Characterization of Bathyarchaeota genomes assembled from metagenomes of biofilms residing in mesophilic and thermophilic biogas reactors.

BACKGROUND: Previous studies on the Miscellaneous Crenarchaeota Group, recently assigned to the novel archaeal phylum Bathyarchaeota, reported on the dominance of these Archaea within the anaerobic carbohydrate cycle performed by the deep marine biosphere. For the first time, members of this phylum were identified also in mesophilic and thermophilic biogas-forming biofilms and characterized in detail. RESULTS: Metagenome shotgun libraries of biofilm microbiomes were sequenced using the Illumina MiSeq system. Taxonomic classification revealed that between 0.1 and 2% of all classified sequences were assigned to Bathyarchaeota. Individual metagenome assemblies followed by genome binning resulted in the reconstruction of five metagenome-assembled genomes (MAGs) of Bathyarchaeota. MAGs were estimated to be 65-92% complete, ranging in their genome sizes from 1.1 to 2.0 Mb. Phylogenetic classification based on core gene sets confirmed their placement within the phylum Bathyarchaeota clustering as a separate group diverging from most of the recently known Bathyarchaeota clusters. The genetic repertoire of these MAGs indicated an energy metabolism based on carbohydrate and amino acid fermentation featuring the potential for extracellular hydrolysis of cellulose, cellobiose as well as proteins. In addition, corresponding transporter systems were identified. Furthermore, genes encoding enzymes for the utilization of carbon monoxide and/or carbon dioxide via the Wood-Ljungdahl pathway were detected. CONCLUSIONS: For the members of Bathyarchaeota detected in the biofilm microbiomes, a hydrolytic lifestyle is proposed. This is the first study indicating that Bathyarchaeota members contribute presumably to hydrolysis and subsequent fermentation of organic substrates within biotechnological biogas production processes.

Microbial communities involved in biogas production from wheat straw as the sole substrate within a two-phase solid-state anaerobic digestion.

Microbial communities involved in biogas production from wheat straw as the sole substrate were investigated. Anaerobic digestion was carried out within an up-flow anaerobic solid-state (UASS) reactor connected to an anaerobic filter (AF) by liquor recirculation. Two lab-scale reactor systems were operated simultaneously at 37 °C and 55 °C. The UASS reactors were fed at a fixed organic loading rate of 2.5 g L(-1) d(-1), based on volatile solids. Molecular genetic analyses of the bacterial and archaeal communities within the UASS reactors (digestate and effluent liquor) and the AFs (biofilm carrier and effluent liquor) were conducted under steady-state conditions. The thermophilic UASS reactor had a considerably higher biogas and methane yield in comparison to the mesophilic UASS, while the mesophilic AF was slightly more productive than the thermophilic AF. When the thermophilic and mesophilic community structures were compared, the thermophilic system was characterized by a higher Firmicutes to Bacteroidetes ratio, as revealed by 16S rRNA gene (rrs) sequence analysis. The composition of the archaeal communities was phase-separated under thermophilic conditions, but rather stage-specific under mesophilic conditions. Family- and order-specific real-time PCR of methanogenic Archaea supported the taxonomic distribution obtained by rrs sequence analysis. The higher anaerobic digestion efficiency of the thermophilic compared to the mesophilic UASS reactor was accompanied by a high abundance of Firmicutes and Methanosarcina sp. in the thermophilic UASS biofilm.

Use of biochars in anaerobic digestion.

This study investigated the behavior of biochars from pyrolysis (pyrochar) and hydrothermal carbonization (hydrochar) in anaerobic digestion regarding their degradability and their effects on biogas production and ammonia inhibition. A batch fermentation experiment (42°C, 63 days) was conducted in 100mL syringes filled with 30 g inoculum, 2g biochar and four levels of total ammonium nitrogen (TAN). For pyrochar, no clear effect on biogas production was observed, whereas hydrochar increased the methane yield by 32%. This correlates with the hydrochar's larger fraction of anaerobically degradable carbon (10.4% of total carbon, pyrochar: 0.6%). Kinetic and microbiota analyses revealed that pyrochar can prevent mild ammonia inhibition (2.1 g TANk g(-1)). Stronger inhibitions (3.1-6.6 g TAN kg(-1)) were not mitigated, neither by pyrochar nor by hydrochar. Future research should pay attention to biochar-microbe interactions and the effects in continuously-fed anaerobic digesters.

Development of a flow-fluorescence in situ hybridization protocol for the analysis of microbial communities in anaerobic fermentation liquor.

BACKGROUND: The production of bio-methane from renewable raw material is of high interest because of the increasing scarcity of fossil fuels. The process of biomethanation is based on the inter- and intraspecific metabolic activity of a highly diverse and dynamic microbial community. The community structure of the microbial biocenosis varies between different biogas reactors and the knowledge about these microbial communities is still fragmentary. However, up to now no approaches are available allowing a fast and reliable access to the microbial community structure. Hence, the aim of this study was to originate a Flow-FISH protocol, namely a combination of flow cytometry and fluorescence in situ hybridization, for the analysis of the metabolically active microorganisms in biogas reactor samples. With respect to the heterogenic texture of biogas reactor samples and to collect all cells including those of cell aggregates and biofilms the development of a preceding purification procedure was indispensable. RESULTS: Six different purification procedures with in total 29 modifications were tested. The optimized purification procedure combines the use of the detergent sodium hexametaphosphate with ultrasonic treatment and a final filtration step. By this treatment, the detachment of microbial cells from particles as well as the disbandment of cell aggregates was obtained at minimized cell loss. A Flow-FISH protocol was developed avoiding dehydration and minimizing centrifugation steps. In the exemplary application of this protocol on pure cultures as well as biogas reactor samples high hybridization rates were achieved for commonly established domain specific oligonucleotide probes enabling the specific detection of metabolically active bacteria and archaea. Cross hybridization and autofluorescence effects could be excluded by the use of a nonsense probe and negative controls, respectively. CONCLUSIONS: The approach described in this study enables for the first time the analysis of the metabolically active fraction of the microbial communities within biogas reactors by Flow-FISH.

Anaerobic digestion of wheat straw--performance of continuous solid-state digestion.

In this study the upflow anaerobic solid-state (UASS) reactor was operated at various conditions to optimize the process parameters for anaerobically digesting wheat straw in a continuous process. Additionally, particle size effects have been studied in the operation at 55 and 60°C. Moreover, the incremental effect of the organic loading rate (OLR) to the system was examined from 2.5 to 8 gVS L(-1) d(-1). It was found that the UASS operating at 60 °C with a small OLR yields highest methane production, but the advantage over thermophilic operation is negligible. The rise in OLR reduces the systems yields, as expected. From OLR=8 gVS L(-1) d(-1) a second stage is necessary to circumvent volatile fatty acids accumulation.

Thermo- and mesophilic anaerobic digestion of wheat straw by the upflow anaerobic solid-state (UASS) process.

In this experimental work, the feasibility of wheat straw as a feedstock for biogas production is investigated using the newly developed upflow anaerobic solid-state (UASS) process. With the analytical emphasis placed on methane and metabolite production, both mesophilic and thermophilic 39 L UASS reactors were operated for 218 days at an organic loading rate of 2.5 g(VS)L(-1)d(-1) using wheat straw as sole substrate. For improved methanization of soluble metabolites, each UASS reactor was connected to an individual 30 L anaerobic filter (AF). During steady state thermophilic straw digestion was found to have a 36% higher methane yield (0.165 L g(VS)(-1)) whereas the hydrolysis rate constant increased by 106% (0.066 d(-1)).