Dibenzothiophene oxidation by horseradish peroxidase in organic media: effect of the DBT:H2O2 molar ratio and H2O2 addition mode

Its is well known that in the biodesulfurization (BDS) process the low water solubility of sulfur compounds hinders its transference from the oil phase to the cells being the rate-limiting step in the metabolism of dibenzothiophenes (DBT). Thus sulfur compounds derivatives with high water solubility could be more easily transported increasing the BDS efficiency. The present work performed a stepwise evaluation of the enzymatic oxidation of DBT by horseradish peroxidase (HRP). Reactions were carried out in monophasic organic media containing 25% (v/v) acetonitrile. The following parameters were evaluated: DBT:H2O2 molar ratio (1:1-1:20); H2O2 addition mode (single or stepwise); pH (6.0-8.0) and temperature (37-50 degrees C). Best results were observed in a reaction medium at pH 8.0 presenting HRP 0.06IUml(-1), DBT 0.267mM, DBT:H2O2 molar ratio of 1:20 (stepwise hydrogen peroxide addition) and incubated at 45 degrees C for 60min. Under these conditions 60% of DBT was converted into dibenzothiophene sulfoxide (12%) and dibenzothiophene sulfone (46%). The DBT oxidation rate observed in this work, of 5mmolmin(-1)g(-1) of HRP, was 250-fold higher than the BDS rate, 20mumolmin(-1)g(-1) of catalyst. As such a combined enzyme-microbial desulfurization process could be envisaged. Products were determined by HPLC RP C-18.     

Thermophilic desulfurization of dibenzothiophene and different petroleum oils by Klebsiella sp. 13T

Purpose

Biodesulfurization (BDS) has the potential to desulfurize dibenzothiophene (DBT) and its alkylated derivatives, the compounds that are otherwise refractory to hydrodesulfurization (HDS). Thermophilic microorganisms are more appropriate to be used for BDS applications following HDS. The aim of the present study was to isolate a thermophilic microorganism and to explore its commercial relevance for BDS process.

Methods

The desulfurizing thermophilic strain was isolated and enriched from various soil and water samples using sulfur free medium (SFM) supplemented with DBT. Microbiological and genomic approach was used to characterize the strain. Desulfurization reactions were carried out using DBT and petroleum oils at 45°C followed by different analytical procedures.

Results

We report the isolation of a thermophilic bacterium Klebsiella sp. 13T from contaminated soils collected from petroleum refinery. HPLC analysis revealed that Klebsiella sp. 13T could desulfurize DBT to 2-hydroxybiphenyl (2-HBP) at 45°C through 4S pathway. In addition, adapted cells of Klebsiella sp. 13T were found to remove 22?C53% of sulfur from different petroleum oils with highest sulfur removal from light crude oil.

Conclusion

Klebsiella sp. 13T is a potential candidate for BDS because of its thermophilic nature and capability to desulfurize petroleum oils.     

Methods for the preparation of a biodesulfurization biocatalyst using Rhodococcus sp

Several methods to prepare a biodesulfurization (BDS) biocatalyst were investigated in this study using a strain of Rhodococcus sp. 1awq. This bacterium could selectively remove sulfur from dibenzothiophene (DBT) via the "4S" pathway. DBT, dimethylsulfoxide (DMSO), sodium sulphate and mixed sulfur sources were used to study their influence on cell density, desulfurization activity, desulfurization ability, and the cost of biocatalyst production. In contrast to that observed from bacteria cultured in DBT, only partial desulfurization activity of strain 1awq was induced by DBT after cultivation in a medium containing inorganic sulfur as the sole sulfur source. The biocatalyst, prepared from culture with mixed sulfur sources, was found to possess desulfurization activity. With DMSO as the sole sulfur source, the desulfurization activity was shown to be similar to that of bacteria incubated in medium with DBT as the sole sulfur source. The biocatalyst prepared by this method with the least cost could remove sulfur from hydrodesulfurization (HDS)-treated diesel oil efficiently, providing a total desulfurization percent of 78% and suggesting its cost-effective advantage.     

Adsorption of DBT and 4,6-DMDBTon nanoporous activated carbons: the role of surface chemistry and the solvent

Environmental Science and Pollution Research - Adsorption of dibenzothiophene (DBT) and 4,6-dimethyldibenzothiophene (4,6-DMDBT) from solutions in hexane and hexadecane respectively as well as in...     

