Root exudates modify bacterial diversity of phenanthrene degraders in PAH-polluted soil but not phenanthrene degradation rates

To determine whether the diversity of phenanthrene‐degrading bacteria in an aged polycyclic aromatic hydrocarbon (PAH) contaminated soil is affected by the addition of plant root exudates, DNA stable isotope probing (SIP) was used. Microcosms of soil with and without addition of ryegrass exudates and with ¹³C‐labelled phenanthrene (PHE) were monitored over 12 days. PHE degradation was slightly delayed in the presence of added exudate after 4 days of incubation. After 12 days, 68% of added PHE disappeared both with and without exudate. Carbon balance using isotopic analyses indicated that a part of the ¹³C‐PHE was not totally mineralized as ¹³CO₂ but unidentified ¹³C‐compounds (i.e. ¹³C‐PHE or ¹³C‐labelled metabolites) were trapped into the soil matrix. Temporal thermal gradient gel electrophoresis (TTGE) analyses of 16S rRNA genes were performed on recovered ¹³C‐enriched DNA fractions. 16S rRNA gene banding showed the impact of root exudates on diversity of PHE‐degrading bacteria. With PHE as a fresh sole carbon source, Pseudoxanthomonas sp. and Microbacterium sp. were the major PHE degraders, while in the presence of exudates, Pseudomonas sp. and Arthrobacter sp. were favoured. These two different PHE‐degrading bacterial populations were also distinguished through detection of PAH‐ring hydroxylating dioxygenase (PAH‐RHD_α) genes by real‐time PCR. Root exudates favoured the development of a higher diversity of bacteria and increased the abundance of bacteria containing known PAH‐RHD_α genes.     

Novel bacteria capable of degrading phenanthrene in activated sludge revealed by stable-isotope probing coupled with high-throughput sequencing

The indigenous microorganisms responsible for degrading phenanthrene (PHE) in activated biosludge were identified using DNA-based stable isotope probing. Besides the well-known PHE degraders Burkholderia, Ralstonia, Sinobacteraceae and Arthrobacter, we for the first time linked the taxa Paraburkholderia and Kaistobacter with in situ PHE biodegradation. Analysis of PAH-RHD_α gene detected in the heavy DNA fraction of ¹³C-PHE treatment suggested the mechanisms of horizontal gene transfer or inter-species hybridisation in PAH-RHD gene spread within the microbial community. Additionally, three cultivable PHE degraders, Microbacterium sp. PHE-1, Rhodanobacter sp. PHE-2 and Rhodococcus sp. PHE-3, were isolated from the same activated biosludge. Among them, Rhodanobacter sp. PHE-2 is the first identified strain in its genus with PHE-degrading ability. However, the involvement of these strains in PHE degradation in situ was questionable, due to their limited enrichment in the heavy DNA fraction of ¹³C-PHE treatment and lack of PAH-RHD_α gene found in these isolates. Collectively, our findings provide a deeper understanding of the diversity and functions of indigenous microbes in PHE degradation.     

Rhizospheric degradation of phenanthrene is a function of proximity to roots

Rhizodegradation of recalcitrant organic pollutants, such as polycyclic aromatic hydrocarbons (PAH), may benefit from the major role that root exudates have on rhizospheric microbial processes. We investigated the influence of the proximity to ryegrass (Lolium perenneL.) roots on microbial populations and their biodegradation of phenanthrene (PHE) using compartmented pots. PAH degrading bacteria, total heterotrophic bacteria and PHE biodegradation were quantified in three consecutive sections at different distance (0–3, 3–6, 6–9 mm) from a mat of actively exuding roots. A bacterial gradient was observed with higher numbers of heterotrophs and PAH degrading bacteria closest to the roots. In parallel, a PHE biodegradation gradient was evident in the presence of roots. The biodegradation reached 86%, 48% and 36% of initially added PHE, respectively, in the layers 0–3 mm, 3–6 and 6–9 mm from the roots. The biodegradation rate was similar throughout the three layers of the non planted control. The present experimental system seems well suited for spatial and dynamic studies of PAH rhizoremediation.     

