Change of sludge consortium in response to sequential adaptation to benzene, toluene, and o-xylene

Activated sludge was sequentially adapted to benzene, toluene, and o-xylene (BTX) to study the effects on the change of microbial community. Sludge adapted to BTX separately degraded each by various rates in the following order; toluene>o-xylene>benzene. Degradation rates were increased after exposure to repeated spikes of substrates. Eleven different kinds of sludge were prepared by the combination of BTX sequential adaptations. Clustering analyses (Jaccard, Dice, Pearson, and cosine product coefficient and dimensional analysis of MDS and PCA for DGGE patterns) revealed that acclimated sludge had different features from nonacclimated sludge and could be grouped together according to their prior treatment. Benzene- and xylene-adapted sludge communities showed similar profiles. The sludge profile was affected from the point of the final adaptation substrate regardless of the adaptation sequence followed. In the sludge adapted to 50 ppm toluene, Nitrosomonas sp. and bacterium were dominant, but these bands were not dominant in benzene and benzene after toluene adaptations. Instead, Flexibacter sp. was dominant in these cultures. Dechloromonas sp. was dominant in the culture adapted to 50 ppm benzene. Thauera sp. was the main band in the sludge adapted to 50 ppm xylene, but became vaguer as the xylene concentration was increased. Rather, Flexibacter sp. dominated in the sludge adapted to 100 ppm xylene, although not in the culture adapted to 250 ppm xylene. Two bacterial species dominated in the sludge adapted to 250 ppm xylene, and they also existed in the sludge adapted to 250 ppm xylene after toluene and benzene.     

Removal of BTEX compounds by industrial sludge microbes in batch systems: statistical analysis of main and interaction effects

Biodegradation is an effective technique to remediate polluted soil and groundwater. In the present experimental study, a mixed microbial culture obtained from the wastewater treatment sludge of a chemical industry was used to degrade liquid phase benzene, toluene, ethyl benzene, and xylene (BTEX), at individual initial concentrations varying between 15 and 75 mg/l. Experiments were conducted according to 2 ^k−1 fractional factorial design at the low (15 mg/l) and high (75 mg/l) levels of BTEX concentrations, to identify the main and interaction effects of parameters and their influence on biodegradation of individual BTEX compounds in mixtures. The individual removals varied between 16% and 75% when the concentrations of B, T, E, and X were sufficiently low in the mixture. However, both synergistic (removal of ethyl benzene) and antagonistic (removal of benzene) behavior were noticed when the concentrations of toluene and xylene was increased to higher levels. The individual removals were greater than 67% at their center point levels. The total BTEX removal values were later statistically analyzed and based on the Fischer’s variance ratio (F) and Probability values (P) it was observed that the main effects for total BTEX removal were significant than the squared and interaction effects.     

The mutagenic effect of benzene, toluene and xylene studied by the SCE technique.

In experiments in vitro, neither benzene, toluene nor xylene changed the number of sister-chromatid exchanges (SCEs) or the number of chromosomal aberrations in human lymphocytes. Toluene and xylene caused a significant cell growth inhibition which was not observed with benzene in the same concentrations.     

The mutagenic effect of benzene,toluene and xylene studied by the SCE technique

In experiments in vitro, neither benzene, toluene nor xylene changed the number of sister-chromatid exchanges (SCEs) or the number of chromosomal aberrations in human lymphocytes. Toluene and xylene caused a significant cell growth inhibition which was not observed with benzene in the same concentrations.     

