Abstract: A microarray spotted with 369 different 16S rRNA gene probes specific to microorganisms involved in the degradation process of organic waste during composting was developed. The microarray was tested with pure cultures, and of the 30,258 individual probe-target hybridization reactions performed, there were only 188 false positive (0.62%) and 22 false negative signals (0.07%). Labeled target DNA was prepared by polymerase chain reaction amplification of 16S rRNA genes using a Cy5-labeled universal bacterial forward primer and a universal reverse primer. The COMPOCHIP microarray was applied to three different compost types (green compost, manure mix compost, and anaerobic digestate compost) of different maturity (2, 8, and 16 weeks), and differences in the microorganisms in the three compost types and maturity stages were observed. Multivariate analysis showed that the bacterial composition of the three composts was different at the beginning of the composting process and became more similar upon maturation. Certain probes (targeting Sphingobacterium, Actinomyces, Xylella/Xanthomonas/Stenotrophomonas, Microbacterium, Verrucomicrobia, Planctomycetes, Low G + C and Alphaproteobacteria) were more influential in discriminating between different composts. Results from denaturing gradient gel electrophoresis supported those of microarray analysis. This study showed that the COMPOCHIP array is a suitable tool to study bacterial communities in composts.
Abstract: The aim of this study was to design a microarray targeting methanogens found in anaerobic digesters, and to apply this chip together with a cloning approach to investigate the methanogenic community present in an anaerobic digester. Oligonucleotide probes were designed based on sequence differences in the 16S rRNA genes in order to target microorganisms in situ. For microarray hybridisations, DNA was subjected to PCR amplification of the 16S rRNA gene and Cy5-labeled. The microarray was tested with pure cultures, and of the 1854 individual probe-target hybridisation reactions performed, there were only 28 false positive (1.5%) and 16 false negative signals (0.86%). The sensitivity of the array was also tested, and it was found that when 0.4pg of DNA from a pure culture was subjected to PCR amplification, signals above the detection limit were obtained. Also, the application of 25ng of PCR product from a pure culture to an array resulted in detectable signals. The ANAEROCHIP was hybridised with DNA from the anaerobic sludge. Strong hybridisation signals were obtained for Methanoculleus, and weaker signals, in decreasing order, were obtained for Methanosarcina, Methanobacterium, Methanobrevibacter, and Methanosphaera. In order to check the results obtained with the microarray, the archaeal community structure of the same digester was analysed by 16S rRNA gene cloning and sequencing. Community structure was determined by restriction digestion of almost 200 clones and by sequencing of the 15 different resulting patterns. Methanoculleus was the dominant (84.1%) microorganism in the anaerobic sludge, and Methanobrevibacter (5.8%), Methanobacterium (3.7%), Methanosarcina (2.1%), Methanosphaera (1.6%), an uncultured archaeon (1.6%) and Methanothermobacter (1%) were also detected. These results showed the microarray to be a suitable tool for studying methanogenic communities in sludge.
Abstract: Prokaryotic diversity was investigated near the inlet and outlet of a plug-flow reactor. After analyzing 800 clones, 50 bacterial and 3 archaeal phylogenetic groups were defined. Clostridia (>92%) dominated among bacteria and Methanoculleus (>90%) among archaea. Significant changes in pH and volatile fatty acids did not invoke a major shift in the phylogenetic groups. We suggest that the environmental filter imposed by the saline conditions (20 g liter(-1)) selected a stable community of halotolerant and halophilic prokaryotes.
Abstract: The aim of this work was to evaluate the impact of different N-rich animal wastes on the composting of ligno-cellulosic wastes by a range of classical and novel methods, with particular emphasis on microbial community composition. Two composting mixtures were prepared by adding to a mixture of cotton carding wastes and wheat straw: (i) meat and bone meal and (ii) blood meal and horn and hoof meal. Composts were analyzed using physico-chemical and biochemical properties, as well as nucleic acid microarrays. Results showed that physico-chemical and biochemical parameters differentiated composts depending on their degree of stability, while microarray hybridization discriminated compost samples according to the starting materials used in the compost production. Microarray analysis indicated not only the presence in the composts of bacteria involved in N(2) fixation and plant disease suppression, but also the presence of Acinetobacter calcoaceticus that is suspected to trigger an autoimmune response related to bovine spongiform encephalopathy. The present work highlights the importance of using parameters addressing different properties of the composting matrix for a proper evaluation of the process performance.
