Growth requirements of hyperthermophilic sulfur-dependent heterotrophic archaea isolated from a shallow submarine geothermal system with reference to their essential amino acids.

Three hyperthermophilic sulfur-dependent heterotrophs were isolated from a shallow submarine hydrothermal system at an inlet of Kodakara-jima island, Kagoshima, Japan. The isolates grew at 60 to 97 degrees C, with the optimum temperatures at 85 to 90 degrees C. Sensitivity to rifampin and the existence of ether lipids indicated that the isolates are hyperthermophilic archaea. Partial sequencing of the genes coding for 16S rRNA showed that the three isolates are closely related to the genus Thermococcus. They grew on proteinaceous mixtures, such as yeast extract, Casamino Acids, and purified proteins (e.g., casein and gelatin), but not on carbohydrates or organic acids as sole carbon and energy sources. Nine amino acids were essential for growth of isolate KS-1 (Thr, Leu, Ile, Val, Met, Phe, His, Tyr, and Arg). Isolate KS-2 required Lys in addition to the nine amino acids, and KS-8 required Lys instead of Tyr. In comparative studies, it was shown that Thermococcus celer DSM 2476 required 10 amino acids (Thr, Leu, Ile, Val, Met, Phe, Tyr, Trp, Lys, and Arg) while Pyrococcus furiosus DSM 3638 required only Ile and Val. The hyperthermophilic fermentative eubacterium Thermotoga neapolitana DSM 4359 did not require any amino acids for growth.     

Minimal Amino Acid Requirements of the Hyperthermophilic Archaeon Pyrococcus abyssi, Isolated from Deep-Sea Hydrothermal Vents

A minimal growth medium containing only nine amino acids and vitamins as the sole carbon and energy sources allowed the growth of Pyrococcus abyssi GE 5, a novel hyperthermophilic sulfur-metabolizing archaeon isolated from deep-sea hydrothermal vents. The generation time in this medium was about 40 min, and cell densities up to 5 x 10(sup8) cells ml(sup-1) were attained. These results are similar to those obtained previously with complex proteinaceous media.     

Oxidation and Assimilation of Atmospheric Methane by Soil Methane Oxidizers

The metabolism of atmospheric methane in a forest soil was studied by radiotracer techniques. Maximum (sup14)CH(inf4) oxidation (163.5 pmol of C cm(sup-3) h(sup-1)) and (sup14)C assimilation (50.3 pmol of C cm(sup-3) h(sup-1)) occurred at the A(inf2) horizon located 15 to 18 cm below the soil surface. At this depth, 31 to 43% of the atmospheric methane oxidized was assimilated into microbial biomass; the remaining methane was recovered as (sup14)CO(inf2). Methane-derived carbon was incorporated into all major cell macromolecules by the soil microorganisms (50% as proteins, 19% as nucleic acids and polysaccharides, and 5% as lipids). The percentage of methane assimilated (carbon conversion efficiency) remained constant at temperatures between 5 and 20(deg)C, followed by a decrease at 30(deg)C. The carbon conversion efficiency did not increase at methane concentrations between 1.7 and 1,000 ppm. In contrast, the overall methane oxidation activity increased at elevated methane concentrations, with an apparent K(infm) of 21 ppm (31 nM CH(inf4)) and a V(infmax) of 188 pmol of CH(inf4) cm(sup-3) h(sup-1). Methane oxidizers from soil depths with maximum methanotrophic activity respired approximately 1 to 3% of the assimilated methane-derived carbon per day. This apparent endogenous respiration did not change significantly in the absence of methane. Similarly, the potential for oxidation of atmospheric methane was relatively insensitive to methane starvation. Soil samples from depths above and below the zone with maximum atmospheric methane oxidation activity showed a dramatic increase in the turnover of the methane assimilated (>20 times increase). Physical disturbance such as sieving or mixing of soil samples decreased methane oxidation and assimilation by 50 to 58% but did not alter the carbon conversion efficiency. Ammonia addition (0.1 or 1.0 (mu)mol g [fresh weight](sup-1)) decreased both methane oxidation and carbon conversion efficiency. This resulted in a dramatic decrease in methane assimilation (85 to 99%). In addition, ammonia-treated soil showed up to 10 times greater turnover of the assimilated methane-derived carbon (relative to untreated soil). The results suggest a potential for microbial growth on atmospheric methane. However, growth was regulated strongly by soil parameters other than the methane concentration. The pattern observed for metabolism of atmospheric methane in soils was not consistent with the physiology of known methanotrophic bacteria.     

