Biodegradation of phenanthrene using adapted microbial consortium isolated from petrochemical contaminated environment

In developing countries like India, there are many industrial areas discharging effluent containing large amount of polyaromatic hydrocarbon (PAH) which causes hazardous effect on the soil-water environment. The objective of this study was to isolate and characterize high-efficiency PAH-degrading microbial consortium from 3 decade old petrochemical refinery field located in Nagpur, Maharashtra with history of PAH disposal. Based on biochemical tests and 16S rDNA gene sequence analysis the consortium was identified as Sphingobacterium sp., Bacillus cereus and a novel bacterium Achromobacter insolitus MHF ENV IV with effective phenanthrene-degrading ability. The biodegradation data of phenanthrene indicates about 100%, 56.9% and 25.8% degradation at the concentration of 100mg/l, 250 mg/l and 500 mg/l respectively within 14 days. The consortium and its monoculture isolates also utilized variety of other hydrocarbons for growth. To best of our knowledge this is the first time that Achromobacter insolitus has been reported to mineralize phenanthrene effectively. GC-MS analysis of phenanthrene degradation confirmed biodegradation by detection of intermediates like salicylaldehyde, salicylic acid and catechol. All the results indicated that the microbial consortium have a promising application in bioremediation of petrochemical contaminated environments and could be potentially useful for the study of PAH degradation and for bioremediation purposes.     

Biodegradation of chlorpyrifos and its hydrolysis product 3,5,6-trichloro-2-pyridinol by Bacillus pumilus strain C2A1

A bacterial strain C2A1 isolated from soil was found highly effective in degrading chlorpyrifos and its first hydrolysis metabolite 3,5,6-trichloro-2-pyridinol (TCP). On the basis of morphology, physiological characteristics, biochemical tests and 16S rRNA sequence analysis, strain C2A1 was identified as Bacillus pumilus. Role of strain C2A1 in the degradation of chlorpyrifos was examined under different culture conditions like pH, inoculum density, presence of added carbon/nutrient sources and pesticide concentration. Chlorpyrifos was utilized by strain C2A1 as the sole source of carbon and energy as well as it was co-metabolized in the presence of glucose, yeast extract and nutrient broth. Maximum pesticide degradation was observed at high pH (8.5) and high inoculum density when chlorpyrifos was used as the sole source and energy. In the presence of other nutrients, chlorpyrifos degradation was enhanced probably due to high growth on easily metabolizable compounds which in turn increased degradation. The strain C2A1 showed 90% degradation of TCP (300 mg L⁻¹) within 8 days of incubation.     

Perspectives of low cost arsenic remediation of drinking water in Pakistan and other countries

Arsenic concentrations above acceptable standards for drinking water have been detected in many countries and this should therefore is a global issue. The presence of arsenic in subsurface aquifers and drinking water systems is a potentially serious human health hazard. The current population growth in Pakistan and other developing countries will have direct bearing on the water sector for meeting the domestic, industrial and agricultural needs. Pakistan is about to exhaust its available water resources and is on the verge of becoming a water deficit country. Water pollution is a serious menace in Pakistan, as almost 70% of its surface waters as well as its groundwater reserves have contaminated by biological, organic and inorganic pollutants. In some areas of Pakistan, a number of shallow aquifers and tube wells are contaminated with arsenic at levels which are above the recommended USEPA arsenic level of 10 ppb (10 μg L⁻¹). Adverse health effects including human mortality from drinking water are well documented and can be attributed to arsenic contamination. The present paper reviews appropriate and low cost methods for the elimination of arsenic from drinking waters. It is recommended that a combination of low cost chemical treatment like ion exchange, filtration and adsorption along with bioremediation may be useful option for arsenic removal from drinking water.     

