Toluene-degrading Bacteria Research Articles

Biochar enhanced the stability of toluene removal in extracted groundwater amended with nitrate under microaerobic conditions

Groundwater pollution caused by the leakage of petroleum and various fuel oils is becoming a serious environmental problem. In this study, carbon-based materials including biochar and hydrochar were applied to investigate the effects of additives on the toluene removal in the extracted groundwater under microaerobic condition with the addition of nitrate. Biochar and hydrochar could adsorb toluene, and thus enhance the toluene removal in the system. The toluene removal efficiency was 8.2–8.9 mg/(g·h) at the beginning, and then decreased with time in the control and the hydrochar treatment, while it remained the stable values in the biochar treatment, owing to the fact that biochar could reduce the NO3−-N loss by partial denitrification. Moreover, biochar could prompt the growth of toluene-degrading bacteria including Thauera, Rhodococcus, Ideonella and Denitratisoma, which had the capability of denitrification. However, hydrochar could stimulated the growth of denitrifiers without toluene-degrading capacity including Candidatus Competibacter and Ferrovibrio, which might play a key role in the partial denitrification of the system. The findings are helpful for developing remediation techniques of contaminated groundwater.

Self-assembled hierarchical porous carbon nanotube sponge bioanode for energy harvesting and gaseous toluene removal

Microbial fuel cells (MFCs) have emerged as a promising technology with potential for volatile organic compounds (VOCs) treatment and energy recovery. However, the low efficiency of electron transfer between microorganisms and electrodes has limited their degradation ability and power output. To address this issue, we designed a macroscopic hierarchical porous super-aligned carbon nanotube (SACNT) sponge bioanode. The hierarchical structure of the SACNT anode exhibited an appropriate distribution of macro/mesopores. The large pores facilitated biofilm formation and mass transfer, enhancing direct electron transfer between bacteria and SACNTs. The abundant mesopores augmented adsorption and electrochemical reaction sites, thereby enhancing flavin-mediated indirect electron transfer. This hierarchical pore structure induced the formation of biofilms mainly composed of electroactive bacteria and toluene-degrading bacteria, promoting toluene removal and power generation. The MFC demonstrated an exceptional power density of 726.18 mW m−2, surpassing existing toluene-fueled MFCs, while achieving the toluene elimination capacity of 61.29 g m−3 h−1.

Promoting biodegradation of toluene and benzene in groundwater using microbial fuel cells with cathodic modification

A tubular microbial fuel cell (T-MFC) was used to remove benzene and toluene from groundwater with a great contaminant removal and power production efficiency. The main results were that T-MFC can completely degrade 200 mg/L toluene in 10 days under closed-circuit (CC) conditions, with a removal efficiency (RE) that is 38.2% greater than under open circuit (OC) conditions. When toluene and benzene were used as co-contaminants and a carbonized porous ceramic ring (CPCR) was added to the T-MFC cathode, the RE reached 100% within 6 days and the power density reached 15.6 W/m3. The radius of influence for 100% contaminant removal was 6 cm when the CPCR was added to the T-MFC cathode with a composite carbon source. The exoelectrogens Geobacter and Thermincola were the main species on the anodic surface, while the toluene-degrading bacteria Rhodocyclaceae dominated in the anode solution. This T-MFC is an innovative technology for the bioremediation of soil and groundwater that is contaminated by aromatic compounds, and has the potential to be used as a green energy device in the field.

Isolation of toluene-degrading bacteria and investigation of the ability to decompose aromatic hydrocarbon mixture in laboratory wastewater

Toluene is an aromatic hydrocarbon commonly used as a solvent in industry and as one of the main components of gasoline. Due to its solubility in water, the compound may accumulate in surface water or reachgroundwater causing harmful effects on human health. Twenty bacterial strains grown on a minimal medium supplemented with toluene as the sole carbon source were isolated from the sediment samples of a laboratory wastewater treatment system. Among these, strains TL5 and TL27 performed toluene degradation capability. After 24 hours of incubation in a minimal medium supplemented with toluene (0.025% v/v) as the only carbon source, chromatographic data recorded that TL5 and TL27 degraded 96.71 and 100% toluene, respectively. In a minimal medium with a mixture of aromatic hydrocarbons added, after 4 days of incubation, toluene and benzaldehyde were completely degraded while phenol, xylene, acetophenone, and pyridine were degraded 88.27, 40.9, 34.67, and 23.26%, respectively. Based on 16S-rRNA sequence analysis, the potential aromatic hydrocarbon degrader was genetically identified as Enterobacter sp. TL27.