Microbiological Comparisons within and across Contiguous Lacustrine, Paleosol, and Fluvial Subsurface Sediments

Twenty-six subsurface samples were collected from a borehole at depths of 173.3 to 196.8 m in the saturated zone at the Hanford Site in south-central Washington State. The sampling was performed throughout strata that included fine-grained lacustrine (lake) sediments, a paleosol (buried soil) sequence, and coarse-grained fluvial (river) sediments. A subcoring method and tracers were used to minimize and quantify contamination to obtain samples that were representative of subsurface strata. Sediment samples were tested for total organic carbon, inorganic carbon, total microorganisms by direct microscopic counts, culturable aerobic heterotrophs by plate counts, culturable anaerobes by most-probable-number enumeration, basal respiration rates, and mineralization of (sup14)C-labeled glucose and acetate. Total direct microscopic counts of microorganisms were low, ranging from below detection to 1.9 x 10(sup5) cells g (dry weight)(sup-1). Culturable aerobes and anaerobes were below minimum levels of detection in most samples. Direct microscopic counts, basal respiration rates, and (sup14)C-glucose mineralization were all positively correlated with total organic carbon and were highest in the lacustrine sediments. In contrast to previous subsurface studies, these saturated-zone samples did not have higher microbial abundance and activities than unsaturated sediments sampled from the same borehole, the fine-textured lacustrine sediment had higher microbial numbers and activities than the coarse-textured fluvial sands, and the paleosol samples did not have higher biomass and activities relative to the other sediments. The results of this study expand the subsurface microbiology database to include information from an environment very different from those previously studied.     

Dissimilatory Nitrate Reduction in Anaerobic Sediments Leading to River Nitrite Accumulation

Recent studies on Northern Ireland rivers have shown that summer nitrite (NO(inf2)(sup-)) concentrations greatly exceed the European Union guideline of 3 (mu)g of N liter(sup-1) for rivers supporting salmonid fisheries. In fast-flowing aerobic small streams, NO(inf2)(sup-) is thought to originate from nitrification, due to the retardation of Nitrobacter strains by the presence of free ammonia. Multiple regression analyses of NO(inf2)(sup-) concentrations against water quality variables of the six major rivers of the Lough Neagh catchment in Northern Ireland, however, suggested that the high NO(inf2)(sup-) concentrations found in the summer under warm, slow-flow conditions may result from the reduction of NO(inf3)(sup-). This hypothesis was supported by field observations of weekly changes in N species. Here, reduction of NO(inf3)(sup-) was observed to occur simultaneously with elevation of NO(inf2)(sup-) levels and subsequently NH(inf4)(sup+) levels, indicating that dissimilatory NO(inf3)(sup-) reduction to NH(inf4)(sup+) (DNRA) performed by fermentative bacteria (e.g., Aeromonas and Vibrio spp.) is responsible for NO(inf2)(sup-) accumulation in these large rivers. Mechanistic studies in which (sup15)N-labelled NO(inf3)(sup-) in sediment extracts was used provided further support for this hypothesis. Maximal concentrations of NO(inf2)(sup-) accumulation (up to 1.4 mg of N liter(sup-1)) were found in sediments deeper than 6 cm associated with a high concentration of metabolizable carbon and anaerobic conditions. The (sup15)N enrichment of the NO(inf2)(sup-) was comparable to that of the NO(inf3)(sup-) pool, indicating that the NO(inf2)(sup-) was predominantly NO(inf3)(sup-) derived. There is evidence which suggests that the high NO(inf2)(sup-) concentrations observed arose from the inhibition of the DNRA NO(inf2)(sup-) reductase system by NO(inf3)(sup-).     

