In situ microbial treatment of landfill leachate using aerated lagoons.

The aim of this study was to assess the efficiency of leachate treatment by microbial oxidation in four connected on-site aerated lagoons at a landfill site. The landfill site was found to be in an ageing methanogenic state, producing leachate with relatively low COD (mean value 1740 mg l(-1)) and relatively high ammonium concentrations (mean value 1241 mg l(-1)). Removal of COD averaged 75%, with retention times varying from 11 to 254 days. Overall 80% of the N load was removed within the plant, some by volatilisation of ammonium. Microbial community profiling of the water from each lagoon showed a divergent community profile, presumably a reflection of the nutrient status in each lagoon. In municipal solid waste landfills under similar conditions, leachate treatment through a facultative aerobic system in which sequential aerobic and anaerobic microbial oxidations occurred can readily be achieved using a simple two-lagoon system, suggesting this technology can be economic to install and simple to run.

Isolation of haloarchaea that grow at low salinities.

Summary Archaea, the third domain of life, were long thought to be limited to environmental extremes. However, the discovery of archaeal 16S rRNA gene sequences in water, sediment and soil samples has called into question the notion of Archaea as obligate extremophiles. Until now, none of these novel Archaea has been brought into culture, a critical step for discovering their ecological roles. We have cultivated three novel halophilic Archaea (haloarchaea) genotypes from sediments in which the pore-water salinity was close to that of sea water. All previously reported haloarchaeal isolates are obligate extreme halophiles requiring at least 9% (w/v) NaCl for growth and are typically the dominant heterotrophic organisms in salt and soda lakes, salt deposits and salterns. Two of these three newly isolated genotypes have lower requirements for salt than previously cultured haloarchaea and are capable of slow growth at sea-water salinity (2.5% w/v NaCl). Our data reveal the existence of Archaea that can grow in non-extreme conditions and of a diverse community of haloarchaea existing in coastal salt marsh sediments. Our findings suggest that the ecological range of these physiologically versatile prokaryotes is much wider than previously supposed.

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

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 C(1)-utilizing Methanolobus and Methanococcoides and the H(2)-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.

Use of 16S rRNA-targeted oligonucleotide probes to investigate function and phylogeny of sulphate-reducing bacteria and methanogenic archaea in a UK estuary.

Abstract Sulphate-reducing bacteria (SRB) and methanogenic archaea (MA) are important anaerobic terminal oxidisers of organic matter. However, we have little knowledge about the distribution and types of SRB and MA in the environment or the functional role they play in situ. Here we have utilised sediment slurry microcosms amended with ecologically significant substrates, including acetate and hydrogen, and specific functional inhibitors, to identify the important SRB and MA groups in two contrasting sites on a UK estuary. Substrate and inhibitor additions had significant effects on methane production and on acetate and sulphate consumption in the slurries. By using specific 16S-targeted oligonucleotide probes we were able to link specific SRB and MA groups to the use of the added substrates. Acetate consumption in the freshwater-dominated sediments was mediated by Methanosarcinales under low-sulphate conditions and Desulfobacter under the high-sulphate conditions that simulated a tidal incursion. In the marine-dominated sediments, acetate consumption was linked to Desulfobacter. Addition of trimethylamine, a non-competitive substrate for methanogenesis, led to a large increase in Methanosarcinales signal in marine slurries. Desulfobulbus was linked to non-sulphate-dependent H(2) consumption in the freshwater sediments. The addition of sulphate to freshwater sediments inhibited methane production and reduced signal from probes targeted to Methanosarcinales and Methanomicrobiales, while the addition of molybdate to marine sediments inhibited Desulfobulbus and Desulfobacterium. These data complement our understanding of the ecophysiology of the organisms detected and make a firm connection between the capabilities of species, as observed in the laboratory, to their roles in the environment.

The distribution and activity of sulphate reducing bacteria in estuarine and coastal marine sediments.