Screening and study of the degradation characteristics of efficient toluene degrading bacteria combinations

ABSTRACT In this paper, three effective toluene-degrading bacteria were obtained through acclimation and screening by using landfill leachate as the initial liquid strain. The three obtained bacteria were denoted as J1, J2 and J3, and identified as Pseudomonas, Bacillus and Staphylococcus, respectively. We then identified the optimal combination of these toluene-degrading bacteria in the laboratory. The combination of J1 + J3 (1:1) exhibited the highest toluene removal efficiency (RE). A vertical bio-trickling filter (BTF) packed with ceramsite was started by inoculation with the effective combination. The performance of the BTF in treating toluene under various operating conditions was investigated. After 17 days of operation, the toluene RE reached about 90% and the maximum elimination capacity reached 42.0 g m−3 h−1. The scanning electron microscope (SEM) showed that after the successful start-up of the BTF, the biofilm on the packing surface primarily consisted of Bacillus and Staphylococcus. During the stable state, the RE of the BTF was maintained above 80%, the shortest empty bed residence time was 34 s and toluene concentrations ranged between 300 and 800 mg m−3. The results indicate that the BTF started using the effective combination of bacteria described here is robust. This paper also provides a preliminary analysis of the mechanism of microbial degradation of pollutants in the BTF.

Toluene degradation via a unique metabolic route in indigenous bacterial species.

Tanneries are the primary source of toluene pollution in the environment and toluene due to its hazardous effects has been categorized as persistent organic pollutant. Present study was initiated to trace out metabolic fingerprints of three toluene-degrading bacteria isolated from tannery effluents of Southern Punjab. Using selective enrichment and serial dilution methods followed by biochemical, molecular and antibiotic resistance analysis, isolated bacteria were subjected to metabolomics analysis. GC-MS/LC-MS analysis of bacterial metabolites helped to identify toluene transformation products and underlying pathways. Three toluene-metabolizing bacteria identified as Bacillus paralicheniformis strain KJ-16 (IUBT4 and IUBT24) and Brevibacillus agri strain NBRC 15538 (IUBT19) were found tolerant to toluene and capable of degrading toluene. Toluene-degrading potential of these isolates was detected to be IUBT4 (10.35 ± 0.084mg/h), IUBT19 (14.07 ± 3.14mg/h) and IUBT24 (11.1 ± 0.282mg/h). Results of GC-MS analysis revealed that biotransformation of toluene is accomplished not only through known metabolic routes such as toluene 3-monooxygenase (T3MO), toluene 2-monooxygenase (T2MO), toluene 4-monooxygenase (T4MO), toluene methyl monooxygenase (TOL), toluene dioxygenase (Tod), meta- and ortho-ring fission pathways. But additionally, confirmed existence of a unique metabolic pathway that involved conversion of toluene into intermediates such as cyclohexene, cyclohexane, cyclohexanone and cyclohexanol. LC-MS analysis indicated the presence of fatty acid amides, stigmine, emmotin A and 2, 2-dinitropropanol in supernatants of bacterial cultures. As the isolated bacteria transformed toluene into relatively less toxic molecules and thus can be preferably exploited for the eco-friendly remediation of toluene.

Draft Genome Sequence of "Candidatus Izimaplasma sp." Strain ZiA1, Obtained from a Toluene-Degrading and Iron-Reducing Enrichment Culture.

Here, we report the draft genome sequence of "Candidatus Izimaplasma sp." strain ZiA1 (1.88 Mb and 29.6% G+C content). Strain ZiA1 was cocultured with iron-reducing and toluene-degrading bacteria in an enrichment culture from tidal flat sediment. Like the genomes of other strains of "Ca. Izimaplasma," the ZiA1 genome contained genes required for anaerobic fermentation.

A novel approach for toluene gas treatment using a downflow hanging sponge reactor.

