Anaerobic degradation of benzene, toluene, ethylbenzene, and o-xylene in sediment-free iron-reducing enrichment cultures

Monoaromatic hydrocarbons such as benzene, toluene, ethylbenzene, and xylene (BTEX) are widespread contaminants in groundwater. We examined the anaerobic degradation of BTEX compounds with amorphous ferric oxide as electron acceptor. Successful enrichment cultures were obtained for all BTEX substrates both in the presence and absence of AQDS (9,10-anthraquinone-2,6-disulfonic acid). The electron balances showed a complete anaerobic oxidation of the aromatic compounds to CO2. This is the first report on the anaerobic degradation of o-xylene and ethylbenzene in sediment-free iron-reducing enrichment cultures.     

Anaerobic Degradation of Benzene, Toluene, Ethylbenzene, and o-Xylene in Sediment-Free Iron-Reducing Enrichment Cultures

Monoaromatic hydrocarbons such as benzene, toluene, ethylbenzene, and xylene (BTEX) are widespread contaminants in groundwater. We examined the anaerobic degradation of BTEX compounds with amorphous ferric oxide as electron acceptor. Successful enrichment cultures were obtained for all BTEX substrates both in the presence and absence of AQDS (9,10-anthraquinone-2,6-disulfonic acid). The electron balances showed a complete anaerobic oxidation of the aromatic compounds to CO₂. This is the first report on the anaerobic degradation of o-xylene and ethylbenzene in sediment-free iron-reducing enrichment cultures.     

Effect of arbuscular mycorrhizal colonization and two levels of compost supply on nutrient uptake and flowering of pelargonium plants

Two challenges frequently encountered in the production of ornamental plants in organic horticulture are: (1) the rate of mineralization of phosphorus (P) and nitrogen (N) from organic fertilizers can be too slow to meet the high nutrient demand of young plants, and (2) the exclusive use of peat as a substrate for pot-based plant culture is discouraged in organic production systems. In this situation, the use of beneficial soil microorganisms in combination with high quality compost substrates can contribute to adequate plant growth and flower development. In this study, we examined possible alternatives to highly soluble fertilizers and pure peat substrates using pelargonium (Pelargonium peltatum L’Her.) as a test plant. Plants were grown on a peat-based substrate with two rates of compost addition and with and without arbuscular mycorrhizal (AM) fungi. Inoculation with three different commercial AM inocula resulted in colonization rates of up to 36% of the total root length, whereas non-inoculated plants remained free of root colonization. Increasing the rate of compost addition increased shoot dry weight and shoot nutrient concentrations, but the supply of compost did not always completely meet plant nutrient demand. Mycorrhizal colonization increased the number of buds and flowers, as well as shoot P and potassium (K) concentrations, but did not significantly affect shoot dry matter or shoot N concentration. We conclude that addition of compost in combination with mycorrhizal inoculation can improve nutrient status and flower development of plants grown on peat-based substrates.     

Bacterial oxygenases: in vivo enzyme biosensors for organic pollutants

An in vivo enzyme-based biosensor platform was developed that uses specific oxygenase enzymes to detect aromatic compounds in water. Bacteria capable of degrading highly reduced hydrocarbons initiate substrate oxidation using well-characterised oxygenase enzymes, which due to their specificity, stability and high activity can be applied in vivo as biosensor components. Oxygenase enzyme activity was determined in vivo using BD Oxygen Biosensor plates to measure oxygenase-mediated oxygen depletion in the presence of specific aromatic analytes. The BTEX family of compounds (benzene, toluene, ethylbenzene and the three isomers of xylene) were used as model compounds. Detection limits and sensitivity achieved using this approach (microM detection range) was similar to levels achieved with oxygen electrode-based and some recombinant DNA-based approaches. No significant signals were detected with hydrocarbons that were not substrates of the initial oxygenases.     

