Biodegradation of α- and β-endosulfan by soil bacteria

Extensive applications of persistent organochlorine pesticides like endosulfan on cotton have led to the contamination of soil and water environments at several sites in Pakistan. Microbial degradation offers an effective approach to remove such toxicants from the environment. This study reports the isolation of highly efficient endosulfan degrading bacterial strains from soil. A total of 29 bacterial strains were isolated through enrichment technique from 15 specific sites using endosulfan as sole sulfur source. The strains differed substantially in their potential to degrade endosulfan in vitro ranging from 40 to 93% of the spiked amount (100 mg l⁻¹). During the initial 3 days of incubation, there was very little degradation but it got accelerated as the incubation period proceeded. Biodegradation of endosulfan by these bacteria also resulted in substantial decrease in pH of the broth from 8.2 to 3.7 within 14 days of incubation. The utilization of endosulfan was accompanied by increased optical densities (OD₅₉₅) of the broth ranging from 0.511 to 0.890. High performance liquid chromatography analyses revealed that endosulfan diol and endosulfan ether were among the products of endosulfan metabolism by these bacterial strains while endosulfan sulfate, a persistent and toxic metabolite of endosulfan, was not detected in any case. The presence of endosulfan diol and endosulfan ether in the bacterial metabolites was further confirmed by GC-MS. Abiotic degradation contributed up to 21% of the spiked amount. The three bacterial strains, Pseudomonas spinosa, P. aeruginosa, and Burkholderia cepacia, were the most efficient degraders of both α- and β-endosulfan as they consumed more than 90% of the spiked amount (100 mg l⁻¹) in the broth within 14 days of incubation. Maximum biodegradation by these three selected efficient bacterial strains was observed at an initial pH of 8.0 and at an incubation temperature of 30°C. The results of this study may imply that these bacterial strains could be employed for bioremediation of endosulfan polluted soil and water environments.     

Biodegradation of organochlorine pesticide, endosulfan, by a fungal soil isolate, Aspergillus niger

Endosulfan is a chlorinated pesticide widely used in India for the protection of cotton, tea, sugarcane and vegetables. The persistence of endosulfan in environment and toxic effects on biota necessitate its removal. The role of soil fungi in recycling organic matter prompted us to attempt biodegradation of endosulfan using fungi. This study aims at enrichment, isolation and screening of fungi capable of metabolizing endosulfan. In all, 16 fungal isolates were obtained by enrichment of soil samples that had seems exposed to endosulfan before. Isolates were screened by a gradient plate assay, and results were confirmed by broth assay. On the basis of tolerance to endosulfan, an isolate, identified as Aspergillus niger was selected for further studies. The culture could tolerate 400 mg ml⁻¹ of technical grade endosulfan. Complete disappearance of endosulfan was seen on 12 days of incubation. Evolution of carbon dioxide during endosulfan metabolism has indicated the complete mineralization of endosulfan. Change in pH of culture broth to acidic range supported the biological transformation. Thin layer chromography (TLC) analyses revealed the formation of various intermediates of endosulfan metabolism including endosulfan diol, endosulfan sulfate, and an unidentified metabolite. The toxic intermediate, endosulfan sulfate, was also metabolized, further resulting in complete mineralization of endosulfan. Direct desulfurization of endosulfan sulfate or a novel pathway could be the mechanism of endosulfan and endosulfan sulfate degradation in Aspergillus niger. The fungal strain isolated by us could prove valuable for bioremediation of endosulfan contaminated soils and waters.     

Biodegradation of α and β endosulfan in broth medium and soil microcosm by bacterial strain Bordetella sp. B9

Bacterial strains were isolated from endosulfan treated soil to study the microbial degradation of this pesticide in broth medium and soil microcosm. The isolates were grown in minimal medium and screened for endosulfan degradation. The strain, which utilized endosulfan and showed maximum growth, was selected for detail studies. Maximum degrading capability in shake flask culture was shown by Bordetella sp. B9 which degraded 80% of α endosulfan and 86% of β endosulfan in 18 days. Soil microcosm study was also carried out using this strain in six different treatments. Endosulfan ether and endosulfan lactone were the main metabolites in broth culture, while in soil microcosm endosulfan sulfate was also found along with endosulfan ether and endosulfan lactone. This bacterial strain has a potential to be used for bioremediation of the contaminated sites.     

