David B. Nedwell

Identification of active methylotroph populations in an acidic forest soil by stable- isotope probing

Stable-isotope probing (SIP) is a culture-independent technique that enables the isolation of DNA from micro-organisms that are actively involved in a specific metabolic process. In this study, SIP was used to characterize the active methylotroph populations in forest soil (pH 3.5) microcosms that were exposed to (13)CH(3)OH or (13)CH(4). Distinct (13)C-labelled DNA ((13)C-DNA) fractions were resolved from total community DNA by CsCl density-gradient centrifugation. Analysis of 16S rDNA sequences amplified from the (13)C-DNA revealed that bacteria related to the genera Methylocella, Methylocapsa, Methylocystis and Rhodoblastus had assimilated the (13)C-labelled substrates, which suggested that moderately acidophilic methylotroph populations were active in the microcosms. Enrichments targeted towards the active proteobacterial CH(3)OH utilizers were successful, although none of these bacteria were isolated into pure culture. A parallel analysis of genes encoding the key enzymes methanol dehydrogenase and particulate methane monooxygenase reflected the 16S rDNA analysis, but unexpectedly revealed sequences related to the ammonia monooxygenase of ammonia-oxidizing bacteria (AOB) from the beta-subclass of the PROTEOBACTERIA: Analysis of AOB-selective 16S rDNA amplification products identified Nitrosomonas and Nitrosospira sequences in the (13)C-DNA fractions, suggesting certain AOB assimilated a significant proportion of (13)CO(2), possibly through a close physical and/or nutritional association with the active methylotrophs. Other sequences retrieved from the (13)C-DNA were related to the 16S rDNA sequences of members of the Acidobacterium division, the beta-Proteobacteria and the order Cytophagales, which implicated these bacteria in the assimilation of reduced one-carbon compounds or in the assimilation of the by-products of methylotrophic carbon metabolism. Results from the (13)CH(3)OH and (13)CH(4) SIP experiments thus provide a rational basis for further investigations into the ecology of methylotroph populations in situ.

Diversity and Abundance of Nitrate Reductase Genes (narG and napA), Nitrite Reductase Genes (nirS and nrfA), and Their Transcripts in Estuarine Sediments

ABSTRACT Estuarine systems are the major conduits for the transfer of nitrate from agricultural and other terrestrial-anthropogenic sources into marine ecosystems. Within estuarine sediments some microbially driven processes (denitrification and anammox) result in the net removal of nitrogen from the environment, while others (dissimilatory nitrate reduction to ammonium) do not. In this study, molecular approaches have been used to investigate the diversity, abundance, and activity of the nitrate-reducing communities in sediments from the hypernutrified Colne estuary, United Kingdom, via analysis of nitrate and nitrite reductase genes and transcripts. Sequence analysis of cloned PCR-amplified narG, napA, and nrfA gene sequences showed the indigenous nitrate-reducing communities to be both phylogenetically diverse and also divergent from previously characterized nitrate reduction sequences in soils and offshore marine sediments and from cultured nitrate reducers. In both the narG and nrfA libraries, the majority of clones (48% and 50%, respectively) were related to corresponding sequences from delta-proteobacteria. A suite of quantitative PCR primers and TaqMan probes was then developed to quantify phylotype-specific nitrate (narG and napA) and nitrite reductase (nirS and nrfA) gene and transcript numbers in sediments from three sites along the estuarine nitrate gradient. In general, both nitrate and nitrite reductase gene copy numbers were found to decline significantly (P < 0.05) from the estuary head towards the estuary mouth. The development and application, for the first time, of quantitative reverse transcription-PCR assays to quantify mRNA sequences in sediments revealed that transcript numbers for three of the five phylotypes quantified were greatest at the estuary head.

Detection and diversity of expressed denitrification genes in estuarine sediments after reverse transcription-PCR amplification from mRNA.

ABSTRACT The expression of five denitrification genes coding for two nitrate reductases (narG and napA), two nitrite reductases (nirS and nirK), and nitrous oxide reductase (nosZ) was analyzed by reverse transcription (RT)-PCR of mRNA extracted from two sediment samples obtained in the River Colne estuary (United Kingdom), which receives high nitrogen inputs and for which high denitrification rates have been observed. The presence of all five genes in both sediment samples was confirmed by PCR amplification from extracted DNA prior to analysis of gene expression. Only nirS and nosZ mRNAs were detected; nirS was detected directly as an RT-PCR amplification product, and nosZ was detected following Southern blot hybridization. This indicated that active expression of at least the nirS and nosZ genes was occurring in the sediments at the time of sampling. Amplified nirS RT-PCR products were cloned and analyzed by sequencing, and they were compared with amplified nirS gene sequences from isolates obtained from the same sediments. A high diversity of nirS sequences was observed. Most of the cloned nirS sequences retrieved were specific to one site or the other, which underlines differences in the compositions of the bacterial communities involved in denitrifrification in the two sediments analyzed.

