Biodegradation of atrazine in surface soils and subsurface sediments collected from an agricultural research farm

The purpose of the present study was to assess atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) mineralization by indigenous microbial communities and to investigate constraints associated with atrazine biodegradation in environmental samples collected from surface soil and subsurface zones at an agricultural site in Ohio. Atrazine mineralization in soil and sediment samples was monitored as ¹⁴CO₂ evolution in biometers which were amended with ¹⁴C-labeled atrazine. Variables of interest were the position of the label ([U-¹⁴C-ring]-atrazine and [2-¹⁴C-ethyl]-atrazine), incubation temperature (25°C and 10°C), inoculation with a previously characterized atrazine-mineralizing bacterial isolate (M91-3), and the effect of sterilization prior to inoculation. In uninoculated biometers, mineralization rate constants declined with increasing sample depth. First-order mineralization rate constants were somewhat lower for [2-¹⁴C-ethyl]-atrazine when compared to those of [U-¹⁴C-ring]-atrazine. Moreover, the total amount of ¹⁴CO₂ released was less with [2-¹⁴C-ethyl]-atrazine. Mineralization at 10°C was slow and linear. In inoculated biometers, less ¹⁴CO₂ was released in [2-¹⁴C-ethyl]-atrazine experiments as compared with [U-¹⁴C-ring]-atrazine probably as a result of assimilatory incorporation of ¹⁴C into biomass. The mineralization rate constants (k) and overall extents of mineralization (P _max ) were higher in biometers that were not sterilized prior to inoculation, suggesting that the native microbial populations in the sediments were contributing to the overall release of ¹⁴CO₂ from [U-¹⁴C-ring]-atrazine and [2-¹⁴C-ethyl]-atrazine. A positive correlation between k and aqueous phase atrazine concentrations (C _eq ) in the biometers was observed at 25°C, suggesting that sorption of atrazine influenced mineralization rates. The sorption effect on atrazine mineralization was greatly diminished at 10°C. It was concluded that sorption can limit biodegradation rates of weakly-sorbing solutes at high solid-to-solution ratios and at ambient surface temperatures if an active degrading population is present. Under vadose zone and subsurface aquifer conditions, however, low temperatures and the lack of degrading organisms are likely to be primary factors limiting the biodegradation of atrazine.Abbreviations C _eq solution phase atrazine concentration at equilibrium - C _s amount of atrazine sorbed - CLA [2-¹⁴C-ethyl]-atrazine - k first-order mineralization rate constant - K _d sorption coefficient - m slope - P _max maximum amount of CO₂ released - RLA [U-¹⁴C-ring]-atrazine     

Biodegradation of atrazine in surface soils and subsurface sediments collected from an agricultural research farm

The purpose of the present study was to assess atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) mineralization by indigenous microbial communities and to investigate constraints associated with atrazine biodegradation in environmental samples collected from surface soil and subsurface zones at an agricultural site in Ohio. Atrazine mineralization in soil and sediment samples was monitored as ¹⁴CO₂ evolution in biometers which were amended with ¹⁴C-labeled atrazine. Variables of interest were the position of the label ([U-¹⁴C-ring]-atrazine and [2-¹⁴C-ethyl]-atrazine), incubation temperature (25°C and 10°C), inoculation with a previously characterized atrazine-mineralizing bacterial isolate (M91-3), and the effect of sterilization prior to inoculation. In uninoculated biometers, mineralization rate constants declined with increasing sample depth. First-order mineralization rate constants were somewhat lower for [2-¹⁴C-ethyl]-atrazine when compared to those of [U-¹⁴C-ring]-atrazine. Moreover, the total amount of ¹⁴CO₂ released was less with [2-¹⁴C-ethyl]-atrazine. Mineralization at 10°C was slow and linear. In inoculated biometers, less ¹⁴CO₂ was released in [2-¹⁴C-ethyl]-atrazine experiments as compared with [U-¹⁴C-ring]-atrazine probably as a result of assimilatory incorporation of ¹⁴C into biomass. The mineralization rate constants (k) and overall extents of mineralization (P _max ) were higher in biometers that were not sterilized prior to inoculation, suggesting that the native microbial populations in the sediments were contributing to the overall release of ¹⁴CO₂ from [U-¹⁴C-ring]-atrazine and [2-¹⁴C-ethyl]-atrazine. A positive correlation between k and aqueous phase atrazine concentrations (C _eq ) in the biometers was observed at 25°C, suggesting that sorption of atrazine influenced mineralization rates. The sorption effect on atrazine mineralization was greatly diminished at 10°C. It was concluded that sorption can limit biodegradation rates of weakly-sorbing solutes at high solid-to-solution ratios and at ambient surface temperatures if an active degrading population is present. Under vadose zone and subsurface aquifer conditions, however, low temperatures and the lack of degrading organisms are likely to be primary factors limiting the biodegradation of atrazine.     