The response of soil Arthrobacter agilis lush13 to changing conditions: Transition between vegetative and dormant state

This work was aimed at studying the response of soil non-spore-forming actinobacterial strain Arthrobacter agilis Lush 13 to changing natural conditions, such as nutrient availability and the presence of degradable and recalcitrant aliphatic and aromatic substrates. The A. agilis strain Lush13 was able to degrade octane, nonane, hexadecane, benzoate, phenol, and 2,3-, 2,4-, 2,5-, 2,6-dichlorophenols, but not grew on 3,4-dichlorophenol, 2,3,4-, 2,4,5-, 2,4,6-trichlorophenol (TCP), pentachlorophenol (PCP), 2-chlorobenzoate, 3-chlorobenzoate, 3,5-dichlorobenzoate, 2,4-dichlorobenzoate. Under growth-arresting conditions due to nitrogen- or multiple starvation or recalcitrant (non-utilizable) carbon source, the studied strain preserved viability for prolonged periods (4–24 months) due to transition to dormancy in the form of conglomerated small and ultrasmall cyst-like dormant cells (CLC). Dormant cells were shown to germinate rapidly (30 min or later) after removal of starvation stress, and this process was followed by breakdown of conglomerates with the eliberation and further division of small multiple actively growing daughter cells. Results of this study shed some light to adaptive capabilities of soil arthrobacters in pure and polluted environments.     

Combined photo-Fenton and biological oxidation for pesticide degradation: effect of photo-treated intermediates on biodegradation kinetics

Biodegradability of a partially photo-oxidized pesticide mixture is demonstrated and the effect of photo-Fenton treatment time on growth and substrate consumption of the bacteria Pseudomonas putida CECT 324 is shown. Four commercial pesticides, laition, metasystox, sevnol and ultracid, usually employed in citric orchards in eastern Spain, were chosen for these experiments. The active ingredients are, respectively, dimethoate, oxydemeton-methyl, carbaryl and methidathion. Judging by biomass measurements, dissolved organic carbon measurements and biodegradation efficiency, it may be concluded that 90min相似文献   

Dibenzothiophene desulfurization by Gordonia alkanivorans strain 1B using recycled paper sludge hydrolyzate

Enzymatic hydrolyzates of recycled paper sludge were tested as suitable feedstock for biological desulfurization by Gordonia alkanivorans strain 1B. Only the hydrolyzate obtained after enzymatic mixture dialysis (dialyzed hydrolyzate) allowed dibenzothiophene (DBT) desulfurization, in spite of faster bacterial growth did occur on non-dialyzed hydrolyzate. For dialyzed hydrolyzate, 250microM DBT was consumed after 96h displaying a maximum specific productivity of 2-hydroxybiphenyl of 1.1micromol g(-1)(dry cell weight) h(-1). A comparison of the kinetics of biodesulfurization was assessed according to the type of hydrolyzate supplementation. Complete consumption of DBT was observed upon the addition of only phosphates and ammonia although further addition of zinc did increase the 2-hydroxybiphenyl production by 14%. Strain 1B was able to desulfurize a model oil containing DBT, 4-methylDBT and 4,6-dimethylDBT, reducing by 63% the total sulfur content in 168h.     

A comparison between toxicity tests using single species and a microbial process.

In this study the sensitivity of the acetate mineralization process performed by five strains of microorganisms in soil for the toxicants Zn2+ or PCP was calculated from the sensitivity of the contributing species. The species used were a fungus (Aspergillus niger CBS 121.49), an actinomycete (Streptomyces lividans 66), two Gram-negative Pseudomonas putida strains (MT-2 and DSM 50026) and a Gram-positive strain Rhodococcus erythropolis A177. For zinc the EC10 of the process performed by the five strains together was 77 mg/kg whereas for pentachlorophenol it was 2 mg/kg. The EC10 of the process was compared with the EC50 of the most sensitive species contributing to the process. P. putida MT2 was the most zinc sensitive strain (EC50 = 22 mg Zn/kg) and A. niger was the most sensitive strain for pentachlorophenol (EC50 = 1.4 mg/kg). This shows that a 10% inhibition of a process can be accompanied by a more than 50% inhibition of the most sensitive species.     

