Enhanced phenanthrene biodegradation in river sediments by harnessing calcium peroxide nanoparticles and minerals in Sphingomonas sp. DSM 7526 cultivation.

Coupling chemical oxidation and biodegradation to remediate polycyclic aromatic hydrocarbon (PAH)-contaminated sediment has recently gained significant attention. In this study, calcium peroxide nanoparticles (nCaO2) were utilized as an innovative oxygen-releasing compound for in-situ chemical oxidation. The study investigates the bioremediation of phenanthrene (PHE)-contaminated sediment inoculated with Sphingomonas sp. DSM 7526 bacteria and treated with either aeration or nCaO2. Using three different culture media, the biodegradation efficiencies of PHE-contaminated anoxic sediment, aerobic sediment, and sediment treated with 0.2% w/w nCaO2 ranged from 57.45% to 63.52%, 69.87% to 71.00%, and 92.80% to 94.67%, respectively. These values were significantly higher compared to those observed in non-inoculated sediments. Additionally, the type of culture medium had a prominent effect on the amount of PHE removal. The presence of minerals in the culture medium increased the percentage of PHE removal compared to distilled water by about 2-10%. On the other hand, although the application of CaO2 nanoparticles negatively impacted the abundance of sediment bacteria, resulting in a 30-42% decrease in colony-forming units after 30 days of treatment, the highest PHE removal was obtained when coupling biodegradation and chemical oxidation. These findings demonstrate the successful application of bioaugmentation and chemical oxidation processes for treating PAH-contaminated sediment.

Genome-resolved analyses of oligotrophic groundwater microbial communities along phenol pollution in a continuous-flow biodegradation model system.

Groundwater pollution is one of the major environmental concerns. The entrance of pollutants into the oligotrophic groundwater ecosystems alters native microbial community structure and metabolism. This study investigated the application of innovative Small Bioreactor Chambers and CaO2 nanoparticles for phenol removal within continuous-flow sand-packed columns for 6 months. Scanning electron microscopy and confocal laser scanning microscopy analysis were conducted to indicate the impact of attached biofilm on sand surfaces in bioremediation columns. Then, the influence of each method on the microbial biodiversity of the column's groundwater was investigated by next-generation sequencing of the 16S rRNA gene. The results indicated that the simultaneous application of biostimulation and bioaugmentation completely eliminated phenol during the first 42 days. However, 80.2% of phenol remained in the natural bioremediation column at the end of the experiment. Microbial diversity was decreased by CaO2 injection while order-level groups known for phenol degradation such as Rhodobacterales and Xanthomonadales dominated in biostimulation columns. Genome-resolved comparative analyses of oligotrophic groundwater prokaryotic communities revealed that Burkholderiales, Micrococcales, and Cytophagales were the dominant members of the pristine groundwater. Six-month exposure of groundwater to phenol shifted the microbial population towards increasing the heterotrophic members of Desulfobacterales, Pseudomonadales, and Xanthomonadales with the degradation potential of phenol and other hydrocarbons.

Genome-resolved analyses show an extensive diversification in key aerobic hydrocarbon-degrading enzymes across bacteria and archaea.

BACKGROUND: Hydrocarbons (HCs) are organic compounds composed solely of carbon and hydrogen that are mainly accumulated in oil reservoirs. As the introduction of all classes of hydrocarbons including crude oil and oil products into the environment has increased significantly, oil pollution has become a global ecological problem. However, our perception of pathways for biotic degradation of major HCs and key enzymes in these bioconversion processes has mainly been based on cultured microbes and is biased by uneven taxonomic representation. Here we used Annotree to provide a gene-centric view of the aerobic degradation ability of aliphatic and aromatic HCs in 23,446 genomes from 123 bacterial and 14 archaeal phyla.  RESULTS: Apart from the widespread genetic potential for HC degradation in Proteobacteria, Actinobacteriota, Bacteroidota, and Firmicutes, genomes from an additional 18 bacterial and 3 archaeal phyla also hosted key HC degrading enzymes. Among these, such degradation potential has not been previously reported for representatives in the phyla UBA8248, Tectomicrobia, SAR324, and Eremiobacterota. Genomes containing whole pathways for complete degradation of HCs were only detected in Proteobacteria and Actinobacteriota. Except for several members of Crenarchaeota, Halobacterota, and Nanoarchaeota that have tmoA, ladA, and alkB/M key genes, respectively, representatives of archaeal genomes made a small contribution to HC degradation. None of the screened archaeal genomes coded for complete HC degradation pathways studied here; however, they contribute significantly to peripheral routes of HC degradation with bacteria. CONCLUSION: Phylogeny reconstruction showed that the reservoir of key aerobic hydrocarbon-degrading enzymes in Bacteria and Archaea undergoes extensive diversification via gene duplication and horizontal gene transfer. This diversification could potentially enable microbes to rapidly adapt to novel and manufactured HCs that reach the environment.

