Means to improve the effect of in situ bioremediation of contaminated soil: an overview of novel approaches

Different aspects of bacterial degradation of organic contaminants in soil, and how to improve the efficiency and reproducibility is discussed in this review. Although bioremediation in principle includes the use of any type of organism in improving the condition of a contaminated site, most commonly bacteria are the degraders and other organisms, such as soil animals or plant roots, play a role in dissemination of bacteria and, indirectly, plasmids between bacteria, and in providing nutrients and co-substrates for the bacteria active in the degradation process. There are a number of different procedures that have been tested more-or-less successfully in attempts to improve reliability, cost efficiency and speed of bioremediation. The methods range from minimal intervention, such as mere monitoring of intrinsic bioremediation, through in situ introduction of nutrients and/or bacterial inocula or improvement of physico-chemical conditions, all the way to excavation followed by on site or ex situ composting in its different varieties. In the past the rule has been that more intervention (leading to higher costs) has been more reliable, but novel ideas are continuously tried out, both as a means to come up with new truly functional applications and also as a line of studies in basic soil microbial ecology. Both approaches generate valuable information needed when predicting outcome of remediation activities, evaluating environmental risks, deciding on cleaning-up approaches, etc. The emphasis of this review is to discuss some of the novel methods for which the value has not been clearly shown, but that in our view merit continued studies and efforts to make them work, separately or in combination.     

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.     

不同处理条件对石油污染土壤植物修复的影响

针对石油烃植物修复过程中的主要影响因素,研究了不同植物种类、不同土壤调理剂和菌剂使用等不同条件对土壤中石油烃植物修复效果的影响.结果表明,不同种类的植物修复可使总石油烃的年降解率达到37.8％ ～ 73.98％,其中大豆和碱蓬具有较好的修复效果;3种不同土壤调理剂对石油烃污染土壤修复的效果为商业添加剂＞牛粪＞蛭石;先微生物修复后种植植物的处理要优于单独的微生物修复及微生物、植物修复同步进行的处理.     

Ecotoxicological evaluation of in situ bioremediation of soils contaminated by the explosive 2,4,6-trinitrotoluene (TNT)

To evaluate the environmental relevance of in situ bioremediation of contaminated soils, effective and reliable monitoring approaches are of special importance. The presented study was conducted as part of a research project investigating in situ bioremediation of topsoils contaminated by the explosive 2,4,6-trinitrotoluene (TNT). Changes in soil toxicity within different experimental fields at a former ordnance factory were evaluated using a battery of five bioassays (plant growth, Collembola reproduction, soil respiration, luminescent bacteria acute toxicity and mutagenicity test) in combination to chemical contaminant analysis. Resulting data reveal clear differences in sensitivities between methods with the luminescent bacteria assay performed with soil leachates as most sensitive toxicity indicator. Complete test battery results are presented in so-called soil toxicity profiles to visualise and facilitate the interpretation of data. Both biological and chemical monitoring results indicate a reduction of soil toxicity within 17 months of remediation.     

Bioremediation of soil contaminated with polynuclear aromatic hydrocarbons (PAHs): a review

Polynuclear aromatic hydrocarbons (PAHs) constitute a group of priority pollutants which are present at high concentrations in the soils of many industrially contaminated sites. Criteria established for the removal or treatment or both of soils contaminated with PAHs vary widely within and between nations. The bioremediation of contaminated soils with in-situ, on-site, and bioreactor techniques is reviewed, together with the factors affecting PAH degradation. Current in-situ remediation techniques are considered ineffective for the removal of most PAHs from contaminated soil. On-site 'landforming' methods have been used successfully (and within a reasonable period of time) to degrade only those PAHs with three or fewer aromatic rings. Bioreactors have proved most effective for soil remediation, since conditions for enhanced degradation can be achieved most readily. However, bioreactors are still at the development stage, and further research is required to optimise their efficiency and economy for routine use. Degradation of the more recalcitrant high-molecular-weight PAHs is contaminated soil has not been particularly successful to date. Further research needs are identified to help develop bioremediation into a most cost-effective technology. The importance of full site assessments and treatability studies for successful application in the field is emphasised.     