Sulphate-reducing bacteria (SRB) play a vital role both the carbon and sulphur cycles and thus are extremely important components of the global microbial community. However, it is clear that the ecology, the distribution and activity of different SRB groups is poorly understood. Probing of rRNA suggests that different sediments have distinctly different patterns of SRB with complex factors controlling the activity of these organisms. The linking of community structure and function using sediment slurry microcosms suggests that certain groups of SRB, e.g., Desulfobacter and Desulfobulbus, can be linked to the use of specific substrates in situ. However, it is still unclear what environmental substrates are utilised by the majority of known SRBs. The work to date has greatly enhanced our understanding of the ecology of these organisms and is beginning to suggest patterns in their distribution and activity that may be relevant to understanding microbial ecology in general.

Rapid assessment of macro algal cover on intertidal sediments in a nutrified estuary.

Macroalgal blooms have been considered to be an indicator of eutrophication. A new and rapid method is described for the assessment of macroalgal cover in the intertidal zone of estuaries. Macroalgal cover in the intertidal of the nutrient-enriched River Deben estuary was found to reach a maximum of 50% coverage, but this varied seasonally with the highest percentage cover during June and July. Macro-algae mats were particularly associated with areas of hard substrata providing suitable attachment points, rather than with the nutrient concentrations along the estuary. The occurrence of macroalgae may be more related to the substrate than to the nutrient status of the estuary.

Use of 16S rRNA-targeted oligonucleotide probes to investigate the distribution of sulphate-reducing bacteria in estuarine sediments.

The distribution of sulphate-reducing bacteria (SRBs) in three anaerobic sediments, one predominantly freshwater and low sulphate and two predominantly marine and high sulphate, on the River Tama, Tokyo, Japan, was investigated using 16S rRNA-targeted oligonucleotide probes. Hybridisation results and sulphate reduction measurements indicated that SRBs are a minor part of the bacterial population in the freshwater sediments. Only Desulfobulbus and Desulfobacterium were detected, representing 1.6% of the general bacterial probe signal. In contrast, the SRB community detected at the two marine-dominated sites was larger and more diverse, representing 10-11.4% of the bacterial signal and with Desulfobacter, Desulfovibrio, Desulfobulbus and Desulfobacterium detected. In contrast to previous reports our results suggest that Desulfovibrio may not always be the most abundant SRB in anaerobic sediments. Acetate-utilising Desulfobacter were the dominant SRB in the marine-dominated sediments, and Desulfobulbus and Desulfobacterium were active in low-sulphate sediments, where they may utilise electron acceptors other than sulphate.

Temperature dependence of inorganic nitrogen uptake: reduced affinity for nitrate at suboptimal temperatures in both algae and bacteria.

Nitrate utilization and ammonium utilization were studied by using three algal isolates, six bacterial isolates, and a range of temperatures in chemostat and batch cultures. We quantified affinities for both substrates by determining specific affinities (specific affinity = maximum growth rate/half-saturation constant) based on estimates of kinetic parameters obtained from chemostat experiments. At suboptimal temperatures, the residual concentrations of nitrate in batch cultures and the steady-state concentrations of nitrate in chemostat cultures both increased. The specific affinity for nitrate was strongly dependent on temperature (Q10 approximately 3, where Q10 is the proportional change with a 10 degrees C temperature increase) and consistently decreased at temperatures below the optimum temperature. In contrast, the steady-state concentrations of ammonium remained relatively constant over the same temperature range, and the specific affinity for ammonium exhibited no clear temperature dependence. This is the first time that a consistent effect of low temperature on affinity for nitrate has been identified for psychrophilic, mesophilic, and thermophilic bacteria and algae. The different responses of nitrate uptake and ammonium uptake to temperature imply that there is increasing dependence on ammonium as an inorganic nitrogen source at low temperatures.

Phylogenetic diversity of Archaea in sediment samples from a coastal salt marsh.

