2,4-D impact on bacterial communities, and the activity and genetic potential of 2,4-D degrading communities in soil

The key role of telluric microorganisms in pesticide degradation is well recognized but the possible relationships between the biodiversity of soil microbial communities and their functions still remain poorly documented. If microorganisms influence the fate of pesticides, pesticide application may reciprocally affect soil microorganisms. The objective of our work was to estimate the impact of 2,4-D application on the genetic structure of bacterial communities and the 2,4-D-degrading genetic potential in relation to 2,4-D mineralization. Experiments combined isotope measurements with molecular analyses. The impact of 2,4-D on soil bacterial populations was followed with ribosomal intergenic spacer analysis. The 2,4-D degrading genetic potential was estimated by real-time PCR targeted on tfdA sequences coding an enzyme specifically involved in 2,4-D mineralization. The genetic structure of bacterial communities was significantly modified in response to 2,4-D application, but only during the intense phase of 2,4-D biodegradation. This effect disappeared 7 days after the treatment. The 2,4-D degrading genetic potential increased rapidly following 2,4-D application. There was a concomitant increase between the tfdA copy number and the 14C microbial biomass. The maximum of tfdA sequences corresponded to the maximum rate of 2,4-D mineralization. In this soil, 2,4-D degrading microbial communities seem preferentially to use the tfd pathway to degrade 2,4-D.     

Glutathione S-transferase-encoding gene as a potential probe for environmental bacterial isolates capable of degrading polycyclic aromatic hydrocarbons.

Homologs of the glutathione S-transferase (GST)-encoding gene were identified in a collection of aromatic hydrocarbon-degrading Sphingomonas spp. isolated from New Zealand, Antarctica, and the United States by using PCR primers designed from the GST-encoding gene of Sphingomonas paucimobilis EPA505. Sequence analysis of PCR fragments generated from these isolates and of the GST gene amplified from DNA extracted from polycyclic aromatic hydrocarbon (PAH)-contaminated soil revealed a high degree of conservation, which may make the GST-encoding gene a potentially useful marker for PAH-degrading bacteria.     

Cultivation and characterization of bacterial isolates capable of degrading pharmaceutical and personal care products for improved removal in activated sludge wastewater treatment

Pharmaceutical and personal care products (PPCPs) discharged with wastewater treatment plant (WWTP) effluents are an emerging surface water quality concern. Biological transformation has been identified as an important removal mechanism during wastewater treatment. The aim of this research was the identification of bacteria with characteristics for potential bioaugmentation to enhance PPCP removal. We report here the cultivation and characterization of bacteria capable of degrading PPCPs to ng/L concentrations. An isolation approach was developed using serial enrichment in mineral medium containing 1 mg/L of an individual PPCP as the sole organic carbon source available to heterotrophs until the original activated sludge inocula was diluted to ~10^?8 of its initial concentration, followed by colony growth on solid R2A agar. Eleven bacteria were isolated, eight that could remove triclosan, bisphenol A, ibuprofen, or 17β-estradiol to below 10 ng/L, one that could remove gemfibrozil to below 60 ng/L, and two that could remove triclosan or E2, but not to ng/L concentrations. Most bacterial isolates degraded contaminants during early growth when grown utilizing rich carbon sources and were only able to degrade the PPCPs on which they were isolated. Seven of the bacterial isolates were sphingomonads, including all the triclosan and bisphenol A degraders and the ibuprofen degrader. The study results indicate that the isolated bacteria may have a positive influence on removal in WWTPs if present at sufficient concentrations and may be useful for bioaugmentation.     

Identification of Rhodococcuserythropolis isolates capable of degrading the fungicide carbendazim

Continuous enrichment cultures were used to identify bacterial isolates capable of degrading the fungicide carbendazim. The bacteria originated from sites that had been repeatedly treated with the structurally related parent fungicide, benomyl, over a period of several years. Six isolates were identified as carbendazim degraders on the basis of their ability to produce diffusion-clearing zones on a minimal salts solid medium spray-coated with a 0.1% solution of carbendazim, their ability to grow in a minimal salts broth supplemented with carbendazim as the sole carbon source, and their ability to reduce carbendazim levels in liquid cultures. All six isolates were identified as Rhodococcus erythropolis or a closely related species by analyses of nutritional utilization and whole-cell fatty acid methyl ester profiles. A chemically induced mutant of R. erythropolis isolate B2E was identified that was no longer capable of degrading carbendazim, as determined by negative results in all three assays. Further characterization of these strains provides an opportunity for their development in bioremediation of the compound. Received: 22 July 1996 / Received revision: 16 December 1996 / Accepted: 10 January 1997     