Mycoremediation of Endosulfan and Its Metabolites in Aqueous Medium and Soil by Botryosphaeria laricina JAS6 and Aspergillus tamarii JAS9

Microbial degradation offers an efficient and ecofriendly approach to remove toxicants from the contaminated environments. Botryosphaeria laricina JAS6 and Aspergillus tamarii JAS9 were capable of degrading endosulfan and their metabolites which were isolated through enrichment technique. Both the strains were able to withstand an exposure of 1300 mg/L and showed luxuriant growth at 1000 mg/L of endosulfan. The change in pH in the culture broth was from 6.8 to 3.4 and 3.8 during growth kinetic studies of JAS6 and JAS9 strains, respectively upon biological degradation of endosulfan. The degradation of endosulfan by JAS6 and JAS9 strains were examined by HPLC. The biodegradation rate constant (k) and the initial concentration were reduced by 50% (DT₅₀) which was determined by first and pseudo first order kinetic models. In the present investigation it has been revealed that Botryosphaeria laricina JAS6 and Aspergillus tamarii JAS9 possessing endosulfan degrading capability are being reported for the first time. These findings confirm the degradation of endosulfan by JAS6 and JAS9 strains which were accompanied by significant reduction in the toxicity and could be used as remedial measure in contaminated environments.     

Distribution and characteristics of aflatoxin-producingAspergillus flavus in the soil in Kanagawa Prefecture,Central Japan

In Kanagawa Prefecture, located in central Japan, aflatoxin-producingAspergillus flavus was isolated in 4 (2.5%) of 160 field soil samples. In the 4 fields, whose soil contained aflatoxin-producingA. flavus, the annual average temperature of the sampling sites of the soil ranged from 13.8 to 15.1°C. Of all the isolated strains of aflatoxin-producingA. flavus, 4 strains, isolated from a single soil sample, produced large amounts of aflatoxin B₁ and B₂ when incubated in coconut agar, peanut agar, peanuts or trilaurin-added rice, although they did not produce aflatoxin when incubated in rice, yeast extract-sucrose broth or sucrose-low salts broth.     

Concentration dependent growth/non-growth linked kinetics of endosulfan biodegradation by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

The rates of biodegradation of endosulfan by P. aeruginosa were determined with different initial endosulfan concentrations (10, 50, 100, 150, 200 and 250 mg l⁻¹) and different growth linked kinetic models were fitted at these concentrations. At 10 mg endosulfan l⁻¹, Monod no growth model was well fitted. Monod with growth model described the biodegradation pattern at an initial concentration of 50, 100 and 150 mg endosulfan l⁻¹. Significant increases of P. aeruginosa MN2B14 density in broth culture during incubation further support this result. Conversely, zero order kinetic model was well fitted into the biodegradation data if initial endosulfan concentration was ≥200 mg endosulfan l⁻¹. The kinetics of endosulfan biodegradation by P. aeruginosa MN2B14 in liquid broth was highly dependent upon its initial concentration. The results of this study could be employed for predicting the persistence of endosulfan in water environment containing P. aeruginosa as an endosulfan degrading bacterium.     

Diversity of fungal communities in saline and acidic soils in the Soos National Natural Reserve,Czech Republic

During 2003–2005, the diversity of culturable filamentous soil microfungi in saline and acidic soils of the Soos National Natural Reserve (Czech Republic) was studied. Altogether, 28 soil samples were collected from four sampling sites and were processed by various approaches. In total, 92 fungal taxa were identified using classical and molecular markers. Several detected species were known from similar substrata worldwide; however, the overall fungal spectrum was distinct, as shown by comparison to similar studies. All methodological approaches increased the observed fungal diversity. The different fungal communities observed on the four sampling sites were influenced by the complex effects of environmental factors. The growth response of selected strains to different salinities and pH values was determined. The results of the growth tests showed high adaptability of all tested species to the extreme conditions of the studied substrate. Two acidophilic species (Acidomyces acidophilus, Sporothrix sp.) were isolated.     

