Effects of Environmental Parameters on the Formation and Turnover of Acetate by Forest Soils

The capacity to form acetate from endogenous matter was a common property of diverse forest soils when incubated under anaerobic conditions. At 15 to 20(deg)C, acetate synthesis occurred without appreciable delay when forest soils were incubated as buffered suspensions or in microcosms at various percentages of their maximum water holding capacity. Rates for acetate formation with soil suspensions ranged from 35 to 220 (mu)g of acetate per g (dry weight) of soil per 24 h, and maximal acetate concentrations obtained in soil suspensions were two- to threefold greater than those obtained with soil microcosms at the average water holding capacity of the soil. Cellobiose degradation in soil suspensions yielded H(inf2) as a transient product. Under anaerobic conditions, supplemental H(inf2) and CO(inf2) were directed towards the acetogenic synthesis of acetate, and enrichments yielded a syringate-H(inf2)-consuming acetogenic consortium. At in situ temperatures, acetate was a relatively stable anaerobic end product; however, extended incubation periods induced acetoclastic methanogenesis and sulfate reduction. Higher mesophilic and thermophilic temperatures greatly enhanced the capacity of soils to form methane. Although methanogenic and sulfate-reducing activities under in situ-relevant conditions were negligible, these findings nonetheless demonstrated the occurrence of methanogens and sulfate-reducing bacteria in these aerated terrestrial soils. In contrast to the protracted stability of acetate under anaerobic conditions at 15 to 20(deg)C with unsupplemented soils, acetate formed by forest soils was rapidly consumed in the presence of oxygen and nitrate, and substrate-product stoichiometries indicated that acetate turnover was coupled to oxygen-dependent respiration and denitrification. The collective results suggest that acetate formed under anaerobic conditions might constitute a trophic link between anaerobic and aerobic processes in forest soils.     

Effect of temperature on composition of the methanotrophic community in rice field and forest soil

Temperature change affects methane consumption in soil. However, there is no information on possible temperature control of methanotrophic bacterial populations. Therefore, we studied CH(4) consumption and populations of methanotrophs in an upland forest soil and a rice field soil incubated at different temperatures between 5 and 45 degrees C for up to 40 days. Potential methane consumption was measured at 4% CH(4). The temporal progress of CH(4) consumption indicated growth of methanotrophs. Both soils showed maximum CH(4) consumption at 25-35 degrees C, but no activity at >40 degrees C. In forest soil CH(4) was also consumed at 5 degrees C, but in rice soil only at 15 degrees C. Methanotroph populations were assessed by terminal restriction fragment length polymorphism (T-RFLP) targeting particulate methane monooxygenase (pmoA) genes. Eight T-RFs with relative abundance >1% were retrieved from both forest and rice soil. The individual T-RFs were tentatively assigned to different methanotrophic populations (e.g. Methylococcus/Methylocaldum, Methylomicrobium, Methylobacter, Methylocystis/Methylosinus) according to published sequence data. Two T-RFs were assigned to ammonium monooxygenase (amoA) gene sequences. Statistical tests showed that temperature affected the relative abundance of most T-RFs. Furthermore, the relative abundance of individual T-RFs differed between the two soils, and also exhibited different temperature dependence. We conclude that temperature can be an important factor regulating the community composition of methanotrophs in soil.     

Anaerobic Capacities of Leaf Litter

Leaf litter displayed a capacity to spontaneously form organic acids, alcohols, phenolic compounds, H(inf2), and CO(inf2) when incubated anaerobically at 20(deg)C either as buffered suspensions or in a moistened condition in microcosms. Acetate was the predominant organic product formed regardless of the degree of litter decomposition. Initial rates of acetate formation in litter suspensions and microcosms approximated 2.6 and 0.53 (mu)mol of acetate per g (dry weight) of litter per h, respectively. Supplemental H(inf2) was directed towards the apparent acetogenic synthesis of acetate. Acetoclastic methanogenesis was induced by partially decomposed litter after extended lag phases; freshly fallen litter did not display this capacity.     