苯酚降解菌的分离及培养特性研究

对南充市郊炼油厂活性污泥进行富集,驯化筛选得到2株能以苯酚作为唯一碳源和能源生长的菌株,编号为S1,S2,两菌株可耐10,000mg/L左右的苯酚浓度,实验得出其最佳生长条件为pH7-8,温度25℃-30℃,在适宜条件下,对苯酚有较好的降解能力,而且苯对两菌株的生长表现为抑制作用。     

Comamonas acidovorans strain MC1: a new isolate capable of degrading the chiral herbicides dichlorprop and mecoprop and the herbicides 2,4-D and MCPA

A gram-negative prototrophic bacterial species, strain MC1, was isolated from the vicinity of herbicide-contaminated building rubble and identified by 16S rDNA sequence analysis, its physiological properties, GC content, and fatty acid composition as Comamonas acidovorans. This strain displays activity for the productive degradation of the two enantiomers of dichlorprop [(RS)-2-(2,4-dichlorophenoxy-)propionate; (RS)-2,4-DP] and mecoprop [(RS)-2-(4-chloro-2-methyl-) phenoxypropionate; (RS)-MCPP] in addition phenoxyacetate herbicides, i.e. 2,4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA), and various chlorophenols were utilized. Rates amounted to 1.2 mmoles/h g dry mass (2,4-D) and 2.7 mmoles/h g dry mass [(RS)-2,4-DP]. Degradation of (RS)-2,4-DP was not inhibited up to concentrations of 500 mg/l, nor of 2,4-D up to 200 mg/l. The optimum pH value of (RS)-2,4-DP degradation was around 8. The application of respective primers for PCR amplification revealed the presence of tfdB and tfdC genes.     

Identification and molecular characterization of a novel Bacillus strain capable of degrading Tween-80

A Tween-80-degrading novel marine Bacillus strain, N10, has recently been isolated in Alexandria University, Egypt. The taxonomic position of this endospore forming bacterium was investigated on the basis of fatty acid analysis and 16S rRNA gene sequencing. Comparative computer database analyses revealed that the bacterium is a Bacillus subtilis strain. The gene encoding the small acid-soluble protein gamma-type (SASP-B), sspE, was successfully utilized in this study as a tool for discrimination between the two B. subtilis subspecies W23 and 168. Based on the alignment of 16S rRNA sequences and analysis of SASP-B relatedness, it has been demonstrated that the novel marine B. subtilis strain N10 is more closely related to the B. subtilis reference strain W23 than to 168. The strain, N10, has been deposited in the Bacillus Genetic Stock Center (BGSC) and assigned the accession number 3A17.     

石油降解菌剂的研制及其在石油污染土壤修复中的应用

用液态和固态相结合的方式对包含2种细菌的石油降解菌剂进行培养,牛肉膏、蛋白胨作为液态培养基培齐初级种子,然后接种到草炭和麸皮的固态培养基中培养：分析温度、接种量、料水比、草炭与麸皮的比例、培养时间对固态培养的影响.制备的BC—E和BC-12种菌剂的活菌量分别达到2．47×10^11个／g和3．6×10^10个／g。采用研制的菌剂对石油污染土壤进行修复实验,1个月污染土壤中的石油降解率可达到45％。     

Isolation and characterization of Pseudomonas sp. strain capable of degrading diethylstilbestrol

Since diethylstilbestrol (DES) interrupts endocrine systems and generates reproductive abnormalities in both wildlife and human beings, methods to remove DES from the environments are urgently recommended. In this study, bacterial strain J51 was isolated and tested to effectively degrade DES. J51 was identified as Pseudomonas sp. based on its nucleotide sequence of 16S rRNA. The quinoprotein alcohol dehydrogenase and isocitrate lyase were identified to be involved in DES degradation by MALDI–TOF–TOF MS/MS analysis. In the presence of 40 mg/l DES, increase of the genes encoding quinoprotein alcohol dehydrogenase and isocitrate lyase in both RNA and protein levels was determined. The HPLC/MS analysis showed that DES was hydrolyzed to a major degrading metabolite DES-4-semiquinone. It was the first time to demonstrate the characteristics of DES degradation by specific bacterial strain and the higher degradation efficiency indicated the potential application of Pseudomonas sp. strain J51 in the treatment of DES-contaminated freshwater and seawater environments.     

