Bioaugmentation of Syntrophic Acetate-Oxidizing Culture in BiogasReactors Exposed to Increasing Levels of Ammonia

Maria Westerholm,a Lotta Levén,a,b and Anna Schnürera

Uppsala Biocenter, Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden,a and Institute of Agricultural and EnvironmentalEngineering, Uppsala, Swedenb

The importance of syntrophic acetate oxidation for process stability in methanogenic systems operating at high ammonia con-centrations has previously been emphasized. In this study we investigated bioaugmentation of syntrophic acetate-oxidizing(SAO) cultures as a possible method for decreasing the adaptation period of biogas reactors operating at gradually increased am-monia concentrations (1.5 to 11 g NH4

�-N/liter). Whole stillage and cattle manure were codigested semicontinuously for about460 days in four mesophilic anaerobic laboratory-scale reactors, and a fixed volume of SAO culture was added daily to two of thereactors. Reactor performance was evaluated in terms of biogas productivity, methane content, pH, alkalinity, and volatile fattyacid (VFA) content. The decomposition pathway of acetate was analyzed by isotopic tracer experiments, and population dynam-ics were monitored by quantitative PCR analyses. A shift in dominance from aceticlastic methanogenesis to SAO occurred simul-taneously in all reactors, indicating no influence by bioaugmentation on the prevailing pathway. Higher abundances of Clostrid-ium ultunense and Tepidanaerobacter acetatoxydans were associated with bioaugmentation, but no influence onSyntrophaceticus schinkii or the methanogenic population was distinguished. Overloading or accumulation of VFA did notcause notable dynamic effects on the population. Instead, the ammonia concentration had a substantial impact on the abun-dance level of the microorganisms surveyed. The addition of SAO culture did not affect process performance or stability againstammonia inhibition, and all four reactors deteriorated at high ammonia concentrations. Consequently, these findings furtherdemonstrate the strong influence of ammonia on the methane-producing consortia and on the representative methanizationpathway in mesophilic biogas reactors.

Biogas has promising potential as a substitute for fossil fuel andcould contribute to the reduction of greenhouse gas emissions

and global warming. Methane is the energy-rich component ofbiogas and is formed as the end product during anaerobic degra-dation of organic material. In addition to valuable production ofrenewable energy, anaerobic degradation also represents a suit-able method for waste and wastewater treatment and may bringindirect environmental benefits such as reduced spontaneousemissions of ammonia and methane otherwise occurring duringcomposting or storage of untreated animal manure (7, 8). Fur-thermore, utilization of the residue as a crop fertilizer contributesto recirculation of nutrients, lowering the use of additional min-eral nitrogen fertilizers (5). Among many different materials thatcan be used for biogas production, protein-rich materials such asslaughterhouse waste, animal manure, and distiller’s waste arehighly interesting due to their high methane potential. Further-more, the digestion residues contain large amounts of plant-avail-able ammonia, thus representing a valuable fertilizer. However,the high content of ammonia is a concern due to its associationwith unstable process performance and increased risk of processfailure (10). The potential inhibitory effects on the biogas-pro-ducing consortia and in particular the acetate-utilizing methano-gens are considered to be the main causes of decline in processperformance (21, 35).

In several studies, investigators have considered strategies forimproving process operations with protein materials, e.g., dilu-tion of substrate, use of additives, change of operational temper-ature, etc. (2, 3, 14, 20). In addition, acclimatization has beenreported to increase tolerance and retain microbial viability atammonia concentrations far exceeding the initial inhibitory con-centrations (4, 21, 22, 43). One possible explanation for this accli-

matization effect is enhanced growth of ammonia-tolerant micro-organisms able to produce methane through syntrophic acetateoxidation (SAO) (39). Methanogenesis through SAO involves atwo-step reaction consisting of acetate oxidation to hydrogen andcarbon dioxide by syntrophic acetate-oxidizing bacteria (SAOB),followed by conversion of these products to methane by hydro-gen-utilizing methanogens (44). In addition to elevated levels ofammonia, high operation temperatures, high acetate concentra-tions, long retention times, and absence of the aceticlastic Metha-nosaetaceae are factors suggested to have an impact on the dy-namic transition from aceticlastic methanogenesis to SAO (1, 15,18, 26, 34).

