Sulfate is the last oxidized form of sulfur which is readily soluble in water, so it occurs naturally in most water supplies and is present in wastewater as well. Under anaerobic condition, sulfate has been known to be reduced into sulfide by sulfate reducing bacteria(SRB) using organic substrates such as volatile fatty acid(VFA) and hydrogen and give large influences on the anaerobic methanogenesis and activities of other microorganisms. Many studies have been done about the role of sulfate and SRB during the anaerobic methanogenesis in various in situ ecosystems but haven't provide common characteristics which can be accurately applied into evaluating anaerobic degradation. In this studies anaerobic sludge which has the same origin was acclimated in laboratory scale(12L) differently under the condition of sulfate-added(S) and non-sulfate-added(N) in the influent media. The investigation relevant to characteristics of VFAs utilization, and effects of nitrogen compounds, 16S rDNA sequence of these sludges was carried out. We intended to show the effects of sulfate on the anaerobic methanogenesis through comparing those above results.
In utilization of VFAs, there was large difference in metabolism. Acetate was a main intermediate of anaerobic organic metabolism under the presence of sulfate. Propionate was also produced but the amount was equivalent to 25% of acetate while conventional fermentation was mainly occurred when sulfate is absent. Propionate known to be main product of fermentation was produced as major intermediate during the non-sulfate methanogenesis. As sulfate reducing activity was disappeared the role of propionate-producing bacteria became to be enlarged. it is possible to elucidate that SRB and sulfate plays a major role in propionate or other substance degradation. However sulfidogenic activity with propionate was very low(16%) than with any other VFAs(43%). The activity was most high on acetate and butyrate. So SRB was thought to play major roles in the β-oxidation of substrate and utilization of acetate. Sulfidogenesis inhibited methaonogenesis up to 48% from all substrate studied, but overall organic removal efficiency was rather increased. Although methanogenesis was suppressed to 14% by butyrate, SRB used much more ratio of organics(43%), thus total removal ratio was increased about 20%. Interestingly sulfate reduction involved in reduction of cell mass. Suspended solid of these two sludge are 26.3(N) and 20.9g/L(S) respectively. The difference was 5.4g/L about 21% lower in S-sludge. Though removal efficiency was almost same between two sludge, mass of sludge produced is rather low in the present of sulfate. The difference has significant meaning in view of sludge disposal. The less sludge production means the cheaper disposal cost. Consequently, effects of sulfate reduction was not negative but mutually benefit on the whole.
As another viewpoint, the differences of effects by nitrogen compounds on anaerobic methanation of VFAs was investigate Increasing concentration of ammonia results in stable decease of methanogenesis from all of the VFAs studied. Until 2000mg-N/L methanogenic activity was not affected, gentle decrease was appeared over that concentration. But nitrate and nitrite have shown large inhibition in high concentration range(15mM over). Most of methanogenic activity was extinguished. The effect of nitrite was rather severe. At 8mM concentration of nitrite methanogenesis was inhibited in all the VFAs mostly(90%). Below 5mM of concentration the effects of nitrate and nitrite on methanogenesis were so little, however, there are sudden decrease was occurred in common. Far from sulfidogenesis was affected negatively with increasing nitrogen compound concentrations, the production activities was slightly increased. Below the level of 4000mg-N/L with ammonia and 10mM nitrate, nitrite, sulfide production was increased about 60%. But there were differences in results between nitrate and nitrite at the level of 15mM over. Sudden decrease and extinction of sulfidogenesis was appeared in case of nitrite. Ammonia and nitrate, nitrite was acted as good nitrogen source for sulfidogenesis but as inhibitor. but in high concentration nitrate and nitrite became to show inhibition to sulfidogenic activity due to the self-toxicity.
Denitrification potential was exist in both sludge when nitrate and nitrite was added, Nitrate showed high nitrogen gas production with all VFAs in proportion to concentration, which was higher about 40% in N-sludge. This difference may be a tribute to sulfidogenesis. In N-sludge, nitrogen gas was produced as that of nitrate. In S-sludge, however, little nitrogen gas was produced. This makes the postulation possible that the inhibition of nitrate and nitrite on methanogenic activity was different Nitrate inhibition was attributed to the out-competition of denitrifier in substrate utilization with methanogens, while nitrite thought to self-toxicity under the presence of sulfate. There was no ammonium ion, the product of assimilatory denitrification was found in the reaction. VFAs might to be used in dissimilatory denitrification only.