Growth,mineral nutrition and mycorrhizal colonization of red clover and cucumber plants grown in a soil amended with composted urban wastes

The utility of an urban solid waste, either freshly composted or vermicomposted, for improvement of plant growth in a soil B horizon was investigated. Growth, mineral nutrition and arbuscular mycorrhizal (AM) colonization of cucumber and red clover plants were studied in an experiment carried out under controlled growing conditions, using different mixtures of soil and composts as plant substrates. Soil inoculation with the AM fungus Acaulospora sp. did not benefit growth of plants when soil was used as the only substrate, possibly due to its poor fertility. Results showed that neither mycorrhizal plant species grew when soil was mixed with composted urban waste or when compost was used as the only substrate. However, amendment of soil with 10 or 50% vermicompost significantly increased dry matter yields of red clover and cucumber plants, compared to treatments where soil was the only substrate. Addition of vermicompost also increased Olsen-P and other mineral elements in soil and shoot P, Ca, Mg, Cu, Mn and Zn concentrations, but caused a significant reduction on root length colonized by AM fungi in red clover plants. It is concluded that application of high amounts of vermicompost from composted urban wastes to soils might cause a significant reduction of activity of AM fungi, which must be taken into account when using these organic amendments in agricultural systems.     

Distribution of Petroleum and Aromatic Hydrocarbons at a Former Crude Oil and Natural Gas Production Facility

Site characterization and remediation activities were performed at a former crude oil and natural gas production facility prior to redevelopment of the site. Field activities included delineation, excavation and segregation of approximately 1,250,000 m³ of soil impacted by total petroleum hydrocarbons (TPH) and the aromatic volatile organic compounds (VOCs) benzene, toluene, ethylbenzene, and xylenes (hereafter, collectively referred to as BTEX). Petroleum hydrocarbon chain length information was used to determine whether remediation was required in impacted areas, because the site-specific cleanup values for TPH compounds, established by the California State Regional Water Quality Control Board (RWQCB), were based on hydrocarbon chain length. Site-specific cleanup levels were also established by the RWQCB for BTEX. Subsurface investigation activities performed at the site indicated that the mean percentage of condensate and TPH compounds in the gasoline range was significantly greater at depths ranging from 4.6 to 18 m than in shallower samples. There was no significant difference in the mean concentration of BTEX compounds and mean percentage of diesel range and heavier hydrocarbons with depth. The occurrence of BTEX, diesel range, and heavier hydrocarbons at depth may result from preferential pathways for downward migration of contaminants, including blown out wells, abandoned wellbores, and the presence of faults. Vapor phase diffusion may also be a major transport mechanism controlling movement of BTEX compounds beneath the site.     

Attenuation of petroleum hydrocarbons by weathering: a case study

Possible alterations in the distribution and composition of total petroleum hydrocarbon (TPH), polycyclic aromatic hydrocarbons (PAHs), and benzene, toluene, ethyl benzene, and xylene isomers (BTEX) in the released oil at Idu-Ekpeye in Niger Delta (Nigeria) were studied within two seasonal variations of two months and six months, with a view to assessing the level of attenuation of these hydrocarbons in impacted soils. Although there were significant contaminations in the kerosene range (n-C10-n-C14) two months after, especially of the n-C12 and n-C13 fractions, the complete disappearance of the n-C8 to n-C23 hydrocarbons, including the acyclic isoprenoids (pristane and phytane), and the reduced amounts of PAHs, and BTEX, six months after, provided substantial evidence of attenuation as indicated in the reduction in total hydrocarbon content (THC) from 61.17 to 42.86%. Soil physicochemical properties such as pH, moisture content, heavy metal, TOC, and TOM, all provided corroborative evidence of hydrocarbon attenuation. The pristane/phytane ratio of the spill samples suggests that the spilled oil was genetically oxic.     

