Construction of an engineered strain free of antibiotic resistance gene markers for simultaneous mineralization of methyl parathion and ortho-nitrophenol

Pseudomonas sp. strain NyZ402, a native soil organism that grows on para-nitrophenol (PNP), was genetically engineered for the simultaneous degradation of methyl parathion (MP) and ortho-nitrophenol (ONP) by integrating mph (methyl parathion hydrolase gene) from Pseudomonas sp. strain WBC-3 and onpAB (ONP 2-monooxygenase and ONP o-benzoquinone reductase genes) from Alcaligenes sp. strain NyZ215 into the genome of strain NyZ402. Methyl parathion hydrolase (MPH), ONP 2-monooxygenase (OnpA) and o-benzoquinone reductase (OnpB) were constitutively expressed in the engineered strain NyZ-MO. Strain NyZ-MO was free of exogenous antibiotic resistance gene markers and the introduced genes were genetically stable. Degradation experiments showed that strain NyZ-MO could utilize MP or ONP as the sole carbon and energy source, and mineralize 0.1 mM MP–0.1 mM ONP simultaneously. This method may serve as a useful strategy for the construction of engineered strains in the degradation of multiple environmental pollutants.     

Characterization of a para-nitrophenol catabolic cluster in Pseudomonas sp. strain NyZ402 and construction of an engineered strain capable of simultaneously mineralizing both para- and ortho-nitrophenols

Pseudomonas sp. strain NyZ402 was isolated for its ability to grow on para-nitrophenol (PNP) as a sole source of carbon, nitrogen, and energy, and was shown to degrade PNP via an oxidization pathway. This strain was also capable of growing on hydroquinone or catechol. A 15, 818 bp DNA fragment extending from a 800-bp DNA fragment of hydroxyquinol 1,2-dioxygenase gene (pnpG) was obtained by genome walking. Sequence analysis indicated that the PNP catabolic gene cluster (pnpABCDEFG) in this fragment shared significant similarities with a recently reported gene cluster responsible for PNP degradation from Pseudomonas sp. strain WBC-3. PnpA is PNP 4-monooxygenase converting PNP to hydroquinone via benzoquinone in the presence of NADPH, and genetic analysis indicated that pnpA plays a key role in PNP degradation. pnpA1 present in the upstream of the cluster (absent in the cluster from strain WBC-3) encodes a protein sharing as high as 55% identity with PnpA, but was not involved in PNP degradation by either in vitro or in vivo analyses. Furthermore, an engineered strain capable of growing on PNP and ortho-nitrophenol (ONP) was constructed by introducing onpAB (encoding ONP monooxygenase and ortho-benzoquinone reductase which catalyzed the transformation of ONP to catechol) from Alcaligenes sp. strain NyZ215 into strain NyZ402.     

Bioaugmentation of a methyl parathion contaminated soil with Pseudomonas sp. strain WBC-3

Pseudomonas sp. strain WBC-3 utilizes methyl parathion (MP) and para-nitrophenol as the sole source of carbon, nitrogen and energy. In this study, strain WBC-3 was inoculated into lab-scale MP-contaminated soil for bioaugmentation. Accelerated removal of MP was achieved in bioaugmentation treatment compared to non-bioaugmentation treatment, with complete removal of 0.536 mg g⁻¹ dry soil in bioaugmentation treatment within 15 days and without accumulation of toxic intermediates. The analysis of denaturing gradient gel electrophoresis and real-time PCR showed that strain WBC-3 existed stably during the entire bioaugmentation period. Simultaneously, redundancy analysis for evaluating the relationships between the environmental factors and microbial community structure indicated that the indigenous bacterial community structure was significantly influenced by strain WBC-3 inoculation (P = 0.002).     