Effect of two types of biosurfactants on phenanthrene availability to the bacterial bioreporter Burkholderia sartisoli strain RP037

Biosurfactants are tensio-active agents that have often been proposed as a means to enhance the aqueous solubility of hydrophobic organic contaminants, such as polycyclic aromatic hydrocarbons (PAHs). Biosurfactant-producing bacteria such as those belonging to the genus Pseudomonas might therefore enhance PAH availability to PAH-degrading bacteria. We tested the effects of two types of biosurfactants produced by Pseudomonas sp., cyclic lipopeptides and rhamnolipids, on phenanthrene bioavailability. Bioavailability was judged from growth rates on phenanthrene and from specific induction of a phenanthrene-responsive GFP-reporter in Burkholderia sartisoli strain RP037. Co-culturing of strain RP037 with the lipopeptide-producing bacterium Pseudomonas putida strain PCL1445 enhanced GFP expression compared to a single culture, but this effect was not significantly different when strain RP037 was co-cultivated with a non-lipopeptide-producing mutant of P. putida. The addition of partially purified supernatant extracts from the P. putida lipopeptide producer equally did not unequivocally enhance phenanthrene bioavailability to strain RP037 compared to controls. In contrast, a 0.1% rhamnolipid solution strongly augmented RP037 growth rates on phenanthrene and led to a significantly larger proportion of cells in culture with high GFP expression. Our data therefore suggest that biosurfactant effects may be strongly dependent on the strain and type of biosurfactant.     

Degradation of phenanthrene by the endophytic fungus <Emphasis Type="Italic">Ceratobasidum stevensii</Emphasis> found in <Emphasis Type="Italic">Bischofia polycarpa</Emphasis>

Some strains of white rot fungi, non-lignolytic fungi and litter-decomposing basidiomycetes have been recognized as PAH degraders. The purpose of our research was to enlarge the scope of PAH-degrading fungi and explore the huge endophytic microorganism resource for bioremediation of PAHs. In this study, phenanthrene was used as a model PAHs compound. Nine strains of endophytic fungi isolated from four kinds of plant from Eupharbiaceae were screened for degradation of phenanthrene. The endophytic fungus Ceratobasidum stevensii (strain B6) isolated from Bischofia polycarpam showed high degradation efficiency and was selected for further studies. Into the fungal culture, 100 mg l⁻¹ phenanthrene was added, and after 10 days of incubation, about 89.51% of the phenanthrene was removed by strain B6. Extracellular ligninolytic enzyme activities of strain B6 were tested. The results showed that manganese peroxidase [MnP] was the predominant ligninolytic enzyme and that its production was greatly induced by the presence of phenanthrene. To confirm the involvement of MnP in phenanthrene degradation, promotion and inhibition studies on MnP in different concentration level of Mn²⁺ and NaN₃ were performed. Additionally, fungal mycelium-free and resuspended experiments were carried out. The results showed no apparent correlation between MnP activity and phenanthrene degradation. The mycelium and fresh medium were the crucial factors affecting the degradation of phenanthrene. To date, this is the first report on PAH degradation by Ceratobasidum stevensii. This study suggests that endophytic fungi might be a novel and important resource for microorganisms that have PAH-degrading capabilities.     

Extracellular polymeric substances enhanced mass transfer of polycyclic aromatic hydrocarbons in the two-liquid-phase system for biodegradation

