Peter Westermann

Mesophilic and thermophilic anaerobic digestion of primary and secondary sludge. Effect of pre-treatment at elevated temperature

Anaerobic digestion is an appropriate technique for the treatment of sludge before final disposal and it is employed worldwide as the oldest and most important process for sludge stabilization. In general, mesophilic anaerobic digestion of sewage sludge is more widely used compared to thermophilic digestion. Furthermore, thermal pre-treatment is suitable for the improvement of stabilization, enhancement of dewatering of the sludge, reduction of the numbers of pathogens and could be realized at relatively low cost especially at low temperatures. The present study investigates (a) the differences between mesophilic and thermophilic anaerobic digestion of sludge and (b) the effect of the pre-treatment at 70 degrees C on mesophilic and thermophilic anaerobic digestion of primary and secondary sludge. The pre-treatment step showed very positive effect on the methane potential and production rate upon subsequent thermophilic digestion of primary sludge. The methane production rate was mostly influenced by the pre-treatment of secondary sludge followed by mesophilic and thermophilic digestion whereas the methane potential only was positively influenced when mesophilic digestion followed. Our results suggest that the selection of the pre-treatment duration as well as the temperature of the subsequent anaerobic step for sludge stabilization should depend on the ratio of primary to secondary sludge.

Specific single-cell isolation and genomic amplification of uncultured microorganisms

We in this study describe a new method for genomic studies of individual uncultured prokaryotic organisms, which was used for the isolation and partial genome sequencing of a soil archaeon. The diversity of Archaea in a soil sample was mapped by generating a clone library using group-specific primers in combination with a terminal restriction fragment length polymorphism profile. Intact cells were extracted from the environmental sample, and fluorescent in situ hybridization probing with Cy3-labeled probes designed from the clone library was subsequently used to detect the organisms of interest. Single cells with a bright fluorescent signal were isolated using a micromanipulator and the genome of the single isolated cells served as a template for multiple displacement amplification (MDA) using the Phi29 DNA polymerase. The generated MDA product was afterwards used for 16S rRNA gene sequence analysis and shotgun-cloned for additional genomic analysis. Sequence analysis showed >99% 16S rRNA gene homology to soil crenarchaeotal clone SCA1170 and shotgun fragments had the closest match to a crenarchaeotal BAC clone previously retrieved from a soil sample. The system was validated using Methanothermobacter thermoautotrophicus as single-cell test organism, and the validation setup produced 100% sequence homology to the ten tested regions of the genome of this organism.

In vitro fermentation of arabinoxylan-derived carbohydrates by Bifidobacteria and mixed fecal microbiota

Bifidobacterium adolescentis ATCC 15703, Bifidobacterium breve ATCC 15700, Bifidobacterium longum ATCC 15707, and human fecal microbiota were cultivated in vitro with d-xylose, l-arabinose, xylo-oligosaccharides (XOS), and arabinoxylo-oligosaccharides (AXOS) as carbon sources. The pH, formation of volatile fatty acids, and carbohydrate utilization profiles were followed. In the pure bifidobacteria cultures optical density and in the fecal slurries pressure and H(2) were also detected. A differing substrate preference was observed among the various bifidobacteria strains. B. adolescentis grew on XOS, slowly on d-xylose, but not on l-arabinose. In contrast, B. longum preferred l-arabinose and did not grow on pure d-xylose or XOS. Both strains were able to utilize AXOS but with differing strategies, since after the cleavage of l-arabinose B. adolescentis consumed the XOS formed, whereas B. longum fermented the l-arabinose released. B. breve grew poorly on all of the substrates provided. A bifidobacterial mixture and the fecal microbiota were able to utilize pure singly substituted AXOS almost completely, but pure AXOS with a doubly substituted xylose residue was fermented only by the fecal microbiota. Thus, AXOS appear to be potential candidates for slowly fermenting prebiotics, but their prebiotic effects may be dependent on the type of arabinose substitution and the presence of other carbohydrates.

Metabolic Interactions Between Methanogenic Consortia and Anaerobic Respiring Bacteria

Most types of anaerobic respiration are able to outcompete methanogenic consortia for common substrates if the respective electron acceptors are present in sufficient amounts. Furthermore, several products or intermediate compounds formed by anaerobic respiring bacteria are toxic to methanogenic consortia. Despite the potentially adverse effects, only few inorganic electron acceptors potentially utilizable for anaerobic respiration have been investigated with respect to negative interactions in anaerobic digesters. In this chapter we review competitive and inhibitory interactions between anaerobic respiring populations and methanogenic consortia in bioreactors. Due to the few studies in anaerobic digesters, many of our discussions are based upon studies of defined cultures or natural ecosystems.

Archaea in protozoa and metazoa

The presence of Archaea is currently being explored in various environments, including extreme geographic positions and eukaryotic habitats. Methanogens are the dominating archaeal organisms found in most animals, from unicellular protozoa to humans. Many methanogens can contribute to the removal of hydrogen, thereby improving the efficiency of fermentation or the reductive capacity of energy-yielding reactions. They may also be involved in tissue damage in periodontal patients. Recent molecular studies demonstrated the presence of Archaea other than methanogens in some animals—but so far, not in humans. The roles of these microorganisms have not yet been established. In the present review, we present the state of the art regarding the archaeal microflora in animals.