Influence of Incubation Temperature on the Microbial Reductive Dechlorination of 2,3,4,6-Tetrachlorobiphenyl in Two Freshwater Sediments

We studied the impact of incubation temperatures on the dechlorination of 2,3,4,6-tetrachlorobiphenyl (2346-CB) in two sediments from different climates: polychlorinated biphenyl (PCB)-free sediment from Sandy Creek Nature Center Pond (SCNC) in Athens, Ga., and PCB-contaminated sediment from Woods Pond (WP) in Lenox, Mass. Sediment slurries were incubated anaerobically with 350 (mu)M 2346-CB for 1 year at temperatures ranging from 4 to 66(deg)C. Most of the 2346-CB was dechlorinated between 12 and 34(deg)C in both sediments and, unexpectedly, between 50 and 60(deg)C in WP sediment. This is the first report of PCB dechlorination at thermobiotic temperatures. The data reveal profound differences in dechlorination rate, extent, and products as a function of sediment and temperature. The highest observed rate of dechlorination of 2346-CB to trichlorobiphenyls occurred at 30(deg)C in both sediments, but the rate was higher for WP than for SCNC sediment (46 versus 16 (mu)mol liter(sup-1) day(sup-1)). For SCNC sediment the rate of dechlorination dropped sharply below 30(deg)C, but for WP sediments it was near optimal from 20 to 34(deg)C and then dropped sharply below 20(deg)C. In WP sediment most of the meta chlorines were removed between 8 and 34(deg)C and between 50 and 60(deg)C. para dechlorination was restricted from 18 to 34(deg)C and was optimal at 20(deg)C. ortho dechlorination occurred between 8 and 30(deg)C, with optima around 15 and 27(deg)C, but the extent was highly variable. In SCNC sediment complete meta dechlorination occurred from 12 to 34(deg)C and para dechlorination occurred from 18 to 30(deg)C; both were optimal at 30(deg)C. No ortho dechlorination was observed. Dechlorination products were 246-CB, 236-CB, and 26-CB (both sediments) and 24-CB, 2-CB, and 4-CB (WP sediment). The data suggest that in SCNC sediment similar factors controlled meta and para PCB dechlorination over a broad temperature range (18 to 30(deg)C) but that in WP sediment there were multiple temperature-dependent changes in the factors controlling ortho, meta, and para dechlorination. We attribute the differences observed in the two sediments to differences in their PCB-dechlorinating communities.     

The Low Biomass Yields of the Acetic Acid Bacterium Acetobacter pasteurianus Are Due to a Low Stoichiometry of Respiration-Coupled Proton Translocation

Growth energetics of the acetic acid bacterium Acetobacter pasteurianus were studied with aerobic, ethanol-limited chemostat cultures. In these cultures, production of acetate was negligible. Carbon limitation and energy limitation were also evident from the observation that biomass concentrations in the cultures were proportional to the concentration of ethanol in the reservoir media. Nevertheless, low concentrations of a few organic metabolites (glycolate, citrate, and mannitol) were detected in culture supernatants. From a series of chemostat cultures grown at different dilution rates, the maintenance energy requirements for ethanol and oxygen were estimated at 4.1 mmol of ethanol (middot) g of biomass(sup-1) (middot) h(sup-1) and 11.7 mmol of O(inf2) (middot) g of biomass(sup-1) (middot) h(sup-1), respectively. When biomass yields were corrected for these maintenance requirements, the Y(infmax) values on ethanol and oxygen were 13.1 g of biomass (middot) mol of ethanol(sup-1) and 5.6 g of biomass (middot) mol of O(inf2)(sup-1), respectively. These biomass yields are very low in comparison with those of other microorganisms grown under comparable conditions. To investigate whether the low growth efficiency of A. pasteurianus might be due to a low gain of metabolic energy from respiratory dissimilation, (symbl)H(sup+)/O stoichiometries were estimated during acetate oxidation by cell suspensions. These experiments indicated an (symbl)H(sup+)/O stoichiometry for acetate oxidation of 1.9 (plusmn) 0.1 mol of H(sup+)/mol of O. Theoretical calculations of growth energetics showed that this low (symbl)H(sup+)/O ratio adequately explained the low biomass yield of A. pasteurianus in ethanol-limited cultures.     

Production of a Novel Extracellular Polysaccharide by Lactobacillus sake 0-1 and Characterization of the Polysaccharide

A novel exopolysaccharide (EPS) produced by Lactobacillus sake 0-1 (CBS 532.92) has been isolated and characterized. When the strain was grown on glucose, the produced EPS contained glucose and rhamnose in a molar ratio of 3:2 and the average molecular mass distribution (M(infm)) was determined at 6 x 10(sup6) Da. At a concentration of 1%, the 0-1 EPS had better viscosifying properties than xanthan gum when measured over a range of shear rates from 0 to 300 s(sup-1), while shear-thinning properties were comparable. Rheological data and anion-exchange chromatography suggested the presence of a negatively charged group in the EPS. Physiological parameters for optimal production of EPS were determined in batch fermentation experiments. Maximum EPS production was 1.40 g (middot) liter(sup-1), which was obtained when L. sake 0-1 had been grown anaerobically at 20(deg)C and pH 5.8. When cultured at lower temperatures, the EPS production per gram of biomass increased from 600 mg at 20(deg)C to 700 mg at 10(deg)C but the growth rate in the exponential phase decreased from 0.16 to 0.03 g (middot) liter(sup-1) (middot) h(sup-1). EPS production started in the early growth phase and stopped when the culture reached the stationary phase. Growing the 0-1 strain on different energy sources such as glucose, galactose, mannose, fructose, lactose, and sucrose did not alter the composition of the EPS produced.     