Assessment of heavy metal bioavailability in contaminated sediments and soils using green fluorescent protein-based bacterial biosensors

A green fluorescent protein (GFP)-based bacterial biosensor Escherichia coli DH5alpha (pVLCD1) was developed based on the expression of gfp under the control of the cad promoter and the cadC gene of Staphylococcus aureus plasmid pI258. DH5alpha (pVLCD1) mainly responded to Cd(II), Pb(II), and Sb(III), the lowest detectable concentrations being 0.1 nmol L(-1), 10 nmol L(-1), and 0.1 nmol L(-1), respectively, with 2h exposure. The biosensor was field-tested to measure the relative bioavailability of the heavy metals in contaminated sediments and soil samples. The results showed that the majority of heavy metals remained adsorbed to soil particles: Cd(II)/Pb(II) was only partially available to the biosensor in soil-water extracts. Our results demonstrate that the GFP-based bacterial biosensor is useful and applicable in determining the bioavailability of heavy metals with high sensitivity in contaminated sediment and soil samples and suggests a potential for its inexpensive application in environmentally relevant sample tests.     

Soil microbial parameters and luminescent bacteria assays as indicators for in situ bioremediation of TNT-contaminated soils

In situ bioremediation is increasingly being discussed as a useful strategy for cleaning up contaminated soils. Compared to established ex situ procedures, meaningful and reliable approaches for monitoring the remediation processes and their efficiency are of special importance. The subject of this study was the significance of two bioassays for monitoring purposes. The work was performed within the scope of a research project on the in situ bioremediation of topsoil contaminated with 2,4,6-trinitrotoluene (TNT). To evaluate changes within different experimental fields during a 17-month remediation period, the results of soil microbial assays and luminescent bacteria assays were compared with chemical monitoring data. The luminescent bacteria assays showed a significant reduction of the water-soluble soil toxicants in the treated fields. This bioassay proved to be a sensitive screening indicator of toxicity and may effectively aid the ecotoxicological interpretation of chemical monitoring data. Microbial biomass (C(mic)), the metabolic quotient (qCO2), and the ratio of microbial to organic carbon (C(mic)/C(org)) showed a highly significant correlation with total concentrations of TNT in the soil. But, in contrast to luminescent bacteria assays, this approach did not reveal any recovery of the soil at the end of the remediation period. There is clear evidence for persistent adverse effects of chronic TNT contamination on the site-specific microbial community and the local carbon cycle in the soil. The study clearly exhibits the differences between, as well as the complementary value of both bioassay approaches for monitoring short-term and long-term effects of soil contamination and the efficiency of remediation.     

Bacterial release of arsenic ions and organoarsenic compounds from soil contaminated by chemical warfare agents

The objective of this paper was to investigate possible participation of microorganisms in the release of soluble arsenical compounds from organoarsenic warfare agents in contaminated soil. A number of bacterial strains were isolated with high resistance against As3+ and As5+ ions which are able to degrade the water insoluble compounds triphenylarsine (TP) and triphenylarsineoxide (TPO). These strains belong to different genera of bacteria. Release of arsenic ions and soluble organoarsenic compounds from soil by the activity of autochthonic soil bacteria and a mixture of the isolated pure cultures was demonstrated by percolation experiments with undisturbed soil samples (core drills) from the contaminated site. This release increased after addition of nutrients (mineral nitrogen and phosphorus, sodium acetate and ethanol) and is nearly independent of the percolation temperature (5 degrees C and 22 degrees C). These results show that bacteria play an important role in the release of arsenical compounds from organoarsenic warfare agent contaminated soil. This release is limited by shortage of water and, above all, of nutrients for the microorganisms in the sandy forest soil. These results are important both for the management and security and possibly for bioremediation of military waste sites containing similar contaminations. Furthermore, this is the first report on bacterial degradation of organoarsenic warfare compounds.     

