Genetic diversity of culturable bacteria in oil-contaminated rhizosphere of Galega orientalis

A collection of 50 indigenous meta-toluate tolerating bacteria isolated from oil-contaminated rhizosphere of Galega orientalis on selective medium was characterized and identified by classical and molecular methods. 16S rDNA partial sequencing showed the presence of five major lineages of the Bacteria domain. Gram-positive Rhodococcus, Bacillus and Arthrobacter and gram-negative Pseudomonas were the most abundant genera. Only one-fifth of the strains that tolerated m-toluate also degraded m-toluate. The inoculum Pseudomonas putida PaW85 was not found in the rhizosphere samples. The ability to degrade m-toluate by the TOL plasmid was detected only in species of the genus Pseudomonas. However, a few Rhodococcus erythropolis strains were found which were able to degrade m-toluate. A new finding was that Pseudomonas migulae strains and a few P. oryzihabitans strains were able to grow on m-toluate and most likely contained the TOL plasmid. Because strain specific differences in degradation abilities were found for P. oryzihabitans, separation at the strain level was important. For strain specific separation (GTG)5 fingerprinting was the best method. A combination of the single locus ribotyping and the whole genomic fingerprinting techniques with the selective partial sequencing formed a practical molecular toolbox for studying genetic diversity of culturable bacteria in oil-contaminated rhizosphere.     

Impacts of organic carbon availability and recipient bacteria characteristics on the potential for TOL plasmid genetic bioaugmentation in soil slurries

The effectiveness of genetic bioaugmentation relies on efficient plasmid transfer between donor and recipient cells as well as the plasmid’s phenotype in the recipient cell. In the present study, the effects of varying organic carbon substrates, initial recipient-to-donor cell density ratios, and mixtures of known recipient bacterial strains on the conjugation and function of a TOL plasmid were tested in sterile soil slurry batch reactors. The presence of soil organic carbon was sufficient in ensuring TOL plasmid transconjugant occurrence (up to 2.1 ± 0.5%) for most recipient strains in soil slurry batch mating experiments. The addition of glucose had limited effects on transconjugant occurrence; however, glucose amendment increased the specific toluene degradation rates of some Enterobacteriaceae transconjugants in soil slurry. Initial cell density ratios and mixtures of recipient strains had smaller impacts on plasmid conjugation and resulting phenotype functionality. These observations suggest that genetic bioaugmentation may be improved by minimal altering of environmental conditions.     

Bioaugmentation of aerobic microbial granules with Pseudomonas putida carrying TOL plasmid

This paper describes results of a successful bioaugmentation experiment on aerobic granular sludge using Pseudomonas putida KT2442 cells bearing the TOL (pWWO) plasmid. The methodology was designed to monitor incorporation of the added donor cells into pre-existent microbial granules and the subsequent plasmid transfer to the autochthonous microbial community using shake flask microcosms. Expression of reporter proteins (GFP and DsRed) allowed in situ monitoring of donor cell attachment and plasmid transfer to the recipient cells using confocal laser scanning microscopy. Concomitant with donor integration and transconjugant proliferation in the granules, a significant increase in degradation of benzyl alcohol (used as sole substrate) was observed in the augmented microcosms. In contrast, control microcosms (with non-augmented granules) did not show any noticeable increase in the degradation of the substrate. This study shows that bioaugmentation of aerobic granular sludge via donor colonization and plasmid transfer is feasible for enhanced biodegradation.     

Evaluation of the Galega-Rhizobium galegae system for the bioremediation of oil-contaminated soil

The bioremediation potential of a nitrogen-fixing leguminous plant, Galega orientalis, and its microsymbiont Rhizobium galegae was evaluated in BTX (benzene, toluene, xylene)-contaminated soils in microcosm and mesocosm scale. To measure the intrinsic tolerance of the organisms to m-toluate, a model compound representing BTX, G. orientalis and R. galegae were cultivated under increasing concentrations of m-toluate alone and in association with Pseudomonas putida pWWO, a bacterial strain able to degrade toluene-derived compounds. The test plants and rhizobia remained viable in m-toluate concentrations as high as 3000 ppm. Plant growth was inhibited in concentrations higher than 500 ppm, but restituted when plants were transferred into m-toluate-free medium. Nodulation was blocked under the influence of m-toluate, but was restored after the plants were transferred into the non-contaminated media. In the mesocosm assay the Galega plants showed good growth, nodulation and nitrogen fixation, and developed a strong rhizosphere in soils contaminated with oil or spiked with 2000 ppm m-toluate. Thus, this legume system has good potential for use on oil-contaminated sites     

