Parallel processing of substrate correlates with greater functional stability in methanogenic bioreactor communities perturbed by glucose

Parallel processing is more stable than serial processing in many areas that employ interconnected activities. This hypothesis was tested for microbial community function using two quadruplicate sets of methanogenic communities, each set having substantially different populations. The two communities were maintained at a mean cell residence time of 16 days and a mean glucose loading rate of 0.34 g/liter-day in variable-volume reactors. To test stability to perturbation, they were subjected to an instantaneous glucose pulse that resulted in a 6.8-g/liter reactor concentration. The pattern of accumulated products in response to the perturbation was analyzed for various measures of functional stability, including resistance, resilience, and reactivity for each product. A new stability parameter, "moment of amplification envelope," was used to compare the soluble compound stability. These parameters indicated that the communities with predominantly parallel substrate processing were functionally more stable in response to the perturbation than the communities with predominantly serial substrate processing. The data also indicated that there was good replication of function under perturbed conditions; the degrees of replication were 0.79 and 0.83 for the two test communities.     

Vertical distribution of structure and function of the methanogenic archaeal community in Lake Dagow sediment

Detailed studies on the relation of structure and function of microbial communities in a sediment depth profile scarcely exist. We determined as functional aspect the vertical distribution of the acetotrophic and hydrogenotrophic CH4 production activity by measuring production rates and stable 13C/12C-isotopic signatures of CH4 in the profundal sediment of Lake Dagow. The structural aspect was determined by the composition of the methanogenic community by quantifying the abundance of different archaeal groups using 'real-time' polymerase chain reaction and analysis of terminal restriction fragment length polymorphism (T-RFLP). Methane production rates in the surface sediment (0-3 cm depth) were higher in August than in May, but strongly decreased with depth (down to 20 cm). The delta13C of the produced CH4 and CO2 indicated an increase in isotopic fractionation with sediment depth. The relative contribution of hydrogenotrophic to total methanogenesis, which was calculated from the isotopic signatures, increased with depth from about 22% to 38%. Total numbers of microorganisms were higher in August than in May, but strongly decreased with depth. The increase of microorganisms from May to August mainly resulted from Bacteria. The Archaea, on the other hand, exhibited a rather constant abundance, but also decreased with depth from about 1 x 10(8) copies of the archaeal 16S rRNA gene per gram of dry sediment at the surface to 4 x 10(7) copies per gram at 15-20 cm depth. T-RFLP analysis combined with phylogenetic analysis of cloned sequences of the archaeal 16S rRNA genes showed that the methanogenic community consisted mainly of Methanomicrobiales and Methanosaetaceae. The relative abundance of Methanosaetaceae decreased with depth, whereas that of Methanomicrobiales slightly increased. Hence, the vertical distribution of the functional characteristics (CH4 production from acetate versus H2/CO2) was reflected in the structure of the community consisting of acetotrophic (Methanosaetaceae) versus hydrogenotrophic (Methanomicrobiales) phenotypes.     

Efficient treatment of garbage slurry in methanogenic bioreactor packed by fibrous sponge with high porosity

Adding a supporting material to a methanogenic bioreactor treating garbage slurry can improve efficiency of methane production. However, little is known on how characteristics (e.g., porosity and hydrophobicity) of the supporting material affect the bioreactor degrading garbage slurry. We describe the reactor performances and microbial communities in bioreactors containing hydrophilic or hydrophobic sheets, or fibrous hydrophilic or hydrophobic sponges. The porosity affected the efficiency of methane production and solid waste removal more than the hydrophilic or hydrophobic nature of the supporting material. When the terminal restriction fragment length polymorphism technique was used at a lower organic loading rate (OLR), microbial diversities in the suspended fraction were retained on the hydrophobic, but not the hydrophilic, sheets. Moreover, real-time quantitative polymerase chain reaction (PCR) performed at a higher OLR revealed that the excellent performance of reactors containing fibrous sponges with high porosity (98%) was supported by a clear increase in the numbers of methanogens on these sponges, resulting in larger total numbers of methanogens in the reactors. In addition, the bacterial communities in fractions retained on both the hydrophobic and hydrophilic fibrous sponges differed from those in the suspended fraction, thus increasing bacterial diversity in the reactor. Thus, higher porosity of the supporting material improves the bioreactor performance by increasing the amount of methanogens and bacterial diversity; surface hydrophobicity contributes to maintaining the suspended microbial community.     

