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     

一株高温高效产氢细菌的分离及产氢特征研究

实验组采用非选择性培养、选择性培养、液体培养和固体培养一整套培养方法,结合平面厌氧操作技术与培养瓶厌氧技术,从混合产氢培养液中分离得到1株较好的产氢细菌。细菌需氧实验证明:此细菌为厌氧细菌;Gram实验和芽孢染色实验表明:此菌为革兰氏阴性杆菌,无芽孢。在不同的pH值、不同金属离子与温度下,于间歇式条件下测定其产氢量。实验结果表明:此菌株对pH值敏感,其最适产氢pH值为5.5,最适温度在45℃左右,在最适宜条件下产气量在1000 mL/(5 g葡萄糖),产气周期在15 d左右。金属离子影响实验表明:缺少铁和镁离子对此产气菌株的产气量有明显影响,此菌株对铜离子(0.1 g/L)无抗性。     

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。     

污泥厌氧消化产酸发酵过程中乙酸累积机制

[目的]研究污泥厌氧消化产挥发性脂肪酸(VFA)过程中的有机物碳流的转化机制,阐明乙酸累积机理。[方法]研究溴乙烷磺酸盐(BES)和氯仿(CHCl3)抑制模型下中间代谢产物和气体的累积,检测各产乙酸功能菌群数量,推断污泥产酸发酵过程中的有机物碳流方向和乙酸累积机理。[结果]BES模型乙酸浓度达27 mmol/L,fhs基因拷贝数比对照组高2-3倍,产氢产乙酸菌略有下降。CHCl3模型乙酸浓度达22 mmol/L,fhs基因拷贝数比BES组低一个数量级,产氢产乙酸菌下降明显。[结论]BES特异性较高,除产甲烷菌外对其他厌氧产酸细菌没有影响,乙酸浓度增加并且其主要来源于水解发酵产酸以及同型产乙酸过程。氯仿除抑制产甲烷菌外,对同型乙酸菌和产氢产乙酸菌也有强烈的抑制作用。     

高效产氢细菌新种--Ethanologenbacterium sp.X-1的分离鉴定及其产氢效能

为获得高效产氢发酵细菌 ,采用改进的厌氧Hungate培养技术 ,从生物制氢反应器CSTR中分离一株产氢细菌X 1。对该株细菌进行了形态学特征、生理生化指标、16SrDNA和 16S 2 3SrDNA间隔区序列分析等研究。结果表明与最相近的种属Clostridiumcellulosi和Acetanaerobacteriumelongatum等的 16SrRNA基因序列同源性为 94 %以下。16S 2 3SrRNA间隔区基因序列比对分析显示保守区域仅为tRNAAla和tRNAIle序列 ,其它可变部位没有同源性区域 ,鉴定为新属Ethanologenbacteriumsp .。该株细菌为专性厌氧杆菌 ,代谢特征为乙醇发酵 ,葡萄糖发酵产物主要为乙醇、乙酸、H2 和CO2 。在pH4 0和 36℃条件下最大产氢速率是 2 8 3mmolH2 (gdrycell·h)。经鉴定和产氢效能分析表明该菌株是一新属的高效产氢细菌     

产氢菌的复合诱变选育及突变株HCM-23的产氢特性

以厌氧产氢细菌Clostridium sp. H-61为原始菌株, 先后经亚硝基胍(NTG)、紫外(UV)诱变, 选育得到1株高产突变株HCM-23。在葡萄糖浓度为10 g/L的条件下, 其产氢量为3024 mL/L, 比原始菌株提高了69.89%; 其最大产氢速率为33.19 mmol H2/g DW·h, 比原始菌株(19.74 mmol H2/g DW·h)提高了68.14%。经过多次传代试验, 稳定性良好。其发酵末端产物以乙醇和乙酸为主, 属于典型乙醇型发酵代谢类型。其最适产氢初始pH为6.5, 最适生长温度为36℃, 以蔗糖为最佳碳源。与原始菌株相比, 突变株HCM-23的产氢特性发生了改变, 如生长延滞期延长, 可利用无机氮源等。     

