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

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

Maus I, Rumming M, Bergmann I, et al. Characterization of Bathyarchaeota genomes assembled from metagenomes of biofilms residing in mesophilic and thermophilic biogas reactors. Biotechnology for Biofuels. 2018;11(1): 167

Monitoring of methane producing microflora in full-scale biogas plants in rual area

Die Produktion von Biogas aus landwirtschaftlichen Primärprodukten oder Reststoffen stellt einen we-sentlichen Beitrag zur Reduktion des CO2-Ausstoßes sowie zur Entwicklung einer nachhaltigen Landbewirtschaftung dar. Gegenwärtig stehen im Fokus der Forschung die Entwicklung und Optimierung von Biogasreaktoren und Betriebstechniken sowie die Optimierung der Substratbereitstellung. Über die Zusammensetzung der an der Biogasbildung beteiligten mikrobiellen Lebensgemeinschaften in landwirt-schaftlichen Biogasanlagen gibt es jedoch bis heute nur wenige Informationen. Im Rahmen dieser Studie wurde die Struktur der methanogenen Biozönose in zehn landwirtschaftlichen Biogasanlagen, welche auf Basis von Nachwachsenden Rohstoffen (NawaRo) betrieben wurden, unter-sucht. Hierzu wurde ein polyphasischer Ansatz mit verschiedenen kultivierungsunabhängigen, molekularen Verfahren gewählt. Primär wurde eine PCR-RFLP Analyse der Nukleotidsequenz der 16S rDNA sowie des mcrA Gens durchgeführt. Die Bestimmung der relativen Häufigkeit der Methan bil-denden Mikroorganismen in den Proben erfolgte mittels quantitativer real-time PCR (Q-PCR) auf Basis gruppenspezifischer Primer für das 16S rDNA Gen. Für ausgewählte Biogasreaktoren wurde ergänzend eine mikroskopische Quantifizierung mittels Fluoreszenz in situ Hybridisierung (FISH) durchgeführt. Für die FISH wurden spezifische Sonden für die Domänen Bacteria (EUB338) und Archaea (ARCH915) so-wie für die Ordnungen Methanomicrobiales (MG1200), Methanobacteriales (MB311 und MB1174) und den Familien Methanosarcinaceae (Ms821) und Methanosaetaceae (Mx825) verwendet. Die Ergebnisse der Analysen zeigten, dass in neun der zehn untersuchten Biogasanlagen die hydroge-notrophen Methanogenen, repräsentiert durch Vertreter der Ordnungen Methanomicrobiales und Methanobacteriales, vorherrschend waren. In diesen Biogasreaktoren wurde die Gattung Methanoculleus (Ordnung Methanomicrobiales) als dominierende Gattung nachgewiesen. Nennenswerte Anteile an ace-toklastischen Methanbildnern, insbesondere der Gattung Methanosaeta, konnten für sechs der untersuchten zehn Anlagen nachgewiesen werden. Jedoch wurde nur in einer Biogasanlage diese Gattung als die dominierende Gruppe von Methanbildnern nachgewiesen. Diese Ergebnisse deuten darauf hin, dass in 90 % der untersuchten Biogasanlagen die hydrogenotrophe Methanogenese der vorrangige Stoff-wechselweg für die Methanproduktion ist. In dieser Studie konnte kein Zusammenhang zwischen den jeweils zur Biogasgewinnung eingesetzten Substraten und der Struktur der Archaea-Biozönose beobachtet werden. Ebenso wenig scheinen unter-schiedliche Verweilzeiten der Substrate sowie die Raumbelastung der Reaktoren einen Einfluss auf die methanogenen Archaea zu haben. Jedoch ließ sich ein negativer Einfluss