Bacterial community structure in experimental methanogenic bioreactors and search for pathogenic clostridia as community members

Microbial conversion of organic waste or harvested plant material into biogas has become an attractive technology for energy production. Biogas is produced in reactors under anaerobic conditions by a consortium of microorganisms which commonly include bacteria of the genus Clostridium. Since the genus Clostridium also harbors some highly pathogenic members in its phylogenetic cluster I, there has been some concern that an unintended growth of such pathogens might occur during the fermentation process. Therefore this study aimed to follow how process parameters affect the diversity of Bacteria in general, and the diversity of Clostridium cluster I members in particular. The development of both communities was followed in model biogas reactors from start-up during stable methanogenic conditions. The biogas reactors were run with either cattle or pig manures as substrates, and both were operated at mesophilic and thermophilic conditions. The structural diversity was analyzed independent of cultivation using a PCR-based detection of 16S rRNA genes and genetic profiling by single-strand conformation polymorphism (SSCP). Genetic profiles indicated that both bacterial and clostridial communities evolved in parallel, and the community structures were highly influenced by both substrate and temperature. Sequence analysis of 16S rRNA genes recovered from prominent bands from SSCP profiles representing Clostridia detected no pathogenic species. Thus, this study gave no indication that pathogenic clostridia would be enriched as dominant community members in biogas reactors fed with manure.     

Microbial biomass and community structure of a phase-separated methanogenic reactor determined by lipid analysis

Summary An anaerobic phase-separation biomass reactor was established on cellulose with the hydrolysis and fermentation steps occurring in the first stage, and acetogenesis and methanogenesis in the second stage. Based upon lipid biomarker analysis, eubacterial and eukaryotic cells accounted for approximately 6% of the volatile solids of the first stage and 17% of the second, while methanogens were approximately 1% of the volatile solids in the first stage and 9% of the second. Clustering the polar lipid fatty acids into groups based upon their distributions between the two stages of the reactor clarified the differences in community structure caused by phase-separated operation. Although inoculated from the same source, the two stages maintained very different microbial communities. Signature fatty acids known as indicators of unbalanced growth in eubacteria were significantly higher in the first stage of the reactor.     

Optimal covalent immobilization of glucose oxidase-containing liposomes for highly stable biocatalyst in bioreactor

The glucose oxidase-containing liposomes (GOL) were prepared by entrapping glucose oxidase (GO) in the liposomes composed of phosphatidylcholine (PC), dimyristoyl L-alpha-phosphatidylethanolamine (DMPE), and cholesterol (Chol) and then covalently immobilized in the glutaraldehyde-activated chitosan gel beads. The immobilized GOL gel beads (IGOL) were characterized to obtain a highly stable biocatalyst applicable to bioreactor. At first, the glutaraldehyde concentration used in the gel beads activation as well as the immobilizing temperature and time were optimized to enhance the immobilization yield of the GOL to the highest extent. The liposome membrane composition and liposome size were then optimized to obtain the greatest possible immobilization yield of the GOL, the highest possible activity efficiency of the IGOL, and the lowest possible leakage of the entrapped GO during the GOL immobilization. As a result, the optimal immobilization conditions were found to be as follows: the liposome composition, PC/DMPE/Chol = 65/5/30 (molar percentage); the liposome size, 100 nm; the glutaraldehyde concentration, 2% (w/v); the immobilizing temperature, 4 degrees C; and the immobilizing time, 10 h. Furthermore, the optimal IGOL prepared were characterized by its rapidly increasing effective GO activity to the externally added substrate (glucose) with increasing temperature from 20 to 40 degrees C, and also by its high stability at 40 degrees C against not only the thermal denaturation in a long-term (7 days) incubation but also the bubbling stress in a bubble column. Finally, compared to the conventionally immobilized glucose oxidase (IGO), the higher operational stability of the optimal IGOL was verified by using it either repeatedly (4 times) or for a long time (7 days) to catalyze the glucose oxidation in a small-scale airlift bioreactor.     