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。     

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。     

产氢菌的复合诱变选育及突变株HCM-23的产氢特性

以厌氧产氢细菌Clostridium sp.H-61为原始菌株,先后经亚硝基胍(NTG)、紫外(UV)诱变,选育得到1株高产突变株HCM-23.在葡萄糖浓度为10 g/L的条件下,其产氢量为3024 mL/L,比原始菌株提高了69.89%;其最大产氢速率为33.19 mmol H2/g DW·h,比原始菌株(19.74 mmolH2/g DW·h)提高了68.14%.经过多次传代试验,稳定性良好.其发酵末端产物以乙醇和乙酸为主,属于典型乙醇型发酵代谢类型.其最适产氢初始pH为6.5,最适生长温度为36℃,以蔗糖为最佳碳源.与原始菌株相比,突变株HCM-23的产氢特性发生了改变,如生长延滞期延长,可利用无机氮源等.     

高效产氢细菌新种——Ethanologenbacterium sp. X-1的分离鉴定及其产氢效能

为获得高效产氢发酵细菌,采用改进的厌氧Hungate培养技术,从生物制氢反应器CSTR中分离一株产氢细菌X1。对该株细菌进行了形态学特征、生理生化指标、16S rDNA和16S23S rDNA 间隔区序列分析等研究。结果表明与最相近的种属Clostridium cellulosi和Acetanaerobacterium elongatum等的16S rRNA基因序列同源性为94％以下。16S23S rRNA 间隔区基因序列比对分析显示保守区域仅为tRNAAla和tRNAIle序列,其它可变部位没有同源性区域,鉴定为新属Ethanologenbacterium sp.。该株细菌为专性厌氧杆菌,代谢特征为乙醇发酵,葡萄糖发酵产物主要为乙醇、乙酸、H2和CO2。在pH4.0和36℃条件下最大产氢速率是28.3mmol H2/(g dry cell·h)。经鉴定和产氢效能分析表明该菌株是一新属的高效产氢细菌。     

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     

Application of pseudomurein endoisopeptidase to fluorescence in situ hybridization of methanogens within the family Methanobacteriaceae

In situ detection of methanogens within the family Methanobacteriaceae is sometimes known to be unsuccessful due to the difficulty in permeability of oligonucleotide probes. Pseudomurein endoisopeptidase (Pei), a lytic enzyme that specifically acts on their cell walls, was applied prior to 16S rRNA-targeting fluorescence in situ hybridization (FISH). For this purpose, pure cultured methanogens within this family, Methanobacterium bryantii, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, and Methanothermobacter thermautotrophicus together with a Methanothermobacter thermautotrophicus-containing syntrophic acetate-oxidizing coculture, endosymbiotic Methanobrevibacter methanogens within an anaerobic ciliate, and an upflow anaerobic sludge blanket (UASB) granule were examined. Even without the Pei treatment, Methanobacterium bryantii and Methanothermobacter thermautotrophicus cells are relatively well hybridized with oligonucleotide probes. However, almost none of the cells of Methanobrevibacter ruminantium, Methanosphaera stadtmanae, cocultured Methanothermobacter thermautotrophicus, and the endosymbiotic methanogens and the cells within UASB granule were hybridized. Pei treatment was able to increase the probe hybridization ratio in every specimen, particularly in the specimen that had shown little hybridization. Interestingly, the hybridizing signal intensity of Methanothermobacter thermautotrophicus cells in coculture with an acetate-oxidizing H(2)-producing syntroph was significantly improved by Pei pretreatment, whereas the probe was well hybridized with the cells of pure culture of the same strain. We found that the difference is attributed to the differences in cell wall thicknesses between the two culture conditions. These results indicate that Pei treatment is effective for FISH analysis of methanogens that show impermeability to the probe.     

A mathematical model of syntrophic cocultures in the chemostat

Abstract A model is presented for syntrophic associations between H₂-producing acetogenic bacteria and H₂-utilizing bacteria. A growth rate expression different from the usual Monod equation is applied to the H₂-producing acetogenic bacterium to take into account the thermodynamics of the metabolic reaction involved as dominant factor. The steady of states of the model system are given as branches in a bifurcation diagram. Numerical experiments on the stability of the branches and on the influence of different values in model parameters are performed. The predictions of our model are discussed with regard to the results obtained from experiments with syntrophic cocultures of different species of H₂-producing acetogenic bacteria in combination with H₂-consuming bacteria.     

