Initial community evenness favours functionality under selective stress.

Owing to the present global biodiversity crisis, the biodiversity-stability relationship and the effect of biodiversity on ecosystem functioning have become major topics in ecology. Biodiversity is a complex term that includes taxonomic, functional, spatial and temporal aspects of organismic diversity, with species richness (the number of species) and evenness (the relative abundance of species) considered among the most important measures. With few exceptions (see, for example, ref. 6), the majority of studies of biodiversity-functioning and biodiversity-stability theory have predominantly examined richness. Here we show, using microbial microcosms, that initial community evenness is a key factor in preserving the functional stability of an ecosystem. Using experimental manipulations of both richness and initial evenness in microcosms with denitrifying bacterial communities, we found that the stability of the net ecosystem denitrification in the face of salinity stress was strongly influenced by the initial evenness of the community. Therefore, when communities are highly uneven, or there is extreme dominance by one or a few species, their functioning is less resistant to environmental stress. Further unravelling how evenness influences ecosystem processes in natural and humanized environments constitutes a major future conceptual challenge.

Bacterial diversity and reductive dehalogenase redundancy in a 1,2-dichloroethane-degrading bacterial consortium enriched from a contaminated aquifer.

BACKGROUND: Bacteria possess a reservoir of metabolic functionalities ready to be exploited for multiple purposes. The use of microorganisms to clean up xenobiotics from polluted ecosystems (e.g. soil and water) represents an eco-sustainable and powerful alternative to traditional remediation processes. Recent developments in molecular-biology-based techniques have led to rapid and accurate strategies for monitoring and identification of bacteria and catabolic genes involved in the degradation of xenobiotics, key processes to follow up the activities in situ. RESULTS: We report the characterization of the response of an enriched bacterial community of a 1,2-dichloroethane (1,2-DCA) contaminated aquifer to the spiking with 5 mM lactate as electron donor in microcosm studies. After 15 days of incubation, the microbial community structure was analyzed. The bacterial 16S rRNA gene clone library showed that the most represented phylogenetic group within the consortium was affiliated with the phylum Firmicutes. Among them, known degraders of chlorinated compounds were identified. A reductive dehalogenase genes clone library showed that the community held four phylogenetically-distinct catalytic enzymes, all conserving signature residues previously shown to be linked to 1,2-DCA dehalogenation. CONCLUSIONS: The overall data indicate that the enriched bacterial consortium shares the metabolic functionality between different members of the microbial community and is characterized by a high functional redundancy. These are fundamental features for the maintenance of the community's functionality, especially under stress conditions and suggest the feasibility of a bioremediation treatment with a potential prompt dehalogenation and a process stability over time.

How to get more out of molecular fingerprints: practical tools for microbial ecology.

Community-level molecular techniques are widely used in comparative microbial ecology to assess the diversity of microbial communities and their response to changing environments. These include among others denaturing and temperature gradient gel electrophoresis (DGGE/TGGE), single-strand conformation polymorphism (SSCP), length heterogeneity-PCR (LH-PCR), terminal-restriction fragment length polymorphism (tRFLP) and 16S rRNA gene clone libraries. The amount of data derived from these techniques available in literature is continuously increasing and the lack of a universal way to interpret the raw fingerprint itself makes it difficult to compare between different results. Taking the DGGE technique as an example, we propose a setting-independent theoretical interpretation of the DGGE pattern, based on a straightforward processing on three levels of analysis: (i) the range-weighted richness (Rr) reflecting the carrying capacity of the system, (ii) the dynamics (Dy) reflecting the specific rate of species coming to significance, and (iii) functional organization (Fo), defined through a relation between the structure of a microbial community and its functionality. These Rr, Dy and Fo values, each representing a score to describe a microbial community, can be plotted in a 3D graph. The latter represents a visual ecological interpretation of the initial raw fingerprinting pattern.

Quantifying community dynamics of nitrifiers in functionally stable reactors.

A sequential batch reactor (SBR) and a membrane bioreactor (MBR) were inoculated with the same sludge from a municipal wastewater treatment plant, supplemented with ammonium, and operated in parallel for 84 days. It was investigated whether the functional stability of the nitrification process corresponded with a static ammonia-oxidizing bacterial (AOB) community. The SBR provided complete nitrification during nearly the whole experimental run, whereas the MBR showed a buildup of 0 to 2 mg nitrite-N liter(-1) from day 45 until day 84. Based on the denaturing gradient gel electrophoresis profiles, two novel approaches were introduced to characterize and quantify the community dynamics and interspecies abundance ratios: (i) the rate of change [Delta(t)((week))] parameter and (ii) the Pareto-Lorenz curve distribution pattern. During the whole sampling period, it was observed that neither of the reactor types maintained a static microbial community and that the SBR evolved more gradually than the MBR, particularly with respect to AOB (i.e., average weekly community changes of 12.6% +/- 5.2% for the SBR and 24.6% +/- 14.3% for the MBR). Based on the Pareto-Lorenz curves, it was observed that only a small group of AOB species played a numerically dominant role in the nitritation of both reactors, and this was true especially for the MBR. The remaining less dominant species were speculated to constitute a reserve of AOB which can proliferate to replace the dominant species. The value of these parameters in terms of tools to assist the operation of activated-sludge systems is discussed.

