Soil Slurry Microcosms Research Articles

The amendment of Organic matters enhances the anaerobic dechlorination of Polychlorinated Biphenyls in Paddy Soil.

The toxicity of high-chlorinated polychlorinated biphenyls (PCBs) can be efficiently reduced through anaerobic dechlorination. However, this approach suffers a lot in face of in situ microbial remediations, like a shortage of biomass. In this study, we showed that the amendment of organic matters could help microbiota in paddy soil with anaerobic dechlorination and greatly shortened the lag period. The presence of organic matters offered a better environment for dechlorinating bacteria. They provided not only a more strictly anaerobic milieu but also copious carbon sources. By using high-throughput 16S rRNA gene sequencing, genera Dehalobacter, Dehalobacterium, and Desulfitobacterium capable of dechlorination were identified in enriched cultures. Taken together, this study proved that extra organic matters can promote anaerobic dechlorination in paddy soil slurry microcosm systems, which provides new insights into the bioremediation of PCB-contaminated soils.

Biotransformation of dimethoate into novel metabolites by bacterial isolate Pseudomonas kilonensis MB490

Bacterial strain (Pseudomonas kilonensis MB490) isolated from agricultural fields of Mianwali, was selected to check its potential to degrade Organophosphate insecticide dimethoate (DM). Strain MB490 was able to degrade dimethoate equally well at given pH range (6.0, 7.0 and 8.0), thus showing its pH independence for dimethoate degradation. Optimum temperature for dimethoate degradation varied from 25–30 °C. There was more dimethoate degradation under shaking conditions with optimum growth. Strain MB490 showed 90% dimethoate degradation in M-9 broth and 90.6% in soil slurry, while exhibited 81.5% dimethoate degradation in soil microcosm within 9 days, based on HPLC analysis of bacterial samples supplemented with 200 mg/L dimethoate. The average half-life (t 1/2) of dimethoate after bacterial degradation ranged from 1.95 days in 1st phase to 5 days in 2nd phase in M-9 broth, soil slurry and soil microcosm, while in control media without bacteria, it ranged from 30 to 64.3 days. GCMS investigation revealed the transformation of dimethoate into 5 metabolic products namely Methyl diethanol amine, Aspartylglycine ethyl ester, Phosphonothioic acid propyl-O, S-dimethyl ester, O, O, O-Trimethyl thiophosphate and omethoate which were ultimately mineralized by the strain MB490, providing energy for its growth.

Novel metabolites of triazophos formed during degradation by bacterial strains Pseudomonas kilonensis MB490, Pseudomonas kilonensis MB498 and pseudomonas sp. MB504 isolated from cotton fields

In the current scenario of overuse of pesticides (resulting in soil and water pollution and ultimately leading to biomagnification), a research project was carried out to study biodegradation of Triazophos. For this purpose, three bacterial strains (Pseudomonas kilonensis MB490, Pseudomonas kilonensis MB498 and Pseudomonas sp. MB504), isolated from cotton fields of Mianwali, Pakistan were investigated for Triazophos degradation and metabolite formation in M-9 broth, soil slurry and soil microcosm after incubation for 9 days. There was 88.4–95.8% Triazophos degradation in M-9 broth, 99.90% degradation in soil slurry and 92.74 to 96% Triazophos degradation in soil microcosm by these bacteria after 9 days. While there was negligible Triazophos degradation (upto 7%) in the controls without bacteria. According to GCMS analysis, 7 unique and novel metabolites (1, 2, 4-Triazole-4—amine, N-(2-Thienylmethyl), Benzene sulfonic acid hydrazide, Benzene sulfonic acid methyl ester, 4H-1,2,4-Triazole-4-benzenesulfonamide, 4, 5 dihydro-N-(O-toyl)-3-furamide, Ethyl 4-phenyldiazenylbenzoate and Dibutyl methanephosphonate) of Triazophos were revealed. Current results strongly suggest the potential of these bacterial strains for the remediation of Triazophos contaminated agricultural soils.

