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Denitrification is a process that reduces nitrogen levels in headwaters and other streams. We compared nirS and nirK abundances with the absolute rate of denitrification, the longitudinal coefficient of denitrification (i.e., Kden, which... more
Denitrification is a process that reduces nitrogen levels in headwaters and other streams. We compared nirS and nirK abundances with the absolute rate of denitrification, the longitudinal coefficient of denitrification (i.e., Kden, which represents optimal denitrification rates at given environmental conditions), and water quality in seven prairie streams to determine if nir-gene abundances explain denitrification activity. Previous work showed that absolute rates of denitrification correlate with nitrate levels; however, no correlation has been found for denitrification efficiency, which we hypothesise might be related to gene abundances. Water-column nitrate and soluble-reactive phosphorus levels significantly correlated with absolute rates of denitrification, but nir-gene abundances did not. However, nirS and nirK abundances significantly correlated with Kden, as well as phosphorus, although no correlation was found between Kden and nitrate. These data confirm that absolute denitrification rates are controlled by nitrate load, but intrinsic denitrification efficiency is linked to nirS and nirK gene abundances.
Rieske non-heme iron oxygenases enzymes have been widely studied, as they catalyse essential reactions initiating the bacterial degradation of organic compounds, for instance aromatic hydrocarbons. The genes encoding these enzymes offer a... more
Rieske non-heme iron oxygenases enzymes have been widely studied, as they catalyse essential reactions initiating the bacterial degradation of organic compounds, for instance aromatic hydrocarbons. The genes encoding these enzymes offer a potential target for studying aromatic hydrocarbon degrading organisms in the environment. However, previously reported primer sets that target dioxygenase gene sequences or the common conserved Rieske-centre of aromatics dioxygenases have limited specificity and/or target non-dioxygenase genes. In this work, an extensive database of dioxygenase α-subunit gene sequences was constructed, and primer sets targeting the conserved Rieske-centre were developed. The high specificity of the primers was confirmed by polymerase chain reaction analysis, agarose gel electrophoresis and sequencing. Quantitative polymerase chain reaction (qPCR) assays were also developed and optimised, following MIQE guidelines (Minimum Information for Publication of Quantitativ...
Bacterial spores are widespread in marine sediments, including those of thermophilic, sulphate-reducing bacteria, which have a high minimum growth temperature making it unlikely that they grow in situ. These Desulfotomaculum spp. are... more
Bacterial spores are widespread in marine sediments, including those of thermophilic, sulphate-reducing bacteria, which have a high minimum growth temperature making it unlikely that they grow in situ. These Desulfotomaculum spp. are thought to be from hot environments and are distributed by ocean currents. Their cells and spores upper temperature limit for survival is unknown, as is whether they can survive repeated high-temperature exposure that might occur in hydrothermal systems. This was investigated by incubating estuarine sediments significantly above (40-80 °C) maximum in situ temperatures (∼23 °C), and with and without prior triple autoclaving. Sulphate reduction occurred at 40-60 °C and at 60 °C was unaffected by autoclaving. Desulfotomaculum sp. C1A60 was isolated and was most closely related to the thermophilic D. kuznetsovii(T) (∼96% 16S rRNA gene sequence identity). Cultures of Desulfotomaculum sp. C1A60, D. kuznetsovii(T)and D. geothermicum B2T survived triple autocla...
... consid-ering microbial populations as V or'K'strategists, with distinct colonisation and survival characteristics (Graham & Curtis 2003) that ... principally members of the genera Nitrosospira and Nitrosomonas, in... more
... consid-ering microbial populations as V or'K'strategists, with distinct colonisation and survival characteristics (Graham & Curtis 2003) that ... principally members of the genera Nitrosospira and Nitrosomonas, in nitrification in a wide range of environments (Kowalchuk & Stephen ...