A novel gas-scrubbing bioreactor based on a downflow hanging sponge (DHS) reactor was developed as a new volatile organic compound (VOC) treatment system. In this study, the effects of varying the space velocity and gas/liquid ratio were investigated to assess the effectiveness of using toluene gas as a model VOC. Under optimal conditions, the toluene removal rate was greater than 80%, and the maximum elimination capacity was observed at approximately 13g-Cm-3h-1. The DHS reactor demonstrated slight pressure loss (20Pa) and a high concentration of suspended solids (up to 30,000mg/L-sponge). Cloning analysis of the 16S rRNA and functional genes of toluene degradation pathways (tmoA, todC, tbmD, xylA, and bssA) revealed that the clones belonging to the toluene-degrading bacterium Pseudomonas putida constituted the predominant species detected at the bottom of the DHS reactor. The toluene-degrading bacteria Pseudoxanthomonas spadix and Pseudomonas sp. were also detected by tmoA- and todC-targeted cloning analyses, respectively. These results demonstrate the potential for the industrial application of this novel DHS reactor for toluene gas treatment.

Potential application of biocover soils to landfills for mitigating toluene emission

Biocover soils have been demonstrated to be a good alternative cover material to mitigate CH4 emission from landfills. To evaluate the potential of biocover soil in mitigating emissions of non-methane volatile organic compounds (NMVOCs) from landfills, simulated cover soil columns with the influx of toluene (chosen as typical of NMVOCs) concentrations of 102–1336mgm−3 in the presence or absence of the major landfill gas components (i.e., CH4 and CO2) were conducted in this study. In the two experimental materials (waste biocover soils (WBS) and landfill cover soils (LCS)), higher toluene reduction was observed in WBS with respect to LCS. After the introduction of landfill gas, an increase of microbial diversity and relative abundance of toluene-degrading bacteria and methanotrophs occurred in WBS. To illustrate the role of toluene-degrading activity in mitigating toluene emissions through landfill covers, an analytical model was developed by incorporating the steady-state vapor transport with the first-order kinetics of aerobic biodegradation limited by O2 availability. This study demonstrated that biocover soils have great potential in applying to landfills for mitigating toluene emission to the atmosphere.

Performance of a suspended biofilter as a new bioreactor for removal of toluene

A suspended biofilter, as a new bioreactor, was constructed for removal of VOCs (volatile organic compounds). The biofilter bed (height of 30cm, diameter 16cm and working volume of 6.0L) was formed by applying suspended carrier with porous surface and the carrier itself has a density slightly lower than water. According to the results, it was found that the suspended biofilter was suitable to treat the toluene-contaminated stream. Since it could startup quickly, tolerate great transient shock loadings, and possess average removal efficiency higher than 90.2% in a toluene loading range of 11.0–58.5gm−3h−1. What is more, it also gained a maximum elimination capacity of 113.6gm−3h−1 at a toluene loading rate of 272.2gm−3h−1. The remarkable performance could be contributed to the rich bacterial communities for toluene degradation. A following 16S rRNA gene sequence analysis reveals that the communities contain high efficient toluene-degrading bacteria, i.e., Acinetobacter sp. Tol 5, Burkholderia, and Comamonadaceae. In addition, after a long-term operation of 141 days, no clogging was observed although there was a high biomass concentration in the biofilter bed. It demonstrated that this novel suspended biofilter overcome clogging and accumulation of biomass, which are the drawback of fixed biofilter.

Study on culture conditions of several strains of toluene-degrading bacteria isolated from common ornamental houseplants