A slurry microcosm study on the interaction between antibiotics and soil bacterial community

Antibiotics released in the environment have attracted great attention. The environmental emission control of antibiotics should be based on the degree of their negative impacts on the environment and ecology. Here, we conducted a series of soil slurry microcosm experiments to investigate the interactions between antibiotics and the soil bacterial community. In the soil slurry, distinctive behaviors were observed for different antibiotics. Beta-lactams (ampicillin and ceftriaxone) experienced fast biodegradation. Kanamycin was adsorbed on soil particles soon after its addition. Nalidixic acid was stable throughout the experimental period (164 h). The main inactivation mechanism of tetracycline was deduced to be hydrolysis. Bacterial communities in slurries with or without antibiotic-treatment were profiled via high-throughput Illumina sequencing of the 16S rRNA gene. Unstable (ceftriaxone) and adsorbed (kanamycin) antibiotics show minor or negligible influences on the soil bacterial community. Stable antibiotics (nalidixic acid and tetracycline) have significantly affected the structure of the bacterial community. Most of enriched bacterial genera by various antibiotics belong to the same phylum, Proteobacteria. Inhibited bacterial phyla by nalidixic acid are Firmicutes and Bacteroidetes, while those inhibited by tetracycline are Firmicutes, Bacteroidetes and Cyanobacteria. According to the PICRUSt prediction of metagenome, influence of antibiotics on overall metabolic function of the bacterial community is rather limited. This study has provided valuable information, from a phylogenetic viewpoint, about the influence of high concentration of antibiotics on soil bacterial community.

Interactions of nanoscale zero valent iron and iron reducing bacteria in remediation of trichloroethene

Absract In this work we investigated the interactions between the nanoscale zero valent iron (nZVI) and iron reducing bacteria (IRB) during the remediation of trichloroethene (TCE). Dehalogenation of TCE was examined using nZVI NANOFER 25 (N25) and the Gram-negative bacterium Shewanella algae CCM 4595 in liquid and soil slurry microcosms. The rates of dehalogenation were determined by gas chromatography. The interactions between N25 and S. algae were investigated using X-Ray diffraction (XRD), ferrozine test, Tandem Scanning Confocal Microscope (TSCM) and viable cell counts. Results showed that S. algae impaired TCE removal by N25. TCE dehalogenation rates in liquid microcosms were 17 times faster in absence than in presence of S. algae, and 8 times faster in soil slurry microcosms. The concentrations of dissolved Fe2+ and Fe3+ ions in the N25 + S. algae microcosms were an order of magnitude higher than that in bacteria-free microcosms. Moreover, the XRD data showed that the presence of S. algae significantly increases the depletion of reactive Fe0 and causes the formation of poorly soluble and insoluble Fe3+ oxide-hydroxides that can inhibit electron transfer from iron to the contaminant. Viable cell counts showed that N25 considerably inhibited microbial growth. Furthermore, TSCM demonstrated that S. algae tends to adhere to the surface of N25 suggesting that direct nanoparticle attachment may impact both the microbial population and the electron transfer from the iron to the contaminant. Data suggested that S. algae contributes significantly to N25 anoxic corrosion through microbial iron respiration. Our findings provide better insights about the fate of nZVI in the subsurface and its interactions with surrounding microorganisms.

Biodegradability of HCH in agricultural soils from Guadeloupe (French West Indies): identification of the lin genes involved in the HCH degradation pathway