hoher Ammonium- bzw. Am-moniakkonzentrationen auf das Wachstum acetoklastischer Methanbildner, insbesondere Methanosaeta spp., in den untersuchten landwirtschaftlichen Biogasanlagen feststellen. Die aus dieser Studie gewonnenen Erkenntnisse dienen dem grundlegenden Verständnis der methanoge-nen Lebensgemeinschaft in landwirtschaftlichen Biogasanlagen, und sollen somit zur weiteren Optimierung der Biogasgewinnung beitragen.Biogas production from agricultural main products or remnants provides a substantial contribution to the reduction of CO2 emissions but also to the development of a sustainable agriculture. Currently, the re-search focuses on development and optimization of biogas reactors and reactor performance as well as optimization of substrate eligibility. However, the knowledge of the structure and dynamics of microbial communities which are involved in the biogas production is still insufficient. Within this study, the structures of methanogenic biocoenosis in ten agricultural biogas plants which are operated on basis of renewable raw materials were analyzed. Therefore a polyphasic approach with differ-ent culture independent molecular genetic techniques was chosen. First, PCR-RFLP analyses of 16S rDNA and mcrA genes were conducted. For determination of the relative abundance of methane pro-ducing microorganisms in the samples a quantitative real-time PCR (Q-PCR) on basis of 16S rDNA group-specific primers was applied. Fluorescence in situ hybridization (FISH) was carried out for direct quantification of methanogenic cells in reactor contents of six of the ten biogas plants. FISH was carried out with specific probes for the domains Bacteria (EUB338) and Archaea (ARCH915), the orders Metha-nomicrobiales (MG1200) and Methanobacteriales (MB311 and MB1174) and the families Methanosarcinaceae (Ms821) and Methanosaetaceae (Mx825). The results of the analyses showed, that in nine of the ten biogas plants the hydrogenotrophic methano-gens, represented by the orders Methanomicrobiales and Methanobacteriales, were prevalent. The genus Methanoculleus (Methanomicrobiales) was the predominating genus in these biogas plants. Acetoclastic methanogens such as organisms of the genus Methanosaeta were found only in six of the ten analyzed biogas plants. However, in one biogas plant Methanosaeta was determined as the predominant methano-genic group. These results indicate that the hydrogenotrophic methanogenesis was the major metabolic pathway for methane production in 90 % of the analyzed biogas plants. In this study, no correlation between used substrates for biogas production and the structure of Archaea biocoenosis could be observed. Also, the retention time of substrates and the organic loading rate of reac-tors seemed to have no obvious effect on the methanogenic Archaea. However, in the analyzed biogas plants a negative influence of high ammonium and ammonia concentrations, respectively, on the growth of acetoclastic methanogens in particular Methanosaeta spp. was determined. The results of this study serve the basic understanding of the methanogen community in agricultural bio-gas plants and are to contribute to further optimization of biogas production

