Soil microbial community characterization is increasingly being used to determine the responses of soils to stress and disturbances and to assess ecosystem sustainability. However, there is little experimental evidence to indicate that predictable patterns in microbial community structure or composition occur during secondary succession or ecosystem restoration. This study utilized a chronosequence of developing jarrah (Eucalyptus marginata) forest ecosystems, rehabilitated after bauxite mining (up to 18 years old), to examine changes in soil bacterial and fungal community structures (by automated ribosomal intergenic spacer analysis [ARISA]) and changes in specific soil bacterial phyla by 16S rRNA gene microarray analysis. This study demonstrated that mining in these ecosystems significantly altered soil bacterial and fungal community structures. The hypothesis that the soil microbial community structures would become more similar to those of the surrounding nonmined forest with rehabilitation age was broadly supported by shifts in the bacterial but not the fungal community. Microarray analysis enabled the identification of clear successional trends in the bacterial community at the phylum level and supported the finding of an increase in similarity to nonmined forest soil with rehabilitation age. Changes in soil microbial community structure were significantly related to the size of the microbial biomass as well as numerous edaphic variables (including pH and C, N, and P nutrient concentrations). These findings suggest that soil bacterial community dynamics follow a pattern in developing ecosystems that may be predictable and can be conceptualized as providing an integrated assessment of numerous edaphic variables.

This biogeochemical, molecular genetic and lipid biomarker study of sediments ( approximately 4 m cores) from the Skagerrak (Denmark) investigated methane cycling in a sediment with a clear sulfate-methane-transition zone (SMTZ) and where CH(4) supply was by diffusion, rather than by advection, as in more commonly studied seep sites. Sulfate reduction removed sulfate by 0.7 m and CH(4) accumulated below. (14)C-radiotracer measurements demonstrated active H(2)/CO(2) and acetate methanogenesis and anaerobic oxidation of CH(4) (AOM). Maximum AOM rates occurred near the SMTZ ( approximately 3 nmol cm(-3) day(-1) at 0.75 m) but also continued deeper, overall, at much lower rates. Maximum rates of H(2)/CO(2) and acetate methanogenesis occurred below the SMTZ but H(2)/CO(2) methanogenesis rates were x 10 those of acetate methanogenesis, and this was consistent with initial values of (13)C-depleted CH(4) (delta(13)C c.-80 per thousand). Areal AOM and methanogenic rates were similar ( approximately 1.7 mmol m(-2) day(-1)), hence, CH(4) flux is finely balanced. A 16S rRNA gene library from 1.39 m combined with methanogen (T-RFLP), bacterial (16S rRNA DGGE) and lipid biomarker depth profiles showed the presence of populations similar to some seep sites: ANME-2a (dominant), ANME-3, Methanomicrobiales, Methanosaeta Archaea, with abundance changes with depth corresponding to changes in activities and sulfate-reducing bacteria (SRB). Below the SMTZ to approximately 1.7 m CH(4) became progressively more (13)C depleted (delta(13)C -82 per thousand) indicating a zone of CH(4) recycling which was consistent with the presence of (13)C-depleted archaeol (delta(13)C -55 per thousand). Pore water acetate concentrations decreased in this zone (to approximately 5 microM), suggesting that H(2), not acetate, was an important CH(4) cycling intermediate. The potential biomarkers for AOM-associated SRB, non-isoprenoidal ether lipids, increased below the SMTZ but this distribution reflected 16S rRNA gene sequences for JS1 and OP8 bacteria rather than those of SRB. At this site peak rates of methane production and consumption are spatially separated and seem to be conducted by different archaeal groups. Also AOM is predominantly coupled to sulfate reduction, unlike recent reports from some seep and gassy sediment sites.

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The methanogen community in sediment from the edge of a small brackish lake connected to the Beaulieu Estuary (Hampshire, UK) was investigated by analysis of 16S rRNA gene diversity using new methanogen-specific primers plus Archaea-specific primers. 16S rRNA gene primers previously used for polymerase chain reaction (PCR) detection of methanogenic Archaea from a variety of environments were evaluated by in silico testing. The primers displayed variable coverage of the four main orders of methanogens, highlighting the importance of this type of primer evaluation. Three PCR primer sets were designed using novel reverse primers to facilitate specific amplification of the orders Methanomicrobiales/Methanosarcinales, Methanobacteriales and Methanococcales. Diversity of the methanogen functional gene, methyl coenzyme M reductase (mcrA), was also studied. All gene libraries constructed from this sediment indicated that Methanomicrobiales and Methanosarcinales were the only methanogens detected. There was good agreement between the relative sequence abundances in the methanogen-specific 16S rRNA gene library and terminal restriction fragment length polymorphism (T-RFLP) profiling, suggesting that the population was dominated by putative H2 CO2 utilizing Methanomicrobiales, although acetate-utilizing methanogens were also present. The methanogen population analyses were in agreement with methanogenic activity measurements, which indicated that bicarbonate methanogenesis was higher than acetate methanogenesis at all depths measured and overall there was a significant difference (P = 0.001) between the rates of the two pathways. This study demonstrates the utility of new 16S rRNA gene PCR primers targeting specific methanogenic orders, and the combined results suggest that the CO2 reduction pathway dominates methanogenesis in the brackish sediment investigated.