Biodegradation of methyl parathion in the presence of goethite: The effect of Pseudomonas sp. Z1 adhesion

In this research, the influence of goethite on biodegradation kinetic of methyl parathion was investigated in the presence of Pseudomonas sp. Z1. Semipermeable membrane experiments were performed to demonstrate the role of adhesion of degrading bacteria to surface of goethite in biodegradation of methyl parathion. Sorption of methyl parathion and bacteria onto goethite particles were also measured to assess the distribution of methyl parathion and bacteria between water and goethite surface. The first-order degradation rate constant of methyl parathion in different concentrations of goethite was in the order of 0.1 g L⁻¹ > 0.01 g L⁻¹ > 0 g L⁻¹ > 1 g L⁻¹ > 20 g L⁻¹, suggesting the presence of low concentrations of goethite accelerated the biodegradation of methyl parathion and high concentrations of goethite inhibited this biodegradation process. According to the result of semipermeable membrane experiment, when no bacterial attachment occurred in the system, the promotive effect of 0.1 g L⁻¹ goethite for microbial degradation was disappeared and the inhibition effect of 20 g L⁻¹ goethite increased. The results clearly demonstrated that the adhesion of bacteria to goethite was beneficial to the biodegradation of methyl parathion. The information obtained is of fundamental significance for the understanding of microbial degradation of organic pollution in soil.     

Degradation of cyclohexylamine by a new isolate of <Emphasis Type="Italic">Pseudomonas plecoglossicida</Emphasis>

A strain utilizing cyclohexylamine as the sole source of carbon and nitrogen, designated NyZ12, was isolated from soil and identified by 16S rDNA sequencing as Pseudomonas plecoglossicida. This bacterium released ammonia into the medium when grown on cyclohexylamine, and also grows readily on cyclohexanone as the sole carbon source, suggesting that degradation involves an initial deamination step.     

Behaviour and recovery of the secondary metabolite batatasin-III from boreal forest humus: influence of temperature,humus type and microbial community

Batatasin-III (3,3′-dihydroxy-5-methoxybibenzyl) produced by Empetrum hermaphroditum has been identified as the main metabolite responsible for the chemical interference exerted by the shrub on the surrounding vegetation in the boreal forest of northern Sweden. In earlier studies, batatasin-III has been found to be present in both soil and soil solutions. However, to understand the actual mechanisms by which batatasin-III may interact, we need to know more about the fate and behaviour of the compound in soil. In order to achieve this, we firstly evaluated the efficiency of different extraction methods in recovering batatasin-III from Empetrum humus and found that the highest yield was obtained using ethyl acetate (6.57 ± 20 μg g⁻¹ HDM, least square mean ± SE). In contrast, no detectable amounts of batatasin-III were obtained when extracting the humus with distilled water, 0.125 M citric acid or 0.05 M NaOH. Secondly, we performed a series of experiments in which the recovery of batatasin-III was determined from humus samples exposed to different treatments. In summary, the recovery of batatasin-III was found to be initially strongly dependent on humus type (i.e. humus collected from under a cover of Empetrum, Vaccinium or forest herbs) as well as the temperature and the length of the incubation period. The amount of recovered batatasin-III was in general highest from the Empetrum humus and lowest from the herb humus, contrary to our hypothesis that microorganisms might be better adapted to batatasin-III in humus of the Empetrum origin than in humus of other origin. Regardless of humus type, the recovery was generally higher at +2 °C than at +18 °C suggesting that microbial degradation of batatasin-III did occur. However, when the recovery of batatasin-III from sterile and non-sterile humus was compared, microbial degradation was found to be of minor importance. In addition, no phenolic degradation products of batatasin-III were detected after batatasin-III had been added to non-sterile humus. The obtained results suggest that batatasin-III, when released into humus, becomes physically trapped by organic matter and metabolized by soil microbes to a less extent.     

Survival of a novel endophytic fungus Phomopsis liquidambari B3 in the indole-contaminated soil detected by real-time PCR and its effects on the indigenous microbial community

The recently isolated fungal strain Phomopsis liquidambari B3 can degrade high concentrations of indole, indicating its potential for the bioremediation of indole-contaminated soil. In this study, a specific real-time PCR was developed to detect the survival of P. liquidambari B3 in soil. Subsequently, degradation activity of strain B3 and its effects on indigenous microbial community were analyzed. Results showed the amount of P. liquidambari B3 genomic DNA increased to a maximum 5.67 log (pg g⁻¹ dry soil) 10 days after inoculation of 5.04 log (pg g⁻¹ dry soil), and then gradually decreased with time and after 40 days it was below the detection limit. By the end of the experiment (day 40), bioaugmented microsoms showed a 93.7% decrease in indole, while the values for biostimulated and control microcosms were much lower. Higher microbial biomass and enzyme activities were observed in bioaugmented soil. Denaturing gradient gel electrophoresis analysis showed bioaugmentation increased richness of resident microbial community. These results indicate that P. liquidambari B3 is effective for the remediation of indole-contaminated soil and also provides valuable information about the behavior of the inoculant population during bioremediation, which could be directly used in the risk assessment of inoculant population and optimization of bioremediation process.     