Banana has been a main agricultural product in the French West Indies (Guadeloupe and Martinique) since the 1960s. This crop requires the intensive use of pesticides to prevent attacks by insect pests. Chlorinated pesticides, such as hexachlorocyclohexane (HCH), chlordecone and dieldrin, were used until the beginning of the 1990s, resulting in a generalized diffuse contamination of the soil and water in the areas of banana production, hence the need to develop solutions for cleanup of the polluted sites. The aims of this work were (i) to assess lindane degradation in soil slurry microcosms treated with lindane at 10 mg/L and (ii) to detect the catabolic genes involved in the HCH degradation pathway. The soil slurry microcosm system showed a 40% lindane degradation efficiency at the end of a 30-day experiment. Lower lindane removal was also detected in the abiotic controls, probably caused by pesticide adsorption to soil particles. Indeed, the lindane concentration decreased from 6000 to 1330 ng/mL and from 800 to 340 ng/mL for the biotic and abiotic soils, respectively. Nevertheless, some of the genes involved in the HCH degradation pathway were amplified by polymerase chain reaction (PCR) from crude deoxyribonucleic acid (DNA) extracted from the Guadeloupe agricultural soil, suggesting that HCH degradation is probably mediated by bacteria closely related to the family Sphingomonadaceae.

Microbial degradation of decabromodiphenyl ether (DBDE) in soil slurry microcosms.

Decabromodiphenyl ether (DBDE), which has been identified as an endocrine disrupting compound, is used as brominated flame retardant, and this can result in serious bioaccumulation within ecological systems. The objective of this study was to explore DBDE bioremediation (25 mg/kg) using laboratory scale soil slurry microcosms. It was found that effective biodegradation of DBDE occurred in all microcosms. Various biometabolites were identified, namely polybrominated diphenyl ethers congeners and hydroxylated brominated diphenyl ether. Reductive debrominated products such as tri-BDE to hepta-BDE congeners were also detected, and their total concentrations ranged from 77.83 to 91.07 ng/g. The mechanism of DBDE biodegradation in soil slurry microcosms is proposed to consist of a series of biological reactions involving hydroxylation and debromination. Catechol 2,3-oxygenase genes, which are able to bring about meta-cleavage at specific unbrominated locations in carbon backbones, were identified as present during the DBDE biodegradation. No obvious effect on the ecological functional potential based on community-level physiological profiling was observed during DBDE biodegradation, and one major facultative Pseudomonas sp. (99 % similarity) was identified in the various soil slurry microcosms. These findings provide an important basis that should help environmental engineers to design future DBDE bioremediation systems that use a practical microcosm system. A bacterial-mixed culture can be selected as part of the bioaugmentation process for in situ DBDE bioremediation. A soil/water microcosm system can be successfully applied to carry out ex situ DBDE bioremediation.

Monitoring aromatic hydrocarbon biodegradation by functional marker genes

The development of biological treatment technologies for contaminated environments requires tools for obtaining direct information about the biodegradation of specific contaminants. The potential of functional gene array analysis to monitor changes in the amount of functional marker genes as indicators of contaminant biodegradation was investigated. A prototype functional gene array was developed for targeting key functions in the biodegradation of naphthalene, toluene and xylenes. Internal standard probe based normalization was introduced to facilitate comparison across multiple samples. Coupled with one-colour hybridization, the signal normalization improved the consistency among replicate hybridizations resulting in better discrimination for the differences in the amount of target DNA. During the naphthalene biodegradation in a PAH-contaminated soil slurry microcosm, the normalized hybridization signals in naphthalene catabolic gene probes were in good agreement with the amount of naphthalene-degradation genes and the production of 14CO 2. Gene arrays provide efficient means for monitoring of contaminant biodegradation in the environment.

A Targeted Real-Time PCR Assay for Studying Naphthalene Degradation in the Environment

A quantitative real-time polymerase chain reaction (PCR) assay was developed for monitoring naphthalene degradation during bioremediation processes. The phylogenetic affiliations of known naphthalene-hydroxylating dioxygenase genes were determined to target functionally related bacteria, and degenerate primers were designed on the basis of the close relationships among dioxygenase genes identified from naphthalene-degrading Proteobacteria. Evaluation of the amplification specificity demonstrated that the developed real-time PCR assay represents a rapid, precise means for the group-specific enumeration of naphthalene-degrading bacteria. According to validation with bacterial pure cultures, the assay discriminated between the targeted group of naphthalene dioxygenase sequences and genes in other naphthalene or aromatic hydrocarbon-degrading bacterial strains. Specific amplification of gene fragments sharing a high sequence similarity with the genes included in the assay design was also observed in soil samples recovered from large-scale remediation processes. The target genes could be quantified reproducibly at over five orders of magnitude down to 3 x 10(2) gene copies. To investigate the suitability of the assay in monitoring naphthalene biodegradation, the assay was applied in enumerating the naphthalene dioxygenase genes in a soil slurry microcosm. The results were in good agreement with contaminant mineralization and dot blot quantification of nahAc gene copies. Furthermore, the real-time PCR assay was found to be more sensitive than hybridization-based analysis.

