Fate of Clostridium botulinum and incidence of pathogenic clostridia in biogas processes.

AIMS: This study aimed to assess the sanitary situation in agricultural biogas plants (BP) regarding pathogenic Clostridium spp. METHODS AND RESULTS: The incidence of Clostridium botulinum, Clostridium difficile, Clostridium novyi, Clostridium haemolyticum, Clostridium septicum and Clostridium chauvoei was investigated in 154 plant and animal substrates, digester sludges and digestates from full-scale BP using a method combining microbial enrichment with Real-Time PCR. The investigated clostridia were absent in the samples, except for Cl. novyi that was barely present (3·9%) and Cl. difficile that was more frequently detected (44·8%). Clostridium botulinum exposed to lab-scale digesters in sentinel chambers was reduced with D-values of 34·6 ± 11·2 days at 38°C and 1·0 ± 0·2 days at 55°C. CONCLUSIONS: These findings indicate minor relevance of clostridial pathogens in BP and an improved sanitary quality of the digestion product compared to untreated substrates concerning Cl. botulinum. However, the frequent detection of Cl. difficile opens questions on the durability of this organism in manure digestion lines. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study providing data on the reduction of Cl. botulinum during biogas processes that scientifically invalidate contrary claims by some media in the public. Furthermore, the results improve the fragmentary knowledge on the prevalence of several clostridial pathogens in agricultural biogas production.

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.

A metabolic quotient for methanogenic Archaea.

Biogas production from renewable resources is an alternative to generate energy and concomitantly save fossil fuels and mitigate greenhouse gas emissions. As methanogenesis is a major bottleneck in the biogas process, the determination of the specific activity of methanogenic Archaea can be a good indicator of the process state. A new parameter, the metabolic quotient (MQ), was developed to evaluate the specific activity of methanogens. A standard was created from mesophilic maize-fed fermenters to calculate the expected concentration of methanogens for a given methane productivity at stable process stages. The MQ, the ratio of the predicted to the actual concentration of methanogens, defines their metabolic activity. The MQ was able to indicate methanogenic cell stress metabolism and imminent process failure before conventional chemical parameters. As a further approach, the methanogenic activity was determined by quantification of mRNA transcripts in relation to the mcrA/mrtA-gene, coding for a key enzyme subunit of methanogenesis. The cDNA/DNA ratio reflected the specific actual process activity of the methanogens. As both methods are potent parameters for the early detection of process failure, biogas plant operators may avoid economical losses by their preventive application.

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.

Biogas production from mono-digestion of maize silage-long-term process stability and requirements.

Biogas production from mono-digestion of maize silage was studied for more than one year in six continuously stirred, daily fed 36 L fermenters. Chemical and microbiological parameters were analysed concomitantly. The reactors acidified already after 8 months of operation at a low organic loading rate (OLR) of 2 g VS*(L*d)(-1). The TVA/TAC ratio was the most reliable parameter to indicate early process instabilities leading to acidification. A TVA/TAC threshold of 0.5 should not be exceeded. After acidification and recovery of the fermenters, propionic acid was no reliable parameter anymore to indicate process failure, since values far below the threshold of 1 g*L(-1) were obtained although the process had collapsed.The acidified reactors recovered better, showed greatly improved stability and allowed a higher OLR when a trace element (TE) cocktail was supplemented. Hydrolysis was obviously not process-limiting, results indicated that methanogens were affected. The most limiting element in long-term mono-digestion of maize silage turned out to be cobalt, but data obtained suggest that molybdenum and selenium should also be provided. TE supplementation should be designed specifically in order to meet the actual needs. TE availability for the biocenosis appears to be a key issue in biogas production, not only in mono-but also in co-digestion processes.

Methanogens in biogas production from renewable resources--a novel molecular population analysis approach.