Factors Limiting Microbial Growth and Activity at a Proposed High-Level Nuclear Repository, Yucca Mountain, Nevada

As part of the characterization of Yucca Mountain, Nev., as a potential repository for high-level nuclear waste, volcanic tuff was analyzed for microbial abundance and activity. Tuff was collected aseptically from nine sites along a tunnel in Yucca Mountain. Microbial abundance was generally low: direct microscopic cell counts were near detection limits at all sites (3.2 x 10(sup4) to 2.0 x 10(sup5) cells g(sup-1) [dry weight]); plate counts of aerobic heterotrophs ranged from 1.0 x 10(sup1) to 3.2 x 10(sup3) CFU g(sup-1) (dry weight). Phospholipid fatty acid concentrations (0.1 to 3.7 pmol g(sup-1)) also indicated low microbial biomasses; diglyceride fatty acid concentrations, indicative of dead cells, were in a similar range (0.2 to 2.3 pmol g(sup-1)). Potential microbial activity was quantified as (sup14)CO(inf2) production in microcosms containing radiolabeled substrates (glucose, acetate, and glutamic acid); amendments with water and nutrient solutions (N and P) were used to test factors potentially limiting this activity. Similarly, the potential for microbial growth and the factors limiting growth were determined by performing plate counts before and after incubating volcanic tuff samples for 24 h under various conditions: ambient moisture, water-amended, and amended with various nutrient solutions (N, P, and organic C). A high potential for microbial activity was demonstrated by high rates of substrate mineralization (as much as 70% of added organic C in 3 weeks). Water was the major limiting factor to growth and microbial activity, while amendments with N and P resulted in little further stimulation. Organic C amendments stimulated growth more than water alone.     

Amino Acid Requirements of Two Hyperthermophilic Archaeal Isolates from Deep-Sea Vents, Desulfurococcus Strain SY and Pyrococcus Strain GB-D

Two sulfur-dependent hyperthermophilic archaea, Desulfurococcus strain SY and Pyrococcus strain GB-D, which were isolated from deep-sea hydrothermal vents, utilized free amino acids and peptides obtained from various molecular size fractions of yeast extract. It was found that 11 amino acids were essential for growth. The metabolic products were acetate, i-butyrate, and i-valerate.     

Population Dynamics of Polychlorinated Biphenyl-Dechlorinating Microorganisms in Contaminated Sediments

The growth dynamics of polychlorinated biphenyl (PCB)-dechlorinating microorganisms were determined for the first time, along with those of sulfate reducers and methanogens, by using the most-probable-number technique. The time course of Aroclor 1248 dechlorination mirrored the growth of dechlorinators; dechlorination ensued when the dechlorinating population increased by 2 orders of magnitude from 2.5 x 10(sup5) to 4.6 x 10(sup7) cells g of sediment(sup-1), at a specific growth rate of 6.7 day(sup-1) between 2 and 6 weeks. During this period, PCB-dechlorinating microorganisms dechlorinated Aroclor 1248 at a rate of 3.9 x 10(sup-8) mol of Cl g of sediment(sup-1) day(sup-1), reducing the average number of Cl molecules per biphenyl from 3.9 to 2.8. The growth yield was 4.2 x 10(sup13) cells mol of Cl dechlorinated(sup-1). Once dechlorination reached a plateau, after 6 weeks, the number of dechlorinators began to decrease. On the other hand, dechlorinators inoculated into PCB-free sediments decreased over time from their initial level, suggesting that PCBs are required for their selective enrichment. The numbers of sulfate reducers and methanogens increased in both PCB-free and contaminated sediments, showing little difference between them. The maximum population size of sulfate reducers was about an order of magnitude higher than that of dechlorinators, whereas that of methanogens was slightly less. Unlike those of dechlorinators, however, numbers of both sulfate reducers and methanogens remained high even when dechlorination ceased. The results of this study imply that PCB concentrations may have to exceed a certain threshold to maintain the growth of PCB dechlorinators.     