Effects of temperature and pH on conjugal transfer of zinc-resistant plasmids residing in Gram-negative bacteria isolated from industrial effluents

Two zinc (Zn)-resistant strains, AnZn-1 and AnZn-2, which were resistant to ZnSO4 up to 12.5 mg ml(-1) were isolated from industrial effluents. Both were Gram-negative with motile cells. They exhibited tolerance to Ba2+, Ni+, Co2+, Mn2+, Cu2+, Fe2+, Ni2+, Cd2+, kanamycin, chloramphenicol, ampillicin and tetracycline, but were sensitive to Hg2+ and streptomycin. For AnZn-1 and AnZn-2, the optimum pH for growth was 7. Both were facultative anaerobes and had cytochrome oxidase and urease enzymes, while catalase was present only in AnZn-2. Both strains had the ability to hydrolyse gelatin, reduce nitrate, and yield acid from arabinose and rhamnose. The two strains shared maximum characters with Vibrionaceae. Each strain carries a single Zn-resistant conjugative plasmid. The plasmid residing in AnZn-1 (pSH1211) displayed a lower level of resistance than the plasmid of AnZn-2 (pSH1212). Both required a minimum of 24 h for mating and showed highest transfer frequency at 25 degrees C. pSH1211 preferred pH 7 and pSH1212 pH 8.5 for their transfer. Both plasmids, when allowed to mate with Escherichia coli at 25 degrees C, alkaline pH values of 8-8.5 (pSH1211) of pH 7.5 (pSH1212), showed increased transfer frequency.     

Tolerance to agricultural pesticides of strains belonging to four genera of Rhizobiaceae

In order to determine their tolerance to pesticides, 122 strains of rhizobia isolated from different geographical regions, and belonging to the genera Rhizobium, Mesorhizobium, Sinorhizobium and Bradyrhizobium were tested against eight herbicides, four fungicides and five insecticides. Sensitivity to the pesticides was measured by using the filter paper disk method at four concentrations, 0.45, 4.5, 45 and 450 μg per disk. When the pesticides were used at 0.45 μg per disk, a concentration similar to that found when pesticides are applied under field conditions, no inhibition was observed. Strains growth was affected at concentrations of 45 and 450 μg pesticide per disk. These higher concentrations can be encountered when seeds are treated with pesticides. Pesticides tolerance level was correlated to pesticide function, i.e rhizobial strains were more tolerant to insecticides, followed by herbicides and then fungicides. Two fungicides, captan and mancozeb, inhibited the highest number of strains. Only one insecticide, carbaryl, affected the growth of some rhizobial strains. Strains isolated from the arctic (Mesorhizobium spp. and R. leguminosarum bv. viciae), a putative pesticides-free environment, were either less or equally affected by pesticides compared to strains isolated from agricultural regions.     

The fate of recombinant plasmids during composting of organic wastes

Composting was investigated as a means for safe disposal of organic waste containing bacteria that carry transgenes in recombinant plasmids. To generate model recombinant plasmids, a mobile IncQ plasmid, RSF1010, and a non-mobile plasmid, pGFP, were genetically modified to carry a DNA segment encoding both green fluorescent protein and kanamycin resistance and were designated as RSF1010-GFPK and pGFPK. Escherichia coli (E. coli) C600 harboring these plasmids were inoculated into chicken manure specimens that were placed in compost at 20 and 60 cm from the bottom of a 1.0-m high compost bin. Control specimens were held at ambient temperature. By day 10, compost temperatures at the lower and upper levels of the bin had reached 45.3 and 61.5°C, respectively, and at both levels the target E. coli had been inactivated and the plasmids had lost their capacity to be transformed or mobilized. Furthermore, based on real time Polymerase chain reaction (PCR), the transgene fragments along with the host chromosomal DNA fragment from specimens at the upper level had been degraded beyond the detection limit. However, at the lower level where temperatures remained below 48°C these fragment persisted to day 21. At ambient temperatures (0–8°C), the E. coli, plasmids and the transgene fragments persisted in manure specimens throughout the 21 day test period. The study showed the potential for composting as a safe procedure for disposal of bacteria carrying transgenes in recombinant plasmids.     

