Microbiological methods for the cleanup of soil and ground water contaminated with halogenated organic compounds

There is growing interest in the enhancement of microbial degradative activities as a means of bringing about the in situ cleanup of contaminated soils and ground water. The halogenated organic compounds are likely to be prime targets for such biotechnological processes because of their widespread utilisation and the biodegradability of many of the most commonly used compounds. The aim of this review is to consider the potential for microbiological cleanup of haloorganic-contaminated sites. The technologies available involve the provision of suitable environmental conditions to facilitate maximum biodegradation rates either in the subsurface or in on-site bioreactors. Methodologies include the supply of inorganic nutrients, the supply of oxygen gas, the addition of degradative microbial inocula and the introduction of co-metabolic substrates. The potential efficiencies and limitations of the methods are critically discussed from a microbiological viewpoint with respect to substrate degradability and population responses to supplementation.     

Copper complexation by natural organic matter in contaminated and uncontaminated ground water

Abstract

Ground-water samples were collected from an uncontaminated and a contaminated site. Copper complexation was characterized by ion- selective electrode (ISE), fluorescence quenching (FQ), and cathodic stripping voltammetric (CSV) titrations. All of the samples were titrated at their natural pH values and some of the samples were also titrated at other pH values. For a total Cu concentration of 10^?6 M, the free Cu²⁺ concentrations in the samples from the uncontaminated site were all less than 10^?7 M, while free Cu²⁺ in the samples from the contaminated site were all less than 10^?8 M. For a particular sample and total Cu concentration, the free Cu²⁺ concentration decreased as the pH increased. Relative to ISE, FQ underestimated and CSV overestimated the degree of Cu²⁺ binding. The Cu²⁺ -complexing properties of the ground waters are similar to many published results for the same pH and for ligand concentrations normalized to T.O.C. Chemical equilibrium computations indicate that organic complexes would dominate Cu speciation in the uncontaminated ground waters for 10^?7 to 10^?5 M total Cu. In the contaminated ground waters, sulfide complexes would be the predominant Cu species for total Cu less than the total S(?11) concentration. Organic complexes would dominate Cu speciation for total Cu greater than total S(?11).     

Microbiological characterization of a fuel-oil contaminated site including numerical identification of heterotrophic water and soil bacteria

Seven soil samples and seven groundwater samples from a site contaminated with fuel-oil were investigated using several chemical and microbiological techniques. In soil samples, 500 to 7,500 mg/kg of total hydrocarbons were found. These samples contained no n-alkanes but iso- and branched chain alkanes. No polychlorinated biphenyls could be detected. Microbiological investigations included estimations of total cell counts, viable cell counts on different media, and numbers of methylotrophic, denitrifying, sulphate reducing, anaerobic (with the exception of methanogenic organisms), and hydrocarbon degrading bacteria. Viable and hydrocarbon degrading bacteria were found in all samples. A total of 1,366 pure cultures was characterized morphologically and physiologically and identified by numerical identification using a data base of more than 4,000 reference strains. Groundwater samples were dominated by gram-negative bacteria of the generaPseudomonas, Comamonas, Alcaligenes, andAcinetobacter, which were also found in soil samples. In addition, more grampositive bacteria belonging to the generaArthrobacter, Nocardia, andBacillus could be isolated from soil samples.     

Method for spiking soil samples with organic compounds

We examined the harmful side effects on indigenous soil microorganisms of two organic solvents, acetone and dichloromethane, that are normally used for spiking of soil with polycyclic aromatic hydrocarbons for experimental purposes. The solvents were applied in two contamination protocols to either the whole soil sample or 25% of the soil volume, which was subsequently mixed with 75% untreated soil. For dichloromethane, we included a third protocol, which involved application to 80% of the soil volume with or without phenanthrene and introduction of Pseudomonas fluorescens VKI171 SJ132 genetically tagged with luxAB::Tn5. For both solvents, application to the whole sample resulted in severe side effects on both indigenous protozoa and bacteria. Application of dichloromethane to the whole soil volume immediately reduced the number of protozoa to below the detection limit. In one of the soils, the protozoan population was able to recover to the initial level within 2 weeks, in terms of numbers of protozoa; protozoan diversity, however, remained low. In soil spiked with dichloromethane with or without phenanthrene, the introduced P. fluorescens VKI171 SJ132 was able to grow to a density 1,000-fold higher than in control soil, probably due mainly to release of predation from indigenous protozoa. In order to minimize solvent effects on indigenous soil microorganisms when spiking native soil samples with compounds having a low water solubility, we propose a common protocol in which the contaminant dissolved in acetone is added to 25% of the soil sample, followed by evaporation of the solvent and mixing with the remaining 75% of the soil sample.     