Slow-Release Inoculation Allows Sustained Biodegradation of γ-Hexachlorocyclohexane

This study investigated the feasibility of a slow-release inoculation approach as a bioaugmentation strategy for the degradation of lindane (gamma-hexachlorocyclohexane [gamma-HCH]). Slow-release inoculation of Sphingomonas sp. gamma 1-7 was established in both liquid and soil slurry microcosms using open-ended silicone tubes in which the bacteria are encapsulated in a protective nutrient-rich matrix. The capacity of the encapsulated cells to degrade lindane under aerobic conditions was evaluated in comparison with inoculation of free-living cells. Encapsulation of cells in tubes caused the removal of lindane by adsorption to the silicone tubes but also ensured prolonged biodegradation activity. Lindane degradation persisted 2.2 and 1.4 times longer for liquid and soil slurry microcosms, respectively, than that for inoculation with free cells. While inoculation of free-living cells led to a loss in lindane-degrading activity in limited time intervals, encapsulation in tubes allowed for a more stable actively degrading community. The loss in degrading activity was linked to the loss of the linA gene, encoding gamma-HCH dehydrochlorinase (LinA), which is involved in the initial steps of the lindane degradation pathway. This work shows that a slow-release inoculation approach using a catabolic strain encapsulated in open-ended tubes is a promising bioaugmentation tool for contaminated sites, as it can enhance pollutant removal and can prolong the degrading activity in comparison with traditional inoculation strategies.

2,4,6-Trinitrotoluene Transformation Using Spinacia Oleracea: Saturation Kinetics of the Nitrate Reductase Enzyme

A series of aqueous phase and soil-slurry phase microcosm studies were conducted on 2,4,6-trinitrotoluene (TNT) to obtain kinetic data for optimizing a treatment protocol using an enzyme extract from spinach (Spinacia oleracea). Crude extract was obtained by homogenization of fresh leaves with a buffered protease inhibitor, and employed as phytoremediation agent. Aqueous phase microcosms containing 20 mg∕L TNT and soil–slurry microcosms containing 1 g of a characterized sandy loam soil contaminated with 2,500 mg∕kg TNT and 1,000 mg∕kg hexahydro-1,3,5-trinitro-1,3,5-triazine were dosed with fixed aliquots of extract and analyzed for TNT transformation over time. The TNT concentration was monitored using a colorimetric method for nitroaromatic compounds based on EPA Method 8515. Nitrate reductase activity of the applied crude extract was simultaneously quantified. The transformation of TNT was described by a pseudofirst-order reaction. Coupling kinetic rate information with enzyme activity allowed for estim...

Monitoring of accelerated naphthalene-biodegradation in a bioaugmented soil slurry

The effect of microbial inoculation on the mineralization of naphthalene in a bioslurry treatment was evaluated in soil slurry microcosms. Inoculation by Pseudomonas putida G7 carrying the naphthalene dioxygenase (nahA) gene resulted in rapid mineralization of naphthalene, whereas indigenous microorganisms in the PAH-contaminated soil required a 28 h adaptation period before significant mineralization occurred. The number of nahA-like gene copies increased in both the inoculated and non-inoculated soil as mineralization proceeded, indicating selection towards naphthalene dioxygenase producing bacteria in the microbial community. In addition, 16S rRNA analysis by denaturing gradient gel electrophoresis (DGGE) analysis showed that significant selection occurred in the microbial community as a result of biodegradation. However, the indigenous soil bacteria were not able to compete with the P. putida G7 inoculum adapted to naphthalene biodegradation, even though the soil microbial community slightly suppressed naphthalene mineralization by P. putida G7.