The conversion from aceticlastic methanogenesis to SAO maycomprise a decline in gas production and methane yield (31).However, as the shift may involve development of an ammonia-tolerant biogas-producing community, the process can continueeven at ammonia levels reported as being inhibitory for the aceti-clastic methanogens (42).

Bioaugmentation, in terms of adding specific microorganisms orenriched consortia to anaerobic processes to enhance a desired activ-ity, has been reported to lead to improvements in degradation ofspecific organic compounds (9, 13, 16), such as cellulose-containingbiomass (6) and manure (25), start-up of new reactors (28), odor

Received 28 May 2012 Accepted 7 August 2012

Published ahead of print 24 August 2012

Address correspondence to Anna Schnürer, anna.schnurer@slu.se.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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reduction (12, 38), and recovery after organic overload (37) and tox-icant exposure (30) at a laboratory scale. Furthermore, improved re-actor performance in terms of increased methane production anddecreased accumulation of volatile fatty acids has been observed afterthe addition of hydrogen-utilizing methanogens to mesophilic (26 to35°C) reactors degrading distillery wastewater (29). To our knowl-edge, bioaugmentation with the intention to improve reactor opera-tion at high ammonia concentrations has not been assessed previ-ously.

Thus, the aim of this study was to examine bioaugmentation ofthe natural biogas-producing consortia with syntrophic acetate oxi-dizers as a method for decreasing the adaptation period of biogasreactors operating at gradually increased ammonia concentrations.The influence of bioaugmentation and potential associations be-tween the acetate conversion pathway, microbial abundance dynam-ics, and operational parameters and process performances were ana-lyzed.

MATERIALS AND METHODSCulture used for bioaugmentation. The syntrophic acetate-oxidizing(SAO) cultures used for bioaugmentation included SAOB isolated at theDepartment of Microbiology, Swedish University of Agricultural Sci-ences. These were Clostridium ultunense sp. Esp JCM16670 (41), Syn-trophaceticus schinkii JCM16669 (41), Tepidanaerobacter acetatoxydansDSM 21804 (42), and the hydrogen-utilizing methanogen Methanoculleussp. strain MAB1 (33). Cultivation was conducted in modified bicarbon-ate-buffered basal medium (41) containing 0.2 M NH4Cl and 100 mMacetate, added on two occasions. Complete degradation of acetate wasconfirmed before addition to the reactors.

The average gene abundances of the SAOB in the SAO culture weredetermined to be about 2.5 � 0.3 � 107, 3 � 0.5 � 109, and 2.7 � 1.1 �1010 per ml for C. ultunense, S. schinkii, and T. acetatoxydans, respectively.

Reactor operation and substrate. Four identical laboratory-scalecontinuously stirred tank reactors (Belach Bioteknik, Stockholm, Swe-den), designated E1, E2, R1, and R2 (E, experimental; R, reference) andwith a working volume of 5 liters, were operated for 458 days under meso-philic conditions (37°C). The reactors operated semicontinuously andwere fed 6 days a week with a mixture of cattle manure and whole stillageproduced from fermentation of cereals at an ethanol production plant.The inoculum used for setting up the processes, the origin and collectionof substrates, and the total solids (TS), volatile solids (VS), carbon/nitro-gen (C/N) ratios, chemical characteristics, and methane potentials of thewhole stillage and the manure are specified by Westerholm et al. (40). Thesubstrate mixtures varied during the experimental period and consisted of16% manure and 84% whole stillage (based on total VS) during the first375 days. Between day 375 and 409, some of the whole stillage was re-placed with egg albumin powder (Källbergs Industries, Sweden). The re-sulting substrate mixture consisted of 16% manure, 59% whole stillage,and 25% egg albumin (based on total VS). In the final operating period,i.e., from day 410, whole stillage was completely omitted from the sub-strate and exchanged for egg albumin. At this stage the mixture consistedof 16% manure and 84% egg albumin based on total VS. Reactor perfor-mance was monitored regularly. Measurements of volumes and compo-sitions of the gases produced were determined daily, whereas pH wasmonitored once a week. Volatile fatty acid (VFA) concentrations, bicar-bonate alkalinity, and ammonium-nitrogen were measured continuously.Initially, the reactors operated at a hydraulic retention time (HRT) of 57days and an organic loading rate (OLR) of 0.8 g VS/day for about 150 days,but later the operation was changed in order to increase the ammoniumlevel in the reactors (Fig. 1).

