File Name: structure and function of biogas plant .zip
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Metrics details. One of the most promising technologies to sustainably produce energy and to mitigate greenhouse gas emissions from combustion of fossil energy carriers is the anaerobic digestion and biomethanation of organic raw material and waste towards biogas by highly diverse microbial consortia. In this context, the microbial systems ecology of thermophilic industrial-scale biogas plants is poorly understood.
Analysis of metagenome-derived 16S rRNA gene sequences revealed that the bacterial genera Defluviitoga 5. Among the Archaea , Methanoculleus 2. By applying 11 different cultivation strategies, 52 taxonomically different microbial isolates representing the classes Clostridia , Bacilli , Thermotogae , Methanomicrobia and Methanobacteria were obtained. The latter three metabolites are substrates for hydrogentrophic and acetoclastic archaeal methanogenesis. Obtained results showed that high abundance of microorganisms as deduced from metagenome analysis does not necessarily indicate high transcriptional or metabolic activity, and vice versa.
Additionally, it appeared that the microbiome of the investigated thermophilic biogas plant comprised a huge number of up to now unknown and insufficiently characterized species. As consequence of the Kyoto Protocol, approved in , and the therein specified urgent demand regarding reduction of greenhouse gas emissions, the policy for energy transition was intensified in Germany [ 1 ].
This strategy essentially implies utilization of renewable biomass for the generation of heat, electricity and fuels. In Germany approximately biogas plants are currently generating permanently more than 3.
The energy production from biogas avoids the emission of approximately In addition, bio-methane production from 'energy crops' and plant residues is the most efficient bioenergy production pathway [ 4 ]. Despite the ecological and economical importance of biogas generation, the microbial networks responsible for anaerobic digestion and biomethanation of biomass are still poorly understood.
The classical concept of understanding the biogas process from biomass to biogas is assuming a more or less linear degradation pathway beginning with the hydrolytic breakdown of complex biomass compounds towards short-chained volatile fatty acids VFA acidogenesis , which are subsequently converted mainly to acetic acid acetogenesis and gases, mainly CO 2 and molecular hydrogen H 2. An alternative pathway for methanogenesis is the H 2 -mediated reduction of CO 2 , released during acidogenesis or as product of the oxidation of acetate hydrogenotrophic methanogenesis.
Furthermore, advanced molecular analysis, such as high-throughput DNA sequencing of microbial 16S rRNA genes and microbial metagenomes derived from biogas plants, showed that the microbial community architecture is much more complex and may include up to several hundreds or even thousands of microbial species [ 8 , 9 ].
As an example, members of the phylum Firmicutes , namely several species of the genus Clostridium such as C. However, molecular studies pointed also to the participation of members of other phyla in the anaerobic degradation process, namely those of the phyla Chloroflexi , Proteobacteria , Synergistetes , and, predominantly, Bacteroidetes [ 11 — 15 ].
Both, microbiological and molecular studies for characterization of biogas communities were mostly applied on anaerobic digesters operated at mesophilic temperatures. Only few plants, i. Thermophilic plants have the reputation to be less stable than mesophilic ones. Due to the limited number of thermophilic biogas plants, studies on the associated microbial trophic networks are still limited and mostly focused on waste, wastewater or manure digesting plants [ 18 ].
Hence, thermophilic microbial consortia appear to be less well understood than mesophilic ones. Despite the undoubted advances in microbial ecology by the introduction of microbial metagenomics, -transcriptomics, and -proteomics, a major drawback of all these approaches is the huge number of un-assignable sequences [ 15 , 19 , 20 ].
This is due to the still highly limited availability of reference strains and their corresponding genomes in public databases. Consequently, for a detailed characterization of complex microbial consortia, commonly a polyphasic approach is recommended involving parallel application of both, traditional cultivation as well as molecular analyses.
In this study, for the first time, such a comprehensive polyphasic approach was applied to unravel the structure and the functionality of the microbial consortium within an industrial-scale thermophilic biogas plant optimized for anaerobic digestion and biomethanation of 'energy crops'.
The overall aim was the compilation of the core microbiome and its functional characterization for a thermophilic biogas plant. In total, every fermenter received 5. Feeding of substrates followed eight times per day. Trace elements, i. Picture a and flow chart b of the sampled thermophilic biogas plant.
