Effect of Growth Media on the Diversity of Neocallimastigomycetes from Non-Rumen Habitats.

Anaerobic fungi (AF), belonging to the phylum Neocallimastigomycota, are a pivotal component of the digestive tract microbiome of various herbivorous animals. In the last decade, the diversity of AF has rapidly expanded due to the exploration of numerous (novel) habitats. Studies aiming at understanding the role of AF require robust and reliable isolation and cultivation techniques, many of which remained unchanged for decades. Using amplicon sequencing, we compared three different media: medium with rumen fluid (RF), depleted rumen fluid (DRF), and no rumen fluid (NRF) to enrich the AF from the feces of yak, as a rumen control; and Przewalski's horse, llama, guanaco, and elephant, as a non-rumen habitats. The results revealed the selective enrichment of Piromyces and Neocallimastix from the feces of elephant and llama, respectively, in the RF medium. Similarly, the enrichment culture in DRF medium explicitly manifested Piromyces-related sequences from elephant feces. Five new clades (MM1-5) were defined from llama, guanaco, yak, and elephant feces that could as well be enriched from llama and elephant samples using non-conventional DRF and NRF media. This study presents evidence for the selective enrichment of certain genera in medium with RF and DRF from rumen as well as from non-rumen samples. NRF medium is suggested for the isolation of AF from non-rumen environments.

Simultaneous Metabarcoding and Quantification of Neocallimastigomycetes from Environmental Samples: Insights into Community Composition and Novel Lineages.

Anaerobic fungi from the herbivore digestive tract (Neocallimastigomycetes) are primary lignocellulose modifiers and hold promise for biotechnological applications. Their molecular detection is currently difficult due to the non-specificity of published primer pairs, which impairs evolutionary and ecological research with environmental samples. We developed and validated a Neocallimastigomycetes-specific PCR primer pair targeting the D2 region of the ribosomal large subunit suitable for screening, quantifying, and sequencing. We evaluated this primer pair in silico on sequences from all known genera, in vitro with pure cultures covering 16 of the 20 known genera, and on environmental samples with highly diverse microbiomes. The amplified region allowed phylogenetic differentiation of all known genera and most species. The amplicon is about 350 bp long, suitable for short-read high-throughput sequencing as well as qPCR assays. Sequencing of herbivore fecal samples verified the specificity of the primer pair and recovered highly diverse and so far unknown anaerobic gut fungal taxa. As the chosen barcoding region can be easily aligned and is taxonomically informative, the sequences can be used for classification and phylogenetic inferences. Several new Neocallimastigomycetes clades were obtained, some of which represent putative novel lineages such as a clade from feces of the rodent Dolichotis patagonum (mara).

A Novel Primer Mixture for GH48 Genes: Quantification and Identification of Truly Cellulolytic Bacteria in Biogas Fermenters.

Genomic studies revealed the glycoside hydrolases of family 48 (GH48) as a powerful marker for the identification of truly cellulolytic bacteria. Here we report an improved method for detecting cellulolytic bacteria in lab-scale biogas fermenters by using GH48 genes as a molecular marker in DNA and RNA samples. We developed a mixture of primers for the specific amplification of a GH48 gene region in a broad range of bacteria. Additionally, we built a manually curated reference database containing GH48 gene sequences directly linked to the corresponding taxonomic information. Phylogenetic correlation analysis of GH48 to 16S rRNA gene sequences revealed that GH48 gene sequences with 94% identity belong with high confidence to the same genus. Applying this analysis, GH48 amplicon reads revealed that at mesophilic fermenter conditions, 50-99% of the OTUs appear to belong to novel taxa. In contrast, at thermophilic conditions, GH48 gene sequences from the genus Hungateiclostridium dominated with 60-91% relative abundance. The novel primer combinations enabled detection and relative quantification of a wide spectrum of GH48 genes in cellulolytic microbial communities. Deep phylogenetic correlation analysis and a simplified taxonomic identification with the novel database facilitate identification of cellulolytic organisms, including the detection of novel taxa in biogas fermenters.

