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1.
The activity of microorganisms often plays an important role in dynamic natural attenuation or engineered bioremediation of subsurface contaminants, such as chlorinated solvents, metals, and radionuclides. To evaluate and/or design bioremediated systems, quantitative reactive transport models are needed. State-of-the-art reactive transport models often ignore the microbial effects or simulate the microbial effects with static growth yield and constant reaction rate parameters over simulated conditions, while in reality microorganisms can dynamically modify their functionality (such as utilization of alternative respiratory pathways) in response to spatial and temporal variations in environmental conditions. Constraint-based genome-scale microbial in silico models, using genomic data and multiple-pathway reaction networks, have been shown to be able to simulate transient metabolism of some well studied microorganisms and identify growth rate, substrate uptake rates, and byproduct rates under different growth conditions. These rates can be identified and used to replace specific microbially-mediated reaction rates in a reactive transport model using local geochemical conditions as constraints. We previously demonstrated the potential utility of integrating a constraint-based microbial metabolism model with a reactive transport simulator as applied to bioremediation of uranium in groundwater. However, that work relied on an indirect coupling approach that was effective for initial demonstration but may not be extensible to more complex problems that are of significant interest (e.g., communities of microbial species and multiple constraining variables). Here, we extend that work by presenting and demonstrating a method of directly integrating a reactive transport model (FORTRAN code) with constraint-based in silico models solved with IBM ILOG CPLEX linear optimizer base system (C library). The models were integrated with BABEL, a language interoperability tool. The modeling system is designed in such a way that constraint-based models targeting different microorganisms or competing organism communities can be easily plugged into the system. Constraint-based modeling is very costly given the size of a genome-scale reaction network. To save computation time, a binary tree is traversed to examine the concentration and solution pool generated during the simulation in order to decide whether the constraint-based model should be called. We also show preliminary results from the integrated model including a comparison of the direct and indirect coupling approaches and evaluated the ability of the approach to simulate field experiment.  相似文献   

2.
Environments with high-salt concentrations are often populated by dense microbial communities. Halophilic microorganisms can be isolated from different saline environments and different strains even belonging to the same genus have various applications. Wastewater and soil rich in both organic matter and salt are difficult to treat using conventional microorganisms typically found in wastewater treatment and soil bioremediation facilities. Studies on decontaminative capabilities and decontamination pathways of organic contaminants (i.e., aromatic compounds benzoate, cinnamate, 3-phenylpropionate, 4-hydroxybenzoic acid), heavy metals (i.e., tellurium, vanadium), and nutrients in the biological treatment of saline wastewater and soil by halophilic microorganisms are discussed in this review.  相似文献   

3.
Copper and iron isotope fractionation by plant uptake and translocation is a matter of current research. As a way to apply the use of Cu and Fe stable isotopes in the phytoremediation of contaminated sites, the effects of organic amendment and microbial addition in a mine-spoiled soil seeded with Helianthus annuus in pot experiments and field trials were studied. Results show that the addition of a microbial consortium of ten bacterial strains has an influence on Cu and Fe isotope fractionation by the uptake and translocation in pot experiments, with an increase in average of 0.99?‰ for the δ65Cu values from soil to roots. In the field trial, the amendment with the addition of bacteria and mycorrhiza as single and double inoculation enriches the leaves in 65Cu compared to the soil. As a result of the same trial, the δ56Fe values in the leaves are lower than those from the bulk soil, although some differences are seen according to the amendment used. Siderophores, possibly released by the bacterial consortium, can be responsible for this change in the Cu and Fe fractionation. The overall isotopic fractionation trend for Cu and Fe does not vary for pot and field experiments with or without bacteria. However, variations in specific metabolic pathways related to metal–organic complexation and weathering can modify particular isotopic signatures.  相似文献   