Microbiological and Geochemical Characterization of Microbial Fe(III) Reduction in Salt Marsh Sediments

Population densities of anaerobic Fe(III)-reducing bacteria (FeRB) and aerobic heterotrophs were inversely correlated in the surficial (0-2 cm) layers of Sapelo Island, Georgia, salt marsh sediments. In surficial sediments where densities of aerobic heterotrophs were low, the density of culturable FeRB correlated positively with the concentration of amorphous Fe(III) oxyhydroxides extractable by ascorbate. High FeRB densities and a decrease with depth of ascorbate-extractable Fe(III) were observed in the upper 6 cm of a tidal creek core. Culturable sulfate-reducing bacteria (SRB) and SRB-targeted rRNA signals were also detected in the upper 6-cm depth. The disappearance of FeRB below 6 cm, however, coincided with a large increase in the abundance of SRB. Thus, when FeRB are not limited by the availability of readily reducible amorphous Fe(III) oxyhydroxides, FeRB may outcompete SRB for growth substrates. Shewanella putrefaciens- and Geobacteraceae-targeted rRNA signals were at or below detection limits in all sediment samples, indicating that these FeRB are not predominant members of the active FeRB populations. The ubiquitous presence of FeRB at the sites studied challenges the traditional view that dissimilatory Fe(III) reduction is not an important pathway of organic carbon oxidation in salt marsh sediments.     

Effects of Temperature, Water Activity, and Incubation Time on Production of Aflatoxins and Cyclopiazonic Acid by an Isolate of Aspergillus flavus in Surface Agar Culture

An experiment with a full factorial design was used to study the effects of and interactions among temperature, water activity (a(infw)), incubation period, and substrate on coproduction of aflatoxins (AF) and cyclopiazonic acid (CPA) by an isolate of Aspergillus flavus. Analysis of variance showed that there was a complex interaction among all of these factors and that this influenced the relative concentrations of the mycotoxins produced. The optimum temperatures for the production of AF and CPA were 30(deg)C and 25(deg)C, respectively. Both mycotoxins were maximally produced (0.306 to 0.330 (mu)g of AF(middot)ml of medium(sup-1), 4.040 to 6.256 (mu)g of CPA(middot)ml of medium(sup-1)) at an a(infw) of 0.996 and after 15 days of incubation. No AF were produced in either yeast extract agar or Czapek yeast autolysate agar medium at an a(infw) of 0.90 at 20 or 37(deg)C after 15 days (minimum conditions), while 0.077 to 0.439 (mu)g of CPA(middot)ml of medium(sup-1) was produced under the same conditions. Yeast extract agar favored maximum AF production, and Czapek yeast autolysate agar favored maximum CPA production.     

Effects of Nitrate Availability and the Presence of Glyceria maxima on the Composition and Activity of the Dissimilatory Nitrate-Reducing Bacterial Community

The effects of nitrate availability and the presence of Glyceria maxima on the composition and activity of the dissimilatory nitrate-reducing bacterial community were studied in the laboratory. Four different concentrations of NO(inf3)(sup-), 0, 533, 1434, and 2,905 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1), were added to pots containing freshwater sediment, and the pots were then incubated for a period of 69 days. Upon harvest, NH(inf4)(sup+) was not detectable in sediment that received 0 or 533 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1). Nitrate concentrations in these pots ranged from 0 to 8 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1) at harvest. In pots that received 1,434 or 2,905 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1), final concentrations varied between 10 and 48 (mu)g of NH(inf4)(sup+)-N g of dry sediment(sup-1) and between 200 and 1,600 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1), respectively. Higher input levels of NO(inf3)(sup-) resulted in increased numbers of potential nitrate-reducing bacteria and higher potential nitrate-reducing activity in the rhizosphere. In sediment samples from the rhizosphere, the contribution of denitrification to the potential nitrate-reducing capacity varied from 8% under NO(inf3)(sup-)-limiting conditions to 58% when NO(inf3)(sup-) was in ample supply. In bulk sediment with excess NO(inf3)(sup-), this percentage was 44%. The nitrate-reducing community consisted almost entirely of NO(inf2)(sup-)-accumulating or NH(inf4)(sup+)-producing gram-positive species when NO(inf3)(sup-) was not added to the sediment. The addition of NO(inf3)(sup-) resulted in an increase of denitrifying Pseudomonas and Moraxella strains. The factor controlling the composition of the nitrate-reducing community when NO(inf3)(sup-) is limited is the presence of G. maxima. In sediment with excess NO(inf3)(sup-), nitrate availability determines the composition of the nitrate-reducing community.     