Influence of temperature and high acetate concentrations on methanogenesis in lake sediment slurries

Methanogenesis from main methane precursors H(2)/CO(2) and acetate was investigated in a temperature range of 2-70 degrees C using sediments from Lake Baldegg, Switzerland. Psychrophilic, psychrotrophic, mesophilic, and thermophilic methanogenic microbial communities were enriched by incubations for 1-3 months of nonamended sediment slurries at 5, 15, 30, and 50 degrees C. Isotope experiments with slurries amended with (14)C-labeled bicarbonate and (14)C-2-acetate showed that in the psychrophilic community (enriched at 5 degrees C), about 95% of methane originated from acetate, in contrast to the thermophilic community (50 degrees C) where up to 98% of methane was formed from bicarbonate. In the mesophilic community (30 degrees C), acetate was the precursor of about 80% of the methane produced. When the hydrogen-carbon dioxide mixture (H(2)/CO(2)) was used as a substrate, it was directly converted to methane under thermophilic conditions (70 and 50 degrees C). Under mesophilic conditions (30 degrees C), both pathways, hydrogenotrophic and acetoclastic, were observed. At low temperatures (5 and 15 degrees C), H(2)/CO(2) was converted into methane by a two-step process; first acetate was formed, followed by methane production from acetate. When slurries were incubated at high partial pressures of H(2)/CO(2), the high concentrations of acetate produced of more than 20 mM inhibited acetoclastic methanogenesis at a temperature below 15 degrees C. However, slow adaptation of the psychrophilic microbial community to high acetate concentrations was observed.     

Mechanistic Analysis of Ammonium Inhibition of Atmospheric Methane Consumption in Forest Soils

Methane consumption by forest soil was studied in situ and in vitro with respect to responses to nitrogen additions at atmospheric and elevated methane concentrations. Methane concentrations in intact soil decreased continuously from atmospheric levels at the surface to 0.5 ppm at a depth of 14 cm. The consumption rate of atmospheric methane in soils, however, was highest in the 4- to 8-cm depth interval (2.9 nmol per g of dry soil per day), with much lower activities below and above this zone. In contrast, extractable ammonium and nitrate concentrations were highest in the surface layer (0 to 2 cm; 22 and 1.6 μmol per g of dry soil, respectively), as was potential ammonium-oxidizing activity (19 nmol per g of dry soil per day). The difference in zonation between ammonium oxidation and methane consumption suggested that ammonia-oxidizing bacteria did not contribute significantly to atmospheric methane consumption. Exogenous ammonium inhibited methane consumption in situ and in vitro, but the pattern of inhibition did not conform to expectations based on simple competition between ammonia and methane for methane monooxygenase. The extent of ammonium inhibition increased with increasing methane concentration. Inhibition by a single ammonium addition remained constant over a period of 39 days. In addition, nitrite, the end product of methanotrophic ammonia oxidation, was a more effective inhibitor of methane consumption than ammonium. Factors that stimulated ammonium oxidation in soil, e.g., elevated methane concentrations and the availability of cosubstrates such as formate, methanol, or β-hydroxybutyrate, enhanced ammonium inhibition of methane oxidation, probably as a result of enhanced nitrite production.     

Acetate Degradation at 70 degrees C in Upflow Anaerobic Sludge Blanket Reactors and Temperature Response of Granules Grown at 70 degrees C