Bioaugmentation of experimental reactors E1 and E2 with the SAOcultures was initiated on day 150, concurrently with the gradual increasein loading rates. A 10-ml portion of the culture was added daily immedi-ately before addition of the substrate. In order to compensate for any

improvement in process performance due to the addition of mediumcomponents to the experimental reactors, 10 ml of sterile medium wasadded to the reference reactors R1 and R2.

Analytical and tracer analyses. Analyses of the VFA composition, TS,VS, C/N ratio, bicarbonate alkalinity, trace element concentrations, totalgas production, and the methane and carbon dioxide content of the gaseswere performed as described previously (40). Ammonium-nitrogen wasanalyzed as described by Westerholm et al. (40) or by Tekniska Verken iLinköping AB according to FOSS Tecator application sub note 3502 witha Kjeltec 8200 auto distillation unit (FOSS, Scandinavia, Sweden). Thepathway of acetate degradation to methane was determined by traceranalysis involving incubation of reactor sludge (20 ml) with [2-14C]ac-etate (final concentration, 0.11 �Ci/ml) and monitoring of labeled gasesby scintillation counting as described by Schnürer and Nordberg (31). A14CO2/14CH4 ratio above 1 indicates dominance of syntrophic acetateoxidation, while aceticlastic methanogenesis is the main pathway at ratiosbelow 1 (31).

Molecular analysis. Samples for molecular analyses were withdrawnfrom the reactors at different time points throughout the operating periodand stored at �20°C until analysis. Extraction of total genomic DNA,construction of DNA standards, and performance of the quantitative PCR(qPCR) analysis were conducted as reported elsewhere (39). For the quan-tification of Thermacetogenium phaeum, the primer pair TH795f/985r wasused (L. Sun, B. Müller, M. Westerholm, and A. Schnürer, unpublisheddata). To target the methanogen MAB species, the primers MAB62f (5=-GGAATGCCCTGTAATCCAAA-3=) and MAB301r (5=-CACCTGAACAGCCTGCATT-3=) were developed as described by Westerholm et al. (39).This primer pair targeted 16S rRNA genes of Methanoculleus bourgensisand Methanoculleus sp. strains MAB1, MAB2, MAB3, and BA1 (33). Tocontrol the specificity, the new primers were used in PCR with genomicDNA from the closely related methanogens Methanoculleus submarinusDSM 15122, Methanoculleus palmolei DSM 4273, and Methanoculleus chi-kugoensis DSM 13459. The quantifications of Methanomicrobiales, Metha-nobacteriales, Methanosaetaceae, and Methanosarcinaceae were conductedwith the primer sets and qPCR protocols assigned by Westerholm et al.(40). The mean similarities of gene abundances were compared usingStudent’s t test, and a P value of �0.05 was regarded as indicating a sta-tistically significant difference. Triplicate samples from each reactor andsampling point were included in the analyses.

RESULTS AND DISCUSSIONReactor performance. According to the chemical analyses, all thereactors operated with similar performances throughout the op-erating period (Fig. 1, 2, and 3 and Table 1). The performanceswere initially stable (days 0 to 150) but later involved two instabil-ity periods (around days 230 to 300 and days 400 to 458). In theinitial operational period (�150 days), the four reactors operatedat an OLR of 0.8 g VS/day and an HRT of 57 days (Fig. 1). The gasproduced was composed of about 61 to 66% CH4, the specific

FIG 1 OLR (A) and HRT (B) of the reference (R) and experimental (E)reactors throughout the operation period of about 460 days.