This article studies the impact of some environmental conditions and the nutrition of culturing medium on the growth of bacteria and theirs capacity of toluene removal. The 5 bacterial strains isolated from leaf samples of three different common houseplants in Vietnam are Gram-negative, rod-shaped bacteria. The cells are single or arranged in chains. The cell size is relatively small and ranged from 0.7 to 2.5mm. These bacteria prefer the incubating temperature from 28°C to 32°C and a neutral pH 6.5 to 7.5. They are able to assimilate different nitrogen and carbon sources. In the liquid SH1 medium containing 200ppm toluene five selected strains have shown the ability to degrade toluene at a rate of 12.8 to 75.2% in comparison with the control at 30°C at a speed of 200rpm for over 120 hours. These 5 studied strains are potentially useful in bioremediation strategies to remove airborne toluene.
 5 chủng vi khuẩn có khả năng phân giải toluene được phân lập từ lá một số cây cảnh phổ biến ở Việt Nam là vi khuẩn G (-), dạng trực khuẩn và kích thước tế bào từ 0,7 – 2,5m. Một số điều kiện môi trường nuôi cấy thích hợp cho 5 chủng vi khuẩn nghiên cứ gồm nhiệt độ 28°C-32°C, pH 6,5-7,5, có khả năng đồng hoá nhiều nguồn nitơ và ba nguồn carbon khác nhau. Trong điều kiện môi trường dịch SH1 chứa 200ppm toluene, 5 chủng vi khuẩn này cho thấy khả năng phân giải toluene từ 12,8 – 75,2%. Đây là các chủng vi khuẩn có tiềm năng ứng dụng để loại bỏ toluene từ không khí ô nhiễm.

Microbial community and function of enrichment cultures with methane and toluene

The interaction effect of co-existence of toluene and CH4 on community and activity of methanotrophs and toluene-degrading bacteria was characterized in three consortia enriched with CH4 and toluene (MT), toluene (T), and CH4 (M), respectively, in this study. The CH4 oxidation activity in the enrichment culture of MT was significantly lower than that of M at the end of the experiment (P = 0.001). The toluene degradation rate could be enhanced by continuous addition of CH4 and toluene in the initial days, but it was inhibited in the later days. Phylogenetic analysis of 16S rRNA genes showed that Proteobacteria and Bacteroidetes were dominant in the three enriched consortia, but the community of methanotrophs and toluene-degrading bacteria was significantly affected by the co-existence of CH4 and toluene. Both Methylosinus (91.8 %) and Methylocystis (8.2 %) were detected in the enrichment culture of MT, while only Methylocystis species were detected in M. The toluene-degrading bacteria including Burkholderia, Flavobacteria, Microbacterium, and Azoarcus were all detected in the enrichment culture of T. However, only Azoarcus was found in the enrichment culture of MT. Significantly higher contents of extracellular polymeric substances polysaccharose and protein in the enrichment culture of MT than that of T and M suggested that a higher environmental stress occurred in the enrichment culture of MT.

Bacterial community diversity and functional gene abundance of structured mixed packing and inert packing materials based biotrickling filters

Packing is the most important factor in biofilter design. A structured mixed packing (SMP) material, combined with various organic and inorganic materials (mineral matter is 80.18 ± 0.48%, w: w), was constructed by ureaformaldehyde resin in order to minimize the disadvantages of these materials when used as stand-alone components. The performance of the toluene biotrickling filter (BTF) packed with SMP was compared with the other BTFs packed with a ceramic raschig ring, ceramic pall ring, and lava rock, respectively, for 217 day under various operating conditions. Real-time PCR and DGGE techniques were applied to reveal the gene coding for the toluene-degrading enzymes and the bacterial community structure in the BTFs. The toluene-degradation gene copies exponentially increased, and bacterial diversity significantly decreased with the improving elimination capacities of the BTFs. The overload and shutdown operations resulted in insignificant fluctuations in the toluene-degradation gene copies at equal levels as well as a slight variation in the bacterial community structures in the BTFs. Various putative toluene-degrading bacteria were found using sequencing bands from the DGGE gels; some bacteria, such as Burkholderia spp., were further confirmed by real-time PCR; other bacteria, such as Alcaligenes spp., might not have been reported. The packing properties of SMP material supported more toluene-degradation gene copies in the biofilm, and higher toluene-degrading bacterial diversity of the BTF, than did inert packing. Thus, the BTF with SMP demonstrated excellent performance, suggesting the suitability of SMP for real applications, whereas the capabilities of inert packing materials are more suited to the treatment of steady low VOC loads.