Biodegradation of trichloroethylene and toluene by indigenous microbial populations in vadose sediments

The unsaturated subsurface (vadose zone) receives significant amounts of hazardous chemicals, yet little is known about its microbial communities and their capacity to biodegrade pollutants. Trichloroethylene (TCE) biodegradation occurs readily in surface soils; however, the process usually requires enzyme induction by aromatic compounds, methane, or other cosubstrates. The aerobic biodegradation of toluene and TCE by indigenous microbial populations was measured in samples collected from the vadose zone at unpolluted and gasoline-contaminated sites. Incubation at field moisture levels showed little activity on either TCE or toluene, so samples were tested in soil suspensions. No degradation occurred in samples suspended in water or phosphate buffer solution; however, both toluene and TCE were degraded in samples suspended in mineral salts medium. TCE degradation depended on toluene degradation, and little loss occurred under sterile conditions. Studies with specific nutrients showed that addition of ammonium sulfate was essential for degradation, and addition of other mineral nutrients further enhanced the rate. Additional studies with vadose sediments amended with nutrients showed similar trends to those observed in sediment suspensions. Initial rates of biodegradation in suspensions were faster in uncontaminated samples than in gasolinecontaminated samples, but the same percentages of chemicals were degraded. Biodegradation was slower and less extensive in shallower samples than deeper samples from the uncontaminated site. Two toluene-degrading organisms isolated from a gasoline-contaminated sample were identified as Corynebacterium variabilis SVB74 and Acinetobacter radioresistens SVB65. Inoculation with 10⁶ cells of C. variabilis ml^–1 of soil solution did not enhance the rate of degradation above that of the indigenous population. These results indicate that mineral nutrients limited the rate of TCE and toluene degradation by indigenous populations and that no additional benefit was derived from inoculation with a toluene-degrading bacterial strain. Correspondence to: K.M. Scow     

Biotechnological Potential of Bacillus salmalaya 139SI: A Novel Strain for Remediating Water Polluted with Crude Oil Waste

Environmental contamination by petroleum hydrocarbons, mainly crude oil waste from refineries, is becoming prevalent worldwide. This study investigates the bioremediation of water contaminated with crude oil waste. Bacillus salamalaya 139SI, a bacterium isolated from a private farm soil in the Kuala Selangor in Malaysia, was found to be a potential degrader of crude oil waste. When a microbial population of 10⁸ CFU ml^-1 was used, the 139SI strain degraded 79% and 88% of the total petroleum hydrocarbons after 42 days of incubation in mineral salt media containing 2% and 1% of crude oil waste, respectively, under optimum conditions. In the uninoculated medium containing 1% crude oil waste, 6% was degraded. Relative to the control, the degradation was significantly greater when a bacteria count of 99 × 10⁸ CFU ml^-1 was added to the treatments polluted with 1% oil. Thus, this isolated strain is useful for enhancing the biotreatment of oil in wastewater.     

Ecological risk assessment of toxic elements,microbial loads,parasites and antinutritional factors in Telfairia occidentalis grown on sewage contaminated soil