The objective was to elucidate the role of extracellular polymeric substances (EPS) in biodegradation of polycyclic aromatic hydrocarbons in two-liquid-phase system (TLPs). Therefore, biodegradation of phenanthrene (PHE) was conducted in a typical TLPs—silicone oil–water—with PHE-degrading bacteria capable of producing EPS, Sphingobium sp. PHE3 and Micrococcus sp. PHE9. The results showed that the presence of both strains enhanced mass transfer of PHE from silicone oil to water, and that biodegradation of PHE mainly occurred at the interfaces. The ratios of tightly bound (TB) proteins to TB polysaccharides kept almost constant, whereas the ratios of loosely bound (LB) proteins to LB polysaccharides increased during the biodegradation. Furthermore, polysaccharides led to increased PHE solubility in the bulk water, which resulted in an increased PHE mass transfer. Both LB-EPS and TB-EPS (proteins and polysaccharides) correlated with PHE mass transfer in silicone oil, indicating that both proteins and polysaccharides favored bacterial uptake of PHE at the interfaces. It could be concluded that EPS could facilitate microbial degradation of PHE in the TLPs.     

Differential composition of bacterial communities as influenced by phenanthrene and dibenzo[a,h]anthracene in the rhizosphere of ryegrass (Lolium perenne L.)

Bioremediation technologies of Polycyclic Aromatic Hydrocarbons (PAH) are often limited by the recalcitrance to biodegradation of high molecular weight (HMW) PAH. Rhizosphere is known to increase the biodegradation of PAH but little is known about the biodegradability of these HMW compounds by␣rhizosphere bacteria. This study compared the effects of a 3 and a 5-ring PAH, phenanthrene (PHE) and␣dibenzo[a,h]anthracene (dBA) respectively, on the composition of bacterial community, the bacterial density and the biodegradation activity. Compartmentalized devices were designed to harvest three consecutive sections of the rhizosphere. Rhizosphere and non-rhizosphere compartments were filled with PHE or dBA spiked or unspiked sand and inoculated with a soil bacterial inoculum. Different bacterial communities and degradation values were found 5 weeks after spiking with PHE (41–76% biodegradation) and dBA (12–51% biodegradation). In sections closer to the root surface, bacterial populations differed as a function of the distance to roots and the PAH added, whereas in further rhizosphere sections, communities were closer to those of the non-planted treatments. Biodegradation of PHE was also a function of the distance to roots, and decreased from 76 to 42% within 9 mm from the roots. However, biodegradation of dBA was significantly higher in the middle section (3–6 mm from roots) than the others. Rhizosphere degradation of PAH varies with the nature of the PAH, and C fluxes from roots could limit the degradation of dBA.     

基于吐温-80下2株功能菌协同降解多环芳烃的特性研究

【目的】研究恶臭假单胞菌B6-2和克雷伯氏菌CW-D3T构建的混合功能菌对多环芳烃的协同修复效能,并探究非离子表面活性剂吐温-80对混菌降解多环芳烃的影响,以期为芳烃化合物的生物修复提供技术参考和理论依据。【方法】通过生长曲线及平板菌落计数法反映混菌生长情况及比例,从而评估混菌降解体系的可行性;通过高效液相色谱法探究各体系以及不同吐温-80浓度下混培体系对多环芳烃的降解效能;最后通过烷烃吸附法测定细胞表面疏水性,以探究吐温-80对混合功能菌降解多环芳烃的影响机制。【结果】等比例混合的2株菌共培养生长状态优于纯培体系,对混合多环芳烃(菲、荧蒽、芘)的降解率分别为33.4%、30.1%、28.6%(7 d),相较于菌CW-D3T,分别提高了1.31倍、1.46倍、1.42倍。混培体系中加入500 mg/L的吐温-80对菲、荧蒽、芘的降解率分别为47.7%、43.2%、38.8%(7 d),相较于对照组各提高了1.55倍、1.38倍、1.31倍,而更高浓度的吐温-80无明显促进作用或轻微抑制。添加吐温-80使菌CW-D3T和混菌的表面疏水性提高,而菌B6-2表面疏水性降低。结合细菌生长量分析...     