Effect of temperature on structure and function of the methanogenic archaeal community in an anoxic rice field soil.

Soil temperatures in Italian rice fields typically range between about 15 and 30 degrees C. A change in the incubation temperature of anoxic methanogenic soil slurry from 30 degrees C to 15 degrees C typically resulted in a decrease in the CH4 production rate, a decrease in the steady-state H2 partial pressure, and a transient accumulation of acetate. Previous experiments have shown that these changes were due to an alteration of the carbon and electron flow in the methanogenic degradation pathway of organic matter caused by the temperature shift (K. J. Chin and R. Conrad, FEMS Microbiol. Ecol. 18:85-102, 1995). To investigate how temperature affects the structure of the methanogenic archaeal community, total DNA was extracted from soil slurries incubated at 30 and 15 degrees C. The archaeal small-subunit (SSU) rRNA-encoding genes (rDNA) of these environmental DNA samples were amplified by PCR with an archaeal-specific primer system and used for the generation of clone libraries. Representative rDNA clones (n = 90) were characterized by terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis. T-RFLP analysis produced for the clones terminally labeled fragments with a characteristic length of mostly 185, 284, or 392 bp. Sequence analysis allowed determination of the phylogenetic affiliation of the individual clones with their characteristic T-RFLP fragment lengths and showed that the archaeal community of the anoxic rice soil slurry was dominated by members of the families Methanosarcinaceae (185 bp) and Methanosaetaceae (284 bp), the kingdom Crenarchaeota (185 or 284 bp), and a novel, deeply branching lineage of the (probably methanogenic) kingdom Euryarchaeota (392 bp) that has recently been detected on rice roots (R. Grosskopf, S. Stubner, and W. Liesack, Appl. Environ. Microbiol. 64:4983-4989, 1998). The structure of the archaeal community changed when the temperature was shifted from 30 degrees C to 15 degrees C. Before the temperature shift, the clones (n = 30) retrieved from the community were dominated by Crenarchaeota (70%), "novel Euryarchaeota" (23%), and Methanosarcinacaeae (7%). Further incubation at 30 degrees C (n = 30 clones) resulted in a relative increase in members of the Methanosarcinaceae (77%), whereas further incubation at 15 degrees C (n = 30 clones) resulted in a much more diverse community consisting of 33% Methanosarcinaceae, 23% Crenarchaeota, 20% Methanosaetaceae, and 17% novel Euryarchaeota. The appearance of Methanosaetaceae at 15 degrees C was conspicuous. These results demonstrate that the structure of the archaeal community in anoxic rice field soil changed with time and incubation temperature.     

Quantification of mcrA by fluorescent PCR in methanogenic and methanotrophic microbial communities

A quantitative fluorogenic PCR method for detecting methanogenic and methanotrophic orders was established using a refined primer set for the methyl coenzyme M reductase subunit A gene (mcrA). The method developed was applied to several microbial communities in which diversity and abundance of methanogens or anaerobic methanotrophs (ANMEs) was identified by 16S rRNA gene clone analysis, and strong correlations between the copy numbers of mcrA with those of archaeal 16S rRNA genes in the communities were observed. The assay can be applied to detecting and assessing the abundance of methanogens and/or ANMEs in anoxic environments that could not be detected by 16S rRNA gene sequence analyses.     