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A laboratory scale reactor operated as a single sludge, denitrification-nitrification bioreactor (DNB), was fed a synthetic wastewater. The effect of the C/N ratio of the influent on the structure of beta-proteobacterial autotrophic... more
A laboratory scale reactor operated as a single sludge, denitrification-nitrification bioreactor (DNB), was fed a synthetic wastewater. The effect of the C/N ratio of the influent on the structure of beta-proteobacterial autotrophic ammonia-oxidizing bacterial (AOB) communities was determined by DGGE analysis of 16S rRNA gene fragments amplified using a range of AOB-selective primers. Fluorescence in situ hybridisation (FISH) was used to determine quantitative changes in the AOB communities. When operated at a C/N ratio of 2 the DNB was effective in nitrogen removal and nitrification was measured at approximately 1.0 mg NH4+-N/g dry wt/h. Altering the C/N ratio to 5 resulted in a 50% reduction in nitrification rates. Nitrification was restored to its original level when the C/N ratio was returned to 2. AOB were detected by DGGE analysis of samples from the DNB under all operating conditions but the changes in C/N ratio and nitrification rates were accompanied by changes in the community structure of the AOB. However, quantitative FISH analysis indicated that beta-proteobacterial AOB were only present in high numbers (ca. 10(8) cells/ml) under the original operating conditions with a C/N ratio of 2. Beta-proteobacterial AOB could not be detected by FISH when the C/N ratio was 5. When nitrification activity was restored by returning the C/N ratio to 2, beta-proteobacterial AOB were still not detected and it is likely that either beta-proteobacterial AOB were not responsible for ammonia oxidation or that beta-proteobacterial AOB that did not contain the target sites for the range of 4 AOB selective probes used, were present in the reactor.
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Lab-scale reactors are commonly used to simulate full-scale plants as they permit the effects of defined experimental perturbations to be evaluated. Ideally, lab- and full-scale reactors should possess similar microbial populations. To... more
Lab-scale reactors are commonly used to simulate full-scale plants as they permit the effects of defined experimental perturbations to be evaluated. Ideally, lab- and full-scale reactors should possess similar microbial populations. To determine this we compared the diversity of the beta-proteobacterial autotrophic ammonia-oxidising bacteria (AOB) in a full-scale and lab-scale biological aerated filter (BAF) using PCR with AOB selective primers combined with denaturing gradient gel electrophoresis (DGGE). PCR amplified 16S rRNA gene fragments from the nitrification unit of the lab-and full-scale BAF were subjected to cloning and sequencing to determine the phylogenetic affiliation of the AOB. A high degree of comparability between the lab-and full-scale BAF was observed with respect to AOB populations. However minor differences were apparent. The importance of these minor constituents in the overall performance of the reactor is unknown. Nonetheless the lab-scale reactor in this study did appear to reflect the dominant AOB community within the full-scale equivalent.
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It is the best of times for biofilm research. Systems biology approaches are providing new insights into the genetic regulation of microbial functions, and sophisticated modelling techniques are enabling the prediction of microbial... more
It is the best of times for biofilm research. Systems biology approaches are providing new insights into the genetic regulation of microbial functions, and sophisticated modelling techniques are enabling the prediction of microbial community structures. Yet it is also clear that there is a need for ecological theory to contribute to our understanding of biofilms. Here, we suggest a concept
Hydrocarbons are common constituents of surface, shallow, and deep-subsurface environments. Under anaerobic conditions, hydrocarbons can be degraded to methane by methanogenic microbial consortia. This degradation process is widespread in... more
Hydrocarbons are common constituents of surface, shallow, and deep-subsurface environments. Under anaerobic conditions, hydrocarbons can be degraded to methane by methanogenic microbial consortia. This degradation process is widespread in the geosphere. In comparison with other anaerobic processes, methanogenic hydrocarbon degradation is more sustainable over geological time scales because replenishment of an exogenous electron acceptor is not required. As a consequence, this process has been responsible for the formation of the world's vast deposits of heavy oil, which far exceed conventional oil assets such as those found in the Middle East. Methanogenic degradation is also a potentially important component of attenuation in hydrocarbon contamination plumes. Studies of the organisms, syntrophic partnerships, mechanisms, and geochemical signatures associated with methanogenic hydrocarbon degradation have identified common themes and diagnostic markers for this process in the subsurface. These studies have also identified the potential to engineer methanogenic processes to enhance the recovery of energy assets as biogenic methane from residual oils stranded in petroleum systems.
