Methanol Promotes Atmospheric Methane Oxidation by Methanotrophic Cultures and Soils

Two methanotrophic bacteria, Methylobacter albus BG8 and Methylosinus trichosporium OB3b, oxidized atmospheric methane during batch growth on methanol. Methane consumption was rapidly and substantially diminished (95% over 9 days) when washed cell suspensions were incubated without methanol in the presence of atmospheric methane (1.7 ppm). Methanotrophic activity was stimulated after methanol (10 mM) but not methane (1,000 ppm) addition. M. albus BG8 grown in continuous culture for 80 days with methanol retained the ability to oxidize atmospheric methane and oxidized methane in a chemostat air supply. Methane oxidation during growth on methanol was not affected by methane deprivation. Differences in the kinetics of methane uptake (apparent K_m and V_max) were observed between batch- and chemostat-grown cultures. The V_max and apparent K_m values (means ± standard errors) for methanol-limited chemostat cultures were 133 ± 46 nmol of methane 10⁸ cells⁻¹ h⁻¹ and 916 ± 235 ppm of methane (1.2 μM), respectively. These values were significantly lower than those determined with batch-grown cultures (V_max of 648 ± 195 nmol of methane 10⁸ cells⁻¹ h⁻¹ and apparent K_m of 5,025 ± 1,234 ppm of methane [6.3 μM]). Methane consumption by soils was stimulated by the addition of methanol. These results suggest that methanol or other nonmethane substrates may promote atmospheric methane oxidation in situ.     

Nitrate Reduction in a Sulfate-Reducing Bacterium, Desulfovibrio desulfuricans, Isolated from Rice Paddy Soil: Sulfide Inhibition, Kinetics, and Regulation

From the second-highest dilution in a most-probable-number dilution series with lactate and sulfate as substrates and rice paddy soil as the inoculum, a strain of Desulfovibrio desulfuricans was isolated. In addition to reducing sulfate, sulfite, and thiosulfate, the strain also reduced nitrate to ammonia. The latter process was studied in detail, since the ability to reduce nitrate was strongly influenced by the presence of sulfide. Sulfide inhibited both growth on nitrate and nitrate reduction. A 70% inhibition of the nitrate reduction rate was obtained at 127 μM sulfide, and growth was inhibited by 50% at approximately 320 μM sulfide and was not detectable above 700 μM sulfide. In contrast, sulfate reduction was not affected at concentrations of up to 5 mM. After growth with sulfate, an induction period of 2 to 4 days was needed before nitrate reduction started. When nitrate and sulfate were present simultaneously, only sulfate was reduced, except when sulfate was present at very low concentrations (4 μM). At higher sulfate concentrations (500 μM), nitrate reduction was temporarily halted. The affinity for nitrate uptake was extremely high (K_m = 0.05 μM) compared with that for sulfate uptake (K_m = 5 μM). Thus, at low nitrate concentrations this bacterium is favored relative to denitrifiers (K_m = 1.8 to 13.7 μM) or other nitrate ammonifiers (e.g., Clostridium spp. [K_m = 500 μM]).     

Kinetics of Formate Metabolism in Methanobacterium formicicum and Methanospirillum hungatei

The kinetics of formate metabolism in Methanobacterium formicicum and Methanospirillum hungatei were studied with log-phase formate-grown cultures. The progress of formate degradation was followed by the formyltetrahydrofolate synthetase assay for formate and fitted to the integrated form of the Michaelis-Menten equation. The K_m and V_max values for Methanobacterium formicicum were 0.58 mM formate and 0.037 mol of formate h⁻¹ g⁻¹ (dry weight), respectively. The lowest concentration of formate metabolized by Methanobacterium formicicum was 26 μM. The K_m and V_max values for Methanospirillum hungatei were 0.22 mM and 0.044 mol of formate h⁻¹ g⁻¹ (dry weight), respectively. The lowest concentration of formate metabolized by Methanospirillum hungatei was 15 μM. The apparent K_m for formate by formate dehydrogenase in cell-free extracts of Methanospirillum hungatei was 0.11 mM. The K_m for H₂ uptake by cultures of Methanobacterium formicicum was 6 μM dissolved H₂. Formate and H₂ were equivalent electron donors for methanogenesis when both substrates were above saturation; however, H₂ uptake was severely depressed when formate was above saturation and the dissolved H₂ was below 6 μM. Formate-grown cultures of Methanobacterium formicicum that were substrate limited for 57 h showed an immediate increase in growth and methanogenesis when formate was added to above saturation.     