Samples of 0. All samples were centrifuged and the supernatant was acidified by addition of ortho-phosphoric acid to obtain a final pH value of 2. Total VFA, i. The FID was used in the automatic and splitless mode. A weekly calibration was performed with a commercial external standard Supelco U, Sigma-Aldrich, Germany. DM and VS content were determined according to the standard guidelines, i. VDI protocols [ 22 ]. Microscopic determinations of cell morphologies and titers were performed with a DMB fluorescence microscope Leica, Germany fitted with a motorized and PC-controlled three-axis cross table.
For each sample, approximately 20 images were captured in succession in the chosen area at fold magnification. Shutter speed was Both values were kept for all experiments. Further details on the procedure were published previously [ 23 ]. Genomic DNA fragmentation into approx. In order to extract the total microbial RNA, the modified protocol published previously [ 19 ] was applied. The modifications are described in the Additional file 1. Obtained metagenomic raw sequences were quality filtered and separated by the different multiplex identifiers.
In total, five metagenome datasets were obtained, one for each DNA extraction method. Overlapping paired-end reads from each dataset were merged together applying the computational tool Flash [ 24 ]. Finally, all metagenome reads were compared with the ribosomal database project RDP database [ 26 ] in order to identify encoded 16S rRNA genes in the metagenome. Due to the lack of Defluviitoga 16S rRNA gene sequences in the RDP database at the point of analysis, additional investigation of Thermotogae sequences from the analyzed biogas plant was performed.
The occurrence of the carbohydrate-active enzymes was predicted using the carbohydrate-active enzyme database annotation web-server dbCAN [ 30 ] also applying MGX standard settings. For taxonomic characterization of the metabolically active biogas community the obtained metatranscriptome sequences were quality filtered and uploaded into MGX. The RDP database was used to identify all 16S rRNA gene transcripts obtained from the sequenced metatranscriptome for taxonomic profiling.
For cultivation and culture-based identification of members of the microbial community within the main digesters of the biogas plant, in total 11 different isolation strategies were applied with respect to the different trophic microbial groups involved in anaerobic digestion of biomass and subsequent biomethanation.
Exemplary work-flow pathways are depicted in the Additional file 1 : Figure S1. Details on the isolation strategies are provided as Additional file 1. Briefly, following protocols for isolation of fermentative Bacteria and for isolation of methanogenic Archaea were used.
The anaerobic culture conditions were generated using the AnaeroGen 2. Further details are given below. Isolation strategy 3 targeting cellulolytic Bacteria: GS2 medium [ 32 ] or mineral medium [ 33 ] supplemented with 0. Isolation strategy 4 targeting cellulolytic Bacteria: Similar to [ 35 ], with some modifications as described in detail in the Additional file 1. Isolation strategy 5 targeting cellulolytic Bacteria: Similar to strategy 3 , with the following modifications: the dilution of the suspended sludge was directly plated on agar plates containing 0.
To obtain pure cultures, the deep agar shake method was applied [ 37 ]. Single colonies were picked and re-streaked for purification. Isolation strategy 10 targeting methanogenic Archaea: A cultivation technique for strictly anaerobic microorganisms was performed in accordance to the recommendations by [ 42 ]. Details on the nutrient media and the applied antibiotics are provided as Additional file 1.
A combination of the antibiotics ampicillin and vancomycin was applied. Therefore, each colony with different morphology was analyzed. The Shimadzu Biotech Launchpad software Shimadzu Europe was used for spectra acquisition and peak detection. All settings and guidelines from the manufacturer for the standard application were used.
Bacteria and Archaea obtained with the isolation strategies 3 — 11 were identified by 16S rRNA gene sequence analysis using the EzTaxon identification tool [ 17 , 43 ]. Details are provided as Additional file 1. To quantify the abundance of the isolated strains within the thermophilic microbial community, combined metagenome and metatranscriptome sequences were mapped against the 16S rRNA gene sequence of the obtained isolates applying the gsMapper 2.
To determine the phylogenetic relationship between the different isolates and the corresponding closest related type strains, a phylogenetic tree was constructed based on 16S rRNA gene sequences applying the ARB program [ 44 ]. All sequences were aligned using the high-throughput multiple sequence alignment tool SINA [ 46 ]. Subsequently, the resulting multiple sequence alignment was introduced into the phylogenetic tree containing selected 16S rRNA gene sequences of previously described bacterial and archaeal type strains as provided by the SILVA database [ 47 ].
