Behaviour of toluene, benzene and naphthalene under anaerobic conditions in sediment columns

The biotransformation of toluene, benzene and naphthalene was examined in anaerobic sediment columns. Five columns filled with a mixture of sediments were operated in the presence of bicarbonate, sulfate, iron, manganese, or nitrate as electron acceptor. The columns were continuously percolated with a mixture of the three organic compounds (individual concentrations 25–200 μM) at 20°C. Toluene was transformed readily (within 1 to 2 months) under all redox conditions tested. Benzene was recalcitrant over the test period of 375–525 days in all five columns. Naphthalene was partly transformed in the column with nitrate or manganese as electron acceptor present; the addition of benzoate had a positive effect in the column with nitrate. In the column with sulfate, the majority of the added naphthalene disappeared. No effect was observed after adding and omitting an easier degradable substrate. [¹⁴C]naphthalene was used to confirm this disappearance to be the result of degradation; two third of the naphthalene was converted to CO₂.     

Chemolithotrophic growth ofDesulfovibrio desulfuricans with hydrogen coupled to ammonification of nitrate or nitrite

Two of nine sulfate reducing bacteria tested,Desulfobulbus propionicus andDesulfovibrio desulfuricans (strain Essex 6), were able to grow with nitrate as terminal electron acceptor, which was reduced to ammonia. Desulfovibrio desulfuricans was grown in chemostat culture with hydrogen plus limiting concentrations of nitrate, nitrite or sulfate as sole energy source. Growth yields up to 13.1, 8.8 or 9.7 g cell dry mass were obtained per mol nitrate, nitrite or sulfate reduced, respectively. The apparent half saturation constants (K _s) were below the detection limits of 200, 3 or 100 mol/l for nitrate, nitrite of sulfate, respectively. The maximum growth rates {ie63-1} raised from 0.124 h^-1 with sulfate and 0.150 h^-1 with nitrate to 0.193 h^-1 with nitrite as electron acceptor. Regardless of the electron acceptor in the culture medium, cell extracts exhibited absorption maxima corresponding to cytochromec and desulfoviridin. Nitrate reductase was found to be inducible by nitrate or nitrite, whereas nitrite reductase was synthesized constitutively. The activities of nitrate and nitrite reductases with hydrogen as electron donor were 0.2 and 0.3 mol/min·mg protein, respectively. If limiting amounts of hydrogen were added to culture bottles with nitrate as electron acceptor, part of the nitrate was only reduced to the level of nitrite. In media containing nitrate plus sulfate or nitrite plus sulfate, sulfate reduction was suppressed.The results demonstrate that the ammonification of nitrate or nitrite can function as sole energy conserving process in some sulfate-reducing bacteria.     

Stoichiometry of proton uptake in isolated pea chloroplasts under different light intensities

The number of protons released inside the chloroplast thylakoids per electron which is transferred through the electron transport chain (H⁺/e^– ratio) was measured in isolated pea chloroplasts at pH 6.0 under continuous illumination and with methyl viologen as an electron acceptor. At saturating light intensity (200 W · m^–2) (strong light) the H⁺/e^– ratio was 3. At low intensity (0.9 W · m^–2) (weak light) the H⁺/e^– ratio was 2 with dark-adapted chloroplasts, but it was close to 3 with chloroplasts that were preilluminated with strong light. It is shown that the presence of azide in the reaction mixture leads to errors in the determination of the H⁺/e^– ratio due to underestimation of the initial rate of H⁺ efflux on switching off the light. To explain the above data, we assume that transformation of the electron transport chain occurs during illumination with strong light, namely, the Q cycle becomes operative.     

