Activated zeolite--suitable carriers for microorganisms in anaerobic digestion processes?

Plant cell wall structures represent a barrier in the biodegradation process to produce biogas for combustion and energy production. Consequently, approaches concerning a more efficient de-polymerisation of cellulose and hemicellulose to monomeric sugars are required. Here, we show that natural activated zeolites (i.e. trace metal activated zeolites) represent eminently suitable mineral microhabitats and potential carriers for immobilisation of microorganisms responsible for anaerobic hydrolysis of biopolymers stabilising related bacterial and methanogenic communities. A strategy for comprehensive analysis of immobilised anaerobic populations was developed that includes the visualisation of biofilm formation via scanning electron microscopy and confocal laser scanning microscopy, community and fingerprint analysis as well as enzyme activity and identification analyses. Using SDS polyacrylamide gel electrophoresis, hydrolytical active protein bands were traced by congo red staining. Liquid chromatography/mass spectroscopy revealed cellulolytical endo- and exoglucanase (exocellobiohydrolase) as well as hemicellulolytical xylanase/mannase after proteolytic digestion. Relations to hydrolytic/fermentative zeolite colonisers were obtained by using single-strand conformation polymorphism analysis (SSCP) based on amplification of bacterial and archaeal 16S rRNA fragments. Thereby, dominant colonisers were affiliated to the genera Clostridium, Pseudomonas and Methanoculleus. The specific immobilisation on natural zeolites with functional microbes already colonising naturally during the fermentation offers a strategy to systematically supply the biogas formation process responsive to population dynamics and process requirements.

Investigation of mircroorganisms colonising activated zeolites during anaerobic biogas production from grass silage.

The colonisation of activated zeolites (i.e. clinoptilolites) as carriers for microorganisms involved in the biogas process was investigated. Zeolite particle sizes of 1.0-2.5mm were introduced to anaerobic laboratory batch-cultures and to continuously operated bioreactors during biogas production from grass silage. Incubation over 5-84 days led to the colonisation of zeolite surfaces in small batch-cultures (500 ml) and even in larger scaled and flow-through disturbed bioreactors (28 l). Morphological insights were obtained by using scanning electron microscopy (SEM). Single strand conformation polymorphism (SSCP) analysis based on amplification of bacterial and archaeal 16S rRNA fragments demonstrated structurally distinct populations preferring zeolite as operational environment. via sequence analysis conspicuous bands from SSCP patterns were identified. Populations immobilised on zeolite (e.g. Ruminofilibacter xylanolyticum) showed pronounced hydrolytic enzyme activity (xylanase) shortly after re-incubation in sterilised sludge on model substrate. In addition, the presence of methanogenic archaea on zeolite particles was demonstrated.

Enhancement of biogas production by addition of hemicellulolytic bacteria immobilised on activated zeolite.

Biogas from agricultural biomass and residues is a valuable source of renewable energy. However, recalcitrant plant cell structures represent a barrier in the fermentative biodegradation process in single- and two-stage reactors. Therefore, approaches concerning a more efficient de-polymerisation of cellulose and hemicellulose to monomeric sugars are required amongst others in order to optimise the fermentation efficiency and to increase methane yields. Here we show a new strategy for the enhancement of biogas production from hemicellulose-rich substrates. Hemicellulolytic populations from a common biogas fermenter consortium were successively enriched in batch-cultures using a synthetic medium containing xylan powder as single carbon source under anaerobic mesophilic conditions. Enriched hemicellulolytic bacteria were immobilised on trace metal activated zeolite to ensure a stable storage and easy application. Xylanase activity increased continuously during subsequent enrichment cycles by up to 162%. In batch-culture experiments we were able to observe an increase of methane by 53% compared to controls without additionally introduced microorganisms immobilised on zeolite. Specific enrichment of hemicellulolytic bacteria during the process was confirmed by using single strand conformation polymorphism (SSCP) analysis based on amplification of the eubacterial 16S rDNA fragments. Using sequence analysis conspicuous bands from SSCP patterns could be identified as belonging to the groups Bacteroides sp., Azospira oryzae (Dechlorosoma sp.) as well as to a wide spectrum of diverse species within the order of Clostridiales (Firmicutes).

Aerobic treatment of a concentrated urea wastewater with simultaneous stripping of ammonia.

An industrial wastewater containing a total Kjeldahl nitrogen (TKN) of 12.80 g l(-1) was treated in a continuously fed activated sludge reactor. The main contaminant was urea (21.52 g l(-1)), together with minor amounts of the nitrification inhibitor dicyandiamide (0.46 g l(-1)) and free ammonia (0.56 g l(-1)). The wastewater was diluted 1:1 with water and treated under alkaline conditions (pH 9.4), enabling the simultaneous hydrolysis of urea and stripping of free ammonia in one aerobic reactor. Experiments were conducted to eliminate the remaining ammonia in a separate treatment unit by nitrification/denitrification. An adapted nitrifying bacterial population was isolated which was able to nitrify at a rate of 0.1 g nitrogen l(-1) day(-1) at a dicyandiamide concentration of 0.22 g l(-1). However, this was found to be too slow for an industrial-scale operation. Therefore, separate stripping with air or steam after pH adjustment to > or =10.5 is proposed. The diluted wastewater was treated with a hydraulic retention time of 6 days, corresponding to a volumetric nitrogen loading rate of 1.1 g nitrogen l(-1) day(-1) with an overall TKN reduction of 78.0%.