To detect phylogenetically different isolates, the 16S rRNA gene sequences of closely related isolates were compared to each other using the ARB distance matrix tool. For genome sequencing and analysis, three bacterial strains originating from the analyzed biogas plant, namely Clostridium resp. Ruminiclostridium cellulosi str. DG5 taxonomic denomination under revision [ 48 ], Herbinix hemicellulosilytica str.
L3 [ 27 , 37 ] were selected. DG5 and H. Obtained sequences were de novo assembled using the GS de novo Assembler software version 2.
Finally, an in silico gap closure approach was performed [ 50 ]. Gene prediction, annotation and pathway reconstruction of the sequenced genomes were accomplished using the GenDB platform [ 51 ]. To predict genes encoding carbohydrate-active enzymes the carbohydrate-active enzyme database CAZy annotation web-server dbCAN was used [ 30 ].
In order to determine species within the biogas plant affiliated to the strains C. DG5, H. L3, the corresponding metagenome sequences were mapped on these three genomes as described recently [ 52 ]. Therefore, the combined metagenome sequencing dataset see above was used, representing the thermophilic biogas-producing microbial community. Afterwards, the FR-HIT software tool [ 53 ] was used to perform a global alignment against the completely sequenced genomes of the strains described above.
Finally, the fragment recruitment was visualized by plotting the identity of the alignment against the alignment position on the corresponding genome sequence. To investigate the process performance of the biogas plant analyzed at the sampling time point, fermentation samples were analyzed regarding their physico-chemical characteristics.
The process fluid of the sampled digester, from which the microbiological analysis was performed, had the following physico-chemical characteristics: pH value 8. These findings correlate with the parameters of agricultural biogas plants previously described by Laaber et al.
It mainly comprises of hydro-carbon which is combustible and can produce heat and energy when burnt. Bio-gas is produced through a bio-chemical process in which certain types of bacteria convert the biological wastes into useful bio-gas. Since the useful gas originates from biological process, it has been termed as bio-gas. Methane gas is the main constituent of biogas. The process of bio-gas production is anaerobic in nature and takes place in two stages. The two stages have been termed as acid formation stage and methane formation stage. In the acid formation stage, the bio-degradable complex organic compounds present in the waste materials are acted upon by a group of acid forming bacteria present in the dung.
Shipping portfolio. How is biogas produced? We have a simple aim: we want to look after the environment and produce local renewable energy for our customers. Read more. Biogas is produced through the processing of various types of organic waste. The circular-economy impact of biogas production is further enhanced by the organic nutrients recovered in the production process.
The complexity of the microbial communities and metabolic pathways involved in the microbiological process of biogas production is poorly understood and numerous microorganisms in the fermentation sample of the biogas plant are still unclassified or unknown. The structure and function of microbial communities and the effects of the addition of trace elements are needed to be known, to control and channel the energy sources microbes produce and to capture and store the useful by-products or for targeted screening of novel enzymes. In this review, we discussed an emerging idea that Metagenome sequence data from a biogas-producing microbial community residing in a fermenter of a biogas plant provide the basis for a rational approach to improve the biotechnological process of biogas production. The composition and gene content of a biogas-producing consortium can be determined through metagenomic approach which allows the design of the optimal microbial community structure for any biogas plant for the significant progress in the efficacy and economic improvement of biogas production and biofertilizer of either balanced nutrition or rich in specific element for plant growth produced from the sludge of biogas plant. Biogas-producing microbial community from different production-scale biogas plants supplied with different raw materials as substrates can be analyzed by polyphasic approach to find out the best raw material composition for biogas production.
Metrics details. One of the most promising technologies to sustainably produce energy and to mitigate greenhouse gas emissions from combustion of fossil energy carriers is the anaerobic digestion and biomethanation of organic raw material and waste towards biogas by highly diverse microbial consortia. In this context, the microbial systems ecology of thermophilic industrial-scale biogas plants is poorly understood. Analysis of metagenome-derived 16S rRNA gene sequences revealed that the bacterial genera Defluviitoga 5. Among the Archaea , Methanoculleus 2. By applying 11 different cultivation strategies, 52 taxonomically different microbial isolates representing the classes Clostridia , Bacilli , Thermotogae , Methanomicrobia and Methanobacteria were obtained. The latter three metabolites are substrates for hydrogentrophic and acetoclastic archaeal methanogenesis.
It mainly comprises of hydro-carbon which is combustible and can produce heat and energy when burnt. Bio-gas is produced through a bio-chemical process in which certain types of bacteria convert the biological wastes into useful bio-gas.