Kinetics of Butyrate, Acetate, and Hydrogen Metabolism in a Thermophilic, Anaerobic, Butyrate-Degrading Triculture

Kinetics of butyrate, acetate, and hydrogen metabolism were determined with butyrate-limited, chemostat-grown tricultures of a thermophilic butyrate-utilizing bacterium together with Methanobacterium thermoautotrophicum and the TAM organism, a thermophilic acetate-utilizing methanogenic rod. Kinetic parameters were determined from progress curves fitted to the integrated form of the Michaelis-Menten equation. The apparent half-saturation constants, K_m, for butyrate, acetate, and dissolved hydrogen were 76 μM, 0.4 mM, and 8.5 μM, respectively. Butyrate and hydrogen were metabolized to a concentration of less than 1 μM, whereas acetate uptake usually ceased at a concentration of 25 to 75 μM, indicating a threshold level for acetate uptake. No significant differences in K_m values for butyrate degradation were found between chemostat- and batch-grown tricultures, although the maximum growth rate was somewhat higher in the batch cultures in which the medium was supplemented with yeast extract. Acetate utilization was found to be the rate-limiting reaction for complete degradation of butyrate to methane and carbon dioxide in continuous culture. Increasing the dilution rate resulted in a gradual accumulation of acetate. The results explain the low concentrations of butyrate and hydrogen normally found during anaerobic digestion and the observation that acetate is the first volatile fatty acid to accumulate upon a decrease in retention time or increase in organic loading of a digestor.     

Effect of Low Sulfate Concentrations on Lactate Oxidation and Isotope Fractionation during Sulfate Reduction by Archaeoglobus fulgidus Strain Z

The effect of low substrate concentrations on the metabolic pathway and sulfur isotope fractionation during sulfate reduction was investigated for Archaeoglobus fulgidus strain Z. This archaeon was grown in a chemostat with sulfate concentrations between 0.3 mM and 14 mM at 80°C and with lactate as the limiting substrate. During sulfate reduction, lactate was oxidized to acetate, formate, and CO₂. This is the first time that the production of formate has been reported for A. fulgidus. The stoichiometry of the catabolic reaction was strongly dependent on the sulfate concentration. At concentrations of more than 300 μM, 1 mol of sulfate was reduced during the consumption of 1 mol of lactate, whereas only 0.6 mol of sulfate was consumed per mol of lactate oxidized at a sulfate concentration of 300 μM. Furthermore, at low sulfate concentrations acetate was the main carbon product, in contrast to the CO₂ produced at high concentrations. We suggest different pathways for lactate oxidation by A. fulgidus at high and low sulfate concentrations. At about 300 μM sulfate both the growth yield and the isotope fractionation were limited by sulfate, whereas the sulfate reduction rate was not limited by sulfate. We suggest that the cell channels more energy for sulfate uptake at sulfate concentrations below 300 to 400 μM than it does at higher concentrations. This could explain the shift in the metabolic pathway and the reduced growth yield and isotope fractionation at low sulfate levels.     