Screening for alkaline keratinolytic activity in fungi isolated from soils of the biosphere reserve “Parque Costero del Sur” (Argentina)

A screening was carried out on 69 fungal strains isolated from alkaline-calcareous, neutral and alkaline-sodic soils, as well as from their associated plant material, to determine their ability to grow at alkaline pH. A total of 32 fungi were selected for their ability to produce alkaline keratinase activity in submerged shaken cultures supplemented with soybean meal (SM) and tryptone and on cow hair (CH) under solid state fermentation conditions. Although several fungal strains produced keratinolytic activity on both SM and CH, they differed in the levels detected. Among them, Aspergillus niger, Cladosporium cladosporioides, Metarrhizium anisopliae, Neurospora tetrasperma and Westerdikella dispersa were the best producers, with levels higher than 1.2 U ml⁻¹. Different fungal species are here reported for the first time for their ability to produce keratinolytic activity at alkaline pH.     

Status of airborne spores and pollen in a coir factory in Kerala,India

The prevalence of airborne fungal spores and pollen grains in the indoor and outdoor environments of a coir factory in Thiruvananthapuram district of Kerala state, India was studied using the Burkard Personal Sampler and the Andersen 2-stage Sampler for 2 years (September 1997 to August 1999). The concentration of pollen grains was remarkably lower than that of fungal spores (ratio of 1:28). There was no large difference in the concentrations and types of fungal spores between the indoor and outdoor environments, with 26 spore types found to be present indoors and 27 types outdoors; of these, 22 were common to both the environments. Aspergillus/Penicillium, Cladosporium, ‘other basidiospores’ and ascospores were the dominant spore types. The total spore concentration was highest in February and lowest in September, and it was significantly higher in 1998–1999 than in 1997–1998. Twenty viable colony-forming types were isolated from inside the coir factory. The most dominant viable fungi isolated were Penicillium citrinum, Aspergillus flavus and Aspergillus niger. The total pollen concentration was higher in the outdoor environment of the coir factory than indoors, with 15 and 17 pollen types, respectively. Grass and Cocos nucifera pollen types were dominant. The dominant spore and pollen types trapped in the two environments of the coir factory are reportedly allergenic and, consequently, workers are at risk of catching respiratory/allergic diseases.     

Biocontrol Potential of Siderophore Producing Heavy Metal Resistant <Emphasis Type="Italic">Alcaligenes</Emphasis> sp. and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> RZS3 vis-à-vis Organophosphorus Fungicide

In present study in vitro phytopathogen suppression activity of siderophoregenic preparations of Ni and Mn resistant Alcaligenes sp. STC1 and Pseudomonas aeruginosa RZS3 SH-94B isolated from soil were found superior over the chemical pesticide. Siderophore rich culture broth and siderophore rich supernatant exerted antifungal activity against Aspergillus niger NCIM 1025, Aspergillus flavus NCIM 650, Fusarium oxysporum NCIM 1281, Alternaria alternata ARI 715, Cercospora arachichola, Metarhizium anisopliae NCIM 1311 and Pseudomonas solanacerum NCIM 5103. Siderophore rich broth and supernatant exhibited potent antifungal activity vis-à-vis oraganophosphorus chemical fungicide; kitazine. The minimum fungicidal concentration required was 25 μl for Aspergillus niger, Aspergillus flavus, Fusarium oxysporum, Cercospora arachichola, Metarhizium anisopliae, Pseudomonas solanacerum and 75 μl for A. alternata.     