Inhibition of atrazine degradation by cyanazine and exogenous nitrogen in bacterial isolate M91-3

A variety of s-triazine herbicides and nitrogen fertilizers frequently occur as co-contaminants at pesticide manufacturing and distribution facilities. The degradation of atrazine and cyanazine by the bacterial isolate M91-3 was investigated in washed-cell suspensions and crude cellular extracts. Cyanazine competitively inhibited atrazine degradation. The maximum atrazine degradation rate (V _max) was 41 times higher and the half-saturation constant for the inhibitor (K _i) was 1.3 times higher in the crude cellular extract than in the washed-cell suspension, suggesting that cellular uptake influenced degradation of the s-triazines. Cultures that had received prior exposure to atrazine and simazine exhibited comparable atrazine degradation rates, while cells exposed to cyanazine, propazine, ametryne, cyanuric acid, 2-hydroxyatrazine, biuret, and urea exhibited a lack of atrazine-degradative activity. Growth in the presence of exogenous inorganic nitrogen inhibited subsequent atrazine-degradative activity in washed-cell suspensions, suggesting that regulation of s-triazine and nitrogen metabolism are linked in this bacterial isolate. These findings have significant implications for the environmental fate of s-triazines in agricultural settings since these herbicides are frequently applied to soils receiving N fertilizers. Furthermore, these results suggest that bioremediation of s-triazine-contaminated sites (common at pesticide distribution facilities in the cornbelt) may be inhibited by the presence of N fertilizers that occur as co-contaminants. Received: 3 March 1998 / Received revision: 24 September 1998 / Accepted: 11 October 1998     

The effect of growth conditions on the biodegradation of tributyl phosphate and potential for the remediation of acid mine drainage waters by a naturally-occurring mixed microbial culture

The biodegradation of tributyl phosphate (Bu₃-P, TBP), releasing phosphate at a high enough concentration locally to precipitate uranium from solution, was demonstrated by a mixed culture consisting primarily of pseudomonads. The effect of various parameters on Bu₃-P biodegradation by growing cells is described. Growth at the expense of Bu₃-P as the carbon and phosphorus source occurred over a pH range from 6.5 to 8, and optimally at pH 7. Bu₃-P biodegradation was optimal at 30 °C, reduced at 20 °C and negligible at 4 °C and 37 °C. Incorporation of Cu or Cd inhibited, and Ni, Co and Mn reduced its degradation. Inorganic phosphate (above 10 mM) and kerosene (up to 1 g/l) reduced Bu₃-P biodegradation significantly, but nitrate had no effect. Sulphate (10–100 mM) was inhibitory. When pregrown biomass was used the fastest rates of tributyl and dibutyl phosphate biodegradation were 25 μmol h⁻¹ mg protein⁻¹ and 37 μmol h⁻¹ mg protein⁻¹ respectively. Microcarrier-immobilised biomass decontaminated uranium-bearing acid mine waste water by uranium phosphate precipitation at the expense of Bu₃-P hydrolysis in the presence of 35 mM SO₄ ²⁻. At pH 4.5, 79% of the UO₂ ²⁺ was removed at a flow rate of 1.4 ml/h on a 7-ml test column. Received: 2 June 1997 / Received revision: 15 September 1997 / Accepted: 19 September 1997     

