Isolation and characterization of an abamectin-degrading <Emphasis Type="Italic">Burkholderia cepacia</Emphasis>-like GB-01 strain

Abamectin is widely used in agriculture as an insecticide and in veterinary as an anti-parasitic agent, and has caused great environmental pollution by posing potential risk to non-target soil invertebrates and nearby aquatic systems. A bacterium designated GB-01, which was capable of degrading abamectin, was isolated from soil by enrichment culture method. On the basis of morphological, physiological and biochemical characteristics, combined with phylogenetic analysis of 16S rRNA gene, the bacterium GB-01 was identified as Burkholderia cepacia-like species. The bacterium GB-01 was able to utilize abamectin as its sole carbon source for growth, and could degrade more than 90% of abamectin at initial concentrations of 50 and 100 mg l⁻¹ in mineral salt medium in 30 and 36 h, respectively. The longer degradation cycle was observed with abamectin concentrations higher than 100 mg l⁻¹. Optimal growth temperatures and pH values with highest degradation rate were 30–35°C and 7–8, respectively. Two new degradation products were identified and characterized by high performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) based mass spectral data and a plausible partial degradation pathway of abamectin was proposed. This is the first report in which an abamectin-degrading Burkholderia species isolated from soil was identified and characterized.     

Biodegradation of fenvalerate and 3-phenoxybenzoic acid by a novel Stenotrophomonas sp. strain ZS-S-01 and its use in bioremediation of contaminated soils

A bacterial strain ZS-S-01, newly isolated from activated sludge, could effectively degrade fenvalerate and its hydrolysis product 3-phenoxybenzoic acid (3-PBA). Based on the morphology, physiological biochemical characteristics, and 16 S rDNA sequence, strain ZS-S-01 was identified as Stenotrophomonas sp. Strain ZS-S-01 could also degrade and utilize deltamethrin, beta-cypermethrin, beta-cyfluthrin, and cyhalothrin as substrates for growth. Strain ZS-S-01 was capable of degrading fenvalerate rapidly without a lag phase over a wide range of pH and temperature, even in the presence of other carbon sources, and metabolized it to yield 3-PBA, then completely degraded it. No persistent accumulative product was detected by HPLC and GC/MS analysis. Studies on biodegradation in various soils showed that strain ZS-S-01 demonstrated efficient degradation of fenvalerate and 3-PBA (both 50 mg·kg⁻¹) with a rate constant of 0.1418–0.3073 d⁻¹, and half-lives ranged from 2.3 to 4.9 days. Compared with the controls, the half-lives for fenvalerate and 3-PBA reduced by 16.9–156.3 days. These results highlight strain ZS-S-01 may have potential for use in bioremediation of pyrethroid-contaminated environment.     

A microcosm study on bioremediation of fenitrothion-contaminated soil using Burkholderia sp. FDS-1

Fenitrothion, a toxic organophosphorus pesticide, can build up the concentration of nitrophenolic compound in soils and hence needs to be removed. Burkholderia sp. FDS-1, a fenitrothion-degrading strain, was used in this work to study factors affecting its growth, and then evaluated for its capacity to degrade fenitrothion in soil microcosms. Minimal salt medium containing 1% (w/v) glucose was found to be a suitable carbon source for inoculum preparation. Various factors, including soil pH, temperature, initial fenitrothion concentration, and inoculum size influenced the degradation of fenitrothion. Microcosm studies performed with varying concentrations (1–200 mg kg⁻¹) of fenitrothion-spiked soils showed that strain FDS-1 could effectively degrade fenitrothion in the range of 1–50 mg kg⁻¹ soil. The addition of Burkholderia sp. FDS-1 at 2×10⁶ colony forming units g⁻¹ soil was found to be suitable for fenitrothion degradation over a temperature range of 20–40 °C and at a slight alkaline pH (7.5). The results indicate that strain FDS-1 has potential for use in bioremediation of fenitrothion and its metabolite-contaminated sites. This is a model study that could be used for decontamination of sites contaminated with other compounds.     

