污泥厌氧消化中同型产乙酸菌富集研究

在市政污泥厌氧消化过程中,采用三阶段选择性富集同型产乙酸菌,通过监测培养过程中pH值、挥发性脂肪酸、关键酶的活性以及不同碳源利用率等,研究同型产乙酸菌在系统中的累积情况.实验数据表明:经过40 d的富集培养,pH值稳定在8.0,乙酸含量逐渐趋于稳定,占COD的比例为46.4％,同型产乙酸菌对底物的利用率达到了87％,乙酸激酶和乙酰磷酸转移酶呈现明显上升的趋势.结果显示,通过本方法可在市政污泥中有效地富集同型产乙酸菌.     

初始pH值对产氢产乙酸/耗氢产乙酸两段耦合工艺定向生产乙酸的影响

以葡萄糖为底物,以经加热预处理并活化过的厌氧污泥为种泥,研究了初始pH值对产氢产乙酸/耗氢产乙酸两段耦合工艺厌氧发酵定向生产乙酸的影响。实验考察了7个初始pH值(5、6、7、8、9、10、11)条件下的底物降解、产物产生和发酵过程pH值的变化。结果表明:产氢产乙酸段初始pH值的变化不仅影响本阶段产酸,而且影响耗氢产乙酸段产酸。初始pH=5时主要进行乙醇型发酵;pH=6和7时主要进行丁酸型发酵;pH=8时混合酸型发酵类型逐渐占优势,pH=8~11时均以乙酸为主要产物,耦合系统生产乙酸最优初始pH值为10。在初始pH=8~11范围内,产氢产乙酸段初期的乙醇浓度一般较高,但到后期因乙醇被微生物进一步代谢转化成乙酸而使其含量下降。     

不同生境微生物转化H2/CO2产乙酸及其在合成气发酵中应用

【目的】合成气发酵对大力开发可再生资源和促进国家可持续发展具有重要意义，研究旨在探究不同生境微生物转化H2/CO2产乙酸及其合成气发酵的潜力。【方法】采集剩余污泥、牛粪、产甲烷污泥和河道底物样品在中温(37 °C)条件下生物转化H2/CO2气体，将来源于牛粪样品的H2/CO2转化富集物用于合成气发酵，通过454高通量技术和定量PCR技术分析复杂微生物群落的组成，GC气相色谱法检测气体转化产生的挥发性脂肪酸(VFAs)浓度。【结果】牛粪和剩余污泥微生物利用H2/CO2气体生成乙酸、乙醇和丁酸等，最高乙酸浓度分别为63 mmol/L和40 mmol/L，明显高于河道底物和产甲烷污泥样品的最高乙酸浓度3 mmol/L和16 mmol/L。牛粪和剩余污泥微生物中含有种类多样化的同型产乙酸菌，剩余污泥中同型产乙酸菌主要为Clostridium spp.、Sporomusa malonica和Acetoanaerobium noterae，牛粪中则为Clostridium spp.、Treponema azotonutricium和Oxobacter pfennigii。【结论】同型产乙酸菌的丰富度和数量两个因素都对复杂微生物群落转化H2/CO2产乙酸效率至关重要；转化H2/CO2得到的富集物可用于合成气发酵产乙酸和乙醇，这为基于混合培养技术的合成气发酵提供了依据。     

颗粒厌氧污泥中的产氢产乙酸细菌研究

本文报道颗粒厌氧污泥中产氢产乙酸细菌的含量及存在方式。在正常运行状态,随着颗粒污泥的培养和生长、产氢产乙酸细菌含量维持在10⁷-10⁸个/ml.一旦厌氧反应器“酸化”,颗粒污泥性能变差,产氢产乙酸细菌急剧下降,减小到约10⁵个/ml.比正常状态低2-3个数级,说明细菌生长受到了不可逆抑制。电镜观察表明,产氢产乙酸细菌的分布不是随机的,它们以微菌落方式存在并排列有序。除了与甲烷短杆菌互营共生外,还发现了一种和甲烷丝菌间的新型互营共生关系,分析     

