Christoph Reitschuler

Impact of protein-, lipid- and cellulose-containing complex substrates on biogas production and microbial communities in batch experiments.

In the present study, nine complex organic substrates from three classes (protein-, lipid-, and cellulose-rich) were investigated in batch experiments and compared with a control in order to evaluate their potential for utilisation as substrates for biogas production. High methane production was observed from protein-rich substrates; problems arose from lipid-containing, lactose and cellulose fermentation. Using DGGE analysis it could be shown that different classes of substrate resulted in different microbial communities, whereupon similar substrates tended to show a similar microbial structure. By means of qPCR Methanoculleus sp., a hydrogenotrophic methanogen was found to be the most abundant organism in the batch experiments. Additionally, it could be demonstrated that methanogenic organisms withstood adverse environmental conditions for at least an incubation period of 55 days, pointing to a high stability of the archaeal community even in times of decreasing or even failing fermenter performance.

Development and evaluation of inocula combating high acetate concentrations during the start-up of an anaerobic digestion

In the present study inocula to combat high acetate (CH(3)COO(-)) concentrations during start-up of an anaerobic digestion were designed and evaluated. Two strategies were followed (i) a stepwise adaptation of the engaged microorganisms within 1, 2, 4 or 6weeks, each at increasing CH(3)COO(-) concentrations of 50, 100, and finally 150mM, and (ii) shock variants, meaning a direct start with 150mM for the same durations. The stepwise adaptation for 4 and 6 weeks resulted in inocula, leading to a significant improved start-up under high CH(3)COO(-) concentrations compared to controls and shock enriched inocula. These results point to the possibility to facilitate the start-up under high CH(3)COO(-) concentrations during anaerobic digestion by addition of specific adapted inocula.

Methanosarcina spp., the key to relieve the start-up of a thermophilic anaerobic digestion suffering from high acetic acid loads.

This paper investigates if it is possible to produce inocula to counteract high acetic acid (CH3COO(-)) concentrations during thermophilic anaerobic digestion. To this end, fermenter sludge was exposed for different durations to either gradually increasing CH3COO(-) concentrations or directly exposed to a high concentration (150 mM). Altogether, these enrichments led to inocula with a distinct decrease of representatives of Methanobacteriales, while those of Methanoculleus spp. were hardly affected by any treatment. After the inoculation, good agreements of the abundance of Methanosarcinales and Methanoculleus spp. with total DNA content and methane production rate were apparent. In addition, a gradual adaptation of the inoculum for at least 4 weeks led to a significant increase of Methanosarcina spp. during the subsequent fermentation. These results demonstrate the potential of bioaugmentation to relieve the start-up of an anaerobic digestion suffering from high CH3COO(-) loads, especially pointing to the robust acetoclastic genus Methanosarcina.

Impact of several antibiotics and 2-bromoethanesulfonate on the volatile fatty acid degradation, methanogenesis and community structure during thermophilic anaerobic digestion.

The main aim of the present study was to gain insight into the stability of an anaerobic digestion process suffering from exposure to antibiotics and the methanogenic inhibitor 2-bromoethanesulfonate (BES). For this purpose, eleven antibiotics and BES were investigated with regard to the degradation of volatile fatty acids (VFAs), methanogenesis, and impact on the microbial community structure. Only neomycin, gentamicin, rifampicin, and BES showed complete inhibitions of VFA degradations. This points to distinct interferences with important trophic degradation cascades. Based upon DGGE and sequencing approaches, Methanosarcina spp. were severely influenced by the treatments while hydrogenotrophic methanogens were less affected. Interestingly, BES and neomycin inhibited the degradation of acetate while only BES inhibited methanogenesis completely. It seems that Methanosarcina spp. were mandatory for the degradation of acetate at high rates. The present results highly emphasize the detrimental effects of antimicrobial compounds with the potential to significantly inhibit the anaerobic digestion.

Cultivation of moonmilk-born non-extremophilic Thaum and Euryarchaeota in mixed culture