Corticosteroid and thyroid responses of larval and juvenile turbot exposed to the water-soluble fraction of crude oil

Turbot larvae (24–590 degree C days; 2–32 days post-hatch) and juveniles (1345 degree C days; 98 days post-hatch), were exposed for 6 h to 25, 33 and 50% water-soluble fraction (WSF) of crude oil in either static or flow-through test systems. Larvae showed generalized primary endocrine responses, identified by elevated whole body cortisol content from as early as 2 days post-hatch. In older larvae and juveniles, the response was related to the WSF concentration. This dose-response relationship was not apparent in younger and yolk-sac larvae. Whole body thyroxine content of turbot larvae exposed to the WSF of crude oil was increased, but triiodothyronine content remained stable. Aromatic hydrocarbon concentrations [benzene, toluene, ethylbenzene and xylene (BTEX) and naphthalenes] remained constant during flow-through tests, but 65% of the initial level of BTEX hydrocarbons and 40% of the naphthalenes were lost during static exposures. Larval mortalities increased with exposure to an increasing concentration of crude oil WSF. Larval activity was significantly reduced even at the lowest WSF concentration.     

Induced plant uptake and transport of mercury in the presence of sulphur-containing ligands and humic acid

The induced accumulation of mercury (Hg) by plants was investigated for the species Phaseolus vulgaris (Bush bean), Brassica juncea (Indian mustard), and Vicia villosa (Hairy vetch). All plants were grown in modified Hg-contaminated mine tailings and were treated with sulphur-containing ligands to induce Hg accumulation. The effects of varied substrate Hg concentration and humic acid (HA) level on the induced plant-Hg accumulation for B. juncea were examined. Thiosulphate salts (ammonium and sodium) mobilised Hg in the substrates and caused an increase in the Hg concentration of roots and shoots of all tested plant species. Root Hg accumulation was positively correlated to extractable Hg for (NH4)2S2O3-treated B. juncea plants grown in HA-amended substrates. However, shoot Hg translocation for this species was inhibited at 1.25 g HA kg(-1) of substrate. Mercury-thiosulphate complexes could be translocated and accumulated in the upper parts of the plants up to 25 times the Hg concentration in the substrate. We conclude that shoot Hg accumulation in the presence of thiosulphate salts is dependent upon plant species characteristics (e.g. root surface area) and humic acid content.     

Treatment of Groundwater Contaminated with PAHs, Gasoline Hydrocarbons, and Methyl tert-butyl Ether in a Laboratory Biomass-Retaining Bioreactor

In this study, we investigated the treatability of co-mingled groundwater contaminated with polycyclic aromatic hydrocarbons (PAHs), gasoline hydrocarbons, and methyl tert-butyl ether (MtBE) using an ex-situ aerobic biotreatment system. The PAHs of interest were naphthalene, methyl-naphthalene, acenaphthene, acenaphthylene, and carbazole. The gasoline hydrocarbons included benzene, toluene, ethyl benzene, and p-xylene (BTEX). Two porous pot reactors were operated for a period of 10 months under the same influent contaminant concentrations. The contaminated groundwater was introduced into the reactors at a flow rate of 4 and 9 l/day, resulting in a hydraulic retention time (HRT) of 32 and 15 h, respectively. In both reactors, high removal efficiencies were achieved for the PAHs (>99%), BTEX and MtBE (>99.7%). All the PAHs of interest and the four BTEX compounds were detected at concentrations less than 1 μg/l throughout the study duration. Effluent MtBE from both reactors was observed at higher levels; nevertheless, its concentration was lower than the 5 μg/l Drinking Water Advisory for MtBE implemented in California.     

Effects of AM colonization on “wild tobacco” plants grown in zinc-contaminated soil

This greenhouse study aimed to determine the effect of colonization by the arbuscular mycorrhizal (AM) fungus (Glomus intraradices Schenck & Smith) on the “wild” tobacco (Nicotiana rustica L. var. Azteca), under soil–zinc (Zn) conditions. Plants of N. rustica were grown in AM or non-AM inoculated substrate and subjected to four soil–[Zn] concentrations (0, 50, 100, and 250 mg Zn kg⁻¹ dry soil). The AM root colonization increased markedly from 14 to 81% with the increasing soil–[Zn] and the mycorrhizal structures were significantly more abundant at the highest soil–[Zn], suggesting that Zn may be involved directly or indirectly in AM root colonization. In addition, total Zn content or Zn concentrations in shoots and roots were shown to increase as soil–[Zn] increased in both AM and non-AM plants. As for the growth parameters studied, there were no significant differences between treatments despite the increase in Zn content or concentration. The AM roots subjected to the highest soil–[Zn] had a significant reduction by about 50% of total Zn content and Zn concentration compared to non-AM roots. Still, the relative extracted Zn percentage decreased dramatically as soil–[Zn] increased. Soil pH was significantly lower in non-AM than AM treatments at the highest soil–[Zn]. In summary, AM plants (particularly roots) showed lower Zn content and concentration than non-AM plants. In this regard, the AM fungi have a protective role for the host plant, thus playing an important role in soil-contaminant immobilization processes; and, therefore, are of value in phytoremediation, especially when heavy metals approach toxic levels in the soil.     

