Molecular assessment of the potential for in situ bioremediation of PCBs from aquatic sediments

Polychlorinated biphenyls (PCBs) are a family of xenobiotic compounds that are ubiquitous and oftentimes persistent environmental pollutants. As such, PCBs are a common target of sediment remediation efforts. Microbial degradation of sediment pollutants such as PCBs offers an environmentally sound and economically favorable alternative to conventional means of remediation such as dredging. This project describes the development of a PCR-based assay to determine the potential for PCB bioremediation by the resident microbial consortium in contaminated sediments. Using PCR and RT-PCR of DNA and RNA, respectively, extracted from aquatic sediments collected from the western basin of Lake Erie and one of its tributaries, we were able to amplify the bphA1 gene that encodes the large subunit of biphenyl dioxygenase. Since other studies have determined that the BphA1 gene product dictates PCB congener specificity, this assay may prove to be a useful screen for endemic catabolic activities for PCB mixtures in aquatic sediments.     

Comparative in situ remediation potential of Pseudomonas putida 710A and Commamonas aquatica 710B using plant (Vigna radiata (L.) wilczek) assay

A greenhouse study was conducted to determine the effect of cadmium (Cd)-resistant Pseudomonas putida strain 710A and Commamonas aquatica strain 710B, used either alone or as a binary mixture on the toxicity of cadmium to mungbean [Vigna radiata (L.) wilczek] plants. The bacterial strains 710A and 710B, isolated from the Semra mines in Ranchi, India, were resistant to 0.5 mM and 1 mM, CdCl₂ respectively. Moreover, both strains grew well at 10 °C and 30 °C. Of the two bacterial strains, strain 710B showed 129.6 and 83.5 times higher P solubilizing activity than strain 710A, when grown in liquid broth supplemented with cadmium at 10 °C and 30 °C, respectively. Furthermore, 16S ribosomal DNA (rDNA) sequencing identified 710A as a Pseudomonas putida strain and 710B as a Commamonas aquatica strain. The strain 710A significantly (p?<?0.05) stimulated the growth of roots (35.2 %) and shoots (30 %) of mungbean plants grown in soil treated with 110 μM CdCl₂. However, the increase in the case of 710B was found to be 15.4 % and 5.17 %, respectively. The loss of chlorophyll content was replenished by up to 80 %, 66 %, and 77.3 % by inoculation of 710A, 710B and binary mixture, respectively. Moreover the strains were able to reduce Cd accumulation in roots and shoots. Most significantly, residual Cd concentration in soil was reduced in the presence of bioinoculants. However, in terms of efficiency, the strains were found to be in the following order: 710A?>?710A +710B?>?710B. The results suggest that P. putida 710A strain is a potentially effective candidate metal to be used for sequestering and as a growth-promoting bioinoculant in Cd polluted soil.     

Characterization and degradation potential of diesel-degrading bacterial strains for application in bioremediation

Bioremediation of polluted soils is a promising technique with low environmental impact, which uses soil organisms to degrade soil contaminants. In this study, 19 bacterial strains isolated from a diesel-contaminated soil were screened for their diesel-degrading potential, biosurfactant (BS) production, and biofilm formation abilities, all desirable characteristics when selecting strains for re-inoculation into hydrocarbon-contaminated soils. Diesel-degradation rates were determined in vitro in minimal medium with diesel as the sole carbon source. The capacity to degrade diesel range organics (DROs) of strains SPG23 (Arthobacter sp.) and PF1 (Acinetobacter oleivorans) reached 17–26% of total DROs after 10 days, and 90% for strain GK2 (Acinetobacter calcoaceticus). The amount and rate of alkane degradation decreased significantly with increasing carbon number for strains SPG23 and PF1. Strain GK2, which produced BSs and biofilms, exhibited a greater extent, and faster rate of alkane degradation compared to SPG23 and PF1. Based on the outcomes of degradation experiments, in addition to BS production, biofilm formation capacities, and previous genome characterizations, strain GK2 is a promising candidate for microbial-assisted phytoremediation of diesel-contaminated soils. These results are of particular interest to select suitable strains for bioremediation, not only presenting high diesel-degradation rates, but also other characteristics which could improve rhizosphere colonization.     