Enhanced degradation of benzo[a]pyrene by Mycobacterium sp. in conjunction with green alga

Previous work in this laboratory has confirmed that the bacteria Mycobacterium sp. strain RJGII.135 and Sphingomonas yanoikuyae strain B1 and the green alga Selanastrum capricornutum strain UTEX 1648 degrade benzo[a]pyrene (BaP) to various BaP intermediates. S. capricornutum was first grown with BaP for 4 days. The organic extract of this media was then introduced into separate cultures of strain RJGII.135 and strain B1; separate cultures were grown with BaP for comparison. Cultures grown with BaP and those grown with the algal/BaP extract showed similar mineralization patterns. The quantity of total metabolites formed was greater in bacterial cultures grown with the algal/BaP extract than those grown with BaP alone. For strain RJGII.135, only 27% of the original BaP remained in cultures grown with the algal/BaP extract; 59% remained in cultures grown with BaP. For strain B1, only 6% of the original BaP remained in cultures grown with the algal/BaP extract; 38% remained in cultures grown with BaP. These results indicate that strategies utilizing organisms together may be necessary in being able to degrade large, recalcitrant polycyclic aromatic hydrocarbons (PAHs) such as BaP.     

Assessment of adsorption behavior of dibutyltin (DBT) to clay-rich sediments in comparison to the highly toxic tributyltin (TBT)

The sorption behavior of dibutyltin (DBT) to four types of natural clay-rich sediments as a function of pH and salinity was studied. The strongest affinity of DBT was found to the montmorillonite-rich sediment, which is characterized by the highest specific surface area and cation exchange capacity of the four used sediments. Kd values range between 12 and 40 (l/kg) on simulated marine conditions (pH 8, salinity 32%). A maximum of DBT adsorption was found at a salinity of 0% and pH 6. Desorption occurred over the entire studied pH range (4-8) when contaminated sediments interact with butyltin-free water. The maximum of desorption coincided with the minimum of adsorption, and vice versa. The results of DBT adsorption are compared with tributyltin (TBT), and the mechanism of the adsorption process is discussed.     

Biodegradation capacity of tributyltin by two Chlorella species

Two microalgal species, Chlorella vulgaris and Chlorella sp., which showed high tributyltin (TBT) tolerant ability were investigated for their capabilities in degrading TBT at sublethal concentration. The distribution of TBT and its degraded products dibutyltin (DBT) and monobutyltin (MBT) in the incubation medium, extracellular surface and intracellular fraction were monitored during an exposure period of 14 days. Results showed that biosorption of TBT by the algal cell wall was the major mechanism in reducing 40% of the initial TBT from the medium in the first 2 days. The half-life of TBT incubated with C. vulgaris was 60 h while that of Chlorella sp. was 80 h. The occurrence of DBT at Day 1 in the culture medium provided direct evidence to the biodegradation of TBT by both Chlorella species. At the end of the experimental period, 27 and 41% of the original TBT were recovered as DBT and MBT in cultures of C. vulgaris, respectively. In contrast, DBT appeared to be the only degradation product of Chlorella sp. and only 26% of the original TBT was transformed to DBT. Despite the same genus, TBT was debutylated to a greater extent to MBT by C. vulgaris, while DBT was the end degradation product by Chlorella sp. The capability of such debutylating process therefore accounted for the higher tolerant ability of C. vulgaris than Chlorella sp.     

In-situ partitioning of butyltin compounds in estuarine sediments

The in-situ solid/pore-water partitioning of tributyltin (TBT), dibutyltin (DBT) and monobutyltin (MBT) was determined for an estuarine sediment profile collected from a commercial marina. Total butyltin levels exceeded sediment quality guideline values, and were 220-8750 microg/kg for TBT, 150-5450 microg/kg for DBT and 130-4250 microg/kg for MBT. Pore-water butyltin concentrations ranged from 0.05 to 2.35 microg/l for TBT, 0.07-3.25 microg/l for DBT, and 0.05-0.53 microg/l for MBT. The partitioning of butyltin compounds between the sediment solid-phase and pore-water was described by an organic carbon normalised distribution ratio (D(OC)). The observed D(OC) values were similar for TBT, DBT and MBT, and were 10(5)-10(6) l/kg. Values for the Butyltin Degradation Index (BDI) were larger than 1 at depths greater than 10 cm below the sediment/water-column interface. This indicates that substantial TBT degradation has occurred in the sediments, and suggests that natural attenuation may be a viable sediment remediation strategy.     