Bioremediation of endosulfan contaminated soil and water -- optimization of operating conditions in laboratory scale reactors

A mixed bacterial culture consisted of Staphylococcus sp., Bacillus circulans-I and -II has been enriched from contaminated soil collected from the vicinity of an endosulfan processing industry. The degradation of endosulfan by mixed bacterial culture was studied in aerobic and facultative anaerobic conditions via batch experiments with an initial endosulfan concentration of 50mg/L. After 3 weeks of incubation, mixed bacterial culture was able to degrade 71.58+/-0.2% and 75.88+/-0.2% of endosulfan in aerobic and facultative anaerobic conditions, respectively. The addition of external carbon (dextrose) increased the endosulfan degradation in both the conditions. The optimal dextrose concentration and inoculum size was estimated as 1g/L and 75mg/L, respectively. The pH of the system has significant effect on endosulfan degradation. The degradation of alpha endosulfan was more compared to beta endosulfan in all the experiments. Endosulfan biodegradation in soil was evaluated by miniature and bench scale soil reactors. The soils used for the biodegradation experiments were identified as clayey soil (CL, lean clay with sand), red soil (GM, silty gravel with sand), sandy soil (SM, silty sand with gravel) and composted soil (PT, peat) as per ASTM (American society for testing and materials) standards. Endosulfan degradation efficiency in miniature soil reactors were in the order of sandy soil followed by red soil, composted soil and clayey soil in both aerobic and anaerobic conditions. In bench scale soil reactors, endosulfan degradation was observed more in the bottom layers. After 4 weeks, maximum endosulfan degradation efficiency of 95.48+/-0.17% was observed in red soil reactor where as in composted soil-I (moisture 38+/-1%) and composted soil-II (moisture 45+/-1%) it was 96.03+/-0.23% and 94.84+/-0.19%, respectively. The high moisture content in compost soil reactor-II increased the endosulfan concentration in the leachate. Known intermediate metabolites of endosulfan were absent in all the above degradation studies.     

Hazardous concentrations of selenium in soil and groundwater in North-West India

Soil and groundwater samples were collected for bulk elemental analyses in particular for selenium (Se) concentrations from six agricultural sites located in states of Punjab and Haryana in North-West India. Toxic concentrations of Se (45-341 μg L(-1)) were present in groundwater (76 m deep) of Jainpur and Barwa villages in Punjab. Selenium enrichments were also found in top soil layers (0-15 cm) of Jainpur (2.3-11.6 mg kg(-1)) and Barwa (3.1 mg kg(-1)). Mineralogical analyses confirmed silicates and phyllosilicates as main components of these soils, also reflected by the high content of SiO(2) (40-62 wt.%), Al(2)O(3) (9-21 wt.%) and K(2)O (2.2-3.2 wt.%). Prevailing intensive irrigation practices in Punjab with Se enriched groundwater may be the cause of Se accumulation in soils. Sequential extraction revealed >50% Se bioavailability in Jainpur soils. Appearance of selenite was observed in some of the batch assays with soil slurries under reducing conditions. Although safe Se concentrations were found in Hisar, Haryana, yet high levels of As, Mo and U present in groundwater indicated its unsuitability for drinking purposes. Detailed biogeochemical studies of Se in sediments or groundwater of Punjab are not available so far; intensive investigations should be started for better understanding of the problem of Se toxicity.     

Biodegradation of 2,4,6-trinitrophenol by Rhodococcus sp. isolated from a picric acid-contaminated soil

A picric acid-degrading bacterium, strain NJUST16, was isolated from a soil contaminated by picric acid and identified as a member of Rhodococcus sp. based on 16S rRNA sequence. The degradation assays suggested that the strain NJUST16 could utilize picric acid as the sole source of carbon, nitrogen and energy. The isolate grew optimally at 30 degrees C and initial pH 7.0-7.5 in the mineral salts medium supplemented with picric acid. It was basically consistent with degradation of picric acid by the isolate. Addition of nitrogen sources such as yeast extract and peptone accelerated the degradation of picric acid. However, the stimulation was concentration dependent. The degradation was accompanied by release of stoichiometric amount of nitrite and acidification. The degradation of picric acid at relatively high concentrations (>3.93 mM) demonstrated that the degradation was both pH and nitrite dependent. Neutral and slightly basic pH was crucial to achieve high concentrations of picric acid degradation by the NJUST16 strain.     