Degradation of phenanthrene,fluorene and fluoranthene by pure bacterial cultures

Summary Bacterial mixed cultures able to degrade the polycyclic aromatic hydrocarbons (PAH) phenanthrene, fluorene and fluoranthene, were obtained from soil using conventional enrichment techniques. From these mixed cultures three pure strains were isolated:Pseudomonas paucimobilis degrading phenanthrene;P. vesicularis degrading fluorene andAlcaligenes denitrificans degrading fluoranthene. The maximum rates of PAH degradation ranged from 1.0 mg phenanthrene/ml per day to 0.3 mg fluoranthene/ml per day at doubling times of 12 h to 35 h for growth on PAH as sole carbon source. The protein yield during PAH degradation was about 0.25 mg/mg C for all strains. Maximum PAH oxidation rates and optimum specific bacterial growth were obtained near pH 7.0 and 30°C. After growth entered the stationary phase, no dead end-products of PAH degradation could be detected in the culture fluid.     

Chemical characterization of root exudates from rice (Oryza sativa) and their effects on the chemotactic response of endophytic bacteria

Root exudates represent an important source of nutrients for microorganisms in the rhizosphere and seem to participate in early colonization inducing chemotactic responses of rhizospheric bacteria. We characterized the root exudates collected from rice plantlets cultured under hydroponic conditions and assessed their effects on the chemotaxis of two strains of endophytic bacteria, Corynebacterium flavescens and Bacillus pumilus, collected from the rice rhizosphere. We compared these chemotactic effects on endophytic bacteria with those on two strains of plant-growth-promoting bacteria, Azospirillum brasilense (isolated from the corn rhizosphere) and Bacillus sp. (from the rice rhizosphere). The root exudates were collected at different time intervals. The highest concentration and diversity of amino acids and carbohydrates were found during the first 2 weeks after seeding. Histidine, proline, valine, alanine, and glycine were the main amino acid residues identified during the 4 weeks of culture. The main carbohydrates identified were glucose, arabinose, mannose, galactose, and glucuronic acid. The chemotactic responses of the analyzed endophytic bacteria to root exudates were 3.9 to 5.1 times higher than those of A. brasilense and 2.2 to 2.8 times higher than Bacillus sp. Our results indicate that rice exudates may induce a higher chemotactic response for endophytic bacteria than for other bacterial strains present in the rice rhizosphere.     

Biochemical and physiological peculiarities of the interactions between <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> and <Emphasis Type="Italic">Sorghum bicolor</Emphasis> in the presence of phenanthrene

The effect of phenanthrene, a polycyclic aromatic hydrocarbon (PAH) at concentrations of 0, 10, and 100 mg/kg and the bacterium Sinorhizobium meliloti P221 on root exudation of Sorghum bicolor L. Moench was studied in laboratory vegetative experiments. Inoculation of the bacterium promoted plant resistance to the pollutant stress and increased their acclimation rate and biomass formation. The ability of this microorganism to produce a phytohormone, indolyl-3-acetic acid, and to degrade phenanthrene, resulted in morphological changes of the plant root system and in the changed intensity of root exudation. In root exudates of sorghum, enzyme activities towards the metabolites formed during microbial degradation of PAH were revealed, which is indicative of a direct involvement of plants in PAH degradation in the rhizosphere as well as of the coupled plant-microbial metabolism in the course of xenobiotic degradation in the root zone. In phenanthrene-contaminated soil, sorghum was found to support selectively the development of the S. meliloti P221 population.     

Effect of the polycyclic aromatic hydrocarbon phenanthrene on root exudation of Sorghum bicolor (L.) Moench