Growth and Nitrogen Uptake Kinetics in Cultured Prorocentrum donghaiense

We compared growth kinetics of Prorocentrum donghaiense cultures on different nitrogen (N) compounds including nitrate (NO₃ ⁻), ammonium (NH₄ ⁺), urea, glutamic acid (glu), dialanine (diala) and cyanate. P. donghaiense exhibited standard Monod-type growth kinetics over a range of N concentraions (0.5–500 μmol N L⁻¹ for NO₃ ⁻ and NH₄ ⁺, 0.5–50 μmol N L⁻¹ for urea, 0.5–100 μmol N L⁻¹ for glu and cyanate, and 0.5–200 μmol N L⁻¹ for diala) for all of the N compounds tested. Cultures grown on glu and urea had the highest maximum growth rates (μ_m, 1.51±0.06 d⁻¹ and 1.50±0.05 d⁻¹, respectively). However, cultures grown on cyanate, NO₃ ⁻, and NH₄ ⁺ had lower half saturation constants (K_μ, 0.28–0.51 μmol N L⁻¹). N uptake kinetics were measured in NO₃ ⁻-deplete and -replete batch cultures of P. donghaiense. In NO₃ ⁻-deplete batch cultures, P. donghaiense exhibited Michaelis-Menten type uptake kinetics for NO₃ ⁻, NH₄ ⁺, urea and algal amino acids; uptake was saturated at or below 50 μmol N L⁻¹. In NO₃ ⁻-replete batch cultures, NH₄ ⁺, urea, and algal amino acid uptake kinetics were similar to those measured in NO₃ ⁻-deplete batch cultures. Together, our results demonstrate that P. donghaiense can grow well on a variety of N sources, and exhibits similar uptake kinetics under both nutrient replete and deplete conditions. This may be an important factor facilitating their growth during bloom initiation and development in N-enriched estuaries where many algae compete for bioavailable N and the nutrient environment changes as a result of algal growth.     

Transport of Ammonium and Methylammonium Ions by Azotobacter vinelandii

Ammonium and methylammonium are rapidly taken up by cultures of Azotobacter vinelandii respiring in the presence of succinate. The rate of methylamine uptake increased with external pH from 5.5 to 7.5 but increasing the pH further to 8.5 had little effect on activity, indicating that methylammonium cation rather than uncharged methylamine is the permeant species. The kinetics of methylammonium entry followed the Michaelis-Menten relationship, yielding a K_m of 25 μM and a V_max of 3.8 nmol/min per mg of cell protein. At saturating concentrations ammonium was taken up at rates 30-fold higher than those for methylammonium. Ammonium was a competitive inhibitor of methylammonium uptake and gave an inhibition constant of 1 μM. Ammonium derivatives were inhibitors of methylammonium entry in order of effectiveness: hydrazine > methylhydrazine > formamidine > guanidine > dimethylamine > ethylamine; amides and amino acids did not block uptake. Likewise, metal cations inhibited in the order Tl⁺ > Cs⁺ > Rb⁺, whereas Na⁺, K⁺, and Li⁺ produced no significant effect. Methylammonium uptake was blocked in cells exposed to an uncoupler, p-trifluorome-thoxycarbonyl cyanide-phenyl hydrazone or gramicidin D, but not with dicyclo-hexylcarbodiimide or arsenate. Valinomycin stimulated methylammonium entry into cells in a K⁺-free medium but prevented entry in the presence of 10 mM K⁺. Monensin and nigericin had little effect on transport. These results indicate that methylammonium and ammonium ions enter A. vinelandii electrogenically via a specific transporter.     

Role of Carboxydobacteria in Consumption of Atmospheric Carbon Monoxide by Soil

The carbon monoxide consumption rates of the carboxydobacteria Pseudomonas (Seliberia) carboxydohydrogena, P. carboxydovorans, and P. carboxydoflava were measured at high (50%) and low (0.5 μl liter⁻¹) mixing ratios of CO in air. CO was only consumed when the bacteria had been grown under CO-autotrophic conditions. As an exception, P. carboxydoflava consumed CO also after heterotrophic growth on pyruvate. At low cell densities the CO consumption rates measured at low CO mixing ratios were similar in cell suspensions and in mixtures of bacteria in soil. CO consumption observed in natural soil (loess, eolian sand, chernozem) as well as in suspensions or soil mixtures of carboxydobacteria showed Michaelis-Menten kinetics. The K_m values for CO of the carboxydobacteria (K_m = 465 to 1,110 μl of CO liter⁻¹) were much higher than those of the natural soils (K_m = 5 to 8 μl of CO liter⁻¹). Considering the difference of the K_m values and the observed V_max values, carboxydobacteria cannot contribute significantly to the consumption of atmospheric CO.     

