Intrinsic capacities of soil microfloræ for gasoline degradation

A methodology to determine the intrinsic capacities of a microflora to degrade gasoline was developed, in particular for assessing the potential of autochtonous populations of polluted and non polluted soils for natural attenuation and engineered bioremediation. A model mixture (GM23) constituted of the 23 most representative hydrocarbons of a commercial gasoline was used. The capacities of the microfloræ (kinetics and extent of biodegradation) were assessed by chromatographic analysis of hydrocarbon consumption and of CO2 production. The degradation of the components of GM23 was assayed in separate incubations of each component and in the complete mixture. For the microflora of an unpolluted spruce forest soil, all hydrocarbons of GM23 except cyclohexane, 2,2,4- and 2,3,4-trimethylpentane isomers were degraded to below detection limit in 28 days. This microflora was reinforced with two mixed microbial communities selected from gasoline-polluted sites and shown to degrade cyclohexane and 2,2,4- trimethylpentane. With the reinforced microflora, complete degradation of GM23 was observed. The degradation patterns of individual components of GM23 were similar when the compounds were present individually or in the GM23 mixture, as long as the concentrations of 2-ethyltoluene and trimethylbenzene isomers were kept sufficiently low ( 35 mg.l-1) to remain below their inhibitory level.     

Selection of microbial populations degrading recalcitrant hydrocarbons of gasoline by monitoring of culture-headspace composition

A methodology was devised and was found useful for the selection of populations degrading recalcitrant hydrocarbons. The work was part of a programme aiming at developing knowledge of the intrinsic capacities of autochtonous microflorae of the environment for gasoline biodegradation. The methodology involved monitoring the progress of degradation in enrichment liquid cultures on the selected hydrocarbon by gas chromatographic analysis of CO2 production and O2 consumption. Populations degrading in particular o-xylene, 1,2,4-trimethylbenzene, cyclohexane were obtained. Concerning 2,2,4-trimethylpentane (isooctane), one microflora (and a pure strain derived from it) growing on this hydrocarbon were obtained from gasoline-polluted water.     

Characterisation of biodegradation capacities of environmental microflorae for diesel oil by comprehensive two-dimensional gas chromatography

In contaminated soils, efficiency of natural attenuation or engineered bioremediation largely depends on biodegradation capacities of the local microflorae. In the present study, the biodegradation capacities of various microflorae towards diesel oil were determined in laboratory conditions. Microflorae were collected from 9 contaminated and 10 uncontaminated soil samples and were compared to urban wastewater activated sludge. The recalcitrance of hydrocarbons in tests was characterised using both gas chromatography (GC) and comprehensive two-dimensional gas chromatography (GC×GC). The microflorae from contaminated soils were found to exhibit higher degradation capacities than those from uncontaminated soil and activated sludge. In cultures inoculated by contaminated-soil microflorae, 80% of diesel oil on an average was consumed over 4-week incubation compared to only 64% in uncontaminated soil and 60% in activated sludge cultures. As shown by GC, n-alkanes of diesel oil were totally utilised by each microflora but differentiated degradation extents were observed for cyclic and branched hydrocarbons. The enhanced degradation capacities of impacted-soil microflorae resulted probably from an adaptation to the hydrocarbon contaminants but a similar adaptation was noted in uncontaminated soils when conifer trees might have released natural hydrocarbons. GC×GC showed that a contaminated-soil microflora removed all aromatics and all branched alkanes containing less than C₁₅. The most recalcitrant compounds were the branched and cyclic alkanes with 15–23 atoms of carbon.     

Biodegradability of volatile hydrocarbons of gasoline

The biodegradability under aerobic conditions of volatile hydrocarbons (4–6 carbons) contained in gasoline and consisting of n-alkanes, iso-alkanes, cycloalkanes and alkenes, was investigated. Activated sludge was used as the reference microflora. The biodegradation test involved the degradation of the volatile fraction of gasoline in closed flasks under optimal conditions. The kinetics of biodegradation was monitored by CO₂ production. Final degradation was determined by gas chromatographic analysis of all measurable hydrocarbons (12 compounds) in the mixture after sampling the headspace of the flasks. The degradation of individual hydrocarbons was also studied with the same methodology. When incubated individually, all hydrocarbons used as carbon sources, except 2,2-dimethylbutane and 2,3-dimethylbutane, were completely consumed in 30 days or less with different velocities and initial lag periods. When incubated together as constituents of the light gasoline fraction, all hydrocarbons were metabolised, often with higher velocities than for individual compounds. Cometabolism was involved in the degradation of dimethyl isoalkanes. Received: 19 October 1999 / Received revision: 21 January 2000 / Accepted: 23 January 2000     