The Archaea present in salt marsh sediment samples from a tidal creek and from an adjacent area of vegetative marshland, both of which showed active methanogenesis and sulfate reduction, were sampled by using 16S rRNA gene libraries created with Archaea-specific primers. None of the sequences were the same as reference sequences from cultured taxa, although some were closely related to sequences from methanogens previously isolated from marine sediments. A wide range of Euryarchaeota sequences were recovered, but no sequences from Methanococcus, Methanobacterium, or the Crenarchaeota were recovered. Clusters of closely related sequences were common and generally contained sequences from both sites, suggesting that some related organisms were present in both samples. Recovery of sequences closely related to those of methanogens such as Methanococcoides and Methanolobus, which can use substrates other than hydrogen, provides support for published hypotheses that such methanogens are probably important in sulfate-rich sediments and identifies some likely candidates. Sequences closely related to those of methanogens such as Methanoculleus and Methanogenium, which are capable of using hydrogen, were also discovered, in agreement with previous inhibitor and process measurements suggesting that these taxa are present at low levels of activity. More surprisingly, we recovered a variety of sequences closely related to those from different halophilic Archaea and a cluster of divergent sequences specifically related to the marine group II archaeal sequences recently shown by PCR and probing to have a cosmopolitan distribution in marine samples.

Rapid Extraction of DNA and rRNA from Sediments by a Novel Hydroxyapatite Spin-Column Method.

We describe a novel hydroxyapatite spin-column method of nucleic acid extraction from natural sediments by which DNA and rRNA can be extracted separately. This very rapid method produces pure nucleic acid that can be utilized in some of the most common molecular biological procedures used in the analysis of natural microbial communities.

Capacity for methane oxidation in landfill cover soils measured in laboratory-scale soil microcosms.

Laboratory-scale soil microcosms containing different soils were permeated with CH(inf4) for up to 6 months to investigate their capacity to develop a methanotrophic community. Methane emissions were monitored continuously until steady states were established. The porous, coarse sand soil developed the greatest methanotrophic capacity (10.4 mol of CH(inf4) (middot) m(sup-2) (middot) day(sup-1)), the greatest yet reported in the literature. Vertical profiles of O(inf2), CH(inf4), and methanotrophic potential in the soils were determined at steady state. Methane oxidation potentials were greatest where the vertical profiles of O(inf2) and CH(inf4) overlapped. A significant increase in the organic matter content of the soil, presumably derived from methanotroph biomass, occurred where CH(inf4) oxidation was greatest. Methane oxidation kinetics showed that a soil community with a low methanotrophic capacity (V(infmax) of 258 nmol (middot) g of soil(sup-1) (middot) h(sup-1)) but relatively high affinity (k(infapp) of 1.6 (mu)M) remained in N(inf2)-purged control microcosms, even after 6 months without CH(inf4). We attribute this to a facultative, possibly mixotrophic, methanotrophic microbial community. When purged with CH(inf4), a different methanotrophic community developed which had a lower affinity (k(infapp) of 31.7 (mu)M) for CH(inf4) but a greater capacity (V(infmax) of 998 nmol (middot) g of soil(sup-1) (middot) h(sup-1)) for CH(inf4) oxidation, reflecting the enrichment of an active high-capacity methanotrophic community. Compared with the unamended control soil, amendment of the coarse sand with sewage sludge enhanced CH(inf4) oxidation capacity by 26%; K(inf2)HPO(inf4) amendment had no significant effect, while amendment with NH(inf4)NO(inf3) reduced the CH(inf4) oxidation capacity by 64%. In vitro experiments suggested that NH(inf4)NO(inf3) additions (10 and 71 (mu)mol (middot) g of soil(sup-1)) inhibited CH(inf4) oxidation by a nonspecific ionic effect rather than by specific inhibition by NH(inf4)(sup+).

Influence of temperature on growth rate and competition between two psychrotolerant Antarctic bacteria: low temperature diminishes affinity for substrate uptake.

