Philipp Lins

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.

Effects of different nitrogen sources on the biogas production - a lab-scale investigation.

For anaerobic digestion processes nitrogen sources are poorly investigated although they are known as possible process limiting factors (in the hydrolysis phase) but also as a source for fermentations for subsequent methane production by methanogenic archaea. In the present study different complex and defined nitrogen sources were investigated in a lab-scale experiment in order to study their potential to build up methane. The outcome of the study can be summarised as follows: from complex nitrogen sources yeast extract and casamino acids showed the highest methane production with approximately 600 ml methane per mole of nitrogen, whereas by the use of skim milk no methane production could be observed. From defined nitrogen sources L-arginine showed the highest methane production with almost 1400 ml methane per mole of nitrogen. Moreover it could be demonstrated that the carbon content and therefore C/N-ratio has only minor influence for the methane production from the used substrates.

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.

Effects of various fatty acid amendments on a microbial digester community in batch culture

Since biogas production is becoming increasingly important the understanding of anaerobic digestion processes is fundamental. However, large-scale digesters often lack online sensor equipment to monitor key parameters. Furthermore the possibility to selectively change fermenting parameter settings in order to investigate methane output or microbial changes is limited. In the present study we examined the possibility to investigate the microbial community of a large scale (750,000 L) digester within a laboratory small-scale approach. We studied the short-term response of the downscaled communities on various fatty acids and its effects on gas production and compared it with data from the original digester sludge. Even high loads of formic acid led to distinct methane formation, whereas high concentrations of other acids (acetic, butyric, propionic acid) caused a marked inhibition of methanogenesis coupled with an increase in hydrogen concentration. Molecular microbial techniques (DGGE/quantitative real-time-PCR) were used to monitor the microbial community changes which were related to data from GC and HPLC analysis. DGGE band patterns showed that the same microorganisms which were already dominant in the original digester re-established again in the lab-scale experiment. Very few microorganisms dominated the whole fermenting process and species diversity was not easily influenced by moderate varying fatty acid amendments--Methanoculleus thermophilus being the most abundant species throughout the variants. MCR-copy number determined via quantitative real-time-PCR--turned out to be a reliable parameter for quantification of methanogens, even in a very complex matrix like fermenter sludge. Generally the downscaled batch approach was shown to be appropriate to investigate microbial communities from large-scale digesters.

Reduction of accumulated volatile fatty acids by an acetate-degrading enrichment culture.

The effects of the addition of an acetate-degrading enrichment culture to an anaerobic digester with a stagnating biogas production were investigated. Initially, a thermophilic batch-operated lab-scale digester was inoculated with the diluted fermenter sludge of a biogas plant, and process parameters including the concentration of volatile fatty acids (VFAs) and gases in the headspace were measured. After a phase of high gas production, a stagnation of biogas production followed for a further 30 days. An acetate enrichment culture was added 34 days after the commencement of the experiment and this resulted in a sharp decrease in the concentrations of accumulated VFAs and an increase in total biogas and CH(4) production. An archaeon with a sequence similarity of 98% to Methanosarcina sp. and the ability to degrade acetic acid was introduced with the enrichment culture and is proposed to have been the driving factor for the changes that occurred within a few days to the process.

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.