Kinetics of biodegradation of gasoline and its hydrocarbon constituents

Aerobic biodegradation of gasoline and its constituents, benzene, toluene and ethylbenzene were studied by an enrichment from soil indigenous microbial population. The enrichment culture completely degraded 16.1–660 mg/l gasoline in 2.5–16 days respectively, without accumulation of any by-products. The kinetics of gasoline as well as benzene, toluene and ethylbenzene biodegradation was investigated with initial gasoline concentrations of 16.1–62.6 mg/l. The maximum specific rates of biodegradation of benzene, toluene and ethylbenzene were 0.12, 0.38 and 0.19 mg mg biomass⁻¹ day⁻¹ respectively. When benzene and toluene were used as sole substrate, the maximum specific rates of their biodegradation were 62.9 and 16.4 times greater than the corresponding values for a mixture (gasoline). The microbial culture was able to mineralize up to 200 mg/l pure toluene and benzene. Maximum mineralization efficiencies of benzene and toluene were 76.7 ± 5.1% and 76.8 ± 1.3% respectively. Self-inhibition and competitive inhibition patterns were observed during the biodegradation of benzene and toluene alone and in the mixture respectively. The observed kinetics was modeled according to Andrews' inhibition model. Received: 6 August 1997 / Received revision: 18 November 1997 / Accepted: 29 November 1997     

Thermophilic biodegradation of BTEX by two consortia of anaerobic bacteria

Two thermophilic anaerobic bacterial consortia (ALK-1 and LLNL-1), capable of degrading the aromatic fuel hydrocarbons, benzene, toluene, ethylbenzene, and the xylenes (BTEX compounds), were developed at 60 °C from the produced water of ARCO'S Kuparuk oil field at Alaska and the subsurface water at the Lawrence Livermore National Laboratory gasoline-spill site, respectively. Both consortia were found to grow at 45–75 °C on BTEX compounds as their sole carbon and energy sources with 50 °C being the optimal temperature. With 3.5 mg total BTEX added to sealed 50-ml serum bottles, which contained 30 ml mineral salts medium and the consortium, benzene, toluene, ethylbenze, m-xylene, and an unresolved mixture of o- and p-xylenes were biodegraded by 22%, 38%, 42%, 40%, and 38%, respectively, by ALK-1 after 14 days of incubation at 50 °C. Somewhat lower, but significant, percentages of the BTEX compounds also were biodegraded at 60 °C and 70 °C. The extent of biodegradation of these BTEX compounds by LLNL-1 at each of these three temperatures was slightly less than that achieved by ALK-1. Use of [ring-¹⁴C]toluene in the BTEX mixture incubated at 50 °C verified that 41% and 31% of the biodegraded toluene was metabolized within 14 days to water-soluble products by ALK-1 and LLNL-1, respectively. A small fraction of it was mineralized to ¹⁴CO₂. The use of [U-¹⁴C]benzene revealed that 2.6%–4.3% of the biodegraded benzene was metabolized at 50 °C to water-soluble products by the two consortia; however, no mineralization of the degraded [U-¹⁴C]benzene to ¹⁴CO₂ was observed. The biodegradation of BTEX at all three temperatures by both consortia was tightly coupled to sulfate reduction as well as H₂S generation. None was observed when sulfate was omitted from the serum bottles. This suggests that sulfate-reducing bacteria are most likely responsible for the observed thermophilic biodegradation of BTEX in both consortial cultures. Received: 12 July 1996 / Received revision: 31 December 1996 / Accepted: 31 January 1997     

The mineralization of chloroform in river sediments

The formation of¹⁴CO₂ from 3 μg l⁻¹ labelled chloroform was studied in anaerobic Dutch river sediments. All incubations were performed under anaerobic conditions. The observed first order mineralization kinetics showed half-lives of 2–37 days at 20°C in 12 muddy sediments. In contrast most of the sandy sediment samples did not show a mineralization of chloroform. Most probable number analysis revealed about 3.10⁴ chloroform mineralizing bacteria per g of dry sediment in a muddy sediment and 1–2.10³ chloroform mineralizing bacteria per g of dry sediment in a sandy sediment. Therefore the persistence of chloroform in sandy sediments is not caused by the absence of chloroform mineralizing bacteria but by the inactivity of these bacteria. This inactivity of the sandy sediments might allow chloroform from infiltrating river water to reach the groundwater. Mud samples from a relatively unpolluted site showed a similar chloroform mineralization rate compared with the polluted sediments from the rivers Rhine and Meuse. The data indicate that the reductive dechlorination of aliphatic compounds is not influenced at the polluted sites.     