Decolorization of sulfonated azo dye Metanil Yellow by newly isolated bacterial strains: Bacillus sp. strain AK1 and Lysinibacillus sp. strain AK2

Two different bacterial strains capable of decolorizing a highly water soluble azo dye Metanil Yellow were isolated from dye contaminated soil sample collected from Atul Dyeing Industry, Bellary, India. The individual bacterial strains Bacillus sp. AK1 and Lysinibacillus sp. AK2 decolorized Metanil Yellow (200 mg L(-1)) completely within 27 and 12h respectively. Various parameters like pH, temperature, NaCl and initial dye concentrations were optimized to develop an economically feasible decolorization process. The maximum concentration of Metanil Yellow (1000 mg L(-1)) was decolorized by strains AK2 and AK1 within 78 and 84 h respectively. These strains could decolorize Metanil Yellow over a broad pH range 5.5-9.0; the optimum pH was 7.2. The decolorization of Metanil Yellow was most efficient at 40°C and confirmed by UV-visible spectroscopy, TLC, HPLC and GC/MS analysis. Further, both the strains showed the involvement of azoreductase in the decolorization process. Phytotoxicity studies of catabolic products of Metanil Yellow on the seeds of chick pea and pigeon pea revealed much reduction in the toxicity of metabolites as compared to the parent dye. These results indicating the effectiveness of strains AK1 and AK2 for the treatment of textile effluents containing azo dyes.     

Assessment of bioremediation possibilities of technical grade hexachlorocyclohexane (tech-HCH) contaminated soils

Hexachlorocyclohexane (HCH) is a broad spectrum insecticide still used in some of the developing countries, though developed countries have banned or curtailed its use. Even in those countries where the use of t-HCH has been discontinued for a number of years, the problem of residues of all isomers of t-HCH remains because of its high persistence. These insecticides in the soil disturb the delicate equilibrium between microorganisms and their environment. Few reports on the degradation of t-HCH isomers in soil are present in literature, and very little information is available on the effect of these t-HCH isomers on soil microflora. In the present study, an attempt has been made to see the microbial diversity in the uncontaminated soils and the effect of application of t-HCH on the soil microflora. The soil was spiked with t-HCH and incubated, at regular time intervals the soil samples were analyzed for microbial diversity as well as t-HCH isomers residues. The results show that at higher concentrations of t-HCH, microbial populations were inhibited and the inhibited populations did not reappear even after prolonged incubation. Potential t-HCH degrading cultures were isolated and subjected to further acclimation in order to enhance their degradation capacity. The results are presented and discussed in this paper.     

The influence of different soil constituents on the reaction kinetics of wet oxidation of the creosote compound quinoline

Creosote contaminated sites have become a widespread problem in industrialized countries. Recently, wet oxidation using high temperature, pressure, water and oxygen followed by activated sludge treatment proved to be an efficient method for removing a wide selection of creosote compounds in contaminated soils. Wet oxidation of the creosote compound quinoline was carried out in the presence of montmorillionite, quartz and humic acid. The products derived from wet oxidation were identified and treated biologically by activated sludge testing their biodegradability. The influence on the oxidation kinetics of quinoline during wet oxidation was pH dependent. Humic acid supported the oxidation of quinoline, whereas the addition of montmorillionite and quartz had either an inhibiting effect or led only to a slight increase in oxidation. In mixtures of soil constituents, especially at low contents of humic acid, the adsorption of quinoline on montmorillionite prevented oxidation at neutral pH. Thus, alkaline extraction of both quinoline and humic acid was needed for an efficient oxidation. A proposed reaction mechanism suggests that quinoline was oxidized by hydroxyl radicals formed during the oxidation of the humic acid. A wide selection of reaction products (mainly carboxylic acids, benzene and pyridine derivatives) derived from the wet oxidation of humic acid and quinoline. The reaction products from humic acid degradation had a rate limiting effect on the wet oxidation of quinoline leaving small residues of quinoline after the treatment. On the contrary, these reaction products also improved the biodegradation of products from the quinoline oxidation due to co-digestion of carboxylic acids. Therefore, the presence of soil components (mainly humic acid) improved the combined wet oxidation and biological activated sludge treatment of quinoline.     