Bioremediation of β-cypermethrin and 3-phenoxybenzaldehyde contaminated soils using Streptomyces aureus HP-S-01

Using laboratory and field experiments, the ability of Streptomyces aureus HP-S-01 to eliminate β-cypermethrin (β-CP) and its metabolite 3-phenoxybenzaldehyde (3-PBA) in soils was investigated. In the laboratory, 80.5% and 73.1% of the initial dose of β-CP and 3-PBA (50 mg kg⁻¹) was removed in sterilized soils within 10 days, respectively, while in the same period, disappearance rate of β-CP and 3-PBA in non-sterilized soils was higher and reached 87.8% and 79.3%, respectively. Furthermore, the disappearance process followed the first-order kinetics and the half-life (T _1/2) for β-CP and 3-PBA reduced by 20.3–52.9 and 133.7–186.8 days, respectively, as compared to the controls. The addition of sucrose to the soils enhanced the ability of strain HP-S-01 to eliminate β-CP and 3-PBA. Similar results were observed in the field experiments. The introduced strain HP-S-01 quickly adapted to the environment and rapidly removed β-CP and 3-PBA without any lag phases in the field experiments. Compared with the controls, 47.9% and 67.0% of applied dose of β-CP and 3-PBA was removed from the soils without extra carbon sources and 52.5% and 73.3% of β-CP and 3-PBA was eliminated in soils supplemented with sucrose within 10 days, respectively. Analysis of β-CP degradation products in soil indicated that the tested strain transform β-CP to 3-PBA and α-hydroxy-3-phenoxy-benzeneacetonitrile. However, both intermediates were transient and they disappeared after 10 days. Therefore, the selected actinomyces strain HP-S-01 is suitable for the efficient and rapid bioremediation of β-CP contaminated soils.     

Adaptation of a <Emphasis Type="Italic">Mycobacterium</Emphasis> strain to phenanthrene degradation in a biphasic culture system: influence on interfacial area and droplet size

A phenanthrene-degrading Mycobacterium sp. strain 6PY1 was grown in an aqueous/organic biphasic culture system with phenanthrene as sole carbon source. Its capacity of degradation was studied during sequential inoculum enrichments, reaching complete phenanthrene degradation at a maximim rate of 7 mg l⁻¹ h⁻¹. Water–oil emulsions and biofilm formation were observed in biphasic cultures after four successive enrichments. The factors influencing interfacial area in the emulsions were: the initial phenanthrene concentration, the initial inoculum size, and the silicone oil volume fraction. The results showed that the interfacial area was mainly dependent on the silicone oil/mineral salts medium ratio and the inoculum size.     

Bioremediation of Fungicides by Spent Mushroom Substrate and Its Associated Microflora

Experiments were conducted both under in vitro and in situ conditions to determine the biodegradation potential of button mushroom spent substrate (SMS) and its dominating microbes (fungi and bacteria) for carbendazim and mancozeb, the commonly used agricultural fungicides. During 6 days of incubation at 30 ± 2°C under broth culture conditions, highest degradation of carbendazim (17.45%) was recorded with B-1 bacterial isolate, while highest degradation of mancozeb (18.05%) was recorded with Trichoderma sp. In fungicide pre-mixed sterilized SMS, highest degradation of carbendazim (100.00–66.50 μg g⁻¹) was recorded with mixed inoculum of Trichoderma sp. and Aspergillus sp., whereas highest degradation of mancozeb (100.00–50.50 μg g⁻¹) was with mixed inoculum of Trichoderma sp., Aspergillus sp. and B–I bacterial isolate in 15 days of incubation at 30 ± 2°C. All these microbes both individually as well as in different combinations grew well and produced extracellular lignolytic enzymes on SMS, which helped in fungicides degradation. Under in situ conditions, among three different proportions of SMS (10, 20 and 30%, w/w) mixed with fungicide pre-mixed soil (100 μg g⁻¹ of soil), the degradation of carbendazim was highest in 30% SMS treatment, while for mancozeb it was in 20% SMS treatment. The residue levels of both fungicides decreased to half of their initial concentration after 1 month of SMS mixing.     