Abstract: Two different case studies concerning potential overload situations of anaerobic digesters were investigated and mathematically modelled by means of the Anaerobic Digestion Model No. 1 (ADM1). The first scenario included a digester failure at a municipal WWTP which occurred during revision works of the upstream digester within a two-step digestion system when the sludge was directly by-passed to the 2nd-step reactor. Secondly, the non-occurrence of a highly expected upset situation in a lab-scale digester fed with cattle manure was investigated. ADM1 was utilized to derive indicators which were used to investigate the relationship between digester stability and biomass population dynamics. Conventional design parameters such as the organic loading rate appeared unsuitable for process description under dynamic conditions. Indicators reflecting the biokinetic state (e.g. F(net)/M(net) or the VFA/alkalinity ratio) are more adequate for the assessment of the stability of reactors in transient situations.
Abstract: In this study, 16S rRNA gene primers were designed to complement the suite of already available PCR primers for the detection of different methanogens involved in biogas production through anaerobic digestion by SYBR Green real-time PCR. Primers designed for use in TaqMan real-time PCR for the organisms Methanosaeta, Methanosarcina, and Methanoculleus have been described previously; however, we found that (i) the Methanoculleus primers were not specific to members of the genus and that (ii) the Methanosarcina primers did not work specifically with SYBR Green real-time PCR. Thus, we designed new primers for these and other methanogens, and we optimized SYBR Green real-time PCR assays. Primers were tested by end-point and real-time PCR, and they were found to work specifically and sensitively. Application of these primers will allow the detection and quantification of Methanoculleus, Methanosarcina, Methanothermobacter, and a group of yet uncultured archaea from anaerobic habitats.
Abstract: The earthworm, Lumbricus rubellus, plays an essential role in soil ecosystems as it affects organic matter decomposition and nutrient cycling. By ingesting a mixture of organic and mineral material, a variety of bacteria and fungi are carried to the intestinal tract of the earthworm. To get a better understanding of the interactions between L. rubellus and the microorganisms ingested, this study tried to reveal if the diet affects the composition of the gut microflora of L. rubellus or if its intestinal tract hosts an indigenous, species-specific microbiota. A feeding experiment with L. rubellus was set up; individuals were collected in the field, transferred to a climate chamber and fed with food sources of different quality (dwarf shrub litter, grass litter or horse dung) for six weeks. DNA was extracted from the guts of the earthworms, as well as from the food sources and the surrounding soil, and further analysed by a molecular fingerprinting method, PCR-DGGE (Polymerase Chain Reaction -- Denaturing Gradient Gel Electrophoresis). We were able to demonstrate that the gut microbiota was strongly influenced by the food source ingested and was considerably different to that of the surrounding soil. Sequencing of dominant bands of the bacterial DGGE fingerprints revealed a strong occurrence of y-Proteobacteria in all gut samples, independent of the food source. A specific microflora in the intestinal tract of L. rubellus, robust against diet changes, could not be found.
Abstract: All organic material eventually is decomposed by microorganisms, and considerable amounts of C and N end up as gaseous metabolites. The emissions of greenhouse relevant gases like carbon dioxide, methane and nitrous oxides largely depend on physico-chemical conditions like substrate quality or the redox potential of the habitat. Manipulating these conditions has a great potential for reducing greenhouse gas emissions. Such options are known from farm and waste management, as well as from wastewater treatment. In this paper examples are given how greenhouse gas production might be reduced by regulating microbial processes. Biogas production from manure, organic wastes, and landfills are given as examples how methanisation may be used to save fossil fuel. Methane oxidation, on the other hand, might alleviate the problem of methane already produced, or the conversion of aerobic wastewater treatment to anaerobic nitrogen elimination through the anaerobic ammonium oxidation process might reduce N2O release to the atmosphere. Changing the diet of ruminants, altering soil water potentials or a change of waste collection systems are other measures that affect microbial activities and that might contribute to a reduction of carbon dioxide equivalents being emitted to the atmosphere.