Rapid atrazine mineralisation in soil slurry and moist soil by inoculation of an atrazine-degrading Pseudomonas sp. strain

The evaluation of pesticide-mineralising microorganisms to clean-up contaminated soils was studied with the widely applied and easily detectable compound atrazine, which is rapidly mineralised by several microorganisms including the Pseudomonas sp. strain Yaya 6. The rate of atrazine removal was proportional to the water content of the soil and the amount of bacteria added to the soil. In soil slurry, 6 mg atrazine kg soil⁻¹ was eliminated within 1 day after application of 0.3 g dry weight inoculant biomass kg soil⁻¹ and within 5 days when 0.003 g kg soil⁻¹ was used. In partially saturated soil (60% of the maximal water-holding capacity) 15 mg atrazine kg soil⁻¹ was eliminated within 2 days by 1 g biomass kg soil⁻¹ and within 25 days when 0.01 g biomass kg soil⁻¹ was used. In unsaturated soil, about 60% [U-ring-¹⁴C]atrazine was converted to ¹⁴CO₂ within 14 days. Atrazine was very efficiently removed by the inoculant biomass, not only in soil that was freshly contaminated but also in soil aged with atrazine for up to 260 days. The bacteria exposed to atrazine in unsaturated sterile soil were still active after a starvation period of 240 days: 15 mg newly added atrazine kg soil⁻¹ was eliminated within 5 days. Received: 31 October 1997 / Received revision: 16 January 1998 / Accepted: 18 January 1998     

Atrazine degradation in saline wastewater by Pseudomonas sp strain ADP

Wastewater from atrazine manufacturing plants contains large amounts of residual atrazine and atrazine synthesis products, which must be removed before disposal. One of the obstacles to biological treatment of these wastewaters is their high salt content, eg, up to 4% NaCl (w/v). To enable biological treatment, bacteria capable of atrazine mineralization must be adapted to high-salinity conditions. A recently isolated atrazine-degrading bacterium, Pseudomonas sp strain ADP, originally isolated from contaminated soils was adapted to biodegradation of atrazine at salt concentrations relevant to atrazine manufacturing wastewater. The adaptation mechanism was based on the ability of the bacterium to produce trehalose as its main osmolyte. Trehalose accumulation was confirmed by natural-abundance ¹H NMR spectral analysis. The bacterium synthesized trehalose de novo in the cells, but could not utilize trehalose added to the growth medium. Interestingly, the bacterium could not produce glycine betaine (a common compatible solute), but addition of 1 mM of glycine betaine to the medium induced salt tolerance. Osmoregulated Pseudomonas sp strain ADP, feeding on citrate decreased the concentration of atrazine in non-sterile authentic wastewater from 25 ppm to below 1 ppm in less than 2 days. The results of our study suggest that salt-adapted Pseudomonas sp strain ADP can be used for atrazine degradation in salt-containing wastewater. Received 26 August 1997/ Accepted in revised form 06 December 1997     

Biodegradation kinetics of monoterpenes in liquid and soil-slurry systems

Batch experiments were conducted to evaluate the biodegradation rates of limonene, α-pinene, γ-terpinene, terpinolene and α-terpineol at 23 °C under aerobic conditions. Biodegradation was demonstrated by the depletion of monoterpene mass, CO₂ production and a corresponding increase in biomass. Monoterpene degradation in liquid cultures devoid of soil followed Monod kinetics. The maximum specific growth rate (μ_max) was 0.02 h⁻¹ and 0.06 h⁻¹ and the half-velocity constant (K _s ) varied from 32 mg/l to 3 mg/l for the limonene and α-terpineol respectively. The recovery of monoterpenes by solvent extraction from autoclaved and azide-amended soil-slurry samples decreased over time and ranged from 69% to 73% for 120 h of incubation period. Although a significant fraction of monoterpene hydrocarbon could not be extracted, mineralization of these compounds in the soil-slurry systems took place, as shown by CO₂ production. The soil-normalized degradation rates for the hydrocarbon monoterpenes ranged from 0.6 μg g⁻¹ h⁻¹ to 2.1 μg g⁻¹ h⁻¹. A kinetic model – which combined monoterpene biodegradation in the liquid phase and net desorption – was developed and applied to data obtained from soil-slurry assays. Received: 10 September 1996 / Received revision: 16 December 1996 / Accepted: 10 January 1997     

Utilization of sorbed compounds by microorganisms specifically isolated for that purpose