4.
The interaction between two autochthonous microorganisms (Brevibacillus brevis and Glomus mosseae) isolated from Cd amended soil increased plant growth, arbuscular mycorrhizal (AM) colonization and physiological characteristics of the AM infection (measured as SDH or ALP activities). The enhanced plant Cd tolerance after coinoculation with native microorganisms seemed to be a consequence of increased P and K acquisition and, simultaneously, of decreased concentration of Cd, Cr, Mn, Cu, Mo, Fe and Ni in plant tissue. Autochthonous microbial strains were more efficient for nutrient uptake, to immobilize metals and decrease their translocation to the shoot than reference G. mosseae (with or without bacteria). Indole acetic acid produced by B. brevis may be related to its ability for improving root growth, nodule production and AM fungal intra and extraradical development. Dehydrogenase, phosphatase and beta-glucosidase activities, indicative of microbial metabolism and soil fertility, were maximized by the coinoculation of autochthonous microorganisms in cadmium polluted conditions. As a consequence, the use of native microorganisms may result very efficient in bioremediation.  相似文献   

5.
In recent years, microbial degradation and bioremediation approaches of polychlorinated biphenyls (PCBs) have been studied extensively considering their toxicity, carcinogenicity and persistency potential in the environment. In this direction, different catabolic enzymes have been identified and reported for biodegradation of different PCB congeners along with optimization of biological processes. A genome analysis of PCB-degrading bacteria has led in an improved understanding of their metabolic potential and adaptation to stressful conditions. However, many stones in this area are left unturned. For example, the role and diversity of uncultivable microbes in PCB degradation are still not fully understood. Improved knowledge and understanding on this front will open up new avenues for improved bioremediation technologies which will bring economic, environmental and societal benefits. This article highlights on recent advances in bioremediation of PCBs in soil. It is demonstrated that bioremediation is the most effective and innovative technology which includes biostimulation, bioaugmentation, phytoremediation and rhizoremediation and acts as a model solution for pollution abatement. More recently, transgenic plants and genetically modified microorganisms have proved to be revolutionary in the bioremediation of PCBs. Additionally, other important aspects such as pretreatment using chemical/physical agents for enhanced biodegradation are also addressed. Efforts have been made to identify challenges, research gaps and necessary approaches which in future, can be harnessed for successful use of bioremediation under field conditions. Emphases have been given on the quality/efficiency of bioremediation technology and its related cost which determines its ultimate acceptability.  相似文献   

6.
Increasingly often soil residual concentrations of pharmaceutical antibiotics are detected, while their ecotoxic relevance is scarcely known. Thus, dose related effects of two antibiotics, sulfapyridine and oxytetracycline, on microorganisms of two different topsoils were investigated. The fumigation-extracted microbial C (E(C)) and ergosterol were determined to indicate soil microbial and fungal biomass, respectively. Microbial activity was tested as basal respiration (BR), dehydrogenase activity (DHA), substrate-induced respiration (SIR), and Fe(III) reduction. The BR and DHA were uninfluenced even at antibiotic concentrations of 1000 microg g(-1). This revealed that an activation of microbial growth through nutrient substrate addition is required to test possible effects of the bacteriostatic antibiotics. In addition, the effects of both antibiotics were time dependent, showing that short-term tests were not suitable. Clear dose-response relations were determined with SIR when the short-term incubation of 4h was extended into the growth phase of the microorganisms (24 and 48 h). The Fe(III) reduction test, with a 7-d incubation, was also found to be suitable for toxicity testing of antibiotics in soils. Effective doses inhibiting the microbial activity by 10% (ED(10)) ranged from total antibiotic concentrations of 0.003-7.35 microg g(-1), depending on the antibiotic compound and its soil adsorption. Effective solution concentrations (EC(10)), calculated from distribution coefficients, ranged from 0.2 to 160 ng g(-1). The antibiotics significantly (p<0.05) reduced numbers of soil bacteria, resulting in dose related shifts in the fungal:bacterial ratio, which increased during 14 d, as determined from analysis of ergosterol and E(C). It was concluded that pharmaceutical antibiotics can exert a temporary selective pressure on soil microorganisms even at environmentally relevant concentrations.  相似文献   

7.
The use of synthetic pesticides has become an indispensable tool in agriculture for the control of pests. Therefore, the search for remedies and techniques for decontamination and detoxification of a pesticide-contaminated environment has become an important part of the research. Currently, bioremediation seems to be one of the most environmentally safe and cost-effective methods. In nature, the existence of abundant material resources can be used to degrade the environmental pesticide pollutants. At present, a number of microorganisms, capable of degrading pesticides, have been isolated and characterised. For insects, insecticide resistance-associated esterases have been purified and characterised from several insect species, and a new family of cytochrome P450 apparently associated with insecticide resistance in the tobacco budworm, was discovered. Generally, two bioremediation approaches have been used one directly based on microorganisms, and the other involved in isolated enzymes. For the former, in addition to using natural microorganism strains, with genetic techniques certain desirable biodegradation pathways from different organisms are brought together in a single host. However, because of their own limits, especially problems associated with releasing genetically altered microorganisms into the environment, the strategy based on enzymes seems more feasible. In the long term, collaborations between microbiologists, biochemists, and engineers will become increasingly important to efficiently dispose of the pesticide pollutants.  相似文献   

