Characterization and rank assignment criteria for the anaerobic fungi (Neocallimastigomycota).

Establishing a solid taxonomic framework is crucial for enabling discovery and documentation efforts. This ensures effective communication between scientists as well as reproducibility of results between laboratories, and facilitates the exchange and preservation of biological material. Such framework can only be achieved by establishing clear criteria for taxa characterization and rank assignment. Within the anaerobic fungi (phylum Neocallimastigomycota), the need for such criteria is especially vital. Difficulties associated with their isolation, maintenance and long-term storage often result in limited availability and loss of previously described taxa. To this end, we provide here a list of morphological, microscopic, phylogenetic and phenotypic criteria for assessment and documentation when characterizing newly obtained Neocallimastigomycota isolates. We also recommend a polyphasic rank-assignment scheme for novel genus-, species- and strain-level designations for newly obtained Neocallimastigomycota isolates.

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.

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.

Towards molecular biomarkers for biogas production from lignocellulose-rich substrates.

Biogas production from lignocellulose-rich agricultural residues is gaining increasingly importance in sustainable energy production. Hydrolysis/acidogenesis (H/A) of lignocellulose as the initial rate-limiting step deserves particular optimization. A mixture of straw/hay was methanized applying two-phase digester systems with an initial H/A reactor and a one-stage system at different, meso- and thermophilic temperatures. H/A was intensified with increasing pH values and increasing temperature. H/A fermenters, however, were prone to switch to methanogenic systems at these conditions. Substrate turnover was accelerated in the bi-phasic process but did not reach the methanation efficiency of the single-stage digestion. There was no indication that two different cellulolytic inocula could establish in the given process. Bacterial communities were analyzed applying conventional amplicon clone sequencing targeting the hypervariable 16S rRNA gene region V6-V8 and by metagenome analyses applying direct DNA pyrosequencing without a PCR step. Corresponding results suggested that PCR did not introduce a bias but offered better phylogenetic resolution. Certain Clostridium IV and Prevotella members were most abundant in the H/A system operated at 38 °C, certain Clostridium III and Lachnospiraceae bacteria in the 45 °C, and certain Clostridium IV and Thermohydrogenium/Thermoanaerobacterium members in the 55 °C H/A system. Clostridium III representatives, Lachnospiraceae and Thermotogae dominated in the thermophilic single-stage system, in which also a higher portion of known syntrophic acetate oxidizers was found. Specific (RT-)qPCR systems were designed and applied for the most significant and abundant populations to assess their activity in the different digestion systems. The RT-qPCR results agreed with the DNA based community profiles obtained at the different temperatures. Up to 10(12) 16S rRNA copies mL(-1) were determined in H/A fermenters with prevalence of rRNA of a Ruminococcaceae subgroup. Besides, Thermohydrogenium/Thermoanaerobacterium rRNA prevailed at thermophilic and Prevotellaceae rRNA at mesophilic conditions. The developed (RT)-qPCR systems can be used as biomarkers to optimize biogas production from straw/hay and possibly other lignocellulosic substrates.

Editorial for Special Issue "Unleashing the Hidden Potential of Anaerobic Fungi".

Anaerobic fungi (AF) of the phylum Neocallimastigomycota are a very peculiar group of microorganisms [...].

Uncovering antimicrobial resistance in three agricultural biogas plants using plant-based substrates.