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methane production varied between 285 and 343 ml CH4/g VS,and the alkalinity and the pH in the digester sludge varied between7.5 and 10 g CaCO3/liter and 7.5 to 7.8, respectively. Ammonium-nitrogen was found to be 1.5 g NH4

�-N/liter (107 mM) in all thereactors. The VFA levels were low, illustrating that the perfor-mance was stable (Fig. 3). A gradual increase in OLR and simul-taneous decrease in HRT, starting at day 150, to 3.6 g VS/day and26 days, initially resulted in an increased gas production but laterin reactor instability, as indicated by a rapid decrease in methaneyield (Fig. 2) and pH (to about 6.5 to 7) and increased VFA(Fig. 3). The operational instability was most likely caused by theincrease in OLR and decrease in HRT, combined with the rela-tively high concentration of ammonia-nitrogen (about 3.0 gNH4

�-N/liter; 214 mM). By decreasing the OLR during the fol-lowing period (days 244 to 305), it was possible to stabilize thereactor operation, as indicated by a reduction in the acetic acidconcentration and a recovery of the methane yield. However, thelevels of propionic acid did not decrease and at day 301 concen-trations of about 50 to 60 mM and 4 to 26 mM were established inthe experimental and reference reactors, respectively. After 305days of operation OLR was once again increased, but at this pointwith extended sludge retention time (HRT 41 to 45 days). Thelonger HRT increased the degree of mineralization, which wasreflected in increased ammonium-nitrogen concentrations (Table1). After the increase in OLR, the specific methane yield stabilizedat lower levels than those during the initial period and variedbetween 164 and 204 ml CH4/g VS between days 367 and 400. Achange in feed composition, with retained OLR, performed byexchanging part of the whole stillage for egg albumin powder (day375) and by the use of manure and egg albumin powder as the sole

substrates (from day 410) further increased the ammonium-ni-trogen content in the reactors, which reached levels around 11 gNH4

�-N/liter (790 mM) at day 449. This operation resulted in acomplete failure of the process, with decreasing methane yield andincreasing VFA levels. At this point, the high ammonia level wasthe likely factor behind the instability, especially since the reactorsoperated with comparatively lower OLR and longer HRT. Thisresult is in line with several previous studies illustrating processinstability as a consequence of increasing ammonia levels (10).The acetic acid concentrations in the reactors were relatively stablebetween days 310 and 392, 11 to 61 mM, but rapidly increased to247 to 274 mM at the final sampling point. In contrast to aceticacid concentrations, the propionic acid concentrations showed notendency toward decrement between these two instability periods.Instead, the concentrations continued to increase and finallyreached relatively high levels at day 442 (120 to 136 mM). Worthconsidering in this regard is that during this period, higher levelsof propionic acid were reached earlier in the experimental reactorsthan in the reference reactors.

Methanogenic pathway. The tracer analyses showed relativelysimilar results for all four reactors, and the labeling recovery onthe different sampling occasions was between 93 and 110%. At day138, the average 14CO2/14CH4 ratio in the reactors was 1.2 � 0.3,indicating activities of both SAO and aceticlastic methanogenesisin the operational phase preceding the first increase in OLR. Thepresence of SAO at this stage was somewhat surprising due to thelow levels of ammonia and acetate but could possibly be explainedby the prevailing long HRT (57 days). This result confirms claimsby Shigematsu et al. (34) that SAO is the primary pathway at lowdilution rates, whereas the aceticlastic pathway dominates athigher dilution rates. In agreement with these results, aceticlasticmethanogenesis in the present study became dominant whenHRT decreased, which was seen at day 241 when a 14CO2/14CH4

ratio of 0.3 � 0.1 was determined. One possible explanation forthese findings is that a relatively short HRT constrains the growthand activity of mesophilic SAOB. At day 374, the monitored ratioswere distinctly higher, 1.8 � 0.4, reflecting a change in the primarypathway for acetate degradation to SAO in both the experimentaland reference reactors. This shift in pathways confirms findings ina previous study in which SAO was activated at ammonia levelsexceeding 3 g NH4

�-N/liter (31). The inhibition of aceticlasticmethanogens by ammonia probably gives ammonia-tolerant syn-trophic acetate oxidizers a competitive advantage (32, 41, 42).Otherwise, these organisms are considered to be less competitivefor acetate than the aceticlastic methanogens (27).

As the results were equal in all digesters, the bioaugmentation

FIG 2 Methane yield of the reference and experimental reactors.

FIG 3 Concentrations of acetic acid (left) and propionic acid (right) in thereference and experimental reactors.

TABLE 1 Average ammonium-nitrogen concentrations in the referenceand experimental reactors

Day of operatingperiod NH4

� (mM) NH3 (mM)

76 107 � 3 4.1 � 0.4170 76.5 � 0.4 2.9 � 0.3185 170 � 4 8.5 � 0.2245 214 � 9 11 � 2295 236 � 12 9.7 � 3350 278 � 6 19 � 9385 336 � 5 20 � 2449 790 � 48 30 � 10

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of the SAO cultures in the biogas reactors seemed to have noinfluence on the dominant pathway for acetate degradation.