Biodegradation of endosulfan and endosulfan sulfate by <Emphasis Type="Italic">Achromobacter xylosoxidans</Emphasis> strain C8B in broth medium

Endosulfan is one of the most widely used wide spectrum cyclodiene organochlorine insecticide. In environment, endosulfan can undergo either oxidation or hydrolysis reaction to form endosulfan sulfate and endosulfan diol respectively. Endosulfan sulfate is as toxic and as persistent as its parent isomers. In the present study, endosulfan degrading bacteria were isolated from soil through selective enrichment technique using sulfur free medium with endosulfan as sole sulfur source. Out of the 8 isolated bacterial strains, strain C8B was found to be the most efficient endosulfan degrader, degrading 94.12% α-endosulfan and 84.52% β-endosulfan. The bacterial strain was identified as Achromobacter xylosoxidans strain C8B on the basis of 16S rDNA sequence similarity. Achromobacter xylosoxidans strain C8B was also found to degrade 80.10% endosulfan sulfate using it as sulfur source. No known metabolites were found to be formed in the culture media during the entire course of degradation. Besides, the bacterial strain was found to degrade all the known endosulfan metabolites. There was marked increase in the quantity of released CO₂ from the culture media with endosulfan as sulfur source as compared to MgSO₄ suggesting that the bacterial strain, Achromobacter xylosoxidans strain C8B probably degraded endosulfan completely through the formation of endosulfan ether.     

Microbial transformation of artemisinin to 5-hydroxyartemisinin by Eurotium amstelodami and Aspergillus niger

Transformation of the anti-malarial drug artemisinin by the fungi Eurotium amstelodami and Aspergillus niger were investigated. Cultures were grown in sucrose/malt broth with artemisinin for 14 days and extracted with ethyl acetate. Extracts were characterized by liquid chromatography. Two metabolites from each fungal extract were isolated and identified using mass spectrometry and nuclear magnetic resonance. 5β-hydroxyartemisinin and 7β-hydroxyartemisinin were isolated in 63 and 32% yields, respectively, from the extract of E. amstelodami, and 80 and 19%, respectively, from the extract of A. niger.     

Xylanase production by fungal strains on spent sulphite liquor

Xylanase production by seven fungal strains was investigated using concentrated spent sulphite liquor (SSLc), xylan and d-xylose as carbon substrates. An SSLc-based medium induced xylanase production at varying levels in all of these strains, with Aspergillus oryzae NRRL 3485 and Aspergillus phoenicis ATCC 13157 yielding activities of 164 and 146 U ml⁻¹, respectively; these values were higher than those obtained on xylan or d-xylose with the same fungal strains. The highest xylanase activity of 322 U ml⁻¹ was obtained with Aspergillus foetidus ATCC 14916 on xylan. Electrophoretic and zymogram analysis indicated three xylanases from A. oryzae with molecular weights of approximately 32, 22 and 19 kDa, whereas A. phoenicis produced two xylanases with molecular weights of about 25 and 21 kDa. Crude xylanase preparations from these A. oryzae and A. phoenicis strains exhibited optimal activities at pH 6.5 and 5.0 and at 65 and 55°C, respectively. The A. oryzae xylanolytic activity was stable at 50°C over the pH range 4.5–10. The crude xylanase preparations from these A. oryzae and A. phoenicis strains had negligible cellulase activity, and their application in the biobleaching of hardwood pulp reduced chlorine dioxide consumption by 20–30% without sacrificing brightness.     

Phosphate solubilization by Penicillium spp. isolated from soil samples of Indian Himalayan region

A total of 246 fungal isolates representing 36 genera and 72 species were isolated from the soil samples collected from Indian Himalayan region. Twenty-one species belonged to the genus Penicillium alone. All the Penicillium species were screened for phosphate solubilizing activity on Pikovskaya agar at 21 °C. Eight species of Penicillium, exhibiting formation of halos (zone of solubilization) around the fungal colonies in qualitative plate assays, were selected for quantitative estimations. In quantitative estimations that were conducted upto day 30 (at 3 days interval), seven species of Penicillium brought maximum solubilization after day 15, while P. oxalicum showed maximum solubilization after day 21 of incubation. The increase in solubilization coincided with decrease in pH of the broth. Acid phosphatase activity was 1.5–2.0 times higher in comparison to alkaline phosphatase. Many of these species showed wide range of tolerance for temperature, pH and salt concentration.     