硝酸盐对牛粪梭菌甲酸脱氢酶缺失型Wood-Ljungdahl途径固碳的影响

【背景】异于同型产乙酸菌通常利用Wood-Ljungdahl途径将2分子CO₂还原为1分子乙酰辅酶A,Clostridium bovifaecis缺失Wood-Ljungdahl途径甲基支路第1步将CO₂还原为甲酸的甲酸脱氢酶,需甲酸存在时将1分子甲酸和1分子CO₂还原为乙酰辅酶A发生葡萄糖的同型产乙酸型发酵。已有报道显示,硝酸盐也可作为同型产乙酸菌的电子受体,而且对不同同型产乙酸菌的代谢影响有所不同,然而硝酸盐对这种独特的甲酸脱氢酶缺失型Wood-Ljungdahl途径固碳的影响尚不清楚。【目的】探究硝酸盐对C.bovifaecis甲酸脱氢酶缺失型Wood-Ljungdahl途径固碳的影响。【方法】硝酸盐浓度分别为10 mmol/L和30 mmol/L时,以未添加硝酸盐为对照实验,研究C.bovifaecis在葡萄糖+甲酸+CO₂为基质条件下的细菌生长、底物消耗和产物生成情况。【结果】10 mmol/L和30 mmol/L硝酸盐存在时,主要产物乙醇浓度分别为5.80 mmol/L和1.66 mmo...     

pH值和产甲烷抑制剂对两相耦合系统污泥发酵定向产乙酸的影响

采用产氢产乙酸/同型产乙酸两相耦合工艺对剩余污泥进行了半连续式厌氧发酵,主要研究了pH值和产甲烷抑制剂2-bromoethanesulphonate(BES)对耦合系统定向产乙酸的影响.结果表明:碱性pH(pH=10.0)和添加BES都能促进A相乙酸的积累,提高乙酸的产率,同时碱性pH比添加BES更有利于污泥的水解.当...     

厌氧生境体系中产氢产乙酸细菌的FISH定量解析

产氢产乙酸细菌是一类在有机物厌氧降解过程中起重要作用的细菌。以基于16S rRNA序列设计的特异性寡核苷酸探针为基础,优化FISH实验条件,确定该技术检测产氢产乙酸细菌的实验条件为样品固定19h、乙醇脱水5min,杂交缓冲液中甲酰胺浓度55%。运用建立的FISH技术检测了几种厌氧消化体系中产氢产乙酸细菌的数量,并与用传统MPN方法的结果进行了比较。结果表明,产氢产乙酸细菌分布广泛,废水处理UASB反应器和动物消化道,特别是反刍动物瘤胃中的产氢产乙酸细菌数量较高,其丰度分别为1.70×109 cells/mL样品,6.50×108 cells/mL样品。湖底沉积物中产氢产乙酸细菌数量较少,仅占整个微生物群落的0.4%,含量为1.20×108 cells/mL样品。     

豆腐废水UASB反应器中的原核生物多样性及主要功能菌群

通过构建16S rRNA基因文库,对豆腐废水UASB反应器中颗粒污泥的原核生物多样性进行了分析,并用MPN法对颗粒污泥中的互养产乙酸细菌和产甲烷菌进行了活菌数量测定。结果表明,33%的16S rRNA基因序列属于产甲烷菌,氢和乙酸盐营养型的产甲烷菌在颗粒污泥中数量最多,分别为1.1×10.9个/mL和4.5×10.8个/mL。低GC革兰氏阳性菌和δ-变形菌纲分支的细菌也是颗粒污泥中的主要菌群,它们的16S rRNA序列分别占22%和9%,其中互养产乙酸细菌在颗粒污泥中的数量可达4.5×10.7个/ml。绿色非硫细菌是另一类丰度很高的细菌,其16S rRNA序列占文库的12%。对各类微生物在颗粒污泥中可能的作用进行了讨论。通过研究不仅了解了特定环境中的微生物组成,还为从中分离特异类群的微生物提供了指导。     