Aerobic denitrification in various heterotrophic nitrifiers

Various heterotrophic nitrifiers have been tested and found to also be aerobic denitrifiers. The simultaneous use of two electron acceptors (oxygen and nitrate) permits these organisms to grow more rapidly than on either single electron acceptor, but generally results in a lower yield than is obtained on oxygen, alone. One strain, formerly known as Pseudomonas denitrificans, was grown in the chemostat and shown to achieve nitrification rates of up to 44 nmol NH₃ min^–1 mg protein^–1 and denitrification rates up to 69 nmol NO _inf3 ^sup–1 min^–1 mg protein^–1.Unlike Thiosphaera pantotropha, this strain needed to induce its nitrate reductase. However, the remainder of the denitrifying pathway was constitutive and, like T. pantotropha, Ps. denitrificans probably possesses the copper nitrite reductase.     

Effect of 2-hydroxybenzoate on the rate of naphthalene mineralization in soil

The effect of 2-hydroxybenzoate (2-OHB, salicylate) on the mineralization rate of [¹⁴C]naphthalene, the population density of naphthalene-degrading bacteria, and the concentration of genes encoding for naphthalene dioxygenase in a soil bacterial community was investigated. Six different concentrations of 2-OHB (10, 20, 50, 100, 150 and 200 g g^–1 soil) were tested in 100-g portions of soil. The addition of 10, 20 or 50 g 2-OHB g^–1 soil produced a general increase in total soil bacterial population density, whereas the addition of 100 g or 200 g 2-OHB g^–1 soil specifically increased the proportion of naphthalene degraders relative to the total population. The addition of 50 g 2-OHB g^–1 soil produced a fourfold increase (the maximum observed) in the rate of naphthalene mineralization relative to the rate in unamended soil. The concentration of 2-OHB ( 100 g/g) added to soil correlated with the population density of naphthalene degraders (r=0.961). Addition of up to 200 g 2-OHB g^–1 correlated with the abundance of DNA sequences homologous to known naphthalene dioxygenase genes (nahAB) (r=0.958). However, mineralization of [¹⁴C]naphthalene was stimulated significantly only by the addition of 50 g 2-OHB g^–1 soil. Results of the mineralization experiments were supported by the detection of nahAB mRNA extracted directly from soil. The specificity of the effect of 2-OHB on naphthalene biodegradation was confirmed in a control experiment using equivalent concentrations of 4-OHB which repressed naphthalene mineralization by about 50%. Addition of ammonium nitrate to the soil also increased the rate of naphthalene mineralization. Ammonium nitrate added together with 2-OHB reduced the mineralization enhancement effect of either compound alone. The study confirmed that specific induction of biodegradative genes can enhance chemical pollutant removal in situ. Correspondence to: O. A. Ogunseitan     

Physiology and kinetics of autotrophic denitrification by Thiobacillus denitrificans

Summary A strain of Thiobacillus denitrificans was isolated after enrichment under anaerobic conditions by the continuous culture technique using thiosulfate as energy source and nitrate as electron acceptor and nitrogen source. The isolate was an active denitrifyer, the optimal conditions being 30°C and pH 7.5–8.0. Denitrification was inhibited by sulfate (the reaction product) above 5 g SO ₄ ⁼ /l, whereas high concentrations of the substrates nitrate and thiosulfate were less harmful; nitrite affected denitrification above 0.2 g NO ₂ ^– /l. During the time course of denitrification in a batch culture growth and substrate consumption slowed down already after only half the substrate was utilized due to product inhibition. The following parameters were determined in continuous culture under nitrate limitation: _max=0.11 h^–1, K _S=0.2 mg NO ₃ ^– /l, maximum denitrification rate=0.78 g NO ₃ ^– /g cells·h, g cells/g NO ₃ ^– , g cells/g S₂O ₃ ⁼ . Nitrite did not accumulate during steady state denitrification; the denitrification gas was almost pure N₂. The concentrations of N₂O and NO were below 1 ppm.     

Isolation and characterization of a thermophilic sulfate-reducing bacterium Desulfotomaculum thermoacetoxidans sp. nov.

