Indicative Marker Microbiome Structures Deduced from the Taxonomic Inventory of 67 Full-Scale Anaerobic Digesters of 49 Agricultural Biogas Plants.

There are almost 9500 biogas plants in Germany, which are predominantly operated with energy crops and residues from livestock husbandry over the last two decades. In the future, biogas plants must be enabled to use a much broader range of input materials in a flexible and demand-oriented manner. Hence, the microbial communities will be exposed to frequently varying process conditions, while an overall stable process must be ensured. To accompany this transition, there is the need to better understand how biogas microbiomes respond to management measures and how these responses affect the process efficiency. Therefore, 67 microbiomes originating from 49 agricultural, full-scale biogas plants were taxonomically investigated by 16S rRNA gene amplicon sequencing. These microbiomes were separated into three distinct clusters and one group of outliers, which are characterized by a specific distribution of 253 indicative taxa and their relative abundances. These indicative taxa seem to be adapted to specific process conditions which result from a different biogas plant operation. Based on these results, it seems to be possible to deduce/assess the general process condition of a biogas digester based solely on the microbiome structure, in particular on the distribution of specific indicative taxa, and without knowing the corresponding operational and chemical process parameters. Perspectively, this could allow the development of detection systems and advanced process models considering the microbial diversity.

Microbiome Diversity and Community-Level Change Points within Manure-based small Biogas Plants.

Efforts to integrate biogas plants into bioeconomy concepts will lead to an expansion of manure-based (small) biogas plants, while their operation is challenging due to critical characteristics of some types of livestock manure. For a better process understanding, in this study, three manure-based small biogas plants were investigated with emphasis on microbiome diversity. Due to varying digester types, feedstocks, and process conditions, 16S rRNA gene amplicon sequencing showed differences in the taxonomic composition. Dynamic variations of each investigated biogas plant microbiome over time were analyzed by terminal restriction fragment length polymorphism (TRFLP), whereby nonmetric multidimensional scaling (NMDS) revealed two well-running systems, one of them with a high share of chicken manure, and one unstable system. By using Threshold Indicator Taxa Analysis (TITAN), community-level change points at ammonium and ammonia concentrations of 2.25 g L-1 and 193 mg L-1 or volatile fatty acid concentrations of 0.75 g L-1were reliably identified which are lower than the commonly reported thresholds for critical process stages based on chemical parameters. Although a change in the microbiome structure does not necessarily indicate an upcoming critical process stage, the recorded community-level change points might be a first indication to carefully observe the process.

Impact of feeding and stirring regimes on the internal stratification of microbial communities in the fermenter of anaerobic digestion plants.

In anaerobic digestion plants (ADs), homogenization of the feed, fermenter content and microbial communities is crucial for efficient and robust biogas production. However, mixing also requires a significant amount of energy. For an 850 m3 agricultural AD equipped with eight sampling ports, we investigated whether different feeding and stirring regimes enable a sufficient homogenization of the microbial community using metaproteomics and terminal restriction fragment length polymorphism (TRFLP) analysis. Systematic comparison of samples taken at the top and the bottom as well as at the rim and the center of the AD using scatter plots and students t-test revealed only a small number of differences in metaproteins, taxonomies and biological processes. Obviously, the applied stirring and feeding conditions were sufficient to largely homogenize the content of the AD.

Effect of a Profound Feedstock Change on the Structure and Performance of Biogas Microbiomes.

In this study the response of biogas-producing microbiomes to a profound feedstock change was investigated. The microbiomes were adapted to the digestion of either 100% sugar beet, maize silage, or of the silages with elevated amounts of total ammonium nitrogen (TAN) by adding ammonium carbonate or animal manure. The feedstock exchange resulted in a short-range decrease or increase in the biogas yields according to the level of chemical feedstock complexity. Fifteen taxa were found in all reactors and can be considered as generalists. Thirteen taxa were detected in the reactors operated with low TAN and six in the reactors with high TAN concentration. Taxa assigned to the phylum Bacteroidetes and to the order Spirochaetales increased with the exchange to sugar beet silage, indicating an affinity to easily degradable compounds. The recorded TAN-sensitive taxa (phylum Cloacimonetes) showed no specific affinity to maize or sugar beet silage. The archaeal community remained unchanged. The reported findings showed a smooth adaptation of the microbial communities, without a profound negative impact on the overall biogas production indicating that the two feedstocks, sugar beet and maize silage, potentially do not contain chemical compounds that are difficult to handle during anaerobic digestion.

Nexus between the microbial diversity level and the stress tolerance within the biogas process.

