Isolation and characterization of indigenous soil bacteria for bioaugmentation of PAH contaminated soil of semiarid Patagonia, Argentina

The aim of this work was to isolate PAH degrading-bacteria from contaminated Patagonia soil with the ability to tolerate the usual environmental stresses (oligotrophic and dryness conditions). Two approaches were utilized to obtain PAH-degrading bacteria from the Patagonian soil. With a traditional enrichment approach only the PAH- degrading strain 36 was isolated. Using a direct isolation approach three PAH-degrading strains (1A, 22A and 22B) were isolated. The phylogenetic analysis revealed that all isolates belonged to Sphingomonas genus. The PAH degrading activity and the resistance to stress conditions of the strains were determined and compared with those of the exogenous PAH-degrading Sphingomonas paucimobilis 20006FA. The strains 1A, 22A and 36 were phylogenetically closely related between them and with the strain 20006FA. The strain 22B, that showed a different phylogenetic position, was more resistant to C-starvation and drying conditions than other Patagonian strains. The effect of the inoculation of these strains on phenanthrene-induced mineralization and elimination was studied in Patagonian soil artificially contaminated, at different environmental conditions. The results suggest that strain 22B is the most suitable strain for bioaugmentation in PAH-contaminated soils of Central Patagonia, due to its adaptation to the usual environmental conditions. Our results show the importance of a detailed physiological characterization of isolates for autochthonous bioaugmentation strategies success.     

Isolation and characterization of four polycyclic aromatic hydrocarbon degrading bacteria from soil near an oil refinery

Four bacterial strains (I-IV) capable of optimum growth on 0·1% naphthalene, anthracene or a mixture of naphthalene and phenanthrene were isolated from soil near an oil refinery. Two isolates (I and II) were identified as belonging to the genus Micrococcus , while strains III and IV were identified as Pseudomonas and Atcaligenes respectively. All the isolates were found to bear high molecular weight plasmid DNA (isolate I and IV 89%, II 67·5% and III 92·1% of Λ DNA), which is presumed to aid in the metabolism of polycyclic aromatic hydrocarbons. The strains also showed appreciable growth at high concentrations of NaCl (up to 7·5%).     

Isolation and characterization of fenamiphos degrading bacteria

The biological factors responsible for the microbial breakdown of the organophosphorus nematicide fenamiphos were investigated. Microorganisms responsible for the enhanced degradation of fenamiphos were isolated from soil that had a long application history of this nematicide. Bacteria proved to be the most important group of microbes responsible for the fenamiphos biodegradation process. Seventeen bacterial isolates utilized the pure active ingredient fenamiphos as a carbon source. Sixteen isolates rapidly degraded the active ingredient in Nemacur 5GR. Most of the fenamiphos degrading bacteria were Microbacterium species, although Sinorhizobium, Brevundimonas, Ralstonia and Cupriavidus were also identified. This array of gram positive and gram negative fenamiphos degrading bacteria appeared to be pesticide-specific, since cross-degradation toward fosthiazate, another organophosphorus pesticide used for nematode control, did not occur. It was established that the phylogenetical relationship among nematicide degrading bacteria is closer than that to non-degrading isolates.     

Isolation and characterization of phorate degrading soil bacteria of environmental and agronomic significance

Phorate [O,O-diethyl-S-(ethylthio)methyl phosphoradiothioate] degrading bacteria were isolated from agricultural soil and characterized based on their morphological and biochemical characteristics. The selected isolates PS-1, PS-2 and PS-3 were presumptively identified as Rhizobium, Pseudomonas and Proteous species, respectively. The HPLC analysis of phorate in bioaugmented soil revealed its complete disappearance within 40 days. The degradation isotherms of the isolates PS-1, PS-2 and PS-3 suggested time-dependent disappearance of phorate following the first order rate kinetics at the corresponding rate constants of 0.04, 0.05 and 0.04 days-1. Besides, the isolates concurrently exhibited substantial phosphate solubilization, indole acetic acid, and siderophore production. The isolate PS-3 also showed anti-fungal activity against a phytopathogen Fusarium oxysporum. As a result of the multifarious biological properties, the isolates have been suggested to be important bioresource for efficient bioinoculant development.     