土壤有机农药污染的降解机理与生物修复技术

通过对有机农药在土壤中的环境行为与迁移转化规律综合分析研究,探讨了有机农药污染土壤的生物修复技术,并对生物修复技术进行了前瞻性研究.     

AhR agonist and genotoxicant bioavailability in a PAH-contaminated soil undergoing biological treatment

Background, aim, and scope  Degradation of the 16 US EPA priority PAHs in soil subjected to bioremediation is often achieved. However, the PAH loss is not always followed by a reduction in soil toxicity. For instance, bioanalytical testing of such soil using the chemical-activated luciferase gene expression (CALUX) assay, measuring the combined effect of all Ah receptor (AhR) activating compounds, occasionally indicates that the loss of PAHs does not correlate with the loss of Ah receptor-active compounds in the soil. In addition, standard PAH analysis does not address the issue of total toxicant bioavailability in bioremediated soil. Materials and methods  To address these questions, we have used the CALUX AhR agonist bioassay and the Comet genotoxicity bioassay with RTL-W1 cells to evaluate the toxic potential of different extracts from a PAH-contaminated soil undergoing large-scale bioremediation. The extracts were also chemically analyzed for PAH16 and PCDD/PCDF. Soil sampled on five occasions between day 0 and day 274 of biological treatment was shaken with n-butanol with vortex mixing at room temperature to determine the bioavailable fraction of contaminants. To establish total concentrations, parts of the same samples were extracted using an accelerated solvent extractor (ASE) with toluene at 100°C. The extracts were tested as inducers of AhR-dependent luciferase activity in the CALUX assay and for DNA breakage potential in the Comet bioassay. Results  The chemical analysis of the toluene extracts indicated slow degradation rates and the CALUX assay indicated high levels of AhR agonists in the same extracts. Compared to day 0, the bioavailable fractions showed no decrease in AhR agonist activity during the treatment but rather an up-going trend, which was supported by increasing levels of PAHs and an increased effect in the Comet bioassay after 274 days. The bio-TEQs calculated using the CALUX assay were higher than the TEQs calculated from chemical analysis in both extracts, indicating that there are additional toxic PAHs in both extracts that are not included in the chemically derived TEQ. Discussion  The response in the CALUX and the Comet bioassays as well as the chemical analysis indicate that the soil might be more toxic to organisms living in soil after 274 days of treatment than in the untreated soil, due to the release of previously sorbed PAHs and possibly also metabolic formation of novel toxicants. Conclusions  Our results put focus on the issue of slow degradation rates and bioavailability of PAHs during large-scale bioremediation treatments. The release of sorbed PAHs at the investigated PAH-contaminated site seemed to be faster than the degradation rate, which demonstrates the importance of considering the bioavailable fraction of contaminants during a bioremediation process. Recommendations and perspectives  It has to be ensured that soft remediation methods like biodegradation or the natural remediation approach do not result in the mobilization of toxic compounds including more mobile degradation products. For PAH-contaminated sites this cannot be assured merely by monitoring the 16 target PAHs. The combined use of a battery of biotests for different types of PAH effects such as the CALUX and the Comet assay together with bioavailability extraction methods may be a useful screening tool of bioremediation processes of PAH-contaminated soil and contribute to a more accurate risk assessment. If the bioremediation causes a release of bound PAHs that are left undegraded in an easily extracted fraction, the soil may be more toxic to organisms living in the soil as a result of the treatment. A prolonged treatment time may be one way to reduce the risk of remaining mobile PAHs. In critical cases, the remediation concept might have to be changed to ex situ remediation methods.     