Abstract: The metabolic diversity of microbial communities is fundamental for the multiple soil functions mediated by microorganisms. Community level physiological profiles (CLPPs) based on sole C source oxidation have been used as a fast and reproducible tool to study soil microbial functional diversity because the utilisation of available carbon is the key factor governing microbial growth in soil. Our aim was to assess the phylogenetic affiliation of the microorganisms responsible for C consumption after inoculating Biolog plates. For this purpose, two semi-arid Mediterranean forest soils with significantly different patterns of C consumption and microbial community structure were used. Following the inoculation of the Biolog plates, suspensions from seven wells were sampled after 1, 2 and 7 d of incubation. DNA was extracted and the microbial communities analysed by polymerase chain reaction followed by denaturing gradient gel electrophoresis (PCR-DGGE) and sequencing of excised bands. Despite major differences in the microbial communities of the soils studied, their DGGE banding patterns after incubation were similar for all the analysed C source suspensions. Microorganisms belonging to beta-Proteobacteria (Ralstonia sp. and Burkholderia sp.) and alpha-Proteobacteria (Rhizobium sp.) were dominant. These opportunists had a competitive advantage under the conditions at which the CLPPs were analysed. This study reveals that significantly different CLPP patterns can be generated on the basis of only 3-4 genera, as reflected by PCR-DGGE analysis. Also for this reason, CLPPs based on incubations of soil suspensions should just be used as a screening method and always be accompanied by other techniques for community analysis.
Abstract: The compost environment consists of complex organic materials that form a habitat for a rich and diverse microbial community. The aim of this research was to study the dynamics of microbial communities during the compost-curing phase. Three different methods based on 16S rRNA gene sequence were applied to monitor changes in the microbial communities: (1) denaturing gradient gel electrophoresis of PCR-generated rRNA gene fragments; (2) partial rRNA gene clone libraries; and (3) a microarray of oligonucleotide probes targeting rRNA gene sequences. All three methods indicated distinctive community shifts during curing and the dominant species prevailing during the different curing stages were identified. We found a successional transition of different bacterial phylogenetic groups during compost curing. The Proteobacteria were the most abundant phylum in all cases. The Bacteroidetes and the Gammaproteobacteria were ubiquitous. During the midcuring stage, Actinobacteria were dominant. Different members of nitrifying bacteria and cellulose and macromolecule-degrading bacteria were found throughout the curing process. In contrast, pathogens were not detected. In the cured compost, bacterial population shifts were still observed after the compost organic matter and other biochemical properties had seemingly stabilized.
Abstract: During composting, the degradation of organic waste is accompanied and driven by a succession of microbial populations exhibiting a broad range of functional capabilities. Detailed inventories of the microbial communities in mature compost, however, are not available. Mature composts, originating from biowaste as well as sewage sludge and anaerobic sludge, were studied by denaturing gradient gel electrophoresis-fingerprints after polymerase chain reaction (PCR) amplification of the 16S rRNA genes using three different universal primer pairs, as well as by differential scanning calorimetry and thermogravimetry. The composts of different origin had different bacterial communities. The influence of different 16S rDNA primer sets on the same batches of compost DNA was evaluated. The clearest separation of different compost types was obtained by using the PCR primer pair 338f + 518r which is suggested for future applications. Communities from the different biowaste compost samples clustered together and could be separated from sewage sludge communities indicating the establishment of different microbial consortia. A similar differentiation of composts was found with the thermogavimetric analyses. It may thus be concluded that the resulting humus quality is closely linked to the microbial communities involved.
Abstract: The existence of anaerobic ammonia-oxidizing (anammox) bacteria was postulated in the late 1970s. Approximately 20 years later, these lithotrophic members of the nitrogen cycle were identified as deep-branching members of the planctomycetes. Recently, full-scale implementation of biological deammonification was successfully achieved in the DEMON reactor at the wastewater treatment plant in Strass, Austria. The sludge of this reactor contains red granules and brownish flocs that can be physically separated. The two fractions yielded different banding patterns in denaturing gradient gel electrophoresis of PCR products obtained with primer sets targeting the 16S rRNA genes of planctomycetes. Comparative analysis of partial sequences of almost full-length 16S rRNA gene clones obtained from the granules and flocs confirms the differences in the community composition of the two fractions. The sequences retrieved from the red granules were 93% similar to those of Candidatus Brocadia anammoxidans, a bacterium known to catalyze the anaerobic ammonia oxidation.
Abstract: Different digestion technologies for various substrates are addressed by the generic process description of Anaerobic Digestion Model No. 1. In the case of manure or agricultural wastes a priori knowledge about the substrate in terms of ADM1 compounds is lacking and influent characterisation becomes a major issue. The actual project has been initiated for promotion of biogas technology in agriculture and for expansion of profitability also to rather small capacity systems. In order to avoid costly individual planning and installation of each facility a standardised design approach needs to be elaborated. This intention pleads for bio kinetic modelling as a systematic tool for process design and optimisation. Cofermentation under field conditions was observed, quality data and flow data were recorded and mass flow balances were calculated. In the laboratory different substrates have been digested separately in parallel under specified conditions. A configuration of four ADM1 model reactors was set up. Model calibration identified disintegration rate, decay rates for sugar degraders and half saturation constant for sugar as the three most sensitive parameters showing values (except the latter) about one order of magnitude higher than default parameters. Finally, the model is applied to the comparison of different reactor configurations and volume partitions. Another optimisation objective is robustness and load flexibility, i.e. the same configuration should be adaptive to different load situations only by a simple recycle control in order to establish a standardised design.