Isolation and characterization of novel atrazine-degrading microorganisms from an agricultural soil

Six previously undescribed microorganisms capable of atrazine degradation were isolated from an agricultural soil that received repeated exposures of the commonly used herbicides atrazine and acetochlor. These isolates are all Gram-positive and group with microorganisms in the genera Nocardioides and Arthrobacter, both of which contain previously described atrazine degraders. All six isolates were capable of utilizing atrazine as a sole nitrogen source when provided with glucose as a separate carbon source. Under the culture conditions used, none of the isolates could utilize atrazine as the sole carbon and nitrogen source. We used several polymerase-chain-reaction-based assays to screen for the presence of a number of atrazine-degrading genes and verified their identity through sequencing. All six isolates contain trzN and atzC, two well-characterized genes involved in the conversion of atrazine to cyanuric acid. An additional atrazine-degrading gene, atzB, was detected in one of the isolates as well, yet none appeared to contain atzA, a commonly encountered gene in atrazine impacted soils and atrazine-degrading isolates. Interestingly, the deoxyribonucleic acid sequences of trzN and atzC were all identical, implying that their presence may be the result of horizontal gene transfer among these isolates.     

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.     

Isolation and characterization of bacteria degrading polychlorinated biphenyls from transformer oil

Polychlorinated biphenyls from transformer oil were degraded in liquid culture under aerobic conditions using a mixed bacterial culture isolated from a transformer oil sample with a high content of polychlorinated biphenyls and other hydrocarbons. Four strains were identified, three of them corresponded to genusBacillus, the other one toErwinia. Bacteria in the transformer oil could remove as much as 65% of polychlorinated biphenyls (88%W/V in the transformer oil). Additional data showed that the two isolated strains ofB. lentus were able to grow on transformer oil and degrade polychlorinated biphenyls by 80 and 83%. Our results provide evidence that microorganisms occurring in transformer oil have the potential to degrade polychlorinated biphenyls.     

一株毒死蜱降解细菌的分离鉴定及其在土壤修复中的应用

从蔬菜大棚土壤中分离到一株能以毒死蜱为唯一碳源和能源生长的菌株DSP3,该菌在含毒死蜱（100mg/L）的酵母膏和蛋白胨与同样毒死蜱含量而无酵母膏蛋白胨的无机盐培养基中,18d对毒死蜱的降解率分别为986%和762%;在土壤实验中20d对毒死蜱（100mg/kg）的降解率接近100%,加入DSP3菌在蔬菜大棚新鲜土壤中能有效促进毒死蜱在土壤中的降解。根据生理生化特征、16S rDNA序列分析、（G+C）mol%测定和DNA同源性分析,将菌株DSP3鉴定为粪产碱杆菌（Alcaligenes faecalis）。     

活性污泥中群体感应淬灭菌的分离纯化及功能验证

【背景】近年来,群体感应淬灭(Quorum Quenching,QQ)技术在膜生物污堵防控中的应用研究受到了广泛关注。然而,目前已成功分离纯化的高效QQ菌有限,更多高效QQ菌资源亟待挖掘。【目的】从实际运行的膜生物反应器(MembraneBioreactor,MBR)活性污泥中采样,分离并富集高效QQ菌。【方法】以根瘤农杆菌(Agrobacterium tumefaciens) A136为报告菌株,使用指示琼脂平板法测定各菌株的N-辛酰基高丝氨酸内酯(N-Octanoyl-DL-Homoserine Lactone,C8-HSL)降解能力。以紫色色杆菌(Chromobacterium violaceum) VIR24为报告菌株,定量测定所得QQ菌降解N-己酰高丝氨酸内酯(N-Hexanoyl-DL-Homoserine Lactone,C6-HSL)信号分子的能力。通过微生物形态、生理生化及16SrRNA基因序列测定、构建系统发育树、扫描电子显微镜形态观测等方法对菌株进行分类学鉴定。用共培养法分析QQ菌对生物膜形成的抑制能力,通过聚乙烯醇和海藻酸钠包埋固定化QQ菌。【结果】筛选出了6株高效QQ菌,其中对C8-HSL分解能力最强的为杆状、革兰氏阴性戴尔福特菌属(Delftia sp.) JL5。定量分析结果表明菌株JL5能在10 h内完全降解C6-HSL。菌株JL5显著抑制铜绿假单胞菌(Pseudomonas aeruginosa) PAO1和菠萝泛菌(Pantoea ananatis) SK-1生物膜的形成。固定化后的JL5微球仍具有高效的C6-HSL和C8-HSL信号分子分解能力,而且分解速度较被广泛报道的红球菌(Rhodococcussp.)BH4更快。【结论】研究分离得到了高效的QQ菌,能够有效抑制N-酰基高丝氨酸内酯(N-Acyl-HomoserineLactones,AHL)型群体感应菌生物膜的形成,固定化后仍然具有强QQ活性,具备广泛的应用前景,为后续QQ膜生物污堵防控技术的实践应用奠定了基础。     