Microbial biodiversity and in situ bioremediation of endosulfan contaminated soil

Molecular characterization based on 16s rDNA gene sequence analysis of bacterial colonies isolated from endosulfan contaminated soil showed the presence of Ochrobacterum sp, Burkholderia sp, Pseudomonas alcaligenes, Pseudomonas sp and Arthrobacter sp which degraded 57–90% of α-endosulfan and 74–94% of β-endosulfan after 7days. Whole cells of Pseudomonas sp and Pseudomonas alcaligenes showed 94 and 89% uptake of α-isomer and 86 and 89% of β-endosulfan respectively in 120 min. In Pseudomonas sp, endosulfan sulfate was the major metabolite detected during the degradation of α-isomer, with minor amount of endosulfan diol while in Pseudomonas alcaligenes endosulfan diol was the only product during α-endosulfan degradation. Whole cells of Pseudomonas sp also utilized 83% of endosulfan sulfate in 120 min. In situ applications of the defined consortium consisting of Pseudomonas alcaligenes and Pseudomonas sp (1:1) in plots contaminated with endosulfan showed that 80% of α-endosulfan and 65% of β-endosulfan was degraded after 12 weeks of incubation. Endosulfan sulfate formed during endosulfan degradation was subsequently degraded to unknown metabolites. ERIC-PCR analysis indicated 80% survival of introduced population of Pseudomonas alcaligenes and Pseudomonas sp in treated plots.     

水稻秸秆还田时间对土壤真菌群落结构的影响

为揭示水稻秸秆还田对土壤真菌群落结构的长期影响,采用荧光定量PCR和PCR-DGGE技术分析了秸秆还田90,180,270 d和360 d的土壤真菌基因丰度和群落结构组成演变趋势,并利用冗余分析(RDA)研究土壤真菌群落结构变化与环境因子的关系。结果表明:随着秸秆还田时间的增加,土壤真菌群体数量和多样性指数(H、R和E)显著增加,在360 d时达到最高。对DGGE图谱的特征条带进行胶回收、测序,系统进化分析表明,土壤真菌主要种群包括:接合菌(Zygomycete sp.)、盐腐霉菌(Pythium salinum)、肉盘菌(Uncultured Sarcosomataceae)、牛粪盘菌(Ascobolus stercorarius)、大链壶菌(Lagenidium giganteum)、青霉菌(Penicillium sp.)、曲霉属真菌(Aspergillus sp.)和疏绵状丝孢菌(Thermomyces lanuginosus)、灰绿曲霉菌(Aspergillus glaucus)、禾谷多粘菌(Polymyxa graminis)和枝顶孢霉菌(Acremonium sp.),其中青霉菌(Penicillium sp.)、曲霉属真菌(Aspergillus sp.)和枝顶孢霉菌(Acremonium sp.)具有纤维素降解能力,而枝顶孢霉菌(Acremonium sp.)在90 d时成为新的优势菌群。RDA分析表明,90 d和180 d秸秆还田与对照土壤的真菌群落结构较为类似,270 d和360 d的秸秆还田与对照土壤的真菌群落结构发生了明显变化。土壤有机碳、pH和速效磷是引起土壤真菌群落结构及多样性变异的主要因素。     

Production of extracellular enzymes by Antarctic fungal strains

 Thirty-three fungal strains, isolated from different sites on Victoria Land (continental Antarctica), were plate-screened for their ability to produce twelve extracellular enzymes. Lipases were generally present and in high quantities in almost all the strains. Polygalacturonase, as well as amylase and phosphatase, was common. Glucose oxidase, protease and DNAase appeared to be generally low or absent. Many strains, producing a limited number of enzymes, appeared to have a low eco-nutritional versatility while a few, such as Verticillium cfr. lecanii no. 1, V. cfr. lecanii no. 3, Aspergillus versicolor and Phoma sp. no. 2, showing a diversified enzymatic competence, are probably advantaged in extreme terrestrial environments characterized by low competition. The possibility of utilizing the enzyme-producing ability of these fungi in applied research is also discussed. Received: 3 November 1995/Accepted: 29 May 1996     