Structure and function of the methanogenic archaeal community in stable cellulose-degrading enrichment cultures at two different temperatures (15 and 30 degrees C)

Methanogenic cultures were enriched from an air-dried rice field soil and incubated under anaerobic conditions at 30 degrees C with cellulose as substrate (ET1). The culture was then transferred and further incubated at either 15 degrees C (E15) or 30 degrees C (E30), to establish stable cultures that methanogenically degrade cellulose. After five transfers, the rates of CH(4) production became reproducible. At 30 degrees C, CH(4) production rates were (mean+/-S.D.) 15.2+/-0.7 nmol h(-1) ml(-1) culture for the next 16 transfers and at 15 degrees C, they were 0.38+/-0.07 nmol h(-1) ml(-1) for the next six transfers. When E30 was assayed at temperatures between 5-50 degrees C, CH(4) production rates increased with the temperature, reached a maximum at 40 degrees C and then decreased. The same temperature optimum was observed in E15, but with a lower maximum CH(4) production rate. The apparent activation energies of CH(4) production were similar (about 120 kJ mol(-1)4 mM at the beginning of the assay. The structure of the archaeal community was analyzed by molecular techniques. Total DNA was extracted from the microbial cultures before the transfer to different temperatures (ET1) and afterwards (E15, E30). The archaeal small subunit (SSU) ribosomal RNA-encoding genes (rDNA) of these DNA samples were amplified by PCR with archaeal-specific primers and characterized by terminal restriction fragment length polymorphism (T-RFLP). After obtaining a constant T-RFLP pattern in the cultural transfers at 15 and 30 degrees C, the PCR amplicons were used for the generation of clone libraries. Representative rDNA clones (n=10 for each type of culture) were characterized by T-RFLP and sequence analysis. In the primary culture (ET1), the archaeal community was dominated by clones representing 'rice cluster I', a novel lineage of methanogenic Euryarchaeota. However, further transfers resulted in the dominance of Methanosarcinaceae and Methanosaetaceae at 30 and 15 degrees C, respectively. This dominance was confirmed by fluorescence in situ hybridization (FISH) of archaeal cells. Obviously, different archaeal communities were established at the two different temperatures, but their activities nevertheless exhibited similar temperature optima.     

Links between archaeal community structure, vegetation type and methanogenic pathway in Alaskan peatlands

Although northern peatlands contribute significantly to natural methane emissions, recent studies of the importance and type of methanogenesis in these systems have provided conflicting results. Mechanisms controlling methanogenesis in northern peatlands remain poorly understood, despite the importance of methane as a greenhouse gas. We used 16S rRNA gene retrieval and denaturing gradient gel electrophoresis (DGGE) to analyse archaeal communities in 15 high-latitude peatland sites in Alaska and three mid-latitude peatland sites in Massachusetts. Archaeal community composition was analysed in the context of environmental, vegetation and biogeochemical factors characterized in a parallel study. Phylogenetic analysis revealed that Alaskan sites were dominated by a cluster of uncultivated crenarchaeotes and members of the families Methanomicrobiaceae and Methanobacteriaceae, which are not acetoclastic. Members of the acetoclastic family Methanosarcinaceae were not detected, whereas those of the family Methanosaetaceae were either not detected or were minor. These results are consistent with biogeochemical evidence that acetoclastic methanogenesis is not a predominant terminal decomposition pathway in most of the sites analysed. Ordination analyses indicated a link between vegetation type and archaeal community composition, suggesting that plants (and/or the environmental conditions that control their distribution) influence both archaeal community activity and dynamics.     

Presence of tRNA-dependent pathways correlates with high cysteine content in methanogenic Archaea

Archeal proteomes can be clustered into two groups based on their cysteine content. One group of proteomes displays a low cysteine content ( approximately 0.7% of the entire proteome), whereas the second group contains twice as many cysteines as the first ( approximately 1.3%). All cysteine-rich organisms belong to the methanogenic Archaea, which generates special cysteine clusters associated with primitive metabolic reactions. Our findings suggest that cysteine plays an important role in early forms of life.     

