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 bacteria from soil contaminated with diesel oil and the possible use of these in autochthonous bioaugmentation

Two bacterial species (isolates N and O) were isolated from a paddy soil microcosm that had been artificially contaminated with diesel oil to which extrinsic Pseudomonas aeruginosa strain WatG, had been added exogenously. One bacterial species (isolate J) was isolated from a similar soil microcosm that had been biostimulated with Luria–Bertani (LB) medium. Isolates N and O, which were tentatively identified as Stenotrophomonas sp. and Ochromonas sp., respectively, by sequencing of their 16 S rRNA genes had no ability to degrade diesel oil on their own in any liquid medium. When each strain was cocultivated with P. aeruginosa strain WatG in liquid mineral salts medium (MSM) containing 1% diesel oil, isolate N enhanced the degradation of diesel oil by P. aeruginosa strain WatG, but isolate O inhibited it. In contrast, isolate J, which was tentatively identified as a Rhodococcus sp., degraded diesel oil contained not only in liquid LB and MSM, but also in paddy soil microcosms supplemented with LB medium. The bioaugmentation capacity of isolate J in soil microcosms contaminated with diesel oil was much higher than that of P. aeruginosa strain WatG. The possibility of using isolate J for autochthonous bioaugmentation is discussed.     

Isolation of denitrifying bacteria from hydrocarbon-contaminated Antarctic soil

In this study, we report the isolation of denitrifiers from hydrocarbon-contaminated Antarctic soils. Seventy-two isolates were obtained from soils that had received a fertilizer treatment to stimulate hydrocarbon degradation. All isolates, except one, belonged to the genus Pseudomonas. The one exception was a member of the Microbacteriaceae, which was also, coincidentally, the only isolate negative for the nirS gene. The diversity of the 16S rRNA and nosZ genes was assessed by denaturing gradient gel electrophoresis and sequencing. There was a slight correlation between the 16S rRNA and nosZ operational taxonomic units. Surprisingly, many isolates contained nosZ on plasmids and, to the best of our knowledge, this is the first report of nosZ being extra-chromosomally present in Pseudomonas spp.     

Effects of nitrobenzene contamination and of bioaugmentation on nitrification and ammonia-oxidizing bacteria in soil

Bioaugmentation of nitrobenzene-contaminated soil was performed by inoculation with Pseudomonas putida ZWL73, which can grow on nitrobenzene as carbon and nitrogen sources and release free ammonium from the aromatic ring via a partial-reductive pathway. Removal of nitrobenzene was effectively enhanced with concurrent accumulation of ammonium in the bioaugmented soil. Moreover, the negative impact of nitrobenzene contamination on culturable bacterial types and soil nitrification was reduced by strain ZWL73. Changes in the community structure of ammonia-oxidizing bacteria, determined by denaturing gradient gel electrophoresis, were associated with changes in environmental factors in nitrobenzene-contaminated soil, including concentrations of nitrobenzene, ammonium, nitrite and nitrate, but their influence was attenuated in the bioaugmented soil. Overall, P. putida ZWL73 shows promising abilities for effective removal of nitrobenzene and for attenuating the negative effects of nitrobenzene contamination on soil functioning.     