Anaerobic acetate degradation at 70 degrees C and at 55 degrees C (as a reference) was studied by running laboratory upflow anaerobic sludge blanket (UASB) reactors inoculated with mesophilic granular sludge. In UASB reactors fed with acetate-containing media (3 g of chemical oxygen demand [COD] per liter, corresponding to 47 mM acetate) approximately 50 days was needed at 70 degrees C and less than 15 days was needed at 55 degrees C to achieve an effluent COD of 500 to 700 mg/liter. In the UASB reactors at both 70 and 55 degrees C up to 90% of the COD was removed. Batch assays showed that sludges from two 70 degrees C UASB reactors, one run at a low effluent acetate concentration and the other run at a high effluent acetate concentration, exhibited slightly different responses to temperatures in the range from 37 to 70 degrees C. Both 70 degrees C sludges, as well as the 55 degrees C sludge, produced methane at temperatures of 37 to 73 degrees C. The 55 degrees C sludge exhibited shorter lag phases than the 70 degrees C sludges and higher specific methane production rates between 37 and 65 degrees C.     

Acetogenic Capacities and the Anaerobic Turnover of Carbon in a Kansas Prairie Soil

To assess the anaerobic capacities of a temperate grassland soil, a Kansas prairie soil was incubated anaerobically as either soil-water (1:2) suspensions or as soil microcosms at 78% soil water-holding capacity. Prairie soil formed acetate and CO(inf2) as the two main initial carbonaceous products from the anaerobic turnover of endogenous organic matter. Metabolic capacities of soil suspensions and microcosms were similar. Rates of acetate formation from endogenous organic matter in soil-water suspensions incubated at 40, 30, and 15(deg)C approximated 3.3, 2.4, and 1.1 (mu)g of acetate per g (dry weight) of soil per h, respectively. Supplemental H(inf2) and CO(inf2) were subject to consumption with the apparent concomitant synthesis of acetate in both soil suspensions and soil microcosms. In soil microcosms, rates of H(inf2)-dependent acetogenesis at 30 and 55(deg)C were nearly equivalent. The uptake of supplemental H(inf2) was not coupled to methanogenesis under any condition examined. These anaerobic activities were relatively stable when soils were subjected to either aerobic drying or alternating periods of O(inf2) enrichment. On the basis of the formation of nitrogen (N(inf2)), denitrification was engaged during anaerobic incubation periods; nitrous oxide (N(inf2)O) was also formed under certain conditions. Although extended incubation of soil induced the delayed methanogenic turnover of acetate, acetate was subject to immediate turnover under either O(inf2)- or nitrate-enriched conditions. These studies support the following concepts: (i) obligately anaerobic bacteria such as acetogenic bacteria are stable to periods of aerobiosis and are active in the anaerobic microsites of oxic soils, and (ii) acetate synthesized in anaerobic microsites of oxic terrestrial soils constitutes a trophic link to both aerobic and anaerobic microbial communities.     

Acetate production from hydrogen and [13C]carbon dioxide by the microflora of human feces.

Fecal suspensions from humans were incubated with 13CO2 and H2. The suspensions were from subjects who harbored 10(8) and 10(10) methanogens per g (dry weight) of feces, respectively, and from a subject who did not harbor methanogens. Quantitative nuclear magnetic resonance spectroscopy showed that acetate labeled in both the methyl and carboxyl groups was formed by suspensions from the subject without methanogens and the subject with the lower concentrations of methanogens. The amounts of labeled acetate formed were in agreement with the amounts expected based on measurements of H2 utilization. No labeled acetate was formed by suspensions from the subject with the higher concentrations of methanogens, and essentially all of the H2 used was accounted for by CH4 production. Suspensions from the subject with lower concentrations of methanogens produced both methane and acetate from H2 and CO2. The results indicate that reduction of CO2 to acetate may be a major pathway for microbial production of acetate in the human colon except when very high concentrations of methanogens (ca. 10(10) per g [dry weight] of feces) are present. Double-labeled acetate was also formed from H2 and 13CO2 by fecal suspensions from nonmethanogenic and moderately methanogenic rats.     