Bioremediation of a polyaromatic hydrocarbon contaminated soil by native soil microbiota and bioaugmentation with isolated microbial consortia

Biodegradation of a mixture of PAHs was assessed in forest soil microcosms performed either without or with bioaugmentation using individual fungi and bacterial and a fungal consortia. Respiratory activity, metabolic intermediates and extent of PAH degradation were determined. In all microcosms the low molecular weight PAH’s naphthalene, phenanthrene and anthracene, showed a rapid initial rate of removal. However, bioaugmentation did not significantly affect the biodegradation efficiency for these compounds. Significantly slower degradation rates were demonstrated for the high molecular weight PAH’s pyrene, benz[a]anthracene and benz[a]pyrene. Bioaugmentation did not improve the rate or extent of PAH degradation, except in the case of Aspergillus sp. Respiratory activity was determined by CO₂ evolution and correlated roughly with the rate and timing of PAH removal. This indicated that the PAHs were being used as an energy source. The native microbiota responded rapidly to the addition of the PAHs and demonstrated the ability to degrade all of the PAHs added to the soil, indicating their ability to remediate PAH-contaminated soils.     

石油烃和酚类物质在土中的生物降解与土壤酶活性

本文通过模拟实验,研究了不同条件下石油烃和酚类物质在土中的降解进程及其与土壤酶活性的关系,并在此基础上,对所述污染物的土地处理提出了若干建议。     

Biochemical features of the degradation of pollutants by Rhodococcus as a basis for contaminated wastewater and soil cleanup

Rhodococcus bacteria are considered to be promising degraders of persistent pollutants and are the basis of biological preparations for contaminated wastewater and soil cleanup. Biotechnological application of this group of bacteria is based on the peculiaraties of their metabolism. This review briefly discusses the following main points: I. Growth of Rhodococcus on various aromatic substrates, II. Chloro/methylcatechol transformation pathways, 3-Chlorocatechol branch of the modified ortho-pathway, 4-Chlorocatechol branch of the modified ortho-pathway, Modified 3-chlorocatechol branch in Rhodococcus opacus 1CP, Meta-cleavage ofchlorocatechols, Modified pathway for methylcatechol degradation, III. Approaches to the enhancement of degradation activity, IV. Rhodococcus-based biopreparations, IV. Prospects.     

Reaction of the soil microflora after contamination with chlorinated aromatic compounds and HCH

Abstract The effect of the pollution of an industrial land site with chlorinated benzenes, chlorinated phenols, hexachlorocyclohexane-isomers (HCH) on the soil microflora was investigated. Cell counts (microscopic and by plate count) as well as respiration rated did not correlate negatively with the concentration of the contaminants. Soil microorganisms grew in the presence of up to 750 μmol 1⁻¹ pf chlorinated compounds in liquid culture. Only 150 μmol l⁻¹ 2,4,5-trichlorophenol (2,4,5-TCP) inhibited growth totally. In enrichment cultures, bacteria used α- and γ-HCH, 3-chlorophenol (3-CP), 2,3-dichlorophenol (2,3-DCP), 2,6-DCP, 2,4,5-TCP, and 1,2,4,5-tetrachlorobenzene (1,2,4,5-TeCB) as a sole source of carbon and energy under aerobic conditions. No growth was observed with β-HCH. Under anaerobic conditions no growth was observed with any of the substances tested     

Recent methods for the determination of volatile and non-volatile organic compounds in natural and purified drinking water

Four analytical protocols for the extraction and preconcentration of organic residues in natural or purified drinking water were investigated and compared: closed loop stripping analysis; simultaneous extraction—distillation; purge and trap analysis; continuous liquid—liquid extraction. Organic extracts were submitted to a variety of separation and identification techniques. Volatiles were determined by conventional capillary column gas chromatography with tandem mass spectrometry, using triple-stage quadrupole instruments. Non-volatile and thermally labile molecules were investigated by several different techniques (high-temperature gas chromatography, capillary column supercritical fluid chromatography, pyrolysis gas chromatography—mass spectrometry, thermospray liquid chromatography with tandem mass spectrometry and conventional fast-atom bombardment with tandem mass spectrometry). Several samples recently examined in the laboratory provide examples of this multitechnique approach for a more complete knowledge of the organic carbon distribution in water-dissolved organic matter, taking into account organic substances with widely different volatilities, polarities and thermal stabilities.     