Specific detection of organotin compounds with a recombinant luminescent bacteria

Organotin compounds are widely used as biocides in marine and terrestrial environments. Several currently used techniques allow either the measurement of the chemicals or their effects on living organisms. Our current research focuses on the development of a complementary method based on a bacterial bioluminescence-based bioassay for the specific detection of organotin compounds. The performance of the bioassay was assessed. The Escherichia coli bacterial strain used in this study is specific for TBT and DBT (with Cl, Br or I as the halogen group) with the central tin atom important for light production. The assay is conducted after overnight culture of the bacterial strain, followed by 60 min of contact time with the organotin compound for significant light production. The detection limits were found to be 0.08 microM for TBT (26 microgl(-1)) and 0.0001 microM for DBT (0.03 microgl(-1)) with a linear range of one logarithm. The repeatability of the bioassay is 8% and the reproducibility for TBT and DBT was approximately 14%. Lyophilization of the strains did not significantly modify the detection limit as well as the range of detection. Applications of the bioassay to environmental samples are discussed.     

Reliable QSAR for estimating Koc for persistent organic pollutants: correlation with molecular connectivity indices

Several recent studies have shown that n-octanol/water partition coefficients may not be a good predictor for estimating soil sorption coefficients of persistent organic pollutants (POPs), defined here as chemicals with log Kow greater than 5. Thus, an alternative QSAR model was developed that seems to provide reliable estimates for the soil sorption coefficients of persistent organic pollutants. This model is based on a set of calculated molecular connectivity indices and evaluated soil sorption data for 18 POPs. The chemical's size and shape, quantified by 1chi, 3chiC and 4chiC(v) indices, have a dominant effect on the soil sorption process of POPs. The developed QSAR model was rationalized in terms of potential hydrophobic interactions between persistent organic pollutants and soil organic matrix. Its high predictive power has been verified by an extensive internal and external validation procedure.     

Mixing-controlled biodegradation in a toluene plume--results from two-dimensional laboratory experiments

Various abiotic and biotic processes such as sorption, dilution, and degradation are known to affect the fate of organic contaminants, such as petroleum hydrocarbons in saturated porous media. Reactive transport modeling of such plumes indicates that the biodegradation of organic pollutants is, in many cases, controlled by mixing and therefore occurs locally at the plume's fringes, where electron donors and electron-acceptors mix. Herein, we aim to test whether this hypothesis can be verified by experimental results obtained from aerobic and anaerobic degradation experiments in two-dimensional sediment microcosms. Toluene was selected as a model compound for oxidizable contaminants. The two-dimensional microcosm was filled with quartz sand and operated under controlled flow conditions simulating a contaminant plume in otherwise uncontaminated groundwater. Aerobic degradation of toluene by Pseudomonas putida mt-2 reduced a continuous 8.7 mg L(-1) toluene concentration by 35% over a transport distance of 78 cm in 15.5 h. In comparison, under similar conditions Aromatoleum aromaticum strain EbN1 degraded 98% of the toluene infiltrated using nitrate (68.5+/-6.2 mg L(-1)) as electron acceptor. A major part of the biodegradation activity was located at the plume fringes and the slope of the electron-acceptor gradient was steeper during periods of active biodegradation. The distribution of toluene and the significant overlap of nitrate at the plume's fringe indicate that biokinetic and/or microscale transport processes may constitute additional limiting factors. Experimental data is corroborated with results from a reactive transport model using double Monod kinetics. The outcome of the study shows that in order to simulate degradation in contaminant plumes, detailed data sets are required to test the applicability of models. These will have to deal with the incorporation of existing parameters coding for substrate conversion kinetics and microbial growth.     

Horizontal distribution of butyltins in surface sediments from an enclosed bay system, Korea

Tributyltin (TBT), dibutyltin (DBT), and monobutyltin (MBT) compounds were quantitatively determined from surface sediment samples (total 59 stations) covering a whole basin where harbors, shipyards, and aquaculture farms were located. Butyltin compounds were detected from all the stations covering 640 km(2) of an enclosed bay system. TBT concentrations ranged from 4 to 382 ng/g as tin on a dry weight basis, and total butyltin concentrations, from 27 to 1763 ng/g. Horizontal distribution of TBT concentration showed apparent negative gradients from harbors and shipyards, indicating that its contamination was closely related to boating and dry-docking activities. However, TBT concentrations were decreased steeply from source areas. Elevated DBT and MBT levels in creeks imply the possible input of DBT from industrial wastewater. Total butyltin concentrations in sediments are significantly correlated with particulate organic carbon concentration for the subset of stations that are distant from source areas.     