An obligately anaerobic thermophilic sporeforming sulfate-reducing bacterium, named strain CAMZ, was isolated from a benzoate enrichment from a 58°C thermophilic anaerobic bioreactor. The cells of strain CAMZ were 0.7 m by 2–5 m rods with pointed ends, forming single cells or pairs. Spores were central, spherical, and caused swelling of the cells. The Gram stain was negative. Electron donors used included lactate, pyruvate, acetate and other short chain fatty acids, short chain alcohols, alanine, and H₂/CO₂. Lactate and pyruvate were oxidized completely to CO₂ with sulfate as electron acceptor. Sulfate was required for growth on H₂/CO₂, and both acetate and sulfide were produced from H₂/CO₂-sulfate. Sulfate, thiosulfate, or elemental sulfur served as electron acceptors with lactate as the donor while sulfite, nitrate, nitrite, betaine, or a hydrogenotrophic methanogen did not. The optimum temperature for growth of strain CAMZ was 55–60°C and the optimum pH value was 6.5. The specific activities of carbon monoxide dehydrogenase of cells of strain CAMZ grown on lactate, H₂/CO₂, or acetate with sulfate were 7.2, 18.1, and 30.8 mol methyl viologen reduced min^–1 [mg protein]^–1, respectively, indicating the presence of the CO/Acetyl-CoA pathway in this organism. The mol%-G+C of strain CAMZ's DNA was 49.7. The new species name Desulfotomaculum thermoacetoxidans is proposed for strain CAMZ.     

Response of coastal phytoplankton to ammonium and nitrate pulses: seasonal variations of nitrogen uptake and regeneration

Seasonal variation in uptake and regeneration of ammonium and nitrate in a coastal lagoon was studied using ¹⁵N incorporation in particulate matter and by measuring changes in particulate nitrogen. Uptake and regeneration rates were two orders of magnitude lower in winter than in summer. Summer uptake values were 2.8 and 2.2 mol N.l^–1.d^–1 for ammonium and nitrate, respectively. Regeneration rates were 2.9 and 2.1 mol N.l^–1.d^–1 for ammonium and nitrate respectively. Regeneration/uptake ratios were often below one, indicating that water column processes were not sufficient to satisfy the phytoplankton nitrogen demand. This implies a role of other sources of nitrogen, such as macrofauna (oysters and epibionts) and sediment. Phytoplankton was well adapted to the seasonal variations in resources, with mixotrophic dinoflagellates dominant in winter, and fast growing diatoms in summer. In winter and spring, ammonium was clearly preferred to nitrate as a nitrogen source, but nitrate was an important nitrogen source in summer because of high nitrification rates. Despite low nutrient levels, the high rates of nitrogen regeneration in summer as well as the simultaneous uptake of nitrate and ammonium allow high phytoplankton growth rates which in turn enable high oyster production.     

Benzene oxidation under sulfate-reducing conditions in columns simulating in situ conditions

The oxidation of benzene under sulfate-reducing conditions was examined in column and batch experiments under close to in situ conditions. Mass balances and degradation rates for benzene oxidation were determined in four sand and four lava granules filled columns percolated with groundwater from an anoxic benzene-contaminated aquifer. The stoichiometry of oxidized benzene, produced hydrogen carbonate and reduced sulfate correlated well with the theoretical equation for mineralization of benzene with sulfate as electron acceptor. Mean retention times of water in four columns were determined using radon (²²²Rn) as tracer. The retention times were used to calculate average benzene oxidation rates of 8–36 μM benzene day⁻¹. Benzene-degrading, sulfide-producing microcosms were successfully established from sand material of all sand filled columns, strongly indicating that the columns were colonized by anoxic benzene-degrading microorganisms. In general, these data indicate a high potential for Natural Attenuation of benzene under sulfate-reducing conditions at the field site Zeitz. In spite of this existing potential to degrade benzene with sulfate as electron acceptor, the benzene plume at the field site is much longer than expected if benzene would be degraded at the rates observed in the column experiment, indicating that benzene oxidation under sulfate-reducing conditions is limited in situ.     