8.
植物生态浮床的制备及其对富营养化水体的净化效果   总被引:2,自引:0,他引:2  
针对福州市闽侯县旗山大学城景观湖某富营养化实验水区,制备了一种应用型植物生态浮床。原位修复结果表明,负载美人蕉(Canna coccinea Mill)的生态浮床对浮床周围的小生境有一定的修复效果;对美人蕉根际圈的微生物数量跟踪显示,在60 d内,微生物数量增加了2个数量级,微生物种类无明显变化,但不同植株根际圈的微生物种类存在一定差异。  相似文献   

9.
A multi-compartment system was used to study the importance of microorganisms for Cd desorption from soil amended with sewage sludge and simultaneous resorption of the mobilized metal by soil constituents. Using this system made it possible to study the participation of microorganisms (Arthrobacter, Trichoderma), montmorillonite, humic acids, and iron oxides in resorption of the released Cd. A filter-sterilized water extract of root-free soil of pH 6.7 (RF) or RF supplemented with glucose (RFG) were used to mobilize Cd from soil at 14 degrees C in 48 h. Cadmium found in those extracts after 48-h incubation was recognized as bioavailable. Changes in pH values and enrichment of soil extracts with organic acids and siderophores resulted from microbial growth. RFG with lower pH and a higher content of ligands mobilized, on average, 40% of Cd introduced with sewage sludge amended soil, whereas RF mobilized only 20% of it. Sequential extractions of Cd at time 0 and Cd remaining in soil showed that RFG had mobilized Cd mostly from the fraction bound with Fe and Mn oxides. Microbial biomass accounted for only up to 3.4% (w/w) of the soil constituents used in the experiments but resorbed 25% of mobilized Cd. The chemical composition of mobilizing soil extracts and the solid-to-mobilizing-extracts volume ratio had a significant effect on the amount of bioavailable Cd. The results of the study suggest that microbial metabolites were involved in Cd mobilization, while the biomass of microorganisms was involved in Cd resorption as a biosorbent.  相似文献   

10.
Although the long incubation time of biochemical oxygen demand (BOD7) measurements has been addressed by the use of microbial biosensors, the resulting sensor-BOD values gained from the measurements with specific industrial wastewaters still underestimates the BOD value of such samples. This research aims to provide fast and more accurate BOD measurements in the dairy wastewater samples. Unlike municipal wastewater, wastewater from the dairy industry contains many substrates that are not easily accessible to a majority of microorganisms. Therefore, a bacterial culture, Microbacterium phyllosphaerae, isolated from dairy wastewater was used to construct a semi-specific microbial biosensor. A universal microbial biosensor based on Pseudomonas fluorescens, which has a wide substrate spectrum but is nonspecific to dairy wastewater, was used as a comparison. BOD biosensors were calibrated with OECD synthetic wastewater, and experiments with different synthetic and actual wastewater samples were carried out. Results show that the semi-specific M. phyllosphaerae-based microbial biosensor is more sensitive towards wastewaters that contain milk derivates and butter whey than the P. fluorescens-based biosensor. Although the M. phyllosphaerae biosensor underestimates the BOD7 value of actual dairy wastewaters by 25–32 %, this bacterial culture is more suitable for BOD monitoring in dairy wastewater than P. fluorescens, which underestimated the same samples by 46–61 %.  相似文献   

11.
The uptake of the triazine herbicides, atrazine and terbutryn, was determined for two freshwater photosynthetic microorganisms, the green microalga Chlorella vulgaris and the cyanobacterium Synechococcus elongatus. An extremely rapid uptake of both pesticides was recorded, although uptake rate was lower for the cyanobacterium, mainly for atrazine. Other parameters related to the herbicide bioconcentration capacity of these microorganisms were also studied. Growth rate, biomass, and cell viability in cultures containing herbicide were clearly affected by herbicide uptake. Herbicide toxicity and microalgae sensitivity were used to determine the effectiveness of the bioconcentration process and the stability of herbicide removal. C. vulgaris showed higher bioconcentration capability for these two triazine herbicides than S. elongatus, especially with regard to terbutryn. This study supports the usefulness of such microorganisms, as a bioremediation technique in freshwater systems polluted with triazine herbicides.  相似文献   