Monitoring granule formation in anaerobic upflow bioreactors using oligonucleotide hybridization probes

The process of granule formation in upflow anaerobic sludge blanket (UASB) reactors was studied using oligonucleotide hybridization probes. Two laboratory-scale UASB reactors were inoculated with sieved primary anaerobic digester sludge from a municipal wastewater treatment plant and operated similarly except that reactor G was fed glucose, while reactor GP was fed glucose and propionate. Size measurements of cell aggregates and quantification of different populations of methanogens with membrane hybridization targeting the small-subunit ribosomal RNA demonstrated that the increase in aggregate size was associated with an increase in the abundance of Methanosaeta concilii in both reactors. In addition, fluorescence in situ hybridization showed that the major cell components of small aggregates collected during the early stages of reactor startup were M. concilii cells. These results indicate that M. concilii filaments act as nuclei for granular development. The increase in aggregate size was greater in reactor GP than in reactor G during the early stages of startup, suggesting that the presence of propionate-oxidizing syntrophic consortia assisted the formation of granules. The mature granules formed in both reactors exhibited a layered structure with M. concilii dominant in the core, syntrophic consortia adjacent to the core, and filamentous bacteria in the surface layer. The excess of filamentous bacteria caused delay of granulation, which was corrected by increasing shear through an increase of the recycling rate.     

Detection of ingested bacteria in benthic ciliates using fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was applied to detect ingested natural bacteria within the food vacuoles of ciliates harvested from the natural sediment. In addition to this important qualitative aspect, FISH was also successfully used to measure the bacterivory of a culture of the ciliate Tetrahymena pyriformis on natural field sediment bacteria. In this feeding experiment, we compared the FISH technique with the only available alternative technique using fluorescently stained sediment (FS-sediment). The ingestion rate of unstained sediment bacteria determined by FISH was 4.6 bacteria per ciliate and hour. In contrast, Tetrahymena pyriformis cells that fed on bacteria from FS-sediment ingested 12.7 bacteria per ciliate and hour. Bacterial abundances in the sediment were equal in both sediment types (4 x 10(8) cells g sediment dry weight(-1)) when determined by DAPI counts. However, when analyzed using DTAF-counts, the number of bacteria in the FS-sediment increased to 9.7 x 10(8) cells g sediment dry weight(-1). From our findings we conclude that bacterivory by ciliates is overestimated when FS-sediment is used because DTAF stains bacteria as well as protein-containing detritus particles, which are also ingested by many ciliates. In contrast, FISH is a direct, a posteriori method that specifically stains phylogenetic lineages, e.g. eubacteria, after ingestion and thereby avoids a false determination of the number of ingested bacteria. Thus this method can also be used for the study of natural ciliate bacterivory in benthic systems.     

Monitoring of Ralstonia eutropha KT1 in groundwater in an experimental bioaugmentation field by in situ PCR.

Ralstonia eutropha KT1, which degrades trichloroethylene, was injected into the aquifer after activation with toluene, and then the number of bacteria was monitored by in situ PCR targeting the phenol hydroxylase gene and by fluorescent in situ hybridization (FISH) targeting 16S rRNA. Before injection of the bacterial suspension, the total concentration of bacteria in the groundwater was approximately 3 x 10(5) cells/ml and the amount of Ralstonia and bacteria carrying the phenol hydroxylase gene as a percentage of total bacterial cells was less than 0.1%. The concentration of bacteria carrying the phenol hydroxylase gene detected by in situ PCR was approximately 3 x 10(7) cells/ml 1 h after injection, and the concentration of Ralstonia detected by FISH was similar. The number of bacteria detected by in situ PCR was similar to that detected by FISH 4 days after the start of the extraction of groundwater. On and after day 7, however, the number of bacterial cells detected by FISH was less than that detected by in situ PCR.     

Successional development of sulfate-reducing bacterial populations and their activities in an activated sludge immobilized agar gel film