Control of microbial souring by nitrate,nitrite or glutaraldehyde injection in a sandstone column

Microbial souring (production of hydrogen sulfide by sulfate-reducing bacteria, SRB) in crushed Berea sandstone columns with oil field-produced water consortia incubated at 60°C was inhibited by the addition of nitrate (NO₃) or nitrite (NO ₂ ^– ). Added nitrate (as nitrogen) at a concentration of 0.71 mM resulted in the production of 0.57–0.71 mM nitrite by the native microbial population present during souring and suppressed sulfate reduction to below detection limits. Nitrate added at 0.36 mM did not inhibit active souring but was enough to maintain inhibition if the column had been previously treated with 0.71 mM or greater. Continuous addition of 0.71–0.86 mM nitrite also completely inhibited souring in the column. Pulses of nitrite were more effective than the same amount of nitrite added continuously. Nitrite was more effective at inhibiting souring than was glutaraldehyde, and SRB recovery was delayed longer with nitrite than with glutaraldehyde. It was hypothesized that glutaraldehyde killed SRB while nitrite provided a long-term inhibition without cell death. Removal of nitrate after as long as 3 months of continuous addition allowed SRB in a biofilm to return to their previous level of activity. Inhibition was achieved with much lower levels of nitrate and nitrite, and at higher temperatures, than noted by other researchers.     

Growth and respiration ofBradyrhizobium japonicum USDA 143 with nitrous oxide as the terminal electron acceptor

Bradyrhizobium japonicum USDA 143 grew chemoorganotrophically under anoxic conditions with exogenous N₂O as the sole terminal electron acceptor. Cell growth and dissimilatory N₂O reduction were significantly inhibited by C₂H₂ when either N₂O or N₂O plus NO ₃ ^– served as terminal electron acceptor(s). Reduction of N₂O accounted for 20% of the energy for cell growth in cultures supplied with NO ₃ ^– as the terminal electron acceptor. Nitrous oxide was produced stoichiometrically in cultures containing NO ₃ ^– and C₂H₂, but cell growth was proportionately reduced when compared with cultures supplied with an equal amount of NO ₃ ^– . Exogenous N₂O delayed the reduction of NO ₃ ^– in cultures supplied with both electron acceptors. Direct amperometric monitoring of N₂O respiration showed a specific activity of 0.082±0.004 moles N₂O/min/mg cell protein, and azide inhibited cell respiration.     

Metal removal by immobilised and non-immobilised Azolla filiculoides

Milled-sieved and epichlorhydrin-immobilised Azolla biosorbed ca. 363 and 320 mol Cu²⁺ g^–1 from a 100 mg l^–1 solution. Efficiency of Cu²⁺ removal by columns was in the order epichlorohydrin-immobilised Azolla>milled-sieved Azolla>untreated Azolla. The 2.5 g epichlorohydrin-immobilised Azolla column demonstrated complete metal sequestration from ca. 12 l of influent 5 mg Cu²⁺ l^–1 and was still at less than 75% saturation even after ca. 22 l had passed through the column. EDTA effectively desorbed Cu²⁺ with a ca. 55-fold decrease in volume.     

Denitrification in the water column of an intermittently anoxic fjord

Denitrification was studied in the water column in the Bunnefjord, inner part of the Oslofjord in southern Norway, using a ¹⁵N-technique (the isotope pairing method). The fjord is 150 m deep and during our surveys in September–December 1998 hydrogen sulphide was present in the deep water below 80 m. No significant denitrification was found in water samples from the surface layer (4 m depth), but high rates were observed within a deep density gradient between 62 and 78 m depth. Oxygen concentration within this layer was low (<21 mmol m^–3), and the concentration of NO₃ decreased from ca. 15 mmolm^–3 at 62 m depth to not detectable below 78 m. Pronounced peaks of NO₂ up to 4.4 mmol m^–3 were observed at 70–78 m depth. The maximum denitrification rate of 1.5 mmol N m^–3 d^–1 was observed at 70 m depth. Integrated for the whole layer, the denitrification rate was 13 mmol N m^–2 d^–1. A significant linear correlation was found between the denitrification rate and the ambient nitrate concentration which indicated that the rate was primarily controlled by the availability of nitrate in the O₂-poor water. Compared to rates reported for coastal water, denitrification in the water column in the Bunnefjord was high and the process appears to be a major sink of bioavailable nitrogen in the fjord.     