Exposure to indoor fungi in different working environments: A comparative study

In this study an attempt was made to evaluate the qualitative and quantitative fungal burden (load) in five different working environments of South Assam (India) and the possible risks of indoor fungi to employees and stored products. Fungal concentrations in different working environments were studied using a Burkard personal petriplate sampler. The survey was done in five different working environments for one year. A total of 76 fungal types were recorded in the indoor air of South Assam during the survey period. The maximum fungal concentration (5,437.6 ± 145.3 CFU m⁻³ air) was recorded in the indoor air of medical wards, followed by the paper-processing industry (3,871.7 ± 93.4 CFU m⁻³ air). However the lowest concentration was observed in the indoor air of a bakery (1,796.8 ± 54.4 CFU m⁻³ air). The most dominant fungal genera were Aspergillus (34.2%) followed by Penicillium (17.8%), Geotrichum (7.0%) and the most dominant fungal species were Aspergillus fumigatus (2,650.4 CFU m⁻³ air) followed by Aspergillus flavus (1,388.2 CFU m⁻³ air), Geotrichum candidum (1,280.3 CFU m⁻³ air), Aspergillus niger (783.3 CFU m⁻³ air), and Penicillium aurantiovirens (774.0 CFU m⁻³ air). The fungal species viz., Aspergillus fumigatus, Penicillium aurantiovirens, Aspergillus flavus, Aspergillus niger, Geotrichum candidum, and Penicillium thomii, which were recorded well above threshold levels, may lead to adverse health hazards to indoor workers. Setting occupational exposure limits for indoor fungal spores as reference values is obligatory for prevention and control of adverse effects of indoor fungal exposure.     

Zn biosorption by Rhizopus arrhizus and other fungi

Biosorption of zinc ions by inactivated fungal mycelia was studied. Of the six fungal species, Rhizopus arrhizus, Mucor racemosus, Mycotypha africana, Aspergillus nidulans, Aspergillus niger and Schizosaccharomyces pombe, R. arrhizus exhibited the highest capacity (Q ^max = 213 μmol g⁻¹ dry weight). Further experiments with different cellular fractions of R. arrhizus showed that Zn was predominantly bound to cell-wall chitin and chitosan (Q ^max = 312 μmol g⁻¹ dry weight). Adsorption data were best modelled by the Langmuir isotherm, although they can be modelled by the Freundlich equation as well at relatively low aqueous concentrations. Biosorption generally decreased with increase in biosorbent particle size and its concentration. Low pH reduced Zn sorption, because of the strong competition from hydrogen ions for binding sites on fungi. The presence of ligands reduced metal uptake, chiefly by forming metal complexes of a less biosorbable nature. Received: 2 November 1998 / Received revision: 12 January 1999 / Accepted: 17 January 1999     

从黑茶中分离和筛选产单宁酶菌株

茶叶中富含单宁化合物。从分离自黑茶的真菌菌株中,筛选高产单宁酶的菌株;进而分离纯化单宁酶,分析单宁酶对茶汤的转溶效果。从不同产地的3个黑茶样品中,共分离获得44个真菌分离物;经初步鉴定,这些真菌分离物以曲霉属（Aspergillus）、青霉属（Penicillium）和散囊菌属（Eurotium）的真菌居多。以单宁酸为底物的鉴别培养基初筛表明,其中26个真菌分离物在鉴别平板上产生透明圈,显示单宁水解酶活性;通过固体发酵复筛,筛选到1株产单宁酶活性较高的菌株,初步鉴定为青霉属（Penicillium）菌株,命名为青霉MP-24菌株。青霉MP-24可以以茶叶、茶梗和麸皮等农副产品作为原料固体发酵产生单宁酶。以麸皮为原料的发酵产物经过硫酸铵分级沉淀、DEAE阴离子交换层析和葡聚糖G-150凝胶层析等分离纯化步骤,得到分子量为70 kDa的单一蛋白质条带,单宁酶活力达到603.68 U/mg。纯化获得的单宁酶对茶汤有良好的转溶效果。研究结果表明,在黑茶相关微生物中含有丰富的产单宁酶菌株,是工业酶制剂的重要资源。