The objective of this work was to study the effect of two concentrations (10 and 100 mg kg⁻¹) of phenanthrene, a ubiquitous polycyclic aromatic hydrocarbon (PAH), on root exudation of the remediating plant Sorghum bicolor (L.) Moench under controlled conditions in a pot experiment. It was found that the phenanthrene concentration of 10 mg kg⁻¹ did not cause significant effects on plant survival and growth but had little stimulating effect on carbohydrate exudation. The contamination with phenanthrene at 100 mg kg⁻¹ inhibited accumulation of plant shoot and root biomass, decreasing the carboxylic acid, carbohydrate, and amino acid amounts released by sorghum root into the rhizosphere. However, root exudation per unit of root surface was not changed significantly with increasing phenanthrene concentration. There were no differences in qualitative composition of root exudates under the influence of PAH were found. The observed alterations in the ratio between the main root-exuded components are assumed to manifest adaptive alterations occurring in the plant as a response to pollutant stress. The activity of three oxidoreductases (oxidase, peroxidase, and tyrosinase) released by sorghum roots was clearly progressive to the increasing phenanthrene concentration in the substrate. Under the influence of phenanthrene, the population of phenanthrene-degrading microorganisms in sorghum root zone increased, and their share in the total number of culturable heterotrophs increased as well. The main promotional factor was the pollutant; however, the stimulating effect of the plant root exudates was also involved. The increased pollutant-degrading microbial population and activity of the extracellular root enzymes are presumed to be important for the rhizodegradation of PAH.     

Peroxidases from alfalfa roots: Catalytic properties and participation in degradation of polycyclic aromatic hydrocarbons

From the roots and root exudates of 3-week-old plants of alfalfa (Medicago sativa L.), anionic and cationic peroxidases differing in principal physicochemical and catalytic properties were isolated and purified. Main features of anionic peroxidases detected in the roots and root exudates were identical. Phenanthrene present in the soil used for alfalfa growing influenced the number of forms and activity of peroxidases in crude enzyme preparations but did not affect the properties of pure enzymes. In the presence of a synthetic mediator, purified peroxidases can oxidize phenanthrene and its derivatives, including potential microbial metabolites of polycyclic aromatic hydrocarbons (PAH). The fact that the enzymes excreted in root exudates in a purified form can oxidase PAH proves their participation in degradation of PAH and their microbial metabolites in alfalfa rhizosphere. These new data indicate that the processes of plant and microbial degradation of pollutants in the rhizosphere are coupled; they are relevant to understanding the molecular mechanisms of degradation of persistent pollutants by plant-microbial complexes.     

Effect of wetland plants and bacterial inoculation on dissipation of phenanthrene

This study attempts to evaluate the capacity of wetland plants' ability to dissipate phenanthrene (PHE) under waterlogged conditions. The results indicate that Typha latifolia and Vetiveria zizanioides may efficiently degrade PHE, and were much more effective when under combined plant cultivation with the inoculation of Pseudomonas frederiksbergensis (ATCC BAA-257) . Concentrations of PHE declined from 200 to less than 52 mg kg^?1 in all treatments with plant cultivation. At the end of the experimental period, PHE was undetectable in combined plant cultivation in the presence of bacteria inoculation. Microbial biomass C(carbon), N(nitrogen), and P(phosphate) were significantly different (p < 0.05) in the presence and absence of bacteria inoculation with bacteria inoculation significantly (p < 0.05) increased microbial biomass P. The presence of bacteria inoculation and different plant species significantly (p < 0.05) decreased the PHE concentrations in the microcosms. The inoculation of bacteria and release of exudates from plant roots further enhanced the dissipation of PHE in sand. Concentrations of citric and malic acids were decreased up to 69% in bacteria-inoculated treatments, showing large citric and malic acids serving as a food source and growth substrate for bacteria.     

Soil Type-Dependent Responses to Phenanthrene as Revealed by Determining the Diversity and Abundance of Polycyclic Aromatic Hydrocarbon Ring-Hydroxylating Dioxygenase Genes by Using a Novel PCR Detection System