The growth kinetics of two psychrotolerant Antarctic bacteria, Hydrogenophaga pseudoflava CR3/2/10 (2/10) and Brevibacterium sp. strain CR3/1/15 (1/15), were examined over a range of temperatures in both batch culture and glycerol-limited chemostat cultures. The maximum specific growth rate (mu max) and Ks values for both bacteria were functions of temperature, although the cell yields were relatively constant with respect to temperature. The mu max values of both strains increased up to an optimum temperature, 24 degrees C for 2/10 and 20 degrees C for 1/15. Strain 1/15 might therefore be considered to be more psychrophilic than strain 2/10. For both bacteria, the specific affinity (mu max/Ks) for glycerol uptake was lower at 2 than at 16 degrees C, indicating a greater tendency to substrate limitation at low temperature. As the temperature increased from 2 to 16 degrees C, the specific affinity of 1/15 for glycerol increased more rapidly than it did for 2/10. Thus 1/15, on the basis of this criterion, was less psychrophilic than was 2/10. The steady-state growth kinetics of the two strains at 2 and 16 degrees C imply that 1/15 would be able to outgrow 2/10 only at relatively low substrate concentrations (< 0.32 g of glycerol.liter-1) and high temperatures (> 12 degrees C), which suggests that 1/15 has a less psychrotolerant survival strategy than does 2/10. Our data were compared with other data in the literature for bacteria growing at low temperatures. They also showed an increase of substrate-specific affinity with increasing temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of changing temperature on growth rate and competition between two psychrotolerant Antarctic bacteria: competition and survival in non-steady-state temperature environments.

Competition between two psychrotolerant bacteria was examined in glycerol-limited chemostat experiments subjected to non-steady-state conditions of temperature. One bacterium, a Brevibacterium sp. strain designated CR3/1/15, responded rapidly to temperature change, while a second, Hydrogenophaga pseudoflava, designated CR3/2/10, exhibited a lag in growth after a shift-down during a square-wave temperature cycle but not after a shift-up. The effects on competition and survival by these bacteria of both sine-wave and square-wave temperature changes between 2 and 16 degrees C over a 24-h cycle time were examined, as well as square-wave cycles over 12 and 96 h. The changing proportion of each bacterium in the chemostat was determined by plate counting at regular intervals. Under a sine-wave temperature cycle H. psedoflava outcompeted the Brevibacterium sp., but under square-wave temperature cycles the two bacteria coexisted because the lag by H. pseudoflava after the temperature shift-down favored the faster-responding Brevibacterium sp. The two bacteria thus exhibited different survival strategies, with H. pseudoflava adapted to effective competition under steady-state conditions and the Brevibacterium sp. adapted to rapid adaptation and survival in a changing environment. The degree of perturbation of the bacteria, expressed as a temperature challenge index (delta temp/delta time), was greater under a square-wave temperature cycle than under a sine-wave cycle of equivalent amplitude and frequency, and higher-temperature challenge favored the Brevibacterium sp. A computer model was developed to examine competition between the bacteria in transient environments. The frequency of the temperature cycle influenced competition, as with a longer cycle (96 h) the significance of the lag by H. pseudoflava decreased compared with that of a 24-h cycle, and H. pseudoflava predominated in a mixed culture with a 96-h cycle. The shift-down lag by H. pseudoflava, during which it adapted to low temperature, disadvantaged it in a changing temperature environment, but at a short cycle time (12 h) this disadvantage was countered by the incomplete loss of low-temperature adaptation between cycles and thus the carryover of some low-temperature adaptation. Also, it was demonstrated that, as well as consideration of the effect of temperature changes on inducing lags in growth, the loss of adaptation to low temperature between cycles had to be taken into account in the computer model if it was to reproduce the trends in the experimental data.

Measurements of Seasonal Rates and Annual Budgets of Organic Carbon Fluxes in an Antarctic Coastal Environment at Signy Island, South Orkney Islands, Suggest a Broad Balance between Production and Decomposition.