Enhanced degradation of carbazole and 2,3-dichlorodibenzo-p-dioxin in soils by Pseudomonas resinovorans strain CA10.

We studied the degradation of carbazole (CAR) and 2,3-dichlorodibenzo-p-dioxin (2,3-DCDD) in soils inoculated with carbazole- and dioxin-degrader Pseudomonas resinovorans strain CA10. By using Tn5-based transposon delivery systems, this bacterium was chromosomally marked with a tandem green fluorescent protein (gfp) gene. Real-time competitive PCR and direct counting using the (gfp) marker were employed to monitor the total number of carbazole 1,9a-dioxygenase gene (carAa) and survival of CA10 cells in the soil and soil slurry microcosms. Bioaugmentation studies indicated that the survival of the marked CA10 cells in soil microcosms was strongly influenced by pH and organic matter. While the number of the marked CA10 cells decreased rapidly in pH 6 with low organic matter, a high cell density was maintained in pH 7.3 with 2.5% organic matters up to 21 days after inoculation. In pH 7.3 soil, the period needed for complete degradation of CAR (100 microg kg(-1)) was markedly shortened from 21 to 7 days by the inoculation with the CA10 cells. Single inoculation of CA10 cells into the soil slurry system of 2,3-DCDD-contaminated soil enhanced the degradation of 2,3-DCDD from 25.0% to 37.0%. In this system, the population density of CA10 cells and the total number of carAa gene were maintained up to 14 days after inoculation. By repeated inoculation (every 2 days) with CA10 cells each at a density of 10(9) CFU g(-1) of soil, almost all of the 2,3-DCDD (1 microg kg(-1)) was degraded within 14 days. Results of these experiments suggest that P. resinovorans strain CA10 may be an important resource for bioremediation of CAR and chlorinated dibenzo-p-dioxin in contaminated soils.

Effect of sorption and desorption resistance on aerobic trichloroethylene biodegradation in soils

Biodegradation of trichloroethylene (TCE) by toluene-degrading bacteria was measured under aerobic conditions in aqueous and soil-slurry batch microcosms. For soil-phase experiments, a freshly contaminated soil and a soil containing only the desorption-resistant fraction of TCE were tested. In both cases, presence of soil resulted in biodegradation rates substantially lower than those determined in the absence of soil. In aqueous-phase experiments, an appreciable increase in the rate and extent of TCE biodegradation was observed in microcosms when toluene was added multiple times. The TCE degradation rates were clearly correlated with toluene dioxygenase (TOD) enzyme activity over time, thus providing an indication of the cometabolic pathway employed by the microbial population. In soil-slurry experiments containing freshly contaminated soil, a TCE degradation rate of approximately 150 microg TCE/kg/h was observed during the first 39-h period, and then the TCE degradation rate slowed considerably to 0.59 and 0.84 microg TCE/kg/h for microcosms receiving one and two additions of toluene, respectively. The TCE degradation rates in soil-slurry microcosms containing the desorption-resistant fraction of TCE-contaminated soil were approximately 0.27 and 0.32 microg TCE/kg/h in microcosms receiving one and two additions of toluene, respectively. It is clear from these results that mass transfer into the aqueous phase limited bioavailability of TCE in the contaminated soil.