The population structure of thermo- and mesophilic biogas reactors digesting maize silage as the sole substrate was investigated employing a novel, highly degenerated PCR-primer pair targeting mcrA/mrtA coding for the key enzyme of methanogens. No sequence affiliating with Methanococcales, Methanopyrales, ANME-, rice or fen soil clusters was detected. Direct MeA PCR-cloning results indicated that Methanobacteriales were the most important methanogens in the thermophilic reactors. 57% and 80% of the analysed sequences affiliated with this order, 14% and 20% with Methanosarcinaceae and 0% and 29% with Methanomicrobiales. Methanomicrobiales dominated in the mesophilic reactors at the given conditions, 69% and 84% of the sequences recovered from direct MeA primed cloning affiliated with this order, 31% and 0% with Methanosarcinaceae and 0% and 16% with Methanobacteriales. No sequence affiliating with Methanosaetaceae was found. MeA primed PCR-single-strand conformation polymorphism indicated that population fluctuations occurred. According to sequence analysis of excised bands, Methanosarcinaceae dominated and Methanobacteriales were significantly represented in the thermophilic fermenter. Only 1 Methanosaetaceae sequence was found. Hydrogenotrophs appear to have a much higher and obligate acetoclastic methanogens a much lower importance than previously thought in biogas production from renewable resources.

Quantification of Cryptosporidium parvum in anaerobic digesters treating manure by (reverse-transcription) quantitative real-time PCR, infectivity and excystation tests.

The survival of Cryptosporidium parvum oocysts in anaerobic digesters treating manure was investigated for mesophilic, thermophilic, and a combined treatment (mesophilic-thermophilic-mesophilic) under different retention times of oocysts in the reactors. C. parvum DNA was extracted with an optimised protocol, and its amount determined by quantitative real-time PCR (qPCR). Results indicated noteworthy differences in DNA content after the different treatments. DNA was not degraded during the process. However, excystation and infectivity tests showed a reduction of viable oocyst numbers of > or = 2 and > or = 5 log units after the thermophilic treatment in two different experiments. Thus qPCR-targeting DNA can overestimate the number of oocysts that survive and remain viable after anaerobic digestion. However, targeting DNA is suitable to indicate the presence or absence of oocysts. Reverse transcription qPCR (RT-qPCR) targeting C. parvum hsp70 mRNA successfully indicated the presence of viable fraction of oocysts.

Mesophilic-thermophilic-mesophilic anaerobic digestion of liquid dairy cattle manure.

The potential of a mesophilic-thermophilic-mesophilic anaerobic digestion system was investigated with respect to improvement of both digestion and sanitation efficiencies during treatment of liquid cattle manure. The pilot plant produced a high methane yield from liquid dairy cattle manure of 0.24 m3 (kg VSfed)(-1) Considering the low system loading rate of 1.4-1.5 kg VS (m3 d)(-1), digestion efficiency compared to conventional processes did not appear improved. The minimum guaranteed retention time in the tubular thermophilic reactor was increased compared to a continuously stirred tank reactor. Levels of intestinal enterococci in raw liquid manure as determined with cultivation methods were reduced by 2.5 -3 log units to a level of around 10(2) cfu/mL. This sanitizing effect was achieved both during mesophilic-thermophilic-mesophilic and thermophilic-mesophilic treatment, provided the thermophilic digester was operated at 53-55 degrees C. A change in feeding interval from 1 h to 4 h did not significantly alter methane yield and sanitation efficiency. It was proposed that a two-stage, thermophilic-mesophilic anaerobic digestion system would be able to achieve the same sanitizing effect and equal or better digestion efficiency at lower costs.

Quantitative detection of enteroviruses in activated sludge by cell culture and real-time RT-PCR using paramagnetic capturing.

We have compared in extracts of activated sludge the number of enteroviruses detectable with buffalo green monkey (BGM) cell-cultures versus the number of enteroviral genomes determined by reverse-transcription quantitative real-time PCR (RT-qPCR). In order to find conditions adequate for quantifying enteroviral RNA isolated from (waste)water we have investigated affinity capture of RNA with polystyrene beads (Dynabeads). The capture efficiency strongly depended on the genomic region chosen for the affinity binding. Capture of the RNA by its 3'-tail was most efficient (almost 100%); other regions within the genome yielded variable but lower results. Indirect capture (first hybridization of the RNA to the oligonucleotides, then attachment of the duplex molecules to the beads) was much more efficient than direct capture (attachment of the oligonucleotides to the beads first, then binding of the RNA), and resulted in RNA capture of maximally 60-80%. At least partly, this was due to incomplete hybridization of the RNA to the complementary oligonucleotides. No correlation was found between the number of cytopathic effects (CPE) determined by cell culture and the number of genomes quantified by RT-qPCR; RT-qPCR values were consistently much higher than the number of CPE. This points to overestimation of infectious enteroviruses by RT-qPCR and/or underestimation by the cell culture approach.