Electrochemical wastewater treatment: influence of the type of carbon and of nitrogen on the organic load removal

Boron-doped diamond (BDD) and Ti/Pt/PbO₂ anodes were utilized to perform the electrodegradation of synthetic samples containing humic acid in the presence of different organic and inorganic carbon-containing and nitrogen-containing compounds. The influence of the chloride ion in the degradation process of the different synthetic samples was also assessed. The results showed that the anodic oxidation process can efficiently degrade recalcitrant compounds such as humic acid. The presence of carbonate in solution enhances the nitrogen removal, whereas it hinders the oxidation of the organic compounds. When organic nitrogen is present, it is converted to NH₄ ⁺, which in turn is oxidized to nitrate and to volatile nitrogen compounds. Hydroxyl radicals are more prone to oxidize the organic nitrogen than the ammonium nitrogen. The presence of chloride enhances the organic matter and nitrogen removal rates, BDD being the anode material that yields the highest removals.     

Real-time reverse transcription PCR analysis of trichloroethylene-regulated toluene dioxygenase expression in Pseudomonas putida F1

Toluene dioxygenase (tod) is a multicomponent enzyme system in Pseudomonas putida F1. Tod can mediate the degradation of Trichloroethylene (TCE), a widespread pollutant. In this study, we try to explore the TCE-regulated tod expression by using real-time qRT-PCR. The minimal culture media were supplemented with glucose, toluene, or a mixture of glucose/toluene respectively as carbon and energy sources. The TCE was injected into each medium after a 12-hour incubation period. The TCE injection severely affected bacterial growth when cultured with toluene or toluene/glucose mixtures. The cell density dropped 61 % for bacteria growing in toluene and 36 % for bacteria in the glucose/toluene mixture after TCE injection, but the TCE treatment had little effect on bacteria supplied with glucose alone. The decrease in cell number was caused by the cytotoxicity of the TCE metabolized by tod. The results from the real-time qRT-PCR revealed that TCE was capable of inducing tod expression in a toluene-dependent manner and that the tod expression level increased 50 times in toluene and 3 times in the toluene/glucose mixture after 6 hours of TCE treatment. Furthermore, validation of the rpoD gene as a reference gene for P. putida F1 was performed in this study, providing a valuable foundation for future studies to use real-time qRT-PCR in the analysis of the P. putida F1 strain.     

Ozone oxidation and aerobic biodegradation with spent mushroom compost for detoxification and benzo(a)pyrene removal from contaminated soil

The combination of ozonation and spent mushroom compost (SMC)-mediated aerobic biological treatment was investigated in the removal of benzo(a)pyrene from contaminated soil. The performances of the process alone and combined were evaluated in terms of benzo(a)pyrene removal efficiency, mineralization efficiency (as total organic carbon removal), and soil residual toxicity (phytotoxicity to Lepidium Sativum and toxicity to Vibrio fischeri). In spite of the removal efficiency (35%) obtained by SMC-mediated biological process as a stand-alone treatment, the combined process showed a benzo(a)pyrene concentration reduction higher than 75%; the best removal (82%) was observed after 10 min pre-ozonation treatment. In particular, ozonation improved the biodegradability of the contaminant, as confirmed by the increase of CO(2) production (close to 70% compared to the control), mineralization (greater than 60%) and bacterial density (which increased by two orders of magnitude). Moreover, according to phytotoxicity tests on L. Sativum, the aerobic biological process of pre-ozonated soil decreased toxicity. According to the results achieved in the present study, ozonation pre-treatment showed an high potential to overcome the limitation of bioremediation of recalcitrant compound, but it should be carefully operated in order to maximize PAH removal efficiency as well as to minimize soil residual toxicity which can result from the formation of the oxidation intermediates.     

Degradation of reactive dyes I. A comparative study of ozonation, enzymatic and photochemical processes

The environmental problems associated with textile activities are represented mainly by the extensive use of organic dyes. A great number of these compounds are recalcitrant and shown carcinogenic or mutagenic character. In this work three processes were studied for degradation of an anthraquinone dye (C.I. reactive blue-19). The ozonation process leads to complete decolorization with a very short reaction time; however, effective mineralization of the dye was not observed. The enzymatic process promotes quick decolorization of the dye; nevertheless, maximum decolorization degrees of about 30% are insignificant in relation to the decolorization degree achieved by the other processes. The best results were found for the photocatalytical process. The use of ZnO or TiO2 as photocatalysts, permits total decolorization and mineralization of the dye with reaction times of about 60 min.