Inhibition of methanogenesis by sulphate reducing bacteria competing for transferred hydrogen.

A methanogenic bacterial consortium was obtained after inoculation of benzoate medium under N2/CO2 atmosphere with intertidal sediment. A hydrogen donating organotroph andMethanococcus mazei were isolated from this enrichment. H2-utilising sulphate reducing bacteria were isolated under H2/CO2 in the absence of organic electron donors. TheMethanococcus was able to produce methane in yeast extract medium under N2/CO2 if the H2 donating organism was present, and sulphate reduction occurred if the hydrogen utilising sulphate reducing bacteria were grown with the H2 donating organism. The ability of the H2 utilising sulphate reducing bacteria to inhibitMethanococcus competitively was shown in cultures containing both of these H2 utilising bacteria.

Changes in benthic denitrification, nitrate ammonification, and anammox process rates and nitrate and nitrite reductase gene abundances along an estuarine nutrient gradient (the Colne estuary, United Kingdom).

ABSTRACT Estuarine sediments are the location for significant bacterial removal of anthropogenically derived inorganic nitrogen, in particular nitrate, from the aquatic environment. In this study, rates of benthic denitrification (DN), dissimilatory nitrate reduction to ammonium (DNRA), and anammox (AN) at three sites along a nitrate concentration gradient in the Colne estuary, United Kingdom, were determined, and the numbers of functional genes (narG, napA, nirS, and nrfA) and corresponding transcripts encoding enzymes mediating nitrate reduction were determined by reverse transcription-quantitative PCR. In situ rates of DN and DNRA decreased toward the estuary mouth, with the findings from slurry experiments suggesting that the potential for DNRA increased while the DN potential decreased as nitrate concentrations declined. AN was detected only at the estuary head, accounting for ∼30% of N2 formation, with 16S rRNA genes from anammox-related bacteria also detected only at this site. Numbers of narG genes declined along the estuary, while napA gene numbers were stable, suggesting that NAP-mediated nitrate reduction remained important at low nitrate concentrations. nirS gene numbers (as indicators of DN) also decreased along the estuary, whereas nrfA (an indicator for DNRA) was detected only at the two uppermost sites. Similarly, nitrate and nitrite reductase gene transcripts were detected only at the top two sites. A regression analysis of log(n + 1) process rate data and log(n + 1) mean gene abundances showed significant relationships between DN and nirS and between DNRA and nrfA. Although these log-log relationships indicate an underlying relationship between the genetic potential for nitrate reduction and the corresponding process activity, fine-scale environmentally induced changes in rates of nitrate reduction are likely to be controlled at cellular and protein levels.

CH4 production, oxidation and emission in a U.K. ombrotrophic peat bog: Influence of SO42− from acid rain

Factors influencing the rates of production and emission of CH4, CH4 oxidation and rates of SO42− reduction, were measured in the peat of an ombrotrophic bog in New Galloway, Scotland. Vertical concentration profiles of CH4 and O2 showed that the water table essentially represented the oxic-anoxic boundary in the peat. This boundary was usually at the surface in the case of peat-bog hollows, but up to 20 cm of oxic peat occurred above the water table in peat-bog hummocks. Penetration of O2 into the peat increased under illumination when photosynthesis was active, but decreased in the dark. Emission of CH4 from the peat surface was faster from peat-bog hollows than from hummocks, where most CH4 was reoxidized before emission. CH4 emission rates also varied seasonally, being greatest during summer. For most of the year the amount of organic C oxidized to CO2 by SO42− reduction by anaerobic bacteria exceeded that being transformed to CH4 by methanogenic bacteria, except during summer when SO42− reduction became SO42− limited. Laboratory experiments showed that the addition of SO42− to peat inhibited CH4 formation, confirming that there was competitive inhibition of CH4 formation by active SO42− reduction, as demonstrated in other environments. The degree of acid rain deposition of SO42− onto peat bogs may therefore be extremely important in regulating the production and emission of CH4 from peat. CH4 formation was most active in the strata of peat 5–15 cm below the water table, although actual rates of CH4 formation were slower in the peat beneath hummocks than that below hollows. In contrast, CH4 oxidation occurred nearer the peat surface (only 3–7 cm below the water table) where the methanotrophic bacteria could intercept vertically migrating CH4. Surprisingly, the peak for CH4 oxidation potential occurred at about 5 cm below the water table, in peat which was apparently anoxic. This may reflect either a transiently oxic peat environment, in which aerobic CH4-oxidizing bacteria persisted, or the presence of a community of facultatively anaerobic CH4-oxidizing bacteria which, in anoxic conditions, metabolized substrates other than CH4. There was no evidence of anaerobic CH4 oxidation.

Analysis of the sulfate-reducing bacterial and methanogenic archaeal populations in contrasting Antarctic sediments.