A procedure for production of adapted bacteria to degrade chlorinated aromatics

Production of biomass adapted to the degradation of a mixture of chlorobenzene (CB) and 1,2-dichlorobenzene (DCB) was investigated in a batch culture with substrates supplied by pulses. CB and o-DCB concentrations which gave the best adapted biomass productivity were determined and found to be 150 and 30 microl l(-1), respectively. The biomass productivity was 51 mg l(-1) h(-1). The biomass yield was 0.38 g of biomass dry weight per gram of substrate. The pulses of 200 microl CB and 40 microl o-DCB, were inhibitory to the bacterial culture. Among the metabolites, muconic acid was found in large quantities in the medium and in the cells. At a time between two pulses of 60 min, adding 150 microl CB and 30 microl o-DCB per each pulse, 7.6g l(-1) of biomass was obtained.The produced biomass served as an inoculum for the biotrickling filter which treated industrial waste gases contaminated by CBs.The method of adapted biomass production was described using CBs, but the degradation of any other toxic volatile pollutant can be improved using this technique.     

Degradation of pentachlorophenol in contaminated soil suspensions by potassium monopersulfate catalyzed oxidation by a supramolecular complex between tetra(p-sulfophenyl)porphineiron(III) and hydroxypropyl-beta-cyclodextrin

To enhance the catalytic oxidation of pentachlorophenol (PCP) in contaminated soil suspensions using tetra(p-sulfophenyl)porphineiron(III) (Fe(III)-TPPS) as a catalyst and potassium monopersulfate (KHSO(5)) as the single-oxygen donor, the effect of added hydroxypropyl-beta-cyclodextrin (HP-beta-CD) was examined. At pH 4 and 6, the percentage of PCP disappearance increased substantially in the presence of HP-beta-CD. In addition, the self-degradation of Fe(III)-TPPS was significantly retarded in the presence of HP-beta-CD. This retarded self-degradation can be attributed to the stabilization of Fe(III)-TPPS via the formation of a supramolecular complex with HP-beta-CD. The kinetic constant for the self-degradation of Fe(III)-TPPS in the presence of HP-beta-CD at pH 6 was much smaller than that at pH 4, indicating that Fe(III)-TPPS is more stable at pH 6. Thus, the amount of Fe(III)-TPPS, KHSO(5) and HP-beta-CD required to degrade PCP in contaminated soil suspensions was optimal at pH 6. When PCP-contaminated soil suspensions were treated under the optimized conditions, 12-18% and 24-28% of the PCP was mineralized to CO(2) in the absence and presence of HP-beta-CD, respectively. These results show that the presence of HP-beta-CD in the Fe(III)-TPPS/KHSO(5) catalytic system is effective in enhancing the degradation of PCP in contaminated soil suspensions.     

Effect of humic substances on Cu(II) solubility in kaolin-sand soil

The type and amount of organic matter present in industrially contaminated soils will influence the risk they pose. Previous studies have shown the importance of humic and fulvic acids (FAs) (important components of soil organic matter) in increasing the solubility of toxic metals but were not carried out using toxic metal levels and the pH range typical of industrially contaminated soils. This study investigated the influence of three humic substances (HSs: humates, fulvates and humins) on the solubility of copper(II) ions in kaolinitic soil spiked with Cu at levels representative of industrially contaminated soil. Humates, fulvates and humin were extracted from Irish moss peat, and controlled pH batch leaching tests were conducted on an artificial kaolin-sand soil that was spiked with each. Further leaching tests were conducted on soil spiked with each HS and copper nitrate. Dissolved organic contents were determined by titration and total and free aqueous copper concentrations in the leachate were measured using AAS and ion selective electrode (ISE) potentiometry respectively (dissolved complexed copper levels were determined by difference). It was found that humates and fulvates are partially sorbed by the soil, probably by chemisorption on positively charged gibbsite (Al-hydroxide) sites in the kaolinite. The addition of 340 mg/kg Cu(II) ions did not significantly affect the amount of humate or fulvate sorbed. Dissolved humates and fulvates form soluble complexes with copper over the pH range 3-11. However, in the presence of kaolinite, soluble copper humates and fulvates are unable to compete with the kaolinite for Cu ions at pH 6-7. Above pH 8, humate and fulvate complexes are the only forms of dissolved Cu. Humin is largely insoluble and has little effect on Cu mobility between pH 2 and 12. The implication of this study is that measurement of total soil organic content and water leaching tests should be a standard part of contaminated site investigation.     

