利用基因工程菌BL21处理有机磷混合农药废水的研究

研究了悬浮状态和固定化状态的基因工程菌BL21对有机磷混合农药废水的降解特性.工程菌能快速、高效地降解有机磷混合农药,其最适底物是对硫磷,而马拉硫磷不能被工程菌降解.不同农药降解速率的差别造成了不同有机磷农药的降解过程需要用不同的动力学模型来描述.比较固定化状态和悬浮状态的工程菌的降解效果可知,固定化工程菌的降解活性较后者明显降低,其比降解速率大约仅为后者的20%.考察固定化工程菌长期运行的效果,发现其降解活性保存良好,工程菌稳定性大大提高,未出现固定化细胞溶涨、破碎现象.固定化后,工程菌的比降解速率虽然比悬浮工程菌降低了,但固定化工程菌更适用于长期运行的废水处理系统.     

Genotoxicity of methyl parathion in short‐term bacterial test systems

Abstract

Genotoxicity of the insecticide methyl parathion was investigated in Salmonella typhimurium and Escherichia coli bacterial test systems for the detection of back mutations and DNA‐damage. Methyl parathion was mutagenic to S. typhimurium strain TA100 after activation with rat liver microsomal and cytosolic enzymes. In DNA repair tests, methyl parathion was effective in inducing damage to the S. typhimurium strain TA1538 which lack excision repair compared to the strain TA1978 which is proficient in excision repair mechanisms. Normal laboratory light conditions had no effect on the mutagenicity tests, however, exposure of methyl parathion in the petri dish containing the tester strain TA100 and rat liver microsomal and cytosolic enzymes reduced the mutagenic activity and increased the toxic effects of methyl parathion.     

Removal of mixed pesticides from drinking water system by photodegradation using suspended and immobilized TiO2

Lindane (1α, 2α, 3β, 4α, 5α, 6β-hexachloro cyclohexane), methyl parathion (O,O-dimethyl-O-4-nitrophenyl phosphorothioate) and dichlorvos (2,2-dichlorovinyl-O-O-dimethyl phosphate) are removed from water individually and as a mixture by photo degradation using suspended and immobilized forms of TiO₂ (Degussa P-25). Studies were conducted to optimize the coating thickness of immobilized photo catalyst. The rate of degradation of pesticides was compared in both suspended and immobilized TiO₂ systems. Degradation studies of mixed pesticides were carried out with low concentrations (1.0 and 2.5 mg/L) of pesticides. Only three intermediate byproducts such as methyl paraoxon, O,O,O-trimethyl phosphonic thionate and p-nitrophenol were observed during the methyl parathion degradation in suspended, immobilized TiO₂ systems and mixed pesticides degradation studies. At the end of the reaction methyl parathion and its by-products were completely degraded. During lindane degradation hexachloro cyclohexane, pentachloro cyclohexane, hexachloro benzene, 1-hydroxy 2,3,4,5,6-chlorocyclohexane, 1-hydroxy 2,3,4,5,6-chlorobenzene, pentachloro cyclopentadiene, 1,2,3,4,5-hydroxy cyclopentene and 1,2,3-hydroxy cyclobutane were identified in suspended and immobilized TiO₂ systems, whereas only hexachloro cyclohexane, pentachloro cyclohexane, hexachloro benzene and pentachloro cyclopentadiene were observed during mixed pesticides degradation. No intermediate by-product was observed during the photo degradation of dichlorvos. Langmuir-Hinshelwood pseudo first order kinetic equation showed that there was not much change in the rates of degradation in both suspended and immobilized TiO₂ systems irrespective of the pesticide. During mixed pesticides degradation, the degradation pattern was not similar to that of single pesticide.     

Biodegradation of mixed pesticides by mixed pesticide enriched cultures

This paper discusses the degradation kinetics of mixed (lindane, methyl parathion and carbofuran) pesticides by mixed pesticide enriched cultures (MEC) under various environmental conditions. The bacterial strains isolated from the mixed microbial consortium were identified as Pseudomonas aeruginosa (MTCC 9236), Bacillus sp. (MTCC 9235) and Chryseobacterium joostei (MTCC 9237). Batch studies were conducted to estimate the biokinetic parameters like the maximum specific growth rate (μ_max), Yield Coefficient (Y_T), half saturation concentration (K_s) and inhibition concentration (Ki) for individual and mixed pesticide enriched cultures. The cultures enriched in a particular pollutant always showed high growth rate and low inhibition in that particular pollutant compared to MEC. After seven weeks of incubation, mixed pesticide enriched cultures were able to degrade 72% lindane, 95% carbofuran and 100% of methyl parathion in facultative co-metabolic conditions. In aerobic systems, degradation efficiencies of lindane methyl parathion and carbofuran were increased by the addition of 2g L^{? 1} of dextrose. Though many metabolic compounds of mixed pesticides were observed at different time intervals, none of the metabolites were persistent. Based on the observed metabolites, a degradation pathway was postulated for different pesticides under various environmental conditions.     