Two 16S rDNA clone libraries of bacteria were constructed from the sludge samples taken from N-reactor and S-reactor after two years operation. The community structure of bacterial and archaeal clones were determined by clone phylogenic analysis of 16S rDNA sequences. A total 120 clones were sequenced after PCR amplification. 36, 31 bacterial clones and 13, 22 archaeal clones were analyzed for the clone library respectively. The bacterial clones were grouped into 13 OTUs(N) and 11 OTUs(S) on the basis of having more than 98% sequence similarity within an OUT. Only two OTUs, Bacteroidetes bacterium PPf50E2 and uncultured bacterium clone R5p5, commonly appeared in both sludge. The OTU distributions of bacterial domain in both sludge are as follows: In N-sludge 36.1% to the Bacteroidetes, 13.9% to the Thermotogae, 19.5% to the Actinomycetes, 8.4% to the Nitrospira, 8.4% to Firmicute, 2.8% to the TM7 and 5.6% to the new division (OP 9) are showed while in S-sludge bacterial domain are composed of 34.6% Bacteroidetes, 16.2% Firmicute, 11.5% δ-Proteobacteria, 19.2% Thermotogae, 3.8% to the Spirochaetes, and 11.5% unknown group. Three out of 31 clones were affiliated with sulfate reducing bacteria, Desulforhabdus amnigena ASRB1. After T-RFLP analysis, 13 clones from N-reactor and 22 clones from S-reactor were sequenced. All of these archaeal clones were assigned to the class Euryarchaeota, and all clones were close relatives of methanogens. The archaeal clones from N- and S- sludge were grouped 3 and 4 OTUs respectively. Though the archaeal clone frequencies were different between two samples, the phylogenetic structures were similar. In the N-sludge, 84% of the archaeal clones were closely affiliated with Methanosaeta concilii(above 99% sequence identity) and 10% of the clones were affiliated with Methanoculleus chikugoens. In the S-sludge, 57% of the clones were affiliated with Methanosaeta concilii instead, the frequency of clones having hydrogenotrophic activity were higher (25% of the clones were affiliated with Methanoculleus chikugoens and 14% of the clones were affiliated with uncultured archaeon TA02).
In this study, the effects of sulfate on the microbial communities and degradation of organic materials about anaerobic digesters were analyzed simultaneously. Both results coincided well in two facts. First, the different hydrogen accumulation rate and frequency of hydrogen-utilizing methanogens. Second the different accumulation rate of propionate and acetate in degradation of organic material and the presence or absence of propionate-producing bacterial clone. Due to this difference, the formation of intermediate came to be shifted from propionate into acetate. Difference in hydrogen utilization activity might affect the population structures of syntrophic bacteria and in turn influence on utilization pathway of the intermediates. This thought to be because sulfate ion acts as electron sink, which enables some bacteria to use hydrogen as an electron donor in metabolism, sulfate anion came to facilitate formation of more oxidized short chain fatty acid such as acetate. Though the informations to get from the microbial community structure were limited for many uncultured clones, the present results would be helpful to understand the complex interactions between anaerobic microorganisms in the presence of sulfate.