Microbial community structure and biomass estimates of a methanogenic Antarctic Lake ecosystem as determined by phospholipid analyses

Phospholipid analyses were performed on water column particulate and sediment samples from Ace Lake, a meromictic lake in the Vestfold Hills, Antarctica, to estimate the viable microbial biomass and community structure in the lake. In the water column, methanogenic bacterial phospholipids were present below 17 m in depth at concentrations which converted to a biomass of between 1 and 7×10⁸ cells/liter. Methanogenic biomass in the sediment ranged from 17.7×10⁹ cells/g dry weight of sediment at the surface to 0.1×10⁹ cells/g dry weight at 2 m in depth. This relatively high methanogenic biomass implies that current microbial degradation of organic carbon in Ace Lake sediments may occur at extremely slow rates. Total microbial biomass increased from 4.4×10⁸ cells/ liter at 2 m in depth to 19.4×10⁸ cells/liter at 23 m, near the bottom of the water column. Total nonarchaebacterial biomass decreased from 4.2 ×10⁹ cells/g dry weight in the surface sediment (1/4 the biomass of methanogens) to 0.06×10⁸ cells/g dry weight at 2 m in depth in the sediment. Phospholipid fatty acid profiles showed that microeukaryotes were the major microbial group present in the oxylimnion of the lake, while bacteria dominated the lower, anoxic zone. Sulfate-reducing bacteria (SRB) comprised 25% of the microbial population at 23 m in depth in the water column particulates and were present in the surface sediment but to a lesser extent. Biomass estimates and community structure of the Ace Lake eco-system are discussed in relation to previously measured metabolic rates for this and other antarctic and temperate ecosystems. This is the first instance, to our knowledge, in which the viable biomass of methanogenic and SRB have been estimated for an antarctic microbial community.     

Mammal community structure correlates with arboreal heterogeneity in faunally and geographically diverse habitats: implications for community convergence

Aim To quantify how mammal community structure relates to heterogeneity of vegetation for palaeoecological reconstructions, and to test whether historical or environmental factors are more important in structuring communities. Location Sixty‐three natural protected areas in Asia, Africa and South and Central America. Methods We defined faunal communities by allocating species to ecological guilds and calculating proportional representation within each guild. Vegetation heterogeneity for each natural protected area was calculated from satellite images. The relationship between these ecospaces was calculated using canonical correlations analysis, redundancy analysis and principal components analysis. We expected that large, herbivorous mammals would be most strongly correlated with open areas. Convergence was tested by independently eliminating the effects of geography and vegetation heterogeneity on the structure of the mammal communities. We expected that vegetation would more strongly structure communities than geographical position. Results We show that the guild structure of communities across habitats is significantly correlated with vegetation heterogeneity. The highest correlation was between small, scansorial‐arboreal secondary consumers and heavy tree cover. The first convergence analysis shows American communities distinguished from Asian and African communities; these latter communities show a remarkable convergence in structure. Historical factors only affected the continent whose mammals had experienced a long period of isolation. The second convergence analysis shows that almost all biomes have the same or very similar community structure regardless of continent. Main conclusions Communities from the same environments in different continents showed remarkable convergence. Communities from the same continents only converged when those continents shared a recent geological and biological history. These results suggest that historical and environmental factors are operating over different timescales. This study confirms that environmental reconstructions made on the basis of whole communities will accurately reflect the environment that the community lived in. However, reconstructions made for fossil sites in deep time need to take historical factors into consideration. Small, arboreal and scansorial secondary consumers show the strongest correlation with vegetation, correlating with continuous tree canopy cover. This relationship allows simple reconstructions of the amount of tree cover occurring in a landscape from the proportion of species from the community falling in this ecological guild.     

Detection of active butyrate-degrading microorganisms in methanogenic sludges by RNA-based stable isotope probing

Butyrate-degrading bacteria in four methanogenic sludges were studied by RNA-based stable isotope probing. Bacterial populations in the (13)C-labeled rRNA fractions were distinct from unlabeled fractions, and Syntrophaceae species, Tepidanaerobacter sp., and Clostridium spp. dominated. These results suggest that diverse microbes were active in butyrate degradation under methanogenic conditions.     