Temperature dependence of the primary electron transfer reaction in pigment-modified bacterial reaction centers

The initial electron transfer steps in pigment modified reaction centers, where bacteriopheophytin is replaced by plant pheophytin (R26.Phe-a RCs) have been investigated over a wide temperature range by femtosecond time-resolved spectroscopy. The experimental data obtained in the maximum of the bacteriochlorophyll anion band at 1020 nm show the existence of a high and long-lived population of the primary acceptor P⁺B_A ^– even at 10 K. The data suggest a stepwise electron transfer mechanism with B_A as primary acceptor also in the low temperature domain. A detailed data analysis suggests that the pigment modification leads to a situation with almost isoenergetic primary and secondary acceptor levels, approximately 450 cm^–1 below P^*. A Gaussian distribution (with = 400 cm ^–1) of the G values has to be assumed to account for the strong dispersive character of the kinetics in this sample. Based on these assumptions, a model is presented that reproduces the observed kinetics, heterogeneity and temperature dependence.     

Autotrophic denitrification by Thiobacillus denitrificans in a packed bed reactor

Summary An upflow packed bed reactor with lava stones as support for the microbial growth proved to be very useful for the denitrification of industrial waste water by Thiobacillus denitrificans. The application of the plug flow principle allowed higher concentrations of nitrate to be employed than in a stirred tank reactor because inhibitory concentrations of sulfate from thiosulfate oxidation built up only in the upper part of the column — if at all. In experiments with synthetic media nitrate solutions of different strength (NO ₃ ^– g/l: 1.8; 3.0; 4.3; 6.1) were tested, each at 5 different residence times (5; 3.3; 2.5; 2.0; 1.7 h). The combination of the two parameters which still allowed 95% denitrification was 3 g NO ₃ ^- /l and 2.5 h residence time; this corresponded to a volumetric nitrate loading of about 25 kg/m³·d. Higher nitrate loadings led to incomplete denitrification coupled with the occurence of nitrite in the outflow. Below the critical loading rate nitrite accumulated only in the lower part of the column and was then gradually reduced. Experiments with simulated middle active waste from processing nuclear fuel which contained numerous heavy metals yielded similar results. — Although pure inorganic media were fed into the reactor the microflora developing as a dense layer covering the lava stones consisted not only of T. denitrificans but also of heterotrophic denitrifiers, mainly Pseudomonas aeruginosa.     

Uptake of sulfate,sulfite and thiosulfate by proton-anion symport in Desulfovibrio desulfuricans