Degradation of microcystins using immobilized microorganism isolated in an eutrophic lake

The final purpose of our series of studies is to establish a biological removal method of cyanobacteria and their toxic products using immobilized microorganisms that can lyse cyanobacteria and decompose microcystins. To establish the biological removal method in non-point areas and water purification plants, as the first step, we explored bacteria active against the cyanobacterial hepatotoxin microcystin in the present study. Eleven active bacteria were isolated from samples taken from Lakes Tsukui and Sagami, Japan. Among 3 strains (B-9 to B-11) with degradative activity, strain B-9 exhibited the strongest activity. The 16S rDNA sequence of the strain B-9 showed the highest similarity to that of Sphingomonas sp. Y2 (AB084247, 99% similarity). Microcystins-RR and -LR were completely degraded by strain B-9 (SC16) within 1d, which led to an immobilized microorganism with a polyester resin. The degradation of microcystin-RR in a bioreactor using the immobilized strain B-9 was observed and microcystin-RR (> 90%) was completely degraded after 24 h. Microcystin-RR was added to the lake water at regular intervals and the degradation after 24 h was observed in the bioreactor over a 72-d period. An over 80% removal efficiency continued for 2 months, showing that the life of the immobilized B-9 in terms of activity was at least 2 months under the optimized conditions. From these results, this immobilized B-9 is feasible for the practical treatment of microcystins in non-point areas and water purification plants.     

Effects of atrazine on acetylcholinesterase activity in midges (Chironomus tentans) exposed to organophosphorus insecticides

Acetylcholinesterase activity was determined for midge larvae (Chironomus tentans) exposed to either organophosphorus insecticides (OPs) alone or OP insecticides in binary combination with atrazine (200 μg/l). Although atrazine by itself did not reduce the level of acetylcholinesterase activity, atrazine in combination with chlorpyrifos significantly decreased acetylcholinesterase activity as compared to chlorpyrifos only treatments. Although similar trends existed for malathion and methyl parathion, differences were not statistically significant. These results match previously published toxicity data where atrazine, although not acutely toxic even at much higher levels, decreased EC₅₀ values for chlorpyrifos by a magnitude of 4, decreased methyl parathion values by a magnitude of 2, and did not decrease values for malathion.     

Genotoxicity of methyl parathion in short-term bacterial test systems

Genotoxicity of the insecticide methyl parathion was investigated in Salmonella typhimurium and Escherichia coli bacterial test systems for the detection of back mutations and DNA-damage. Methyl parathion was mutagenic to S. typhimurium strain TA100 after activation with rat liver microsomal and cytosolic enzymes. In DNA repair tests, methyl parathion was effective in inducing damage to the S. typhimurium strain TA1538 which lack excision repair compared to the strain TA1978 which is proficient in excision repair mechanisms. Normal laboratory light conditions had no effect on the mutagenicity tests, however, exposure of methyl parathion in the petri dish containing the tester strain TA100 and rat liver microsomal and cytosolic enzymes reduced the mutagenic activity and increased the toxic effects of methyl parathion.     

Isolation of a selected microbial consortium capable of degrading methyl parathion and p-nitrophenol from a contaminated soil site

A bacterial consortium with the ability to degrade methyl parathion and p-nitrophenol, using these compounds as the only carbon source, was obtained by selective enrichment in a medium with methyl parathion. Samples were taken from Moravia, Medellin; an area that is highly contaminated, owing to the fact that it was used as a garbage dump from 1974 to 1982. Acinetobacter sp, Pseudomonas putida, Bacillus sp, Pseudomonas aeruginosa Citrobacter freundii, Stenotrophomonas sp, Flavobacterium sp, Proteus vulgaris, Pseudomonas sp, Acinetobacter sp, Klebsiella sp and Proteus sp were the microorganisms identified within the consortium. In culture, the consortium was able to degrade 150 mg L?1 of methyl-parathion and p-nitrophenol in 120 h, but after adding glucose or peptone to the culture, the time of degradation decreased to 24 h. In soil, the consortium was also able to degrade 150 mg L?1 of methyl parathion in 120 h at different depths and also managed to decrease the toxicity.     