12.
Chung K  Lee I  Han JI 《Chemosphere》2012,86(4):415-419
As an effort to better utilize the microbial fuel cell (MFC) technology, we previously proposed an innovative MFC system named M2FC consisting of ferric-based MFC part and ferrous-based fuel cell (FC) part. In this reactor, ferric ion, the catholyte in the MFC part, was efficiently regenerated by the FC part with the generation of additional electricity. When both units were operated separately, the ferric-based MFC part produced approximately 1360 mW m−2 of power density with FeCl3 as catholyte and Fe-citrate as anolyte. The ferrous-based FC part with FeCl3 as catholyte and Fe-EDTA as anolyte displayed the highest power density (1500 mW m−2), while that with ferricyanide as catholyte and Fe-noligand as anolyte had the lowest power density (380 mW m−2). The types of catholytes and chelating complexes as anolyte were found to play important roles in the reduction of ferric ions and oxidation of ferrous ion. Linear sweep voltammetry results supported that the cathode electrolytes were electrically active and these agreed well with the M2FC reactor performance. These results clearly showed that ligands played critical role in the efficiency and rate for recycling iron ion and thus the M2FC performance.  相似文献   

13.
Doong RA  Chang SM 《Chemosphere》2000,40(12):1427-1433
An investigation involving the supplement of different concentrations of substrates and microorganisms was carried out under anaerobic condition to assess the feasibility of bioremediation of carbon tetrachloride (CCl4) with the amendment of low concentrations of auxiliary substrate and microorganisms. The concentrations of substrate and microorganisms ranged from 10 to 100 mg/l and from 3.7 × 104 to 3.7 × 106 cell/ml, respectively. The biotransformation rate of CCl4 increased progressively with the increase in the concentrations of the substrate and microorganisms. In the low biomass-amended system (3.7 × 104cells/ml), 28–71% and 57–96% of CCl4 removals were exhibited when 10–100 mg/l of acetate or glucose was supplemented, respectively, whereas nearly complete degradation of CCl4 was observed in the heavily inoculated systems (3.7 × 106 cells/ml). An addition of electron donor in the low microbial activity batches enhanced greater efficiency in dechlorination than in the high microbial activity batches. The second-order rate constants ranged from 0.0059 to 0.0092 l/mg/day in high biomass input system, while a two- to four-fold increase in rate constant was obtained in the low microbial activity system. This study indicates that biomass was the more important environmental parameter than substrate affecting the fate of CCl4. The addition of auxiliary substrates was effective only in low biomass-amended batches (0.56 mg-VSS/l) and diminished inversely with the increase of microbial concentration.  相似文献   

14.
Tetracyclines and sulfonamides used in human and animal medicine are released to terrestrial ecosystems from wastewater treatment plants or by direct manure application. The interactions between plants and these antibiotics are numerous and complex, including uptake and accumulation, phytometabolism, toxicity responses, and degradation in the rhizosphere. Uptake and accumulation of antibiotics have been studied in plants such as wheat, maize, potato, vegetables, and ornamentals. Once accumulated in plant tissue, organic contaminants can be metabolized through a sequential process of transformation, conjugation through glycosylation and glutathione pathways, and ultimately sequestration into plant tissue. While studies have yet to fully elucidate the phytometabolism of tetracyclines and sulfonamides, an in-depth review of plant and mammalian studies suggest multiple potential transformation and conjugation pathways for tetracyclines and sulfonamides. The presence of contaminants in the vicinity or within the plants can elicit stress responses and defense mechanisms that can help tolerate the negative effects of contaminants. Antibiotics can change microbial communities and enzyme activity in the rhizosphere, potentially inducing microbial antibiotic resistance. On the other hand, the interaction of microbes and root exudates on pharmaceuticals in the rhizosphere can result in degradation of the parent molecule to less toxic compounds. To fully characterize the environmental impacts of increased antibiotic use in human medicine and animal production, further research is essential to understand the effects of different antibiotics on plant physiology and productivity, uptake, translocation, and phytometabolism of antibiotics, and the role of antibiotics in the rhizosphere.  相似文献   