ABSTRACT The distribution and activity of communities of sulfate-reducing bacteria (SRB) and methanogenic archaea in two contrasting Antarctic sediments were investigated. Methanogenesis dominated in freshwater Lake Heywood, while sulfate reduction dominated in marine Shallow Bay. Slurry experiments indicated that 90% of the methanogenesis in Lake Heywood was acetoclastic. This finding was supported by the limited diversity of clones detected in a Lake Heywood archaeal clone library, in which most clones were closely related to the obligate acetate-utilizing Methanosaeta concilii. The Shallow Bay archaeal clone library contained clones related to the C1-utilizing Methanolobus and Methanococcoides and the H2-utilizing Methanogenium. Oligonucleotide probing of RNA extracted directly from sediment indicated that archaea represented 34% of the total prokaryotic signal in Lake Heywood and that Methanosaeta was a major component (13.2%) of this signal. Archaea represented only 0.2% of the total prokaryotic signal in RNA extracted from Shallow Bay sediments. In the Shallow Bay bacterial clone library, 10.3% of the clones were SRB-like, related to Desulfotalea/Desulforhopalus, Desulfofaba, Desulfosarcina, and Desulfobacter as well as to the sulfur and metal oxidizers comprising the Desulfuromonas cluster. Oligonucleotide probes for specific SRB clusters indicated that SRB represented 14.7% of the total prokaryotic signal, with Desulfotalea/Desulforhopalus being the dominant SRB group (10.7% of the total prokaryotic signal) in the Shallow Bay sediments; these results support previous results obtained for Arctic sediments. Methanosaeta and Desulfotalea/Desulforhopalus appear to be important in Lake Heywood and Shallow Bay, respectively, and may be globally important in permanently low-temperature sediments.

The Input and Mineralization of Organic Carbon in Anaerobic Aquatic Sediments

The bottom sediments in both marine and freshwater ecosystems are important sites of mineralization and nutrient recycling, particularly where there is shallow water together with high productivity so that there is rapid input of organic carbon to the sediment. In most coastal and intertidal areas and in eutrophic lakes, productivity is relatively high and detrital input to the bottom sediments is appreciable, with the result that much of the sediments in these regions is anaerobic and reduced, apart perhaps from a thin aerobic surface layer. Therefore, at least potentially, a considerable portion of the organic carbon mineralization in these aquatic ecosystems may go on in the sediment under anaerobic rather than aerobic conditions.

Hydrogen as a substrate for methanogenesis and sulphate reduction in anaerobic saltmarsh sediment

Hydrogen gas stimulated sulphate reduction in a saltmarsh sediment and the importance of H2 transferred from organotrophic bacteria to the sulphate-reducers is discussed. β-fluorolactate inhibited sulphate reduction whether lactate, ethanol or hydrogen was being used as growth substrate. When added to sediment β-fluorolactate inhibited sulphate reduction with a consequent increase in methane production.Addition of H2 stimulated methanogenesis in sediment and this stimulation was greater if CO2 was also present. Hydrogen availability was the primary limitation of methanogenesis but the low concentration of dissolved CO2 in seawater may limit methane production even if H2 is available.The removal of inhibition of methanogenesis by the use of fluorolactate to suppress sulphate reduction or by the provision of hydrogen indicates competitive inhibition of methanogens by sulphate reducers utilizing transferred hydrogen.

Oxidation of methane in peat: Kinetics of CH4 and O2 removal and the role of plant roots

Vertical profiles of oxygen uptake potential were measured in peat. When both O2 and CH4 were in excess, methanotrophy accounted for 85% of the O2 uptake potential, showing a high capacity for CH4 oxidation in the peat. In the absence of CH4, maximum O2 uptake potential was near the peat surface where available labile organic matter was present and decreased with depth as organic matter became more refractory. The oxidation of CH4 followed saturation kinetics with respect to both CH4 and O2 when they were at limiting concentrations. For CH4 oxidation in the peat, the kO2 was 32 μm O2 and kCH4 was 57.9 μm CH4. The Vmax for O2 was 209 nmol O2 ml−1 peat h−1, which was approximately double that for CH4, correctly reflecting the stoichiometry of aerobic CH4 oxidation. The CH4 oxidation kinetics were used in a mathematical model to examine the effect of plant roots on increasing the vertical transport rate of CH4 out of and O2 into the peat, by gas phase transport through the roots. In the absence of roots, CH4 oxidation was confined to a narrow layer near the peat surface where vertical gradients of O2 and CH4 overlapped. Little CH4 diffused through this surface layer. With roots present, the model confirmed the possibility of sub-surface peaks of aerobic CH4 oxidation potential below the water table in an apparently anoxic peat. These were due to active CH4 oxidation in the oxic rhizosphere maintained by the plant roots in the otherwise anoxic peat. The extrusion of O2 from the root tips also tended to diminish in situ CH4 formation by inhibition of the anaerobic methanogenic bacteria. The presence of plant roots increased the flux of CH4 out of the peat to the atmosphere, by-passing the surface oxic layer in which active CH4 oxidation mopped up vertically diffusing CH4.