Transformation of microflora during degradation of gaseous toluene in a biofilter detected using PCR-DGGE

A laboratory-scale biofiltration system, the rotatory-switching biofilter (RSB), was operated for 199 days using toluene as a model pollutant. The target gaseous pollutant for the biofiltration experiment was ∼300 ppmv of toluene. Toluene removal efficiency (RE, %) was initially approximately 20% with a 247-ppmv concentration (0.9 g m−3) of toluene during the first 10 days. Although the RE decreased several times whenever nitrogen was consumed, it again reached almost 100% when the nitrogen source was in sufficient supply. Denaturing gradient gel electrophoresis (DGGE) analysis was employed to assess the transformation of microflora during operation of the biofilter. The results based on a 16S rRNA gene profile showed that the microbial community structure changed with operation time. Although the microflora changed during the initial period (before day 40), transformation of the bacterial component was hardly observed after day 51. Statistical analyses of the DGGE profiles indicated that the bacterial community was almost unaffected by the environmental factors, such as adding ozone, high-level nitrogen supply, increase of loading toluene, and the shutdown of the RSB. The DGGE profile using tmoA-like genes, which encode proteins belonging to the hydroxylase component mono-oxygenases involved in the initial attack of aerobic benzene, toluene, ethylbenzene, and xylene degradation, confirmed the existence of toluene-degrading bacteria. There were at least four kinds of toluene-degradable bacteria having tmoA-like genes up to day 36, which decreased to two species after day 40. Sequence analysis after DGGE profiling revealed that Burkholderia cepacia, Sphingobacterium multivorum, and Pseudomonas putida were present in the biofilter. Only Alicycliphilus denitrificans was present throughout the whole operation period. In the initial stage of operating the RSB, many types of bacteria may have tried to adapt to the conditions, and subsequently, only selected bacteria were able to grow and to degrade toluene. Implications: Biofiltration technology, which is more cost-effective and environmentally friendly than any other physicochemical method, has been applied to the treatment of volatile organic compounds (VOCs). It was difficult to maintain a high level of activity because the performance of the biofilter depends on microbial degradation of pollutants. However, there is little knowledge about the transformation of bacteria during long-term biofiltration operations. This study revealed the transformation of microflora in a biofilter using a molecular biological method, PCR-DGGE.

Simultaneous removal of hydrogen sulfide and toluene in a bioreactor: Performance and characteristics of microbial community

We investigated the correlation between performance and the bacteria community composition by H 2S and toluene co-treatment. Operation of the bioreactor was divided into four stages, in which the inlet concentration of toluene and H 2S were gradually increased. In Stage I, toluene was the sole target compound with an average removal efficiency of 86.49%. After adding H 2S in Stage II, removal efficiency of toluene decreased immediately and recovered gradually to 85.96%. When the inlet concentration of toluene and H 2S was increased in Stage III and Stage IV, respectively, the average removal efficiency for toluene increased continuously from 86.31% to 87.24%. The elimination capacities of toluene increased with increasing inlet loading rates of toluene and H 2S. Results of the PCR-DGGE analysis showed a turnover growth and decline of the microbial populations in the bioreactor. In Stage I, the dominant toluene-degrading bacteria mainly contained Pseudomonas sp. strain PS+ and Hydrogenophaga sp. In Stage IV, however, the dominant toluene-degrading bacteria was aciduric bacteria (Clostridiumpopulen). The dominant microbial community in the bioreactor enhanced the elimination capacity of toluene, and adding H 2S changed the environment of microbial growth, thus resulted in an evolution of dominant microorganisms. Analyses of microbial community and their activities provides valuable information to efficiently enhance simultaneous removal of toluene and H 2S in the bioreactor.

Identification of Putative Biomarkers for Toluene-DegradingBurkholderiaand Pseudomonads by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry and Peptide Mass Fingerprinting

The use of peptide mass fingerprinting with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was demonstrated to identify and phenotypically characterize toluene-degrading bacteria via biomarkers of degradation and taxonomical classification. Pseudomonas putida F1, P. mendocina KR1, and Burkholderia sp. JS150 were grown on toluene, extracted, electrophoretically separated, and analyzed by MALDI-TOF MS. Catabolic enzymes were identified and results substantiated using tandem MS.