Abstract: Malodorous emissions and potentially pathogenic microorganisms which develop during domestic organic waste collection are not only a nuisance but may also pose health risks. The aim of the present study was to determine whether the presence of specific microorganisms in biowastes is directly related to the composition of the emitted volatile organic compounds (VOCs). The succession of microbial communities during 16 days of storage in organic waste collection bins was studied by denaturing gradient gel electrophoresis (DGGE) of amplified 16S ribosomal DNA in parallel with a classical cultivation and isolation approach. Approximately 60 different bacterial species and 20 different fungal species were isolated. Additionally, some bacterial species were identified through sequencing of excised DGGE bands. Proton transfer reaction mass spectrometry (PTR-MS) was used to detect VOCs over the sampling periods, and co-inertia analyses of VOC concentrations with DGGE band intensities were conducted. Positive correlations, indicating production of the respective VOC or enhancement of microbial growth, and negative correlations, indicating the use of, or microbial inhibition by the respective compound, were found for the different VOCs. Measurement of the VOC emission pattern from a pure culture of Lactococcus lactis confirmed the positive correlations for the protonated masses 89 (tentatively identified as butyric acid), 63 (tentatively identified as dimethylsulfide), 69 (likely isoprene) and 73 (likely butanone).
Abstract: Different labeling methods were studied to compare various approaches to the preparation of labeled target DNA for microarray experiments. The methods under investigation included a post-PCR labeling method using the Klenow fragment and a DecaLabel DNA labeling kit, the use of a Cy3-labeled forward primer in the PCR, generating either double-stranded or single-stranded PCR products, and the incorporation of Cy3-labeled dCTPs in the PCR. A microarray that had already been designed and used for the detection of microorganisms in compost was used in the study. PCR products from the organisms Burkholderia cepacia and Staphylococcus aureus were used in the comparison study, and the signals from the probes for these organisms analyzed. The highest signals were obtained when using the post-PCR labeling method, although with this method, more non-specific hybridizations were found. Single-stranded PCR products that had been labeled by the incorporation of a Cy3-labeled forward primer in the PCR were found to give the next highest signals upon hybridization for a majority of the tested probes, with less non-specific hybridizations. Hybridization with double-stranded PCR product labeled with a Cy3-labeled forward primer, or labeled by the incorporation of Cy3-labeled dCTPs resulted in acceptable signal to noise ratios for all probes except the UNIV 1389a and Burkholderia genus probes, both located toward the 3' end of the 16S rRNA gene. The comparison of the different DNA labeling methods revealed that labeling via the Cy3-forward primer approach is the most appropriate of the studied methods for the preparation of labeled target DNA for our purposes.
Abstract: Metabolic abilities and micrfiobial community structure were investigated through three semiarid Mediterranean soils of SE Spain. The soils were (1) a Typic Calcixerept under an adult pine plantation (PP), growing on abandoned agricultural terraces; (2) a Typic Calcixeroll under a native pinewood (NP); and (3) a Typic Haploxerept covered with a grass steppe (GS). PP and NP were similar as regards their genesis, but the former used to be tilled. NP and GS were undisturbed and supported natural and seminatural vegetation, respectively. Seven samples in 10-cm depth increments were taken in triplicate along each soil profile. Community-level physiological profiles based on sole-C-source use were determined to characterize the metabolic abilities. A 16S rDNA polymerase chain reaction-denaturing gradient gel electrophoresis analysis was performed to investigate the microbial genetic structure. Plant cover and land-use history were major determinants of microbial community structure. Microbial communities residing in soils under a native pinewood, the most diverse and stable plant cover, were the most complex both metabolically and genetically. The microbial community structure distinctly changed with depth, related to the quantity and quality of total organic carbon. Both undisturbed soils showed falling gradients of metabolic and genetic complexity, which were invariably of a greater magnitude in the mature woodland than in the grass steppe. In the planted pinewood, however, the substrate-use diversity increased with depth, apparently a response to the depleted metabolic abilities within its upper layer (0-30 cm). Tilling and plant cover removal might be responsible for such a perturbation. In the same profile, molecular fingerprint patterns of the topsoil layer (0-10 cm) indicated a disturbed genetic structure that might underlie the loss of metabolic abilities. However, the genetic structure of the deeper layers of the planted and native pinewoods was not dissimilar, revealing that equivalent genetic resources perform different environmental functions under changing soil scenarios.
