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 β-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 β-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

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

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

Theuerl S, Klang J, Hülsemann B, Mächtig T, Hassa J. Microbiome Diversity and Community-Level Change Points within Manure-based small Biogas Plants. Microorganisms. 2020;8(8).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

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 β-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 β-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. © 2018, The Author(s)

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.DFG, KL 2069/3-

The Future Agricultural Biogas Plant in Germany: A Vision

After nearly two decades of subsidized and energy crop-oriented development, agricultural biogas production in Germany is standing at a crossroads. Fundamental challenges need to be met. In this article we sketch a vision of a future agricultural biogas plant that is an integral part of the circular bioeconomy and works mainly on the base of residues. It is flexible with regard to feedstocks, digester operation, microbial communities and biogas output. It is modular in design and its operation is knowledge-based, information-driven and largely automated. It will be competitive with fossil energies and other renewable energies, profitable for farmers and plant operators and favorable for the national economy. In this paper we discuss the required contribution of research to achieve these aims.Peer Reviewe

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

Process Disturbances in Agricultural Biogas Production—Causes, Mechanisms and Effects on the Biogas Microbiome: A Review

Disturbances of the anaerobic digestion process reduce the economic and environmental performance of biogas systems. A better understanding of the highly complex process is of crucial importance in order to avoid disturbances. This review defines process disturbances as significant changes in the functionality within the microbial community leading to unacceptable and severe decreases in biogas production and requiring an active counteraction to be overcome. The main types of process disturbances in agricultural biogas production are classified as unfavorable process temperatures, fluctuations in the availability of macro- and micronutrients (feedstock variability), overload of the microbial degradation potential, process-related accumulation of inhibiting metabolites such as hydrogen (H2), ammonium/ammonia (NH4+/NH3) or hydrogen sulphide (H2S) and inhibition by other organic and inorganic toxicants. Causes, mechanisms and effects on the biogas microbiome are discussed. The need for a knowledge-based microbiome management to ensure a stable and efficient production of biogas with low susceptibility to disturbances is derived and an outlook on potential future process monitoring and control by means of microbial indicators is provided

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. © 2020 by the authors. Licensee MDPI, Basel, Switzerland