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。     

Influence of aromatic hydrocarbons on growth,plant growth promoting activities and survival in soil of Azotobacter chroococcum strain JL104

This study was undertaken to determine the effect of aromatic hydrocarbons on growth and plant growth promoting activities of Azotobacter chroococcum strain JL104. The organism was grown on Jensen’s media without sucrose, supplemented with different concentrations of aromatic hydrocarbons. Azotobacter chroococcum strain JL104 was able to grow in the presence of benzene, toluene, aniline and benzoic acid and was able to utilize these as sole carbon source as well. The culture showed the highest growth in presence of 0.5% concentrations of aniline and benzoic acid and 0.01% concentrations of benzene and toluene. Maximum indole acetic acid (IAA) production and acetylene reduction activity (ARA) were recorded with benzene and benzoic acid, respectively. Among other substituted benzene derivatives such as xylene, p-hydroxybenzoic acid, di-nitrophenol and di-chlorophenol, xylene was observed to be the least toxic and di-nitrophenol the most toxic hydrocarbon. The highest soil survival was found in soil amended with 1% sucrose however, the population of A. chroococccum strain JL104 declined continuously in unamended soil. Amongst various hydrocarbons, 0.1% toluene amended soil supported the maximum survival, indicating it to be least toxic aromatic hydrocarbon carbon in soil.     

Immobilization materials mixed with activated sludge as column biofilters for the treatment of gaseous stream containing benzene and toluene

Activated sludge has been utilized for the treatment of volatile organic compounds (VOCs) which are emitted from industrial processes. Nevertheless, activated sludge systems often suffer from the problem caused by concentration gradients as well as pressure drops. Channeling is also a major problem in the treatment process. As the bed height of the packed activated sludge system increases, the pressure drop increases accordingly. To solve these problems, we proposed immobilized activated sludge column reactors for treating VOCs in air. The immobilization material used to mix with activated sludge was properly selected in this work. Elemental compositions of these materials were analyzed. In this study, we also proposed a VOC feed system so that more stable inlet concentrations could be achieved. Hence, the equipment and operating costs were reduced and the problem of VOCs leaking from peristaltic pumps was avoided. The moisture content of the system was well maintained and better VOC removal efficiency was achieved. With an operation condition of progressive VOC inlet concentrations, better removal efficiency of benzene and toluene was then obtained. In conclusion, by the utilization of immobilization materials selected from wastes as well as immobilized activated sludge column reactors, significant removal efficiency for both benzene and toluene was demonstrated.     

Application of aerobic microorganisms in bioremediation in situ of soil contaminated by petroleum products

Aerobic microorganisms able to biodegrade benzene, toluene, ethylbenzene, xylene (BTEX) have been isolated from an area contaminated by petroleum products. The activity of the isolated communities was tested under both laboratory and field conditions. Benzene, toluene, ethylbenzene and xylene were added to the cultures as the sole carbon source, at a concentration of 500 mg/L. In batch cultures under laboratory conditions, an 84% reduction of benzene, 86% of toluene and 82% of xylene were achieved. In cultures with ethylbenzene as the sole carbon source, the reduction was around 80%. Slightly lower values were observed under field conditions: 95% reduction of benzene and toluene, 81% of ethylbenzene and 80% of xylene. A high biodegradation activity of benzene (914 μM/L/24 h), toluene (771 μM/L/24 h), xylene (673 μM/L/24 h) and ethylbenzene (644 μM/L/24 h) was observed in the isolated communities.     

Isolation and characterization of BTEX tolerant and degrading Pseudomonas putida BCNU 106

Bacterial strains growing in river sediments were screened to identify an organic solvent-tolerant strain of Pseudomonas. Using this screen, Pseudomonas sp. BCNU 106 was isolated on the basis of its ability to grow on benzene, toluene, ethylbenzene, and three xylene isomers, o-, m- and p-xylene, as its sole carbon source. BCNU 106 was identified as a gram-negative, rod-shaped aerobic and mesophilic bacterium, which grew in liquid media containing high concentrations of organic solvents. 16S rDNA analysis classified BCNU 106 as a new member of the genus Pseudomonas. BCNU 106 was distinguishable from other Pseudomonas strains that are tolerant to organic solvents in that the isolate had the ability to utilize all three xylene isomers as well as benzene, toluene and ethylbenzene. The unique properties of the isolate such as solvent-tolerance and the ability to degrade xylene isomers may have important implications for the efficient treatment of solvent wastes.     