IMPACT OF FOUR PESTICIDES ON THE GROWTH AND METABOLIC ACTIVITIES OF TWO PHOTOSYNTHETIC ALGAE

The acute toxicity was determined for soil algae Chlorella kesslerei and Anabaena inaequalis, exposed to pesticides lindane, pentachlorophenol (PCP), isoproturon (IPU), and methyl parathion (MP). Toxicity markers included growth inhibition, chlorophyll biosynthesis, and total carbohydrate content, as a function of dose and time. Concentration response functions (EC₅₀) were estimated by probit data transformation and weighted linear regression analyses. Lindane's toxicity to Chlorella increased sharply with time (EC₅₀=7490, 10.3, 0.09 mg L^?1; 24, 48, 72 h), but remained nearly constant through 72 h with Anabaena (8.7?6.7 mg L^?1; 24–72 h). PCP at low concentrations stimulated algal growth and chlorophyll a production, an effect reversed at higher doses. Anabaena was less tolerant of PCP and MP than was Chlorella. The 96-h static EC₅₀ values for Chlorella were: 0.003, 34, 0.05, and 291 mg L^?1 for lindane, PCP, isoproturon, and MP, respectively; for Anabaena, these were 4.2, 0.13, 0.21, and 19 mg L^?1. Carbohydrate production responses were similar to those of cell density (growth) and chlorophyll biosynthesis, with MP having the lowest adverse impact. The overall relative toxicity among the four tested pesticides was: for Chlorella, lindane>IPU?PCP?MP; and for Anabaena, PCP>IPU>lindane>MP. The results confirm that toxicants such as these pesticides may affect individual (though related) species to significantly different degrees.     

The metabolic fate of 14C‐parathion by some fresh water phytoplankton and its possible effects on the algal metabolism

Abstract

The growth and total carbohydrate contents of Nostoc muscorum and Tolypothrix tenuis were greatly and significantly reduced by the application of parathion. “Chlorophyll a”, carotene biosynthesis and the rate of glucose absorption were enhanced after supplementation of parathion to the culture media of both cyanobacteria. Nitrogen released to the media, total nitrogen content and total nitrogen fixed were increased in both organisms‐ Increase in protein content was accompanied by remarkable drop in amino, peptide and ammonia fractions‐ Phosphorus uptake, RNA, DNA and total phosphorus content were accelerated to reach maximum accumulation at the highest insecticide level. In metabolism study using ¹⁴C‐labelled compound, parathion was readily degraded by Nostoc and Tolypothrix. Following ten days incubation, the aqueous fractions contained 21.1% and 18.1% of the initial activity in Nostoc and Tolypothrix respectively. TLC analysis of the hydrolytic products revealed the presence of three metabolites: p‐aminophenol, p‐nitrophenol and aminoparathion.     

油污土中降解柴油细菌的分离鉴定及降解能力研究

从所采集柴油污染土壤样品中富集、分离得到柴油降解优势菌株,命名为B-3和B-4.根据其生理生化性质及16S rDNA序列比对分析,确定2株菌分别属于Tetrathiobacter kashmirensis、假单胞菌属(Pseudomonas sp.).由于实验中,B-3的生长曲线较特殊,故以B-3和典型石油烃降解菌假单...     

The degradation of parathion and DDT in aqueous systems containing organic additives

Abstract

Laboratory studies were conducted to investigate some of the factors influencing pesticide degradation in‐ aqueous systems. Parathion added to natural water in ethanol, acetone or without organic solvent, was completely degraded within 2 wk. While most of the parathion was reduced to amino‐parathion when added in ethanol, no amino‐parathion was detected in the presence of acetone or when no solvent was added, suggesting that in the latter two cases the insecticide was aerobically degraded to other metabolites. No paraoxon was detected. When ethanol concentration was increased from 1% to 2 and 4%, the rate of parathion degradation was inversely related to the ethanol concentration. In the presence of glucose as a carbon source, approximately 50% of the parathion was reduced to aminoparathion. DDT degradation in natural water was more rapid when it was added in ethanol than when added in acetone. The only DDT metabolite detected was TDE, with about 36% conversion in presence of ethanol, and 20% when the DDT was added in acetone.     