To investigate whether there is a nexus between the microbial diversity level (taxonomic, functional and ecological) and the stress tolerance potential of the microbial community, the impact of different ammonium sources was evaluated. Therefore reactors adapted either to the anaerobic digestions of sugar beet silage or maize silage (SBS/MS) were supplemented with animal manure (M) or ammonium carbonate (A). The results showed that increasing concentrations of total ammonium nitrogen (TAN) were not the only reason for community changes: the bacterial community in the reactors given animal manure became more similar over time compared to the reactors given ammonium carbonate. However, this study revealed that a bacterial community with a few dominant members led to a functional more flexible archaeal community (SBS reactors) which was more stress resistant under the experimental conditions. This indicates that a higher functional diversity within a certain part of the community, in the present study the archaeal community, is one important factor for process stability due to a higher tolerance to increasing amounts of process-inhibiting metabolites such as TAN. Compared to this a bacterial community with higher amount of more evenly distributed community members combined with a more rigid archaeal community (MS reactors) showed a lower stress tolerance potential. Moreover it was observed that the disappearance of members of the phylum Cloacimonetes can be used as an indicator for an upcoming process disturbance due to increasing TAN concentrations.

Terminal restriction fragment length polymorphism is an "old school" reliable technique for swift microbial community screening in anaerobic digestion.

The microbial community in anaerobic digestion has been analysed through microbial fingerprinting techniques, such as terminal restriction fragment length polymorphism (TRFLP), for decades. In the last decade, high-throughput 16S rRNA gene amplicon sequencing has replaced these techniques, but the time-consuming and complex nature of high-throughput techniques is a potential bottleneck for full-scale anaerobic digestion application, when monitoring community dynamics. Here, the bacterial and archaeal TRFLP profiles were compared with 16S rRNA gene amplicon profiles (Illumina platform) of 25 full-scale anaerobic digestion plants. The α-diversity analysis revealed a higher richness based on Illumina data, compared with the TRFLP data. This coincided with a clear difference in community organisation, Pareto distribution, and co-occurrence network statistics, i.e., betweenness centrality and normalised degree. The ß-diversity analysis showed a similar clustering profile for the Illumina, bacterial TRFLP and archaeal TRFLP data, based on different distance measures and independent of phylogenetic identification, with pH and temperature as the two key operational parameters determining microbial community composition. The combined knowledge of temporal dynamics and projected clustering in the ß-diversity profile, based on the TRFLP data, distinctly showed that TRFLP is a reliable technique for swift microbial community dynamics screening in full-scale anaerobic digestion plants.

Marker microbiome clusters are determined by operational parameters and specific key taxa combinations in anaerobic digestion.

In this study, microbiomes of 36 full-scale anaerobic digesters originated from 22 different biogas plants were compared by terminal restriction fragment length polymorphism (TRFLP) analysis. Regarding the differences in microbial community composition, a weighting of the environmental parameters could be derived from higher to lower importance as follows: (i) temperature, (ii) TAN and NH3 concentrations and conductivity, and (iii) the chemical composition of the supplied feedstocks. Biotic interactions between specific bacterial and archaeal community arrangements were revealed, whereby members of the phyla Bacteroidetes and Cloacimonetes combined with the archaeal genus Methanothrix dominated the conversion of homogeneous feedstocks, such as waste water sludge or industrial waste. As most of the detected TRFs were only found in a certain number of anaerobic digestion plants, each plant develops its unique microbiome. The putative rare species, the specialists, are potentially hidden drivers of microbiome functioning as they provide necessary traits under, e.g., process-inconvenient conditions.

Prokaryote community dynamics in anaerobic co-digestion of swine manure, rice straw and industrial clay residuals.

The aim of this study was to analyze the effect of the addition of rice straw and clay residuals on the prokaryote methane-producing community structure in a semi-continuously stirred tank reactor fed with swine manure. Molecular techniques, including terminal restriction fragment length polymorphism and a comparative nucleotide sequence analyses of the prokaryotic 16S rRNA genes, were performed. The results showed a positive effect of clay addition on methane yield during the co-digestion of swine manure and rice straw. At the digestion of swine manure, the bacterial phylum Firmicutes and the archaeal family Methanosarcinaceae, particularly Methanosarcina species, were predominant. During the co-digestion of swine manure and rice straw the microbial community changed, and with the addition of clay residual, the phylum Bacteroidetes predominated. The new nutritional conditions resulted in a shift in the archaeal family Methanosarcinaceae community as acetoclastic Methanosaeta species became dominant.

Reorganisation of a mesophilic biogas microbiome as response to a stepwise increase of ammonium nitrogen induced by poultry manure supply.