Biodegradation of benzene,toluene, ethylbenzene,and o-xylene by a coculture of Pseudomonas putida and Pseudomonas fluorescens immobilized in a fibrous-bed bioreactor

A fibrous-bed bioreactor containing the coculture of Pseudomonas putida and P. fluorescens immobilized in a fibrous matrix was developed to degrade benzene (B), toluene (T), ethylbenzene (E), and o-xylene (X) in synthetic waste streams. The kinetics of BTEX biodegradation by immobilized cells adapted in the fibrous-bed bioreactor and free cells grown in serum bottles were studied. In general, the BTEX biodegradation rate increased with increasing substrate concentration and then decreased after reaching a maximum, showing substrate-inhibition kinetics. However, for immobilized cells, the degradation rate was much higher than that of free cells. Compared to free cells, immobilized cells in the bioreactor tolerated higher concentrations (>1000 mg l⁻¹) of benzene and toluene, and gave at least 16-fold higher degradation rates for benzene, ethylbenzene, and o-xylene, and a 9-fold higher degradation rate for toluene. Complete and simultaneous degradation of BTEX mixture was achieved in the bioreactor under hypoxic conditions. Cells in the bioreactor were relatively insensitive to benzene toxicity; this insensitivity was attributed to adaptation of the cells in the bioreactor. Compared to the original seeding culture, the adapted cells from the fibrous-bed bioreactor had higher specific growth rate, benzene degradation rate, and cell yield when the benzene concentration was higher than 100 mg l⁻¹. Cells in the fibrous bed had a long, slim morphology, which is different from the normal short-rod shape found for suspended cells in solution.     

Acute Toxicity for some Evaporating Aromatic Hydrocarbons for Freshwater Snails and Crustaceans

Some typical and more frequent freshwater invertebrates of running waters were studied to examine the influence of styrene, xylene and benzene on their mortality. Snails Amphimelania holandri FÉR. and Lymnaea stagnalis L. and crustacenas Asellus aquaticus L. and Gammarus fossarum KOCH. , were used in the semi-static test. Compounds were added in volume concentrations of 0.005 to 0.4% v/v. For all concentrations LC₅₀ was calculated by probit method, which demonstrated that mortality depends much more on increased concentrations (depending on the initial concentration) than on the length of exposure. Styrene was the most toxic, followed by xylene and then benzene. The species G. fossarum showed marked sensitivity, followed by A. aquaticus, and the species A. holandri and L. stagnalis showed less sensitivity.     

Changes in the rat's motor behaviour during 4-hr inhalation exposure to prenarcotic concentrations of benzene and its derivatives

Group motility was recorded continuously in male rats during the inhalation of benzene, toluene, ethylbenzene, o-, m- and p-xylene vapours. The solvents were applied in at least six concentrations, up to those inducing anaesthesia. Minimum narcotic concentrations (ppm) were: 5940 (benzene), 3590 (toluene), 2180 (ethyl-benzene), 2180 (0-xylene), 2100 (m-xylene), and 1940 (p-xylene). The results indicate that prenarcotic concentrations of these structurally related aromatic hydrocarbons and also the xylene isomers elicit qualitatively and quantitatively different acute behavioral effects. Except o-xylene which caused depression only the agents produced bell-shaped concentration-action curves characteristic of the biphasic effect, i.e., activation at lower and depression at higher concentrations. The curves differed in form and magnitude depending on the stimulatory potency and on the range of effective concentrations. Based on arbitrary assessment of central excitation, the five aromatics may be ranked as follows: benzene and toluene (striking activation), p-xylene (marked activation), ethylbenzene (moderate activation), m-xylene (slight activation). At the same time, high degree of motor incoordination, and in the case of benzene and p-xylene, also marked tremor could be seen.     