Acetate production from hydrogen and [13C]carbon dioxide by the microflora of human feces

Fecal suspensions from humans were incubated with 13CO2 and H2. The suspensions were from subjects who harbored 10(8) and 10(10) methanogens per g (dry weight) of feces, respectively, and from a subject who did not harbor methanogens. Quantitative nuclear magnetic resonance spectroscopy showed that acetate labeled in both the methyl and carboxyl groups was formed by suspensions from the subject without methanogens and the subject with the lower concentrations of methanogens. The amounts of labeled acetate formed were in agreement with the amounts expected based on measurements of H2 utilization. No labeled acetate was formed by suspensions from the subject with the higher concentrations of methanogens, and essentially all of the H2 used was accounted for by CH4 production. Suspensions from the subject with lower concentrations of methanogens produced both methane and acetate from H2 and CO2. The results indicate that reduction of CO2 to acetate may be a major pathway for microbial production of acetate in the human colon except when very high concentrations of methanogens (ca. 10(10) per g [dry weight] of feces) are present. Double-labeled acetate was also formed from H2 and 13CO2 by fecal suspensions from nonmethanogenic and moderately methanogenic rats.     

Metabolism of H2-CO2, methanol, and glucose by Butyribacterium methylotrophicum.

The fermentative metabolism of Butyribacterium methylotrophicum grown on either H2-CO2, methanol, glucose, or CO is described. The following reaction stoichiometries were obtained: 1.00 H2 + 0.52 CO2 leads to 0.22 acetate + 0.06 cell C; 1 methanol + 0.18 CO2 + 0.01 acetate leads to 0.24 butyrate + 0.29 cell C; and 1.00 glucose leads to 0.31 CO2 + 1.59 acetate + 0.21 butyrate + 0.13 H2 + 1.58 cell C. Cell yields of 1.7 g (dry weight) per mol of H2, 8.2 g (dry weight) per mol of methanol, 42.7 g (dry weight) per mol of glucose, and 3.0 g (dry weight) per mol of CO were obtained from linear plots of cell synthesis and substrate consumption. Doubling times of 9.0, 9.0, and 3 to 4 h were observed during batch growth on H2-CO2, methanol, and glucose, respectively. Indicative of a growth factor limitation, glucose fermentation in defined medium displayed a lower cell synthesis efficiency than when yeast extract (0.05%) was present. B. methylotrophicum fermentation displayed atypically high substrate/cell carbon synthesis conversion ratios for an anaerobe, as greater than 24% of the carbon was assimilated into cells during growth on methanol or glucose. The data indicate that B. methylotrophicum conserves carbon-bound electrons during growth on single-carbon or multicarbon substrates.     

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.     

Carotenoid Formation by Staphylococcus aureus

The carotenoid pigments of Staphylococcus aureus U-71 were identified as phytoene; zeta-carotene; delta-carotene; phytofluenol; a phytofluenol-like carotenoid, rubixanthin; and three rubixanthin-like carotenoids after extraction, saponification, chromatographic separation, and determination of their absorption spectra. There was no evidence of carotenoid esters or glycoside ethers in the extract before saponification. During the aerobic growth cycle the total carotenoids increased from 45 to 1,000 nmoles per g (dry weight), with the greatest increases in the polar, hydroxylated carotenoids. During the anaerobic growth cycle, the total carotenoids increased from 20 nmoles per g (dry weight) to 80 nmoles per g (dry weight), and only traces of the polar carotenoids were formed. Light had no effect on carotenoid synthesis. About 0.14% of the mevalonate-2-(14)C added to the culture was incorporated into the carotenoids during each bacterial doubling. The total carotenoids did not lose radioactivity when grown in the absence of (14)C for 2.5 bacterial doublings. The total carotenoids did not lose radioactivity when grown in the absence of (14)C for 2.5 bacterial doublings. The incorporation and turnover of (14)C indicated the carotenes were sequentially desaturated and hydroxylated to form the polar carotenoids.     