A novel PCR primer system that targets a wide range of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHD_α) genes of both Gram-positive and Gram-negative bacteria was developed and used to study their abundance and diversity in two different soils in response to phenanthrene spiking. The specificities and target ranges of the primers predicted in silico were confirmed experimentally by cloning and sequencing of PAH-RHD_α gene amplicons from soil DNA. Cloning and sequencing showed the dominance of phnAc genes in the contaminated Luvisol. In contrast, high diversity of PAH-RHD_α genes of Gram-positive and Gram-negative bacteria was observed in the phenanthrene-spiked Cambisol. Quantitative real-time PCR based on the same primers revealed that 63 days after phenanthrene spiking, PAH-RHD_α genes were 1 order of magnitude more abundant in the Luvisol than in the Cambisol, while they were not detected in both control soils. In conclusion, sequence analysis of the amplicons obtained confirmed the specificity of the novel primer system and revealed a soil type-dependent response of PAH-RHD_α gene-carrying soil bacteria to phenanthrene spiking.Polycyclic aromatic hydrocarbons (PAHs) are hydrophobic compounds composed of two or more fused aromatic rings. Although PAHs are ubiquitous in the environment (from natural oil seeps, brush fires, and plant derivatives), anthropogenic activities, such as disposal of coal-processing waste, mining accidents, petroleum wastes, and vehicle exhaust, have drastically increased their occurrence in the environment. The fate of PAHs in soil is of great interest due to their potential for bioaccumulation, persistence, transport, and toxicity. Microbe-driven aerobic degradation of PAHs is well documented (15-17). The diversity of PAH-degrading genes in soils is assumed to be huge, but the extent of diversity and how it is influenced by different soil types or their history and type of pollution are not yet fully explored. Knowledge of the genes coding for dioxygenase enzymes that catalyze the primary step of PAH degradation by incorporating molecular oxygen into the aromatic nucleus is an essential prerequisite to unraveling the contributions of microbial population networks to transformation, assimilation, and degradation of organic chemicals in soil. Recently, the complete genomes of several PAH-degrading bacteria became available and allowed new insights into degradative pathways (6, 18, 36). Organic pollutants also serve as nutrients for those microbes that have the appropriate genetic makeup to utilize them, resulting in their increased metabolic activity and abundance (4, 14). In the last decade, impressive progress was seen in techniques that allow cultivation-independent analysis of microbial communities and thus overcome the most severe limitations in studying microbial communities in natural habitats, namely, that only a rather small portion of microbes are accessible to standard cultivation conditions (1, 29). For more than a decade, cultivation-independent approaches have also been employed to unravel the responses of microbial communities in soils and sediments to PAH pollution. In all these studies, PCR amplification of PAH-degrading gene fragments from nucleic acids directly extracted from environmental samples was used to explore the abundance and diversity of PAH ring-hydroxylating dioxygenase (PAH-RHD_α) genes (4, 8, 9, 13, 14, 22, 34, 37). Despite the known biases of PCR amplification from mixed templates, these techniques allow highly sensitive and specific detection even from minute amounts of nucleic acids. In order to select suitable primer systems, previously published primer systems were analyzed for their ranges of target sequences. The existing primer systems were found to have limitations, as they often target only a rather narrow range of sequences, e.g., nahAc- or phnAc-type sequences (21, 34) or only PAH-RHD_α genes from Gram-negative bacteria (3, 13). In other studies, two-primer systems were used to target PAH-RHD_α genes of both Gram-positive and Gram-negative bacteria (4, 37). Only one primer system targeting the Rieske gene fragment was described that amplified a small fragment from PAH-RHD_α genes from both Gram-negative and Gram-positive bacteria (24). However, the amplicon size was only 78 bp and the primer might also target genes coding for dioxygenases that attack nonpolar aromatic compounds, such as benzene, toluene, and xylene. Therefore, this work aimed to design an improved primer system that targets PAH-RHD_α genes from both Gram-positive and Gram-negative bacteria and provides larger amplicon sizes. The novel primer system was tested in silico and validated by sequencing cloned PAH-RHD_α genes amplified from total-community (TC) DNA and was used in endpoint and quantitative real-time PCR (qPCR) formats. The primer system was also applied to study the responses of soil microbial communities in two different soils (a Cambisol and a Luvisol representing typical arable soils in Central Europe with different texture compositions) to artificial phenanthrene pollution.     