Genotoxic effects of a sub-acute low-level inhalation exposure to a mixture of carcinogenic chemicals

A study was conducted using a combined testing protocol (CTP), to determine whether short-term biological end-points, singly or in combination, are sufficiently sensitive to identify damage induced by exposure to ambient levels of industrial chemicals. A small-scale inhalation set-up which is both economical and easy to assemble was designed. Mice were exposed to 4 concentrations of a custom-blend mixture of benzene, chloroprene, epichlorohydrin and xylene in a ratio of 2:2:1:2, respectively. The concentrations for benzene, chloroprene and xylene were 0, 0.1, 1.0 and 10 ppm each. Concentrations for epichlorohydrin were half those for the other components. Groups of 22 males and 22 female mice were exposed to each concentration of the mixture for 3 and 6 weeks. Selected biological end-points including urine mutagenesis, bone marrow cell aberrations and micronuclei, spleen lymphocyte aberrations and liver enzyme induction were monitored. The spleen lymphocyte aberrations and liver enzyme induction were the most sensitive end-points. The lymphocytes showed a significant induction of chromosome aberrations from exposure for 3 weeks to all 3 concentrations of the mixtures. After 6 weeks of exposure, significant induction of aberrations was observed after exposure to low and medium concentrations but not to the high concentration. This lack of response at the high concentration after 6 weeks exposure, appeared to correlate with a significant induction of glutathione S-transferase in the liver. Since this enzyme is known to detoxify 3 of the 4 chemicals in our mixture, it may indicate a detoxification mechanism after enzyme induction. The present study indicates that the CTP is sufficiently sensitive to identify toxicological effects after exposure to ambient levels of a gas mixture.     

Specific plant DNA adducts as molecular biomarkers of genotoxic atmospheric environments

The general purpose of this study was to determine whether the formation of DNA addition products ('adducts') in plants could be a valuable biomarker of genotoxic air pollution. Plants from several species were exposed to ambient atmosphere at urban and suburban sites representative of different environmental conditions. The levels of NO2 and of the quantitatively major genotoxic air pollutants benzene, toluene, and xylene were monitored in parallel with plant exposure. DNA adducts were measured in bean (Phaseolus vulgaris), rye-grass (Lolium perenne), and tobacco (Nicotiana tabacum) seedlings by means of the [32P]-postlabeling method. Whereas, no correlation was found between the levels of the major genotoxic air pollutants and the total amounts of DNA adducts, individual analyses revealed site-specific and plant species-specific adduct responses, both at the qualitative and quantitative level. Among these, the amount of a specific rye-grass DNA adduct (rgs1) correlated with benzene/toluene/xylene levels above a threshold. For further characterization, rye-grass seedlings were treated in controlled conditions with benzene, toluene, xylene or their derivatives. On the other hand, in vitro DNA adduct formation assays were developed involving benzene, toluene, xylene, or their derivatives, and plant microsomes or purified peroxidase. Although in some cases, these approaches produced specific adduct responses, they failed to generate the rgs1 DNA adduct, which appeared to be characteristic for on-site test-plant exposure. Our studies have thus identified an interesting candidate for further analysis of environmental biomarkers of genotoxicity.     

Detection of benzene, toluene, ethyl benzene, and xylenes (BTEX) using toluene dioxygenase-peroxidase coupling reactions

We have developed a simple, whole-cell bioassay for the detection of bioavailable benzene, toluene, ethyl benzene, and xylenes (BTEX) and similar compounds. A genetically engineered E. coli strain expressing toluene dioxygenase (TDO) and toluene dihydrodiol dehydrogenase (TodD) was constructed, enabling the conversion of BTEX into their respective catechols, which were quickly converted into colored products by a horseradish peroxidase (HRP)-coupled reaction. The intensity of the color formation was correlated to concentrations of the BTEX compounds. Under the optimized conditions, a detection limit (defined as three times the standard deviation of the response obtained from the blank) of 10, 10, 20, and 50 microM was observed for benzene, toluene, ethyl benzene, and xylene, respectively. The bioassay was selective toward BTEX-related compounds with no interference observed with commonly used pesticides, herbicides, and organic solvent. The bioassay was very stable with little change in response over a 10-week period. The excellent stability suggests that the reported bioassay may be suitable for field monitoring of BTEX to identify and track contaminated water and follow the bioremediation progress.     