Isolation and characterization of 2,4-dichlorophenoxyacetic acid-catabolizing bacteria and their biodegradation efficiency in soil

Bacterial isolates (NJ 10 and NJ 15) capable of degrading the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were isolated from agricultural soil by enrichment culture technique. The isolates exhibited substantial growth in mineral salt medium supplemented with 0.1–0.5% of 2,4-D as a sole source of carbon and energy. Based on their morphological, cultural and biochemical characteristics, the isolates NJ 10 and NJ 15 have been identified as Pseudomonas species and Pseudomonas aeruginosa, respectively. Biodegradation studies in a soil microcosm enriched with pure cultures of the isolates demonstrated a time-dependent disappearance of 2,4-D from the 100 mg/kg herbicide-amended soil. The HPLC data analysis revealed 96.6 and 99.8% degradation in the soil inoculated with the pure cultures of isolates NJ 10 and NJ 15, respectively with in 20 days of incubation at 30 °C. Both the isolates showed significant solubilization of inorganic phosphate [Ca₃(PO₄)₂] on the specific Pikovskaya's medium.     

Isolation and characterization of lignin‐degrading bacteria from rainforest soils

The deconstruction of lignin to enhance the release of fermentable sugars from plant cell walls presents a challenge for biofuels production from lignocellulosic biomass. The discovery of novel lignin‐degrading enzymes from bacteria could provide advantages over fungal enzymes in terms of their production and relative ease of protein engineering. In this study, 140 bacterial strains isolated from soils of a biodiversity‐rich rainforest in Peru were screened based on their oxidative activity on ABTS, a laccase substrate. Strain C6 (Bacillus pumilus) and strain B7 (Bacillus atrophaeus) were selected for their high laccase activity and identified by 16S rDNA analysis. Strains B7 and C6 degraded fragments of Kraft lignin and the lignin model dimer guaiacylglycerol‐β‐guaiacyl ether, the most abundant linkage in lignin. Finally, LC–MS analysis of incubations of strains B7 and C6 with poplar biomass in rich and minimal media revealed that a higher number of compounds were released in the minimal medium than in the rich one. These findings provide important evidence that bacterial enzymes can degrade and/or modify lignin and contribute to the release of fermentable sugars from lignocellulose. Biotechnol. Bioeng. 2013; 110: 1616–1626. © 2013 Wiley Periodicals, Inc.     

Isolation and characterization of endophytic bacteria from soybean (Glycine max) grown in soil treated with glyphosate herbicide