The rate of soil reduction as affected by levels of methyl parathion and 2, 4‐D

Abstract

A simple technique was demonstrated for determining the potential for synthetic organics to stress microbial populations. Oxidized Crowley and Cecil soil materials were amended with varying concentrations of 2,4‐D and methyl parathion, flooded, and then analyzed for changes in pH, redox potential, and levels of soluble plus exchangeable Fe, Mn, and Zn, all of which may be directly or indirectly influenced by the activity of soil microorganisms. At the concentrations tested (up to 75 ppm), there was little effect of 2,4‐D, but methyl parathion apparently did affect microbial activity contributing to changes in the measured soil properties upon flooding. This approach may be a useful technique for screening various compounds for their potential to stress microbial activity that, for many researchers, would be easier than direct observations of microbial parameters such as population numbers and classifications, and enzyme levels.     

A method for the analysis of organophosphorus pesticide residues in Mexican axolotl

A method based on matrix solid-phase dispersion (MSPD) was developed for quantitative extraction of three organophosphorus pesticides (OPPs) from the Mexican axolotl, Ambystoma mexicanum. The determination was carried out using high- performance liquid chromatography (HPLC) with diode array spectrophotometric UV detection (DAD). The MSPD extraction with octadecylsilyl (C18) sorbent combined with a silica gel clean-up and acetonitrile elution was optimised for chlorpyrifos, fenthion and methyl parathion. The method was validated, yielding recovery values higher than 90%. The precision, expressed as the relative standard deviation (RSD), was less than or equal to 6% in muscle samples at spiking levels of 10 and 5 ppm. Linearity was studied from 15 to 60 ppm for chlorpyrifos and fenthion, and from 7.5 to 30 ppm for methyl parathion. The limits of detection (LODs) were found to be less than or equal to 0.5 ppm_.This method was applied to the analysis of samples from a chlorpyrifos-exposed axolotl, demonstrating its use as an analytical tool for toxicological studies.     

Photochemical nitro-nitrite rearrangement in methyl parathion decay under tropical conditions

This work presents a study of the abiotic degradation of commercially available methyl parathion in aqueous solution at two different concentrations (88 mg/L and 200 μg/L). The effects of solar irradiation and the presence of humic acids were evaluated and revealed a synergistic response between them. The half-life of methyl parathion ranged from 4.9 to 37 days, and the experimental data also show that photochemical processes were the most relevant in this case. The only byproduct found in samples submitted to shadowed conditions was 4-nitrophenol. On the other hand, 4-nitrophenol, methyl paraoxon and a new degradation product (O,O-dimethyl O-p-hydroxyphenyl phosphorothioate) were detected when the samples were exposed directly to sunlight. This newly identified compound was prepared in the laboratory by thiophosphorylation of hydroquinone, and coelution experiments with authentic samples provided unambiguous confirmation of the presence of O,O-dimethyl O-p-hydroxy phenylphosphorothioate in samples.     

Biodegradation of malathion, α- and β-endosulfan by bacterial strains isolated from agricultural soil in Veracruz,Mexico

The objective of this study was to evaluate the capacity of two bacterial strains isolated, cultivated, and purified from agricultural soils of Veracruz, Mexico, for biodegradation and mineralisation of malathion (diethyl 2-(dimethoxyphosphorothioyl) succinate) and α- and β-endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6-9-methano-2,4,3-benzodioxathiepine-3-oxide). The isolated bacterial strains were identified using biochemical and morphological characterization and the analysis of their 16S rDNA gene, as Enterobacter cloacae strain PMM16 (E1) and E. amnigenus strain XGL214 (M1). The E1 strain was able to degrade endosulfan, whereas the M1 strain was capable of degrading both pesticides. The E1 strain degraded 71.32% of α-endosulfan and 100% of β-endosulfan within 24 days. The absence of metabolites, such as endosulfan sulfate, endosulfan lactone, or endosulfan diol, would suggest degradation of endosulfan isomers through non-oxidative pathways. Malathion was completely eliminated by the M1 strain. The major metabolite was butanedioic acid. There was a time-dependent increase in bacterial biomass, typical of bacterial growth, correlated with the decrease in pesticide concentration. The CO₂ production also increased significantly with the addition of pesticides to the bacterial growth media, demonstrating that, under aerobic conditions, the bacteria utilized endosulfan and malathion as a carbon source. Here, two bacterial strains are shown to metabolize two toxic pesticides into non-toxic intermediates.     