红树林土壤中产聚羟基脂肪酸酯细菌的分离及其评估

[背景] 细菌能通过合成聚羟基脂肪酸酯（Polyhydroxyalknoates,PHA）在细胞内储存物质和能量,提高对环境的适应能力。在红树林中,由于土壤周期性受海水浸没,形成营养物质种类丰富和含量波动大的特殊生境,为细菌进化出特殊的PHA合成途径提供了条件。[目标] 为了增加对红树林产PHA细菌资源的了解,获得产PHA细菌,使用纯培养方法分离和鉴定细菌,并评估菌株的产PHA能力。[方法] 采集红树植物海桑根系和红树滩涂土壤样品,连续5周培养、分离纯化获得细菌菌株;通过16S rRNA基因相似性及系统进化分析鉴定细菌分类地位,利用PHA合成酶基因（phaC）鉴定细菌合成PHA的能力;通过基因组草图测序,分析细菌的phaC基因种类、代谢通路及系统进化关系;通过气相色谱分析细菌产PHA的累积量及组成。[结果] 从红树林土壤样品中分离得到97株细菌,其中13株带有phaC基因,包括坚强芽孢杆菌（Cytobacillus firmus）、弯曲芽孢杆菌（Bacillus flexus）、除烃海杆菌（Marinobacter hydrocarbonoclasticus）和酯香微杆菌（Microbacterium esteraromaticum）。B. flexus MN15-19以丙酮酸盐为碳源,可累积细胞干重11%的PHA,同时具有固碳功能的还原性三羧酸循环通路,有开发成为固碳产PHA工程菌株的潜力。酯香微杆菌可产PHA,但是其phaC基因结构特殊,基因组注释未能识别出任何已知phaC基因。[结论] 研究发现红树林土壤可培养细菌中存在未知的PHA合成途径,说明红树林生态系统中的细菌具有资源挖掘的重要价值。     

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.     

Phytoremediation of lead-contaminated soil by Sinapis arvensis and Rapistrum rugosum

Nowadays, public concern relating to ecological deleterious effects of heavy metals is on the rise. To evaluate the potential of Rapistrum rugosum and Sinapis arvensis in lead- contaminate phytoremediate, a pot culture experiment was conducted. The pots were filled by soil treated with different rates of leadoxide (PbO) including 0 (control), 100, 200, 300, 400, and 500 mg Pb per 1 kg soil. Germinated seeds were sown. Surprisingly, with increasing concentration of Pb, dry weight of R. rugosum and S. arvensis did not decrease significantly. In both of species, the concentration of Pb was higher in roots than shoots. In general, S.arvensis was absorbed more Pb compared to R. rugosum. The results revealed high potential of R. rugosum and S. arvensis in withdrawing Pb from contaminated soil. For both species, a positive linear relation was observed between Pb concentration in soil and roots. However, linear relationship was not observed between Pb concentration in the soil and shoots. Although both species test had low ability in translocation Pb from roots to shoots but they showed high ability in uptake soil Pb by roots. Apparently, these plants are proper species for using in phytoremediation technology.     

Isolation and study of azobenzene-transforming soil bacteria

Heterotrophic bacteria were isolated from soil and glass slides and classified as Bacillus cereus SNK12, Paenibacillus polymyxa SNK2, Azotobacter chroococcum ANKII, and Ochrobactrum intermedium ANKI. Their cultures could degrade azobenzene under the conditions of co-metabolism. A rapid test for the ability of bacteria to transform azobenzenes is proposed.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 2, 2005, pp. 185–188.Original Russian Text Copyright © 2005 by Wackerow-Kouzova.     

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.     

Isolation of Gemmata-like and Isosphaera-like planctomycete bacteria from soil and freshwater.

New cultured strains of the planctomycete division (order Planctomycetales) of the domain Bacteria related to species in the genera Gemmata and Isosphaera were isolated from soil, freshwater, and a laboratory ampicillin solution. Phylogenetic analysis of the 16S rRNA gene from eight representative isolates showed that all the isolates were members of the planctomycete division. Six isolates clustered with Gemmata obscuriglobus and related strains, while two isolates clustered with Isosphaera pallida. A double-membrane-bounded nucleoid was observed in Gemmata-related isolates but not in Isosphaera-related isolates, consistent with the ultrastructures of existing species of each genus. Two isolates from this study represent the first planctomycetes successfully cultivated from soil.     