Biodegradation and ecotoxicity of soil contaminated by pentachlorophenol applying bioaugmentation and addition of sorbents

Biodegradation of pentachlorophenol (PCP) in soil by autochthonous microorganisms and in soil bioaugmented by the bacterial strain Comamonas testosteroni CCM 7530 was studied. Subsequent addition of organomineral complex (OMC) or lignite as possible sorbents for PCP immobilization has been investigated as well. The OMC was prepared from humic acids (HAs) isolated from lignite by binding them onto zeolite. Biodegradation of PCP and number of colony forming units (CFUs) were determined in the three types of soil, Chernozem, Fluvisol, and Regosol, freshly spiked with PCP and amended separately with tested sorbents. The enhancing effect of sorbent addition and bioaugmentation on PCP biodegradation depended mainly on the soil type and the initial PCP concentration. Microbial activity resulted in biotransformation of PCP into certain toxic substances, probably lower chlorinated phenols that are more soluble than PCP, and therefore more toxic to present biota. Therefore, it was necessary to monitor soil ecotoxicity during biodegradation. Addition of the OMC resulted in a more significant decrease of soil toxicity in comparison with addition of lignite. Lignite and OMC appear to be good traps for PCP with potential application in remediation technology.     

Biodegradation of slop oil from a petrochemical industry and bioreclamation of slop oil contaminated soil

Slop oil, i.e. waste oil from a petrochemical complex, contains at least 240 hydrocarbon components, of which 54% are from C₅ to C₁₁ and the rest from C₁₂ to C₂₃. Of 22 isolated bacterial cultures that were able to degrade slop oil, seven could each degrade about 40% of the slop oil, and a mixture of all seven could degrade 50% in liquid medium. Bioaugmentation of soil contaminated with slop oil with the mixed bacterial culture gave up to 70% degradation of slop oil after 30 days. This compares with 40% degradation without bioaugmentation. Bioaugmentation led to a significant increase in counts of bacteria able to degrade slop oil. Wheat sown on bioaugmented soil germinated and grew better than on non-augmented soil and led to increased degradation of slop oil (up to 80%). This indicates the potential of mixed culture for bioremediation.     

Spatial distribution of total halogenated organic compounds (TX), adsorbable organic halogens (AOX), and heavy metals in wetland soil irrigated with pulp and paper wastewater

Long-term irrigation using wastewater from paper industry may cause seriously problems to the receiving soil. This work surveyed and monitored the soil quality of a wastewater irrigation wetland system in Yancheng City, Jiangsu Province in China in 2014 and 2015. Τhe wetland soil showed different soil properties and TX, AOX, heavy metal contents after long-term wastewater irrigation. Long-term irrigation also accumulated the heavy metals such as Cu, Cd, Zn, and Pb in the wetland soil. Compared to the control, TX in the irrigated soil increased by 47.7–69.8% (2014) and 61.5–83.1% (2015). AOX varied in concentration from 1.7 to 55.0 mg kg^?1 (2014) and 11.0 to 53.0 mg kg^?1 (2015). The long-term irrigation of wastewater to wetland systems caused the accumulations of heavy metals, TX, and AOX in the soil and the levels of accumulations were related to several factors including soil properties, wastewater quality, and irrigation time.     