Measurement of Nitrous Oxide Reductase Activity in Aquatic Sediments

Denitrification in aquatic sediments was measured by an N₂O reductase assay. Sediments consumed small added quantities of N₂O over short periods (a few hours). In experiments with sediment slurries, N₂O reductase activity was inhibited by O₂, C₂H₂, heat treatment, and by high levels of nitrate (1 mM) or sulfide (10 mM). However, ambient levels of nitrate (<100 μM) did not influence activity, and moderate levels (about 150 μM) induced only a short lag before reductase activity began. Moderate levels of sulfide (<1 mM) had no effect on N₂O reductase activity. Nitrous oxide reductase displayed Michaelis-Menten kinetics in sediments from freshwater (K_m = 2.17 μM), estuarine (K_m = 14.5 μM), and alkaline-saline (K_m = 501 μM) environments. An in situ assay was devised in which a solution of N₂O was injected into sealed glass cores containing intact sediment. Two estimates of net rates of denitrification in San Francisco Bay under approximated in situ conditions were 0.009 and 0.041 mmol of N₂O per m² per h. Addition of chlorate to inhibit denitrification in these intact-core experiments (to estimate gross rates of N₂O consumption) resulted in approximately a 14% upward revision of estimates of net rates. These results were comparable to an in situ estimate of 0.022 mmol of N₂O per m² per h made with the acetylene block assay.     

Sulfate-Dependent Interspecies H2 Transfer between Methanosarcina barkeri and Desulfovibrio vulgaris during Coculture Metabolism of Acetate or Methanol

We compared the metabolism of methanol and acetate when Methanosarcina barkeri was grown in the presence and absence of Desulfovibrio vulgaris. The sulfate reducer was not able to utilize methanol or acetate as the electron donor for energy metabolism in pure culture, but was able to grow in coculture. Pure cultures of M. barkeri produced up to 10 μmol of H₂ per liter in the culture headspace during growth on acetate or methanol. In coculture with D. vulgaris, the gaseous H₂ concentration was ≤2 μmol/liter. The fractions of ¹⁴CO₂ produced from [¹⁴C]methanol and 2-[¹⁴C]acetate increased from 0.26 and 0.16, respectively, in pure culture to 0.59 and 0.33, respectively, in coculture. Under these conditions, approximately 42% of the available electron equivalents derived from methanol or acetate were transferred and were utilized by D. vulgaris to reduce approximately 33 μmol of sulfate per 100 μmol of substrate consumed. As a direct consequence, methane formation in cocultures was two-thirds that observed in pure cultures. The addition of 5.0 mM sodium molybdate or exogenous H₂ decreased the effects of D. vulgaris on the metabolism of M. barkeri. An analysis of growth and carbon and electron flow patterns demonstrated that sulfate-dependent interspecies H₂ transfer from M. barkeri to D. vulgaris resulted in less methane production, increased CO₂ formation, and sulfide formation from substrates not directly utilized by the sulfate reducer as electron donors for energy metabolism and growth.     

Terminal Oxidases of Chlorella pyrenoidosa

In studies of the kinetics of oxygen uptake by glucose-stimulated Chlorella pyrenoidosa, two terminal oxidases could be distinguished. The cytochrome oxidase of Chlorella has a Km (O₂) of 2.1 ± 0.3 μm, while the second oxidase has a Km (O₂) of 6.7 ± 0.5 μm, and a maximum capacity about one-quarter of that of the cytochrome system. The identity of the second oxidase is unknown, but it is not inhibited by carbon monoxide, 1 mm cyanide, 0.1 mm thiocyanate, or 1 mm 8-hydroxyquinoline. In fresh cultures, the second oxidase accounts for at most 35% of the total oxygen uptake.     