The application of bioassays as indicators of petroleum-contaminated soil remediation

Bioremediation has proven successful in numerous applications to petroleum contaminated soils. However, questions remain as to the efficiency of bioremediation in lowering long-term soil toxicity. In the present study, the bioassays Spirotox, Microtox, Ostracodtoxkit F, umu-test with S-9 activation, and plant assays were applied, and compared to evaluate bioremediation processes in heavily petroleum contaminated soils. Six higher plant species (Secale cereale L., Lactuca sativa L., Zea mays L., Lepidium sativum L., Triticum vulgare L., Brassica oleracea L.) were used for bioassay tests based on seed germination and root elongation. The ecotoxicological analyses were made in DMSO/H2O and DCM/DMSO soil extracts. Soils were tested from two biopiles at the Czechowice oil refinery, Poland, that have been subjected to different bioremediation applications. In biopile 1 the active or engineered bioremediation process lasted four years, while biopile 2 was treated passively or non-engineered for eight months. The test species demonstrated varying sensitivity to soils from both biopiles. The effects on test organisms exposed to biopile 2 soils were several times higher compared to those in biopile 1 soils, which correlated with the soil contaminants concentration. Soil hydrocarbon concentrations indeed decreased an average of 81% in biopile 1, whereas in biopile 2 TPH/TPOC concentrations only decreased by 30% after eight months of bioremediation. The bioassays were presented to be sensitive indicators of soil quality and can be used to evaluate the quality of bioremediated soil. The study encourages the need to combine the bioassays with chemical monitoring for evaluation of the bioremediation effectiveness and assessing of the contaminated/remediated soils.     

Isolation and physiological characterization of the pentachlorophenol degrading bacterium Sphingomonas chlorophenolica

Many chlorophenols tend to persist in the environment, and they may become public health hazards. Among chlorophenols, pentachlorophenol (PCP) is a priority pollutant that has been used widely as a general biocide in commercial wood treatment. Owing to the rapid industrial growth, serious soil and water pollutions by chlorophenols has been reported in Taiwan. In this study, 10 indigenous PCP-degrading bacterial strains were isolated from a PCP-degrading mixed culture, and the potential of both the pure and mixed cultures for PCP degradation compared. Moreover, the physiological characteristics and optimum growth conditions of the PCP-degrading bacteria were investigated. One of the isolated bacterial strains with good potential for PCP degradation was characterized and identified as Sphingomonas chlorophenolica by 16S rDNA gene analysis. The result of the optimum growth temperatures revealed that this organism was a mesophile. The optimum pH for PCP removal by S. chlorophenolica was between 6.9 and 7.6. Increase in concentration of PCP has a negative effect on the biodegradation potential of S. chlorophenolica and PCP concentration above 600 mg l(-1) was inhibitory to its growth. The results of this study indicate that this S. chlorophenolica strain has a better potential for PCP degradation compared to the enriched mixed culture. The physiological characterization of the isolates also indicates the possible application of this strain for bioremediation of sites contaminated with PCP.     

Evaluation of TCDD biodegradability under different redox conditions

Polychlorinated dibenzo-p-dioxins have been generated as unwanted by-products in many industrial processes. Although their widespread distribution in different environmental compartments has been recognized, little is known about their fate in the ultimate environment sinks. The highly stable dioxin isomer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been called the most toxic compound known to man. In this laboratory microcosm study, TCDD bioavailability was evaluated under five reduction/oxidation (redox) conditions including aerobic biodegradation, aerobic cometabolism, methanogenesis, iron reduction, and reductive dechlorination. Activated sludge and aquifer sediments from a TCDD and a pentachlorophenol (PCP) contaminated site were used as the inocula. Acetate, sludge cake, and cane molasses were used as the primary substrates (carbon sources) in cometabolism and reductive dechlorination microcosms. After a 90-day incubation period, microcosms constructed under reductive dechlorination conditions were the only treatment showing promising remediation results. The highest TCDD degradation rate [up to 86% of TCDD removal (with an initial concentration of 96 microg/kg of soil)] was observed in the microcosms with anaerobic activated sludge as the microbial inocula and sludge cakes as the primary substrates. Except for reductive dechlorination microcosms, no significant TCDD removal was observed in the microcosms prepared under other conditions. Thus, application of an effective primary substrate to enhance the reductive dechlorination process is a feasible method for TCDD bioremediation. Bioremediation expense can be significantly reduced by the supplement of some less expensive alternative substrates (e.g., sludge cakes, cane molasses). Results would be useful in designing a scale-up in situ or on-site bioremediation system such as bioslurry reactor for field application.     