Isolation and characterization of thermophilic bacteria capable of degrading dehydroabietic acid.

Using a semi-continuous enrichment method, we isolated two thermophilic bacterial strains, which could completely degrade abietane resin acids, including dehydroabietic acid (DhA). Strain DhA-73, isolated from a laboratory-scale bioreactor treating bleached kraft mill effluent at 55 degrees C, grew on DhA as sole carbon source; while DhA-71, isolated from municipal compost, required dilute tryptic soy broth for growth on DhA. DhA-71 grew on DhA from 30 degrees C to 60 degrees C with maximum growth at 50 degrees C; while, DhA-73 grew on DhA from 37 degrees C to 60 degrees C with maximum growth at 55 degrees C. At 55 degrees C, the doubling times for DhA-71 and DhA-73 were 3.3 and 3.7 h, respectively. DhA-71 and DhA-73 had growth yields of 0.26 and 0.19 g of protein per g of DhA, respectively. During growth on DhA, both strains converted DhA to CO2, biomass, and dissolved organic carbon. Analyses of the 16S-rDNA sequences of these two strains suggest that they belong to two new genera in the Rubrivivax subgroup of the beta subclass of the Proteobacteria. Strains DhA-71 and DhA-73 are the first two bacteria isolated and characterized that are capable of biodegradation of resin acids at high temperatures. This study provided direct evidence for biodegradation of resin acids and feasibility for biotreatment of pulp mill effluent at elevated temperatures.     

Mineral and carbon usage of two synthetic pyrethroid degrading bacterial isolates

AIMS: To investigate the biodegrading ability and cometabolism of synthetic pyrethroid (SP) utilizing bacteria in cultures with various minerals and carbon sources. METHODS AND RESULTS: Previously isolated SP-degrading Pseudomonas sp. and Serratia sp. were used in cultures containing either flumethrin SP or cypermethrin SP formulations. The culture media consisted of either (i) water only, (ii) water and sucrose, (iii) mineral broth or (iv) mineral broth and sucrose. The growth of both organisms was greatest in the mineral broth and sucrose medium, but the growth-limiting factor for Pseudomonas sp. strain Circle was the mineral content whereas for Serratia sp. strain White it was the carbon substrate. CONCLUSION: The greatest extent of degradation of both SP-based compounds occurred with Pseudomonas sp. strain Circle but was dependant on the medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This investigation could lead to the development of a relatively inexpensive medium supplement to enhance the microbial biodegradation of undesirable compounds, either in situ or ex situ. In this particular case, for the biodegradation of SPs used in sheep dip.     

Colonisation of seedling roots by 2,4-D degrading bacteria: A plant-microbial model

The development of model plant-microbial associations between Gram negative soil microbes capable of degrading phenoxyacetate herbicides, such as 2,4-D and 2,4-D methyl ester, and the crops canola and wheat was described. Both an Acinetobacter baumannii pJP4 transconjugant and Alcaligenes eutrophus JMP 134 colonised non-parasitically on the roots of sterilised seedlings in a hydroponic system. Laser scanning confocal microscopy has shown that colonisation occurred both on the root surface and deeper inside the mucilage layer or inside some surface root cells. When 2,4-D was added to the hydroponic medium supporting the growth of those seedlings colonised by 2,4-D degrading bacteria, the gas chromatographic analysis showed a rapid decrease in the concentration of this herbicide. These bacteria colonising the root system were shown to be responsible for the degradation of 2,4-D. Plants inoculated with the 2,4-D degrading microbes were subsequently found to be less susceptible to damage by the herbicide in such hydroponic systems.     