Alternative stable states in communities with intraguild predation

Intraguild predation (IGP) is a widespread ecological phenomenon in which two consumers that share a resource also engage in a predator-prey interaction. Theory on IGP predicts the occurrence of alternative stable states, but empirical evidence of such states is scarce. This raises the question of whether alternative states are a rare phenomenon that is unlikely to be observed in nature. Here we analyze a model in which the resource exhibits logistic or chemostat dynamics and consumers have saturating (Type II) functional responses. We show that alternative states can arise for a wide range of biological scenarios and that environmental constraints can make their detection difficult. Our analysis identifies three possible combinations of alternative states: (i) IG prey or IG predator, (ii) coexistence or IG predator, and (iii) coexistence or IG prey. Bifurcation diagrams reveal that alternative states are possible over large regions of the parameter space. However, they can be limited to narrow ranges along the resource productivity axis, which may make it difficult to observe the occurrence of alternative states in communities with IGP. Microcosm experiments provide a promising avenue for detecting combinations of asymptotically stable states along a productivity gradient.     

历山森林群落物种多样性与群落结构研究

采用多样性指数、丰富度指数和均匀度指数对山西历山森林群落物种多样性进行了研究.结果表明,各群落类型的物种多样性指数的顺序为：青榨槭＋五角枫林>青榨槭＋鹅耳枥林>辽东栎林>栓皮栎林>华山松＋红桦林>油松＋栎林>白桦林>侧柏＋栓皮栎林>红桦林>华山松林>侧柏林>油松林>红桦＋山杨林;森林群落灌木层多样性指数和丰富度指数大于草本层和乔木层,而均匀度指数在乔木层、灌木层和草本层则表现出多样化的趋势;海拔1 000～1 920 m之间,Shannon-Wiener指数、Hill多样性指数及物种丰富度与海拔梯度均呈正相关(P<0.05),海拔大于1 920 m则呈现负相关;这些指数沿海拔梯度的变化呈现出“中间高度膨胀”的规律,即中等海拔高度上物种多样性高而低海拔和高海拔物种多样性低.这主要是由于在中海拔（约1 900 m）地段水热条件组合较好,人类活动干扰较少所致.     

Activity, structure and dynamics of the methanogenic archaeal community in a flooded Italian rice field

Methane production was studied in an Italian rice field over two consecutive years (1998, 1999) by measuring the rates of total and acetate-dependent methanogenesis in soil and root samples. Population dynamics of methanogens were followed by terminal restriction fragment length polymorphism and real-time PCR targeting archaeal SSU rRNA genes. Rates of total and acetate-dependent methanogenesis in soil increased during the season, reached a maximum at about 70-80 days after flooding and then decreased again. In contrast, the size of the archaeal community remained relatively constant. Therefore, the seasonal changes in the methanogenic processes were probably not caused by changes in the size of the methanogenic community but in its activity. During the 1998/1999 winter period, a slight decrease in archaeal cell numbers was found. In both years, the dominant groups were methanogens affiliated with Rice cluster I, Methanosaetaceae, Methanosarcinaceae and Methanobacteriaceae. Correspondence analysis showed, however, that the archaeal community structure was different in 1998 and 1999. Methanogens with potential acetoclastic activity made up a larger fraction of the total archaeal community in 1999 (32-53%) than in 1998 (20-32%). Furthermore, the frequency of Methanosaetaceae relative to Methanosarcinaceae was significantly higher in 1999 than in 1998. This difference could be explained by the much lower soil acetate concentrations in 1999, to which Methanosaetaceae are physiologically better adapted than Methanosarcinaceae. Over the season, however, the composition of the archaeal community remained relatively constant and thus did not reflect the observed seasonal change in CH(4) production activity. The analysis of rice root samples in 1999 showed that the archaeal community structure on the roots was similar to that in soil but with acetoclastic methanogens being relatively less common. This observation is in agreement with domination of CH(4) production by H(2)/CO(2)-dependent methanogenesis on roots. Our study provided a link between size, structure and function of the methanogenic community in an Italian rice field.