황산염은 용해성이 높아 자연계 어디에서나 널리 존재하는 이온물질로서 혐기성조건에서 지방산등의 유기물질 산화에서 얻어진 전자를 받아 황산염 환원균에 의해 sulfide로 환원되어진다. 이 과정에서 메탄생성균과 다른 혐기성미생물의 활성은 큰 영향을 받는 것으로 알려져 있으나 sulfide의 독성과 기질경쟁을 중심으로 제한적인 연구가 이루어져 왔고 과거 연구결과 대부분이 황산염이 존재하는 in situ에서 얻어진 것으로 정확히 그 영향을 파악하는데 부족한 부분이 많았다. 따라서 본 연구는 황산염이 미치는 영향을 황산염이 존재하는 조건과 존재하지 않는 조건으로 구분하여 비교 분석함으로서 좀더 분명한 역할을 규명하고자 하였으며 이를 위해 가장 대표적인 혐기성 미생물군집을 가진 혐기성소화조의 슬러지를 연구대상으로 정하고 황산염(2mM)의 첨가유무로 구분된 두 가지 다른 배지공급 조건에서 glucose를 기질로 하여 lab-scale규모로 이년이상 순응시켜 실험에 이용하였다. 황산염이 전혀 공급되지 않은 슬러지는 실험에 이용된 대부분의 지방산에서 고르게 높은 메탄 생성력을 보이며 분해과정에서 프로피온산을 주요 중간산물로 형성하면서 메탄으로 전환되는 전형적인 발효분해경로를 거치게 되는 반면 황산염이 계속 공급된 슬러지는 각기 다른 기질에서 높은 메탄생성(lactate,formate)과 황산염 환원(acetate,buryrate)활성 보여 기존의 단일기질에 대한 경쟁과는 다른 양상을 나타냈으며 특이한 것은 유기물 분해과정이 거의 대부분 아세트산을 중간대사산물로 거치는 특징을 보여 주었다. 황산염의 존재는 유기물 분해과정에서 발생하는 수소의 양에도 영향을 미쳐 반응중간에 발생하여 축적되는 양이 황산염이 존재하면서 최고 80%감소하였고 반응조에서 발생하는 메탄의 수율도 약 40%정도 감소되었다. 그러나 황산염환원에 이용된 유기물의 양은 이보다 더 많아짐으로서 전체적인 유기물 저감율은 더 높아짐을 볼 수 있었으며 이로 인해 슬러지 발생량이 약 21%감소되는 것으로 나타났다. 질소화합물로 인한 기질이용특성은 황산염의 유무와 큰 관련성이 없었으나 유독 Nitrite의 경우 황산염 존재시 메탄생성과 탈질활성 그리고 sulfidogenesis에 강한 저해현상을 보여 반응성이 높은 이온물질의 독성작용에 영향을 미칠 수 있음을 보여 주었으며 특히 황산염 환원은 Nitrate 존재시 거의 모든 지방산에서 아주 높은 활성증가를 보여 탈질보다는 유기물이용에 있어 월등히 높은 경쟁력을 가짐을 알 수 있었다. 유기물 분해과정에서 나타난 특징들을 좀 더 구체적으로 확인하기 위하여 슬러지의 16S rDNA clone library를 조사하여 미생물 군집구조를 비교분석한 결과 황산염은 박테리아 군집구조에도 큰 영향을 미쳐 120개의 clone중 Bacteroidetes bacterium PPf50E2 and uncultured bacterium clone R5p5 두개의 clone만이 공통으로 나타나고 나머지 clone은 모두 다르게 나타났다. 황산염과 황산염 환원균의 영향을 받지않은 슬러지의 피로피온산 생성관련 미생물인 Actinobacteria (19.5 %) Propionibacterium(2.5%) clone의 출현과 황산염이 공급된 슬러지에서 수소와 아세트산의 이용력이 높은 것으로 알려진 Desulforhabdus amnigena ASRB clone의 등장은 중간대사 산물로서 프로피온산과 아세트산이 형성을 잘 보여 주고 있었다. archaea 박테리아 역시 황산염으로 인해 영향을 받았으나 군집의 종류보다는 군집의 구성 비율에 미친 영향이 더 컸다. 황산염의 영향이 없는 슬러지에는 아세트산 분해를 통해 메탄을 생성하는 Methanosaeta concilii, 수소와 이산화탄소를 이용하여 메탄생성력을 가진 Methanoculleus chikugoen이 각각 84%, 10%인 반면 황산염이 공급되면서 이들의 비율이 57%, 36%로 변화하였다. 황산염으로 인해 수소 이용력이 월등히 증가한 것을 알 수 있었으며 이로 인하여 형성된 낮은 수소분압은 유기물 분해과정의 중간대사물이 프로피온산에서 아세트산으로 변경되는 데 결정적 영향을 미친 것으로 판단된다. 미생물 군집구조분석에서 나타난 대부분의 clone이 아직 분리가 이루지지 않음으로 인해 얻을 수 있는 정보의 양은 제한되었지만 수소의 이용에 황산염이 미친 영향을 명백히 보여줌으로 황산염이 존재하는 복잡한 혐기성대사과정의 향후연구에 큰 도움이 될 것으로 판단된다.