Rapid biodegradation of metsulfuron-methyl by a soil fungus in pure cultures and soil

Summary Four strains of bacteria, 9 strains of fungi and 20 strains of actinomycetes capable of utilizing metsulfuron-methyl as sole carbon and energy source were isolated from a metsulfuron-methyl-treated soil by the enrichment culture method. A fungus named DS11F was selected as the most highly effective one according to the maximum tolerance concentration of 1,200 mg l⁻¹ and metsulfuron-methyl-degrading rate of 0.0716 g g⁻¹ cells h⁻¹, and was identified as an unknown strain of Penicillium sp. on the basis of colony growth, morphology and biochemical characteristics.␣Through liquid pure culture, the optimal metsulfuron-methyl-degrading conditions of DS11F were determined to be metsulfuron-methyl concentration 22.6 mg l⁻¹, inoculum concentration 12.25 mg l⁻¹, pH 7.0 and temperature 30°C. As additional C sources, supernatant of soaked compost could increase metsulfuron-methyl degradation by 8%, but glucose was ineffective. DS11F inoculation was found to significantly enhance the degradation of metsulfuron-methyl in soil, with the reduction of the concentration reaching 50% in 6 days. Admixture of compost could promote metsulfuron-methyl degradation to some extent. The growth of the inocula in the soils remained dominant and degradation resumed immediately when metsulfuron-methyl was applied again. The results show that addition of the isolated Penicillium sp. enhances the degradation of metsulfuron-methyl in water and soil.     

Effect of nitrate and glucose addition on denitrification and nitric oxide reductase (<Emphasis Type="Italic">cnorB</Emphasis>) gene abundance and mRNA levels in <Emphasis Type="Italic">Pseudomonas mandelii</Emphasis> inoculated into anoxic soil

The effect of glucose addition (0 and 500 μg C g⁻¹ soil) and nitrate (NO₃) addition (0, 10, 50 and 500 μg NO₃–N g⁻¹ soil) on nitric oxide reductase (cnorB) gene abundance and mRNA levels, and cumulative denitrification were quantified over 48 h in anoxic soils inoculated with Pseudomonas mandelii. Addition of glucose-C significantly increased cnorB _p (P. mandelii and related species) mRNA levels and abundance compared with soil with no glucose added, averaged over time and NO₃ addition treatments. Without glucose addition, cnorB _p mRNA levels were higher when 500 μg NO₃–N g⁻¹ soil was added compared with other NO₃ additions. In treatments with glucose added, addition of 50 μg NO₃–N g⁻¹ soil resulted in higher cnorB _p mRNA levels than soil without NO₃ but was not different from the 10 and 500 μg NO₃–N g⁻¹ treatments. cnorB _p abundance in soils without glucose addition was significantly higher in soils with 500 μg NO₃–N g⁻¹ soil compared to lower N-treated soils. Conversely, addition of 500 μg NO₃–N g⁻¹ soil resulted in lower cnorB _p abundance compared with soil without N-addition. Over 48 h, cumulative denitrification in soils with 500 μg glucose-C g⁻¹ soil, and 50 or 500 μg NO₃–N g⁻¹ was higher than all other treatments. There was a positive correlation between cnorB _p abundance and cumulative denitrification, but only in soils without glucose addition. Glucose-treated soils generally had higher cnorB _p abundance and mRNA levels than soils without glucose added, however response of cnorB _p abundance and mRNA levels to NO₃ supply depended on carbon availability.     

Use of sugarcane molasses “B” as an alternative for ethanol production with wild-type yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> ITV-01 at high sugar concentrations