An anaerobic digestion experiment was investigated to evaluate the impact of increasing amounts of ammonium nitrogen due to poultry manure addition on the reactor performance, especially on the microbiome response. The microbial community structure was assessed by using a 16S rRNA gene approach, which was further correlated with the prevalent environmental conditions by using statistical analyses. The addition of 50% poultry manure led to a process disturbance indicated by a high VFA content (almost 10 g(HAc-Eq) L(-1)) in combination with elevated concentrations of ammonium nitrogen (5.9 g NH4(+)-N kg(FM)(-1)) and free ammonia (0.5 g NH3 kg(FM)(-1)). Simultaneously the microbiome, changed from a Bacteroidetes-dominated to a Clostridiales-dominated community accompanied by a shift from the acetoclastic to the hydrogenotrophic pathway. The "new" microbial community was functional redundant as the overall process rates were similar to the former one. A further increase of poultry manure resulted in a complete process failure.

Community shifts in a well-operating agricultural biogas plant: how process variations are handled by the microbiome.

This study provides a comprehensive, long-term microbiological study of a continuously operated, mesophilic, agricultural biogas plant fed with whole-crop silages of maize and rye, cattle manure and cattle slurry. The microbial community structure was accessed by high-throughput 16S rRNA gene amplicon sequencing. For the characterisation of the microbial dynamics, the community profiling method terminal restriction fragment length polymorphism (TRFLP) in combination with a cloning-sequencing approach as well as a LC-MS/MS approach for protein identification were applied. Our results revealed that the anaerobic digestion is a highly sensitive process: small variations in the process performance induce fluctuations in the microbial community composition and activity. In this context, it could be proven that certain microbial species were better adapted to changing process condition such as temperature (interspecies competition) and that there is a physiological compensation between different microorganisms so that the reactor efficiency was not adversely affected despite of structural and functional changes within the microbial community.

Dynamic variation of the microbial community structure during the long-time mono-fermentation of maize and sugar beet silage.

This study investigated the development of the microbial community during a long-term (337 days) anaerobic digestion of maize and sugar beet silage, two feedstocks that significantly differ in their chemical composition. For the characterization of the microbial dynamics, the community profiling method terminal restriction fragment length polymorphism (TRFLP) in combination with a cloning-sequencing approach was applied. Our results revealed a specific adaptation of the microbial community to the supplied feedstocks. Based on the high amount of complex compounds, the anaerobic conversion rate of maize silage was slightly lower compared with the sugar beet silage. It was demonstrated that members from the phylum Bacteroidetes are mainly involved in the degradation of low molecular weight substances such as sugar, ethanol and acetate, the main compounds of the sugar beet silage. It was further shown that species of the genus Methanosaeta are highly sensitive against sudden stress situations such as a strong decrease in the ammonium nitrogen (NH4(+)-N) concentration or a drop of the pH value. In both cases, a functional compensation by members of the genera Methanoculleus and/or Methanosarcina was detected. However, the overall biomass conversion of both feedstocks proceeded efficiently as a steady state between acid production and consumption was recorded, which further resulted in an equal biogas yield.

Response of recalcitrant soil substances to reduced N deposition in a spruce forest soil: integrating laccase-encoding genes and lignin decomposition.

A long-term field experiment conducted in a Norway spruce forest at Solling, Central Germany, was used to verify and compare the response of lignin-decomposing fungal communities in soils receiving current and preindustrial atmospheric nitrogen (N) input for 14.5 years. Therefore, we investigated the decomposition of lignin compounds in relation to phenol oxidase activity and the diversity of basidiomycetes containing laccase genes in organic and mineral horizons. Lignin-derived CuO oxidation products and enzyme activity decreased with soil depth, while the degree of oxidative transformation of lignin increased. These patterns did not change with reduced atmospheric N input, likely reflecting a lasting saturation in available N. The laccase gene diversity decreased with soil depth in spring. In autumn, this pattern was only found in the control plot, receiving current N input. Principal component analysis confirmed the depth profile and distinguished a response of the fungal community to reduced N deposition for most organic layers in spring and a roof effect for the Oe layer in autumn. These responses of the fungal community did not translate into changes in enzyme activity and lignin content and decomposition, suggesting that transformation processes in soils are well buffered despite the rapid response of the microbial community to environmental factors.

Towards a universally adaptable method for quantitative extraction of high-purity nucleic acids from soil.

A universally adaptable protocol for quantitative extraction of high-purity nucleic acids from soil is presented. A major problem regarding the extraction of nucleic acids from soil is the presence of humic substances, which interfere with the extraction process itself and in subsequent analytical manipulations. By the approach described here, the humic compounds are precipitated prior to cell lysis with Al(2)(SO(4))(3), and thus eliminated prior to the nucleic acid extraction. The protocol allows for removing of a considerable content and range of humic acids and should therefore be applicable for a wide spectrum of soil types. Accordingly, reproducible results in analyses of different soil types are made possible, inclusively for quantitative comparisons.