Staphylococcus sp. KW-07 contains nahH gene encoding catechol 2,3-dioxygenase for phenanthrene degradation and a test in soil microcosm

We isolated three species of phenanthrene-degrading bacteria from oil-contaminated soils and marine sediment, and assessed the potential use of these bacteria for bioremediation of soils contaminated by polycyclic aromatic hydrocarbons (PAHs). Based on 16S rDNA sequences, these bacteria were Staphylococcus sp. KW-07 and Pseudomonas sp. CH-11 from soil, and Ochrobactrum sp. CH-19 from the marine sediment. By PCR amplification, catechol 2,3-dioxygenase genes (nahH genes) mediating PAH degradation in the chromosome of Staphylococcus sp. KW-07 and Ochrobactrum sp. CH-19, and in plasmid DNA of Pseudomonas sp. CH-11 were detected. All isolates had a similar optimal growth temperature (25 °C) and optimal growth pH (7.0) in a minimal salt medium (MSM) with 0.1% (w/v) phenanthrene as the sole source of carbon and energy. Pseudomonas sp. CH-11 and Staphylococcus sp. KW-07 degraded 90% of added phenanthrene in 3 days and Ochrobactrum sp. CH-19 degraded 90% of the phenanthrene in 7 days under laboratory batch culture conditions. However, Staphylococcus sp. KW-07 was the most effective among the three strains in degradation of phenanthrene in soil. After inoculation of 1 × 10¹¹ cells of Staphylococcus sp. KW-07, over 90% degradation of 0.1% phenanthrene (0.1 g/100 g soil) was achieved after 1 month at 25 °C. The results collectively suggest that the Staphylococcus sp. KW-07 strain isolated may be useful in bioremediation of PAH-contaminated soils.     

The Bioadsorption of Cadmium and Lead by Bacteria in Root Exudates Culture

Microorganisms are important for phytoremediation of soil contaminated with heavy metals. In the present study, bacteria Bacillus sp., Pseudomonas sp., Alcaligenes sp., and Flavobacterium sp. isolated from the Zhangshi Irrigation Area were applied to bioadsorbed Cd and Pb in liquid cultures with root exudates of sunflower as a sole carbon source. The experimental data demonstrated that these bacteria had a high potential of enrichment of Cd and Pb, and Bacillus sp. and Alcaligenes sp. had better ability to accumulate Cd or Pb than the others; the distinct bioadsorption of Cd and Pb by bacteria might depend on the physiology of bacteria, categories of heavy metals, and environmental factors (such as pH). In addition, root exudates of sunflower could not only support the growth of these bacteria, but also influence the toxicity and bioavailability of Cd and Pb. Our results indicated that amendment with bacteria isolated from heavy-metal-polluted soil and root exudates could be considered as a potential approach to enhance the phytoremediation of Cd- or Pb-contaminated soil.     

Degradation of phenanthrene by different bacteria: evidence for novel transformation sequences involving the formation of 1-naphthol