Trichloroethylene cometabolic degradation by Rhodococcus sp. L4 induced with plant essential oils

Cometabolic degradation of TCE by toluene-degrading bacteria has the potential for being a cost-effective bioremediation technology. However, the application of toluene may pose environmental problems. In this study, several plant essential oils and their components were examined as alternative inducer for TCE cometabolic degradation in a toluene-degrading bacterium, Rhodococcus sp. L4. Using the initial TCE concentration of 80 microM, lemon and lemongrass oil-grown cells were capable of 20 +/- 6% and 27 +/- 8% TCE degradation, which were lower than that of toluene-grown cells (57 +/- 5%). The ability of TCE degradation increased to 36 +/- 6% when the bacterium was induced with cumin oil. The induction of TCE-degrading enzymes was suggested to be due to the presence of citral, cumin aldehyde, cumene, and limonene in these essential oils. In particular, the efficiency of cumin aldehyde and cumene as inducers for TCE cometabolic degradation was similar to toluene. TCE transformation capacities (T (c)) for these induced cells were between 9.4 and 15.1 microg of TCE mg cells(-1), which were similar to the known toluene, phenol, propane or ammonia degraders. Since these plant essential oils are abundant and considered non-toxic to humans, they may be applied to stimulate TCE degradation in the environment.

Fluorogenic surrogate substrates for toluene-degrading bacteria—Are they useful for activity analysis?

Cultivated bacterial toluene degraders use one or several of four described pathways for the aerobic degradation of this priority groundwater contaminant. To be able to identify un-cultivated toluene-degrading bacteria within enriched or natural consortia, we attempted to develop a set of staining techniques that invariably label toluene-degrading bacteria while differentiating between the different degradation pathways. In the literature, we found suggestions for pathway-specific labels of individual cells that rely on the conversion of toluene surrogates into specific colored and fluorescent products. These surrogate substrates were phenylacetylene (PA), cinnamonitrile, 3-hydroxyphenylacetylene (3-HPA), and indole. We were able to confirm that the chromogenic reactions reliably verified the pathway-specific reactions of well-characterized toluene-degrading bacterial species. However, it was most surprising to find out that three (PA, 3-HPA and cinnamonitrile) of the four supplied surrogate substrates did not lead to any product fluorescence above the cultures' autofluorescence, neither inside of cells nor in supernatants. More disturbingly, the original surrogate compound 3-HPA was inherently fluorescent and found to stain cells at intensities that depended on their states in the cell cycle. Indoxyl originating from the surrogate substrate indole was the only fluorescent product that was formed. It was detected intracellularly when the cells were sealed with para-formaldehyde, but its appearance was unrelated to the presence of expressed toluene degradation pathways. These findings were scrutinized by fluorescence spectroscopy, fluorescence microscopy, and flow cytometry. Activity and growth of the test bacteria were determined by analyzing chromosome numbers and membrane integrity. Our results contradict literature reports that propose the surrogate fluorogenic substrates for the identification of toluene degraders and the identification of specific pathways used by them.

Correlation of TCE cometabolism with growth characteristics on aromatic substrates in toluene-degrading bacteria

We have constructed a bacterial library consisting of 97 strains of toluene-degrading bacteria from soil and activated sludge samples for examining their physiological properties in terms of cometabolism of TCE. Large variation of TCE degradation ability was observed in Gram-positive and Gram-negative strains, as well as diverse patterns of availability of aromatics as a growth substrate. No clear correlation was observed between the number of available substrates for growth and TCE degradation ability. However, the growth on some of the aromatics showed positive or negative correlations with TCE degradation ability. Kendall correlation constants (tau) for the growth on cumene, m-xylene, p-xylene, and m-cresol with TCE degradation ability were statistically significant ( P < 0.001): their values were −0.44, −0.31, −0.26, 0.37, respectively. Among 12 of aromatics, only m-cresol showed positive correlation with TCE degradation ability. These findings would be useful for enrichment and isolation of the microbes that have TCE-cometabolism ability, which is a selective disadvantage through the toxicity or competitive inhibition against growth substrates.

A resazurin-based biosensor for organic pollutants

A new rapid biosensor method employing the dye resazurin as an indicator of bacterial respiration has been developed to provide a rapid, facile and specific biosensor for environmental contaminants that does not rely on genetic modification techniques, is suitable for a high-throughput multiwell format, and is ideally suited to resource-constrained environmental monitoring situations. This whole-cell biosensor has been applied to the test analyte toluene using natural toluene-degrading bacteria as the biological component and is competitive with more complex recombinant approaches. The redox-driven biosensor is dependent on the catabolism of a specific compound, concomitantly reducing the redox indicator resazurin to provide the analytical signal in a whole-cell biosensor assay.