PCR-DGGE, qPCR and sequencing highlighted a quite homogenous archaeal community prevailing in secondary calcite deposits, so-called moonmilk, within the cold alpine Hundalm cave in Tyrol (Austria). Furthermore, the depth profile of this moonmilk could prove that the Archaea are located in oxygen-rich near- and oxygen-depleted sub-surface layers. To gather these communities we therefore applied an aerobic and anaerobic cultivation approach in oligotrophic and methanotrophic media. The mixed moonmilk community was analyzed with a combination of molecular methods using qPCR, PCR-DGGE and sequencing. Anaerobic and aerobic cultures were additionally investigated with GC and HPLC analyses. It was possible to initially cultivate and enrich the supposed aerobic/microaerophilic and anaerobic archaeal fraction, representing the natural archaeal community. While the naturally less abundant near-surface Archaea are closely related to members of the Thaumarchaeota (Nitrosopumilus maritimus), the highly abundant anaerobic Archaea are more distantly related to members within the Euryarchaeota. It is possible that these cultivable moonmilk-born Archaea represent new ecotypes or are so far undescribed. Based on the sequencing results and the production of very low amounts of methane, a corresponding methanogenic community is thought to represent only a minor abundant archaeal fraction. On a physiological level the cultivated moonmilk community is cold-adapted and basically of oligotrophic and organotrophic character.

Methanogenic potential of formate in thermophilic anaerobic digestion

In the present study the methanogenic potential of formate (HCOO−) during thermophilic anaerobic digestion was investigated. After appropriate conditions for methanogenesis (HCOO− and inoculum concentration, pH and duration of incubation) were assessed, an experiment with initial 31 replicates was run. Diluted fermenter sludge was used as inoculum, and process parameters including the pH, quality and quantity of the produced biogas and the concentrations of volatile fatty acids and HCO3− were determined. Remarkably, after 5 days of incubation the highest CH4 production was calculated for a HCOO− concentration of 200 mmol L−1, a concentration, however, which might not occur in situ. During the phase of high CH4 production HCOO− was degraded with a rate of 1.5 mmol L−1 h−1, and distinct changes of Gibbs free energy for several reactions were observed. Based on denaturing high-performance liquid chromatography, denaturing gradient gel electrophoresis, and additional subsequent sequencing approaches the hydrogenotrophic Methanothermobacter wolfeii was the dominant methanogen responsible for CH4 production. Further confirmation was achieved due to the detection of autofluorescing rods with a size of up to ~3 µm, which were often arranged in pairs and chains. It was shown that even high concentrations of HCOO− are readily degraded, which might lead to an underestimation of both, the concentration and thus, the importance of HCOO− in anaerobic digestion.

Microbial succession during thermophilic digestion: the potential of Methanosarcina sp.

A distinct succession from a hydrolytic to a hydrogeno- and acetotrophic community was well documented by DGGE (denaturing gradient gel electrophoresis) and dHPLC (denaturing high performance liquid chromatography), and confirmed by qPCR (quantitative PCR) measurements and DNA sequence analyses. We could prove that Methanosarcina thermophila has been the most important key player during the investigated anaerobic digestion process. This organism was able to terminate a stagnation phase, most probable caused by a decreased pH and accumulated acetic acid following an initial hydrolytic stage. The lack in Methanosarcina sp. could not be compensated by high numbers of Methanothermobacter sp. or Methanoculleus sp., which were predominant during the initial or during the stagnation phase of the fermentation, respectively.

New Undescribed Lineages of Non-extremophilic Archaea Form a Homogeneous and Dominant Element Within Alpine Moonmilk Microbiomes

The moonmilk deposits within the alpine Hundsalm cave in Austria offered the opportunity to investigate anthropogenically uninfluenced microbiomes. Via cultivation experiments we were able to show that the communities were cold-adapted and oligotrophic. Combined qPCR, DGGE, cloning and sequencing data further highlighted that the archaeal community basically comprises a low number of species, though highly abundant. These organisms are assumed to form new lineages within the Euryarchaeota, while the detected Thaumarchaeota, closely related to ammonium oxidizers, form a second, but minor, abundant group within the moonmilk deposits. Moreover, in terms of abundance the archaeal community clearly outnumbers bacteria (e.g., genera Pseudomonas, Flavobacterium and Rhodococcus) and fungi within the investigated microbiomes. In contrast to the highly complex bacterial and fungal communities, only a low number of archaeal species form a constant and essential element within the moonmilk speleothems and other cave-internal habitats.

The use of FAME analyses to discriminate between different strains of Geotrichum klebahnii with different viabilities

A considerable decline in viability of spray dried cells of Geotrichum klebahnii was observed and was attributed to an undefined alteration of the used strain. As common techniques were not able to distinguish the altered from the still viable strains, we used the fatty acid methyl ester (FAME) analysis. On the basis of FAME data we were able to discriminate the three strains under investigation. Especially the ratios of cis/trans fatty acid ratios and of saturated/unsaturated fatty acid were significantly reduced in the less viable strain, pointing to an increased stress level in this strain. These findings clearly show the applicability of the FAME analysis to detect strain alterations and that this method is therefore a suitable, fast and feasible tool for quality assurance.