Molasses “B” is a rich co-product of the sugarcane process. It is obtained from the second step of crystallization and is richer in fermentable sugars (50–65%) than the final molasses, with a lower non-sugar solid content (18–33%); this co-product also contains good vitamin and mineral levels. The use of molasses “B” for ethanol production could be a good option for the sugarcane industry when cane sugar prices diminish in the market. In a complex medium like molasses, osmotolerance is a desirable characteristic for ethanol producing strains. The aim of this work was to evaluate the use of molasses “B” for ethanol production using Saccharomyces cerevisiae ITV-01 (a wild-type yeast isolated from sugarcane molasses) using different initial sugar concentrations (70–291 g L⁻¹), two inoculum sizes and the addition of nutrients such as yeast extract, urea, and ammonium sulphate to the culture medium. The results obtained showed that the strain was able to grow at 291 g L⁻¹ total sugars in molasses “B” medium; the addition of nutrients to the culture medium did not produce a statistically significant difference. This yeast exhibits high osmotolerance in this medium, producing high ethanol yields (0.41 g g⁻¹). The best conditions for ethanol production were 220 g L⁻¹ initial total sugars in molasses “B” medium, pH 5.5, using an inoculum size of 6 × 10⁶ cell mL⁻¹; ethanol production was 85 g L⁻¹, productivity 3.8 g L⁻¹ h⁻¹ with 90% preserved cell viability.     

The reserve of weatherable primary silicates impacts the accumulation of biogenic silicon in volcanic ash soils

Banana plantlets (Musa acuminata cv Grande Naine) cultivated in hydroponics take up silicon proportionally to the concentration of Si in the nutrient solution (0–1.66 mM Si). Here we study the Si status of banana plantlets grown under controlled greenhouse conditions on five soils developed from andesitic volcanic ash, but differing in weathering stage. The mineralogical composition of soils was inferred from X-ray diffraction, elemental analysis and selective chemical/mineralogical extractions. With increasing weathering, the content of weatherable primary minerals decreased. Conversely, clay content increased and stable secondary minerals were increasingly dominant: gibbsite, Fe oxides, allophane, halloysite and kaolinite. The contents of biogenic Si in plant and soil were governed by the reserve of weatherable primary minerals. The largest concentrations of biogenic Si in plant (6.9–7 g kg⁻¹) and soil (50–58 g kg⁻¹) occurred in the least weathered soils, where total Si content was above 225 g kg⁻¹. The lowest contents of biogenic Si in plant (2.8–4.3 g kg⁻¹) and soil (8–31 g kg⁻¹) occurred in the most weathered desilicated soils enriched with secondary oxides and clay minerals. Our data imply that soil weathering stage directly impacted the soil-to-plant transfer of silicon, and thereby the stock of biogenic Si in a soil–plant system involving a Si-accumulating plant. They further imply that soil type can influence the silicon soil–plant cycle and its hydrological output.     

137Cs and 40K isotopes in forest and wasteland soils in a selected region of eastern Poland 20 years after the Chernobyl accident

The vertical ¹³⁷Cs profile of forest and wasteland soils was analyzed in the south of the Podlasie Lowland area (Eastern Poland) about 20 years after the Chernobyl accident. In addition, the concentration of ⁴⁰K in soils of the investigated area was measured. Below the litter layer (mean thickness 3 cm), the soil samples were collected up to a depth of 12 cm and then divided into three layers: 0–3, 3–7, 7–12 cm. The behavior of ¹³⁷Cs and ⁴⁰K isotopes in soils was analyzed depending on the depth from which the soil samples were collected, as well as on the content of organic carbon, pH of soil and its granulometric composition. It was established that the density of ¹³⁷Cs in the litter layer equals 2.17 kBq m⁻²; it is the highest in layer 0–3 cm where it equals 3.44 kBq m⁻², and it decreases with the depth to the value of 0.76 kBq m⁻² in layer 7–12 cm. No similar pattern was observed in wasteland soils. The concentrations of ⁴⁰K in forest and wasteland soils did not change significantly with depth.     

Characterization of a strain of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> isolated from agricultural soil that degrades cadusafos (an organophosphorus pesticide)

Bacteria capable of degrading the pesticide, cadusafos, were isolated from agricultural soil using an enrichment method. In this way, five distinct cadusafos-degrading strains of Pseudomonas putidia were isolated, and were characterized using morphological and biochemical analysis, as well as 16S rRNA sequencing. Strain PC1 exhibited the greatest cadusafos degradation rate and was consequently selected for further investigation. Degradation of cadusafos by strain PC1 was rapid at 20 and 37°C, but was greatly reduced (~1.5-fold) by the presence of carbon sources. Strain PC1 was able to effectively degrade cadusafos in sterilized soil using low inoculum levels. The maximum degradation rate of cadusafos (V _max ) was calculated as 1.1 mg l⁻¹ day⁻¹, and its saturation constant (K _s ) was determined as 2.5 mg l⁻¹. Bacteria such as strain PC1, that use cadusafos as a carbon source, could be employed for the bioremediation of sites contaminated with pesticides.     