Four polycyclic aromatic hydrocarbon (PAH)- degrading bacteria, namely Arthrobacter sulphureus RKJ4, Acidovorax delafieldii P4-1, Brevibacterium sp. HL4 and Pseudomonas sp. DLC-P11, capable of utilizing phenanthrene as the sole source of carbon and energy, were tested for its degradation using radiolabelled phenanthrene. [9-¹⁴C]Phenanthrene was incubated with microorganisms containing 100 mg/l unlabelled phenanthrene and the evolution of ¹⁴CO₂ was monitored: within 18 h of incubation, 30.1, 35.6, 26.5 and 2.1% of the recovered radiolabelled carbon was degraded to ¹⁴CO₂ by RKJ4, P4-1, HL4 and DLC-P11, respectively. When mixtures of other PAHs such as fluorene, fluoranthene and pyrene, in addition to phenanthrene, were added as additional carbon sources, there was a 36.1 and 20.6% increase in ¹⁴CO₂ production from [9-¹⁴C]phenanthrene in the cases of RKJ4 and HL4, respectively, whereas P4-1 and DLC-P11 did not show any enhancement in ¹⁴CO₂ production. Although, a combination of many bacteria enhances the degradation of organic compounds, no enhancement in the degradation of [9-¹⁴C]phenanthrene was observed in mixed culture involving all four microorganisms together. However, when different PAHs, as indicated above, were used in mixed culture, there was a 68.2% increase in ¹⁴CO₂ production. In another experiment, the overall growth rate of P4-1 on phenanthrene could be enhanced by adding the non-ionic surfactant Triton X-100, whereas RKJ4, HL4 and DLC-P11 did not show any enhancement in growth. Pathways for phenanthrene degradation were also analysed by thin-layer chromatography, gas chromatography and gas chromatography-mass spectrometry. Common intermediates such as o-phthalic acid and protocatechuic acid were detected in the case of RKJ4 and o-phthalic acid was detected in the case of P4-1. A new intermediate, 1-naphthol, was detected in the cases of HL4 and DLC-P11. HL4 degrades phenanthrene via 1-hydroxy-2-naphthoic acid, 1-naphthol and salicylic acid, whereas DLC-P11 degrades phenanthrene via the formation of 1-hydroxy-2-naphthoic acid, 1-naphthol and o-phthalic acid. Both transformation sequences are novel and have not been previously reported in the literature. Mega plasmids were found to be present in RKJ4, HL4 and DLC-P11, but their involvement in phenanthrene degradation could not be established. Received: 25 May 1999 / Received revision: 16 July 1999 / Accepted: 1 August 1999     

Effect of plant growth promoting bacterium; Pseudomonas putida UW4 inoculation on phytoremediation efficacy of monoculture and mixed culture of selected plant species for PAH and lead spiked soils

Plant growth promoting bacteria (PGPB) enhanced phytoremediation (PEP) is an attractive remedial strategy for the remediation of polycyclic aromatic hydrocarbon (PAH) and heavy metal (HM) contaminated sites. The effect of PGPB; Pseudomonas putida UW4 inoculation on the phytoremediation efficiency of Medicago sativa, Festuca arundinacea, Lolium perenne, and mixed plants (L. perenne and F. arundinacea) was assessed. This involved two contaminant treatments; “PAH” (phenanthrene; 300?mg·kg^?1, fluoranthene; 200?mg·kg^?1, and benzo[a]pyrene; 5?mg·kg^?1) and “PAH?+?HM” (‘PAH’ treatments +100?mg of Pb/kg). PGPB inoculation significantly enhanced root biomass yield of F. arundinacea in PAH treatment, and the mixed plant shoot biomass and L. perenne root biomass yields of the PAH?+?HM treatment. PGPB significantly enhanced dissipation of phenanthrene and fluoranthene for M. sativa-PAH?+?PGPB treatment and fluoranthene for F. arundinacea-PAH?+?HM?+?PGPB treatment. In others, PGPB inoculation either had no impact or inhibited PAH dissipation. PAH dissipation for the single and mixed plant treatments with PGPB inoculation were not different. The efficiency of PEP is dependent on different factors such as PGPB inoculum biomass, plant species, plant–microbe specificity and type of contaminants. Exploiting PEP technology would require proper understanding of plant tolerance and growth promoting mechanisms, and rhizosphere activities.     

Phenanthrene biodegradation and the interaction of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> BS3701 and <Emphasis Type="Italic">Burkholderia</Emphasis> sp. BS3702 in plant rhizosphere

The interaction between the strains degrading polycyclic aromatic hydrocarbons, Pseudomonas putida BS3701 and Burkholderia sp. BS3702, was studied in the course of phenanthrene degradation in plant rhizosphere. Strain BS3702 was shown to accumulate 1-hydroxy-2-naphthoic acid (which is toxic for plants); it was then utilized by strain BS3701, which thereby increased the resistance of plants to the pollutant and to the toxic intermediate. With this type of interaction (cooperation), the efficiency of phenanthrene degradation was noted to decrease.