Spatial and temporal distribution of a bioluminescent‐marked Pseudomonas putida on soybean root

The ability of rhizobacteria to compete with other microorganisms for root colonization may be critical for its establishment on a root. Over a 6 day period, visualization of the spatial and temporal rhizosphere distribution of a bioluminescent‐marked rhizobacterium, Pseudomonas putida, strain GR7.4lux, was examined on soybean grown in non‐sterile soil conditions. Luminometry technologies showed a rapid root distribution of rhizobacteria where bioluminescence was particularly intense on the seed and upper root parts. The results provide new information on rhizobial root distribution, where, using enrichment broth, 50% of the root tips were still colonized by rhizobacteria up to 6 days after sowing. This suggests that rhizobial enrichment is required to detect low populations at the root tip. Bioluminescent technology represents a promising alternative to previous methods for studying rhizobial growth and distribution on roots. Copyright © 2003 John Wiley & Sons, Ltd.     

Isolation and characterization of altered plasmids in mutant strains of Pseudomonas putida NCIB 9816

The ability of P. putida NCIB 9816 to grow with naphthalene (Nah+) and salicylate (Sal+) is correlated with the presence of an 83 kilobase (kb) conjugative plasmid, pDTG1. Derivatives of pDTG1 were obtained from cells after exposure to halogenated analogs of naphthalene or salicylate. The selection of mutants having a Nah-Sal- or a Nah-Sal+ phenotype could be enhanced by the addition of triphenyltetrazolium chloride to the indicator medium. Structurally modified plasmids were characterized by restriction endonuclease digestion and Southern hybridization experiments. The region of pDTG1 DNA that encodes the enzymes responsible for the conversion of naphthalene to salicylate was identified. The structural changes in mutant plasmids were correlated with the absence of essential enzymatic activities.     

Development and assessment of a preliminary kinetic model for in situ pah bioremediation

Polycyclic aromatic hydrocarbons (PAHs) are a group of organic nonaqueous‐phase liquid (NAPL) contaminants of critical environmental concern. The treatment method used for a contaminated soil depends primarily on the nature and extent of the contamination as well as the cost effectiveness of the method. Current research has shown that bioremediation is perhaps the simplest and most economic process for the treatment of large contaminated areas. Although bioremediation feasibility and effectiveness has been well documented, additional information is required to fully understand subsurface kinetics. Specifically, the importance and effect of interactions between bacteria, supplemental nutrients, oxygen source, contaminant, and soil type must be understood. Preliminary respirometer experiments have been conducted to address these factors for the development of a kinetic model for both steady‐state and unsteady‐state conditions.     

Factors influencing the potential use of Aliquat 336 for the in situ extraction of carboxylic acids from cultures of Pseudomonas putida

The use of extraction techniques to alleviate product inhibition in bioprocesses is one of a number of potential separation methods. However, the intimate contact of an organic phase with the broth implies that the organic components of this phase may be present in the aqueous phase at saturation levels. The quaternary amine Aliquat 336 (trioctyl/decylmethylammonium entity), dissolved in octan-1-ol showed no inhibition on the growth of Pseudomonas putida, at least with respect to molecular toxicity. Nevertheless, it is important to point out two main effects of Aliquat 336 associated with its ion exchange properties. It is able (1) to complex hydroxyl ions and therefore drastically lower the pH of the broth and (2) release its counter ion through these exchanges. Therefore, a strict control of the pH of the cultivation must be conducted, with the constraint that Ps. putida has an optimal pH growth of 7.4-7.5. The pH range tolerated by this strain is, however, between 5.0 and 9.0. In addition, the counter ion of Aliquat 336 needs to be carefully chosen and HSO4- should be preferred to Cl-.     