Evaluation of haloalkaliphilic sulfur-oxidizing microorganisms with potential application in the effluent treatment of the petroleum industry

Haloalkaliphilic sulfur-oxidizing mixed cultures for the treatment of alkaline–saline effluents containing sulfide were characterized and evaluated. The mixed cultures (IMP-PB, IMP-XO and IMP-TL) were obtained from Mexican alkaline soils collected in Puebla (PB), Xochimilco (XO) and Tlahuac (TL), respectively. The Ribosomal Intergenic Spacer Analysis (RISA) revealed bacteria related to Thioalkalibacterium and Thioalkalivibrio in IMP-XO and IMP-PB mixed cultures. Halomonas strains were detected in IMP-XO and IMP-TL. In addition, an uncultured Bacteroides bacterium was present in IMP-TL. Mixed cultures were evaluated at different pH and NaCl concentrations at 30°C. IMP-PB and IMP-TL expressed thiosulfate-oxidizing activity in the 7.5–10.5 pH range, whereas IMP-XO presented its maximal activity with 19.0 mg O₂ g_protein⁻¹ min⁻¹, at pH 10.6; it was not affected by NaCl concentrations up to 1.7 M. In continuous culture, IMP-XO showed a growth rate of 15 day⁻¹, productivity of 433.4 mg_protein l⁻¹ day⁻¹ and haloalkaliphilic sulfur-oxidizing activity was also detected up to 170 mM by means of N-methyl-diethanolamine (MDEA). Saline–alkaline soil samples are potential sources of haloalkaliphilic sulfur-oxidizing bacteria and the mixed cultures could be applied in the treatment of inorganic sulfur compounds in petroleum industry effluents under alkaline–saline conditions.     

Isolation of Fenitrothion-degrading Strain Burkholderia sp. FDS-1 and Cloning of mpd Gene

A short rod shaped, gram-negative bacterium strain Burkholderia sp. FDS-1 was isolated from the sludge of the wastewater treating system of an organophosphorus pesticides manufacturer. The isolate was capable of using fenitrothion as the sole carbon source for its growth. FDS-1 first hydrolyzed fenitrothion to 3-methyl-4-nitrophenol, which was further metabolized to nitrite and methylhydroquinone. The addition of other carbon source and omitting phosphorus source had little effect on the hydrolysis of fenitrothion. The gene encoding the organophosphorus hydrolytic enzyme was cloned and sequenced. The sequence was similar to mpd, a gene previously shown to encode a parathion-methyl-hydrolyzing enzyme in Plesiomonas sp. M6. The inoculation of strain FDS-1 (10⁶ cells g⁻¹) to soil treated with 100 mg fenitrothion emulsion kg⁻¹ resulted in a higher degradation rate than in noninoculated soils regardless of the soil sterilized or nonsterilized. These results highlight the potential of this bacterium to be used in the cleanup of contaminated pesticide waste in the environment. Zhonghui Zhang, Qing Hong: Both authors contributed equally to this work     

Bioremediation of polychlorinated biphenyl-contaminated soil using carvone and surfactant-grown bacteria

Partial bioremediation of polychlorinated biphenyl (PCB)-contaminated soil was achieved by repeated applications of PCB-degrading bacteria and a surfactant applied 34 times over an 18-week period. Two bacterial species, Arthrobacter sp. strain B1B and Ralstonia eutrophus H850, were induced for PCB degradation by carvone and salicylic acid, respectively, and were complementary for the removal of different PCB congeners. A variety of application strategies was examined utilizing a surfactant, sorbitan trioleate, which served both as a carbon substrate for the inoculum and as a detergent for the mobilization of PCBs. In soil containing 100 μg Aroclor 1242 g⁻¹ soil, bioaugmentation resulted in 55–59% PCB removal after 34 applications. However, most PCB removal occurred within the first 9 weeks. In contrast, repeated addition of surfactant and carvone to non-inoculated soil resulted in 30–36% PCB removal by the indigenous soil bacteria. The results suggest that bioaugmentation with surfactant-grown, carvone-induced, PCB-degrading bacteria may provide an effective treatment for partial decontamination of PCB-contaminated soils. Received: 9 March 2000 / Received revision: 27 June 2000 / Accepted: 16 July 2000     