氯仿处理厌氧污泥发酵制氢中微生物多样性的解析

研究了不同浓度氯仿对厌氧污泥产氢及其微生物多样性的影响。在氯仿浓度为0.050%时,累积氢、氢气产率、VFA和总糖降解率均达到最大,分别为639mL、1.71molH2/mol消耗葡萄糖、2880mg/L和85%。利用PCR-DGGE技术对不同浓度氯仿处理的污泥样品中微生物多样性和种群结构进行分析显示,4个细菌克隆属于Clostridia,2个细菌克隆分别属于Acidobacteria和δ-proteobacteria,其他4个均属于不可培养细菌。Clostridia中的4个菌群均属产氢菌群,条带7中含有的细菌可能属于HPB。发酵产氢后的污泥样品C3(氯仿浓度=0.05%)中细菌类型主要有:Megasphaera sueciensis、Megasphaera paucivorans、Clostridium cellulosi、Clostridium sp.和不可培养细菌,为最适产氢群落结构。     

酸碱调控污泥厌氧发酵实现乙酸累积及微生物种群变化

摘要：【目的】通过对污泥厌氧发酵pH调控,研究挥发性脂肪酸的累积、产酸微生物种群变化及产氢产乙酸菌群对乙酸产生的贡献。【方法】测定不同pH条件下污泥厌氧发酵过程中挥发性脂肪酸的累积;分别应用末端限制性片段长度多态性（T-RFLP）和荧光原位杂交技术（FISH）分析产酸系统中微生物种群结构的变化及产氢产乙酸菌的数量。【结果】 pH为10.0时,有机酸和乙酸的产率在发酵结束时分别达到652.6 mg COD/g-VS和322.4 mg COD/g-VS,显著高于其它pH条件。T-RFLP结果表明,pH值为12     

Trophic link between syntrophic acetogens and homoacetogens during the anaerobic acidogenic fermentation of sewage sludge

Acetate production during anaerobic sludge treatment has significant economic and environmental benefits. In this study, trophic links between syntrophic acetogens and homoacetogens in the anaerobic acidogenic fermentation of sewage sludge were investigated using methanogenic inhibitor 2-bromoethanesulfonate (BES) to block the methanogenesis pathway and butyrate to enhance syntrophic acetogenesis. The Gibbs free energies (ΔG) of the butyrate-degrading and homoacetogenic processes were close to the thermodynamic threshold of the reaction activity (−15 kJ/mol). In addition, microbial quantification analysis revealed that the growth of syntrophic acetogenic bacteria and homoacetogens in the treatment incubations was higher than that of the control. The results indicated that hydrogen-producing butyrate degraders are stimulated with homoacetogens when methanogenesis was specifically inhibited.     

Dichloromethane as the sole carbon source for an acetogenic mixed culture and isolation of a fermentative, dichloromethane-degrading bacterium.

Dichloromethane (DCM) is utilized by the strictly anaerobic, acetogenic mixed culture DM as a sole source of carbon and energy for growth. Growth with DCM was linear, and cell suspensions of the culture degraded DCM with a specific activity of 0.47 mkat/kg of protein. A mass balance of 2 mol of chloride and 0.42 mol of acetate per mol of DCM was observed. The dehalogenation reaction showed similar specific activities under both anaerobic and aerobic conditions. Radioactivity from [14C]DCM in cell suspensions was recovered largely as 14CO2 (58%), [14C]acetate (23%), and [14C]formate (11%), which subsequently disappeared. This suggested that formate is a major intermediate in the pathway from DCM to acetate. Efforts to isolate from culture DM a pure culture capable of anaerobic growth with DCM were unsuccessful, although overall acetogenesis and the partial reactions are thermodynamically favorable. We then isolated bacterial strains DMA, a strictly anaerobic, gram-positive, endospore-forming rod, and DMB, a strictly anaerobic, gram-negative, endospore-forming homoacetogen, from culture DM. Both strain DMB and Methanospirillum hungatei utilized formate as a source of carbon and energy. Coculture of strain DMA with either M. hungatei or strain DMB in solid medium with DCM as the sole added source of carbon and energy was observed. These data support a tentative scheme for the acetogenic fermentation of DCM involving interspecies formate transfer from strain DMA to the acetogenic bacterium DMB or to the methanogen M. hungatei.     