Antimicrobial resistance (AMR) is becoming an increasing global concern and the anaerobic digestion (AD) process represents a potential transmission route when digestates are used as fertilizing agents. AMR contaminants, e.g. antibiotic-resistant bacteria (ARB) and plasmid-mediated antibiotic resistance genes (ARGs) have been found in different substrates and AD systems, but not yet been investigated in plant-based substrates. AMR transfer from soils to vegetable microbiomes has been observed, and thus crop material potentially represents a so far neglected AMR load in agricultural AD processes, contributing to AMR spread. In order to test this hypothesis, this study examined the AMR situation throughout the process of three biogas plants using plant-based substrates only, or a mixture of plant-based and manure substrates. The evaluation included a combination of culture-independent and -dependent methods, i.e., identification of ARGs, plasmids, and pathogenic bacteria by DNA arrays, and phylogenetic classification of bacterial isolates and their phenotypic resistance pattern. To our knowledge, this is the first study on AMR in plant-based substrates and the corresponding biogas plant. The results showed that the bacterial community isolated from the investigated substrates and the AD processing facilities were mainly Gram-positive Bacillus spp. Apart from Pantoea agglomerans, no other Gram-negative species were found, either by bacteria culturing or by DNA typing array. In contrast, the presence of ARGs and plasmids clearly indicated the existence of Gram-negative pathogenic bacteria, in both substrate and AD process. Compared with substrates, digestates had lower levels of ARGs, plasmids, and culturable ARB. Thus, digestate could pose a lower risk of spreading AMR than substrates per se. In conclusion, plant-based substrates are associated with AMR, including culturable Gram-positive ARB and Gram-negative pathogenic bacteria-associated ARGs and plasmids. Thus, the AMR load from plant-based substrates should be taken into consideration in agricultural biogas processing.

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).

Effects of CO2 enrichment on the anaerobic digestion of sewage sludge in continuously operated fermenters.

The effect of CO2 enrichment in sewage sludge anaerobic digestion (AD) as a potential strategy to improve the biogas yield was assessed at increasing organic loading rates (OLR). Effects on process performance and resilience were evaluated in long-term continuous AD experiments at lab-scale. The specific methane production (SMP) was sustainably enhanced in the test digester compared to a control at elevated OLRs, reaching an increase of 6 ± 12% on average at the highest OLR tested (4.0 kgVS/(m3·d)). The reduction of CO2 via homoacetogenesis, facilitating acetoclastic CH4 formation is proposed as the dominant conversion pathway. Results suggest that sufficient load of easily degradable substances is a prerequisite for intrinsic formation of the reduction equivalent H2 via acidogenesis. The enhanced resilience of the process under CO2-enriched conditions in response to acid accumulation further qualifies this approach as a viable option for improving AD performance by converting a waste stream into a valuable product.

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.

Aestipascuomyces dupliciliberans gen. nov, sp. nov., the First Cultured Representative of the Uncultured SK4 Clade from Aoudad Sheep and Alpaca.

We report on the isolation of the previously-uncultured Neocallimastigomycota SK4 lineage, by two independent research groups, from a wild aoudad sheep rumen sample (Texas, USA) and an alpaca fecal sample (Baden-Württemberg, Germany). Isolates from both locations showed near-identical morphological and microscopic features, forming medium-sized (2-5 mm) white filamentous colonies with a white center of sporangia, on agar roll tubes and a heavy biofilm in liquid media. Microscopic analysis revealed monocentric thalli, and spherical polyflagellated zoospores with 7-20 flagella. Zoospore release occurred through an apical pore as well as by sporangial wall rupturing, a duality that is unique amongst described anaerobic gut fungal strains. Isolates were capable of growing on a wide range of mono-, oligo-, and polysaccharide substrates as the sole carbon source. Phylogenetic assessment based on the D1-D2 28S large rRNA gene subunit (D1-D2 LSU) and internal transcribed spacer-1 (ITS-1) regions demonstrated high sequence identity (minimum identity of 99.07% and 96.96%, respectively) between all isolates; but low sequence identity (92.4% and 86.7%, respectively) to their closest cultured relatives. D1-D2 LSU phylogenetic trees grouped the isolates as a new monophyletic clade within the Orpinomyces-Neocallimastix-Pecoramyces-Feramyces-Ghazallamyces supragenus group. D1-D2 LSU and ITS-1 sequences recovered from the obtained isolates were either identical or displayed extremely high sequence similarity to sequences recovered from the same aoudad sheep sample on which isolation was conducted, as well as several sequences recovered from domestic sheep and few other herbivores. Interestingly, members of the SK4 clade seem to be encountered preferably in animals grazing on summer pasture. We hence propose accommodating these novel isolates in a new genus, Aestipascuomyces (derived from the Latin word for "summer pasture"), and a new species, A. dupliciliberans. The type strain is Aestipascuomycesdupliciliberans strain R4.