15.
Increased use of ethanol-blended gasoline (gasohol) and its potential release into the subsurface have spurred interest in studying the biodegradation of and interactions between ethanol and gasoline components such as benzene, toluene, ethylbenzene and xylene isomers (BTEX) in groundwater plumes. The preferred substrate status and the high biological oxygen demand (BOD) posed by ethanol and its biodegradation products suggests that anaerobic electron acceptors (EAs) will be required to support in situ bioremediation of BTEX. To develop a strategy for aromatic hydrocarbon bioremediation and to understand the impacts of ethanol on BTEX biodegradation under strictly anaerobic conditions, a microcosm experiment was conducted using pristine aquifer sand and groundwater obtained from Canadian Forces Base Borden, Canada. The initial electron accepter pool included nitrate, sulfate and/or ferric iron. The microcosms typically contained 400 g of sediment, 600 approximately 800 ml of groundwater, and with differing EAs added, and were run under anaerobic conditions. Ethanol was added to some at concentrations of 500 and 5000 mg/L. Trends for biodegradation of aromatic hydrocarbons for the Borden aquifer material were first developed in the absence of ethanol, The results showed that indigenous microorganisms could degrade all aromatic hydrocarbons (BTEX and trimethylbenzene isomers-TMB) under nitrate- and ferric iron-combined conditions, but not under sulfate-reducing conditions. Toluene, ethylbenzene and m/p-xylene were biodegraded under denitrifying conditions. However, the persistence of benzene indicated that enhancing denitrification alone was insufficient. Both benzene and o-xylene biodegraded significantly under iron-reducing conditions, but only after denitrification had removed other aromatics. For the trimethylbenzene isomers, 1,3,5-TMB biodegradation was found under denitrifying and then iron-reducing conditions. Biodegradation of 1,2,3-TMB or 1,2,4-TMB was slower under iron-reducing conditions. This study suggests that addition of excess ferric iron combined with limited nitrate has promise for in situ bioremediation of BTEX and TMB in the Borden aquifer and possibly for other sites contaminated by hydrocarbons. This study is the first to report 1,2,3-TMB biodegradation under strictly anaerobic condition. With the addition of 500 mg/L ethanol but without EA addition, ethanol and its main intermediate, acetate, were quickly biodegraded within 41 d with methane as a major product. Ethanol initially present at 5000 mg/L without EA addition declined slowly with the persistence of unidentified volatile fatty acids, likely propionate and butyrate, but less methane. In contrast, all ethanol disappeared with repeated additions of either nitrate or ferric iron, but acetate and unidentified intermediates persisted under iron-enhanced conditions. With the addition of 500 mg/L ethanol and nitrate, only minor toluene biodegradation was observed under denitrifying conditions and only after ethanol and acetate were utilized. The higher ethanol concentration (5000 mg/L) essentially shut down BTEX biodegradation likely due to high EA demand provided by ethanol and its intermediates. The negative findings for anaerobic BTEX biodegradation in the presence of ethanol and/or its biodegradation products are in contrast to recent research reported by Da Silva et al. [Da Silva, M.L.B., Ruiz-Aguilar, G.M.L., Alvarez, P.J.J., 2005. Enhanced anaerobic biodegradation of BTEX-ethanol mixtures in aquifer columns amended with sulfate, chelated ferric iron or nitrate. Biodegradation. 16, 105-114]. Our results suggest that the apparent conservation of high residual labile carbon as biodegradation products such as acetate makes natural attenuation of aromatics less effective, and makes subsequent addition of EAs to promote in situ BTEX biodegradation problematic.  相似文献   