Anaerobic Biodegradation of Alkylbenzenes in Laboratory Microcosms Representing Ambient Conditions

A microcosm study was performed to document the anaerobic biodegradation of benzene, toluene, ethylbenzene, m- xylene, and/or o-xylene in petroleum-contaminated aquifer sediment from sites in Michigan (MI) and North Carolina (NC) and relate the results to previous field investigations of intrinsic bioremediation. Laboratory microcosms, designed to simulate ambient conditions, were constructed under anaerobic conditions with sediment and groundwater from source, mid-plume, and end-plume locations at each site. The general patterns of biodegradation and electron acceptor utilization in the microcosms were consistent with field data. At the MI site, methane was produced after a moderate lag period, followed by toluene degradation in all sets of microcosms. At the NC site, biodegradation of the target compounds was not evident in the source area microcosms. In the mid-plume microcosms, toluene and o-xylene biodegraded first, followed by m-xylene and benzene, a pattern consistent with contaminant decay along the plume length. Chemical extraction of microcosm sediment at the beginning and end of me incubation indicated that iron-reducing conditions were dominant and iron reduction occurred on a sediment fraction not extracted by 0.5N HC1. In the end-plume microcosms, degradation of benzene, toluene, and xylene isomers occurred but was variable between replicates. Consistent with field data, dissolved concentrations of the target contaminant(s) persisted at low but detectable levels (0.05 to 0.25 μM) in microcosms from both sites where biodegradation was measured.     

Isolation and characterization of a novel organic solvent-tolerant Anoxybacillus sp. PGDY12, a thermophilic Gram-positive bacterium

Aims: To isolate and characterize new bacteria capable of tolerating high concentrations of organic solvents at high temperature. Methods and Results: A solvent‐tolerant, thermophilic bacterium was isolated from hot spring samples at 55°C. The strain PGDY12 was characterized as a Gram‐positive bacterium. It was able to tolerate 100% solvents, such as toluene, benzene and p‐xylene on plate overlay and high concentrations of these solvents in liquid cultures. A comparison of growth showed that 0·2% (v/v) benzene and 0·15% (v/v) p‐xylene were capable of enhancing the final cell yields. Transmission electron micrographs showed the incrassation of electron‐transparent intracellular material and the distorted cytoplasm in case of the cells grown in toluene. A phylogenetic analysis based on 16S rRNA sequence data indicated that the strain PGDY12 was member of the genus Anoxybacillus. Conclusions: The thermophilic, Gram‐positive Anoxybacillus sp. PGDY12 exhibited a unique and remarkable ability to tolerate solvents at 55°C. Significance and Impact of the Study: The solvent tolerance properties are less known in thermophilic bacteria. The Anoxybacillus sp. PGDY12 is the first strictly thermophilic bacterium able to tolerate a broad range of solvents. This strain is a promising candidate for use as a high temperature biocatalyst in the biotechnological applications.     

Utilization of toluene and xylenes by a nitrate-reducing strain of Pseudomonas maltophilia under low oxygen and anoxic conditions

Abstract A nitrate-reducing strain of Pseudomonas maltophilia isolated from sewage sludge degrades toluene, and at least two isomers of mixed xylenes, either in the presence of 2% oxygen or under anoxic conditions when nitrate is present. When individual isomers of xylene are provided only meta and para -xylene are utilized. When mixed xylenes are provided all three isomers may be utilized. In cultures limited by electron acceptor availability, succinate, when present as the major carbon source, does not prevent hydrocarbon utilization. Toluene and xylenes continued to be utilized either with limiting nitrate alone, or with limiting nitrate and oxygen present simultaneously when a hundred-fold excess of succinate is present in the medium. The data suggest that in groundwater containing low levels of oxygen and nitrate, or nitrate only as the electron acceptor, aromatic hydrocarbons may continue to be utilized even in the presence of an excess of readily-degradable non-hydrocarbon organic substrates. These data have implications for bioremediation studies. The strain of Pseudomonas maltophilia used in this study does not degrade benzene, and the presence of benzene does not affect toluene utilization.     