A bacterium obtained by enrichment on nonsorbed phenanthrene was unable to degrade phenanthrene sorbed to polyacrylic beads and had little activity on phenanthrene sorbed to lake-bottom sediment. A bacterium obtained by enrichment on phenanthrene sorbed to polyacrylic beads readily mineralized the compound sorbed to the beads or the sediment. Degradation by the second bacterium of phenanthrene sorbed to beads 38–63 μm or 63–150 μm in diameter was more rapid than the rate of desorption of the hydrocarbon in the absence of the bacterium. Little degradation of sorbed, nonleachable phenanthrene in soil was effected by another isolate obtained by enrichment with the nonsorbed hydrocarbon, but a mixed culture and the bacterium obtained by enrichment on the sorbed compound extensively degraded phenanthrene. Because microorganisms specifically obtained for their capacity to degrade sorbed phenanthrene are more active than species not specialized for use of the bound compound, we suggest that microorganisms enriched on nonsorbed compounds may not be appropriate for evaluation of biodegradation and bioremediation of sorbed compounds. Received: 3 June 1997 / Received revision: 2 September 1997 / Accepted: 15 September 1997     

Microbial degradation of tetraethyl lead in soil monitored by microcalorimetry

Sieved agricultural soil samples were treated with the anti-knock agent tetraethyl lead (Et₄Pb), and the resulting effects were analyzed by microcalorimetry. Et₄Pb additions resulted in an increase of the heat production rate, provided that oxygen was present and that the soil was not autoclaved. The increased heat production rate was accompanied by degradation of Et₄Pb, as verified by speciation analysis (GC-MS) of the remaining Et₄Pb and its ionic degradation products (triethyl lead and diethyl lead cations). Conclusive evidence was obtained that these transformations were mediated mainly by microbes. At an initial Et₄Pb concentration of 2 g Pb/kg dry weight the biodegradation rate was about 780 μmol day⁻¹ kg dry weight⁻¹, whilst the chemical decomposition was only 50 μmol day⁻¹ kg dry weight⁻¹. A fivefold rise of the initial Et₄Pb concentration resulted in a decrease of the biodegradation rate to 600 μmol day⁻¹ kg dry weight⁻¹ and an increase of the chemical decomposition to 200 μmol day⁻¹ kg dry weight⁻¹. The biodegradation rate was not influenced by the addition of glucose, which means that no indication for a cometabolic attack of Et₄Pb was found. Received: 25 February 1997 / Received revision: 22 April 1997 / Accepted: 27 April 1997     

Treatment of cyanide-containing wastewater from the food industry in a laboratory-scale fixed-bed methanogenic reactor

During the process of producing cassava starch from Manihot esculenta roots, large amounts of cyanoglycosides were released, which rapidly decayed to CN⁻ following enzymatic hydrolysis. Depending on the varying cyanoglycoside content of the cassava varieties, the cyanide concentration in the wastewater was as high as 200 mg/l. To simulate anaerobic stabilization, a wastewater with a chemical oxygen demand (COD) of about 20 g/l was prepared from cassava roots and was fermented in a fixed-bed methanogenic reactor. The start-up phase for a 99% degradation of low concentrations of cyanide (10 mg/l) required about 6 months. After establishment of the biofilm, a cyanide concentration of up to 150 mg CN⁻/l in the fresh wastewater was degraded during anaerobic treatment at a hydraulic retention time of 3 days. All nitrogen from the degraded cyanide was converted to organic nitrogen by the biomass of the effluent. The cyanide-degrading biocoenosis of the anaerobic reactor could tolerate shock concentrations of cyanide up to 240 mg CN⁻/l for a short time. Up to 5 mmol/l NH₄Cl (i.e. 70 mg N/l = 265 mg NH₄Cl/l) in the fresh wastewater did not affect cyanide degradation. The bleaching agent sulphite, however, had a negative effect on COD and cyanide removal. For anaerobic treatment, the maximum COD space loading was 12 g l⁻¹ day⁻¹, equivalent to a hydraulic retention time of 1.8 days. The COD removal efficiency was around 90%. The maximum permanent cyanide space loading was 50 mg CN⁻ l⁻¹ day⁻¹, with tolerable shock loadings up to 75 mg CN⁻ l⁻¹day⁻¹. Under steady-state conditions, the cyanide concentration of the effluent was lower than 0.5 mg/l. Received: 15 August 1997 / Received revision: 10 October 1997 / Accepted: 14 October 1997     

Methanogenic and perchloroethylene-dechlorinating activity of anaerobic granular sludge