The aim of this study was to evaluate the ecological health risk assessment of toxic elements, microbial load, and anti-nutrient constituent build-up in vegetables, Talfairia occidentalis, grown on sewage contaminated soil. Samples of soil and vegetables were taken from farms near the dumpsites (sites A). As a control, samples were taken from an area with no dump sites (sites B). Atomic Absorption Spectrophotometry was used to determine the potentially toxic elements. The basic titrimetric method was used to estimate Phytate and Oxalate in the vegetables in order to assess anti-nutrient constituents. The content of cyanogenic glycosides, tannins, and alkaloids in vegetable samples was determine using a standard method. Total heterotrophic bacteria, E. coli, total coliform, faecal coliform, Staphylococcus aureus, Salmonella, and intestinal parasites were all determined using APHA standard methods. For all samples analyzed, the concentrations of metals – Cr, Zn, Pb, Cu, and Ni in site A were higher than those in site B. The soil and vegetable samples differed from the controls by a significant amount (P <0.05). Vegetable samples from site A were mainly infected with faecal coliform TO (9.7 × 10⁵cfu/g) and other bacteria at levels higher than the recommended levels. In contrast to samples from site B, which were not infected with human intestinal helminth parasites, Ascaris lumbricoides ova and Entamoeba histolytica cryst were present in vegetables in site A samples. The majority of the values obtained for the sewage dumpsite were significantly higher than the values recorded by the World Health Organization (WHO). However, sewage wastes, anthropogenic sources, and atmospheric depositions cause higher concentrations of heavy metals, anti-nutrients, and microbial load around dumpsites, which bioaccumulate in vegetables through uptake from the soil and eventual entry into the food chain. The overall evaluation found that consumption of vegetables grown on sewage soil poses a risk of heavy metals, microbial loads, and anti-nutrients adversely affecting human health.     

Association of Microbial Community Composition and Activity with Lead, Chromium, and Hydrocarbon Contamination

Microbial community composition and activity were characterized in soil contaminated with lead (Pb), chromium (Cr), and hydrocarbons. Contaminant levels were very heterogeneous and ranged from 50 to 16,700 mg of total petroleum hydrocarbons (TPH) kg of soil⁻¹, 3 to 3,300 mg of total Cr kg of soil⁻¹, and 1 to 17,100 mg of Pb kg of soil⁻¹. Microbial community compositions were estimated from the patterns of phospholipid fatty acids (PLFA); these were considerably different among the 14 soil samples. Statistical analyses suggested that the variation in PLFA was more correlated with soil hydrocarbons than with the levels of Cr and Pb. The metal sensitivity of the microbial community was determined by extracting bacteria from soil and measuring [³H]leucine incorporation as a function of metal concentration. Six soil samples collected in the spring of 1999 had IC₅₀ values (the heavy metal concentrations giving 50% reduction of microbial activity) of approximately 2.5 mM for CrO₄²⁻ and 0.01 mM for Pb²⁺. Much higher levels of Pb were required to inhibit [¹⁴C]glucose mineralization directly in soils. In microcosm experiments with these samples, microbial biomass and the ratio of microbial biomass to soil organic C were not correlated with the concentrations of hydrocarbons and heavy metals. However, microbial C respiration in samples with a higher level of hydrocarbons differed from the other soils no matter whether complex organic C (alfalfa) was added or not. The ratios of microbial C respiration to microbial biomass differed significantly among the soil samples (P < 0.05) and were relatively high in soils contaminated with hydrocarbons or heavy metals. Our results suggest that the soil microbial community was predominantly affected by hydrocarbons.     

Effect of Dissemination of 2,4-Dichlorophenoxyacetic Acid (2,4-D) Degradation Plasmids on 2,4-D Degradation and on Bacterial Community Structure in Two Different Soil Horizons