Molecular characterization of conjugative plasmids in pesticide tolerant and multi-resistant bacterial isolates from contaminated alluvial soil

A total of 35 bacteria from contaminated soil (cultivated fields) near pesticide industry from Chinhat, Lucknow, (India) were isolated and tested for their tolerance/resistance to pesticides, heavy metals and antibiotics. Bacterial isolates were identified by 16S rDNA sequencing. Gas Chromatography analysis of the soil samples revealed the presence of lindane at a concentration of 547 ng g⁻¹ and α-endosulfan and β-endosulfan of 422 ng g⁻¹ and 421 ng g⁻¹ respectively. Atomic Absorption Spectrophotometry analysis of the test sample was done and Cr, Zn, Ni, Fe, Cu and Cd were detected at concentrations of 36.2, 42.5, 43.2, 241, 13.3 and 11.20 mg kg⁻¹ respectively. Minimum inhibitory concentrations of all the isolates were determined for pesticides and heavy metals. All the multi-resistant/tolerant bacterial isolates were also tested for the presence of incompatibility (Inc) group IncP, IncN, IncW, IncQ plasmids and for rolling circle plasmids of the pMV158-family by PCR. Total community DNA was extracted from pesticide contaminated soil. PCR amplification of the bacterial isolates and soil DNA revealed the presence of IncP-specific sequences (trfA2 and oriT) which was confirmed by dot blot hybridization with RP4-derived DIG-labelled probes. Plasmids belonging to IncN, IncW and IncQ group were neither detected in the bacterial isolates nor in total soil DNA. The presence of conjugative or mobilizable IncP plasmids in the isolates indicate that these bacteria have gene transfer capacity with implications for dissemination of heavy metal and antibiotic resistance genes. We propose that IncP plasmids are mainly responsible for the spread of multi-resistant bacteria in the contaminated soils.     

城市生活污水中志贺氏菌ipaH毒力基因的定量PCR检测

基于ipaH毒力基因的实时荧光定量PCR检测,建立适合城市污水中志贺氏菌的定量检测方法。使用从临床分离出的志贺氏菌构建重组质粒作为实时荧光定量PCR的标准品。在ipaH基因模板量2.58×100～2.58×106copy范围内具有良好的线性关系,每100 mL水样中含有2.58×101copy以上的ipaH基因即可被检出。从西安市生活污水分离得到2株野生型志贺氏菌,分析ipaH基因数量和菌体数量的关系,从而确定ipaH基因定量检测志贺氏菌的可行性。该方法灵敏、快速、特异性好,适用于城市生活污水中志贺氏菌的检测。     

Aquatic environments polluted with antibiotics and heavy metals: a human health hazard

Aquatic environments often receive wastewater containing pollutants such as antibiotics and heavy metals from hospital sewage, as well as contaminants from soil. The presence of these pollutants can increase the rate of exchange of resistant genes between environmental and pathogenic bacteria, which can make the treatment of various types of bacterial infections in humans and animals difficult, in addition to causing environmental problems such as ecological risk. In this study, two tetracycline-resistant Pseudomonas aeruginosa (EW32 and EW33), isolated from aquatic environments close to industries and a hospital in southeastern Brazil, were investigated regarding the possible association between tetracycline and heavy metal resistance. The isolate EW32 presented a conjugative plasmid with coresistance to tetracycline and copper, reinforcing the concern that antibiotic resistance by acquisition of plasmids can be induced by the selective pressure of heavy metals in the environment.     

Illuminating reactive microbial transport in saturated porous media: demonstration of a visualization method and conceptual transport model

We demonstrate a method to study reactive microbial transport in saturated translucent porous media using the bacteria Pseudomonas fluorescens 5RL genetically engineered to carry a plasmid with bioluminescence genes inducible by salicylate. Induced bacteria were injected into a cryolite grain filled chamber saturated with a sterile non-growth-promoting (phosphorus limited) chemical mixture containing salicylate as an aromatic hydrocarbon analogue. The amount of light produced by the bacteria serves as an estimator of the relative efficiency of aerobic biodegradation since bioluminescence is dependent on both salicylate and oxygen but only consumes oxygen. Bioluminescence was captured with a digital camera and analyzed to study the evolving spatial pattern of the bulk oxygen consuming reactions. As fluid flow transported the bacteria through the chamber, bioluminescence was observed to initially increase until an oxygen depletion zone developed behind the advective front. Bacterial transport was modeled with the advection dispersion equation and oxygen concentration was modeled assuming bacterial consumption via Monod kinetics with consideration of additional effects of rate-limited mass transfer from residual gas bubbles. Consistent with previous measurements, bioluminescence was considered proportional to oxygen consumed. Using the observed bioluminescence, model parameters were fit that were consistent with literature values and produced results in good agreement with the experimental data. These findings demonstrate potential for using this method to investigate the complex spatial and temporal dynamics of reactive microbial transport in saturated porous media.     