Biodegradation of hydrocarbon cuts used for diesel oil formulation

The biodegradability of various types of diesel oil (DO), such as straight-run DO, light-cycle DO, hydrocracking DO, Fischer–Tropsch DO and commercial DO, was investigated in biodegradation tests performed in closed-batch systems using two microflorae. The first microflora was an activated sludge from an urban wastewater treatment plant as commonly used in biodegradability tests of commercial products and the second was a microflora from a hydrocarbon-polluted soil with possible specific capacities for hydrocarbon degradation. Kinetics of CO₂ production and extent of DO biodegradation were obtained by chromatographic procedures. Under optimised conditions, the polluted-soil microflora was found to extensively degrade all the DO types tested, the degradation efficiencies being higher than 88%. For all the DOs tested, the biodegradation capacities of the soil microflora were significantly higher than those of the activated sludge. Using both microflora, the extent of biodegradation was highly dependent upon the type of DO used, especially its hydrocarbon composition. Linear alkanes were completely degraded in each test, whereas identifiable branched alkanes such as farnesane, pristane or phytane were degraded to variable extents. Among the aromatics, substituted mono-aromatics were also variably biodegraded.     

Die-Away Kinetic Analysis of the Capacity of Epilithic and Planktonic Bacteria from Clean and Polluted River Water To Biodegrade Sodium Dodecyl Sulfate

The capacities of epilithic and planktonic river bacterial populations to degrade sodium dodecyl sulfate (SDS) in samples taken at two times during 1987 from one clean and four polluted sites in a South Wales river were estimated in die-away tests under simulated environmental conditions. There was a relatively slow disappearance of SDS in die-away tests for both planktonic and epilithic populations taken from the clean source site, as compared with those taken from the downstream polluted sites, for which the rate of biodegradation was accelerated, sometimes after an apparent initial lag period. The kinetic components contributing to the die-away curves were quantified by nonlinear regression analysis in which the experimental data were fitted to a variety of possible kinetic models. All samples except for one from the polluted sites best fitted a model which describes the biodegradation of SDS at concentrations well below its K_m by bacteria whose growth is exponential and unaffected by the addition of a test substrate. Each sample from the clean source site fitted a different model, but there was generally little or no growth on endogenous carbon. A consideration of the numerical values of constants derived from the modeling of epilithic and planktonic populations from polluted sites indicated clearly that the biodegradative capacity of epilithic bacterial populations towards SDS is more stable than that of planktonic bacterial populations.     

Cometabolic biodegradation of methyl tert-butyl ether by a soil consortium: Effect of components present in gasoline

A soil consortium was tested for its ability to degrade reformulated gasoline, containing methyl tert-butyl ether (MTBE). Reformulated gasoline was rapidly degraded to completion. However, MTBE tested alone was not degraded. A screening was carried out to identify compounds in gasoline that participate in cometabolism with MTBE. Aromatic compounds (benzene, toluene, xylenes) and compounds structurally similar to MTBE (tert-butanol, 2,2-dimethylbutane, 2,2,4-trimethylpentane) were unable to cometabolize MTBE. Cyclohexane was resistant to degradation. However, all n-alkanes tested for cometabolic activity (pentane, hexane, heptane) did enable the biodegradation of MTBE. Among the alkanes tested, pentane was the most efficient (200 &mgr;g/day). Upon the depletion of pentane, the consortium stopped degrading MTBE. When the consortium was spiked with pentane, MTBE degradation continued. When the ratio of MTBE to pentane was increased, the amount of MTBE degraded by the consortium was higher. Finally, diethylether was tested for cometabolic degradation with MTBE. Both compounds were degraded, but the process differed from that observed with pentane.     

