells methanogenic of number total the of 90% than more Sample S romLk eiet4()4 B(X G C C(NO) MC MC) MG, (MX, MB 49 (P) 4 sediment Lake Orholm LS1 7Vge Manure Vegger M7 4SdytW lde3 M X(O X(O .30.50 0.13 (NO) MX (NO) MX 8 (M) 37 sludge WW Sydkyst S4 6Vester M6 3FkeW lde3 M X(O X(O .41.50 0.04 (NO) MX (NO) MX 6 (M) 37 sludge WW Fakse S3 2LntfeW lde3 M 0M N)M N)00 1.00 0.02 (NO) MX (NO) MX 10 (M) 37 sludge WW Lundtofte S2 5SusadManure Studsgard M5 4Fne Manure Fangel M4 5HligrW lde3 M X(B G X(O .60.84 0.06 (NO) MX MG) (MB, MX 6 (M) 37 sludge WW Helsingør S5 1Hleø Wsug 5()5M M)M N)0.13 (NO) MX (MS) MX 5 (T) 55 sludge WW Hillerød S1 3Lmi Manure Lemvig M3 8SnigMnr 5()8M N)M N)0.41 (NO) MS (NO) MS 8 (T) 55 Manure Sinding M8 2Hsø Manure Hashøj M2 1Nse aue3 M S(O G(S 2.7 (MS) MG (NO) MS 9 (M) 38 Manure Nysted M1 e d c b a Radioisotope analyses. The liquid and headspace of the bot- ID ctt eoa sgvna h aiu aeo eoa.Teln a hs a bevdbfr ctt removal. acetate before observed was phase lag long The removal. of rate maximum the as given is removal Acetate MS, 0C;M eohlc(5Ct 0C;T hrohlc( Ͼ 50°C). thermophilic T, 40°C); to (35°C mesophilic M, ( Ͻ 20°C); psychrophilic P, W wastewater. WW, tnaddvaini ae ntilct analysis. triplicate on based is deviation Standard tles were sparged with approximately 2 liters of N2 through a 5

14 14 MX, Methanosarcinaceae ; M NaOH trap to collect the CO2. The CH4 collected after 14

trapping was combusted to CO2 in a tube furnace at 800°C. Hjermitslev Reactor

14 name The CO2 generated in this furnace was then trapped in a carbon dioxide absorber for liquid scintillation counting (Car- bosorb-E; Packard Bioscience Company). Radioactivity mea-

surements of liquid samples were performed using a liquid Downloaded from Manure

scintillation counter (Tri-Carb 1600; PerkinElmer). type Feed

Simulation of methane production rates. A simple kinetic MB, Methanosaetaceae ; batch model, based on Monod kinetics with zero-order lag, for conversion of acetate to methane was implemented with a AQUASIM 2.1d (11). The maximum acetate removal rate and ep(°C) Temp 5(T) 55 7()1 S(NO) MS 16 (M) 37 2()1 nD lmns(O G(MS) MG (NO) ﬁlaments UnIDd 10 (T) 52 7()1 B(MC) MB 10 (M) 37 25()8M (NO) MS 8 (T) 52.5 7()1 nutrdarchaeon Uncultured 13 (M) 37

lag phase were estimated by ﬁtting measured cumulative meth- (type) ane to modeled cumulative methane. The Secant method, with b

an objective function of residual sum of squares, was used to ﬁt http://aem.asm.org/ MG, Methanobacteriales ; the data. An overview of the results from the acetate oxidation survey Incubation period (days)

experiment is given in Table 1. (NO) MS 8 Rates of methane production and acetate removal. Methane production rates varied considerably, with fast samples (such as Lundtofte and Hillerød) stopping methane production in 3 Q034(MC) DQ409324 AL .Rslsfo ctt xdto survey oxidation acetate from Results 1. TABLE eoeicbto fe incubation After incubation Before oiat(odmnn)methanogen (nondominant) Dominant