EFFECT OF SORPTION AND DESORPTION RESISTANCE ON AEROBIC TRICHLOROETHYLENE BIODEGRADATION IN SOILS

Biodegradation of trichloroethylene(TCE) by toluene-degrading bacteria was measured under aerobic conditions in aqueous and soil-slurry batch microcosms. For soil-phase experiments, a freshly contaminated soil and a soil containing only the desorption-resistant fraction of TCE were tested. In both cases, presence of soil resulted in biodegradation rates substantially lower than those determined in the absence of soil. In aqueous-phase experiments, an appreciable increase in the rate and extent of TCE biodegradation was observed in microcosms when toluene was added multiple times. The TCE degradation rates were clearly correlated with toluene dioxygenase (TOD) enzyme activity over time, thus providing an indication of the cometabolic pathway employed by the microbial population. In soil-slurry experiments containing freshly contaminated soil, a TCE degradation rate of approximately 150 μg TCE/kg/h was observed during the first 39-h period, and then the TCE degradation rate slowed considerably to 0.59 and 0.84 μg TCE/kg/h for microcosms receiving one and two additions of toluene, respectively. The TCE degradation rates in soil-slurry microcosms containing the desorption-resistant fraction of TCE-contaminated soil were approximately 0.27 and 0.32 μg TCE/kg/h in microcosms receiving one and two additions of toluene, respectively. It is clear from these results that mass transfer into the aqueous phase limited bioavailability of TCE in the contaminated soil.

Stress Survival of a Genetically Engineered Pseudomonas in Soil Slurries: Cytochrome P-450cam-Catalyzed Dehalogenation of Chlorinated Hydrocarbons.

Biological treatment of hazardous chemical wastes has potential as an effective, practical, and economically viable process in above the ground treatment systems that consist of both genetically engineered microorganisms (GEMs) and bioreactors with process control instruments to create ideal conditions for biodegradation. A strain of Pseudomonas putida coexpressing cytochrome P-450cam and luciferase (lux) that provides both the reductive detoxification potential of the hemoprotein and a mechanism for its reduction in the absence of "normal" P-450 redox partners was evaluated for its ability to survive and remain metabolically competent under nutrient stress in soil slurry microcosms. More than 74% of the cells of engineered Pseudomonas were culturable after 7 days of multiple nutrient (C,N,P) starvation. The diagnostic luminescence and carbon monoxide-difference spectra for the two engineered traits could be detected in a significant fraction of the surviving population. The GEM could be revived after repeated desiccation and starvation using Luria broth, benzoate, or citrate as nutrients. Soil slurries inoculated with the GEM transformed hexachloroethane (HCE) to tetrachloroethylene (tetraCE) 8-10-fold faster than uninoculated slurries. The GEM also transformed the insecticide, gamma-HCH (gamma-3,4,5,6-hexachlorocyclhexene), to gamma-3,4,5,6-tetrachlorocyclohexene (gammatetraCH) in soil slurries under subatmospheric conditions. These results indicate that GEMs can be constructed with broad substrate range detoxification catalysts such as cytochrome P-450 for remediation.

Anaerobic biodegradation of pentachlorophenol in a contaminated soil inoculated with a methanogenic consortium or with Desulfitobacterium frappieri strain PCP-1.

Anaerobic biodegradation of pentachlorophenol (PCP) in a contaminated soil from a wood-treating industrial site was studied in soil slurry microcosms inoculated with a PCP-degrading methanogenic consortium. When the microcosms containing 10%-40% (w/v) soil were inoculated with the consortium, more than 90% of the PCP was removed in less than 30 days at 29 degrees C. Less-chlorinated phenols, mainly 3-chlorophenol were slowly degraded and accumulated in the cultures. Addition of glucose and sodium formate to the microcosms was not necessary, suggesting that the organic compounds in the soil can sustain the dechlorinating activity. Inoculation of Desulfitobacterium frappieri strain PCP-1 along with a 3-chlorophenol-degrading consortium in the microcosms also resulted in the rapid dechlorination of PCP and the slow degradation of 3-chlorophenol. Competitive polymerase chain reaction experiments showed that PCP-1 was present at the same level throughout the 21-day biotreatment. D. frappieri, strain PCP-1, inoculated into the soil microcosms, was able to remove PCP from soil containing up to 200 mg PCP/kg soil. However, reinoculation of the strain was necessary to achieve more than 95% PCP removal with a concentration of 300 mg and 500 mg PCP/kg soil. These results demonstrate that D. frappieri strain PCP-1 can be used effectively to dechlorinate PCP to 3-chlorophenol in contaminated soils.