Plasma transthyretin. Tissue sites of degradation and turnover in the rat

Transthyretin (TTR) is involved in the plasma transport of both retinol and thyroid hormones. TTR is synthesized in the liver and choroid plexus, and in small amounts in several other tissues. A study was conducted to determine the tissue sites of degradation and turnover of TTR in the rat. The study employed TTR labeled with tyramine cellobiose (TC) and the trapped ligand method. Samples of purified rat TTR were labeled either with 125I-TC or directly with 131I. A mixture of the two labeled TTRs was injected intravenously into six rats. Blood samples were collected via a venous catheter for kinetic (turnover) analysis. After 24 or 48 h, the rats were killed, and 23 different tissues/organs were assayed as possible sites of TTR degradation. Derivatization of TTR with TC did not appreciably alter TTR plasma kinetics. Plasma turnover data were best fit by a three-pool model. The mean fractional turnover of plasma TTR was 0.15/h, and of total body TTR 0.04/h. The major sites of TTR degradation were the liver (36-38% of total body TTR degradation, almost all in hepatocytes), muscle (12-15%), and skin (8-10%). Tissues that were sites of 1-8% of body TTR degradation included kidneys, adipose tissue, testes, and the gastrointestinal tract. Less than 1% of total TTR degradation occurred in the other tissues examined. A second study was conducted in which labeled TTR was injected intraventricularly into the cerebrospinal fluid in order to explore the degradation of TTR of choroid plexus origin. The kinetics of the appearance and disappearance of such labeled TTR in plasma were physiologically reasonable, with an estimated turnover of cerebrospinal fluid TTR of the order of 0.33/h. The major tissue sites of degradation of labeled TTR injected into cerebrospinal fluid and into plasma were approximately the same. No specific degradation of TTR was found in the nervous system tissues. The most active organs of TTR catabolism, per gram wet weight, were liver and kidneys. These studies demonstrate that many tissues participate in TTR turnover and degradation; the studies provide quantitative information about the tissue sites of TTR catabolism.     

Characterization and degradation potential of diesel-degrading bacterial strains for application in bioremediation

Bioremediation of polluted soils is a promising technique with low environmental impact, which uses soil organisms to degrade soil contaminants. In this study, 19 bacterial strains isolated from a diesel-contaminated soil were screened for their diesel-degrading potential, biosurfactant (BS) production, and biofilm formation abilities, all desirable characteristics when selecting strains for re-inoculation into hydrocarbon-contaminated soils. Diesel-degradation rates were determined in vitro in minimal medium with diesel as the sole carbon source. The capacity to degrade diesel range organics (DROs) of strains SPG23 (Arthobacter sp.) and PF1 (Acinetobacter oleivorans) reached 17–26% of total DROs after 10 days, and 90% for strain GK2 (Acinetobacter calcoaceticus). The amount and rate of alkane degradation decreased significantly with increasing carbon number for strains SPG23 and PF1. Strain GK2, which produced BSs and biofilms, exhibited a greater extent, and faster rate of alkane degradation compared to SPG23 and PF1. Based on the outcomes of degradation experiments, in addition to BS production, biofilm formation capacities, and previous genome characterizations, strain GK2 is a promising candidate for microbial-assisted phytoremediation of diesel-contaminated soils. These results are of particular interest to select suitable strains for bioremediation, not only presenting high diesel-degradation rates, but also other characteristics which could improve rhizosphere colonization.     

Mobility and Persistence of Petroleum Hydrocarbons in Peat Soils of Southeastern Mexico

Spilled crude petroleum from oil wells contains numerous hydrocarbons, some of which are toxic and threaten life. We have studied the mobility and persistence of hydrocarbons in waterlogged soils that contain large proportions of fermented organic matter (Histosols) and large concentrations of dissolved organic carbon (DOC) in the State of Tabasco, Mexico. We sampled soil and phreatic water at sites polluted by oil spills for several decades, as well as at sites that had only recently (few weeks) been polluted, and compared their hydrocarbon contents with those of unaffected sites in the same area. Samples were analyzed for 16 non-alkylated polyaromatic hydrocarbons (PAHs) and n-alkanes from nC9 to nC34. The spilled hydrocarbons had remained predominantly in the organic surface horizons of the soil where spillage occurred; there was little evidence of movement within the soil. The fraction of low molecular weight compounds was larger at sites of recent spills than where spills happened several decades ago. Nevertheless, sites of old spills still contained large concentrations of hydrocarbons, among which those of low molecular weight represented from 30 to 49% of total PAHs and from 50 to 84% of total n-alkanes, indicating that volatilization or microbial degradation is slow in these soils. In the peat horizons the measured organic carbon partition coefficients (K _oc ) for the higher molecular weight PAHs were consistently smaller than those estimated by empirical equations by up to two orders of magnitude. The dissolved organic carbon of these peat soils seems to influence this behavior. At sites of old spills, partition coefficients for the PAHs were larger than at sites of recent spills.     