Importance of Defluviitalea raffinosedens for Hydrolytic Biomass Degradation in Co-Culture with Hungateiclostridium thermocellum.

Bacterial hydrolysis of polysaccharides is an important step for the production of sustainable energy, for example during the conversion of plant biomass to methane-rich biogas. Previously, Hungateiclostridium thermocellum was identified as cellulolytic key player in thermophilic biogas microbiomes with a great frequency as an accompanying organism. The aim of this study was to physiologically characterize a recently isolated co-culture of H. thermocellum and the saccharolytic bacterium Defluviitalea raffinosedens from a laboratory-scale biogas fermenter. The characterization focused on cellulose breakdown by applying the measurement of cellulose hydrolysis, production of metabolites, and the activity of secreted enzymes. Substrate degradation and the production of volatile metabolites was considerably enhanced when both organisms acted synergistically. The metabolic properties of H. thermocellum have been studied well in the past. To predict the role of D. raffinosedens in this bacterial duet, the genome of D. raffinosedens was sequenced for the first time. Concomitantly, to deduce the prevalence of D. raffinosedens in anaerobic digestion, taxonomic composition and transcriptional activity of different biogas microbiomes were analyzed in detail. Defluviitalea was abundant and metabolically active in reactor operating at highly efficient process conditions, supporting the importance of this organism for the hydrolysis of the raw substrate.

Influence of nitrogen-rich substrates on biogas production and on the methanogenic community under mesophilic and thermophilic conditions.

Grass silage was evaluated as a possible substrate in anaerobic digestion for generation of biogas in mesophilic and thermophilic long-term operation. Furthermore, the molecular biological parameter Metabolic Quotient (MQ) was evaluated as early warning system to predict process disturbance. Since this substrate is rich in nitrogen, high ammonia concentration of up to 2.2 g * kgFM-1 emerged. The high buffer capacity of the ammonium/ammonia system can disguise upcoming process acidification. At organic loading rates (OLR) below 1.0 kgVS * m-3 * d-1 (VS: volatile solids) for thermophilic and below 1.5 kgVS * m-3 * d-1 for mesophilic reactors, stable processes were established. With increasing OLR, the process was stressed until it broke down in the thermophilic reactors at an OLR of 3.5 kgVS * m-3 * d-1 or was stopped at an OLR of 4.5 kgVS * m-3 * d-1 in the mesophilic reactors. Mainly propionic acid accumulated in concentrations of up to 6.5 g * kgFM-1. Due to the high buffer capacity of the reactor sludge, the chemical parameter TVA/TIC (ratio of total volatile acids to total inorganic carbon) did not clearly indicate process disturbance in advance. In contrast, the MQ indicated metabolic stress of the methanogens before process breakdown and thus showed its potential as early warning system for process breakdown. During the whole experiment, hydrogenotrophic methanogens dominated. In the thermophilic reactors, Methanoculleus IIA-2 sp. 2 and Methanothermobacter wolfeii were dominant during stable process conditions and were displaced by Methanobacterium III sp. 4, a possible new bioindicator for disturbances at these conditions. In the mesophilic reactors, mainly Methanobacterium III sp. 4 was dominant at stable, stressed and acidified processes. A hitherto uncultivated genospecies, Methanobacteriaceae genus IV(B) sp. 3 was determined as possible new bioindicator for mesophilic process disturbance.

DNA and RNA Extraction and Quantitative Real-Time PCR-Based Assays for Biogas Biocenoses in an Interlaboratory Comparison.