Introduction of anaerobic dechlorinating bacteria into soil slurry microcosms and nested-PCR monitoring

Desulfomonile tiedjei and Desulfitobacterium dehalogenans were chosen as model bacteria to demonstrate the introduction of an anaerobic microbia reductive dechlorination activity into nonsterile soil slurry microcosms by inoculation. De novo 3-chlorobenzoate dechlorination activity was established with the bacterium D. tiedjei in microcosms normally devoid of this dechlorination capacity. The addition of D. tiedjei to microcosms supplemented with 20 mM pyruvate as the cosubstrate resulted in total biotransformation of 1.5 mM 3-chlorobenzoate within 7 days. The introduction of the bacterium Desulfitobacterium dehalogenans into nonsterile microcosms resulted in a shortening of the period required for dechlorination activity to be established. In microcosms inoculated with Desulfitobacterium dehalogenans, total degradation of 6 mM 3-chloro-4-hydroxy phenoxyacetic acid (3-Cl-4-OHPA) was observed after 4 days in contrast to the result in noninoculated microcosms, where the total degradation of 3-Cl-4-OHPA by indigenous microorganisms was observed after 11 days. Both externally introduced bacterial strains were detected in soil slurry microcosms by a nested-PCR methodology.

Introduction and PCR detection of Desulfomonile tiedjei in soil slurry microcosms.

The aim of this work was to test the feasibility of introducing an anaerobic microbial reductive dechlorination activity into non sterile soil slurry microcosms by inoculation with the pure anaerobic bacterial strain Desulfomonile tiedjei, which is capable of dechlorinating 3-chlorobenzoate to benzoate. To show that the bacterium was established in the microcosms we followed the expression of the reductive dechlorination activity and a molecular probe based on PCR amplification of the 16S rDNA gene was developed. However, the success of PCR amplification of the 16S rDNA gene depends on the DNA extraction and purification methodologies applied, as shown through the use of several protocols. In this study we report a DNA extraction and purification method which generates sufficient and very clean DNA suitable for PCR amplification of the D. tiedjei 16S rDNA gene. The threshold of detection was about 5.10(3) bacteria per gram of soil slurry. Introduction of D. tiedjei in soil slurry microcosms proved successful since 3-chlorobenzoate dechlorination activity was established with this bacterium in microcosms normally devoid of this dechlorination capacity. Indeed, the addition of D. tiedjei to microcosms supplemented with acetate plus formate as cosubstrate, at their respective concentrations of 5 and 6 mM, led to a total biotransformation of 2.5 mM of 3-chlorobenzoate within 12 days. After complete 3-chlorobenzoate dechlorination, the 16S rDNA gene of this bacterium was specifically detected only in the inoculated microcosms as shown by PCR amplification followed by restriction mapping confirmation.

The effect of inorganic and organic supplements on the microbial degradation of phenanthrene and pyrene in soils.

The effects of several bioremediation stimulants, including potential metabolism pathway inducers, inorganic/organic nutrients, and surfactants on the metabolism of phenanthrene and pyrene, as well as the population dynamics of PAH degrading microorganisms was examined in five soils with differing background PAH concentrations, exposure histories and physical properties. Most of the supplements either had no significant effect or decreased the mineralization of [14C]-phenanthrene and [14C]-pyrene in soil slurry microcosms. The effect of a particular supplement, however, was often not uniform within or across soils. Decreased mineralization of [14C]-phenanthrene and [14C]-pyrene was usually due to either preferential use of the supplement as carbon source and/or stimulation of non-PAH degrading microorganisms. Many of the supplements increased populations of heterotrophic microorganisms, as measured by plate counts, but did not increase populations of phenanthrene degrading microorganisms, as measured by the [14C]-PAH mineralization MPN analysis or cellular incorporation of [14C]-PAH. These results suggest that the PAH degrading community at each site may be unique in their response to materials added in an attempt to stimulate PAH degradation. The characteristics of the site, including exposure history, soil type, and temporal variation may all influence their response.