Kinetics of transformation of 1,1,1-trichloroethane by Fe(II) in cement slurries

This study examines the applicability of the iron-based degradative solidification/stabilization (DS/S-Fe(II)) process to 1,1,1-trichloroethane (1,1,1-TCA), which is one of common chlorinated aliphatic hydrocarbons (CAHs) of concern at contaminated sites. DS/S-Fe(II) combines contaminant degradation by Fe(II) and immobilization by the hydration reactions of Portland cement. The transformation of 1,1,1-TCA by Fe(II) in 10% Portland cement slurries was studied using a batch slurry reactor system. The effects of Fe(II) dose, pH, and initial concentration of 1,1,1-TCA on the kinetics of 1,1,1-TCA degradation were evaluated. Degradation of 1,1,1-TCA in cement slurries including Fe(II) was very rapid and could be described by a pseudo-first-order rate law. The half-lives for 1,1,1-TCA were measured between 0.4 and 5h when Fe(II) dose ranged from 4.9 to 39.2mM. The pseudo-first-order rate constant increased with pH to a maximum near pH 12.5. A saturation rate equation was able to predict degradation kinetics over a wide range of target organic concentrations and at higher Fe(II) doses. The major transformation product of 1,1,1-TCA in mixtures of Fe(II) and cement was 1,1-dichloroethane (1,1-DCA), which indicates that degradation occurred by a hydrogenolysis pathway. A small amount of ethane was observed. The conversion of 1,1,1-TCA to ethane was better described by a parallel reaction model than by a consecutive reaction model.     

Bioremediation of anthracene contaminated soil in bio-slurry phase reactor operated in periodic discontinuous batch mode

Bioremediation of soil-bound anthracene was studied in a series of bio-slurry phase reactors operated in periodic discontinuous/sequencing batch mode under anoxic-aerobic-anoxic microenvironment using native soil microflora. Five reactors were operated for a total cycle period of 144 h (6 days) at soil loading rate of 16.66 kg soil/m(3)/day at 30 +/- 2 degrees C temperature. The performance of the bioreactors was studied at various substrate loading rates (volumetric substrate loading rate (SLR), 0.1, 0.2 and 0.3g anthracene/kg soil/day) with and without bioaugmentation (domestic sewage inoculum; 2 x 10(6) CFU/g of soil). Control reactor (without microflora) showed negligible degradation of anthracene due to the absence of biological activity. The performance of the bio-slurry system with respect to anthracene degradation was found to depend on both substrate loading rate and bioaugmentation. Application of bioaugmentation showed positive influence on the rate of degradation of anthracene. Anthracene degradation data was analysed using different kinetic models to understand the mechanism of bioremediation process in the bio-slurry phase system. Variation in pH/oxidation-reduction potential (ORP), soil microflora and oxygen consumption rate correlated well with the substrate degradation pattern observed during soil slurry phase anthracene degradation.     

Identification of metabolites during biodegradation of pendimethalin in bioslurry reactor

Bioslurry phase reactor was used for the degradation of pendimethalin, a pre-emergence herbicide in the contaminated soil under aerobic environment. More than 91% degradation of pendimethalin was observed for 5 days of reactor operation augmented with sewage from effluent treatment plant (ETP). The performance of the reactor was monitored regularly by measuring pH and colony forming units (CFU). The metabolites of pendimethalin formed during degradation were identified using various analytical techniques, viz., thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and liquid chromatography-mass spectroscopy (LC-MS/MS). Four metabolites were formed and identified as N-(1-ethylpropyl)-3,4-dicarboxy 2,6-dinitrobenzenamine-N-oxide, N-(1-ethylpropyl)-3,4-dimethoxy-2,6-dinitrobenzenamine and benezimadazole-7-carboxyaldehyde. The reactions involved were monohydrolysis of 2-methyl groups followed by dihydrolysis. Further oxidation of amine groups and hydroxylation of propyl groups produced the above said metabolites. Degradation pathway of pendimethalin has been proposed in the bioslurry phase reactor.     

Combined effects of bacterial-feeding nematodes and prometryne on the soil microbial activity

Microcosm experiments were carried out to study the effects of bacterial-feeding nematodes and indigenous microbes and their interactions on the degradation of prometryne and soil microbial activity in contaminated soil. The results showed that soil indigenous microbes could degrade prometryne up to 59.6-67.9%; bacterial-feeding nematodes accelerated the degradation of prometryne in contaminated soil, and prometryne degradation was raised by 8.36-10.69%. Soil microbial biomass C (C_mic), basal soil respiration (BSR), and respiratory quotient (qCO₂) increased in the beginning of the experiment and decreased in the later stage of the experiment. Nematodes grew and reproduced quite fast, and did increase the growth of soil microbes and enhance soil microbial activity in prometryne contaminated soil during the incubation period.     