Functional gene analysis suggests different acetogen populations in the bovine rumen and tammar wallaby forestomach

Reductive acetogenesis via the acetyl coenzyme A (acetyl-CoA) pathway is an alternative hydrogen sink to methanogenesis in the rumen. Functional gene-based analysis is the ideal approach for investigating organisms capable of this metabolism (acetogens). However, existing tools targeting the formyltetrahydrofolate synthetase gene (fhs) are compromised by lack of specificity due to the involvement of formyltetrahydrofolate synthetase (FTHFS) in other pathways. Acetyl-CoA synthase (ACS) is unique to the acetyl-CoA pathway and, in the present study, acetyl-CoA synthase genes (acsB) were recovered from a range of acetogens to facilitate the design of acsB-specific PCR primers. fhs and acsB libraries were used to examine acetogen diversity in the bovine rumen and forestomach of the tammar wallaby (Macropus eugenii), a native Australian marsupial demonstrating foregut fermentation analogous to rumen fermentation but resulting in lower methane emissions. Novel, deduced amino acid sequences of acsB and fhs affiliated with the Lachnospiraceae in both ecosystems and the Ruminococcaeae/Blautia group in the rumen. FTHFS sequences that probably originated from nonacetogens were identified by low "homoacetogen similarity" scores based on analysis of FTHFS residues, and comprised a large proportion of FTHFS sequences from the tammar wallaby forestomach. A diversity of FTHFS and ACS sequences in both ecosystems clustered between the Lachnospiraceae and Clostridiaceae acetogens but without close sequences from cultured isolates. These sequences probably originated from novel acetogens. The community structures of the acsB and fhs libraries from the rumen and the tammar wallaby forestomach were different (LIBSHUFF, P < 0.001), and these differences may have significance for overall hydrogenotrophy in both ecosystems.     

Response surface methodology analysis of anaerobic syntrophic degradation of volatile fatty acids in an upflow anaerobic sludge bed reactor inoculated with enriched cultures

Anaerobic oxidation of volatile fatty acids (VFAs) as the key intermediates is restricted thermodynamically. Presently, enriched acetogenic and methanogenic cultures were used for syntrophic anaerobic digestion of VFAs in an upflow anaerobic sludge bed reactor fed with acetic, propionic, and butyric acids at maximum concentrations of 5.0, 3.0, and 4.0 g/L, respectively. Interactive effects of propionate, butyrate and acetate were analyzed. Hydraulic retention time (HRT) and acetate oxidizing syntrophs and methanogen (hydrogenotrophs) to syntrophic bacteria (propionate- and butyrate-oxidizing bacteria) population ratio (M/A) were investigated as key microbiological and operating variables of VFA anaerobic degradations. M/A did not affect the size distribution and had little effect on extracellular polymer contents of the granules. Granular sludge with close spatial microbial proximity enhanced syntrophic degradation of VFAs compared to other cultures, such as suspended cultures. Optimum conditions were found to be propionate = 1.93 g/L, butyrate = 2.15 g/L, acetate = 2.50 g/L, HRT = 22 h, and M/A = 2.5 corresponding to maximum VFA removal and biogas production rate. Results of verification experiments and predicted values from fitted correlations were in close agreement at the 95% confidence interval. Granules seemed to be smaller particles and less stable in construction with an irregular fractured surface compared to the original granules.     