A rapid and sensitive immunoassay for the detection of gasoline and diesel fuel in contaminated soil

An enzyme immunoassay (EIA) was developed using a monoclonal antibody (MAb) reagent that detects gasoline and diesel fuel. Xylene and toluene derivatives, which are common components of gasoline, were synthesized with various types of spacers and conjugated to either bovine serum albumen or bovine thyroglobulin. A total of 16 different hapten conjugates were used for immunizing both Balb/c and Swiss Webster mice. A panel of MAbs were produced that recognized xylene and toluene in a competitive EIA. An enzyme‐hapten conjugate was prepared for the MAb (F12–3C8) that demonstrated the most suitable characteristics for sensitivity, cross‐reactivity, and compatibility with extraction buffers. The resulting EIA gave ED₅₀ values for m‐xylene of less than 1 ppm and values of less than 500 ppb for gasoline. Diesel fuel was also detected, with ED₃₀ values in the range of 300 ppb. When samples of gasoline were tested, the EIA gave consistent ED₃₀ values that were independent of manufacturer or octane rating. The EIA was compatible with simplified methods for the extraction of petroleum products from soil. The EIA detected gasoline in spiked soil samples, but was not affected by extracts of negative soil samples. Commercialization of this assay will offer speed, cost effectiveness, and other significant advantages over current testing methods of gasoline and diesel fuel contamination levels in soil.     

基于大型底栖动物群落的上海市河道水质生物学评价

2011年夏秋季,在上海市全境83个河道断面开展了河道底栖动物采样,共获取底栖动物20个分类单位（种）。9个极严重污染断面未采集到活体生物,生境基本丧失。其余74个有活体生物断面,采用三种常用生物指数：Shannon-Wiener多样性指数、Hilsenhoff耐污指数、Goodnight修正指数分别进行计算及评价。Shannon-Wiener多样性指数判别为25个严重污染和49个重污染断面;Hilsenhoff耐污指数划分38个重污染、5个中污染和31个轻污染断面;Goodnight修正指数划分33个重污染、2个中污染和39个轻污染断面。与典型河道断面的水质理化指标监测值进行对照,Shannon-Weiner多样性指数对河道水质评价的准确度较低,Hilsenhoff生物指数和Goodnight修正指数的水质评价效率及准确度均较高,断面污染等级与水质理化指标基本对应。     

Degradation of a Mixture of Hydrocarbons,Gasoline, and Diesel Oil Additives by Rhodococcus aetherivorans and Rhodococcus wratislaviensis