The phylogeny of the genus Nitrobacter based on comparative rep-PCR, 16S rRNA and nitrite oxidoreductase gene sequence analysis.

Strains of Nitrobacter mediate the second step in the nitrification process by oxidizing nitrite to nitrate. The phylogenetic diversity of the genus is currently not well investigated. In this study, a rep-PCR profile and the nearly complete 16S rRNA gene sequence of 30 strains, comprising a wide physiological as well as ecological diversity and encompassing representatives of the four species, were determined. The sequence diversity of the 16S rRNA gene between different species was low, indicating the need for additional phylogenetic markers. Therefore, primers were developed for amplifying the complete nxrX gene and a 380bp fragment of the nxrB1 gene, which are both genes involved in the nitrite oxidation process. These genes confirmed the division into phylogenetic groups revealed by the 16S rRNA gene but showed a better discriminatory power. They can be a valuable additional tool for phylogenetic analysis within the genus Nitrobacter and can assist in the identification of new Nitrobacter isolates.

The inoculum effect on the ammonia-oxidizing bacterial communities in parallel sequential batch reactors.

Three identical sequential batch reactors (SBRs) were each inoculated with sludge from a full-scale wastewater treatment plant (WWTP) treating a waste stream of different origin, i.e. a hospital, a meat processing company, and a municipal WWTP. The SBRs were run in parallel for 84 consecutive days to investigate whether the reactors would become more phylogenetically similar or stay separated concerning their functionality and microbial communities. Overall, the nitrification functionality was high throughout the experiment, and the size and structure of the sludge flocs were very similar. The total bacterial and ammonia-oxidizing bacterial (AOB) communities were analyzed by PCR-DGGE. Cluster analysis demonstrated very distinct bacterial communities in the three SBRs, not showing any trend becoming more similar. The carrying capacity, dynamics and functional organization of the communities were assessed by DGGE analysis and based on these patterns the range-weighted richness, moving window analysis, and constructing Pareto-Lorenz evenness distribution curves were calculated. Between the SBRs, highly comparable internal structure and dynamics of the AOB communities were observed, although they had only one AOB DGGE band in common. These observations indicate that community characteristics such as the extent of biodiversity and dynamics are more important indicators of good microbial functionality than the presence of certain specific species.

Environmental factors shaping the ecological niches of ammonia-oxidizing archaea.

For more than 100 years it was believed that bacteria were the only group responsible for the oxidation of ammonia. However, recently, a new strain of archaea bearing a putative ammonia monooxygenase subunit A (amoA) gene and able to oxidize ammonia was isolated from a marine aquarium tank. Ammonia-oxidizing archaea (AOA) were subsequently discovered in many ecosystems of varied characteristics and even found as the predominant causal organisms in some environments. Here, we summarize the current knowledge on the environmental conditions related to the presence of AOA and discuss the possible site-related properties. Considering these data, we deduct the possible niches of AOA based on pH, sulfide and phosphate levels. It is proposed that the AOA might be important actors within the nitrogen cycle in low-nutrient, low-pH, and sulfide-containing environments.

Nitric oxide reductase (norB) gene sequence analysis reveals discrepancies with nitrite reductase (nir) gene phylogeny in cultivated denitrifiers.

Gene sequence analysis of cnorB and qnorB, both encoding nitric oxide reductases, was performed on pure cultures of denitrifiers, for which previously nir genes were analysed. Only 30% of the 227 denitrifying strains rendered a norB amplicon. The cnorB gene was dominant in Alphaproteobacteria, and dominantly coexisted with the nirK gene, coding for the copper-containing nitrite reductase. Both norB genes were equally present in Betaproteobacteria but no linked distributional pattern of nir and norB genes could be observed. The overall cnorB phylogeny was not congruent with the widely accepted organism phylogeny based on 16S rRNA gene sequence analysis, with strains from different bacterial classes having identical cnorB sequences. Denitrifiers and non-denitrifiers could be distinguished through qnorB gene phylogeny, without further grouping at a higher taxonomic resolution. Comparison of nir and norB phylogeny revealed that genetic linkage of both genes is not widespread among denitrifiers. Thus, independent evolution of the genes for both nitrogen oxide reductases does also occur.

Real-time PCR assay for the simultaneous quantification of nitrifying and denitrifying bacteria in activated sludge.