不适宜生长条件对混菌降解苯胺的影响

分别从台州和衢州某化工厂的好氧池中分离筛选得到2株苯胺降解菌TZ1和JH1,经16S rDNA测序鉴定为Comamonas sp.TZ1和Pseudomonas sp.JH1,均具有较强的苯胺降解能力,培养24 h后,可使初始浓度为800 mg/L的苯胺去除率达到96.4%~98.4%。在此基础上,按体积比1∶1将2株菌液进行混合构建了混合菌体系,进而对比考察了苯胺初始浓度、pH、盐度和重金属等环境因子对单一菌和混合菌生长量及降解苯胺效果的影响,重点探讨混合菌对不适宜生长环境的适应性及其对苯胺的降解特性。通过单一菌和混合菌对比实验发现,在适宜苯胺初始浓度、pH和盐度条件下,混合菌的生长量略高于单一菌;在不适宜生长的高浓度苯胺、pH和盐度条件下,混合菌也表现出了更强的适应性和苯胺矿化能力。Zn2+和Cr6+耐受实验则表明,对于Cr6+,混合菌表现出了更强的耐受能力,而对于Zn2+并没有表现出更强的耐受能力。     

Enhanced degradation of 2-nitrotoluene by immobilized cells of Micrococcus sp. strain SMN-1

Nitrotoluenes are the toxic pollutants of the environment because of their large scale use in the production of explosives. Biodegradation of such chemicals by microorganisms may provide an effective method for their detoxification. We have studied the degradation of 2-nitrotoluene by cells of Micrococcus sp. strain SMN-1 immobilized in various matrices such as polyurethane foam (PUF), sodium alginate (SA), sodium alginate–polyvinyl alcohol (SA–PVA), agar and polyacrylamide. The rate of degradation of 15 and 30 mM 2-nitrotoluene by freely suspended cells and immobilized cells in batches and fed-batch with shaken cultures were compared. The PUF-immobilized cells achieved higher degradation of 15 and 30 mM 2-nitrotoluene than freely suspended cells and the cells immobilized in SA–PVA, polyacrylamide, SA and agar. The PUF-immobilized cells could be reused more than 24 cycles without loosing their degradation capacity and showed more tolerance to pH and temperature changes than freely suspended cells. These results revealed the enhanced rate of degradation of 2-nitrotoluene by PUF-immobilized cells of Micrococcus sp. strain SMN-1.     

The influence of cosolvent and heat on the solubility and reactivity of organophosphorous pesticide DNAPL alkaline hydrolysis

The presented research concerned the compatibility of cosolvents with in situ alkaline hydrolysis (ISAH) for treatment of organophosphorous (OPP) pesticide contaminated sites. In addition, the influence of moderate temperature heat increments was studied as a possible enhancement method. A complex dense non-aqueous phase liquid (DNAPL) of primarily parathion (~50 %) and methyl parathion (~15 %) obtained from the Danish Groyne 42 site was used as a contaminant source, and ethanol and propan-2-ol (0, 25, and 50 v/v%) was used as cosolvents in tap water and 0.34 M NaOH. Both cosolvents showed OPP solubility enhancement at 50 v/v% cosolvent content, with slightly higher OPP concentrations reached with propan-2-ol. Data on hydrolysis products did not show a clear trend with respect to alkaline hydrolysis reactivity in the presence of cosolvents. Results indicated that the hydrolysis rate of methyl-parathion (MP3) decreased with addition of cosolvent, whereas the hydrolysis rate of ethyl-parathion (EP3) remained constant, and overall indications were that the hydrolysis reactions were limited by the rate of hydrolysis rather than NAPL dissolution. In addition to cosolvents, the influence of low-temperature heating on ISAH was studied. Increasing reaction temperature from 10 to 30 °C provided an average rate of hydrolysis enhancement by a factor of 1.4–4.8 dependent on the base of calculation. When combining 50 v/v% cosolvent addition and heating to 30 °C, EP3 solubility was significantly enhanced and results for O,O-diethyl-thiophosphoric acid (EP2 acid) showed a significant enhancement of hydrolysis as well. However, this could not be supported by para-nitrophenol (PNP) data indicating the instability of this product in the presence of cosolvent.

     

Effects of the herbicide molinate on the metabolic activities of a degradative Streptomyces griseus strain

Abstract

Cometabolic degradation of the herbicide molinate was tested using two microorganisms, Arthrobacter sp., strain M3 and Streptomyces griseus strain M2; the latter classified on the basis of the presence of the enzymatic cofactor SF‐420. The strains M3 and M2, inoculated in a basic salts medium with glucose as carbon source and added with 100 mg L^‐1 of molinate, degraded respectively 35 and 51% of the herbicide in 36 days.

Increasing concentrations of molinate, ranging from 50 to 200 mg L^‐1 in glucose medium, did not affect the final ATP yield of the strain M2, but decreased the final growth yield and the ATP synthesis rate. Moreover, the onset of coenzyme SF‐420 synthesis was progressively delayed.

In contrast, surprisingly, SF‐420 final yield and production rate were increased by progressive increasing concentrations of molinate in the mineral medium.