Autotrophic, Hydrogen-Oxidizing, Denitrifying Bacteria in Groundwater, Potential Agents for Bioremediation of Nitrate Contamination

Addition of hydrogen or formate significantly enhanced the rate of consumption of nitrate in slurried core samples obtained from an active zone of denitrification in a nitrate-contaminated sand and gravel aquifer (Cape Cod, Mass.). Hydrogen uptake by the core material was immediate and rapid, with an apparent K_m of 0.45 to 0.60 μM and a V_max of 18.7 nmol cm^-3 h^-1 at 30°C. Nine strains of hydrogen-oxidizing denitrifying bacteria were subsequently isolated from the aquifer. Eight of the strains grew autotrophically on hydrogen with either oxygen or nitrate as the electron acceptor. One strain grew mixotrophically. All of the isolates were capable of heterotrophic growth, but none were similar to Paracoccus denitrificans, a well-characterized hydrogen-oxidizing denitrifier. The kinetics for hydrogen uptake during denitrification were determined for each isolate with substrate depletion progress curves; the K_ms ranged from 0.30 to 3.32 μM, with V_maxs of 1.85 to 13.29 fmol cell^-1 h^-1. Because these organisms appear to be common constituents of the in situ population of the aquifer, produce innocuous end products, and could be manipulated to sequentially consume oxygen and then nitrate when both were present, these results suggest that these organisms may have significant potential for in situ bioremediation of nitrate contamination in groundwater.     

Reinvestigation of the steady-state kinetics and physiological function of the soluble NiFe-hydrogenase I of Pyrococcus furiosus

Pyrococcus furiosus has two types of NiFe-hydrogenases: a heterotetrameric soluble hydrogenase and a multimeric transmembrane hydrogenase. Originally, the soluble hydrogenase was proposed to be a new type of H₂ evolution hydrogenase, because, in contrast to all of the then known NiFe-hydrogenases, the hydrogen production activity at 80°C was found to be higher than the hydrogen consumption activity and CO inhibition appeared to be absent. NADPH was proposed to be the electron donor. Later, it was found that the membrane-bound hydrogenase exhibits very high hydrogen production activity sufficient to explain cellular H₂ production levels, and this seems to eliminate the need for a soluble hydrogen production activity and therefore leave the soluble hydrogenase without a physiological function. Therefore, the steady-state kinetics of the soluble hydrogenase were reinvestigated. In contrast to previous reports, a low K_m for H₂ (~20 μM) was found, which suggests a relatively high affinity for hydrogen. Also, the hydrogen consumption activity was 1 order of magnitude higher than the hydrogen production activity, and CO inhibition was significant (50% inhibition with 20 μM dissolved CO). Since the K_m for NADP⁺ is ~37 μM, we concluded that the soluble hydrogenase from P. furiosus is likely to function in the regeneration of NADPH and thus reuses the hydrogen produced by the membrane-bound hydrogenase in proton respiration.     

Biofilm Dynamics and Kinetics during High-Rate Sulfate Reduction under Anaerobic Conditions

The sulfate kinetics in an anaerobic, sulfate-reducing biofilm were investigated with an annular biofilm reactor. Biofilm growth, sulfide production, and kinetic constants (K_m and V_max) for the bacterial sulfate uptake within the biofilm were determined. These parameters were used to model the biofilm kinetics, and the experimental results were in good agreement with the model predictions. Typical zero-order volume rate constants for sulfate reduction in a biofilm without substrate limitation ranged from 56 to 93 μmol of SO²₄-cm⁻³ h⁻¹ at 20°C. The temperature dependence (Q₁₀) of sulfate reduction was equivalent to 3.4 at between 9 and 20°C. The measured rates of sulfate reduction could explain the relatively high sulfide levels found in sewers and wastewater treatment systems. Furthermore, it has been shown that sulfate reduction in biofilms just a few hundred micrometers thick is limited by sulfate diffusion into biofilm at concentrations below 0.5 mM. This observation might, in some cases, be an explanation for the relatively poor capacity of the sulfate-reducing bacteria to compete with the methanogenic bacteria in anaerobic wastewater treatment in submerged filters.     