Transfer of the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids pEMT1 and pJP4 from an introduced donor strain, Pseudomonas putida UWC3, to the indigenous bacteria of two different horizons (A horizon, depth of 0 to 30 cm; B horizon, depth of 30 to 60 cm) of a 2,4-D-contaminated soil was investigated as a means of bioaugmentation. When the soil was amended with nutrients, plasmid transfer and enhanced degradation of 2,4-D were observed. These findings were most striking in the B horizon, where the indigenous bacteria were unable to degrade any of the 2,4-D (100 mg/kg of soil) during at least 22 days but where inoculation with either of the two plasmid donors resulted in complete 2,4-D degradation within 14 days. In contrast, in soils not amended with nutrients, inoculation of donors in the A horizon and subsequent formation of transconjugants (10⁵ CFU/g of soil) could not increase the 2,4-D degradation rate compared to that of the noninoculated soil. However, donor inoculation in the nonamended B-horizon soil resulted in complete degradation of 2,4-D within 19 days, while no degradation at all was observed in noninoculated soil during 89 days. With plasmid pEMT1, this enhanced degradation seemed to be due only to transconjugants (10⁵ CFU/g of soil), since the donor was already undetectable when degradation started. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes showed that inoculation of the donors was followed by a shift in the microbial community structure of the nonamended B-horizon soils. The new 16S rRNA gene fragments in the DGGE profile corresponded with the 16S rRNA genes of 2,4-D-degrading transconjugant colonies isolated on agar plates. This result indicates that the observed change in the community was due to proliferation of transconjugants formed in soil. Overall, this work clearly demonstrates that bioaugmentation can constitute an effective strategy for cleanup of soils which are poor in nutrients and microbial activity, such as those of the B horizon.     

深渊沉积物中盐单胞菌(Halomonas sp.)NyZ771菌株的分离培养及其降解芳香酸的研究

【背景】海洋是地球上最大的碳库,也是地球生物最大的栖息地。在这个庞大的生态系统中拥有多种多样的微生物,它们在全球碳循环中扮演了重要的角色。海斗深渊(海平面6 000 m以下的海域)由于高静水压和表层沉积汇集了大量有机质,形成了包含丰富生物资源的特殊生境。【目的】从马里亚纳海沟海斗深渊沉积物样品中分离培养能够以芳香酸为唯一碳源和能源生长的微生物,并研究其降解特性。【方法】通过模拟原位高压环境富集培养和常压条件下芳香酸选择性分离培养获得深渊来源的纯培养细菌,并根据形态学观察和16S rRNA基因序列系统发育分析进行种属鉴定,利用不同芳香酸进行培养和生物转化,通过HPLC和LC/MS鉴定芳香酸代谢中间产物。【结果】从马里亚纳海沟6 300 m沉积物样本中分离获得了一株盐单胞菌(Halomonas sp.)NyZ771。该菌株能够利用苯甲酸和4-羟基苯甲酸作为唯一碳源生长。其代谢4-羟基苯甲酸的中间产物鉴定为原儿茶酸。【结论】从深渊沉积物样本分离得到一株能降解苯甲酸和4-羟基苯甲酸的盐单胞菌NyZ771,丰富了深渊来源的微生物资源,为今后研究深渊中微生物的芳香酸降解及海洋微生物驱动的碳循环提供了一定的理论基础。     

The microbially mediated soil organic carbon loss under degenerative succession in an alpine meadow

Land‐cover change has long been recognized as having marked effect on the amount of soil organic carbon (SOC). However, the microbially mediated processes and mechanisms on SOC are still unclear. In this study, the soil samples in a degenerative succession from alpine meadow to alpine steppe meadow in the Qinghai–Tibetan Plateau were analysed using high‐throughput technologies, including Illumina sequencing and geochip functional gene arrays. The soil microbial community structure and diversity were significantly (p < .05) different between alpine meadow and alpine steppe meadow; the microbial ɑ‐diversity in alpine steppe meadow was significantly (p < .01) higher than in alpine meadow. Molecular ecological network analysis indicated that the microbial community structure in alpine steppe meadow was more complex and tighter than in the alpine meadow. The relative abundance of soil microbial labile carbon degradation genes (e.g., pectin and hemicellulose) was significantly higher in alpine steppe meadow than in alpine meadow, but the relative abundance of soil recalcitrant carbon degradation genes (e.g., chitin and lignin) showed the opposite tendency. The Biolog Ecoplate experiment showed that microbially mediated soil carbon utilization was more active in alpine steppe meadow than in alpine meadow. Consequently, more soil labile carbon might be decomposed in alpine steppe meadow than in alpine meadow. Therefore, the degenerative succession of alpine meadow because of climate change or anthropogenic activities would most likely decrease SOC and nutrients medicated by changing soil microbial community structure and their functional potentials for carbon decomposition.     