Molecular analysis. All standard dilution series provided highcorrelation coefficients (�0.99), similar calibration slopes (be-tween �3.3 and �4.0), and qPCR efficiencies above 86%. Thequantitative values of 16S rRNA genes affiliated with the SAOBand the methanogens are displayed in Fig. 4.

Abundances of SAOB. The gene abundances of the differentSAOB were below the detection limits in the substrates used forfeeding the reactors, and T. phaeum was not detected in any of thereactor samples. The reason for the absence or low abundance ofT. phaeum is probably that the mesophilic conditions in the reac-tor did not support this thermophilic bacterium, which requires atemperature range of 40 to 65°C for growth.

The average gene abundance of S. schinkii was initially (days 0to 266) between 0.6 � 107 and 1.9 � 108/ml but increased to 6.1 �108 to 1.4 � 1010/ml after about 300 days of operation. The rela-tively similar abundances in both the reference (R) and experi-mental (E) reactors throughout the operational period illustratedthat at this level this species was evidently unaffected by bioaug-mentation. In contrast, the quantitative assessment of SAOB inthe reactors established distinctly higher levels of C. ultunense andT. acetatoxydans in the experimental reactors than in the reference

reactors in the operational phase following bioaugmentation (day150 to 400) (Fig. 4B and C). The C. ultunense-related species werenot abundant above the detection limits in the reference reactorsbefore day 250 (except in R1 at day 153), but were prominent inreactors E1 and E2 just after the initiation of bioaugmentation(�150 days of operation), with average gene abundances around0.7 to 1.4 � 105/ml. In the reference reactors, C. ultunense wasdetected first at day 301 (average gene abundance, 4.7 � 102/ml)in R1 and day 392 (average gene abundance, 1.3 � 106/ml) in R2.In the experimental reactors, T. acetatoxydans had already beendistinguished before bioaugmentation was initiated (average geneabundance, 1.5 � 104 to 2.0 � 106/ml) but was not detected in R1and R2. However, the average gene abundances increased signifi-cantly (P � 0.05) in the E1 and E2 reactors after day 212 and thenvaried between 1.1 � 105 and 1.2 � 108/ml. In R1 and R2, thespecies was first detected only after 300 days of operation. Thesefindings indicate that bioaugmentation might be consideredsuccessful for C. ultunense and T. acetatoxydans. However, con-sidering the expected theoretical levels of SAOB, based on theamount of added microorganisms and the dilution rates, itappears that the organisms did not become established andgrow in the process during the first 250 days of operation. Thelevels from the addition were lower than expected, possiblysuggesting that the bacteria were broken down to some degree.

Remarkably, a significant increase (P � 0.05) in genes affiliatedwith all mesophilic and thermotolerant SAOB occurred in all thereactors, both the experimental and the reference reactors, afterapproximately 250 to 300 days of operation, i.e., during the firstinstability period. At this stage of the operation, the ammonium-nitrogen concentration had reached 3 g NH4

�-N/liter (215 mM),a level previously shown to induce increased numbers of SAOB(39). The selective pressure of ammonia in combination with thelower HRT at this time probably allowed the establishment of therelatively slow-growing SAOB. The introduction of SAO was alsofurther supported by the tracer analysis, illustrating dominance ofSAO after 374 days of operation. The levels of SAOB in the exper-imental reactors increased by about two log units, concurrentlywith the increase in abundance in the reference reactors. Thus, atthe end of the operating period, the abundances of C. ultunense, T.acetatoxydans, and S. schinkii were at equal levels in the referencereactors and in the experimental reactors. Apparently, these spe-cies were present in the nonbioaugmented reactors and were ableto grow and become established as important components in themicrobial community under appropriate operational conditions.Possibly, in the early phase of operation, when aceticlastic metha-nogenesis was still predominant, the SAOB grew with organiccompounds other than acetate, like amino acids, alcohols, andsugars, and produced acetate as the main degradation product(41, 42). However, the low abundances of these organisms (belowthe detection limits) indicate inferior competitiveness for thesesubstrates compared to that of other, likely more efficient, fer-mentative bacteria. Alternatively, the SAOB performed acetate ox-idation but at very low activity levels. Interestingly, the increase ofC. ultunense and T. acetatoxydans to the final levels occurred ear-lier in the experimental reactors than in the reference reactors,probably due to the relatively higher abundances at the startingpoints for the increase (day 250; Fig. 4B and C). This suggests thatthe added organisms to some extent actually had become estab-lished in the process and, when conditions allowed, quickly in-creased in number. However, this rapid increase in abundances