In order to improve wastewater treatment processes, a need exists for tools that rapidly give detailed insight into the community structure of activated sludge, supplementary to chemical and physical data. In this study, the advantages of microarrays and quantitative polymerase chin reaction (PCR) methods were combined into a real-time PCR assay that allows the simultaneous quantification of phylogenetic and functional genes involved in nitrification and denitrification processes. Simultaneous quantification was possible along a 5-log dynamic range and with high linear correlation (R (2) > 0.98). The specificity of the assay was confirmed by cloning and sequencing analyses of PCR amplicons obtained from activated sludge. The real-time assay was validated on mixed liquid samples of different treatment plants, which varied in nitrogen removal rate. The abundance of ammonia oxidizers was in the order of magnitude of 10(6) down to 10(4) ml(-1), whereas nitrite oxidizers were less abundant (10(3)-10(1) order of magnitude). The results were in correspondence with the nitrite oxidation rate in the sludge types. As for the nirS, nirK, and nosZ gene copy numbers, their abundance was generally in the order of magnitude of 10(8)-10(5). When sludge samples were subjected to lab-scale perturbations, a decrease in nitrification rate was reflected within 18 h in the copy numbers of nitrifier genes (decrease with 1 to 5 log units), whereas denitrification genes remained rather unaffected. These results demonstrate that the method is a fast and accurate tool for the analysis of the (de)nitrifying community structure and size in both natural and engineered environmental samples.

The incidence of nirS and nirK and their genetic heterogeneity in cultivated denitrifiers.

Gene sequence analysis of nirS and nirK, both encoding nitrite reductases, was performed on cultivated denitrifiers to assess their incidence in different bacterial taxa and their taxonomical value. Almost half of the 227 investigated denitrifying strains did not render an nir amplicon with any of five previously described primers. NirK and nirS were found to be prevalent in Alphaproteobacteria and Betaproteobacteria, respectively, nirK was detected in the Firmicutes and Bacteroidetes and nirS and nirK with equal frequency in the Gammaproteobacteria. These observations deviated from the hitherto reported incidence of nir genes in bacterial taxa. NirS gene phylogeny was congruent with the 16S rRNA gene phylogeny on family or genus level, although some strains did group within clusters of other bacterial classes. Phylogenetic nirK gene sequence analysis was incongruent with the 16S rRNA gene phylogeny. NirK sequences were also found to be significantly more similar to nirK sequences from the same habitat than to nirK sequences retrieved from highly related taxa. This study supports the hypothesis that horizontal gene transfer events of denitrification genes have occurred and underlines that denitrification genes should not be linked with organism diversity of denitrifiers in cultivation-independent studies.

Cultivation of denitrifying bacteria: optimization of isolation conditions and diversity study.

An evolutionary algorithm was applied to study the complex interactions between medium parameters and their effects on the isolation of denitrifying bacteria, both in number and in diversity. Growth media with a pH of 7 and a nitrogen concentration of 3 mM, supplemented with 1 ml of vitamin solution but not with sodium chloride or riboflavin, were the most successful for the isolation of denitrifiers from activated sludge. The use of ethanol or succinate as a carbon source and a molar C/N ratio of 2.5, 20, or 25 were also favorable. After testing of 60 different medium parameter combinations and comparison with each other as well as with the standard medium Trypticase soy agar supplemented with nitrate, three growth media were highly suitable for the cultivation of denitrifying bacteria. All evaluated isolation conditions were used to study the cultivable denitrifier diversity of activated sludge from a municipal wastewater treatment plant. One hundred ninety-nine denitrifiers were isolated, the majority of which belonged to the Betaproteobacteria (50.4%) and the Alphaproteobacteria (36.8%). Representatives of Gammaproteobacteria (5.6%), Epsilonproteobacteria (2%), and Firmicutes (4%) and one isolate of the Bacteroidetes were also found. This study revealed a much more diverse denitrifying community than that previously described in cultivation-dependent research on activated sludge.

Impact of agricultural practices on the Zea mays L. endophytic community.

Agricultural practices are known to alter bulk soil microbial communities, but little is known about the effect of such practices on the plant endophytic community. We assessed the influence of long-term applications (20 years) of herbicides and different fertilizer types on the endophytic community of maize plants grown in different field experiments. Nested PCR-denaturing gradient gel electrophoresis (DGGE) analyses targeting general bacteria, type I or II methanotrophs, actinomycetes, and general fungi were used to fingerprint the endophytic community in the roots of Zea mays L. Low intraplant variability (reproducible DGGE patterns) was observed for the bacterial, type I methanotroph, and fungal communities, whereas the patterns for endophytic actinomycetes exhibited high intraplant variability. No endophytic amplification product was obtained for type II methanotrophs. Cluster and stability analysis of the endophytic type I methanotroph patterns differentiated maize plants cultivated by using mineral fertilizer from plants cultivated by using organic fertilizer with a 100% success rate. In addition, lower methanotroph richness was observed for mineral-fertilized plants than for organically fertilized plants. The use of herbicides could not be traced by fingerprinting the endophytic type I methanotrophs or by evaluating any other endophytic microbial group. Our results indicate that the effect of agrochemicals is not limited to the bulk microbial community but also includes the root endophytic community. It is not clear if this effect is due to a direct effect on the root endophytic community or is due to changes in the bulk community, which are then reflected in the root endophytic community.