Accessing the Black Box of Microbial Diversity and Ecophysiology: Recent Advances Through Polyphasic Experiments

156

Knowledge of the microbial consortia participating in the generation of biogas, especially in methane formation, is still limited. To overcome this limitation, the methanogenic archaeal communities in six full-scale biogas plants supplied with different liquid manures and renewable raw materials as substrates were analyzed by a polyphasic approach. Fluorescence in situ hybridization (FISH) was carried out to quantify the methanogenic Archaea in the reactor samples. In addition, quantitative real-time PCR (Q-PCR) was used to support and complete the FISH analysis. Five of the six biogas reactors were dominated by hydrogenotrophic Methanomicrobiales. The average values were between 60 to 63% of archaeal cell counts (FISH) and 61 to 99% of archaeal 16S rRNA gene copies (Q-PCR). Within this order, Methanoculleus was found to be the predominant genus as determined by amplified rRNA gene restriction analysis. The aceticlastic family Methanosaetaceae was determined to be the dominant methanogenic group in only one biogas reactor, with average values for Q-PCR and FISH between 64% and 72%. Additionally, in three biogas reactors hitherto uncharacterized but potentially methanogenic species were detected. They showed closest accordance with nucleotide sequences of the hitherto unclassified CA-11 (85%) and ARC-I (98%) clusters. These results point to hydrogenotrophic methanogenesis as a predominant pathway for methane synthesis in five of the six analyzed biogas plants. In addition, a correlation between the absence of Methanosaetaceae in the biogas reactors and high concentrations of total ammonia (sum of NH 3 and NH 4 ؉ ) was observed.During the last decade the production of biogas from organic materials and residues has increased continuously in order to reduce the greenhouse gas emission resulting from the use of fossil energy sources. The energy-bearing substance of biogas is methane, which is produced as an end product of microbial anaerobic degradation of organic substrates, such as energy crops like maize, grains, grasses, or beets. Research for optimization of biogas production from renewable materials was initially focused on the evaluation of substrate eligibility and on the development and optimization of technical systems. However, biogas formation primarily depends on the structure and activity of the microbial community (28).The key microorganisms in the biogas formation process are the methane-generating microorganisms (methanogens). The capacity for methanogenesis is limited to members of the domain Archaea and, within this domain, on the phylum Euryarchaeota. With respect to the main metabolic precursors used, methanogens are usually divided into two groups: the aceticlastic methanogens that strictly metabolize acetate and the hydrogenotrophic methanogens that use H 2 or formate as an electron donor and CO 2 as a carbon source for their metabolism. Besides these major groups, certain methanogens are also able to convert methyl groups, methylamines, or methanol to methane (23,40). The substrates for the me...

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confidence: 99%

Polyphasic Analyses of Methanogenic Archaeal Communities in Agricultural Biogas Plants

Nettmann

Bergmann

Pramschüfer

et al. 2010

156

“…This probably reflects the metabolic flexibility of bacteria and the range of available substrates in complex input materials. However, several recent discoveries pose the question as to whether archaeal diversity and physiological versatility are greater than currently thought: that is, the huge diversity of yet-to-be cultured archaea (4,6), the detection of energy metabolisms not known previously in archaea (e.g., chemoorganotrophy [1]), and the unexpected predominance of archaeal groups among prokaryotes in unstressed environments, such as ammonia oxidizers in soils (19).…”

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confidence: 99%

“…Hypothetically, this is due to the functional redundancy among diverse phylogenetic groups allowing oscillations of their populations with no effects on the reactor function (2). Archaeal communities are less dynamic than bacterial communities (32), their shifts being related to changes in reactor performance (6) and correlated with important process parameters such as volatile fatty acids (VFAs) (13,16).…”

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confidence: 99%

Effect of Biowaste Sludge Maturation on the Diversity of Thermophilic Bacteria and Archaea in an Anaerobic Reactor