pH changes and sulfide production upon addition of sulfate, sulfite or thiosulfate to non-buffered H₂-saturated cell suspensions of Desulfovibrio desulfuricans were studied by means of electrodes. The addition of these electron acceptors resulted in a rapid alkalinization of the suspension which was accompanied by sulfide production. At-2° C, alkalinization without immediate sulfide production could be obtained. After addition of ³⁵S-labelled sulfate at-2° C, the label was found to be concentrated 7,500-fold in the cells, while 2 protons per sulfate molecule had disappeared from the outer bulk phase. Alkalinization and sulfide production from micromolar electron acceptor additions depended on the transmembraneous proton gradient ( pH), and were reversibly inhibited in alkaline solution (pH>8.0) or by the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP). Protonophore-inhibited sulfide production from sulfite or thiosulfate could be restored if the cell membranes were permeabilized by the detergent cetyltrimethylammonium bromide (CTAB), or if downhill transport was made possible by the addition of electron acceptors at millimolar concentrations. Sulfate was not reduced under these conditions, presumably because the cells did not contain ATP for its activation. K⁺-and Na⁺-ionophores such as nigericin, valinomycin or monensin appeared to be of limited efficiency in D. desulfuricans. In most experiments, sulfate reduction was inhibited by the K⁺–H⁺ antiporter nigericin in the presence of K⁺, but not by the thiocyanate anion or the K⁺-transporter valinomycin. The results indicate that sulfate, sulfite and thiosulfate are taken up by proton-anion symport, presumably as undissociated acids with an electroneutral mechanism, driven by the transmembraneous pH gradient ( pH) or by a solute gradient. Kinetics of alkalinization and sulfide production in cells grown with different electron acceptors revealed that D. desulfuricans has different specific uptake systems for sulfate and thiosulfate, and obviously also for sulfite. It is proposed that the electron acceptor transport finally will not consume net energy during growth in buffered medium: The protons taken up during active electron acceptor transport leave the cell with the reduced end-product by simple passive diffusion of H₂S.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - FCCP carbonyl cyanide p-trifluoromethoxy phenylhydrazone - CTAB cethyltrimethylammonium bromide     

Denitrification potential of epilithic communities in a lotic environment

Denitrification potentials of epilithic microbial populations were assessed using the acetylene inhibition method, in which acetylene is used to block the reduction of nitrous oxide (N₂O) to nitrogen (N₂). Samples of the epilithic community were incubated in filtered river water containing modified Bushnell-Haas salts, glycerol, and yeast extract—under aerobic (0.2 atm O₂) and anaerobic (0.2 atm He) acetylene atmospheres. N₂O was produced under both atmospheres only if exogenous nitrate of nitrite was added. Denitrification potentials were typically higher when nitrite was the added electron acceptor. The rates of denitrification were temperature-and carbon-dependent and the maximum rate, 8.53 g N₂O–N per cm² per day occurred at 23°C when nitrite was the electron acceptor.     

Application of N-15 dilution for simultaneous estimation of nitrification and nitrate reduction in soil-water columns

Nitrification and denitrification rates were estimated simultaneously in soil-floodwater columns of a Crowley silt loam (Typic Albaqualfs) rice soil by an¹⁵N isotopic dilution technique. Labeled NO ₃ ^– was added to the floodwater of soil-water columns, half were treated with urea fertilizer. The (NO ₃ ^– +NO ₂ ^– )–N and (NO ₃ ^– +NO ₂ ^– )–N concentrations in the floodwater were measured over time and production and reduction rates for NO ₃ ^– calculated. Nitrate reduction in the urea amended columns averaged 515 mol N m^–2h^–1 and nitrification averaged 395 mol N m^–2h^–1 over the 35–153 d incubation. The nitrification rate for 4–19 d sampling period (1,560 mol N m^–2h^–1) in the urea amended columns was almost 9 times greater than the reduction rate (175 mol N m^–2h^–1) over the same period. Without the addition of urea the NO ₃ ^– production rate averaged 32 mol N m^–2h^–1 and reduction 101 mol N m^–2h^–1.     

The role of nitrate in osmoregulation of Italian ryegrass

Summary The role of nitrate in osmotic control was studied with Italian ryegrass grown in a nutrient solution in a climate room. Quantum-flux density, osmotic potential of the nutrient solution and availability of nitrate and chloride were varied independently. Plants at high quantum flux density (650 mol m^–2 s^–1) had a lower osmotic potential, a higher carbohydrate concentration and a lower nitrate concentration than plants at low quantum flux density (310 mol m^–2 s^–1), the decrease in nitrate concentration was osmotically equivalent to the increase in carbohydrate concentration. When nitrate in the nutrient solution was partly replaced by chloride, the chloride taken up substituted an equivalent part of the nitrate in the plant. It is concluded that nitrate plays a role in osmoregulation of the plant and compensates for a shortage of other solutes.