丛枝菌根真菌与高羊茅协同修复镉污染土壤

本研究采用温室盆栽试验,利用丛枝菌根（AM）真菌摩西管柄囊霉Funneliformis mosseae进行接种试验,研究了在Cd胁迫下（0、5、15和30mg/kg）接种AM真菌对高羊茅Festuca elata ‘Crossfire II’的生物量、防御酶活性、磷和镉（Cd）含量的影响。结果表明,随着Cd浓度的增加,高羊茅的菌根侵染率和菌根相对依赖性有所增加。接种AM真菌改善了磷从植株根系向地上部的转运,有助于植株在地上部积累更多的磷。此外,AM真菌和Cd胁迫对高羊茅植株抗氧化酶活性都有显著影响,在镉胁迫下,与未接种植株相比,接种AM真菌显著提高了植株的过氧化氢酶活性,而显著降低了植株的丙二醛含量。与未接种植株相比,接种摩西管柄囊霉显著提高了寄主植物对Cd的富集能力,有利于重金属在根部的积累,同时降低了地上部的Cd含量。本研究表明,高羊茅-丛枝菌根共生体在Cd污染土壤的修复中具有潜在应用价值。     

Evaluation of the Soil Contamination of Tangier (Morocco) by the Determination of BTEX,PCBs, and PAHs

Benzene, toluene, ethylbenzene, and xylenes (BTEX), twelve polycyclic aromatic hydrocarbons (PAHs) and seven polychlorinated biphenyls (PCBs), were selected as pollutants to evaluate the contamination of soils in the urban and industrial areas of Tangier (Morocco). PAHs and PCBs were determined by gas chromatography-mass spectrometry (GC-MS) after a microwave-assisted extraction (MAE) and gel permeation chromatography (GPC) clean-up. BTEX were directly determined by head-space GC-MS. Results obtained in this study show the presence of high levels of BTEX and PAHs in the soil near the urban waste deposit. However, the analysis of pollutants in the other sampling sites provided comprehensive evidence that soils of Tangier city are not contaminated.     

Spin state transitions of liver microsomal cytochrome P-450.

Spin state transitions of membrane-bound cytochrome P-450 were investigated by difference spectrophotometry using the 'D'-charge transfer absorbance band at 645 nm as a measure of the amount of hemin iron present in the 5-coordinated state. The magnitude of the 'D'-absorbance band in the absence of exogenous substrates, e.g., the concentration of native high spin cytochrome P-450, was evaluated from the difference in absorbance at 645 nm between ferric cytochrome P-450 and the carbon monoxide derivative of the pigment in its ferrous state. The contribution of the native high spin species to the total cytochrome P-450 content of microsomes was calculated to be between 40% and 65% after induction with phenobarbital and polycyclic hydrocarbons, respectively. Up to 80% of the cytochrome P-450 was found to be present in the high spin state after the addition of exogenous substrates. Further, the steady state concentrations of high spin cytochrome P-450, observed in the presence of reduced pyridine nucleotides, suggest that the rate limiting step for microsomal mixed function oxidation reactions is variable and dependent on the substrate under investigation.     