FIG 4 Average log gene abundance of SAOB (A to C) and methanogens (D toH) in reference (dashed line) and experimental (continuous line) reactors.

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did not seem to influence the performance of the experimentalreactors. Possibly, the slightly higher levels of propionic acid in E1and E2 may have been associated with the higher amounts ofSAOB observed in these bioaugmented reactors.

Despite this initial divergence in abundances between the re-actors, the levels of SAOB were similar in all of the reactors at thefinal sampling points. This indicates a strong dependence on cer-tain operating conditions, most likely high acetate concentration,high ammonia concentration, and/or the methanogenic popula-tion structure for the abundances of the different SAOB. The find-ings in the present study together with results from recent publi-cations indicate that the levels of specific abundant SAOB areindicative of the prevalent pathway for the methanization of ace-tate. Corresponding levels of SAOB-related species have been re-vealed in studies of several laboratory- and full-scale biogas reac-tors operating through SAO or the aceticlastic pathway (19, 39; L.Sun, B. Müller, M. Westerholm, and A. Schnürer, unpublisheddata). However, to fully establish specific levels of SAOB geneabundance as likely indicators for SAO, further studies are re-quired.

Abundance of methanogens. Examination of the methano-genic populations (Methanosaetaceae, Methanosarcinaceae, andMethanobacteriales) revealed similar abundances in both the ex-perimental and reference reactors. Despite changes in operationalparameters and reactor performances in the present study, speciesbelonging to the families Methanosaetaceae and Methanosarci-naceae remained at relatively constant levels during the initial 200days of operation. This is in accordance with an earlier study,where apparently stable methanogenic abundance was demon-strated in biogas reactors despite altered OLR, HRT, and substratecomposition (40). In addition, the results of the present studyshowed that the addition of the acetate-oxidizing culture did notcause observable influences on the abundances of Methanosaeta-ceae and Methanosarcinaceae. However, the abundance of Metha-nosaetaceae decreased significantly (P � 0.05) concurrently withthe increase in SAOB, with average gene abundances of 1.4 to3.7 � 1010/ml (day 212) to 0.7 to 2.3 � 108/ml during the late stageof operation (days 345 to 445). The levels of Methanosarcinaceaewere more stable, but a small decrease was observed from 4.6 to6.5 � 105 at day 63 to 0.8 to 2.3 � 104 at day 392. However, thelevels again increased to 0.7 to 3.3 � 105/ml at the final samplingpoint at day 442. This pattern in abundance is likely to be attrib-utable to the elevated levels of ammonia, causing specific inhibi-tion of the aceticlastic methanogens (39). Furthermore, membersof the Methanosaetaceae have been proven to be more sensitive toammonia than members of the Methanosarcinaceae (17, 24). Am-monia concentrations exceeding 3 g NH4

�-N/liter (215 mM)have been reported to inhibit Methanosaeta species, whereasMethanosarcina species have been shown to tolerate concentra-tions up to 7 g NH4

�-N/liter (500 mM) (11).Interestingly, the relatively high abundances of Methanosarci-

naceae coincided with high abundances of the SAOB, in particularS. schinkii and T. acetatoxydans, even after inception of SAO as thedominant pathway for methane production. Since both thesegroups use acetate for growth, they are probably strongly compet-itive with one another, which was not reflected in the presentresults. However, there have been recent indications that mem-bers belonging to the order Methanosarcinales may act as hydro-gen-utilizing methanogens during SAO (19). Molecular studies ofthe abundant methanogenic population in biogas reactors, with

SAO as the main mechanism for methane formation, revealed thatmore than 90% of the methanogenic population consisted ofMethanosarcinales. In addition, it has been suggested that Metha-nosarcinaceae may be able to perform acetate oxidation (23), in-dicating that these methanogens could mediate the entire processof acetate oxidation and subsequent methanogenesis (18).