Endophytic bacteria are ubiquitous in most plant species influencing the host fitness by disease suppression, contaminant degradation, and plant growth promotion. This endophytic bacterial community may be affected by crop management such as the use of chemical compounds. For instance, application of glyphosate herbicide is common mainly due to the use of glyphosate-resistant transgenic plants. In this case, the bacterial equilibrium in plant–endophyte interaction could be shifted because some microbial groups are able to use glyphosate as a source of energy and nutrients, whereas this herbicide may be toxic to other groups. Therefore, the aim of this work was to study cultivable and noncultivable endophytic bacterial populations from soybean (Glycine max) plants cultivated in soil with and without glyphosate application (pre-planting). The cultivable endophytic bacterial community recovered from soybean leaves, stems, and roots included Acinetobacter calcoaceticus, A. junii, Burkholderiasp., B. gladioli, Enterobacter sakazaki, Klebsiella pneumoniae, Pseudomonas oryzihabitans, P. straminea, Ralstonia pickettii,and Sphingomonassp. The DGGE (Denaturing Gradient Gel Electrophoresis) analysis from soybean roots revealed some groups not observed by isolation that were exclusive for plants cultivated in soil with pre-planting glyphosate application, such as Herbaspirillum sp., and other groups in plants that were cultivated in soil without glyphosate, such as Xanthomonas sp. and Stenotrophomonas maltophilia. Furthermore, only two bacterial species were recovered from soybean plants by glyphosate enrichment isolation. They were Pseudomonas oryzihabitans and Burkholderia gladioliwhich showed different sensibility profiles to the glyphosate. These results suggest that the application at pre-planting of the glyphosate herbicide may interfere with the endophytic bacterial communitys equilibrium. This community is composed of different species with the capacity for plant growth promotion and biological control that may be affected. However, the evaluation of this treatment in plant production should be carried out by long-term experiments in field conditions.     

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。     

Isolation and molecular characterization of thiosulfate-oxidizing bacteria from an Italian rice field soil

In rice paddy soils an active cycling of sulfur compounds takes place. To elucidate the diversity of thiosulfate-oxidizing bacteria these organisms were enriched from bulk soil and rice roots by the most probable number method in liquid medium. From the MPN enrichment cultures 21 bacterial strains were isolated on solid mineral medium, and could be further shown to produce sulfate from thiosulfate. These strains were characterized by 16S rDNA analyses. The isolates were affiliated to seven different phylogenetic groups within the alpha- and beta-subclass of Proteobacteria. Two of these phylotypes were already described as S-oxidizers in this environment (Xanthobacter sp. and Bosea sp. related strains), but five groups represented new S-oxidizers in rice field soil. These isolates were closely related to Mesorhizobium loti, to Hydrogenophaga sp., to Delftia sp., to Pandoraea sp. or showed sequence similarity to a strain of Achromobacter sp.     

Isolation and characterization of surfactant degrading bacteria in a marine environment

Abstract Sea water and sediment samples taken near the coasts of Hyeres bay (France) were used for anionic surfactant titrations with surface and bottom waters and the finest part of sediments. The capacity for surfactant degradation by the 'in situ' microflora was evaluated. By using a selective plating technique 26 strains able to utilize anionic surfactant were isolated from the selected bacterial communities. Their ability to degrade anionic surfactant was verified according to the biodegradation standard method. Isolated strains were characterized by morphological and physiological properties using API 20 NE micro-method. All tested strains were Gram negative, strictly aerobic, rod or helical shaped. Their weak utilization of phenolic substrates suggests that they degrade preferentially the alkyl chain of the surfactant molecule. Biodegradation was more efficient with bacterial communities rather than with any isolated strains. Such observations indicate that complete mineralization involves several other so far non-isolated strains which complete the degradation initiated by the isolated strains.     

Isolation from agricultural soil and characterization of a Sphingomonas sp. able to mineralize the phenylurea herbicide isoproturon.

A soil bacterium (designated strain SRS2) able to metabolize the phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea (IPU), was isolated from a previously IPU-treated agricultural soil. Based on a partial analysis of the 16S rRNA gene and the cellular fatty acids, the strain was identified as a Sphingomonas sp. within the alpha-subdivision of the proteobacteria. Strain SRS2 was able to mineralize IPU when provided as a source of carbon, nitrogen, and energy. Supplementing the medium with a mixture of amino acids considerably enhanced IPU mineralization. Mineralization of IPU was accompanied by transient accumulation of the metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, and 4-isopropyl-aniline identified by high-performance liquid chromatography analysis, thus indicating a metabolic pathway initiated by two successive N-demethylations, followed by cleavage of the urea side chain and finally by mineralization of the phenyl structure. Strain SRS2 also transformed the dimethylurea-substituted herbicides diuron and chlorotoluron, giving rise to as-yet-unidentified products. In addition, no degradation of the methoxy-methylurea-substituted herbicide linuron was observed. This report is the first characterization of a pure bacterial culture able to mineralize IPU.     