We report here the first comprehensive seasonal study of benthic microbial activity in an Antarctic coastal environment. Measurements were made from December 1990 to February 1992 of oxygen uptake and sulfate reduction by inshore coastal sediments at Signy Island, South Orkney Islands, Antarctica. From these measurements the rate of benthic mineralization of organic matter was calculated. In addition, both the deposition rate of organic matter to the bottom sediment and the organic carbon content of the bottom sediment were measured during the same period. Organic matter input to the sediment was small under winter ice cover, and the benthic respiratory activity and the organic content of the surface sediment declined during this period as available organic matter was depleted. On an annual basis, about 32% of benthic organic matter mineralization was anoxic, but the proportion of anoxic compared with oxic mineralization increased during the winter as organic matter was increasingly buried by the amphipod infauna. Fresh organic input occurred as the sea ice melted and ice algae biomass sedimented onto the bottom, and input was sustained during the spring after ice breakup by continued primary production in the water column. The benthic respiratory rate and benthic organic matter content correspondingly increased towards the end of winter with the input of this fresh organic matter. The rates of oxygen uptake during the southern summer (80 to 90 mmol of O(2) m day) were as high as those reported for other sediments at much higher environmental temperatures, and the annual mineralization of organic matter was equally high (12 mol of C m year). Seasonal variations of benthic activity in this antarctic coastal sediment were regulated by the input and availability of organic matter and not by seasonal water temperature, which was relatively constant at between -1.8 and 0.5 degrees C. We conclude that despite the low environmental temperature, organic matter degradation broadly balanced organic matter production, although there may be significant interrannual variations in the sources of the organic matter inputs.

A note on 'plotless' methods for estimating bacterial cell densities.

' Plotless ' techniques for determining population densities have been developed for, and applied to, higher plant populations. They can often be carried out more rapidly than techniques involving total counts of individuals in plots, or quadrants, but such plotless techniques have not been generally applied to the estimation of densities of bacterial cells. Direct microscopical counting of cell numbers in a field of view, an example of a plot-related method, has been traditionally used for microbial cell counts. In this study 'plot' and ' plotless ' methods on a variety of bacterial samples are compared. Estimates of bacterial cell density were obtained by measuring the distance of cells from a fixed point in a field of view. The values, which were more rapidly obtained, were directly correlated with total cell counts. Although there was some apparent deviation from a perfect 1:1 relationship with total counts, as indicated by a correlation coefficient less than 1.0, there were no significant differences between the replicated counts of bacteria on samples of tissue from the surface of Hypholoma basidiocarps (P less than 0.05). This indicated that the methods of enumeration were comparable. The distance-related estimates could readily be obtained from fields of view with cell densities varying over several orders of magnitude. It was more rapidly applied, particularly at high density, and the method was applicable not only to random cell distributions but also to the non-random distributions encountered when microbial cells aggregated into microcolonies. The method appears to be particularly well-suited for automated, digitized, direct counting procedures, as well as to estimating bacterial numbers on membrane filters and natural substrates.

The use of multiple-vessel, open flow systems to investigate carbon flow in anaerobic microbial communities.

Five vessels, connected in series, were used for a continuous flow system to model carbon flow in anaerobic microbial communities. Two such 5-vessel systems were constructed, the inflows containing 10 mM sulfate and either 10 mM glucose or benzoate. Dilution was slow (D=0.0018 h(-1) for the whole system).Analyses of dissolved organic and inorganic carbon, and of CO2 and CH4, showed that the systems attained steady states in which biomass was constant, although there was net biosynthesis in the early vessels and net mineralization in succeeding vessels.Examination of the distributions of sulfate reduction, methanogenesis, and of H2+CO2-utilizing fatty acid-forming bacteria revealed spatial separation of these functional groups of bacteria in different vessels of the array, resembling the vertical spatial separation found in many natural sediments. Such model systems should, therefore, prove valuable in investigating the many microbial activities that contribute to the flow of carbon in anaerobic microbial communities.