Insecticide-tolerant and plant growth promoting <Emphasis Type="Italic">Bradyrhizobium</Emphasis> sp. (<Emphasis Type="Italic">vigna</Emphasis>) improves the growth and yield of greengram [<Emphasis Type="Italic">Vigna radiata</Emphasis> (L.) Wilczek] in insecticide-stressed soils

This study was designed to identify rhizobial strains specific to greengram expressing higher tolerance against insecticides, fipronil and pyriproxyfen, and synthesizing plant growth regulators even amid insecticide-stress. Of the 50 bradyrhizobial isolates, the Bradyrhizobium sp. strain MRM6 showed tolerance up to 1,600 μg mL⁻¹ against each of fipronil and pyriproxyfen. The tolerant Bradyrhizobium sp. (vigna) produced plant growth promoting substances in substantial amounts, both in the presence and absence of insecticides. The strain MRM6 was further used to investigate its impact on greengram grown in soils treated with 200 (the recommended dose), 400 and 600 μg kg⁻¹ soil of fipronil and 1,300 (the recommended dose), 2,600 and 3,900 μg kg⁻¹ soil of pyriproxyfen. Fipronil at 600 μg kg⁻¹ soils and pyriproxyfen at 3,900 μg kg⁻¹ soils had greatest toxic effects and decreased plant biomass, symbiotic efficiency, nutrient uptake and seed yield of greengram plants. The Bradyrhizobium sp. (vigna) inoculant when used with fipronil and pyriproxyfen significantly increased the measured parameters compared to the plants grown in soils treated solely with the same concentration of each insecticide. This study inferred that the Bradyrhizobium sp. (vigna) strain MRM6 may be exploited as bio-inoculant to increase the productivity of greengram exposed to insecticide-stressed soils.     

Bioremediation of atrazine-contaminated soil by repeated applications of atrazine-degrading bacteria

Bioaugmentation has previously been unreliable for the in situ clean-up of contaminated soils because of problems with poor survival and the rapid decline in activity of the bacterial inoculum. In an attempt to solve these problems, a 500-l batch fermenter was investigated for its ability to deliver inoculum repeatedly to contaminated soils via irrigation lines. In a field experiment, mesocosms were filled with 350 kg soil containing 100 mg kg⁻¹ atrazine, and inoculated one, four or eight times with an atrazine-degrading bacterial consortium that was produced in the fermenter. After 12 weeks, no significant degradation of atrazine had occurred in soil that was inoculated only once; whereas, mesocosms inoculated four and eight times mineralized 38% and 72% of the atrazine respectively. Similar results were obtained in a laboratory experiment using soil contaminated with 100 mg kg⁻¹ [¹⁴C]atrazine. After 35 days, soil that was inoculated once with 10⁸ cfu ml⁻¹ of the consortium or with the atrazine-degrading bacterium, Pseudomonas sp. strain ADP, mineralized 17% and 35% of the atrazine respectively. In comparison, microcosms inoculated every 3 days with the consortium or with Pseudomonas sp. (ADP) mineralized 64% or 90% of the atrazine over this same period. Results of these experiments suggest that repeated inoculation from an automated fermenter may provide a strategy for bioaugmentation of contaminated soil with xenobiotic-degrading bacteria. Received: 20 November 1998 / Received revision: 8 February 1999 / Accepted: 12 February 1999     

Studies on non-symbiotic diazotrophic bacterial populations of coastal arable saline soils of India