Uptake of Glyphosate by an Arthrobacter sp

The uptake of glyphosate (N-[phosphonomethyl]glycine) by an Arthrobacter sp. which can utilize this herbicide as its sole source of phosphorus was investigated. Orthophosphate suppressed the expression of the uptake system for glyphosate and also competed with glyphosate for uptake. The K_m for glyphosate uptake was 125 μM, and the K_i for orthophosphate was 24 μM. Organophosphonates as well as organophosphates inhibited glyphosate uptake, but only organophosphates and orthophosphate suppressed the uptake system. Glyphosate uptake was energy dependent, had a pH optimum of 6 to 7, and was differentially affected by divalent cations.     

Two Nucleotide Transport Proteins in Chlamydia trachomatis, One for Net Nucleoside Triphosphate Uptake and the Other for Transport of Energy

The genome of Chlamydia trachomatis, one of the most prominent human pathogens, contains two structural genes coding for proteins, herein called Npt1_Ct and Npt2_Ct (nucleoside phosphate transporters 1 and 2 of C. trachomatis), exhibiting 68 and 61% similarity, respectively, to the ATP/ADP transporter from the intracellular bacterium Rickettsia prowazekii at the deduced amino acid level. Hydropathy analysis and sequence alignments suggested that both proteins have 12 transmembrane domains. The putative transporters were expressed as histidine-tagged proteins in Escherichia coli to study their biochemical properties. His₁₀-Npt1_Ct catalyzed ATP and ADP transport in an exchange mode. The apparent K_m values were 48 (ATP) and 39 (ADP) μM. ATP and ADP transport was specific since AMP, GTP, CTP, UTP, dATP, dCTP, dGTP, and dTTP did not inhibit uptake. In contrast, His₁₀-Npt2_Ct transported all four ribonucleoside triphosphates with apparent K_m values of 31 μM (GTP), 302 μM (UTP), 528 μM (CTP), and 1,158 μM (ATP). Ribonucleoside di- and monophosphates and deoxyribonucleotides were not substrates. The protonophore m-chlorocarbonylcyanide phenylhydrazone abolished uptake of all nucleoside triphosphates by Npt2_Ct. This observation indicated that His₁₀-Npt2_Ct acts as a nucleosidetriphosphate/H⁺ symporter energized by the proton motive force across the Escherichia coli cytoplasmic membrane. We conclude that Npt1_Ct provides chlamydiae with energy whereas Npt2_Ct catalyzes the net uptake of ribonucleoside triphosphates required for anabolic reactions.     

Uptake of glycylglycine by the scutellum of germinating barley grain

The scutella separated from germinating barley grains (Hordeum vulgare L. cv. Himalaya) took up the dipeptide [¹⁴C]glycylglycine (Gly-Gly) rapidly from incubation media. The pH optimum of the process was about 4.5, and the rate of uptake conformed to Michaelis-Menten kinetics with an apparent K_m of 2.3 mm and V_max of 41 μmole gram⁻¹ hour⁻¹. The uptake was strongly inhibited by dinitrophenol and cyanide and by lack of O₂.     

Role of periplasmic trehalase in uptake of trehalose by the thermophilic bacterium Rhodothermus marinus