Hygienization by anaerobic digestion: comparison between evaluation by cultivation and quantitative real-time PCR.

In order to assess hygienization by anaerobic digestion, a comparison between evaluation by cultivation and quantitative real-time PCR (qPCR) including optimized DNA extraction and quantification was carried out for samples from a full-scale fermenter cascade (F1, mesophilic; F2, thermophilic; F3, mesophilic). The system was highly effective in inactivating (pathogenic) viable microorganisms, except for spore-formers. Conventionally performed cultivation underestimated viable organisms particularly in F2 and F3 by a factor of at least 10 as shown by data from extended incubation times, probably due to the rise of sublethally injured (active but not cultivable) cells. Incubation should hence be extended adequately in incubation-based hygiene monitoring of stressed samples, in order to minimize contamination risks. Although results from qPCR and cultivation agreed for the equilibrated compartments, considerably higher qPCR values were obtained for the fermenters. The difference probably corresponded to DNA copies from decayed cells that had not yet been degraded by the residual microbial activity. An extrapolation from qPCR determination to the quantity of viable organisms is hence not justified for samples that had been exposed to lethal stress.

Evaluating real-time PCR for the quantification of distinct pathogens and indicator organisms in environmental samples.

We evaluated quantitative real-time PCR (qPCR) and RTqPCR (for RNA species) for their ability to quantify microorganisms and viruses in problematic environmental samples such as cattle manure, digester material, wastewater and soil. Important developments included a standard spiking approach which compensated for methodological bias and allowed sample-to-sample comparison and reliable quantification. Programme CeTe was developed to calculate endogenous concentrations of target organisms (nucleic acid copies) for each sample separately from the generated standard curves. The approach also permitted assessment of the detection limit of the complete method, including extraction. It varied from sample to sample, due to different extraction efficiencies and variable co-extraction of PCR inhibitors. False negative results were thereby avoided. By using this approach we were able to optimise a DNA extraction protocol from the different tested sample types. Protocols for the extraction of RNA species from environmental samples were also optimised. DNA was (almost) not degraded after lethal shock (autoclaving) in the sterile environment. In contrast, the parallel selective cultivation and qPCR results for various microbial parameters from an anaerobic digester chain suggested that DNA from decaying organisms was readily recycled in metabolically active environments. It may, therefore, be used to determine viable organisms in samples exhibiting substantial metabolic turnover. It is proposed that our standard spiking approach, including data evaluation by the program CeTe, should be considered in future standardisation and norms for the quantification of nucleic acid containing organisms in environmental and product samples.

Using quantitative real-time PCR to determine the hygienic status of cattle manure.

We developed a suitable system of DNA extraction and real-time quantitative polymerase chain reaction (qPCR) for the specific and sensitive quantification of pathogens and other relevant (indicator) organisms in recalcitrant material such as cattle manure. PCR inhibition by coextraction of humic compounds was minimized in this system, resulting in detection sensitivity of one target DNA copy per reaction well. Data from qPCR analysis for Escherichia coli agreed with cultivation based results, but orders of magnitude more fecal enterococci, Enterobacteriaceae and Campylobacter jejuni, were determined by qPCR than by cultivation. These bacteria may have been in a potentially hazardous active but non-cultivable state. The qPCR system is much less time consuming than conventional cultivation, highly specific, can detect non-cultivable organisms, provides high measurement throughput, and is cost attractive. It should be considered as an alternative in various application areas for (prescribed routine) cultivation based assays, e.g. for biosafety and hygiene monitoring.

Ecology and evolution of bacterial microdiversity.