16.
Bacteria and fungi in pristine and oily desert soil samples were counted on inorganic medium aliquots containing 0.5% hexadecane, hexadecanol, hexadecanal or hexadecanoic acid, as sole sources of carbon and energy. It was found that the carbon and energy source most commonly utilized by soil bacteria was the alkane n-hexadecane, and by soil fungi hexadecanoic acid. Representative microorganisms were isolated and identified. The most predominant bacteria in all soil samples belonged to the genera Micrococcus and Pseudomonas; less dominant bacteria belonged to the group of nocardioforms. The most frequent fungal genera were Aspergillus and Penicillium, while Microsporium and Ulocladium were minor fungi. Irrespective of the substrate on which the microbial strains had initially been isolated, the majority of the isolated microorganisms could grow, albeit to a varying degree, on an inorganic medium containing any of the remaining three substrates as sole carbon and energy sources. Bacterial strains preferred the alkane as a carbon and energy source over any of its oxidation products, while fungal strains preferred to grow mainly on the fatty acids. Quantitative analysis by gas liquid chromatography revealed that the predominant bacterial and fungal isolates had a potential for the attenuation of the alkane and its immediate oxidation products in the medium. In view of the continuous release of hydrocarbon oxidation products by oil-utilizing microorganisms in oily environments, it is interesting that the indigenous microflora contribute to the uptake and utilization of all such intermediate compounds, thus, having a potential for efficient self-cleaning and bioremediation of oily soils.  相似文献   

17.
随着拟除虫菊酯类农药使用量不断增加,产生的农药残留问题对生态环境和人类健康造成了危害.对降解拟除虫菊酯类农药的微生物种类、降解酶和降解机制及降解酶基因克隆和构建工程菌等方面进行综述,旨在为研究和开发微生物降解拟除虫菊酯类农药残留提供参考.  相似文献   

18.
Research on the influence of biosurfactants on the efficiency of in situ bioremediation of contaminated soil is continuously growing. Despite the constant progress in understanding the mechanisms involved in the effects of biosurfactants, there are still many factors that are not sufficiently elucidated. There is a lack of research on autochthonous or exogenous microbial metabolism when biostimulation or bioaugmentation is carried out to produce biosurfactants at contaminated sites. In addition, studies on the application of techniques that measure the biosurfactants produced in situ are needed. This is important because, although the positive influence of biosurfactants is often reported, there are also studies where no effect or negative effects have been observed. This review aimed to examine some studies on factors that can improve the production of biosurfactants in soils during in situ bioremediation. Moreover, this work reviews the methodologies that can be used for measuring the production of these biocomposts. We reviewed studies on the potential of biosurfactants to improve the bioremediation of hydrocarbons, as well as the limitations of methods for the production of these biomolecules by microorganisms in soil.  相似文献   

19.
There are strong drivers to increasingly adopt bioremediation as an effective technique for risk reduction of hydrocarbon impacted soils. Researchers often rely solely on chemical data to assess bioremediation efficiently, without making use of the numerous biological techniques for assessing microbial performance. Where used, laboratory experiments must be effectively extrapolated to the field scale. The aim of this research was to test laboratory derived data and move to the field scale. In this research, the remediation of over thirty hydrocarbon sites was studied in the laboratory using a range of analytical techniques. At elevated concentrations, the rate of degradation was best described by respiration and the total hydrocarbon concentration in soil. The number of bacterial degraders and heterotrophs as well as quantification of the bioavailable fraction allowed an estimation of how bioremediation would progress. The response of microbial biosensors proved a useful predictor of bioremediation in the absence of other microbial data. Field-scale trials on average took three times as long to reach the same endpoint as the laboratory trial. It is essential that practitioners justify the nature and frequency of sampling when managing remediation projects and estimations can be made using laboratory derived data. The value of bioremediation will be realised when those that practice the technology can offer transparent lines of evidence to explain their decisions.  相似文献   

20.
Olaniran AO  Igbinosa EO 《Chemosphere》2011,83(10):1297-1306
Chlorophenols are chlorinated aromatic compound structures and are commonly found in pesticide preparations as well as industrial wastes. They are recalcitrant to biodegradation and consequently persistent in the environment. A variety of chlorophenols derivatives compounds are highly toxic, mutagenic and carcinogenic for living organisms. Biological transformation by microorganisms is one of the key remediation options that can be exploited to solve environmental pollution problems caused by these notorious compounds. The key enzymes in the microbial degradation of chlorophenols are the oxygenases and dioxygenases. These enzymes can be engineered for enhanced degradation of highly chlorinated aromatic compounds through directed evolution methods. This review underscores the mechanisms of chlorophenols biodegradation with the view to understanding how bioremediation processes can be optimized for cleaning up chloroaromatic contaminated environments.  相似文献   

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