The effect of fluctuations of salinity in three different seasons on diazotrophic populations and N₂ fixation in six mono cropped rice field soils of the coastal region of the Gangetic delta of West Bengal, India, was studied. The average pH, ECe, organic carbon and total nitrogen of the soils ranged from 4.99–7.08, 2.02–19.58 dSm⁻¹, 4.68–12.03 g kg⁻¹ and 0.44–1.70 g kg ⁻¹, respectively. The average log colony forming units of the bacterial populations and N₂-fixation in the soils varied from 4.61 to 5.86 and 2.74 to 4.52 mg N₂ fixed 50 ml ⁻¹ culture media respectively, with the lowest value recorded in summer. Recovery of microorganisms and N₂- fixation gradually decreased with extraneous addition of NaCl in the culture media. All the eight isolates were Gram positive, spore and capsule formers. They could utilize glucose, sucrose, mannitol, starch, citrate and nitrate, and were catalase and gelatinase positive, but indole, methyl red and Vogues Proskauer reaction negative. The organisms produced alkaline reaction on TSI agar slant. The acetylene reduction assay of the isolates at 0 and 1% NaCl in the culture media were 4.51–164.52 and 1.72–100.6 nmole C₂H₄ ml⁻¹ culture media in 72 h, respectively. The isolates could fix 2.42–4.45 and 2.04–4.08 mg N₂ fixed 50 ml⁻¹ culture media at 0 and 1% NaCl in the culture media respectively. 16S rDNA sequences of the isolates were similar to the species: Bacillus sp. isolate 28A, Bacillus sp. MOLA 87, Bacillus sp. By113 (B)Ydz-dh, Bacillus sp. PN13, Bacillus licheniformis strain RH101, Bacterium Antarctica 14, Bacillus sp. PN13 and Bacillus megaterium.     

Degradation of chloroform by immobilized cells of <Emphasis Type="Italic">Bacillus</Emphasis> sp. in calcium alginate beads

A Bacillus sp., capable of degrading chloroform, was immobilized in calcium alginate. The beads in 20 g alginate l⁻¹ (about 2 × 10⁸ cells/bead) could be re-used nine times for degradation of chloroform at 40 μM. The immobilized cells had a higher range of tolerance (pH 6.5–9 and 20–41°C) than free cells (pH 7–8.5 and 28–32°C). At 5 g alginate l⁻¹, leakage of the cells from the beads was 0.51 mg dry wt ml⁻¹. This species is the first reported Bacillus that can degrade chloroform as the sole carbon source.     

Hydrolytic Dechlorination of Chlorothalonil by Ochrobactrum sp. CTN-11 Isolated from a Chlorothalonil-Contaminated Soil

A bacterial strain, designated as CTN-11, capable of degrading chlorothalonil (CTN), was isolated from a chlorothalonil-contaminated soil in China. Based on the morphological, biochemical characteristics and comparative analysis of the 16S rRNA genes, strain CTN-11 was identified as Ochrobactrum sp. Strain CTN-11 could degrade 50 mg l⁻¹ CTN to a non-detectable level within 48 h, and efficiently degrade CTN in a relatively broad range of temperatures from 20 to 40°C and initial pH values from 6.0 to 9.0. The new isolate differed from those previously reported CTN co-metabolic degraders by transforming CTN in the absence of other carbon sources. A glutathione S-transferase (GST) coding gene, which showed 88% sequence similarity with that from Ochrobactrum anthropi SH35B, was cloned from strain CTN-11. However, the gene was not functionally expressed in the presence of glutathione, indicating that CTN was not reductively dechlorinated by thiolytic substitution catalyzed by GST in strain CTN-11. The metabolite hydroxyl-trichloroisophthalonitrile (CTN-OH) produced during CTN anaerobic degradation was identified based on tandem MS/MS, confirming that hydrolytic dechlorination was involved in the CTN degradation. The removal of CTN by strain CTN-11 in sterile and non-sterile soils was also studied. In both soil samples, 50 mg kg⁻¹ CTN could be degraded to an undetectable level within 3 days. This study highlights an important potential use of strain CTN-11 for the cleanup of CTN-contaminated sites and presents a hydrolytic dechlorination reaction of CTN by a pure culture.