The biodegradation and toxicity of tetrachloroethylene (C₂Cl₄) and trichloroethylene (C₂HCl₃) were studied with different anaerobic enrichment cultures using the following electron donors: acetate, propionate, butyrate, methanol, formate and hydrogen. All of them sustained dechlorination except propionate, for which C₂Cl₄ biodegradation rates were not significant. The best results were obtained with butyrate. Hydrogen appeared to be a relevant electron donor for dechlorination with the present cultures. In the presence of specific inhibitors such as bromoethanesulphonate or molybdate, a slight inhibition of dechlorination was observed. According to dechlorination kinetics, Monod-type behaviour was observed up to 120 μM C₂Cl₄ or 200 μM C₂HCl₃ with K _s values around 7 μM for both compounds. Dechlorination was partially inhibited at higher concentrations. In contrast, methanogens, or at least methane production, were more sensitive to the presence of chlorinated ethylenes and inhibition of methanogenesis was observed to different extents over all the C₂Cl₄/C₂HCl₃ concentration range tested, even at the lowest concentrations. Received: 17 April 1998 / Received revision: 18 June 1998 / Accepted: 19 June 1998     

Removal of tetrachloroethylene in an anaerobic column bioreactor

Removal of tetrachloroethylene (perchloroethylene; C₂Cl₄) by microbial consortia from two sites with different C₂Cl₄ exposure histories was examined in a bench-scale anaerobic column bioreactor. It was hypothesized that optimal removal would be observed in the reactor packed with sediments having an extensive exposure history. Microbial consortia were enriched from hyporheic-zone (HZ) sediments from the Portneuf aquifer near Pocatello, Idaho, and from industrial-zone (IZ) sediments from a highly contaminated aquifer in Portland, Oregon. Lactate and acetate were the electron donors during experiments conducted over 9 and 7 months for HZ and IZ sediments, respectively. In the HZ bioreactor, the retention time ranged from 31 h to 81 h, and inlet C₂Cl₄ concentrations ranged from 0.1 ppm to 1.0 ppm. Dechlorination of C₂Cl₄ averaged 60% and reached a maximum of 78%. An increase in C:N from 27:1 to 500:1 corresponded to an 18% increase in removal efficiency. Trichloroethylene production corresponded to decreased effluent C₂Cl₄; further intermediates were not detected. In the IZ bioreactor, the retention time varied from 34 h to 115 h; the inlet C₂Cl₄ concentration was 1.0 ppm. C₂Cl₄ removal averaged 70% with a maximum of 98%. Trichloroethylene and cis-dichloroethylene were detected in the effluent. Increases in C:N from 50:1 to 250:1 enhanced dechlorination activity. Received: 3 February 1997 / Received revision: 15 May 1997 / Accepted: 1 June 1997     

Hydrogen production with high yield and high evolution rate by self-flocculated cells of Enterobacter aerogenes in a packed-bed reactor

Continuous hydrogen gas evolution by self-flocculated cells of Enterobacter aerogenes, a natural isolate HU-101 and its mutant AY-2, was performed in a packed-bed reactor under glucose-limiting conditions in a minimal medium. The flocs that formed during the continuous culture were retained even when the dilution rate was increased to 0.9 h⁻¹. The H₂ production rate increased linearly with increases in the dilution rate up to 0.67 h⁻¹, giving maximum H₂ production rates of 31 and 58 mmol l⁻¹ h⁻¹ in HU-101 and AY-2 respectively, at a dilution rate of more than 0.67 h⁻¹. The molar H₂ yield from glucose in AY-2 was maintained at about 1.1 at dilution rates between 0.08 h⁻¹ and 0.67 h⁻¹, but it decreased rapidly at dilution rates more than 0.8 h⁻¹. Received: 27 August 1997 / Received revision: 11 November 1997 / Accepted: 14 December 1997     

Screening for fungi intensively mineralizing 2,4,6-trinitrotoluene

Within a screening program, 91 fungal strains belonging to 32 genera of different ecological and taxonomic groups (wood- and litter-decaying basidiomycetes, saprophytic micromycetes) were tested for their ability to metabolize and mineralize 2,4,6-trinitrotoluene (TNT). All these strains metabolized TNT rapidly by forming monoaminodinitrotoluenes (AmDNT). Micromycetes produced higher amounts of AmDNT than did wood- and litter-decaying basidiomycetes. A significant mineralization of [¹⁴C]TNT was only observed for certain wood- and litter-decaying basidiomycetes. The most active strains, Clitocybula dusenii TMb12 and Stropharia rugosa-annulata DSM11372 mineralized 42 % and 36 % respectively of the initial added [¹⁴C]TNT (100 μM corresponding to 4.75 μCi/l) to ¹⁴CO₂ within 64 days. Micromycetes (deuteromycetes, ascomycetes, zygomycetes) proved to be unable to mineralize [¹⁴C]TNT significantly. Received: 8 August 1996 / Received revision: 16 December 1996 / Accepted: 20 December 1996     