Temperature effects and substrate interactions during the aerobic biotransformation of BTEX mixtures by toluene-enriched consortia and Rhodococcus rhodochrous

A microbial consortium derived from a gasoline-contaminated aquifer was enriched on toluene (T) in a chemostat at 20 degrees C and was found to degrade benzene (B), ethylbenzene (E), and xylenes (X). Studies conducted to determine the optimal temperature for microbial activity revealed that cell growth and toluene degradation were maximized at 35 degrees C. A consortium enriched at 35 degrees C exhibited increased degradation rates of benzene, toluene, ethylbenzene, and xylenes in single-substrate experiments; in BTEX mixtures, enhanced benzene, toluene, and xylene degradation rates were observed, but ethylbenzene degradation rates decreased. Substrate degradation patterns over a range of BTEX concentrations (0 to 80 mg/L) for individual aromatics were found to differ significantly from patterns for aromatics in mixtures. Individually, toluene was degraded fastest, followed by benzene, ethylbenzene, and the xylenes. In BTEX mixtures, degradation followed the order of ethylbenzene, toluene, and benzene, with the xylenes degraded last. A pure culture isolated from the 35 degrees C-enriched consortium was identified as Rhodococcus rhodochrous. This culture was shown to degrade each of the BTEX compounds, individually and in mixtures, following the same degradation patterns as the mixed cultures. Additionally, R. rhodochrous was shown to utilize benzene, toluene, and ethylbenzene as primary carbon and energy sources. Studies conducted with the 35 degrees C-enriched consortium and R. rhodochrous to evaluate potential substrate interactions caused by the concurrent presence of multiple BTEX compounds revealed a range of substrate interaction patterns including no interaction, stimulation, competitive inhibition, noncompetitive inhibition, and cometabolism. In the case of the consortium, benzene and toluene degradation rates were slightly enhanced by the presence of o-xylene, whereas the presence of toluene, benzene, or ethylbenzene had a negative effect on xylene degradation rates. Ethylbenzene was shown to be the most potent inhibitor of BTEX degradation by both the mixed and pure cultures. Attempted quantification of these inhibition effects in the case of the consortium suggested a mixture of competitive and noncompetitive inhibition kinetics. Benzene, toluene, and the xylenes had a negligible effect on the biodegradation of ethylbenzene by both cultures. Cometabolism of o-, m-, and p-xylene was shown to be a positive substrate interaction.     

Bioremediation Assessment of a BTEX‐Contaminated Soil

The elimination of BTEX (benzene, toluene, ethylbenzene, o‐xylene) compounds from soil was studied. After 18 days at 20 °C, 21% of the initial total BTEX contamination (400 mg/kg soil) was lost due to sorption onto soil. Biodegradation decreased in the order ethylbenzene > toluene > benzene > o‐xylene. NPK fertilisation stimulated biodegradation, particularly that of benzene and toluene, significantly, and oleophilic fertilisation inhibited biodegradation. After 18 days, the residual contamination in the NPK‐fertilised, unfertilised and with oleophilic nutrients amended soil was 96, 166 and 196 mg total BTEX/kg soil, respectively. The presence of BTEX initially inhibited the biological activity of the soil (fluorescein diacetate hydrolysis) considerably. This short‐term, reversible inhibition was significantly higher in the unfertilised soil than in the fertilised soil.