days and slow samples (e.g., Nysted and Vegger) requiring MC, Methanomicrobiales ; more than 10 days. The anaerobic lake sediment sample (Or- holm) had a lag phase of 31.5 Ϯ 0.8 days. Acetate removal on November 10, 2015 by University of Queensland Library rates also varied within a factor of approximately 10 (Table 1). These rates were higher (Ͼ4mM·dayϪ1) for cultures with a low degree of acetate oxidation than for cultures with a high S(NO) MS nutrdarchaeon Uncultured B(MC) MB B C(MS) MC MB, degree of acetate oxidation (acetate utilization rates lower archaeon Uncultured Ϫ Q036(MS) DQ409326 than 4 mM · day 1). Our rates compare with acetate removal (NO) DQ409325 O o bevd nD,uietﬁd(ybt IHadTG) oiatmethanogen, Dominant TGGE). and FISH both (by unidentiﬁed UnIDd, observed; not NO, Methanococcales ; rates in pure culture for mesophilic (12) and thermophilic (4) acetate-oxidizing cultures. Anaerobic acetate conversion pathways and environmental conditions. In all cases, populations dominated by Methano- c 14 14 Ͻ saetaceae had low levels of acetate oxidation ( CO2/ CH4

0.1), while populations dominated by other methanogenic liter HAc (g 0.77 0.22 1.81 2.33 0.6 Archaea and without Methanosaetaceae had high levels of acetate 1.9 VFA

14 14 0.01 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ͼ Ϯ oxidation ( CO2/ CH4 1) (Table 1). Results obtained .42.34 0.04 .64.4 0.06 .0 2.20 0.003 .24.5 0.12 .0 1.44 0.001 .12.6 0.01 .12.5 0.01 .74 0.07 clearly showed a strong correlation between the absence of 0.11 Ϫ 1 Methanosaetaceae and the involvement of the acetate oxidation d ) pathway. Other factors (e.g., source and inoculum tempera- gNliter N (g 5.6 ture) had no inﬂuence. Acetate cleavage has been generally Ammonia .31.4 0.03 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ regarded as a bimodal system, dominated by Methanosarci- Ϯ .73.5 0.07 .10.7 0.11 .0 4.8 0.001 .3710.065 7.1 0.03 0.09 .49.9 0.04 .01.5 0.10 .510 0.05 .85.3 0.08 .36.1 0.03 .6341.1 3.4 0.06 . 1.8 0.1 naceae at high acetate concentrations and by Methanosaetaceae 0.5 0.12 Ϫ 1 ee (mean Level at low acetate concentrations (2, 14). From the data presented ) here, we propose instead a different bimodal system in mixed ctt removal Acetate cultures, with aceticlastic methanogenesis in the presence of day (mM e e e 2.7 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Methanosaetaceae and acetate oxidation in their absence. The Ϯ Ϯ Ϯ Ϯ . 1.61 0.4 .31.9 0.03 . 0.08 0.6 . 0.11 0.5 . 2.2 0.1 . 0.025 0.7 . 2.45 0.6 . 2.75 0.1 0.4 0.2 0.1 absence of this methanogenic phylogenetic group has been of SD) Ϫ 1 previously investigated in the systems analyzed here and was ) linked to the presence of high ammonia and VFA levels (5). d : 14 2.11 Most probably, the high ammonia concentrations inhibit the 0.04 CO aceticlastic methanogens much more than the hydrogenotro- 1.4 2.9 2 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ / 14 .194 0.11 .892.2 0.18 .0 91 0.004 .0 98.1 0.003 .699 0.16 .497.4 0.14 .0 95.4 0.001 .0 97 0.007 .592.5 0.15 .6101 0.06 .197.5 0.11 phic methanogens, and methane is formed mainly by hydro- 92 0.002 96.2 0.09 0.18 CH gen-utilizing methanogens. This idea is supported by previous d 4 studies (3) indicating that acetate-utilizing methanogens are eoeyof Recovery

more sensitive to ammonia than are hydrogenotrophic methano- 14 96 (%) C Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ gens. The high degree of acetate oxidation in digested manure Ϯ 3.5 3.3 2.5 3 2 4.2 2 3.2 1 3.4 4 0.8 4.9 at high ammonia and VFA levels is also in agreement with 5.2 other results (13). However, a large potential for syntrophic d 5140 KARAKASHEV ET AL. APPL.ENVIRON.MICROBIOL. Downloaded from http://aem.asm.org/