Anaerobic biodegradability of alkylphenols and fuel oxygenates in the presence of alternative electron acceptors

Alkylphenols and fuel oxygenates are important environmental pollutants produced by the petrochemical industry. A batch biodegradability test was conducted with selected ortho-substituted alkylphenols (2-cresol, 2,6-dimethylphenol and 2-ethylphenol), fuel oxygenates (methyl tert-butyl ether, ethyl tert-butyl ether and tert-amylmethyl ether) and tert-butyl alcohol (TBA) as model compounds. The ortho-substituted alkylphenols were not biodegraded after 100 days of incubation under methanogenic, sulfate-, or nitrate-reducing conditions. However, biodegradation of 2-cresol and 2-ethylphenol (150 mg l⁻¹) was observed in the presence of Mn (IV) as electron acceptor. The biodegradation of these two compounds took place in less than 15 days and more than 90% removal was observed for both compounds. Mineralization was indicated since no UV-absorbing metabolites accumulated after 23 days of incubation. These alkylphenols were also slowly chemically oxidized by Mn (IV). No biodegradation of fuel oxygenates or TBA (1 g l⁻¹) was observed after 80 or more days of incubation under methanogenic, Fe (III)-, or Mn (IV)-reducing conditions, suggesting that these compounds are recalcitrant under anaerobic conditions. The fuel oxygenates caused no toxicity towards acetoclastic methanogens activity in anaerobic granular sludge. Received: 8 February 2000 / Received revision: 15 May 2000 / Accepted: 19 May 2000     

Mineralization of synthetic humic substances by manganese peroxidase from the white-rot fungus Nematoloma frowardii

A manganese peroxidase preparation from the white-rot fungus Nematoloma frowardii was found to be capable of releasing up to 17% ¹⁴CO₂ from ¹⁴C-labelled synthetic humic substances. The latter were prepared from [U-¹⁴C]catechol by spontaneous oxidative polymerization or laccase-catalysed polymerization. The ex-tent of humic substance mineralization was considerably enhanced in the presence of the thiol mediator glutathione (up to 50%). Besides the evolution of ¹⁴CO₂, the treatment of humic substances with Mn peroxidase resulted in the formation of lower-molecular-mass products. Analysis of residual radioactivity by gel-permeation chromatography demonstrated that the predominant molecular masses of the initial humic substances ranged between 2 kDa and 6 kDa; after treatment with Mn peroxidase, they were reduced to 0.5–2 kDa. The extracellular depolymerization and mineralization of humic substances by the Mn peroxidase system may play an important role in humus turnover of habitats that are rich in basidiomycetous fungi. Received: 25 September 1997 / Received revision: 12 January 1998 / Accepted: 13 January 1998     

Determination of the kinetic parameters during continuous cultivation of the lipase-producing thermophile Bacillus sp. IHI-91 on olive oil

A thermostable lipase was produced in continuous cultivation of a newly isolated thermophilic Bacillus sp. strain IHI-91 growing optimally at 65 °C. Lipase activity decreased with increasing dilution rate while lipase productivity showed a maximum of 340 U l⁻¹ h⁻¹ at a dilution rate of 0.4 h⁻¹. Lipase productivity was increased by 50% compared to data from batch fermentations. Up to 70% of the total lipase activity measured was associated to cells and by-products or residual substrate. Kinetic and stoichiometric parameters for the utilisation of olive oil were determined. The maximal biomass output method led to a saturation constant K _S of 0.88 g/l. Both batch growth data and a washout experiment yielded a maximal specific growth rate, μ_max, of 1.0 h⁻¹. Oxygen uptake rates of up to 2.9 g l⁻¹h⁻¹ were calculated and the yield coefficient, Y _X/O, was determined to be 0.29 g dry cell weight/g O₂. From an overall material balance the yield coefficient, Y _X/S, was estimated to be 0.60 g dry cell weight/g olive oil. Received: 8 January 1997 / Received revision: 30 April 1997 / Accepted: 4 May 1997