Andreas Otto Wagner

Removal of Free Extracellular DNA from Environmental Samples by Ethidium Monoazide and Propidium Monoazide

ABSTRACT Recently, new DNA extraction techniques (using ethidium monoazide and propidium monoazide) have been developed to discriminate between alive and dead bacterial cells. Nevertheless, for complex environmental samples, no data are available yet. In the present study, these new methods were applied to anaerobic-fermentor sludge and the results were compared to a conventional microbiological approach.

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.

Application of Denaturing High-Performance Liquid Chromatography in Microbial Ecology: Fermentor Sludge, Compost, and Soil Community Profiling

ABSTRACT Genetic fingerprinting methods, such as denaturing gradient gel electrophoresis (DGGE), are used in microbial ecology for the analysis of mixed microbial communities but are associated with various problems. In the present study we used a new alternative method: denaturing high-performance liquid chromatography (dHPLC). This method was previously shown to work with samples from water and gut flora but had not yet been applied to complex environmental samples. In contrast to other publications dealing with dHPLC, we used a commonly available HPLC system. Samples from different origins (fermentor sludge, compost, and soil), all ecologically significant, were tested, and the 16S rRNA gene was amplified via PCR. After optimization of the HPLC elution conditions, amplicons of pure cultures and mixed microbial populations could be separated successfully. Systematic differentiation was carried out by a cloning approach, since fraction collection of the peaks did not result in satisfactory fragment separation. dHPLC was evaluated as a tool for microbial community analysis on a genetic level and demonstrated major improvements compared to gel-based fingerprinting methods, such as DGGE, that are commonly used in microbial ecology.

Survival of selected pathogens in diluted sludge of a thermophilic waste treatment plant and in NaCl-solution under aerobic and anaerobic conditions

Decimal reduction times (DRT or D-value) of Campylobacter jejuni, Salmonella enterica (formerly Salmonella choleraesuis) serovar Senftenberg, Escherichia coli, and Listeria monocytogenes were determined in two different matrices, diluted fermenter sludge (DFS) and 0.95% NaCl-solution (NaCl) at 50 degrees C, both under aerobic and anaerobic conditions. Depending on aeration, matrix composition, and the respective organism, the D-values varied between 10min and more than 15h. Generally the viability of bacteria decreased faster in DFS compared to NaCl-solution and under aerobic compared to anaerobic conditions. After 24h no viable cells could be detected in DFS, both under aerobic as well as under anaerobic conditions, whereas viable cells were still found in NaCl solutions. In both matrices the detection limits determined by means of PCR-based and classical microbiological methods were compared and pointed to lower detection limits of the latter methods. Results of the present investigation show that test organisms were far from surviving several days in DFS whereas hydraulic retention times normally used for thermophilic anaerobic digestion are in the range of 2 weeks. However, an underestimation of survival rates of the test organisms seems probable when applying aerobic standard methods.

Survival of bacterial pathogens during the thermophilic anaerobic digestion of biowaste: laboratory experiments and in situ validation.

Anaerobic digestion is continually gaining importance for the processing of the organic fraction of municipal solid wastes. Although methods for studying the survival of pathogen exist, these methods often need adaptations, are expensive, time consuming or generally not well suited for the harsh conditions within an anaerobic digestion system. In the present study we investigated the applicability of commercially available, mechanically stable and inexpensive pathogen carriers to validate in situ pathogen inhibition within a 750,000l thermophilic, bio-waste treating anaerobic digester. None of the pathogens investigated (Listeria monocytogenes, Salmonella enterica, Escherichia coli, and Campylobacter jejuni) was capable of survival under the conditions of the biogas reactor for more than 24 h indicating that the temperature and physico-chemical properties of the sludge of the fermenter were effective in inhibiting the survival of these microorganisms.

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.

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.

Utilisation of single added fatty acids by consortia of digester sludge in batch culture.

Inocula derived from an anaerobic digester were used to study (i) their potential for methane production and (ii) the utilisation rates of different short chain fatty acids (SCFAs) by the microbial community in defined media with mono-carbon sources (formic-, acetetic-, propionic-, butyric acid) in batch culture. It could be demonstrated that the microbial reactor population could be transferred successfully to the lab, and its ability to build up methane was present even with deteriorating biogas plant performance. Therefore, this reduction in performance of the biogas plant was not due to a decrease in abundance, but due to an inactivity of the microbial community. Generally, the physico-chemical properties of the biogas plant seemed to favour hydrogenotrophic methanogens, as seen by the high metabolisation rates of formate compared with all other carbon sources. In contrast, acetoclastic methanogenesis could be shown to play a minor role in the methane production of the investigated biogas plant, although the origin of up to 66% of methane is generally suggested to be generated through acetoclastic pathway.

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.

Process parameters within a 750,000 litre anaerobic digester during a year of disturbed fermenter performance

A 750,000litre fermenter was studied throughout one entire year by investigating the concentrations of volatile fatty acids (acetic, butyric, i-butyric, propionic, valeric and i-valeric acids), pH, concentrations of total C, N, S and NH(4)(+)-N, amounts of chemical and biological oxygen demand, and abundance of acetogenic microorganisms. Additionally several process parameters such as temperature, retention time, dry weight and input of substrate and liquids, and the concentrations and amounts of CH(4), H(2), CO(2) and H(2)S within the biogas were monitored continuously. Various volatile fatty acids and the ratio of acetic to propionic acid were shown to allow a rough indication on the fermentation but were not sufficiently precise to describe the fermenter performance. Nutrient compounds and special fractions, such as easily extractable carbohydrates or the concentration of total fats were more strongly correlated to the gas production of the fermenter. Results of an MPN-method for the determination of acetogenic microorganisms point to an important role of these microorganisms during the phase of restoration of the fermenter performance.