The abundance of the hydrogenotrophic methanogens belong-ing to Methanomicrobiales, Methanobacteriales, and Methanocul-leus species fluctuated somewhat during the operating period, butoverall no significant changes were observed between the reactors(Fig. 4D to F). An initial increase in the abundances of Metha-noculleus species at day 183 indicated that the establishment ofthese methanogens may have occurred by the time of the initiationof bioaugmentation, but this was contradicted by the rapid de-crease at day 212. After day 212 a more persistent increase inabundances occurred, and at day 253 the levels had increased sig-nificantly by about one to two log units (Fig. 4F). From day 392,the abundances seemed to decrease rapidly, but the changes werecalculated to be insignificant (P � 0.05). These changes were alsoreflected in the number of abundant Methanomicrobiales, com-prising hydrogenotrophic methanogens, among other species be-longing to the genus Methanoculleus. Comparing the gene abun-dances of Methanomicrobiales and Methanoculleus suggested aproportional increase in the latter in line with the increase in am-monia levels. This result is in line with results of a previous study,which suggested that Methanoculleus is an important partner or-ganism during SAO under mesophilic conditions (33).

The similar performances of the reactors showed that the bio-augmentation did not improve their operations during periods ofincreasing ammonia levels. Apparently the positive effects of bio-augmentation illustrated in previous studies were not applicablein this experiment. The fact that the bioaugmentation here wasperformed in a continuous stirred tank reactor (CSTR) while themajority of previous studies were performed with batch reactorsystems might be one explanation for the difference in results.However, in accordance with these results, a review regarding bio-augmentation reported low or no benefit of bacterial augmenta-tion, biomass enhancement, or inoculum addition (36). A varietyof reasons for unsuccessful application were suggested, such aslimitations of available substrate, overrestriction of the amountsof added microorganisms, and growth inhibition due to compe-tition with other microorganisms. These factors may also be ap-plicable here as potential reasons for the absence of apparent ef-fects of bioaugmentation with SAO culture. However, aninvestigation of two-phase anaerobic reactors suggested that ashort retention time and acidic conditions could favor SAO in thefirst-stage reactor. The constant input of SAOB (affiliated with C.ultunense, T. phaeum, and S. schinkii) from the first-stage reactorwas then proposed to promote the prevalence of SAO in the sec-ond-stage reactor and to allow the bacteria to compete success-fully with the aceticlastic methanogens (32).

Conclusions. This study further emphasizes the strong impactof ammonia on the occurrence of SAO and the increased appear-ance of SAOB and the partner hydrogenotrophic methanogens.Previous studies have revealed a significant increase in the abun-dance of SAOB concurrently with an ammonia-induced shiftfrom aceticlastic methanogenesis to SAO in a laboratory-scale re-actor (39). Furthermore, the lack of influence of the syntrophsthat were added to the pathway for acetate degradation in thereactors and the identification of ammonia as the deciding factor

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for the appearance of SAOB indicate that only microbes with theability to thrive and proliferate in the reactor will have any impacton the process in a longer-term perspective. This also highlightsthe need to establish optimized growth conditions for SAOB andthe hydrogenotrophic partner in order to optimize methane pro-duction at high ammonia concentrations. However, informationabout syntrophic acetate oxidation, the organisms responsible,and their role in the methanogenic environment is currently lim-ited. A greater understanding of their response to different envi-ronmental conditions would assist further development and op-timization of the anaerobic treatment processes proceedingthrough the SAO pathway.

The results of the present study also indicate that the SAOBknown today are important components of the biogas-producingmicrobiota and are abundant, although at relatively low levels,even in processes dominated by aceticlastic methanogenesis. Highabundances and supplies of SAO microorganisms are not crucialfactors for the onset of the SAO pathway. Instead, operation undercertain environmental conditions such as high ammonia levelspromotes the dynamic transition to SAO.

ACKNOWLEDGMENTS

We thank Maria Erikson, Li Sun, and Kajsa Risberg for their help withreactor operation and Johnny Ascue for technical support. We also thankMaria Erikson for valuable assistance with DNA extraction.

This work was supported by the thematic research program Micro-drive (http://microdrive.slu.se) at the Swedish University of AgriculturalSciences.

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