Two strains, identified as Rhodococcus wratislaviensis IFP 2016 and Rhodococcus aetherivorans IFP 2017, were isolated from a microbial consortium that degraded 15 petroleum compounds or additives when provided in a mixture containing 16 compounds (benzene, toluene, ethylbenzene, m-xylene, p-xylene, o-xylene, octane, hexadecane, 2,2,4-trimethylpentane [isooctane], cyclohexane, cyclohexanol, naphthalene, methyl tert-butyl ether [MTBE], ethyl tert-butyl ether [ETBE], tert-butyl alcohol [TBA], and 2-ethylhexyl nitrate [2-EHN]). The strains had broad degradation capacities toward the compounds, including the more recalcitrant ones, MTBE, ETBE, isooctane, cyclohexane, and 2-EHN. R. wratislaviensis IFP 2016 degraded and mineralized to different extents 11 of the compounds when provided individually, sometimes requiring 2,2,4,4,6,8,8-heptamethylnonane (HMN) as a cosolvent. R. aetherivorans IFP 2017 degraded a reduced spectrum of substrates. The coculture of the two strains degraded completely 13 compounds, isooctane and 2-EHN were partially degraded (30% and 73%, respectively), and only TBA was not degraded. Significant MTBE and ETBE degradation rates, 14.3 and 116.1 μmol of ether degraded h⁻¹ g⁻¹ (dry weight), respectively, were measured for R. aetherivorans IFP 2017. The presence of benzene, toluene, ethylbenzene, and xylenes (BTEXs) had a detrimental effect on ETBE and MTBE biodegradation, whereas octane had a positive effect on the MTBE biodegradation by R. wratislaviensis IFP 2016. BTEXs had either beneficial or detrimental effects on their own degradation by R. wratislaviensis IFP 2016. Potential genes involved in hydrocarbon degradation in the two strains were identified and partially sequenced.The pollution of soils by petroleum compounds is of great concern mainly because of the solubilities of the different molecules in water, which can endanger aquifers in contact with polluted zones. Petroleum storage facilities are frequently the source of pollution due to leaks and spills during fuel transfer and storage. For example, in the United States in 2007, the EPA indicated that nearly 110,000 old leaks have not yet been cleaned up, and there are an unknown number of petroleum brownfield sites (estimated to be over 200,000) that are predominately old abandoned gas stations (http://www.epa.gov/OUST/pubs/OUST_FY07_Annual_Report-_Final_4-08.pdf). In these locations, the contamination can be generated by diesel oil and/or gasoline leaks from storage tanks, resulting in a complex mixture of compounds with different water solubilities and different biodegradabilities. Among all the phenomena occurring at polluted sites, (i) the interactions between the different compounds can result in enhanced solubility for low-solubility compounds, (ii) the differences in biodegradability levels between the dissolved molecules can lead to dispersion of the poorly biodegradable or nonbiodegradable compounds, and (iii) in the presence of mixtures of compounds, interactions between some of them can lead to detrimental or beneficial effects. For example, methyl tert-butyl ether [MTBE] could enhance the mobility of dissolved benzene, toluene, ethylbenzene, and xylenes [BTEXs] by exerting a cosolvent effect that decreases sorption-related retardation (30). The impact of additive use after petroleum refining to meet specific requirements is a point that deserves more study. MTBE was used extensively in the United States and elsewhere in the world. Several states banned the use of MTBE because of numerous reports of groundwater pollution, but this compound is still used in Europe. Although its use is decreasing, it still remains high, and by the end of 2007, global MTBE production was about 15 million tons. Ethyl tert-butyl ether (ETBE) is used in Europe (France, Spain, Belgium, and Germany), with European production reaching 626,300 tons in 2004 (http://www.agriculture.total.fr). MTBE and ETBE can be added at up to 15% to gasoline in order to reach the octane index requirement; their use was shown to limit noncombusted hydrocarbon release in exhaust pipe fumes. 2-Ethylhexyl nitrate (2-EHN) is the nitric ester of 2-ethyl-1-hexanol, and it is added to diesel formulations at up to 0.4%. The 2-EHN market is about 100,000 tons/year. Alkylates are native components of petroleum products, but in view of the use of ethanol in gasoline, alkylates, like 2,2,4-trimethylpentane (isooctane), are among the few high-octane alternatives that have been proposed, if only the minimal volume of ethanol required to meet oxygenate requirements are used in reformulated gasoline. In this case, other fuel constituents would be needed to make up the resulting 5% gap and the octane shortfall of about 1.5 octane points. Isooctane has an octane rating of 100 and would be attractive for refiners as an octane enhancer since it can be produced by former MTBE production plants (35).Data concerning the use of additives have to be taken into account to assess the impact of petroleum products on polluted sites, such as gas stations, where leaks from different storage tanks can occur, leading to contamination by complex mixtures of petroleum products. The biodegradation of monoaromatic (BTEX) compounds and alkanes has been studied extensively, and both are generally quite biodegradable under aerobic conditions (44, 46). Regarding the biodegradability of MTBE, several microorganisms have been isolated with specific degradation capacities, and some of the genes involved in the biodegradation pathway have been characterized (28). The first-order attenuation rates for MTBE in the plumes in which biodegradation occurred varied from 0.56 to 4.3 year⁻¹, a rate of biodegradation not sufficient to contain the plume (7). In addition, there are numerous sites for which no biodegradation was observed (3, 10), and the presence of BTEXs and MTBE has been shown in the case of Methylibium petroleiphilum PM1 to delay the onset of MTBE biodegradation (13).The behavior of ETBE when spilled in the environment has not been as well studied as that of MTBE, and the extent of contamination has not been documented sufficiently. Similar to that of MTBE, the biodegradation of ETBE is not always observed in microcosms with soils or aquifers derived from contaminated sites (3). Microorganisms able to grow on ETBE have been isolated, and the first monooxygenase system able to degrade ETBE was identified as a cytochrome P450 monooxygenase (encoded by the ethRABCD genes) in Rhodococcus ruber IFP 2001 (6, 21). Highly similar eth gene clusters were also isolated from Rhodococcus zopfii IFP 2005 and Gordonia sp. strain IFP 2009 (4, 16). R. ruber IFP 2001, R. zopfii IFP 2005, and Gordonia sp. strain IFP 2009 were able to grow on ETBE at the expense of the C₂ moiety being released by the cleavage of the ether bond with the accumulation of tert-butyl alcohol (TBA) in the growth culture (C. Malandain, F. Fayolle-Guichard, and T. Vogel, submitted for publication). Interestingly, other microorganisms belonging to the genus Rhodococcus were reported to have biodegradation capacities toward ether fuels. Mo et al. (31) isolated a Rhodococcus sp. strain able to degrade MTBE to a low extent; R. aetherivorans, a new species that belongs to MTBE-degrading actinomycetes (20) was characterized, but the enzymatic system responsible for the MTBE oxidation was not elucidated; Rhodococcus sp. strain EH831 was able to degrade MTBE (27).There are few data in the literature regarding the biodegradability of isooctane; Mycobacterium austroafricanum IFP 2173 was the only strain described for its ability to use isooctane as the sole carbon and energy source (42), and more recently, Cho et al. (9) demonstrated the biodegradability of isooctane using previously acclimated biomass. Regarding the biodegradability of 2-EHN, only M. austroafricanum IFP 2173 was recently reported to degrade 2-EHN to 4-ethyldihydrofuran-2(3H)-one (36).The biodegradation of complex mixtures of hydrocarbons has generally been studied only under the highest-performing conditions using different processes (e.g., biofilters) in which the microorganisms and the role played by each of them have not necessarily been elucidated. Individual microorganisms have generally been characterized for their ability to degrade individual petroleum compounds or classes of compounds, i.e., monoaromatics. There is much less work addressing the issue of the biodegradation by individual, characterized microorganisms of complex mixtures generally found in sites polluted by hydrocarbons, even though some bacterial genera (Pseudomonas and Rhodococcus, for example) are known to degrade a wide range of xenobiotics (17, 25, 41). Some authors have investigated the range of biodegradation capacities of given individual strains. Solano-Serena et al. (42) previously isolated M. austroafricanum IFP 2173 from gasoline-contaminated groundwater, and this strain, tested on a mixture of petroleum compounds, showed extended biodegradation capacities toward various hydrocarbons. More recently, Rhodococcus sp. strain EC1 was shown to degrade BTEXs, short-chain alkanes, pyrene, and MTBE (26).The selection and the study of strains with capacities to use a broad spectrum of various hydrocarbons is of great interest because it could facilitate the study of the effect of selective pressure in terms of gene acquisition. From a bacterial consortium, including bacteria from soil at a gas station polluted by leaking tanks and enriched on a mixture of various hydrocarbons, we isolated two strains of Rhodococcus wratislaviensis and Rhodococcus aetherivorans and studied their biodegradation capacities toward hydrocarbons or additives added individually or in mixtures.     