一株苯系物降解真菌筛选及降解特性

采用逐量分批驯化的方法以污水处理厂污泥作为菌源,苯、甲苯、二甲苯为唯一碳源,驯化、分离、筛选能够有效降解苯系物的真菌,命名为B1。采用单因素以及正交实验方法并对真菌降解环境影响因素及降解效率进行了测定和研究。结果表明:真菌B1对苯系物降解的最佳条件为C:N=5:1,pH5,温度30℃,菌种接种量为5.5ml(50ml培养基)。采用GC对初始液相浓度0~90mg/L范围内的苯系物降解效果进行测定,未发现苯系物对真菌降解活性产生抑制作用。真菌对苯系物的降解效率为:甲苯>苯>二甲苯,最高降解效率分别达到87.39%,85.21%,81.47%。混合物降解效果略高于单一底物的降解效果。     

Phytoremediation of petroleum hydrocarbons by using a freshwater fern species Azolla filiculoides Lam

In this study, the phytoremediation capacity of Azolla filiculoides Lam. for the water resources contaminated with petroleum hydrocarbons was investigated. The plants were grown in nitrogen-free Hoagland nutrient solution containing 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% crude oil under greenhouse conditions for 15 days. Although the growth rate of the plants were not negatively influenced by the presence of crude oil in the media for the concentration of 0.005% and 0.01% v/v, a gradual impeding effect of crude oil in the growth media has been observed at concentrations 0.05–0.1%. More than 0.1% crude oil in the growth medium ostensibly retarded the growth. For example, 0.2% oil in the media reduced growth approximately 50% relative to the control, and the presence of crude oil at concentrations 0.3% or more were lethal. The data about the percentage of plant growth, fresh weight increase and root growth clearly indicated that the tolerance level of A. filiculoides plants to crude oil ranges between 0.1% and 0.2%. In comparison to control samples, the biodegradation rate of total aliphatic and aromatic (phenathrene) hydrocarbons at 0.05–0.2% oil concentrations, was 94–73% and 81–77%, respectively. On the other hand, in case of further increases in oil concentration in media, i.e.; 0.3–0.5%, the biodegradation rate was still higher in the experimental samples, respectively 71–63% and 75–71%. The high biodegradation rates of petroleum hydrocarbons in the experimental samples suggested that A. filiculoides plants could be a promising candidate to be used for the phytoremediation of low crude oil contaminated precious freshwater resources.     

Effect of Benzene, Toluene, Ethylbenzene, and p-Xylene (BTEX) Mixture on Biodegradation of Methyl tert-Butyl Ether (MTBE) and tert-Butyl Alcohol (TBA) by Pure Culture UC1

The effect of a BTEX mixture on the biodegradation of methyl tert-butyl ether (MTBE) and its degradation intermediate, tert-butyl alcohol (TBA) was investigated in the pure bacterial culture UC1, which has been identified to be a strain of the known MTBE-degrader PM1 based on greater than 99% 16S rDNA similarity. Several degradation studies were carried out on UC1 at three initial concentration levels of MTBE or TBA: 6-7; 15-17; and 40-45 mg/l, both with and without BTEX present cumulatively at about half of the MTBE or TBA molar mass in the system. The BTEX mixture was observed not to affect either the rate or the degradation lag period of MTBE or TBA degradation, except that the TBA degradation rate actually increased when BTEX was present initially in the highest concentration studies. When serving as the sole substrate, the MTBE degradation rate ranged from 48 +/- 1.2 to 200 +/- 7.0 mg(MTBE)/g(dw) h, and the TBA degradation rate from 140 +/- 18 to 530 +/- 70 mg(TBA)/g(dw) h. When present with BTEX, MTBE and TBA rates ranged from 46 +/- 2.2 to 210 +/- 14 and 170 +/- 28 to 780 +/- 43 mg(TBA)/g(dw) h, respectively. In studies where varying concentrations of TBA were present with 5 mg/l MTBE, both compounds were degraded simultaneously with no obvious preference for either substrate. In the highest concentration study of TBA with 5 mg/l MTBE, BTEX was also observed to increase the ultimate rate of TBA degradation. In addition to exploring the affect of BTEX, this study also provides general insight into the metabolism of MTBE and TBA by pure culture UC1.     