Using high resolution molecular fingerprinting techniques like random amplification of polymorphic DNA, repetitive extragenic palindromic PCR and multilocus enzyme electrophoresis, a high bacterial diversity below the species and subspecies level (microdiversity) is revealed. It became apparent that bacteria of a certain species living in close association with different plants either as associated rhizosphere bacteria or as plant pathogens or symbiotic organisms, typically reflect this relationship in their genetic relatedness. The strain composition within a population of soil bacterial species at a given field site, which can be identified by these high resolution fingerprinting techniques, was markedly influenced by soil management and soil features. The observed bacterial microdiversity reflected the conditions of the habitat, which select for better adapted forms. In addition, influences of spatial separation on specific groupings of bacteria were found, which argue for the occurrence of isolated microevolution. In this review, examples are presented of bacterial microdiversity as influenced by different ecological factors, with the main emphasis on bacteria from the natural environment. In addition, information available from some of the first complete genome sequences of bacteria (Helicobacter pylori and Escherichia coli) was used to highlight possible mechanisms of molecular evolution through which mutations are created; these include mutator enzymes. Definitions of bacterial species and subspecies ranks are discussed in the light of detailed information from whole genome typing approaches.

Taxonomic characterization of Ochrobactrum sp. isolates from soil samples and wheat roots, and description of Ochrobactrum tritici sp. nov. and Ochrobactrum grignonense sp. nov.

A large collection of bacterial strains, immunotrapped from soil and from the wheat rhizoplane, was subjected to polyphasic taxonomy by examining various pheno- and genotypic parameters. Strains were grouped on (inter) repetitive extragenic palindromic DNA (REP) PCR profiles at the intraspecies level. Pheno- and genotypic characters were assessed for representatives from 13 different REP groups. Strains of nine REP groups constituting two physiological BIOLOG clusters fell in the coherent DNA-DNA reassociation group of Ochrobactrum anthropi. Strains of two REP groups constituting a separate BIOLOG cluster fell in the coherent DNA-DNA reassociation group of Ochrobactrum intermedium. Additional phenotypic characters differentiating O. anthropi and O. intermedium were found. REP group K strains constituted a different BIOLOG cluster, a separate DNA-DNA reassociation group and a distinct phylogenetic lineage in 165 rDNA homology analysis, indicating that REP group K strains represent a new species. Diagnostic phenotypic characters were found. Closest relatives were Ochrobactrum species. The name Ochrobactrum grignonense sp. nov. is proposed (type strain OgA9aT = LMG 18954T = DSM 13338T). REP group J strains again constituted a different BIOLOG cluster, a separate DNA-DNA reassociation group and showed, as a biological particularity, a strict preference for the rhizoplane as habitat. Diagnostic phenotypic characters were found. This indicated that REP group J strains represent a further new species, although phylogenetic analyses using 16S rDNA homology were not able to separate the cluster of REP group J sequences significantly from 16S rDNA sequences of Ochrobactrum anthropi. The name Ochrobactrum tritici sp. nov. is proposed (type strain SCII24T = LMG 18957T = DSM 13340T).

Significance of NADH-vanadate-oxidoreductase of cardiac and erythrocyte cell membranes.

Vanadate(V), which has positive inotropic, natriuretic and vasoconstrictive effects, is taken up by cardiac cells and erythrocytes in large quantities. Most of the intracellular vanadium is shown to exist as protein-bound vanadyl(IV), however Vanadate (VO3) is a powerful inhibitor of the (Na+ rK+)-ATPase and the Ca++-ATPase, whereas it stimulates adenylate cyclase of cardiac tissue. Vanadyl (VO2+) has no or much less effects on these enzymes. Plasma membranes of cardiac tissue (cat, calf, human) as well as erythrocytes contain an enzyme that converts vanadate(V) to vanadyl(IV) in the presence of NADH but not NADPH. The optimal conditions for this NADH-vanadate-oxidoreductase are: pH 6.8, 1 mM, NADH, 1.5 mM Va3VO4. Mg++ inhibits the enzyme half-maximally at 3 mM, Ca++ stimulates at low and inhibits at high concentrations (half-maximally at 0.8 mM). The enzyme is supposed to be located at the inner side of the cell membrane. Vanadate has been proposed as an ideal regulator of active cation transport across the cell membrane. The finding of a HADH-vanadate-oxidoreductase converting vanadate into the rather inactive vanadyl further supports this hypotheses. The amount of vanadate at active sites of the target enzymes might be responsible for the known vanadate effects.