A combination of fluorescence in situ hybridization (FISH), microprofiles, and denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rDNA fragments followed by hybridization analysis with specific probes was applied to investigate successional development of sulfate-reducing bacteria (SRB) community structure and in situ sulfide production activity within an activated sludge immobilized agar gel film. In this model biofilm system, since biases arising from biofilm heterogeneity can be ignored, the population dynamics of SRB in the agar gel is directly related to physiological capability and in situ activity of SRB. Microelectrode measurements showed that an anoxic zone was already developed at the beginning (0 day), a first sulfide production of 0.054 mumol H2S m(-2) x s(-1) was detected during the first week, and the rate increased gradually to 0.221 mumol H2S m(-2) x s(-1) in the fifth week. The most active sulfide production zone moved upward to the chemocline and intensified with time to form a narrow zone with high volumetric sulfide production rates. This result coincided with the shift of the spatial distributions of SRB populations determined by FISH. In situ hybridization with probe SRB385 for mainly general SRB of the delta Proteobacteria plus some gram-positive bacteria and probe 660 for Desulfobulbus indicated that the most abundant populations of SRB were primarily restricted to near the oxic/anoxic interface (chemocline). A close observation of the development of the vertical distributions of SRB populations revealed that the cell numbers of Desulfobulbus tripled (from 0.5 x 10(8) to 1.5 x 10(8) cells cm(-3)) near the oxic/anoxic interface. Similar growth (from 1.0 x10(8) to 4.5 x 10(8) cells cm(-3)) of Desulfovibrio-like SRB that hybridized with probe SRB385 was observed. PCR-DGGE followed by hybridization analysis revealed that one Desulfobulbus strain was detected from the beginning, and another strain appeared after 1 week, coinciding with the first detected sulfide production. In addition, three strains hybridizing with probe 687 (possibly Desulfovibrio) were also dominant SRB in the agar gel.     

Relationship of species-specific filament levels to filamentous bulking in activated sludge

To examine the relationship between activated-sludge bulking and levels of specific filamentous bacteria, we developed a statistics-based quantification method for estimating the biomass levels of specific filaments using 16S rRNA-targeted fluorescent in situ hybridization (FISH) probes. The results of quantitative FISH for the filament Sphaerotilus natans were similar to the results of quantitative membrane hybridization in a sample from a full-scale wastewater treatment plant. Laboratory-scale reactors were operated under different flow conditions to develop bulking and nonbulking sludge and were bioaugmented with S. natans cells to stimulate bulking. Instead of S. natans, the filament Eikelboom type 1851 became dominant in the reactors. Levels of type 1851 filaments extending out of the flocs correlated strongly with the sludge volume index, and extended filament lengths of approximately 6 x 10(8) micro m ml(-1) resulted in bulking in laboratory-scale and full-scale activated-sludge samples. Quantitative FISH showed that high levels of filaments occurred inside the flocs in nonbulking sludge, supporting the "substrate diffusion limitation" hypothesis for bulking. The approach will allow the monitoring of incremental improvements in bulking control methods and the delineation of the operational conditions that lead to bulking due to specific filaments.     

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.     

Application of Pseudomurein Endoisopeptidase to Fluorescence In Situ Hybridization of Methanogens within the Family Methanobacteriaceae

In situ detection of methanogens within the family Methanobacteriaceae is sometimes known to be unsuccessful due to the difficulty in permeability of oligonucleotide probes. Pseudomurein endoisopeptidase (Pei), a lytic enzyme that specifically acts on their cell walls, was applied prior to 16S rRNA-targeting fluorescence in situ hybridization (FISH). For this purpose, pure cultured methanogens within this family, Methanobacterium bryantii, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, and Methanothermobacter thermautotrophicus together with a Methanothermobacter thermautotrophicus-containing syntrophic acetate-oxidizing coculture, endosymbiotic Methanobrevibacter methanogens within an anaerobic ciliate, and an upflow anaerobic sludge blanket (UASB) granule were examined. Even without the Pei treatment, Methanobacterium bryantii and Methanothermobacter thermautotrophicus cells are relatively well hybridized with oligonucleotide probes. However, almost none of the cells of Methanobrevibacter ruminantium, Methanosphaera stadtmanae, cocultured Methanothermobacter thermautotrophicus, and the endosymbiotic methanogens and the cells within UASB granule were hybridized. Pei treatment was able to increase the probe hybridization ratio in every specimen, particularly in the specimen that had shown little hybridization. Interestingly, the hybridizing signal intensity of Methanothermobacter thermautotrophicus cells in coculture with an acetate-oxidizing H₂-producing syntroph was significantly improved by Pei pretreatment, whereas the probe was well hybridized with the cells of pure culture of the same strain. We found that the difference is attributed to the differences in cell wall thicknesses between the two culture conditions. These results indicate that Pei treatment is effective for FISH analysis of methanogens that show impermeability to the probe.