In situ sequenced bioremediation of mixed contaminants in groundwater

A mixture of chlorinated solvents (about 0.5-10 mg/l), including tetrachloroethene (PCE) and carbon tetrachloride (CT), together with a petroleum hydrocarbon, toluene (TOL), were introduced into a 24 m long x 2 m wide x 3 m deep isolated section (henceforth called a gate) of the Borden aquifer and subjected to sequential in situ treatment. An identical section of aquifer was similarly contaminated and allowed to self-remediate by natural attenuation, thus serving as a control. The control presents a rare opportunity to critically assess the performance of the treatment systems, and represents the first such study for sequenced in situ remediation. The first treatment step was anaerobic bioremediation. This was accomplished using a modified nutrient injection wall (NIW) to pulse benzoate and a nutrient solution into the aquifer, maximizing mixing by dispersion and minimizing fouling near the injection wells. In the anaerobic bioactive zone that developed, PCE, CT and chloroform (CF), a degradation product of CT, degraded with a half-lives of about 59, 5.9 and 1.7 days, respectively. The second step was aerobic bioremediation, using a biosparge system. TOL and cis-1,2 dichloroethene (cDCE), from PCE degradation, were found to degrade aerobically with half-lives of 17 and 15 days, respectively. Compared to natural attenuation, PCE and TOL removal rates were significantly better in the sequenced treatment gate. However, CT and CF were similarly and completely attenuated in both gates. It is believed that the presence of TOL helped sustain the reducing environment needed for the reduction of these two compounds.     

Diversity of biphenyl degraders in a chlorobenzene polluted aquifer

Biphenyl degrading bacteria (40 strains) have been isolated along a gradient of chlorobenzene pollution from an aquifer which did not contain any PCB to answer the question of how metabolic/catabolic abilities exist in ecosystems that have not been stressed with the relevant substrates is important for intrinsic bioremediations. Only few of the isolates were characterized by 16S rRNA gene sequence analyses as Pseudomonas species while the majority were Gram-positive, belonging to the order Actinomycetales and representing the genera Rhodococcus and Arthrobacter. The strains could grow on a variety of chlorobenzoates but no pattern of substrate usage and phylogeny or pollution gradient could be found. Strains which were able to grow on 2,5-dichlorobenzoate were often also able to use 3,4- and 3,5-dichloro- and 2,3,5-trichlorobenzoate or those using 2-chlorobenzoate could usually use 2,6-dichlorobenzoate as well. From that results, it is concluded that a highly diverse, basic metabolic activity for PCB degradation existed at this site despite the absence of PCB.     

同步硝化反硝化好氧颗粒污泥微生物相和氮去除能力的研究

对颗粒污泥中的微生物菌群进行了分离和鉴定，并确定纯种微生物的脱氮能力。颗粒污泥中含假单胞菌属5株。亚硝化单胞菌属7株，硝化杆菌属4株，气单胞菌属3株，黄单胞菌属2株，孢杆菌属4株，黄色杆菌属2株和产碱菌属2株。这些菌属中包含了参与硝化反应的亚硝化单胞菌属和硝化杆菌属微生物，以及有好氧反硝化能力的Pseudomonas和Alcaligenes菌。29株微生物摇瓶混合培养的脱氮试验结果表明，30h内有机物去除率为75％，氮去除率近50％。     

Waste recombinant DNA: Effectiveness of thermo-treatment to manage potential gene pollution

Heating at 100 °C for 5–10 min is a common method for treating wastewater containing recombinant DNA in many bio-laboratories in China. In this experiment, plasmid pET-28b was used to investigate decay efficiency of waste recombinant DNA during thermo-treatment. The results showed that the decay half-life of the plasmid was 2.7–4.0 min during the thermo-treatment, and even heating for 30 min the plasmids still retained some transforming activity. Low pH promoted the decay of recombinant DNA, but NaCl, bovine serum albumin and EDTA, which existed in the most wastewater from bio-laboratories, protected DNA from degradation. Thus, the decay half-life of plasmid DNA may be longer than 2.7–4.0 min practically. These results suggest that the effectiveness of heating at 100 °C for treating waste recombinant DNA is low and a gene pollution risk remains when those thermo-treated recombinant DNAs are discharged into the environment. Therefore other simple and effective methods should be developed.     