PAH Degradation Capacity of Soil Microbial Communities—Does It Depend on PAH Exposure?

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants of the environment. But is their microbial degradation equally wide in distribution? We estimated the PAH degradation capacity of 13 soils ranging from pristine locations (total PAHs ≈ 0.1 mg kg^?1) to heavily polluted industrial sites (total PAHs ≈ 400 mg kg^?1). The size of the pyrene- and phenanthrene-degrading bacterial populations was determined by most probable number (MPN) enumeration. Densities of phenanthrene degraders reflected previous PAH exposure, whereas pyrene degraders were detected only in the most polluted soils. The potentials for phenanthrene and pyrene degradation were measured as the mineralization of ¹⁴C-labeled spikes. The time to 10% mineralization of added ¹⁴C phenanthrene and ¹⁴C pyrene was inversely correlated with the PAH content of the soils. Substantial ¹⁴C phenanthrene mineralization in all soils tested, including seven unpolluted soils, demonstrated that phenanthrene is not a suitable model compound for predicting PAH degradation in soils. ¹⁴C pyrene was mineralized by all Danish soil samples tested, regardless of whether they were from contaminated sites or not, suggesting that in industrialized areas the background level of pyrene is sufficient to maintain pyrene degradation traits in the gene pool of soil microorganisms. In contrast, two pristine forest soils from northern Norway and Ghana mineralized little ¹⁴C pyrene within the 140-day test period. Mineralization of phenanthrene and pyrene by all Danish soils suggests that soil microbial communities of inhabited areas possess a sufficiently high PAH degradation capacity to question the value of bioaugmentation with specific PAH degraders for bioremediation.     