Lentinus (Panus) tigrinus augmentation of a historically contaminated soil: matrix decontamination and structure and function of the resident bacterial community

The ability of Lentinus tigrinus to grow and to degrade persistent aromatic hydrocarbons in aged contaminated soil was assessed in this study. L. tigrinus extensively colonized the soil; its degradation activity after 60 d incubation at 28°C, however, was mostly limited to dichloroaniline isomers, polychlorinated benzenes and diphenyl ether while the fungus was unable to deplete 9,10-anthracenedione and 7-H-benz[DE]anthracene-7-one which were the major soil contaminants. Although clean-up levels were limited, both density of cultivable heterotrophic bacteria and richness of the resident bacterial community in L. tigrinus microcosms (LtM) increased over time to a significantly larger extent than the respective amended incubation controls (1.9×10(9) CFU g(-1) vs. 1.0×10(9) CFU g(-1) and 37 vs. 16, respectively). Naphthalene- and catechol 2,3-dioxygenase gene copy numbers, however, decreased over time at a higher rate in LtM than in incubation controls likely due to a higher stimulation on heterotrophs than xenobiotics-degrading community members.     

Stratagems employed for 2,4-dichlorophenoxyacetic acid removal from polluted water sources

2,4-Dichlorophenoxyacetic acid is a selective herbicide used all over the world for both agricultural and horticultural activities for controlling broadleaf weeds. The herbicide is known for exhibiting strong toxicity to humans and animals, and also known for its water contamination potential at varying levels depending on its field application rates. The main route of 2,4-D exposure to humans is through agricultural runoff owing to its excess application. Although several efforts had been taken for its removal from contaminated water bodies via the development of efficient technology, its water contamination continues to be more problematic. In this context, the present study was aimed to arouse the scientific community in developing a more efficient method for complete (100%) removal of 2,4-D from the contaminated sites. This is a qualitative review, which summarizes the various methods used thus far for removing 2,4-D from contaminated aqueous systems and their varying levels of success.     

Underground Nuclear Explosion Sites in Orenburg Oblast: Current Radiation Situation

The sites of underground nuclear explosions conducted in the Orenburg oblast (Magistral' and Sapfir objects) comprise local areas contaminated predominantly with ¹³⁷Cs, with contaminants spreading outside the fenced area. The radioactive contamination levels in soils, water, and locally produced agricultural foodstuffs in the nearest localities do not exceed the characteristic background level and the maximal permissible concentrations. The regional soils exhibit a high retention power with respect to the main contaminating radionuclide, ¹³⁷Cs, whose minor proportion occurs, however, in a mobile form.     

The operation characteristics and electrochemical reactions of a specific circulation-enhanced electrokinetics

For electrokinetics remediation, the acid produced at the anode due to the water electrolysis will cause the soil acidification and destroy the soil constituents. Especially, the contaminated soils in Taiwan are usually agricultural lands; it is necessary to improve the performance of EK system to maintain the soils nature after remediation. In this study, a circulation-enhanced EK system (CEEK) was designed to neutralize the pH of the working solution and soils. Experiments were conducted by the control of different electrolyte species (sodium and potassium salts) and concentrations (10(-3) to 5x10(-2)M), respectively. Experimental results show the operational characteristics include: the CEEK system can effectively stabilize the pH of processing solution at neutral range and the current can be maintained at stable status with carbonate salts; the pH buffering range of working solution in the CEEK system depends on the electrolyte species and their concentration significantly; the water content remains roughly as their original nature in the CEEK system. For consideration of electrochemical reactions, the water electrolysis is the predominating electrochemical reaction in the CEEK system, which not only influences the pH but also the conductivity of the working solution. In the application of practical engineering, there exist linear relationships between the pH, conductivity, current and the electrolyte concentration, respectively, which can serve as a means to assist engineers to select operational parameters of CEEK.