Regulation of the utilization of glucose and aromatic substrates in four strains of Pseudomonas putida

During the oxidation of various mixtures of glucose and aromatic substrates by four strains of Pseudomonas putida, diauxic growth was not observed. Strain A3.12 grew faster on benzoate than on glucose, whereas three other strains showed faster growth on glucose than on the aromatic test substrates. Growth rates on mixtures of glucose and aromatics were intermediate between those on the single substrates.The presence of glucose in media containing aromatic substrates accelerated in the bacteria the appearance of the ability to oxidize aromatic substrates. During growth of the organisms on binary mixtures of aromatics, simultaneous utilization of these compounds occurred, the utilization ratio depending on the quality of the compounds as carbon and energy sources. Addition of glucose to dual aromatic substrate media greatly increased the utilization ratio in favour of the better aromatic substrate.With decreasing concentration of glucose in relation to that of aromatic substrates, the rate of carbon assimilation from glucose increased. Enzymological and radiochemical studies demonstrated that even in the presence of an excess of aromatic substrates, glucose was exclusively catabolized via the 2-keto-3-deoxy-6-phosphogluconate pathway. In contrast, the rate of carbon assimilation from ¹⁴C-ring-labelled benzoate and anisate was unaffected by the presence of an excess of glucose.Abbreviations KDPG 2-keto-3-deoxy-6-phosphogluconate - PP pentose-phosphate - OD optical density     

Structural and functional variability of genetic systems for catabolizing polycyclic aromatic hydrocarbons in Pseudomonas putida strains

The genetic control of naphthalene, phenanthrene, and anthracene biodegradation was studied in three Pseudomonas putida strains isolated from coal tar- and oil-contaminated soils. These strains isolated from different geographical locations contained similar catabolic plasmids controlling the first steps of naphthalene conversion to salicylate (the nah1 operon), functionally inoperative salicylate hydroxylase genes, and genes of the metha-pathway of catechol degradation (the nah2 operon). Salicylate oxidation in these strains is determined by genes located in trans-position relative to the nah1 operon: in strains BS202 and BS3701, they are located on the chromosome, and in the strain BS3790, on the second plasmid.     

Biotization of Arabidopsis thaliana with Pseudomonas putida and assessment of its positive effect on in vitro growth

Determination of the optimal inoculation method and concentration to use for plant-bacteria interaction studies is important in many cases, such as the phytoremediation of heavy metals and other toxic compounds in contaminated areas. The aim of this study was to compare different concentrations and times of inoculation of Pseudomonas putida with various growth stages of Arabidopsis thaliana in 14-d in vitro cultures. A significant beneficial impact of the bacterium was detected in the shoot length and root weight of seedlings. The highest shoot length and root fresh and dry weights were detected in 14-d and 2 × 10³ cfu mL⁻¹ inoculated samples. In addition, the increase in root weight could be visualized with crystal violet staining, as relatively more root hair and lateral root formation occurred in seedlings inoculated with moderate concentrations of bacteria, possibly due to the ability of P. putida to produce indole-acetic acid. Moreover, the highest photosynthetic pigment accumulation was obtained with the highest bacterial inoculum (2 × 10⁶ cfu mL⁻¹), which was tested in 0- or 3-d-old seedlings. Rhizospheric bacterial colonization was also visualized with GFP-labeled bacteria by confocal microscopy. These results showed that biotization of A. thaliana with P. putida KT2440 did not cause severe oxidative stress in seedlings, because H₂O₂ accumulation levels together with CAT and POX activities were not significantly induced. Therefore, this strain could be used for several applications based on plant-bacteria interactions.

     

Growth and survival of Pseudomonas putida strains degrading naphthalene in soil model systems with different moisture levels

The effect of different moisture levels (from 20 to 70%) on the growth and survival of Pseudomonas putida strains G7 and BS3701 degrading naphthalene was studied in soil model systems. P. putida G7 contains plasmid NAH7 and P. putida BS3701 harbours plasmids pBS1141 and pBS1142. A mathematical model is proposed to describe the observed dynamics of the number of viable bacterial cells. Naphthalene and soil organic matter were considered as substrates available to bacteria. Data fitting allowed the estimation of model parameters characterizing microbial growth rate, utilization rate of substrates, specific maintenance rate and yield coefficient. Both the maximum bacterial concentration and the highest yield coefficient were observed at a soil moisture level of 40%. This optimal moisture level is close to but less than the water capacity (48%) of the soil used.     