The decomposition of organic compounds in soil

Summary The course of the CO₂ evolution rates of soil samples has been followed continuously in the absence and in the presence of various organic compounds. After an incubation period of 300 hours at 13 and 20°C the CO₂ evolution from pasture soil (containing 1.76% soil organic carbon) amounted to 0.13 and 0.44g CO₂–C.g soil^–1.h^–1, respectively. For arable soil (containing 1.20% soil organic carbon) the rates amounted to 0.04 and 0.09 g CO₂–C.g soil^–1.h^–1, respectively.At 20°C larger amounts of the organic substrates added to the soil supplied with 20 g NH₄NO₃–N.g soil^–1 were lost as CO₂ than at 13°C, indicating a higher efficiency of the growth of microorganisms at lower temperatures. In the absence of NH₄NO₃ the respiration rates were initially higher than in its presence, suggesting that a part of the soil microflora is inhibited by low concentrations of NH₄NO₃. The amounts of carbon lost were low for phenolcarboxylic acids with OH groups in the ortho position. The replacement of one of these groups by a methoxyl group resulted in a larger amount of the C lost as CO₂. The replacement of the COOH group by a C=C–COOH group had a decreasing effect on the decomposition of the phenolic acids tested. The decomposition of vanillic acid,p-hydroxybenzoic acid, and of the benzoic acids with OH groups in the meta position was as complete as that of glucose, amino acids or casein. The decomposition of bacterial cells to CO₂ was considerably less than that of glucose.No evidence could be obtained that the low percentage of substrate converted to CO₂ at the time of maximal respiration rate was due to the decreasing diffusion rate of substrate to the microbial colonies in the soil during the consumption of substrate.     

Ethanol, BTEX and microbial community interactions in E-blend contaminated soil slurry

Degradation of benzene, toluene, ethylbenzene, m-, p- and o-xylenes (BTEX) and microbial community shifts in soil slurries contaminated with ethanol–gasoline blends (E-blends), containing 10, 50 or 90% (v/v) ethanol (E10, E50 and E90) were studied in soil slurries previously uncontaminated, contaminated by E-blends or ethanol. BTEX originating from E50 degraded fastest whereas from E10 slowest. Among the individual compounds, ethylbenzene degraded fastest (max 30% d⁻¹), and o-xylene slowest (min 1% d⁻¹) during aerobic conditions in previously not contaminated soils. Previous contamination by E-blends increased BTEX degradation significantly (3–19 times) compared with previously uncontaminated soils, whereas previous contamination with ethanol did not show significant difference in BTEX degradation. At least one type of the E-blends during aerobic conditions had a positive effect on total PLFAs (phospholipid fatty acids) and specific PLFAs, i.e. 10Me18:0, 16:1ω6 and cy17:0, but had a negative effect on cy19:0 and 18:2ω6,9c. The effects on total PLFAs, as well as the individual PLFAs, were particularly strong after repeated contamination. The single most affected PLFA was 16:1ω6, which increased 23 times during E10 treatment in soil slurries previously contaminated by E-blends. Altogether, the various E-blends had significantly different effects on BTEX degradation and also on individual PLFAs under aerobic conditions.     

Transformations of 1- and 2-carbon halogenated aliphatic organic compounds under methanogenic conditions

Several 1- and 2-carbon halogenated aliphatic organic compounds present at low concentrations (less than 100 micrograms/liter) were degraded under methanogenic conditions in batch bacterial cultures and in a continuous-flow methanogenic fixed-film laboratory-scale column. Greater than 90% degradation was observed within a 2-day detention time under continuous-flow methanogenic conditions with acetate as a primary substrate. Carbon-14 measurements indicated that chloroform, carbon tetrachloride, and 1,2-dichloroethane were almost completely oxidized to carbon dioxide, confirming removal by biooxidation. The initial step in the transformations of tetrachloroethylene and 1,1,2,2-tetrachloroethane to nonchlorinated end products appeared to be reductive dechlorination to trichloroethylene and 1,1,2-trichloroethane, respectively. Transformations of the brominated aliphatic compounds appear to be the result of both biological and chemical processes. The data suggest that transformations of halogenated aliphatic compounds can occur under methanogenic conditions in the environment.     

Dechlorination of trichlorofluoromethane (CFC-11) by sulfate-reducing bacteria from an aquifer contaminated with halogenated aliphatic compounds.