一株能高效降解几种有机磷农药的菌株JS018的鉴定

从福建三明农药厂附近的土壤分离、筛选获得一株能够高效降解甲基1605、辛硫磷等有机磷农药的菌株JS018,在LB培养基中发酵36h,对甲基1605、辛硫磷、三唑磷、敌敌畏的降解率分别为96%、99%、98.9%和69.0%。该菌在LB平板培养基上形成的菌落为粉红色,圆形,有光泽;经电镜观察,为小球状菌,直径0.5μm~0.75μm;革兰氏染色为阴性;能够在30℃~38℃温度范围内和pH7.0~9.0范围内很好的生长,最适生长温度为32℃,最适pH7.5~8.0;在含有6%NaCl以上的培养基中,不能生长。抗生素敏感性实验表明:JS018菌对安比西林、青霉素、林肯霉素有抗性;对卡那霉素、四环素、庆大霉素等敏感。碳源发酵实验表明:该菌株能发酵葡萄糖、海藻糖、松三糖、乙醇;不能发酵阿拉伯糖、蔗糖、甘露糖、木糖、果糖、半乳糖、麦芽糖、乳糖等;不能利用棕檬酸盐,不能液化明胶,不产生硫化氢,能还原硝酸盐,不产生吲哚,接触酶阳性,脲酶阳性。根据其形态特征,生理生化特性1、6S rDNA序列分析,初步鉴定JS018为Roseomonas(玟瑰单胞菌属)。     

Characterization of a microbial consortium capable of degrading lignocellulose

A microbial consortium, designated WCS-6, was established by successive subcultivation in the presence of rice straw under static conditions. The degradation efficiencies of WSC-6 for 0.5 g filter paper, cotton and rice straw after 3 days of cultivation were 99.0±0.7%, 76.9±1.5% and 81.3±0.8%, respectively as determined by gravimetrical methods. Nine bacterial isolates were obtained from WCS-6 plated under aerobic conditions, and sequencing of their 16S rDNA indicated that these bacteria were related to Bacillus thermoamylovorans BTa, Paenibacillus barengoltzii SAFN-016, Proteobacterium S072, Pseudoxanthomonas taiwanensis CB-226, Rhizobiaceae str. M100, Bacillus sp. E53-10, Beta proteobacterium HMD444, Petrobacter succinimandens 4BON, and Tepidiphilus margaritifer N2-214. DGGE (denaturing gradient gel electrophoresis) and sequencing of 16S rDNA sequences amplified from total consortium DNA revealed the presence of sequences related to those of Ureibacillus thermosphaericus, uncultured bacterium clone GC3, uncultured Clostridium sp. clone A1-3, Clostridium thermobutyricum, and Clostridium thermosuccinogenes in addition to the sequences identified from the cultured bacteria. The microbial community identified herein is a potential candidate consortium for the degradation of waste lignocellulosic biomass.     

一株十二烷基苯磺酸钠降解菌的分离鉴定及降解特性研究

从受污染河水中筛选分离得到一株能以十二烷基苯磺酸钠(Sodiumdodecylbenzenesulfonate,SDBS)为唯一碳源和能源生长的菌株WZR-A。经对其形态特征、生理生化、以及16SrDNA序列分析,将WZR-A初步鉴定为人苍白杆菌(Ochrobactrumanthropi)。研究表明该菌株利用SDBS生长的最适温度为30℃,最适pH为7·0。SDBS浓度低于400mg/L时菌株对SDBS的降解率可在80%以上。菌株细胞蛋白SDS-PAGE结果显示,菌株在SDBS诱导前后的细胞蛋白组成有明显差异。酶的定域试验表明,该菌株的相关降解酶为胞内酶。相关降解酶活性及降解底物测试结果表明,该菌株可能通过邻位途径裂解芳环并具有对多种芳香族化合物降解的能力。此外,利用质粒检测和消除试验发现菌株WZR-A中含有大质粒且该菌株的相关降解基因初步定位于该质粒。     

Purification and characterization of an intracellular esterase from a Fusarium species capable of degrading dimethyl terephthalate

Esterase is the key enzyme involved in microbial degradation of phthalate esters (PAEs). In this study, an intracellular esterase was purified from a coastal sediment fungus Fusarium sp. DMT-5-3 capable of utilizing dimethyl terephthalate (DMT) as a substrate. The purified enzyme is a polymeric protein consisting of two identical subunits with a molecular mass of about 84 kDa. The enzyme showed a maximum esterase activity at 50 °C and was stable below 30 °C. The optimal pH was 8.0 and the enzyme was stable between pH 6.0 and 10.0. The esterase activity was inhibited by Cr³⁺, Hg²⁺, Cu²⁺, Zn²⁺, Ni²⁺, and Cd²⁺. Substrate specificity analysis showed that the enzyme was specific to DMT hydrolysis, but had no effect on other isomers of dimethyl phthalate esters (DMPEs) or monomethyl phthalate esters (MMPEs). These findings suggest that the phthalate esterase produced by Fusarium sp. DMT-5-3 is inducible and distinctive esterases involved in hydrolysis of the two carboxylic ester linkages of DMPEs.     