Comparison of two-stage thermophilic (68 degrees C/55 degrees C) anaerobic digestion with one-stage thermophilic (55 degrees C) digestion of cattle manure

A two-stage 68 degrees C/55 degrees C anaerobic degradation process for treatment of cattle manure was studied. In batch experiments, an increase of the specific methane yield, ranging from 24% to 56%, was obtained when cattle manure and its fractions (fibers and liquid) were pretreated at 68 degrees C for periods of 36, 108, and 168 h, and subsequently digested at 55 degrees C. In a lab-scale experiment, the performance of a two-stage reactor system, consisting of a digester operating at 68 degrees C with a hydraulic retention time (HRT) of 3 days, connected to a 55 degrees C reactor with 12-day HRT, was compared with a conventional single-stage reactor running at 55 degrees C with 15-days HRT. When an organic loading of 3 g volatile solids (VS) per liter per day was applied, the two-stage setup had a 6% to 8% higher specific methane yield and a 9% more effective VS-removal than the conventional single-stage reactor. The 68 degrees C reactor generated 7% to 9% of the total amount of methane of the two-stage system and maintained a volatile fatty acids (VFA) concentration of 4.0 to 4.4 g acetate per liter. Population size and activity of aceticlastic methanogens, syntrophic bacteria, and hydrolytic/fermentative bacteria were significantly lower in the 68 degrees C reactor than in the 55 degrees C reactors. The density levels of methanogens utilizing H2/CO2 or formate were, however, in the same range for all reactors, although the degradation of these substrates was significantly lower in the 68 degrees C reactor than in the 55 degrees C reactors. Temporal temperature gradient electrophoresis profiles (TTGE) of the 68 degrees C reactor demonstrated a stable bacterial community along with a less divergent community of archaeal species.     

Effects of temperature and incubation period on production of fumonisin B1 by Fusarium moniliforme

The kinetics of the production of fumonisin B1 (FB1) by Fusarium moniliforme MRC 826 in corn cultures was investigated as a function of fungal growth at various incubation temperatures. The growth rate of F. moniliforme, as measured by ergosterol concentration, was higher at 25 degrees C than at 20 degrees C, reaching a stationary phase after 4 to 6 weeks in both cases. FB1 production commenced after 2 weeks during the active growth phase, continued to increase during the stationary phase, and decreased after 13 weeks. The overall maximal yield of FB1 (17.9 g/kg, dry weight) was obtained in corn cultures incubated at 20 degrees C for 13 weeks, but it was not significantly (P greater than 0.05) higher than the maximum yield (16.5 g/kg, dry weight) obtained at 25 degrees C after 11 weeks. However, a significantly (P less than 0.05) higher mean yield was detected at 25 degrees C (9.5 g/kg, dry weight) than at 20 degrees C (8.7 g/kg, dry weight). Production reached a plateau after 7 weeks of incubation at 25 degrees C or 9 weeks of incubation at 20 degrees C. The maximal production of FB1 at 30 degrees C was very low (0.6 g/kg, dry weight). FB1 was also found to be heat stable, as there was no reduction in the FB1 concentration after boiling culture material of F. moniliforme MRC 826.     

Effects of temperature and incubation period on production of fumonisin B1 by Fusarium moniliforme.

The kinetics of the production of fumonisin B1 (FB1) by Fusarium moniliforme MRC 826 in corn cultures was investigated as a function of fungal growth at various incubation temperatures. The growth rate of F. moniliforme, as measured by ergosterol concentration, was higher at 25 degrees C than at 20 degrees C, reaching a stationary phase after 4 to 6 weeks in both cases. FB1 production commenced after 2 weeks during the active growth phase, continued to increase during the stationary phase, and decreased after 13 weeks. The overall maximal yield of FB1 (17.9 g/kg, dry weight) was obtained in corn cultures incubated at 20 degrees C for 13 weeks, but it was not significantly (P greater than 0.05) higher than the maximum yield (16.5 g/kg, dry weight) obtained at 25 degrees C after 11 weeks. However, a significantly (P less than 0.05) higher mean yield was detected at 25 degrees C (9.5 g/kg, dry weight) than at 20 degrees C (8.7 g/kg, dry weight). Production reached a plateau after 7 weeks of incubation at 25 degrees C or 9 weeks of incubation at 20 degrees C. The maximal production of FB1 at 30 degrees C was very low (0.6 g/kg, dry weight). FB1 was also found to be heat stable, as there was no reduction in the FB1 concentration after boiling culture material of F. moniliforme MRC 826.     