Groundwater samples were obtained from a deep aquifer contaminated with halogenated aliphatic compounds. One-milliliter samples contained 9.2 x 10(5) total bacteria (by acridine orange microscopic counts) and 2.5 x 10(3) sulfate-reducing bacteria (by most probable number analysis). Samples were incubated anaerobically in a basal salts medium with acetate as the electron donor and nitrate and sulfate as the electron acceptors. Residual levels of trichlorofluoromethane (CFC-11) in samples were biotically degraded, while trichloroethylene was not. When successively higher levels of CFC-11 were added, increasingly rapid degradation rates were observed. Concomitant with CFC-11 degradation was the near stoichiometric production of fluorodichloromethane (HCFC-21); the production of HCFC-21 was verified by mass spectrometry. CFC-11 degradation was dependent on the presence of acetate (or butyrate) and sulfate but was independent of nitrate. Other carbon sources such as lactate and isopropanol did not support the degradation. The addition of 1 mM sodium sulfide completely inhibited CFC-11 degradation; however, degradation occurred in the presence of 2 mM 2-bromoethanesulfonic acid. These results indicate that the anaerobic dechlorination of CFC-11 is carried out by sulfate-reducing bacteria and not by denitrifying or methanogenic bacteria.     

Fungal degradation of chlorpyrifos by Verticillium sp. DSP in pure cultures and its use in bioremediation of contaminated soil and pakchoi

Pesticides residues in soils and on vegetables are a public safety concern. Pretreatment with microorganisms degrading pesticides has the potential to alleviate the conditions. For this purpose, the degradation characteristics of chlorpyrifos by an isolated fungal strain Verticillium sp. DSP in pure cultures, soil, and on pakchoi (Brassica chinensis L.) were investigated. Degradation rate of chlorpyrifos in the mineral salts medium was proportional to the concentrations of chlorpyrifos ranging from 1 to 100 mg l⁻¹. The rate of degradation for chlorpyrifos (1 mg l⁻¹) in the mineral salts medium was 1.12 and 1.04 times faster at pH 7.0 than those at pHs 5.0 and 9.0, and the degradation at 35 °C was 1.15 and 1.12 times faster, respectively, than those at 15 and 20 °C. The addition of the fungal strain DSP into the contaminated soils was found to significantly increase the degradation of chlorpyrifos. Degradation rates of chlorpyrifos in inoculated soils were 3.61, 1.50 and 1.10 times faster in comparison with the sterilized soil, previously chlorpyrifos-untreated soil, and previously chlorpyrifos-treated soil under laboratory conditions. In contrast to the controls, the half-lives of chlorpyrifos were significantly shortened by 10.9% and 17.6% on treated pakchoi, 12.0% and 37.1% in inoculated soils, respectively, in the greenhouse and open field. The results indicate that the fungal strain DSP can be used successfully for the removal or detoxification of chlorpyrifos residues in/on contaminated soil and vegetable.     

Gas chromatography-mass spectrometry with headspace for the analysis of volatile organic compounds in waste water

Headspace analysis combined with high-resolution gas chromatography and detection by mass spectrometry was evaluated for the analysis of 53 volatile organic compounds (VOCs) in river waters, waste waters and treated water samples down to 0.1 microgl(-1) concentration levels. The conditions optimised included sample thermostatting time and temperature, autosampler parameters and the nature of salt, added to the sample. The pollutions origin and their seasonal rippling have been done. It was shown that the content of VOCs in river water mainly correlates to the content of these compounds in waste waters, which shows the anthropogenic character of the pollutions.     

Mass production of bacterial communities adapted to the degradation of volatile organic compounds (TEX)

This study focuses on the mass cultivation of bacteria adapted to the degradation of a mixture composed of toluene, ethylbenzene, o-, m- and p-xylenes (TEX). For the cultivation process Substrate Pulse Batch (SPB) technique was adapted under well-automated conditions. The key parameters to be monitored were handled by LabVIEW software including, temperature, pH, dissolved oxygen and turbidity. Other parameters, such as biomass, ammonium or residual substrate concentrations needed offline measurements. SPB technique has been successfully tested experimentally on TEX. The overall behavior of the mixed bacterial population was observed and discussed along the cultivation process. Carbon and nitrogen limitations were shown to affect the integrity of the bacterial cells as well as their production of exopolymeric substances (EPS). Average productivity and yield values successfully reached the industrial specifications, which were 0.45 kg_DW m⁻³ d⁻¹ and 0.59 g_DW g_C⁻¹, respectively. Accuracy and reproducibility of the obtained results present the controlled SPB process as a feasible technique.