Evaluation of two methods for quantification of hsp70 mRNA from the waterborne pathogen Cryptosporidium parvum by reverse transcription real-time PCR in environmental samples.

We optimized and evaluated two mRNA extraction methods to quantify induced hsp70 mRNA from viable and injured Cryptosporidium parvum oocysts by reverse transcription quantitative real-time PCR (RT-qPCR) in raw and treated manure. Methods based on guanidinium isothiocyanate/phenol/chloroform (GITC-PC) purification and direct mRNA extraction with magnetic oligo(dT)25-coated beads were evaluated for applicability and sensitivity. Both methods proved to be suitable for processing manure samples. With washed manure samples and oocyst disruption by bead beating for 165 s in time intervals with cumulative pooling of the lysate fractions, optimum RT-qPCR results were achieved. On average, 2.6 times more hsp70 mRNA was detected with the oligo(dT)25 method in comparison to the GITC-PC based method using fresh oocysts, whereas less mRNA was detected in aged oocysts. For fresh oocysts, analytical and method detection limits for the oligo(dT)25 based method were 1.7 cDNA copies/qPCR reaction and 5150 oocysts/mL manure, and for the GITC-PC based method 17 cDNA copies/qPCR reaction and 4950 oocysts/mL, respectively. In 12 months old oocysts with reduced viability, mRNA was occasionally detected only by the GITC-PC based method. Failure of or reduced detection with the oligo(dT)25 based method was apparently a result of weakened oocyst walls leading to quicker release of mRNA and therefore mRNA shredding by bead beating in the relatively long stretch between the capture sequence and the RT-qPCR target sites.

Load change capability of an anaerobic thermophilic trickle bed reactor for dynamic H2/CO2 biomethanation.

Increasing shares of energy production originating from fluctuating renewable sources require measures that are able to balance power production for a stable electricity grid. H2/CO2 biomethanation is a suitable approach to convert fluctuating excess renewable energy into the storable substitute natural gas. This study investigated the rapid load change capability of an anaerobic thermophilic trickle bed reactor while maintaining a high methane content. The return to full load (62.1 m3H2/m3trickle bed/d) after a 30-min operational off-cycle was possible almost immediately, while 24-h interruptions required a 60-min stepwise load increase. To accelerate this delayed microbial conversion activity, non-steady state substrate gas conversion can be controlled via substrate and product gas flow rates, allowing to reactivate the entire microbial community and produce high quality product gas. Reactor design might be further improved to avoid short-circuiting and use the entire trickle bed gas phase as high quality gas buffer during initial load increases.

Accelerated biogas production from lignocellulosic biomass after pre-treatment with Neocallimastix frontalis.

Two Neocallimastix frontalis strains, isolated from rumen fluid of a cow and of a chamois, were assessed for their ability to degrade lignocellulosic biomass. Two independent batch experiments were performed. Each experiment was split into two phases: hydrolysis phase and batch fermentation phase. The hydrolysis process during the N. frontalis incubation led to an initial increase of biogas production, an accelerated degradation of dry matter and an increased concentration of volatile fatty acids. As monitored by quantitative PCR, the applied N. frontalis strains were present and transcriptionally active during the hydrolysis phase but were fading during the batch fermentation phase. Thus, a separate hydrolytic pretreatment phase with anaerobic fungi, such as N. frontalis, represents a feasible strategy to improve biogas production from lignocellulosic substrates.

Isolation, identification and characterization of lignocellulolytic aerobic and anaerobic fungi in one- and two-phase biogas plants.