Benzoate fermentation by the anaerobic bacterium Syntrophus aciditrophicus in the absence of hydrogen-using microorganisms.

The anaerobic bacterium Syntrophus aciditrophicus metabolized benzoate in pure culture in the absence of hydrogen-utilizing partners or terminal electron acceptors. The pure culture of S. aciditrophicus produced approximately 0.5 mol of cyclohexane carboxylate and 1.5 mol of acetate per mol of benzoate, while a coculture of S. aciditrophicus with the hydrogen-using methanogen Methanospirillum hungatei produced 3 mol of acetate and 0.75 mol of methane per mol of benzoate. The growth yield of the S. aciditrophicus pure culture was 6.9 g (dry weight) per mol of benzoate metabolized, whereas the growth yield of the S. aciditrophicus-M. hungatei coculture was 11.8 g (dry weight) per mol of benzoate. Cyclohexane carboxylate was metabolized by S. aciditrophicus only in a coculture with a hydrogen user and was not metabolized by S. aciditrophicus pure cultures. Cyclohex-1-ene carboxylate was incompletely degraded by S. aciditrophicus pure cultures until a free energy change (DeltaG') of -9.2 kJ/mol was reached (-4.7 kJ/mol for the hydrogen-producing reaction). Cyclohex-1-ene carboxylate, pimelate, and glutarate transiently accumulated at micromolar levels during growth of an S. aciditrophicus pure culture with benzoate. High hydrogen (10.1 kPa) and acetate (60 mM) levels inhibited benzoate metabolism by S. aciditrophicus pure cultures. These results suggest that benzoate fermentation by S. aciditrophicus in the absence of hydrogen users proceeds via a dismutation reaction in which the reducing equivalents produced during oxidation of one benzoate molecule to acetate and carbon dioxide are used to reduce another benzoate molecule to cyclohexane carboxylate, which is not metabolized further. Benzoate fermentation to acetate, CO(2), and cyclohexane carboxylate is thermodynamically favorable and can proceed at free energy values more positive than -20 kJ/mol, the postulated minimum free energy value for substrate metabolism.     

Studies on plasma free-fatty-acid metabolism and triglyceride synthesis of brown adipose tissue in vivo during cold-induced thermogenesis of the newborn rabbit.

Parameters of plasma free fatty acid metabolism (pool size, half time, disappearance rate, turnover time and absolute turnover rate), the influx of plasma free fatty acids into the glycerides of brown adipose tissue and the pathway of triglyceride synthesis in brown adipose tissue (glycerol-1-phosphate versus monoglyceride pathway) were examined after intravenous injection of [1-14C]palmitate in newborn rabbits. In the thermoneutral environment of 35 degrees C the turnover rate of plasma free fatty acids was 10.20 mumol/min per 100 g body weight and its flux into the glycerides of brown adipose tissue 0.367 mumol/min per 100 g body weight. Cold exposure at an ambient temperature of 20 degrees C caused a decrease to 5.84 mumol/min and 0.207 mumol/min per 100 g body weight, respectively. Both under basal conditions at an ambient temperature of 35 degrees C and under cold-induced thermogenesis at an ambient temperature of 20 degrees C triglyceride synthesis in brown adipose tissue ran through the glycerol 1-phosphate pathway.     