海州香薷（Elsholtzia splendens）根际铜抗性细菌的筛选及生物多样性

摘要：【目的】重金属耐性植物海州香薷根际铜抗性细菌的筛选及生物多样性研究将有助于了解微生物－超富集植物相互关系和植物修复机理、开发微生物－香薷重金属修复新技术。【方法】采用稀释平板涂布法从海州香薷根际筛选铜抗性菌株,测定菌株溶磷和产生吲哚乙酸、铁载体、1-氨基环丙烷-1-羧酸（ACC）脱氨酶的特性,采用16S rDNA限制性酶切多态性分析（amplified rDNA restriction analysis, ARDRA）研究铜抗性细菌的遗传多样性,根据16S rDNA相似性对产ACC脱氨酶的菌株进行了     

Effects of rhizosphere remediation and bioaugmentation on carbofuran removal from soil

Rhizosphere soil contains important sources of nutrients for microorganisms resulting in high number of microorganisms capable of degrading various types of chemicals in the soil. Thus, this study investigated a carbofuran dissipation in rhizosphere soils of 6 weeds namely, umbrella sedge (Cyperus iria L.), fuzzy flatsedge (C. pilosus V.), small flower umbrella plant (C. difformis L.), tall-fringe-rush hoorah grass (Fimbristylis miliacea V.), cover fern (Marsilea crenata P.), and water primrose (Jussiaea linifolia V.). Rhizosphere soil of fuzzy flatsedge showed the shortest half-life (t_1/2) of carbofuran (15 days) among other soils. So, it was selected to be used in the bioaugmentation experiment using carbofuran degrader namely Burkholderia cepacia, PCL3, as inoculum in order to examine whether they would improve carbofuran degradation in soil. The results showed that the addition of PCL3 into rhizosphere soil did not improve carbofuran degradation suggesting that microorganisms in rhizosphere soil might be capable enough to remove carbofuran from soil. The number of carbofuran degraders in the rhizosphere soils was greater than in bulk soil 10–100 times which might be responsible to a rapid degradation of carbofuran in rhizosphere soils without the addition of PCL3. The ability of PCL3 to degrade carbofuran was evident in bulk soil (t_1/2 of 12 days) and autoclaved soils (t_1/2 13–14 days) when compared to soils without an inoculation (t_1/2 of 58 days) indicated that the addition of a degrader was useful in improving carbofuran degradation in soil.     

Isolation and study of azobenzene converting soil bacteria

Heterotrophic bacteria were isolated from soil and glass slides and classified as Bacillus cereus SNK12, Paenibacillus polymyxa SNK2, Azotobacter chroococcum ANKII, and Ochrobacterium intermedium ANKI. Their cultures could degrade azobenzene under the conditions of co-metabolism. A rapid test for the ability of bacteria to convert azobenzenes is proposed.     

Isolation and characterization endophytic bacteria from hyperaccumulator Sedum alfredii Hance and their potential to promote phytoextraction of zinc polluted soil

The aim of this study was to isolate and characterize endophytic bacteria from roots, stems and leaves of Zn/Cd hyperaccumulator Sedum alfredii. Endophytic bacteria were observed in roots, stems and leave of S. alfredii, with a significantly higher density in roots, followed by leave and stems. A total of fourteen bacterial endophytes were isolated and are closely related phylogenetically to Pseudomonas, Bacillus, Stenotrophomonas, Acinetobacte by 16S rRNA sequence analysis. Most of the endophytic bacteria were found to exhibit high Zn and Cd resistance characteristics, but difference existed among this isolates. The fourteen endophytic bacteria all had the capacity to produce IAA. Moreover, strains VI₈L₁, VI₈L₂, VI₈L₄, VI₈R₂, VI₈R₃ and II₂R₃ could solubilize Ca₃(PO₄)₂, strains VI₈L₂, II₈L₄ and VI₈R₂ could produce siderophore, and strains VI₈L₂ and VI₈R₃ had the capacity of nitrogen fixation. Both plate and broth assay proved that strain VI₈L₁, VI₈L₂, II₈L₄ and VI₈R₂ were able to effectively solubilize ZnCO₃ and Zn₃(PO₄)₂. The filtrate liquid media after growth of strains VI₈L₁, VI₈L₂, II₈L₄ and VI₈R₂ extracted much higher Zn from artificially ZnCO₃ and Zn₃(PO₄)₂ contaminated soils than those extracted by axenic SMS broth, and the filtrates of the culture media supporting growth of strains VI₈L₂, II₈L₄ and VI₈R₂ also extracted significantly greater quantities of Zn from the Dabaoshan contaminated soils. This Zn mobilizing, plant growth promoting and metal resistant endophytic bacteria may offer promise as inoculants to increase soil Zn bioavailability and improve growth and Zn accumulation by S. alfredii.     