ABSTRACT The diversity of autotrophic ammonia-oxidizing bacteria (AOB) of the b-subdivision of the class Proteobacteria was investigated in a laboratory-scale denitrification-nitrification bioreactor (DNB) treating a synthetic waste... more
ABSTRACT The diversity of autotrophic ammonia-oxidizing bacteria (AOB) of the b-subdivision of the class Proteobacteria was investigated in a laboratory-scale denitrification-nitrification bioreactor (DNB) treating a synthetic waste stream. 16S ribosomal RNA (rRNA) gene sequences were amplified from DNA extracted from the oxic DNB sludge. Comparative analysis of the rRNA sequences revealed considerable diversity among the AOB-like sequences. The majority of sequences recovered were related to Nitrosomonas spp. but a smaller number of Nitrosospira-like sequences were obtained. Since different AOB may have different kinetic properties the high diversity of AOB, even in a simple laboratory biotreater treating a simple waste stream, has important implications for the operation of nitrifying wastewater treatment processes.
Quantitative real-time PCR is a valuable tool for microbial ecologists. To obtain accurate absolute quantification it is essential that PCR efficiency for pure standards is close to amplification efficiency for test samples. Counter to... more
Quantitative real-time PCR is a valuable tool for microbial ecologists. To obtain accurate absolute quantification it is essential that PCR efficiency for pure standards is close to amplification efficiency for test samples. Counter to normal expectation that PCR efficiency might be lower in environmental DNA, due to the presence of PCR inhibitors, we report the counterintuitive observation that PCR efficiency of pure standards can be lower than for environmental DNA. This can lead to overestimation of gene abundances if not corrected. SYBR green-based qPCR assays of 16S rRNA genes targeting Bacteria, Syntrophus and Smithella spp., Marinobacter spp., Methanomicrobiales, Methanosarcinaceae, and Methanosaetaceae in samples from methanogenic crude oil biodegradation enrichments were tested. In five out of the six assays, PCR efficiency was lower with pure standards than with environmental DNA samples. We developed a solution to this problem based on amending pure clone standards with a background of non-target environmental 16S rRNA genes which significantly improved PCR efficiency of standards in the qPCR assays that exhibited this phenomenon. Overall this method of qPCR standard preparation achieved a more reliable and robust quantification of 16S rRNA genes. We believe this may be a potentially common issue in microbial ecology that often goes unreported, as intuitively one would not expect standards to have poorer PCR efficiency than samples.
To provide deeper insights into nitrification process within aerobic bioreactors containing supplemental physical support media (hybrid bioreactors). Three bench-scale hybrid bioreactors with different media size and one control... more
To provide deeper insights into nitrification process within aerobic bioreactors containing supplemental physical support media (hybrid bioreactors). Three bench-scale hybrid bioreactors with different media size and one control bioreactor were operated to assess how biofilm integrity influences microbial community conditions and bioreactor performance. The systems were operated initially at a 5-day hydraulic retention time (HRT), and all reactors displayed efficient nitrification and chemical oxygen demand (COD) removal (>95%). However, when HRT was reduced to 2.5 days, COD removal rates remained high, but nitrification efficiencies declined in all reactors after 19 days. To explain reduced performance, nitrifying bacterial communities (ammonia-oxidizing bacteria, AOB; nitrite-oxidizing bacteria, NOB) were examined in the liquid phase and also on the beads using qPCR, FISH and DGGE. Overall, the presence of the beads in a reactor promoted bacterial abundances and diversity, but as bead size was increased, biofilms with active coupled AOB-NOB activity were less apparent, resulting in incomplete nitrification. Hybrid bioreactors have potential to sustain effective nitrification at low HRTs, but support media size and configuration type must be optimized to ensure coupled AOB and NOB activity in nitrification. This study shows that AOB and NOB coupling must be accomplished to minimize nitrification failure.
In ecotoxicology, standard biological assays are used to determine the effects of chemicals on microorganisms. One assay, the microbial multiplication inhibition test, measures the degree of growth inhibition of a population of... more
In ecotoxicology, standard biological assays are used to determine the effects of chemicals on microorganisms. One assay, the microbial multiplication inhibition test, measures the degree of growth inhibition of a population of microorganisms when exposed to a chemical. ...