Goberna

Insam

Franke‐Whittle

2009

110

Prokaryotic diversity was investigated near the inlet and outlet of a plug-flow reactor. After analyzing 800 clones, 50 bacterial and 3 archaeal phylogenetic groups were defined. Clostridia (>92%) dominated among bacteria and Methanoculleus (>90%) among archaea. Significant changes in pH and volatile fatty acids did not invoke a major shift in the phylogenetic groups. We suggest that the environmental filter imposed by the saline conditions (20 g liter ؊1 ) selected a stable community of halotolerant and halophilic prokaryotes.The anaerobic digestion of organic wastes constitutes a major research focus due to the global needs for waste recycling and renewable energy production. Currently, the linkage between digester performance and the diversity and dynamics of anaerobic prokaryotes is still not fully understood (2). Bacterial diversity in anaerobic reactors has always been judged to be greater than archaeal diversity (9,13,30). This probably reflects the metabolic flexibility of bacteria and the range of available substrates in complex input materials. However, several recent discoveries pose the question as to whether archaeal diversity and physiological versatility are greater than currently thought: that is, the huge diversity of yet-to-be cultured archaea (4, 6), the detection of energy metabolisms not known previously in archaea (e.g., chemoorganotrophy [1]), and the unexpected predominance of archaeal groups among prokaryotes in unstressed environments, such as ammonia oxidizers in soils (19).Several surveys have investigated the shifts in prokaryotic diversity occurring with waste maturation or under different reactor operating conditions. Some evidence demonstrates bacterial phylogenetic stability under constant operation conditions (18). Generally, however, the dominant bacterial communities are very dynamic, showing chaotic shifts even with stable reactor performance (9, 32). Hypothetically, this is due to the functional redundancy among diverse phylogenetic groups allowing oscillations of their populations with no effects on the reactor function (2). Archaeal communities are less dynamic than bacterial communities (32), their shifts being related to changes in reactor performance (6) and correlated with important process parameters such as volatile fatty acids (VFAs) (13,16).We aimed to analyze the change in prokaryotic diversity in a plug-flow reactor associated with the maturation of biowastes. In a previous study, stable bacterial and archaeal denaturing gradient gel electrophoresis patterns were found in the sludge collected close to the outlet over a year of unstable reactor performance (23). This temporal pattern contradicts the general idea of extremely dynamic bacterial communities proliferating in bioreactors. Here, we investigated the phylogenetic identity of the organisms in sludge samples collected near the inlet and outlet pipes after a period of stable operation and performance in terms of pH and biogas production.Operation of the bioreactor and sampling of sludges. Samples were taken ...

“…However, studies elucidating the population dynamics of both acetotrophic and hydrogenotrophic methanogens during the anaerobic digestion of particulate solid biomass for biogas production are rather scarce. These population processes remain somewhat of a "black box" (12) due to the lack of data concerning the microbial consortia involved therein. Molecular biological techniques such as those targeting the 16S rRNA gene represent a valuable addition to culture-based techniques for studying the biodiversity and structure of complex microbial communities.…”

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confidence: 99%

Anaerobic Digestion of Renewable Biomass: Thermophilic Temperature Governs Methanogen Population Dynamics

Krakat

Westphal

Schmidt

et al. 2010

113

Beet silage and beet juice were digested continuously as representative energy crops in a thermophilic biogas fermentor for more than 7 years. Fluorescence microscopy of 15 samples covering a period of 650 days revealed that a decrease in temperature from 60°C to 55°C converted a morphologically uniform archaeal population (rods) into a population of methanogens exhibiting different cellular morphologies (rods and coccoid cells). A subsequent temperature increase back to 60°C reestablished the uniform morphology of methanogens observed in the previous 60°C period. In order to verify these observations, representative samples were investigated by amplified rRNA gene restriction analysis (ARDRA) and fluorescence in situ hybridization (FISH). Both methods confirmed the temperature-dependent population shift observed by fluorescence microscopy. Moreover, all samples investigated demonstrated that hydrogenotrophic Methanobacteriales dominated in the fermentor, as 29 of 34 identified operational taxonomic units (OTUs) were assigned to this order. This apparent discrimination of acetoclastic methanogens contradicts common models for anaerobic digestion processes, such as anaerobic digestion model 1 (ADM1), which describes the acetotrophic Euryarchaeota as predominant organisms.The replacement of fossil fuels by renewable energy sources such as agricultural crops is gaining momentum internationally as a means to decrease emissions from conventional fuel sources impacting global warming (39). Thereby, biogasification using energy crops is the only fuel-producing process with a closed CO 2 and nutrient cycle (8). The production of biogas from plant waste or other organic materials is a feasible strategy in view of both ecology and economy (63). Fodder beet was chosen as the renewable biomass source for a thermophilic biogas fermentor because the European Union decreased the regulatory price for sugar beets in 2006, and therefore many farmers are looking for an alternative use. Furthermore, fodder beet was considered an attractive renewable energy crop due to its high methane yield per hectare (67), as well as the ideal ensiling conditions enabling the storage of beet silage for many years. Furthermore, the sugar beet was only recently identified as one of the most sustainable energy crops with regard to its water footprint when used for biofuel production (22).A long-term experiment was started on 4 July 2001 (see reference 48 for startup details), and the same biogas fermentors are still running stable due to the use of fuzzy logic control (16,48). During the conversion of biomass to methane, four different microbial processes can be distinguished: hydrolysis, acidogenesis, acetogenesis, and methanogenesis (17, 69). Population changes might therefore impact the entire community by triggering an imbalance that is reflected in the bioreactor performance via accumulation of intermediates such as volatile fatty acids (mainly C 2 and C 3 ), via pH changes, or via reduced efficiency (52). This work focused on the methanogens w...