Five institutional partners participated in an interlaboratory comparison of nucleic acid extraction, RNA preservation and quantitative Real-Time PCR (qPCR) based assays for biogas biocenoses derived from different grass silage digesting laboratory and pilot scale fermenters. A kit format DNA extraction system based on physical and chemical lysis with excellent extraction efficiency yielded highly reproducible results among the partners and clearly outperformed a traditional CTAB/chloroform/isoamylalcohol based method. Analytical purpose, sample texture, consistency and upstream pretreatment steps determine the modifications that should be applied to achieve maximum efficiency in the trade-off between extract purity and nucleic acid recovery rate. RNA extraction was much more variable, and the destination of the extract determines the method to be used. RNA stabilization with quaternary ammonium salts was an as satisfactory approach as flash freezing in liquid N2. Due to co-eluted impurities, spectrophotometry proved to be of limited value for nucleic acid qualification and quantification in extracts obtained with the kit, and picoGreen® based quantification was more trustworthy. Absorbance at 230 nm can be extremely high in the presence of certain chaotropic guanidine salts, but guanidinium isothiocyanate does not affect (q)PCR. Absolute quantification by qPCR requires application of a reliable internal standard for which correct PCR efficiency and Y-intercept values are important and must be reported.

Microbiology and Molecular Biology Tools for Biogas Process Analysis, Diagnosis and Control.

Many biotechnological processes such as biogas production or defined biotransformations are carried out by microorganisms or tightly cooperating microbial communities. Process breakdown is the maximum credible accident for the operator. Any time savings that can be provided by suitable early-warning systems and allow for specific countermeasures are of great value. Process disturbance, frequently due to nutritional shortcomings, malfunction or operational deficits, is evidenced conventionally by process chemistry parameters. However, knowledge on systems microbiology and its function has essentially increased in the last two decades, and molecular biology tools, most of which are directed against nucleic acids, have been developed to analyze and diagnose the process. Some of these systems have been shown to indicate changes of the process status considerably earlier than the conventionally applied process chemistry parameters. This is reasonable because the triggering catalyst is determined, activity changes of the microbes that perform the reaction. These molecular biology tools have thus the potential to add to and improve the established process diagnosis system. This chapter is dealing with the actual state of the art of biogas process analysis in practice, and introduces molecular biology tools that have been shown to be of particular value in complementing the current systems of process monitoring and diagnosis, with emphasis on nucleic acid targeted molecular biology systems.

Process diagnosis using methanogenic Archaea in maize-fed, trace element depleted fermenters.

A mesophilic maize-fed pilot-scale fermenter was severely acidified due to trace element (TE) deficiency. Mainly cobalt (0.07 mg * kg(-1) fresh mass (FM)), selenium (0.007 mg * kg(-1) FM) and sodium (13 mg * kg(-1) FM) were depleted. From this inoculum, three lab-scale flow-through fermenters were operated to analyse micronutrient deficiencies and population dynamics in more detail. One fermenter was supplemented with selenium, one with cobalt, and one served as control. After starvation and recovery of the fermenters, the organic loading rate (OLR) was increased. In parallel, the concentration (Real-Time PCR) of methanogens and their population composition (amplicon sequencing) was determined at the DNA and mRNA level. The parameters Metabolic Quotient (MQ) and cDNA/DNA were calculated to assess the activity of the methanogens. The control without TE supplementation acidified first at an OLR of 4.0 kg volatile solids (VS) * m(-3) * d(-1) while the singular addition of selenium and of cobalt positively influenced the fermenter stability up to an OLR of 4.5 or 5.0 kg VS * m(-3) * d(-1), respectively. In the stable process, the methanogenic populations were dominated by probably residual hydrogenotrophic Methanoculleus sp. (DNA-level), but representatives of versatile Methanosarcina sp. were most active (cDNA-level). When the TE supplemented fermenters began to acidify, Methanosarcina spp. were dominant in the whole (DNA-level) and the active (cDNA-level) community. The acidified control fermenter was dominated by Methanobacteriaceae genus IV. Until acidification, the concentration of methanogens increased with higher OLRs. The MQ indicated stress metabolism approximately one month before the TVA/TIC ratio reached a critical level of 0.7, demonstrating its suitability as early warning parameter of process acidification. The development of the cDNA/DNA ratio also reflected the increasing methanogenic activity with higher OLRs. Highest cDNA/DNA values (ca. 2) were obtained at metabolic strain of the methanogens, at the onset of acidification.