Aerobic and anaerobic fungi are among the most effective plant biomass degraders known and have high potential to increase the efficiency of lignocellulosic biomass utilization, such as for biogas generation. However, limited information is available on their contribution to such industrial processes. Therefore, the presence of fungi along the biogas production chain of one-phase and two-phase biogas plants in Germany was analyzed. Seventeen aerobic species of Zygomycota, Ascomycota and Basidiomycota were identified, including efficient producers of lignocellulases, such as Trichoderma capillare isolated from a hydrolysis tank and Coprinopsis cinerea from fibers separated from pressed digestate. Five anaerobic fungal species of the phylum Neocallimastigomycota (comprising two novel clades) were present in an slightly acidic fermenter of a biogas plant fed with cow manure displaying endoglucanase transcriptional activity. The broad fungal presence demonstrated in this study can serve developing bioaugmentation systems with relevant lignocellulolytic fungi to improve biogas production from recalcitrant fiber material.

High performance biological methanation in a thermophilic anaerobic trickle bed reactor.

In order to enhance energy efficiency of biological methanation of CO2 and H2, this study investigated the performance of a thermophilic (55°C) anaerobic trickle bed reactor (ATBR) (58.1L) at ambient pressure. With a methane production rate of up to 15.4m3CH4/(m3trickle bed·d) at methane concentrations above 98%, the ATBR can easily compete with the performance of other mixed culture methanation reactors. Control of pH and nutrient supply turned out to be crucial for stable operation and was affected significantly by dilution due to metabolic water production, especially during demand-orientated operation. Considering practical applications, inoculation with digested sludge, containing a diverse biocenosis, showed high adaptive capacity due to intrinsic biological diversity. However, no macroscopic biofilm formation was observed at thermophilic conditions even after 313days of operation. The applied approach illustrates the high potential of thermophilic ATBRs as a very efficient energy conversion and storage technology.

Presence and transcriptional activity of anaerobic fungi in agricultural biogas plants.

Bioaugmentation with anaerobic fungi (AF) is promising for improved biogas generation from lignocelluloses-rich substrates. However, before implementing AF into biogas processes it is necessary to investigate their natural occurrence, community structure and transcriptional activity in agricultural biogas plants. Thus, AF were detected with three specific PCR based methods: (i) Copies of their 18S genes were found in 7 of 10 biogas plants. (ii) Transcripts of a GH5 endoglucanase gene were present at low level in two digesters, indicating transcriptional cellulolytic activity of AF. (iii) Phylogeny of the AF-community was inferred with the 28S gene. A new Piromyces species was isolated from a PCR-positive digester. Evidence for AF was only found in biogas plants operated with high proportions of animal feces. Thus, AF were most likely transferred into digesters with animal derived substrates. Additionally, high process temperatures in combination with long retention times seemed to impede AF survival and activity.

Genetic and phenotypic microdiversity of Ochrobactrum spp.

The diversity of Ochrobactrum anthropi, Ochrobactrum intermedium, Ochrobactrum tritici and Ochrobactrum grignonense in agricultural soil and on the wheat rhizoplane was investigated. O. anthropi was isolated both from soil and from the rhizoplane, O. intermedium and grignonense only from bulk soil, and O. tritici only from the wheat rhizoplane. On the genetic level, the immunotrapped isolates and a number of strains from culture collection mainly of clinical origin were compared with rep-PCR profiling using BOX primers, and a subset of these isolates and strains using REP primers. The isolates clustered according to their species affiliation. There was no correlation between rep clusters of O. anthropi isolates and habitat (place of isolation). The genetic diversity of Ochrobactrum at the species level as well as microdiversity of O. anthropi (number of BOX groups) was higher in soil than on the rhizoplane. Similarity values from genetic rep-PCR profiles correlated positively with DNA-DNA reassociation percentages. Isolates with >80.7% similarity in BOX profile and >86.4% in rep profile clustered within the same species. Similarity analysis of rep-PCR profiles is hence an alternative to DNA-DNA hybridization as a genomic criterion for species delineation within the genus Ochrobactrum. We used the substrate utilization system BIOLOG-GN to compare the immunotrapped isolates on the phenetic level. For the isolates from bulk soil, substrate utilization versatility (number of utilized substrates) and substrate utilization capacity (mean conversion rate of substrates) were slightly but significantly higher than for the isolates from the rhizoplane. This trend was also seen using API 20E and 20NE systems. Plate counts of total bacteria and the number of immunotrapped Ochrobactrum isolates per gram dry weight were higher for the rhizoplane than for the soil samples. The results of genetic and phenotypic analyses indicated a 'rhizosphere effect'; the diversity and metabolic capacity of Ochrobactrum isolates were higher in bulk soil, and the population density was higher on the wheat rhizoplane.