Isolation and characterization of a diverse group of phenylacetic acid degrading microorganisms from pristine soil

A diverse range of microorganisms capable of growth on phenylacetic acid as the sole source of carbon and energy were isolated from soil. Sixty six different isolates were identified and grouped according to 16S rRNA gene RFLP analysis. Subsequent sequencing of 16S rDNA from selected strains allowed further characterization of the phenylacetic acid degrading population isolated from soil. Nearly half (30) of the isolates are Bacillus species while the rest of the isolates are strains from a variety of genera namely, Arthrobacter, Pseudomonas, Rhodococcus, Acinetobacter, Enterobacter, Flavobacterium, and Paenibacillus. Sixty-one of the sixty-six strains reproducibly grew in defined minimal liquid culture medium (E2). All strains isolated grew when at least one hydroxylated derivative of phenylacetic acid was supplied as the carbon source, while 59 out of the 61 strains tested, accumulated ortho-hydroxyphenylacetic acid in the assay buffer; when pulsed with phenylacetic acid. Oxygen consumption experiments failed to indicate a clear link between phenylacetic acid and hydroxy-substituted phenylacetic acid in isolates from a broad range of genera.     

Detection of dioxygenase genes present in various activated sludge

GOAL, SCOPE AND BACKGROUND: Activated sludge from refineries contains various microorganisms that could utilize aromatics under aerobic conditions due to the oxygenase enzymes. Dioxygenase enzymes are oxygenases, which are involved in the ring cleavage step of aromatic hydrocarbons. In this study, the selected catabolic loci involved in ring cleavage have been monitored in the activated sludge samples at different time intervals. The investigation of the dioxygenase genes in the Effluent Treatment Plants (ETPs) and evaluation of their presence at different time points provides a clue for the aromatic utilizing potential of the inherent microbial flora. METHODS: The catabolic gene loci pheB, xylE, tod-isp, bed and nahG responsible for the enzymes catechol 1,2-dioxygenase, catechol 2,3-dioxygenase, toluene dioxygenase-iron-sulphur protein component, benzene dioxygenase and naphthalene dioxygenase were used respectively. The time dependent change in eubacterial population was demonstrated by the amplification of 16S rDNA product, followed by restriction digestion. The template DNA was obtained from the activated sludge collected from ETPs. The supporting physiological data for the overall performance of sludge was developed using respirometric analysis. The on-site COD and MLSS analysis for ETP was used in final evaluation. The study was carried out with samples collected from three different ETPs and also from a selected ETP at different time intervals. RESULTS AND DISCUSSION: The respirometric studies were carried out with phenol, catechol, toluene, and naphthalene to arrive at the target genotypes for further study by PCR protocol. The respirometric analysis coupled with the COD and MLSS analysis represented the physiological capacity of the various sludges. Initially, the tracking protocol was optimized by using different sludge samples, which were collected from refineries. The selected genotypes were amplified and their presence has been confirmed using Southern analysis. The gene loci tod-isp, bed and xylE were commonly observed at various time intervals of the sludge from the same source. The gene loci pheB and nahG were found to be relatively rare. CONCLUSION: The 16S rDNA PCR products after restriction digestion produced different DNA fingerprint patterns, suggesting that the microbial community composition was diverse in the three sources. Similarly, the presence of the catechol 2,3-dioxygenase, benzene dioxygenase and toluene dioxygenase genes confirmed the aromatic degrading potential in the various sludges. The probes could not pick the nahG and pheB genes. However, the respirometeric assay suggested that the oxidative capacity to use naphthalene as a substrate exists. RECOMMENDATION AND PERSPECTIVE: Our study of the diversity at various time points from the ETP provided an overview of the shifts of the catabolic composition of the sludge. This also depends on the influential parameters like the incoming pollutant level and the environmental conditions that are prevailing and often changing from time to time. The results of direct DNA extraction and PCR amplification do reflect the relative abundance of a particular catabolic genotype, which could be used to monitor the efficiency of treatment.     

Phylogenetic changes in soil microbial and diazotrophic diversity with application of butachlor

We investigated changes in population and taxonomic distribution of cultivable bacteria and diazotrophs with butachlor application in rice paddy soils. Population changes were measured by the traditional plate-count method, and taxonomic distribution was studied by 16S rDNA sequencing, then maximum parsimony phylogenic analysis with bootstrapping (1,000 replications). The bacterial population was higher after 39 than 7 days of rice cultivation, which indicated the augmentation of soil microbes by rice root exudates. The application of butachlor increased the diazotrophic population in both upper (0–3 cm) and lower (3–15 cm) layers of soils. Especially at day 39, the population of diazotrophs was 1.8 and 1.6 times that of the control in upper and lower layer soils, respectively. We found several bacterial strains only with butachlor application; examples are strains closest to Bacillus arsenicus, B. marisflavi, B. luciferensis, B. pumilus, and Pseudomonas alvei. Among diazotrophs, three strains closely related to Streptomyces sp. or Rhrizobium sp. were found only with butachlor application. The population of cultivable bacteria and the species composition were both changed with butachlor application, which explains in part the contribution of butachlor to augmenting soil nitrogen-fixing ability.