A Method for Detection of Pseudobactin, the Siderophore Produced by a Plant-Growth-Promoting Pseudomonas Strain, in the Barley Rhizosphere

Detection in the rhizosphere of the siderophore produced by an inoculated microorganism is critical to determining the role of microbial iron chelators on plant growth promotion. We previously reported the development of monoclonal antibodies (MAb) to ferric pseudobactin, the siderophore of plant-growth-promoting Pseudomonas strain B10. One of these MAb reacted less strongly to pseudobactin than to ferric pseudobactin. The MAb reacted to Al(III), Cr(III), Cu(II), and Mn(II) complexes of pseudobactin at a level similar to the level at which it reacted to ferric pseudobactin and reacted less to the Zn(II) complex, but these metals would make up only a small fraction of chelated pseudobactin in soil on the basis of relative abundance of metals and relative binding constants. Fourteen-day-old barley plants grown in limed and autoclaved soil were inoculated with 10⁹ CFU of Pseudomonas strain Sm1-3, a strain of Pseudomonas B10 Rif^r Nal^r selected for enhanced colonization, and sampled 3 days later. Extraction and analysis of the roots and surrounding soil using the MAb in an immunoassay indicated a concentration of 3.5 × 10^-10 mol of ferric pseudobacting g^-1 (wet weight). This is the first direct measurement of a pseudobactin siderophore in soil or rhizosphere samples.     

Degradation of 3-Phenoxybenzoic Acid by a Bacillus sp

3-Phenoxybenzoic acid (3-PBA) is of great environmental concern with regards to endocrine disrupting activity and widespread occurrence in water and soil, yet little is known about microbial degradation in contaminated regions. We report here that a new bacterial strain isolated from soil, designated DG-02, was shown to degrade 95.6% of 50 mg·L⁻¹ 3-PBA within 72 h in mineral salt medium (MSM). Strain DG-02 was identified as Bacillus sp. based on the morphology, physio-biochemical tests and 16S rRNA sequence. The optimum conditions for 3-PBA degradation were determined to be 30.9°C and pH 7.7 using response surface methodology (RSM). The isolate converted 3-PBA to produce 3-(2-methoxyphenoxy) benzoic acid, protocatechuate, phenol, and 3,4-dihydroxy phenol, and subsequently transformed these compounds with a q _max, K _s and K _i of 0.8615 h⁻¹, 626.7842 mg·L⁻¹ and 6.7586 mg·L⁻¹, respectively. A novel microbial metabolic pathway for 3-PBA was proposed on the basis of these metabolites. Inoculation of strain DG-02 resulted in a higher degradation rate on 3-PBA than that observed in the non-inoculated soil. Moreover, the degradation process followed the first-order kinetics, and the half-life (t _1/2) for 3-PBA was greatly reduced as compared to the non-inoculated control. This study highlights an important potential application of strain DG-02 for the in situ bioremediation of 3-PBA contaminated environments.     

Biodegradation of the Allelopathic Chemical m-Tyrosine by Bacillus aquimaris SSC5 Involves the Homogentisate Central Pathway

m-Tyrosine is an amino acid analogue, exuded from the roots of fescue grasses, which acts as a potent allelopathic and a broad spectrum herbicidal chemical. Although the production and toxic effects of m-tyrosine are known, its microbial degradation has not been documented yet. A soil microcosm study showed efficient degradation of m-tyrosine by the inhabitant microorganisms. A bacterial strain designated SSC5, that was able to utilize m-tyrosine as the sole source of carbon, nitrogen, and energy, was isolated from the soil microcosm and was characterized as Bacillus aquimaris. Analytical methods such as HPLC, GC-MS, and ¹H-NMR performed on the resting cell samples identified the formation of 3-hydroxyphenylpyruvate (3-OH-PPA), 3-hydroxyphenylacetate (3-OH-PhAc), and homogentisate (HMG) as major intermediates in the m-tyrosine degradation pathway. Enzymatic assays carried out on cell-free lysates of m-tyrosine-induced cells confirmed transamination reaction as the first step of m-tyrosine degradation. The intermediate 3-OH-PhAc thus obtained was further funneled into the HMG central pathway as revealed by a hydroxylase enzyme assay. Subsequent degradation of HMG occurred by ring cleavage catalyzed by the enzyme homogentisate 1, 2-dioxygenase. This study has significant implications in terms of understanding the environmental fate of m-tyrosine as well as regulation of its phytotoxic effect by soil microorganisms.     