降解菌ZQ5与紫茉莉对芘污染土壤的联合修复

在温室盆栽条件下,通过单独种植紫茉莉、单独接种多环芳烃(PAHs)模式化合物芘的专性降解菌ZQ5和两者的联合修复的3种处理,对芘污染土壤的修复效果进行了研究。结果表明,经90 d修复后,植物-微生物联合修复可将人工污染土壤中的芘降解81.1%,将石油污染土壤中的芘降解50.3%,其修复效率明显高于其他2种处理,是紫茉莉修复的1.98倍,是降解菌ZQ5修复的1.39倍。ZQ5的不同接菌量对于修复60 d后的降解率影响不大。外源生物修复条件下,10～20 cm土壤的修复效率要高于5 cm土壤;自然降解条件下,5 cm土层降解率略高于其他土层。     

Advanced multivariate analysis to assess remediation of hydrocarbons in soils

Accurate monitoring of degradation levels in soils is essential in order to understand and achieve complete degradation of petroleum hydrocarbons in contaminated soils. We aimed to develop the use of multivariate methods for the monitoring of biodegradation of diesel in soils and to determine if diesel contaminated soils could be remediated to a chemical composition similar to that of an uncontaminated soil. An incubation experiment was set up with three contrasting soil types. Each soil was exposed to diesel at varying stages of degradation and then analysed for key hydrocarbons throughout 161 days of incubation. Hydrocarbon distributions were analysed by Principal Coordinate Analysis and similar samples grouped by cluster analysis. Variation and differences between samples were determined using permutational multivariate analysis of variance. It was found that all soils followed trajectories approaching the chemical composition of the unpolluted soil. Some contaminated soils were no longer significantly different to that of uncontaminated soil after 161 days of incubation. The use of cluster analysis allows the assignment of a percentage chemical similarity of a diesel contaminated soil to an uncontaminated soil sample. This will aid in the monitoring of hydrocarbon contaminated sites and the establishment of potential endpoints for successful remediation.     

Biostimulation and bioaugmentation enhances aerobic biodegradation of dichloroethenes

The accumulation of dichloroethenes (DCEs) as dominant products of microbial reductive dechlorination activity in soil and water represent a significant obstacle to the application of bioremediation as a remedial option for chloroethenes in many contaminated systems. In this study, the effects of biostimulation and/or bioaugmentation on the biodegradation of cis- and trans-DCE in soil and water samples collected from contaminated sites in South Africa were evaluated in order to determine the possible bioremediation option for these compounds in the contaminated sites. Results from this study indicate that cis- and trans-DCE were readily degraded to varying degrees by natural microbial populations in all the soil and water samples tested, with up to 44% of cis-DCE and 41% of trans-DCE degraded in the untreated soil and water samples in two weeks. The degradation rate constants ranged significantly (P<0.05) between 0.0938 and 0.560 wk(-1) and 0.182 and 0.401 wk(-1), for cis- and trans-DCE, respectively, for the various treatments employed. A combination of biostimulation and bioaugmentation significantly increased the biodegradation of both compounds within two weeks; 14% for cis-DCE and 18% for trans-DCE degradation, above those observed in untreated soil and water samples. These findings support the use of a combination of biostimulation and bioaugmentation for the efficient biodegradation of these compounds in contaminated soil and water. In addition, the results clearly demonstrate that while naturally occurring microorganisms are capable of aerobic biodegradation of cis- and trans-DCE, biotransformation may be affected by several factors, including isomer structure, soil type, and the amount of nutrients available in the water and soil.     