Impact of nickel and cobalt on biogas production and process stability during semi-continuous anaerobic fermentation of a model substrate for maize silage.

The importance of nickel and cobalt on anaerobic degradation of a defined model substrate for maize was demonstrated. Five semi-continuous reactors were operated for 250 days at 35 °C and a well-defined trace metal solution was added to all reactors. Two reactors each were limited regarding the concentration of Ni(2+) and Co(2+), respectively, for certain time intervals. The required nickel concentration was depending on the organic loading rates (OLR) while, for example, above 2.6 g ODM L(-1) d(-1) nickel concentrations below 0.06 mg kg(-1) FM in the process significantly decreased biogas production by up to 25% compared to a control reactor containing 0.8 mg Ni(2+) kg(-1) FM. Similarly, limitation of cobalt to 0.02 mg kg(-1) FM decreased biogas production by about 10%. Limitations of nickel as well as cobalt lead to process instability. However, after gradual addition of nickel till 0.6 mg and cobalt till 0.05 mg kg(-1) FM the OLR was again increased to 4.3 g ODM L(-1) d(-1) while process stability was recovered and a fast metabolisation of acetic and propionic acid was detected. An increase of nickel to 0.88 mg kg(-1) FM did not enhance biogas performance. Furthermore, the increase of cobalt from 0.05 mg kg(-1) FM up to 0.07 mg kg(-1) FM did not exhibit a change in anaerobic fermentation and biogas production.

Characterization of an anaerobic population digesting a model substrate for maize in the presence of trace metals.

The influence of a defined trace metal solution and additionally Ni(2+) on anaerobic digestion of biomass was investigated. A novel synthetic model substrate was designed consisting of cellulose, starch and urea as carbon and nitrogen source in a ratio mimicking the basic composition of maize silage. Two independent batch fermentations were carried out over 21 d with the synthetic model substrate in the presence of the trace metal solution. Particularly an increase in nickel concentrations (17 and 34 microM) enhanced methane formation by up to 20%. This increased activity was also corroborated by fluorescence microscopy measurements based on cofactor F(420). The eubacterial and methanogenic population was characterized with the single strand conformational polymorphism analysis and the amplified 16S rDNA restriction analysis of 16S rRNA genes amplified by different primer systems. Nearly the half of the analyzed bacteria were identified as Firmicutes while 70% in this phylum belonged to the class of Clostridiales and 30% to the class of Bacilli. Bacteroides and uncultured bacteria represented each a quarter of the analyzed community. Methanogenic archaea were investigated with ARDRA, too. The hydrogenotrophic Methanoculleus sp. was the dominant genus which is commonly described for maize digestion thus confirming the value of the model substrate.

Influence of trace elements on methane formation from a synthetic model substrate for maize silage.

The effect of a well-defined trace element solution and the elements nickel, cobalt and molybdenum on anaerobic digestion of a synthetic model substrate for maize silage was studied in batch reactor experiments at 35 degrees C. The defined substrate (dS) consisted of xylan and starch as the main carbon source, urea as nitrogen source and phosphorus from a 0.1M potassium phosphate buffer. Batch reactors were operated for 30 days with 1.5% organic dry matter (ODM) of inoculum sludge from a mono-maize biogas plant and 1% ODM of the defined substrate. Results showed an increase of methane yield of up to 30% upon addition of the trace element solution. With an addition of nickel at 10.6 microM, a final yield of 407 l kg(-1) ODM was reached and an enhanced methane production by 25% at day 25 of operation was observed. Total elimination of nickel from the trace element solution highly decreased methane formation and process stability. Cobalt in a concentration range of 0.4 up to 2.0 microM increased the methane production by 10% approximately. Interestingly, addition of molybdenum did not significantly effect methane production.