Isolation of isoproturon-degrading bacteria from treated soil via three different routes

Three different isolation routes (flask enrichment/flask degradation assay, flask enrichment/microplate degradation assay, MPN assay/microplate degradation assay) were used to obtain pure cultures of bacteria which degraded isoproturon (3-(4-isopropylphenyl)-1,1-dimethylurea) as sole carbon and nitrogen source in a mineral salts medium from a field soil treated with isoproturon in the laboratory. All three isolation routes were successful, but the microplate assay of degradation was more successful than the flask assay. Characterization of 36 isolates indicated that they formed 16 distinct phenotypes (10 Gram-positive phenotypes, six Gram-negative phenotypes) which are likely to represent distinct species. Low concentrations of the degradation product 3-(4-isopropylphenyl)-1-methylurea (IPPMU) were occasionally found in the culture solutions. When provided as the sole source of carbon and nitrogen, the monomethyl degradation product was itself rapidly degraded by several of the isolates. Some isolates were also able to use the demethylated degradation product 3-(4-isopropylphenyl)-urea (IPPU) as sole source of carbon and nitrogen, although there was occasionally an extended lag-phase before rapid degradation commenced. One isolate was particularly active and degraded isoproturon, the monomethyl and demethylated degradation products of isoproturon, and demethylated the related phenylureas diuron and linuron.     

盐碱土壤PAHs 降解菌的筛选鉴定及其降解特性

采用富集培养的方法,从天津大港油田PAHs污染盐碱化土壤中分离出一株能以菲、芘为唯一碳源和能源的优势菌TJB5。经形态观察和16S rDNA序列分析结果表明,该菌株为成团泛菌(Pantoea agglomerans)。采用液体培养的方法,研究了pH、盐度、菲芘的初始浓度对TJB5菌株降解菲芘效果的影响,确定了最佳降解条件。结果表明,该菌对菲、芘的降解具有较广泛的pH、盐度范围和良好的降解效果。在菲、芘浓度分别为50 mg/L、pH 6.8-9.5、盐度2%-3%、温度30°C条件下,接种15 d后菲降解率在93.3%以上,芘降解率在20%以上。     

Isolation and characterization of entomopathogenic bacteria from soil samples from the western region of Cuba

The use of insect pathogens is a viable alternative for insect control because of their relative specificity and lower environmental impact. The search for wild strains against dipterans could have an impact on mosquito control programs. We have made an extensive screening of soil in western Cuba to find bacteria with larvicidal activity against mosquitoes. A total of 150 soil samples were collected and isolates were identifying using the API 50 CHB gallery. Phenotypic characteristics were analyzed by hierarchical ascending classification. Quantitative bioassays were conducted under laboratory conditions following the World Health Organization protocol in order to ascertain the toxicity and efficacy of isolates. The protein profiles of the crystal components were determined by SDS‐PAGE. Eight hundred and eighty‐one bacterial isolates were obtained, and 13 isolates with entomopathogenic activity were isolated from nine samples. Nine isolates displayed higher entomopathogenic activity against both Cx. quinquefasciatus and Ae. aegypti compared with the reference strain 266/2. All toxic isolates showed higher biological potency than the 266/2 strain. These isolates with high entomopathogenic activity displayed a protein pattern similar to the B. thuringiensis var. israelensis IPS‐82 and 266/2 strains. These results are a valuable tool for the control of Diptera of medical importance.