When is a soil remediated? Comparison of biopiled and windrowed soils contaminated with bunker-fuel in a full-scale trial

A six month field scale study was carried out to compare windrow turning and biopile techniques for the remediation of soil contaminated with bunker C fuel oil. End-point clean-up targets were defined by human risk assessment and ecotoxicological hazard assessment approaches. Replicate windrows and biopiles were amended with either nutrients and inocula, nutrients alone or no amendment. In addition to fractionated hydrocarbon analysis, culturable microbial characterisation and soil ecotoxicological assays were performed. This particular soil, heavy in texture and historically contaminated with bunker fuel was more effectively remediated by windrowing, but coarser textures may be more amendable to biopiling. This trial reveals the benefit of developing risk and hazard based approaches in defining end-point bioremediation of heavy hydrocarbons when engineered biopile or windrow are proposed as treatment option.     

Enhanced degradation of fluorene in soil slurry by Absidia cylindrospora and maltosyl-cyclodextrin

This study investigates the fungal biodegradation of fluorene, a polycyclic aromatic hydrocarbon, in liquid medium and soil slurry. Fungal strains and cyclodextrins were used in order to degrade fluorene and optimize fluorene bioavailability and degradation in soil slurries. After a procedure of selection in solid and liquid media, maltosyl-cyclodextrin, a branched cyclodextrin was chosen. 47 fungal strains isolated from a contaminated site were tested for biodegradation. Results showed the greater efficiency of "adapted" fungi isolated from contaminated soil vs reference strains belonging to the collection of the laboratory. These assays allowed us to select the most efficient strain, Absidia cylindrospora, which was used in a bioaugmentation process. Bioaugmentation tests were performed in an artificially contaminated non-sterile soil. In the presence of A. cylindrospora, more than 90% of the fluorene was degraded within 288 h, while 576 h were necessary in the absence of fungal bioremediation. It also appeared that biodegradation was enhanced by amendment with previously selected maltosyl-cyclodextrin. The results of this study indicate that A. cylindrospora and maltosyl-cyclodextrin could be used successfully in fluorene bioremediation systems.     

Biodegradation of endosulfan by Mortieralla sp. strain W8 in soil: Influence of different substrates on biodegradation

To examine the bioremediation potential of Mortierella sp. strain W8 in endosulfan contaminated soil, the fungus was inoculated into sterilized and unsterilized soil spiked with endosulfan. Wheat bran and cane molasses were used as substrates to understand the influence of different organic materials on the degradation of endosulfan in soil. Strain W8 degraded α- and β-endosulfan in both sterilized and unsterilized soil. In unsterilized soil with wheat bran+W8, α- and β- endosulfan were degraded by approximately 80% and 50%, respectively after 28 d incubation against the initial endosulfan concentration (3 mg kg(-1) dw). The corresponding values for α- and β-endosulfan degradation with wheat bran only were 50% and 3%. Endosulfan diol metabolite was detected after 14 d incubation in wheat bran+W8 whereas it was not found with wheat bran only. Production of endosulfan sulfate, the main metabolite of endosulfan, was suppressed with wheat bran+W8 treatment compared with wheat bran only. It was demonstrated that wheat bran is a more suitable substrate for strain W8 than cane molasses. Wheat bran+W8 is a superior fungus and substrate mix for bioremediation in soil contaminated with endosulfan.     

Bioavailability and biodegradation of prosulfocarb in soil

Active microbial degraders of the herbicide prosulfocarb (PSC) were isolated to evaluate their performance in soil with a view to their use for bioremediation. The isolated cultures (a microbial consortium and a Pseudomonas sp. strain) were active when tested in mineral medium with PSC as the only carbon source, but had an adverse effect on the soil indigenous microflora. Biodegradation in the inoculated soils was thus lower than in the uninoculated soil when only the indigenous microflora was present. Further tests showed that the strong affinity of PSC for soil organic matter affected its bioavailability and hence its biodegradation by the inocula. Bioremediation of PSC contaminated soils could thus be undertaken by biostimulation of indigenous microflora.