Monitoring der methanbildenden Mikroflora in Praxis-Biogasanlagen im ländlichen Raum : Analyse des Ist-Zustandes und Entwicklung eines quantitativen Nachweissystems

Die Produktion von Biogas aus landwirtschaftlichen Primärprodukten oder Reststoffen stellt einen wesentlichen Beitrag zur Reduktion des Co2-Ausstoßes sowie zur Entwicklung einer nachhaltigen Landbewirtschaftung dar. Im Rahmen dieses Projektes soll daher die Artenzusammensetzung der methanogenen Mikroflora in ausgewählten Praxis-Biogasanlagen anhand ihrer 16S rDNA analysisert werden

Development of a flow-fluorescence in situhybridization 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

Microspheres as Surrogate Helminth Eggs: A Comparative Labscale Sedimentation Study for Tap- and Wastewater

Re-use of water containing helminth eggs during irrigation for agricultural purposes poses health risks, and likewise during research, due to the potential of spreading on contact. Therefore, polystyrene latex microspheres could be used as surrogates for chemical or biological species during colloidal transport. The aim here is to compare the settling velocities of microspheres having varied surface coatings—that is, proteins A, G and A/G; with that of real helminth eggs obtained from literature. The settling velocities of the microspheres were experimentally determined in tap- and wastewater, as well as theoretically in tap water; which was found to be within the range of mean values for those experimentally determined. There were no differences amongst the microspheres types used for settling in wastewater (i.e., A = 0.072 ± 0.02; G = 0.060 ± 0.03; A/G = 0.053 ± 0.01 mm/s). The same applied for settling in tap water (i.e., A = 0.068 ± 0.02; G = 0.047 ± 0.004; A/G = 0.095 ± 0.02 mm/s), except for microsphere G being different from microsphere A/G. All three types of microspheres settled at velocities lower than that of the wastewater particles (=0.118 ± 0.03). T-test analyses of settling velocities of microspheres in both tap- and wastewater, versus that from literature (i.e., Ascaris, Trichuris and Oesophagostomum), showed that microsphere A and A/G may surrogate for Ascaris in tap water, the same as A/G for Oesophagostomum. In wastewater however, both microspheres A and G are a good fit for Trichuris

Effects of the Antibiotics Chlortetracycline and Enrofloxacin on the Anaerobic Digestion in Continuous Experiments

Significant quantities of antibiotics are used in modern livestock husbandry and are found in livestock waste. Such waste has been reported to exert inhibitory effects if used as a substrate in biogas facilities. The goal of this study is to analyze the inhibitory effect of the antibiotics chlortetracycline (CTC) and enrofloxacin (EFX) on biogas production with pig slurry. Antibiotic concentrations up to 8,000 mg kg−1 dry matter (DM) pig slurry were added in continuous fermentation tests. Impacts on methane production and on the microbial community structure were analyzed. The results clearly show that chlortetracycline and enrofloxacin negatively affect biogas production. Higher concentrations of antibiotics led to lower methane production. The addition of 200 mg kg−1 DM of CTC or EFX reduced the specific methane yields up to 49 and 44 %, respectively. The microbial community did not show any changes at this concentration. When chlortetracycline was added at a concentration of 8,000 mg kg−1 DM, the biodiversity changed slightly compared to the control without antibiotics

Erratum to: Effects of the Antibiotics Chlortetracycline and Enrofloxacin on the Anaerobic Digestion in Continuous Experiments(Bioenerg. Res., (2014), DOI 10.1007/s12155-014-9458-0)

The original version of this article unfortunately contained mistakes in the authorship. The author’s name, “ Christoph Winckler ” as the 10th author, is missing. The correct version is presented above

Slow sand filtration of raw wastewater using biochar as an alternative filtration media

The efficiency of anaerobic biofilters (AnBF) as low-cost wastewater treatment systems was investigated. $\it Miscanthus$-biochar was used as filtration media and compared with sand as a common reference material. Raw sewage from a municipal wastewater treatment plant was stored in a sedimentation tank for two days to allow pre-settlement of wastewater particles. Subsequently, wastewater was treated by AnBFs at 22 °C room temperature at a hydraulic loading rate of 0.05 m∙h$^{−1}$ with an empty bed contact time of 14.4 h and a mean organic loading rate of 509 $\pm$ 173 gCOD∙m$^{−3}$∙d$^{−1}$. Mean removal of chemical oxygen demand (COD) of biochar filters was with 74 $\pm$ 18% significantly higher than of sand filters (61 $\pm$ 12%). In contrast to sand filters with a mean reduction of 1.18 $\pm$ 0.31 log-units, $\textit {E. coli}$ removal through biochar was with 1.35 $\pm$ 0.27 log-units significantly higher and increased with experimental time. Main removal took place within the $\it schmutzdecke$, a biologically active dirt layer that develops simultaneously on the surface of filter beds. Since the $\textit {E. coli}$ contamination of both filter materials was equal, the higher removal efficiency of biochar filters is probably a result of an improved biodegradation within deeper zones of the filter bed. Overall, performance of biochar filters was better or equal compared to sand and have thus demonstrated the suitability of $\it Miscanthus$-biochar as filter media for wastewater treatment

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

Abstract 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