Comparative sequence analysis of the internal transcribed spacer 1 of Ochrobactrum species.

The internal 16S/23S rDNA (rrs/rrl) internal spacer region 1 (ITS1) of 54 Ochrobactrum strains and close relatives was analysed. Separation of ITS1 containing PCR products by gel-electrophoresis, DGGE, cloning and sequencing revealed ITS1 length and sequence heterogeneity. We found up to 5 different allelic ITS1 stretches within a single strain (Ochrobactrum intermedium LMG 3301T), and 2-3 different ITS1 alleles in O. tritici. Within ITS1, ITS1c, being part of the conserved double-stranded rrn processing stem dsPS1, produced the most reliable segment tree. The overall ITS1, ITS1c and rrs phylogenetic tree topologies were generally consistent, but there was evidence for horizontal rrn (segment) transfer in O. tritici LMG 2134 (formerly O. anthropi). Good correlations were found between ITS1, ITS1c and rrs sequence similarity and DNA-DNA hybridization values indicating that phylogenetic analysis of ITS1 and ITS1c both can be used to preliminarily deduce the phylogenetic affiliation if HGT was excluded. Strains sharing > 96.19% ITS1c (> 95.11% ITS1) similarity fell within a species, and < or = 68.42% ITS1c (< or = 70.33% ITS1) similarity outside a genus. Both ITS1 and ITS1c analysis resolved microdiversity more profoundly than rrs analysis and revealed clades (genomovars) within O. anthropi that were also produced in rep cluster analysis. There was no evidence for habitat-specific ITS1 genomovars within Ochrobactrum species. Diversity of Ochrobactrum was higher in soil than at the rhizoplane below and at the species level. Isolates from soil contained only 1 rrn type whereas isolates from human clinical, animal and rhizoplane specimens could contain more.

Development of three specific PCR-based tools to determine quantity, cellulolytic transcriptional activity and phylogeny of anaerobic fungi.

Anaerobic fungi (AF) decompose plant material with their rhizoid and multiple cellulolytic enzymes. They disintegrate the complex structure of lignocellulosic substrates, making them more accessible and suitable for further microbial degradation. There is also much interest in their use as biocatalysts for biotechnological applications. Here, three novel polymerase chain reaction (PCR)-based methods for detecting AF and their transcriptional activity in in vitro cultures and environmental samples were developed. Two real-time quantitative PCR (qPCR)-based methods targeting AF were developed: AF-SSU, was designed to quantify the 18S rRNA genes of AF. AF-Endo, measuring transcripts of an endoglucanase gene from the glycoside hydrolase family 5 (GH5), was developed to quantify their transcriptional cellulolytic activity. The third PCR based approach was designed for phylogenetical analysis. It targets the 28S rRNA gene (LSU) of AF revealing their phylogenetic affiliation. The in silico-designed primer/probe combinations were successfully tested for the specific amplification of AF from animal and biogas plant derived samples. In combination, these three methods represent useful tools for the analysis of AF transcriptional cellulolytic activity, their abundance and their phylogenetic placement.