Effect of Parental Growth on Dynamics of Conjugative Plasmid Transfer in the Pea Spermosphere

Plasmid transfer rates for the conjugative plasmid R388::Tn1721 from Pseudomonas cepacia (donor) to Pseudomonas fluorescens (recipient) on agar media, in broth, and in microcosms containing sterile or nonsterile soil, in the presence or absence of germinating pea seeds, were determined. Donors, recipients, and transconjugants were enumerated on selective media after 1 day on agar or in broth culture and over a 7-day period in soil or pea spermosphere microcosms. Donor and recipient growth rates and plasmid transfer rate constants [(gamma), where (gamma) = transconjugants (middot) (donors (middot) recipients)(sup-1) (middot) h(sup-1)] were calculated for three initial parental densities (10(sup4), 10(sup6), or 10(sup8) CFU/g or ml) in each system. For all initial density levels, values of (gamma) in agar and broth matings were higher than those in soil or in the pea spermosphere-rhizosphere microcosms. Values of (gamma) were not influenced by the pea spermosphere or by sterile or nonsterile conditions of the soil. However, (gamma) values in microcosm experiments were inversely related to initial parental density and were directly related to donor growth rates. Values of (gamma) averaged 4 x 10(sup-10), 4 x 10(sup-12), and 3 x 10(sup-14) when initial donor and recipient cell densities were 10(sup4), 10(sup6), and 10(sup8) CFU/g, respectively. These results suggest that the plasmid transfer rate constant is independent of parental cell density only when parental growth is not limited. In a resource-limited environment, intra- or interspecific competition may reduce the transfer rate by limiting parental growth.     

A method for the analysis of acetate turnover in a coastal marine sediment

The concentrations of volatile fatty acids were measured in the pore water of sediment from the Limfjorden, Denmark. The pore water was freeze-dried and the acids, which were redissolved in formic acid, were analyzed by gas chromatography on a Carbopack column. The limit of detection was 0.1 mol l^–1 pore water. The concentration ranges (mol l^–1 pore water) were as follows: 0.1 to 6.0 for acetate; <0.1 to 0.6 for propionate, and <0.1 to 0.5 for butyrate. The rate constants for the disappearance of injected tracer concentrations of U-¹⁴C-acetate were measured at 2 cm depth intervals in sediment strata (0 to 10 cm). The rate constant for acetate turnover at 4 to 6 cm depth did not vary greatly with season, 2.1 h^–1, SD 0.6 for 7 values. In spring, the rate constants were highest in the 0 to 2 cm stratum and decreased with sediment depth. The calculated rates for acetate turnover of 7.2 mmol m^–2 day^–1 for early spring (2°C) and of 19.6 mmol m^–2 day^–1 for late autumn (7°C) were higher than would be expected from published values for carbon oxidation by sulfate in these sediments.     

Use of Radiolabelled Thymidine and Leucine To Estimate Bacterial Production in Soils from Continental Antarctica

Tritiated thymidine incorporation (TTI) into DNA was used to examine bacterial production in two soil types from the Robertskollen group of nunataks in northwestern Dronning Maud Land, providing the first estimates of bacterial production in soil habitats on the Antarctic continent. Although estimates of bacterial productivity in soils near to bird nests (344 (plusmn) 422 ng of C g [dry weight](sup-1) h(sup-1)) were higher than those for soils from beneath mosses (175 (plusmn) 90 ng of C g [dry weight](sup-1) h(sup-1); measured by TTI at 10(deg)C), these differences were not significant because of patchiness of bacterial activity (P > 0.05). TTI- and [(sup14)C]leucine ([(sup14)C]Leu)-derived estimates of bacterial production were similar when incubations of 3 h were used, although incubations as short as 1 h were sufficient for measurable uptake of radiolabel. Dual-label incorporation of [(sup3)H]thymidine ([(sup3)H]TdR) into DNA and [(sup14)C]Leu into protein indicated that TTI did not reflect bacterial production of in situ assemblages when incubations were longer than 3 h. Isotope dilution analysis indicated that dilution of the specific activity of exogenously supplied [(sup3)H]TdR by de novo synthesis of TdR precursor could be limited by additions of [(sup3)H]TdR at a concentration of 1 nmol per ca. 115 mg of soil. TTI exhibited a psychrotrophic response to variation in temperature, with a temperature optimum of ca. 15(deg)C and a Q(inf10) value for 0 to 10(deg)C of 2.41.     