Detection of recombinant Pseudomonas putida in the wheat rhizosphere by fluorescence in situ hybridization targeting mRNA and rRNA

A method was developed to detect a specific strain of bacteria in wheat root rhizoplane using fluorescence in situ hybridization and confocal microscopy. Probes targeting both 23S rRNA and messenger RNA were used simultaneously to achieve detection of recombinant Pseudomonas putida (TOM20) expressing toluene o-monooxygenase (tom) genes and synthetic phytochelatin (EC20). The probe specific to P. putida 23S rRNA sequences was labeled with Cy3 fluor, and the probe specific to the tom genes was labeled with Alexa647 fluor. Probe specificity was first determined, and hybridization temperature was optimized using three rhizosphere bacteria pure cultures as controls, along with the P. putida TOM20 strain. The probes were highly specific to the respective targets, with minimal non-specific binding. The recombinant strain was inoculated into wheat seedling rhizosphere. Colonization of P. putida TOM20 was confirmed by extraction of root biofilm and growth of colonies on selective agar medium. Confocal microscopy of hybridized root biofilm detected P. putida TOM20 cells emitting both Cy3 and Alexa647 fluorescence signals.     

Effects of lead and cadmium on photosynthesis in Amaranthus spinosus and assessment of phytoremediation potential

Abstract

This study assessed the effects of Pb (0, 200, 500, 1000?mg kg^?1) and Cd (0, 5, 15, 30, 50?mg kg^?1) on photosynthesis in Amaranthus spinosus (A. spinosus), as well as the potential for phytoremediation by pot-culture experiment. Exposure to Pb/Cd produced a concentration-dependent decrease in biomass and all photosynthesis parameters, except for non-photochemical quenching, which increased with the metal concentration. The metals accumulated more in roots compared to shoots. The bioconcentration factor (BCF) of Pb was <1 in shoots at all Pb levels, whereas the BCF was <1 in roots at all but the lowest concentration of Pb. Roots extracted Cd from soil at all treatments. The translocation factor of Cd was larger than that of Pb suggesting that Cd is more mobile than Pb in A. spinosus. Amaranthus spinosus displays a high tolerance for both Pb and Cd with regards to growth and photochemical efficiency, but it is more sensitive to Cd than Pb. Amaranthus spinosus accumulates Pb and Cd primarily in the roots and Cd is more bioconcentrated and translocated in comparison to Pb. This investigation shows that A. spinosus has good potential for phytoremediation of soils contaminated by low levels of Cd and Pb.     

Effect of arsenite on the physiological and cytological properties of Pseudomonas putida strains capable of degrading polycyclic aromatic hydrocarbons]

Some physiological and cytological properties of Pseudomonas putida strains resistant to arsenite and capable of degrading polycyclic aromatic hydrocarbons were studied. The resistance of P. putida BS202 (NPL-1) to arsenite proved to be determined by chromosomal genes, while the arsenite resistance of P. putida BS238 (pBS2; pBS3031) was by plasmid-borne genes. Arsenite affected the pattern and rate of growth of strain BS202 (NPL-1) in media with naphthalene or salicylate as carbon sources; particularly, it lengthened the lag phase. Electron-microscope analysis of the strains studied did not reveal any arsenite-induced destructive changes in the cell envelope. At the same time, arsenite in the growth medium induced some alterations in the structure of the outer membrane of strain BS202 (NPL-1) and the cytoplasmic membrane of strain BS238 (pBS2; pBS3031) and, in both strains, led to an increase in the density of intramembrane particles on the EF face of the freeze-fractured cytoplasmic membrane. Arsenite resistance probably evidently protects cells of both strains from greater damage. Physiological and cytological data suggest that the mechanisms of arsenite resistance in the strains studied are different.