Biostimulation and bioaugmentation for on-site treatment of weathered diesel fuel in Arctic soil

Bioremediation of weathered diesel fuel in Arctic soil at low temperature was studied both on-site in small-scale biopiles and in laboratory microcosms. The field study site was on Ellesmere Island (82°30'N, 62°20'W). Biostimulation was by fertilization with phosphorous and nitrogen. Bioaugmentation was with an enrichment culture originating from the field site. In biopiles, total petroleum hydrocarbons (TPH) were reduced from 2.9 to 0.5 mg/g of dry soil over a period of 65 days. In microcosms at 7 °C, TPH were reduced from 2.4 to 0.5 mg/g of dry soil over a period of 90 days. Inoculation had no effect on hydrocarbon removal in biopiles or in microcosms. Maximum TPH removal rates in the biopiles were approximately 90 μg of TPH g^–1 of soil day^–1, occurring during the first 14 days when ambient temperature ranged from 0 to 10 °C. The fate of three phylotypes present in the inoculum was monitored using most-probable-number PCR, targeting 16S rRNA genes. Populations of all three phylotypes increased more than 100-fold during incubation of both uninoculated and inoculated biopiles. The inoculum increased the initial populations of the phylotypes but did not significantly affect their final populations. Thus, biostimulation on site enriched populations that were also selected in laboratory enrichment cultures. Electronic Publication     

Clostridium thermobutyricum: growth studies and stimulation of butyrate formation by acetate supplementation

Clostridium thermobutyricum produces butyrate as the main fermentation product from glucose, and from yeast extract, which is required for substantial growth. After sequential transfer in the presence of increasing butyrate concentrations, strain JW 171 K grew in the presence of up to 350 mM butyrate either at pH 5.5 or at pH 8.0 and at 40 degrees C as well as at 60 degrees C. This result indicated that butyrate-dependent growth inhibition was independent from the concentration of undissociated butyric acid. Increased butyrate concentration decreased the level of tolerated glucose from above 15% to below 10%. At 0.05 and 2.0% (wt/vol) yeast extract, the Y(Glucose) was 30 and 55 g dry weight cells per mole glucose, respectively. Y(ATP) values between 18 and 21 g weight cells per mole ATP, obtained after growth in the presence of 2% yeast extract, indicate that the butyrate fermentation under thermophilic growth conditions is as energy efficient as it is under mesophilic conditions. Externally added acetate stimulated the production of butyrate. Supplemented 14C-acetate was converted to butyrate, resulting in the formation of 44% labeled butyrate (i.e. formed from 14C-acetate) and 56% unlabeled butyrate (formed from glucose and yeast extract). Continuous removal of H2 in batch cultures led to a shift in the fermentation products from more butyrate to the more oxidized and more energy yielding acetate.     

Denitrifying Bacteria in the Earthworm Gastrointestinal Tract and In Vivo Emission of Nitrous Oxide (N(inf2)O) by Earthworms

Earthworms (Lumbricus rubellus and Octolasium lacteum) and gut homogenates did not produce CH(inf4), and methanogens were not readily culturable from gut material. In contrast, the numbers of culturable denitrifiers averaged 7 x 10(sup7) and 9 x 10(sup6) per g (dry weight) of gut material for L. rubellus and O. lacteum, respectively; these values were 256- and 35-fold larger than the numbers of culturable denitrifiers in the soil from which the earthworms were obtained. Anaerobically incubated earthworm gut homogenates supplemented with nitrate produced N(inf2)O at rates exceeding that of soil homogenates. Furthermore, living earthworms emitted N(inf2)O under aerobic conditions, and N(inf2)O emission was stimulated by acetylene. For earthworms collected from a mildly acidic (pH 6) beech forest soil, the rates of N(inf2)O emission for earthworms and soil averaged 884 and 2 pmol per h per g (fresh weight), respectively. In contrast, for earthworms collected from a more acidic (pH 4.6) oak-beech forest soil, N(inf2)O emission by earthworms and soil averaged 145 and 45 pmol per h per g (fresh weight), respectively. Based on the extrapolation of this data, earthworms accounted for an estimated 16 and 0.25% of the total N(inf2)O produced at the stand level of these beech and oak-beech forest soils, respectively.