Trehalose uptake at 65°C in Rhodothermus marinus was characterized. The profile of trehalose uptake as a function of concentration showed two distinct types of saturation kinetics, and the analysis of the data was complicated by the activity of a periplasmic trehalase. The kinetic parameters of this enzyme determined in whole cells were as follows: K_m = 156 ± 11 μM and V_max = 21.2 ± 0.4 nmol/min/mg of total protein. Therefore, trehalose could be acted upon by this periplasmic activity, yielding glucose that subsequently entered the cell via the glucose uptake system, which was also characterized. To distinguish the several contributions in this intricate system, a mathematical model was developed that took into account the experimental kinetic parameters for trehalase, trehalose transport, glucose transport, competition data with trehalose, glucose, and palatinose, and measurements of glucose diffusion out of the periplasm. It was concluded that R. marinus has distinct transport systems for trehalose and glucose; moreover, the experimental data fit perfectly with a model considering a high-affinity, low-capacity transport system for trehalose (K_m = 0.11 ± 0.03 μM and V_max = 0.39 ± 0.02 nmol/min/mg of protein) and a glucose transporter with moderate affinity and capacity (K_m = 46 ± 3 μM and V_max = 48 ± 1 nmol/min/mg of protein). The contribution of the trehalose transporter is important only in trehalose-poor environments (trehalose concentrations up to 6 μM); at higher concentrations trehalose is assimilated primarily via trehalase and the glucose transport system. Trehalose uptake was constitutive, but the activity decreased 60% in response to osmotic stress. The nature of the trehalose transporter and the physiological relevance of these findings are discussed.     

Estimation of Bacterial Nitrate Reduction Rates at In Situ Concentrations in Freshwater Sediments

A method was developed to follow bacterial nitrate reduction in freshwater sediments by using common high-performance liquid chromatographic equipment. The low detection limit (14 pmol) of the method enabled us to study concentration profiles and reaction kinetics under natural conditions. Significant nitrate concentrations (1 to 27 μM) were observed in the sediment of Lake Vechten during the nonstratified period; the concentration profiles showed a successive depletion of oxygen, nitrate, and sulfate with depth. The profiles were restricted to the upper 3 cm of the sediment which is rich in organics and loosely structured. Nitrate reduction in the sediment-water interface followed first-order reaction kinetics at in situ concentrations. Remarkably high potential nitrate-reducing activity was observed in the part of the sediment in which nitrate did not diffuse. This activity was also observed throughout the whole year. Estimates of K_m varied between 17 and 100 μM and V_max varied between 7.2 and 36 μmol cm⁻³ day⁻¹ for samples taken at different depths. The diffusion coefficient of nitrate ([10 ± 0.4] × 10⁻⁶ cm² s⁻¹) across the sediment-water interface was estimated by a constant-source technique and applied to a mathematical model to estimate the net nitrate reduction during the nonstratified period. In this period, observed nitrate reduction rates by the model, 0.2 to 0.4 mmol m⁻² day⁻¹, were lower than those found for oxygen (27 mmol m⁻² day⁻¹) and sulfate (0.4 mmol m⁻² day⁻¹). During the summer stratification, nitrate was absent in the sediment and reduction could not be estimated by the model.     

Denitrification in San Francisco Bay Intertidal Sediments

The acetylene block technique was employed to study denitrification in intertidal estuarine sediments. Addition of nitrate to sediment slurries stimulated denitrification. During the dry season, sediment-slurry denitrification rates displayed Michaelis-Menten kinetics, and ambient NO₃⁻ + NO₂⁻ concentrations (≤26 μM) were below the apparent K_m (50 μM) for nitrate. During the rainy season, when ambient NO₃⁻ + NO₂⁻ concentrations were higher (37 to 89 μM), an accurate estimate of the K_m could not be obtained. Endogenous denitrification activity was confined to the upper 3 cm of the sediment column. However, the addition of nitrate to deeper sediments demonstrated immediate N₂O production, and potential activity existed at all depths sampled (the deepest was 15 cm). Loss of N₂O in the presence of C₂H₂ was sometimes observed during these short-term sediment incubations. Experiments with sediment slurries and washed cell suspensions of a marine pseudomonad confirmed that this N₂O loss was caused by incomplete blockage of N₂O reductase by C₂H₂ at low nitrate concentrations. Areal estimates of denitrification (in the absence of added nitrate) ranged from 0.8 to 1.2 μmol of N₂ m⁻² h⁻¹ (for undisturbed sediments) to 17 to 280 μmol of N₂ m⁻² h⁻¹ (for shaken sediment slurries).