稻田除草剂二氯喹啉酸降解菌15#的分离、鉴定及降解特性

[背景] 二氯喹啉酸（Quinclorac,QNC）是一种高选择性、激素类、低毒性除草剂,主要用于防治稻田稗草,持效期长,易于在土壤中积累而影响后茬作物的生长发育,而且环境中残留的QNC可对动物生长发育产生不良影响,并影响微生物的群落结构和丰度。[目的] 从稻田土壤中分离筛选出一株可降解除草剂QNC的菌株,鉴定并明确其降解特性。[方法] 通过形态学、生理生化试验、磷脂脂肪酸（Phospholipid Fatty Acid,PLFA）微生物鉴定、16S rRNA基因测序及分析鉴定菌株。通过单因素实验探究菌株的降解特性。[结果] 筛选得到一株编号为15^#的QNC降解菌,被鉴定为无色杆菌属菌株（Achromobacter sp.）。降解特性研究结果表明,菌株15^#的最佳培养条件为：30℃、pH为6.0、初始QNC浓度为100 mg/L、接种量为7%、添加质量分数为0.1%的酵母浸粉、氮源为蛋白胨,在此条件下培养21 d后QNC的降解率可达43.0%。[结论] 筛选到降解QNC新菌株并为该菌株的进一步研究提供理论基础。     

Isolation and characterization of novel bacteria degrading polycyclic aromatic hydrocarbons from polluted Greek soils

Three bacterial strains, designated as Wphe1, Sphe1, and Ophe1, were isolated from Greek soils contaminated with polycyclic aromatic hydrocarbon (PAH)-containing waste from the wood processing, steel, and oil refinery industries. Wphe1, Sphe1, and Ophe1 were characterized and identified as species of Pseudomonas, Microbacterium, and Paracoccus, respectively, based on Gram staining, biochemical tests, phospholipid analysis, FAME analysis, G+C content and 16S rRNA gene sequence analysis. The results of gas chromatography showed that strain Wphe1 degraded naphthalene, phenanthrene, and m-cresol over a wide temperature range; strain Sphe1 was a degrader of phenanthrene and n-alkanes; most interestingly, strain Ophe1 degraded anthracene, phenanthrene, fluorene, fluoranthene, chrysene, and pyrene, as well as cresol compounds and n-alkanes as sole carbon source. This is the first report of a representative of the genus Paracoccus capable of degrading PAHs with such versatility. These three strains may be useful for bioremediation applications.     

Isolation and characterization of polyphenols-degrading bacteria from olive-mill wastewaters polluted soil

In this study 28 bacterial strains, isolated from greenwaters-polluted-soil, were investigated for their ability to grow in presence of phenols added to Mineral Basal Medium (MBM) in aerobic conditions. In particular, three of them were found to be able to use as sole carbon source phenol, cathecol, caffeic acid and ferulic acid with efficiency ranging from 76% (phenol in 5 days, millimolar concentration from 3.7 10^?2 to 9 10^?3) to 95% (ferulic acid in 2 days millimolar concentration from 6.8 10^?1 to 3 10^?2). For these strains the taxonomic position was studied by amplification and sequencing of 16S rRNA genes. The isolated strains were classified belonging to Arthrobacter sulfureus, Pseudomonas synxantha and Pseudomonas oryzihabitans. Noteworthy, for the first time such Pseudomonas strains have been shown to be able to use polyphenols as the only carbon source in vitro. In fact, to the best of our knowledge, this kind of study were not done on Ps. Synxantha, while it was recently shown the ability of P. oryzihabitans to degrade catechol. These findings may open to new biotechnological applications for the degradation of polyphenols.