Anaerobic biodegradation of BTEX and gasoline in various aquatic sediments

We examined the extent of biodegradation of benzene, toluene, ethylbenzene and the three isomers of xylene (BTEX) as a mixture and from gasoline in four different sediments: the New York/New Jersey Harbor estuary (polluted); Tuckerton, N.J. (pristine); Onondaga Lake, N.Y. (polluted) and Blue Mtn. Lake, N.Y. (pristine). Enrichment cultures were established with each sediment using denitrifying, sulfidogenic, methanogenic and iron reducing media, as well as site water. BTEX loss, as measured by GC-FID, was extensive in the sediments which had a long history of pollution, with all compounds being utilized within 21–91 days in the most active cultures, and was very slight or non-existent in the pristine sediments. Also, the pattern of loss was different under the various reducing conditions within each sediment and between sediments. For example benzene loss was only observed in sulfidogenic cultures from the NY/NJ Harbor sediments while toluene was degraded under all redox conditions. The loss of BTEX was correlated to the reduction of the various electron acceptors. In cultures amended with gasoline the degradation was much slower and incomplete. These results show that the fate of the different BTEX components in anoxic sediments is dependent on the prevailing redox conditions as well as on the characteristics and pollution history of the sediment.     

Aerobic Biodegradation of Methyl tert-Butyl Ether by Aquifer Bacteria from Leaking Underground Storage Tank Sites

The potential for aerobic methyl tert-butyl ether (MTBE) degradation was investigated with microcosms containing aquifer sediment and groundwater from four MTBE-contaminated sites characterized by oxygen-limited in situ conditions. MTBE depletion was observed for sediments from two sites (e.g., 4.5 mg/liter degraded in 15 days after a 4-day lag period), whereas no consumption of MTBE was observed for sediments from the other sites after 75 days. For sediments in which MTBE was consumed, 43 to 54% of added [U-¹⁴C]MTBE was mineralized to ¹⁴CO₂. Molecular phylogenetic analyses of these sediments indicated the enrichment of species closely related to a known MTBE-degrading bacterium, strain PM1. At only one site, the presence of water-soluble gasoline components significantly inhibited MTBE degradation and led to a more pronounced accumulation of the metabolite tert-butyl alcohol. Overall, these results suggest that the effects of oxygen and water-soluble gasoline components on in situ MTBE degradation will vary from site to site and that phylogenetic analysis may be a promising predictor of MTBE biodegradation potential.     

The total synthesis of ganglioside GM3

Previous syntheses of ganglioside GM3 (NeuAc alpha3Gal beta4Glc beta1Cer) are reviewed, and both chemoenzymatic and chemical total synthetic approaches were investigated. In a chemoenzymatic approach, (2S,3R,4E)-5'-acetyl-alpha-neuraminyl-(2' --> 3')-beta-galactopyranosyl-(1' --> 4')-beta-glucopyranosyl-(1' <--> 1)-2-azido-4-octadecene-1,3-diol (azidoGM3) was readily prepared utilizing recombinant beta-Gal-(1' --> 3'/4')-GlcNAc alpha-(2' --> 3')-sialyltransferase enzyme, and was evaluated as a synthetic intermediate to ganglioside GM3. The chemical total synthesis of ganglioside GM3 was performed on one of the largest scales yet reported. The highlights of this synthesis include minimizing the steps necessary to prepare the lactosyl acceptor as a useful anomeric mixture, which was present in excess for the highly regioselective and fairly stereoselective sialylation with a known neuraminyl donor to give the protected GM3 trisaccharide. The synthetic methodology maximized convergence by a subsequent glycosidic coupling of the well-characterized GM3 trisaccharide trichloroacetimidate derivative with protected ceramide. The ganglioside GM3 was nearly homogeneous as the two glycosidic couplings utilized preparative HLPC purifications, and variations in the sphingosine base and fatty acyl group were under 0.1 and 0.2%, respectively.     

Microbial degradation and fate in the environment of methyl tert-butyl ether and related fuel oxygenates