Isolation and characterization of Bradyrhizobium sp. 224 capable of degrading sulfanilic acid

A bacterial strain (strain 224), which has the ability to utilize sulfanilic acid as a sole source of carbon, was isolated from soil. 16S rRNA gene sequence obtained from strain 224 exhibited 100% identical to that of species in the genus Bradyrhizobium. Strain 224 degraded 4.7 mM of sulfanilic acid and released almost the same molar concentration of sulfate ion     

Physiological and cellular responses of the 2,4-D degrading bacterium,Burkholderia cepacia YK-2, to the phenoxyherbicides 2,4-D and 2,4,5-T

Our previous research has demonstrated that novel 43-kDa DnaK and 41-kDa GroEL proteins are synthesized in Burkholderia sp. YK-2 in response to sublethal concentrations of 2,4-D stress [Cho et al. (2000) Curr Microbiol 41:33-38]. In this study, we have extended this work to examine the cellular responses of strain YK-2 to stresses induced in response to the phenoxyherbicides 2,4-D or 2,4,5-T. Strain YK-2 exhibited a more sensitive response to 2,4,5-T stress than to 2,4-D stress, as shown in physiological and morphological changes, suggesting a greater cytotoxic effect of 2,4,5-T. SEM analyses revealed the presence of perforations and irregular rod forms with wrinkled surfaces for cells treated with either herbicide. These irregularities were found more frequently for 2,4,5-T-treated cells than for 2,4-D-treated cells. Analysis of cellular fatty acids showed similar effects in the shifts of total cellular fatty acid composition in response to 2,4-D and 2,4,5-T. Strain YK-2 could degrade 2.25 m M 2,4-D completely during 28 h of incubation with transient production of 2,4-dichlorophenol as a metabolite; however, 2,4,5-T was not catabolized at any of the concentrations tested. BIOLOG and 16S rDNA analyses revealed that strain YK-2 was 98% similar to the Burkholderia cepacia species cluster; therefore, we have designated this strain as B. cepacia YK-2.     

Selection and characterization of aerobic bacteria capable of degrading commercial mixtures of low-ethoxylated nonylphenols

Aims:  Isolation and characterization of new bacterial strains capable of degrading nonylphenol ethoxylates (NP n EO) with a low ethoxylation degree, which are particularly recalcitrant to biodegradation.
Methods and Results:  Seven aerobic bacterial strains were isolated from activated sludges derived from an Italian plant receiving NP n EO-contaminated wastewaters after enrichment with a low-ethoxylated NP n EO mixture. On the basis of 16S rDNA sequence, the strains were positioned into five genera: Ochrobactrum , Castellaniella , Variovorax , Pseudomonas and Psychrobacter . Their degradation capabilities have been evaluated on two commercial mixtures, i.e. Igepal CO-210 and Igepal CO-520, the former rich in low ethoxylated congeners and the latter containing a broader spectrum of NP n EO, and on 4- n -nonylphenol (NP). The strains degraded Igepal CO-210, Igepal CO-520 and 4- n -NP all applied at the initial concentration of 100 mg l⁻¹, by 35–75%, 35–90% and 15–25%, respectively, after 25 days of incubation.
Conclusions:  Some of the isolated strains, in particular the Pseudomonas strains BCb12/1 and BCb12/3, showed interesting degradation capabilities towards low ethoxylated NP n EO congeners maintaining high cell vitality.
Significance and Impact of the Study:  Increased knowledge of bacteria involved in NP n EO degradation and the possibility of using the isolated strains in tailored process for a tertiary biological treatment of effluents of wastewater treatment plants.     

Isolation and characterization of an anaerobic ruminal bacterium capable of degrading hydrolyzable tannins.

An anaerobic diplococcoid bacterium able to degrade hydrolyzable tannins was isolated from the ruminal fluid of a goat fed desmodium (Desmodium ovalifolium), a tropical legume which contains levels as high as 17% condensed tannins. This strain grew under anaerobic conditions in the presence of up to 30 g of tannic acid per liter and tolerated a range of phenolic monomers, including gallic, ferulic, and p-coumaric acids. The predominant fermentation product from tannic acid breakdown was pyrogallol, as detected by high-performance liquid chromatography and mass spectrometry. Tannic acid degradation was dependent on the presence of a sugar such as glucose, fructose, arabinose, sucrose, galactose, cellobiose, or soluble starch as an added carbon and energy source. The strain also demonstrated resistance to condensed tannins up to a level of 4 g/liter.