Abstract: A microarray consisting of oligonucleotide probes targeting variable regions of the 16S rRNA gene was designed and tested for the investigation of microbial communities in compost. Probes were designed for microorganisms that have been previously reported in the composting process and for plant, animal and human pathogens. The oligonucleotide probes were between 17 and 25 bp in length and included mostly species-specific sequences. Validation of probe specificity and optimization of hybridization conditions were conducted using fluorescently labeled 16S rRNA gene PCR products of pure culture strains. A labeling method employing a Cy3 or Cy5-labeled forward primer together with a phosphate-conjugated reverse primer for the production of single stranded DNA after a digestion step was optimised and used to label target DNA. A combination of two different DNA extraction methods using both physical and chemical lysis was found to give the best DNA yields. Increased hybridization signal intensities were obtained for probes modified with a 12 mer T-spacer. The microarray was found to have a detection limit of 10(3) cells, although in compost spiking experiments, the detection limit was reduced to 10(5) cells. The application of the microarray to compost samples indicated the presence of Streptococcus, Acinetobacter lwoffii, and Clostridium tetani in various compost samples. The presence of A. lwoffii in those compost samples was confirmed by PCR using primers specific for the organism. The aim of this study was to develop a molecular tool that would allow screening for the presence or absence of different microorganisms within compost samples.
Abstract: Plots of an alpine grassland in the Swiss Alps were treated with elevated (680 microl l(-1)) and ambient CO2 (355 microl l(-1)) in open top chambers (OTC). Several plots were also treated with NPK-fertilizer. Community level physiological profiles (CLPPs) of the soil bacteria were examined by Biolog GN microplates and enzyme activities were determined through the release of methylumbelliferyl (MUF) and methylcoumarin (MC) from MUF- or MC-labelled substrates. A canonical discriminant analysis (CDA) followed by multivariate analysis of variance showed a significant effect of elevated CO2 on the CLPPs both under fertilized and unfertilized conditions. Further, the installation of the OTCs caused significant shifts in the CLPPs (chamber effect). Of the four enzyme activities tested, the beta-D-cellobiohydrolase (CELase) and N-acetyl-beta-D-glucosaminidase (NAGase) activity were enhanced under elevated CO2. L-Leucin-7-aminopeptidase (APEase) activity decreased, when the plots received fertilizer. Beta-D-glucosidase (GLUase) remained unaffected. The results suggest effects of elevated CO2 on specific microbial activities even under low mineral nutrient conditions and when bulk parameters like microbial biomass or respiration, which have been investigated on the same site, remain unaffected. The observed medium-term changes point at possible long-term consequences for the ecosystem that may not be specified yet.
Abstract: Plants in artificial tropical ecosystems were grown under ambient (340 microl l(-1)) and elevated (610 microl l(-1)) atmospheric CO2 for 530 d under low-nutrient conditions on a substrate free of organic C. At the end of the experiment a number of soil chemical and microbiological variables were determined. Although we found no changes in total soil organic matter under elevated CO2, we did find that after physical fractionation the amount of organic C in the supernatant (< 0.2 microm) and the amount of water extractable organic C (WEOC) was lower under elevated CO2. The extractable optical density (OD) indicated a higher degree of humification for the elevated than for the ambient CO2 samples (P = 0.032). Microbial biomass C was not significantly altered under high CO2, but total bacterial counts were significantly higher. The microbial biomass C-to-N ratio was also higher at elevated (15.0) than at ambient CO2 (10.0). The number of mycorrhizal spores was lower at high CO2, but ergosterol contents and fungal hyphal lengths were not significantly affected. Changes were found neither in community level physiological profiles (CLPPs) nor in the structural attributes (phospholipid fatty acids, PLFAs) of the microbial community. Overall, the effects on the soil microbiota were small, perhaps as a result of the low nutrient supply and low organic matter content of the soil used in our study. The few significant results showing changes in specific, though relatively minor, organic matter pools may point to possible long-term changes of the more major pools. Furthermore, the data suggest increased competition between plants and microbes for N at high CO2.