FIG. 1. Distribution of the dominant methanogens observed in the samples as a function of the degree of acetate oxidation versus acetate removal rate. Error bars indicate standard deviations. MX, Methanosaetaceae; MS, Methanosarcinaceae; MB, Methanobacteriales; MG, Methano- microbiales; MC, Methanococcales; UnIDd, unidentified. acetate oxidation was also observed at low acetate concentra- known hydrogen consumers (Methanobacteriales, Methano- tions (in the Orholm sample). It is likely that inhibition or microbiales,orMethanococcales) or uncultured archaea other factors prevent growth of Methanosaetaceae and allow (samples M2 and M6). on November 10, 2015 by University of Queensland Library dominance by acetate oxidation by default. Methanogenic communities in several samples (M2 and M5 The bimodality of the system is also highlighted in Fig. 1, which before incubation and M2 and M6 after incubation) were not shows two distinct groups, with hydrogen-utilizing methanogens identified by FISH. This was due to the limitations of visual in (Methanobacteriales, Methanococcales, Methanomicrobiales, pos- situ hybridization. FISH is very convenient for the rapid anal- sibly Methanosarcinaceae, uncultured archaea [Hashøj and ysis of a large number of environmental samples but is limited Lemvig], and unidentified archaea [Studsgard]) in syntrophic when carried beyond the limits of oligonucleotide probes. cooperation with acetate oxidizers at low maximum acetate ARC915 is an effective general probe, and order-level probes removal rates and the strict aceticlastic methanogen Methano- have been used in a wide range of systems; however, in com- saetaceae at maximum acetate removal rates. The presence of plicated systems, such as manure, they might fail to detect all Methanosarcinaceae as a hydrogen-utilizing syntrophic partner methanogens. Therefore, unidentified methanogens were phy- in the acetate-oxidizing cultures is not surprising. In contrast to logenetically characterized by PCR-TGGE. Samples not iden- the Methanosaetaceae species, which are strict aceticlastic tified by FISH (e.g., M5, Studsgard before inoculation) were methanogens, most Methanosarcinaceae species are mixotro- found by PCR-TGGE to be far outside known phylogenetic phic, utilizing not only acetate but also hydrogen and carbon groupings for methanogens. It is likely that these microbes are dioxide, methanol, and methylamines (2). In addition, Methano- still methanogens, since bacterial methanogenesis is unknown. sarcinaceae are capable themselves of acetate oxidation (7) and These unknown microbial groups are interesting scientifically could be therefore be mediating the entire process of acetate and deserve further investigation. oxidation, and subsequent methanogenesis, rather than acting Nucleotide sequence accession numbers. Sequence data for as an acetate sink via aceticlastic reaction. microbes identified in this study have been submitted to the Methanogenic populations. The FISH observations showed GenBank database under accession numbers DQ409324 to that dominant methanogenic populations (see Figure S1 in the DQ409326. supplemental material) of wastewater sludge samples were consistently Methanosaetaceae, as previously reported (5), We thank Lene Kirstejn Jensen, Birthe Ebert, and Hector Garcia for while manure samples were phylogenetically more diverse. In technical assistance with the experiments. every case, dominance of specific groups as observed by FISH This work was supported by a Danish Government Scholarship and the Danish Research Programme (EFP 05). was clear, and they constituted more than 90% of the archaeal population, as described previously (5). Diversity in subdomi- REFERENCES nant methanogens was limited, except in the Orholm sample 1. American Public Health Association. 1985. Standard methods for the exam- ination of waste and wastewater. American Public Health Association, (sediment sample), where archaea belonging to Methanosaeta- Washington, D.C. ceae, Methanomicrobiales, and Methanococcales were ob- 2. Ferry, J. 1993. Fermentation of acetate, p. 305–334. In J. G. Ferry (ed.), served. Methanogenic population changes were observed Methanogenesis. Ecology, physiology, biochemistry and genetics. Chapman and Hall, New York, N.Y. during growth on acetate in the incubations. For the samples 3. Garcia, J. L., B. K. C. Patel, and B. Ollivier. 2000. Taxonomic, phylogenetic, in which Methanosaetaceae were dominant, the only change and ecological diversity of methanogenic Archaea. Anaerobe 6:205–226. 4. Hattori, S., Y. Kamagata, S. Hanada, and H. Shoun. 2000. Thermacetoge- observed during incubation was the elimination of subdomi- nium phaeum gen. nov., sp. nov., a strictly anaerobic, thermophilic, syntro- nant populations. In the other samples, there was a shift to phic acetate-oxidizing bacterium. Int. J. Syst. Evol. Microb. 50:1601–1604. VOL. 72, 2006 ACETATE OXIDATION DOMINATES WITHOUT METHANOSAETACEAE 5141

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