Effect of vegetation on alpha cellulose decomposition in littoral lake sediments

The effect of vegetation cover on the decomposition of organic matter (alpha cellulose) was studied at three sediment depths (5, 15 and 25 cm) in the littoral area of a small Lake Kiruvere (Estonia). The experiment was carried out in two adjacent sites, with and without vegetation, using the litter bag method. At all sediment depths decomposition was faster at the site covered with vegetation, and was highest at 5 cm sediment depth (decomposition rate k = 0.0037 day⁻¹) and lower at 15 and 25 cm depths (k = 0.0014 day⁻¹ and k = 0.0013 day⁻¹). Higher decomposition rates coincided with higher root mass in the sediment. Decomposition rates were similar at all sediment depths in the site without a vegetation cover (k = 0.0007-0.0009 day⁻¹). The presence of a vegetation cover also affected temperatures in sediments. Temperatures were several degrees higher at all sediment depths in the area with vegetation cover, than in the area without. Mean sediment temperature differences between the two experiment areas were 1.4 °C at 5 cm sediment depth, 2.5 °C at 15 cm depth and 3.1 °C at 25 cm depth. Higher decomposition rates in the site covered with vegetation can be explained by oxygen dispersion from young roots in the higher sediment layers and by higher sediment temperatures due to the internal gas flow enhancing the microbial activity in the lower sediment layers.     

Adaptation of Psychrophilic and Psychrotrophic Sulfate-Reducing Bacteria to Permanently Cold Marine Environments

The potential for sulfate reduction at low temperatures was examined in two different cold marine sediments, Mariager Fjord (Denmark), which is permanently cold (3 to 6(deg)C) but surrounded by seasonally warmer environments, and the Weddell Sea (Antarctica), which is permanently below 0(deg)C. The rates of sulfate reduction were measured by the (sup35)SO(inf4)(sup2-) tracer technique at different experimental temperatures in sediment slurries. In sediment slurries from Mariager Fjord, sulfate reduction showed a mesophilic temperature response which was comparable to that of other temperate environments. In sediment slurries from Antarctica, the metabolic activity of psychrotrophic bacteria was observed with a respiration optimum at 18 to 19(deg)C during short-term incubations. However, over a 1-week incubation, the highest respiration rate was observed at 12.5(deg)C. Growth of the bacterial population at the optimal growth temperature could be an explanation for the low temperature optimum of the measured sulfate reduction. The potential for sulfate reduction was highest at temperatures well above the in situ temperature in all experiments. The results from sediment incubations were compared with those obtained from pure cultures of sulfate-reducing bacteria by using the psychrotrophic strain ltk10 and the mesophilic strain ak30. The psychrotrophic strain reduced sulfate optimally at 28(deg)C in short-term incubations, even though it could not grow at temperatures above 24(deg)C. Furthermore, this strain showed its highest growth yield between 0 and 12(deg)C. In contrast, the mesophilic strain ak30 respired and grew optimally and showed its highest growth yield at 30 to 35(deg)C.     

Kinetic Analyses of Desulfurization of Dibenzothiophene by Rhodococcus erythropolis in Continuous Cultures

Rhodococcus erythropolis N1-36, a desulfurization strain, was grown in continuous culture at 10 different dilution rates with 50 (mu)M dibenzothiophene sulfone (DBTO(inf2)) as the growth-limiting nutrient. The steady-state biomass, concentrations of substrate (DBTO(inf2)) and product (monohydroxybiphenyl), saturation constant (0.39 (mu)M DBTO(inf2)), and cell yield coefficient (9 mg of biomass(middot)(mu)M(sup-1) DBTO(inf2)) were measured. Continuous cultures at five temperatures allowed calculation of activation energy (0.84 kcal(middot)mol(sup-1) [ca. 3.5 kJ(middot)mol(sup-1)]) near the optimal temperature (30(deg)C) for growth. A washout technique was used to calculate the maximum specific growth rate (0.235 h(sup-1)), a value equivalent to a minimum generation time of 2.95 h.