Metabolic diversity of aromatic hydrocarbon-degrading bacteria from a petroleum-contaminated aquifer

We characterized bacteria from contaminated aquifers for their ability to utilize aromatic hydrocarbons under hypoxic (oxygen-limiting) conditions (initial dissolved oxygen concentration about 2 mg/l) with nitrate as an alternate electron acceptor. This is relevant to current intense efforts to establish favorable conditions forin situ bioremediation. Using samples of granular activated carbon slurries from an operating groundwater treatment system, we isolated bacteria that are able to use benzene, toluene, ethylbenzene, orp-xylene as their sole source of carbon under aerobic or hypoxic-denitrifying conditions. Direct isolation on solid medium incubated aerobically or hypoxically with the substrate supplied as vapor yielded 10³ to 10⁵ bacteria ml^–1 of slurry supernatant, with numbers varying little with respect to isolation substrate or conditions. More than sixty bacterial isolates that varied in colony morphology were purified and characterized according to substrate utilization profiles and growth condition (i.e., aerobic vs. hypoxic) specificity. Strains with distinct characteristics were obtained using benzene compared with those isolated on toluene or ethylbenzene. In general, isolates obtained from direct selection on benzene minimal medium grew well under aerobic conditions but poorly under hypoxic conditions, whereas many ethylbenzene isolates grew well under both incubation conditions. We conclude that the conditions of isolation, rather than the substrate used, will influence the apparent characteristic substrate utilization range of the isolates obtained. Also, using an enrichment culture technique, we isolated a strain ofPseudomonas fluorescens, designated CFS215, which exhibited nitrate dependent degradation of aromatic hydrocarbons under hypoxic conditions.Abbreviations BTEX benzene, toluene, ethylbenzene, andp-xylene - HPLC high performance liquid chromatography - GAC granular activated carbon     

Persistence of Toxic Activity and Recycling of Bacillus thuringiensis var. israelensis in Cold Water: Field Experiments Using Diffusion Chambers in a Pond

The persistence of the larvicidal activity of Bacillus thuringiensis var. israelensis (Bti) was tested over a five-month period in a low-temperature aquatic environment. Diffusion chambers filled with a suspension of Bti (100 mg l - 1) and different experimental substrates (pond water, periphyton, sediments and vegetation), but without mosquito larvae, were placed near the bottom of a large pond and removed at various intervals to measure residual toxic activity to mosquito larvae, spore concentration and proteolytic activity. Within the pond water substrate, 50% of the initial toxicity was still present after one month in cold water, while with the periphyton substrate, 30% remained in the liquid fraction after the same period of exposure. Within the vegetation substrate (blue-joint grass, Calamagrostis canadensis), an average of 30% of the initial toxicity was still present in the liquid fraction between day 84 to day 154. Solid fractions of vegetation became toxic very early and remained toxic for five months. At the end (day 154), there was still 54% of the original toxic activity put in the chambers associated with the vegetation samples. In the absence of mosquito larvae, spore recycling was observed in the chambers especially with sediments and vegetation. But spore recycling did not appear to play a major role in the observed persistence, but rather rapid absorption onto vegetation substrates was responsible for the persistence of Bti in a cold climate.     

Phosphodiesterase activities in transgenic tobacco plants associated with the movement protein of tobacco mosaic virus

Summary Hydrolytic activities of leaf extracts from normal and transgenic plants, with (+ MP) and without (-MP) the movement protein of tobacco mosaic virus, were examined. In the + MP transgenic plants, as compared with non-transgenic and — MP plants, higher hydrolytic activities were found on the following substrates: bis-(nitrophenyl)-phosphate (BPNPP, phosphodiesterase), p-nitrophenyl-(phenyl)-phosphate (PNPPP, nucleotidephosphodiesterase) and thymidine-3-monophosphate p-nitrophenyl ester (T₃MPP; 3nucleotide phosphodiesterase.) The + MP plant lines, as compared with other transgenic plants, exhibited higher nucleotide-phosphodiesterase activity in the soluble as well as in the membrane fraction. Substrate concentration kinetic studies revealed the presence of a nucleotide-phospho-diesterase with a high substrate affinity in the +MP extracts in addition to the enzyme with a relatively low substrate affinity present also in the — MP transgenic plants. This high affinity enzyme could be removed from the soluble fraction by precipitation with anti-MP serum, indicating its possible association with the movement protein.