Molecular Ecology of Anaerobic Reactor Systems

Anaerobic reactor systems are essential for the treatment of solid and liquid wastes and constitute a core facility in many waste treatment plants. Although much is known about the basic metabolism in different types of anaerobic reactors, little is known about the microbes responsible for these processes. Only a few percent of Bacteria and Archaea have so far been isolated, and almost nothing is known about the dynamics and interactions between these and other microorganisms. This lack of knowledge is most clearly exemplified by the sometimes unpredictable and unexplainable failures and malfunctions of anaerobic digesters occasionally experienced, leading to sub-optimal methane production and wastewater treatment. Using a variety of molecular techniques, we are able to determine which microorganisms are active, where they are active, and when they are active, but we still need to determine why and what they are doing. As genetic manipulations of anaerobes have been shown in only a few species permitting in-situ gene expression studies, the only way to elucidate the function of different microbes is to correlate the metabolic capabilities of isolated microbes in pure culture to the abundance of each microbe in anaerobic reactor systems by rRNA probing. This chapter focuses on various molecular techniques employed and problems encountered when elucidating the microbial ecology of anaerobic reactor systems. Methods such as quantitative dot blot/fluorescence in-situ probing using various specific nucleic acid probes are discussed and exemplified by studies of anaerobic granular sludge, biofilm and digester systems.

Production of 1,3-PDO and butanol by a mutant strain of Clostridium pasteurianum with increased tolerance towards crude glycerol

The production of biodiesel results in a concomitant production of crude glycerol (10% w/w). Clostridium pasteurianum can utilize glycerol as sole carbon source and converts it into 1,3-propanediol, ethanol, butanol, and CO2. Reduced growth and productivities on crude glycerol as compared to technical grade glycerol have previously been observed. In this study, we applied random mutagenesis mediated by ethane methyl sulfonate (EMS) to develop a mutant strain of C. pasteurianum tolerating high concentrations of crude glycerol. At an initial crude glycerol concentration of 25 g/l the amount of dry cell mass produced by the mutant strain was six times higher than the amount produced by the wild type. Growth of the mutant strain was even detected at an initial crude glycerol concentration of 105 g/l. A pH controlled reactor with in situ removal of butanol by gas-stripping was used to evaluate the performance of the mutant strain. Utilizing stored crude glycerol, the mutant strain showed significantly increased rates compared to the wild type. A maximum glycerol utilization rate of 7.59 g/l/h was observed along with productivities of 1.80 g/l/h and 1.21 g/l/h of butanol and 1,3-PDO, respectively. These rates are higher than what previously has been published for C. pasteurianum growing on technical grade glycerol in fed batch reactors. In addition, high yields of the main products (butanol and 1,3-PDO) were detected and these two products were efficiently separated in two steams using gas-stripping.

Effects of cations on Methanosarcina thermophila TM-1 growing on moderate concentrations of acetate: production of single cells

SummaryThe effects of different concentrations of Mg2+, Ca2+, or Na+ on the morphology and growth of Methanosarcina thermophila TM-1 growing on acetate at concentrations comparable with those found in anaerobic digestors was studied. At 30 mm Mg2+ or less, M. thermophila grew as large aggregates that settled rapidly. At 100 mm Mg2+ or more, the bacteria grew as single cells or a mixture of single cells and small aggregates is suspended culture. Mg2+ was necessary for growth and could not be substituted by addition of either Ca2+ or Na+. The optimal Mg2+ concentration was 30 mm and no growth was observed at 400 mm Mg2+. Cultures could be adapted to 300 mm Mg2+ without a change in growth rate. Added Ca2+ was not required for growth and had no effect on cell morphology. Inhibition by Na+ was directly related to the Mg2+ concentration. When the Mg2+ was 0.05 mm or less, 0.35 m Na+ completely inhibited growth. However, more Na+ was required for inhibition at higher Mg2+ concentrations. The same inhibitory effect of Na+ was observed when the temperature was 52°C or 45°C. The potential for disaggregation of Methanosarcina aggregates in anaerobic digestor environments was discussed.

Coproduction of Bioethanol with Other Biofuels

Large scale transformation of biomass to more versatile energy carriers has most commonly been focused on one product such as ethanol or methane. Due to the nature of the biomass and thermodynamic and biological constraints, this approach is not optimal if the energy content of the biomass is supposed to be exploited maximally. In natural ecosystems, biomass is degraded to numerous intermediary compounds, and we suggest that this principle is utilized in biorefinery concepts, which could provide different fuels with different end use possibilities. In this chapter we describe one of the first pilot-scale biorefineries for multiple fuel production and also discuss perspectives for further enhancement of biofuel yields from biomass. The major fuels produced in this refinery are ethanol, hydrogen, and methane. We also discuss the applicability of our biorefinery concept as a bolt-on plant on conventional corn- or grain-based bioethanol plants, and suggest that petroleum-base refineries and biorefineries appropriately can be coupled during the transition period from a fossil fuel to a renewable fuel economy.

Butyric acid fermentation from pretreated and hydrolysed wheat straw by an adapted Clostridium tyrobutyricum strain

Butyric acid is a valuable building‐block for the production of chemicals and materials and nowadays it is produced exclusively from petroleum. The aim of this study was to develop a suitable and robust strain of Clostridium tyrobutyricum that produces butyric acid at a high yield and selectivity from lignocellulosic biomasses. Pretreated (by wet explosion) and enzymatically hydrolysed wheat straw (PHWS), rich in C6 and C5 sugars (71.6 and 55.4 g l−1 of glucose and xylose respectively), was used as substrate. After one year of serial selections, an adapted strain of C. tyrobutyricum was developed. The adapted strain was able to grow in 80% (v v−1) PHWS without addition of yeast extract compared with an initial tolerance to less than 10% PHWS and was able to ferment both glucose and xylose. It is noticeable that the adapted C. tyrobutyricum strain was characterized by a high yield and selectivity to butyric acid. Specifically, the butyric acid yield at 60–80% PHWS lie between 0.37 and 0.46 g g−1 of sugar, while the selectivity for butyric acid was as high as 0.9–1.0 g g−1 of acid. Moreover, the strain exhibited a robust response in regards to growth and product profile at pH 6 and 7.