Simultaneous degradation of the pesticides methyl parathion and chlorpyrifos by an isolated bacterial consortium from a contaminated site

The simultaneous degradation of the pesticide methyl parathion and chlorpyrifos was tested using a bacterial consortium obtained by selective enrichment from highly contaminated soils in Moravia (Medellin, Colombia). Microorganisms identified in the consortium were 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. In culture medium enriched with each of the pesticides, the consortium was able to degrade 150 mg l⁻¹ of methyl parathion and chlorpyrifos in 120 h. When a mixture of 150 mg l⁻¹ of both pesticides was used the percentage decreased to 72% for methyl parathion and 39% for chlorpyrifos. With the addition of glucose to the culture medium, the consortium simultaneously degraded 150 mg l⁻¹ of the pesticides in the mixture. 4 treatments were carried out in soil that included the addition of glucose with microorganisms, the addition of sugar cane with microorganisms, microorganisms without nutrient addition and without the addition of any item. In the treatment in which glucose was used, degradation percentages of methyl parathion and chlorpyrifos of 98% and 97% respectively were obtained in 120 h. This treatment also achieved the highest percentage of reduction in toxicity, monitored with Vibrio fischeri.     

Evaluation of Methyl Bromide Alternatives Efficacy against Soil-Borne Pathogens,Nematodes and Soil Microbial Community

Methyl bromide (MB) and other alternatives were evaluated for suppression of Fusarium spp., Phytophthora spp., and Meloidogyne spp. and their influence on soil microbial communities. Both Fusarium spp. and Phytophthora spp. were significantly reduced by the MB (30.74 mg kg^-1), methyl iodide (MI: 45.58 mg kg^-1), metham sodium (MS: 53.92 mg kg^-1) treatments. MS exhibited comparable effectiveness to MB in controlling Meloidogyne spp. and total nematodes, followed by MI at the tested rate. By contrast, sulfuryl fluoride (SF: 33.04 mg kg^-1) and chloroform (CF: 23.68 mg kg^-1) showed low efficacy in controlling Fusarium spp., Phytophthora spp., and Meloidogyne spp. MB, MI and MS significantly lowered the abundance of different microbial populations and microbial biomass in soil, whereas SF and CF had limited influence on them compared with the control. Diversity indices in Biolog studies decreased in response to fumigation, but no significant difference was found among treatments in PLFA studies. Principal component and cluster analyses of Biolog and PLFA data sets revealed that MB and MI treatments greatly influenced the soil microbial community functional and structural diversity compared with SF treatment. These results suggest that fumigants with high effectiveness in suppressing soil-borne disease could significantly influence soil microbial community.     

Novel Phenanthrene-Degrading Bacteria Identified by DNA-Stable Isotope Probing

Microorganisms responsible for the degradation of phenanthrene in a clean forest soil sample were identified by DNA-based stable isotope probing (SIP). The soil was artificially amended with either ¹²C- or ¹³C-labeled phenanthrene, and soil DNA was extracted on days 3, 6 and 9. Terminal restriction fragment length polymorphism (TRFLP) results revealed that the fragments of 219- and 241-bp in HaeIII digests were distributed throughout the gradient profile at three different sampling time points, and both fragments were more dominant in the heavy fractions of the samples exposed to the ¹³C-labeled contaminant. 16S rRNA sequencing of the ¹³C-enriched fraction suggested that Acidobacterium spp. within the class Acidobacteria, and Collimonas spp. within the class Betaproteobacteria, were directly involved in the uptake and degradation of phenanthrene at different times. To our knowledge, this is the first report that the genus Collimonas has the ability to degrade PAHs. Two PAH-RHD_α genes were identified in ¹³C-labeled DNA. However, isolation of pure cultures indicated that strains of Staphylococcus sp. PHE-3, Pseudomonas sp. PHE-1, and Pseudomonas sp. PHE-2 in the soil had high phenanthrene-degrading ability. This emphasizes the role of a culture-independent method in the functional understanding of microbial communities in situ.