This study was based on the hypothesis that groundwater-derived biofilms may provide a reservoir for coliform or pathogenic bacteria as has been observed in drinking water distribution systems. Escherichia coli, labelled with green fluorescent protein, was found to colonize all layers of mixedpopulation biofilms developed in association with indigenous groundwater micro-organisms in a laboratory-scale reactor. Biofilm-associated E. coli was removed at a slower rate from the reactor flasks than planktonic E. coli under a continuous flow regime. During flow-through of groundwater, planktonic E. coli removal was slower in flasks containing coverslips for enhanced biofilm development compared to a control flask without coverslips. Conversely, during flow-through of treated effluent, planktonic E. coli removal was faster in flasks with coverslips compared to without. Removal of attached E. coli was also fastest in the coverslip-containing flasks with effluent flowthrough. This suggests that an increase in available nutrients may reduce E. coli survival potential due to either enhanced competition for nutrients or enhanced antagonism by the indigenous microbial population. Under identical conditions, GFP-labelled Pseudomonas aeruginosa was found to persist in the biofilms for longer than E. coli, most notably when exposed to flow-through of treated effluent. However, prolonged persistence of P. aeruginosa in the effluent could not be attributed to an association with the biofilms. This study has shown that under certain conditions the presence of mixed-population biofilms may limit the survival potential of enteric bacterial pathogens introduced into groundwater.

Ammonia-oxidising archaea (AOA) and bacteria (AOB) are responsible for the rate limiting step in nitrification; a key nitrogen (N) loss pathway in agricultural systems. Dominance of AOA relative to AOB in the amoA gene pool has been reported in many ecosystems, although their relative contributions to nitrification activity are less clear. Here we examined the distribution of AOA and AOB with depth in semi-arid agricultural soils in which soil organic matter content or pH had been altered, and related their distribution to gross nitrification rates. Soil depth had a significant effect on gene abundances, irrespective of management history. Contrary to reports of AOA dominance in soils elsewhere, AOA gene copy numbers were four-fold lower than AOB in the surface (0–10 cm). AOA gene abundance increased with depth while AOB decreased, and sub-soil abundances were approximately equal (10–90 cm). The depth profile of total archaea did not mirror that of AOA, indicating the likely presence of archaea without nitrification capacity in the surface. Gross nitrification rates declined significantly with depth and were positively correlated to AOB but negatively correlated to AOA gene abundances. We conclude that AOB are most likely responsible for regulating nitrification in these semi-arid soils.

Sustainable plant establishment on ore processing residues requires development of a functional soil, which includes the introduction of organic matter and reestablishment of active microbial communities. This study investigated the development of microbial diversity and function in residue sand generated from alumina refining of mined bauxite ore. The residue sand embankments underwent rehabilitation with native vegetation over time, allowing study of a 3-year chronosequence using space-for-time substitution. A coastal dune ecosystem was used as a natural alkaline sand analog with which to compare residue properties. Microbial biomass carbon in the residue sands were typically well below that of the coastal sand analog (<50 mg kg −1 in residue sand compared to circa 450 mg kg −1 in coastal sand). Although the size of the microbial biomass appeared to be limited by the low organic matter content of the residue sand, a decline in microbial metabolic quotient indicated a potential alleviation of microbial stress with rehabilitation age. Despite the low microbial biomass, the ability of the residue sand microbial community to function with respect to the metabolism of added amino acids developed rapidly. Contrary to our original hypothesis, the diversity of the bacterial and fungal community also developed rapidly, and was similar to, or higher than, the coastal sand analog in 0.5-year-old rehabilitation. However, the bacterial, and in particular the fungal, community structure within residue sands were significantly different to that of the coastal sand analog with shifts in community structure driven, in part, by changing physicochemical conditions.

Aims: The aim of this study was to deterimine the survival of an enteric bacterium in anaerobic groundwater and effluent microcosms using the green fluorescent protein (GFP) marker gene in combination with the viability indicator propidium iodide (PI). Methods and Results: The pEGFP vector (Clontech) was transformed into Escherichia coli DH5a and was stable for at least 100 generations of growth in nonselective medium at 28°C and 37°C. Using an epifluorescent microscope, GFP cells could be detected under blue light (450-490 nm) and the numbers of PI-positive GFPs could be detected under green light (530-560 nm). GFP-tagged E. coli could be detected for at least 132 d in sterilized water microcosms. GFP fluorescence was not lost from the culturable cell population for the duration of the experiment. However, a slow decline in the number of GFP-fluorescent cells in sterilized groundwater was observed. Escherichia coli die-off and loss of green fluorescence was more rapid in nonsterilized waters than in sterilized. Viable numbers of the GFP-tagged E. coli determined by PI counterstaining were compatible with numbers of colony-forming units. Conclusions:The long-term survival of E. coli and maintainance of GFP-conferred fluorescence in these cells was demonstrated in both groundwater and effluent, under sterilized conditions. However, severe starvation and/or the presence of indigenous microorganisms were found to be factors affecting the maintenance of fluorescence in dead or dying cells. Significance and Impact of the Study: This study demonstrates the successful application of PI with GFP-tagging to monitor long-term bacterial survival in nutrient-limited conditions and mixed microbial populations.