Oxygenates, mainly methyl tert-butyl ether (MTBE), are commonly added to gasoline to enhance octane index and improve combustion efficiency. Other oxygenates used as gasoline additives are ethers such as ethyl tert-butyl ether (ETBE), tert-amyl methyl ether (TAME), and alcohols such as tert-butyl alcohol (TBA). As a result of its wide use, MTBE has been detected, mainly in the USA, in groundwater and surface waters, and is a cause of concern because of its possible health effects and other undesirable consequences. MTBE is a water-soluble and mobile compound that generates long pollution plumes in aquifers impacted by gasoline releases from leaking tanks. Field observations concur in estimating that, because of recalcitrance to biodegradation, natural attenuation is slow (half-life of at least 2 years). However, quite significant advances have been made in recent years concerning the microbiology of the degradation of MTBE and other oxygenated gasoline additives. The recalcitrance of these compounds results from the presence in their structure of an ether bond and of a tertiary carbon structure. For the most part, only aerobic microbial degradation systems have been reported so far. Consortia capable of mineralizing MTBE have been selected. Multiple instances of the cometabolism of MTBE with pure strains or with microflorae, growing on n-alkanes, isoalkanes, cyclohexane or ethers (diethyl ether, ETBE), have been described. MTBE was converted into TBA in all cases and was sometimes further degraded, but it was not used as a carbon source by the pure strains. However, mineralization of MTBE and TBA by several pure bacterial strains using these compounds as sole carbon and energy source has recently been reported. The pathways of metabolism of MTBE involve the initial attack by a monooxygenase. In several cases, the enzyme was characterized as a cytochrome P-450. After oxygenation, the release of a C -unit as formaldehyde or formate leads to the production of TBA, which can be converted to 2-hydroxyisobutyric acid and further metabolized. Developments in microbiology make biological treatment of water contaminated with MTBE and other oxygenates an attractive possibility. Work concerning ex situ treatment in biofilters by consortia and by pure strains, and involving or not cometabolism, is under way. Furthermore, the development of in situ treatment processes is a promisinggoal.     

Kinetics of Polycyclic Aromatic Hydrocarbon (PAH) Degradation in Long-term Polluted Soils during Bioremediation

Bioremediation experiments with ten different soil samples from former industrial sites which were long-term polluted with polycyclic aromatic hydrocarbons (PAHs) were carried out using outdoor pot trials. The degradation of 15 PAHs according to the US EPA was investigated for 168 weeks through repeated soil sampling and determination of the total PAH concentration. On average, degradation was largest for acenaphthene (88%; 63 to 99%) and smallest for anthracene (22%; no significant degradation to 89%). For most of the PAH single substances, degradation kinetics were characterised by a first initial phase of fast degradation. In a subsequent second phase, degradation diminished and residual PAH concentrations were approached within 168 weeks, resulting in a similar PAH pattern in the ten soil samples. Degradation kinetics was calculated through the selection of the appropriate differential rate equation from a set of seven equations. Kinetics of PAH degradation was best fitted by single and two coupled first order exponential equations with median R2 of 0.71 (0.01 to 1.00). Degradation rate constants of the rapid phase (k ₁) ranged from 0.05×10⁻² week⁻¹ for benzo[k]fluoranthene to 18.3 week⁻¹ for naphthalene and for the subsequent slow degradation phase (k ₂) they ranged from 0.01×10⁻² week⁻¹ for benzo[a]anthracene to 2.3×10⁻² week⁻¹ for fluoranthene. Degradation was governed by desorption and diffusion processes of different rates, while microbial activity did not influence the kinetics. Median disappearance times (DT₅₀) ranged from 6.1 weeks for naphthalene to 522 weeks for benzo[k]fluoranthene. With the exception of the 6-ring PAHs dibenzo[ah]anthracene and indeno[1,2,3-cd]pyrene, this sequence followed the PAHs’ degree of condensation. The total initial PAH concentration and the residual concentration were correlated with R2 of 0.69, with larger initial PAH concentrations leading to larger residual concentrations and degradation rates.     

Microbial degradation of petroleum hydrocarbons in a polluted tropical stream

Summary Crude oil degradation was observed in water samples from three sites along the course of a polluted stream in Lagos, Nigeria. Consistent increase and decrease in the total viable counts (TVCs) of indigenous organisms occurred in the test and control experiments, respectively. Enrichments of the water samples with crude oil resulted in the isolation of nine bacteria belonging to seven genera. A mixed culture was developed from the assemblage of the nine species. The defined microbial consortium utilized a wide range of pure HCs including cycloalkane and aromatic HCs. Utilization of crude oil and petroleum cuts, i.e., kerosene and diesel resulted in an increase in TVC (till day 10) concomitant with decreases in pH and residual oil concentration. Crude oil, diesel and kerosene were degraded by 88, 85 and 78%, respectively, in 14 days. Substrate uptake studies with axenic cultures showed that growth was not sustainable on either cyclohexane or aromatics while degradation of the petroleum fractions fell below 67% in spite of extended incubation period (20 day). From the GC analysis of recovered oil, while reductions in peaks of n-alkane fractions and in biomarkers namely n-C₁₇/pristane and n-C₁₈/phytane ratios were observed in culture fluids of pure strains, complete removal of all the HC components of kerosene, diesel and crude oil including the isoprenoids was obtained with the consortium within 14 days.