In vitro H2 utilization by a ruminal acetogenic bacterium cultivated alone or in association with an archaea methanogen is stimulated by a probiotic strain of Saccharomyces cerevisiae.

The effects of a live strain of Saccharomyces cerevisiae on hydrogen utilization and acetate and methane production by two hydrogenotrophic ruminal microorganisms, an acetogenic bacterial strain and an archaea methanogen, were investigated. The addition of yeast cells enhanced by more than fivefold the hydrogenotrophic metabolism of the acetogenic strain and its acetate production. In the absence of yeasts, and in a coculture of the acetogen and the methanogen, hydrogen was principally used for methane synthesis, but the presence of live yeast cells stimulated the utilization of hydrogen by the acetogenic strain and enhanced acetogenesis.     

Anaerobic microbial activities including hydrogen mediated acetogenesis within natural gas transmission lines

Microbially influenced corrosion (MIC) is being increasingly recognised as a serious problem. To investigate the role of MIC, radiotracer activity and lipid biomass measurements were performed on samples from offshore and on‐shore natural gas transmission systems. These measurements evaluated the biomass and metabolism of microbial communities residing inside transmission pipelines. Aqueous and nonaqueous hydrocarbon samples from liquid separators, sludge catchers and nodules attached to pipe walls were aseptically recovered and inoculated into anaerobic tubes for radiotracer time course experiments or preserved with chloroform‐methanol for total lipid analyses. MPN enrichments and phospholipid biomass determinations estimated microbial populations of 10⁴—10⁷ cells per gram in several samples. General microbial metabolism was demonstrated by [l‐¹⁴C]acetate incorporation into lipids and by [¹⁴C]CO₂ production from [U‐¹⁴C]glucose. [¹⁴C]Acetate was slowly mineralised to ¹⁴CO₂ without significant methane production. [¹⁴C]Acetate was produced by fermentation of [¹⁴C]glucose, [¹⁴C]palmitate and by hydrogen mediated acetogenesis in the presence of [^I4C]CO₂. In one location acetogenesis from hydrogen and carbon dioxide accounted for 0–7 mmol.l^‐1 of acetate production per week. These results demonstrated that microorganisms could utilise natural gas impurities to produce organic acids. This activity could adversely affect the structural integrity (MIC) of high pressure natural gas pipelines.     

Origin and fate of acetate in an acidic fen

Acetate is a central intermediate in the anaerobic degradation of organic matter, and the resolution of its metabolism necessitates integrated strategies. This study aims to (1) estimate the contribution of acetogenesis to acetate formation in an acidic fen (pH ~ 4.9), (2) assess the genetic potential for acetogenesis targeting the fhs gene encoding formyltetrahydrofolate synthetase (FTHFS) and (3) unravel the in situ turnover of acetate using stable carbon isotope pore-water analysis. H(2)/CO(2)-supplemented peat microcosms yielded (13)C-depleted acetate (-37.2‰ vs. VPDB (Vienna Peedee belemnite standard) compared with -14.2‰ vs. VPDB in an unamended control), indicating the potential for H(2)-dependent acetogenesis. Molecular analysis revealed a high diversity and depth-dependent distribution of fhs phylotypes with the highest number of operational taxonomic units in 0-20 cm depth, but only few and distant relationships to known acetogens. In pore waters, acetate concentrations (0-170 μM) and δ(13)C-values varied widely (-17.4‰ to -3.4‰ vs. VPDB) and did not indicate acetogenesis, but pointed to a predominance of sinks, which preferentially consumed (12)C-acetate, like acetoclastic methanogenesis. However, depth profiles of methane and δ(13)C(CH4) revealed a temporarily and spatially restricted role of this acetate sink and suggest other processes like sulfate and iron reduction played an important role in acetate turnover.