Sulfate reduction and methanogenesis in the sediment of a saltmarsh on the East coast of the United kingdom.

The rates of sulfate reduction, methanogenesis, and methane loss were measured in saltmarsh sediment at monthly intervals. In addition, dissolved methane and sulfate concentrations together with pS and pH were determined. Methane formation from carbon dioxide, but not from acetate, was detected within the same horizon of sediment where sulfate reduction was most active. Sulfate reduction was about three orders of magnitude greater than annual methanogenesis. The two processes were not separated either spatially or temporally, but occurred within the same layer of sediment at the same time of the year. Their coexistence did not seem to be the result of sulfate-depleted microenvironments within which methanogenesis could occur, but the methanogenic bacteria persisted at very low rates of activity within the same environment as the sulfate reducers.

Evidence for coexistence of two distinct functional groups of sulfate-reducing bacteria in salt marsh sediment.

Oxidation of acetate in salt marsh sediment was inhibited by the addition of fluoroacetate, and also by the addition of molybdate, an inhibitor of sulfate-reducing bacteria. Molybdate had no effect upon the metabolism of acetate in a freshwater sediment in the absence of sulfate. The inhibitory effect of molybdate on acetate turnover in the marine sediment seemed to be because of its inhibiting sulfate-reducing bacteria which oxidized acetate to carbon dioxide. Sulfide was not recovered from sediment in the presence of molybdate added as an inhibitor of sulfate-reducing bacteria, but sulfide was recovered quantitatively even in the presence of molybdate by the addition of the strong reducing agent titanium chloride before acidification of the sediment. Reduction of sulfate to sulfide by the sulfate-reducing bacteria in the sediment was only partially inhibited by fluoroacetate, but completely inhibited by molybdate addition. This was interpreted as showing the presence of two functional groups of sulfate-reducing bacteria-one group oxidizing acetate, and another group probably oxidizing hydrogen.

Hydrogen as an electron donor for sulfate-reducing bacteria in slurries of salt marsh sediment.

Experiments with a Warburg respirometer showed that a sediment slurry consumed hydrogen from a hydrogen atmosphere, and this consumption was not due to the activity of methanogenic bacteria. The hydrogren uptake was inhibited by the addition of 20 mM molybdate. Further experiments with sediment slurry held in conical flasks under an atmosphere of nitrogen showed that hydrogen accumulated in the headspace when bacterial sulfate reduction was inhibited either by the addition of 20 mM molybdate or by low (<5 mM) sulfate concentrations in the slurry. Methanogenesis was stimulated in the presence of a hydrogen atmosphere or by the addition of 20 mM molybdate. The results confirmed that hydrogren was an important electron donor for sulfate-reducing bacteria present in the sediment. The stimulation of methanogenesis by molybdate could be explained in part by a competition for hydrogen between sulfate-reducing bacteria and hydrogen-metabolizing methanogenic bacteria, but competition for another common substrate, possibly acetate, could also be significant.

Serological characteristics within the genus Desulfovibrio.

Antisera were prepared against one strain each of Desulfovibrio desulfuricans, D. vulgaris and D. salexigens. The antisera were tested for cross reactivity against 36 heterologous Desulfovibrio strains by both agglutination titration and by double immunodiffusion percipitin plates. Generally no cross-reaction was demonstrated by agglutination even between heterologous strains of the same species, suggesting that the surface antigens of Desulfovibrio are highly specific. In immunodiffusion plates a single apparently genus-specific surface antigen could be shown to be present in all but two of the strains tested. Although other common precipitin bands showed the presence of some antigens common between heterologous strains these appeared to be randomly distributed among the strains tested, with the exception of one band shown to be generally specific to strains of D. salexigens. With this exception no other precipitin band could be shown to be consistently specific to any other species, nor consistently common to more than one species.