Distinct microbial community along the chronic oil pollution continuum of the Persian Gulf converge with oil spill accidents.

The Persian Gulf, hosting ca. 48% of the world's oil reserves, has been chronically exposed to natural oil seepage. Oil spill studies show a shift in microbial community composition in response to oil pollution; however, the influence of chronic oil exposure on the microbial community remains unknown. We performed genome-resolved comparative analyses of the water and sediment samples along Persian Gulf's pollution continuum (Strait of Hormuz, Asalouyeh, and Khark Island). Continuous exposure to trace amounts of pollution primed the intrinsic and rare marine oil-degrading microbes such as Oceanospirillales, Flavobacteriales, Alteromonadales, and Rhodobacterales to bloom in response to oil pollution in Asalouyeh and Khark samples. Comparative analysis of the Persian Gulf samples with 106 oil-polluted marine samples reveals that the hydrocarbon type, exposure time, and sediment depth are the main determinants of microbial response to pollution. High aliphatic content of the pollution enriched for Oceanospirillales, Alteromonadales, and Pseudomonadales whereas, Alteromonadales, Cellvibrionales, Flavobacteriales, and Rhodobacterales dominate polyaromatic polluted samples. In chronic exposure and oil spill events, the community composition converges towards higher dominance of oil-degrading constituents while promoting the division of labor for successful bioremediation.

Metagenomic investigation of bacteria associated with dental lesions: a cross-sectional study

BACKGROUND: Dental caries is considered as one of the most significant global health problem over the world. Dental caries initiates from bacterial shifts within the supragingival biofilm, then a polymicrobial biofilm is formed on the surface of tooth, and finally various bacterial species aggregate in a complex-organized manner. The exploiting variability in 16S rRNA gene sequence has been considered as a cost-efficient high-throughput characterization approach in human oral microbiome investigations. The aim of this study is to characterize bacterial species associated with superficial dental biofilm, underlying carious dentine and root caries lesion by16S rRNA gene-based metagenomic analysis. MATERIAL AND METHODS: Herein, the bacterial communities in carious dentin lesion, biofilm and root canal samples of 30 subjects (aged 4-76 years) admitted to a clinic in Tehran during 2017 were investigated using a culture independent approach. Total genomic DNA of each tissue was subjected to metagenomic identification of bacteria using a nested PCR assay and 16S rRNA library construction method. RESULTS: 31 samples collected from 30 consenting patients (29 samples from 29 patients ant two biofilm samples from one patient). Bioinformatics analyses of a-800bp sequences of the second step of Nested-PCR revealed presence of 156 bacterial isolates in carious (n = 45), biofilm (n = 81) and root canal (n = 30) specimens. Prevotella spp., Lactobacillus vaginalis, and streptococcus spp. showed higher prevalence in carious dentin, root and biofilm samples, respectively. CONCLUSIONS: Exploring the dental microbiota and comparing them in health or diseased conditions is critical step in the determination of human general health. The method applied in this study could identify bacteria related to the three dental lesions. However, due to lack of data for comparison in Genbank or because of the sequence similarity lower than 98% for most identified bacteria, the use of more powerful approaches like NGS platforms or typing of multiple loci (MLST) in future studies is recommended

No disponible

The impact of calcium peroxide on groundwater bacterial diversity during naphthalene removal by permeable reactive barrier (PRB).

Oxygen-releasing compounds (ORCs) have recently gained much attention in contaminated groundwater remediation. We investigated the impact of calcium peroxide nanoparticles on the groundwater indigenous bacteria in a bioremediation process by permeable reactive barrier (PRB). Three sand-packed columns were applied, including (1) control column (fresh groundwater), (2) natural remediation column (contaminated groundwater), and (3) biostimulation column (contaminated groundwater amended with CaO2). Actinobacteria and Proteobacteria constituted the main phyla among the identified isolates. According to the results of next-generation sequencing, Proteobacteria was the dominant phylum (81% relative abundance) in the natural remediation condition. But, it was declined to 38.1% in the biostimulation column. Meanwhile, the abundance of Actinobacteria and Bacteroidetes were increased to 25.9% and 15.4%, respectively, by exposing the groundwater microbial structure to CaO2 nanoparticles. Furthermore, orders Chlamydiales, Nitrospirales, and Oceanospirillales existing in the control column were detected in the presence of naphthalene. Shannon index was 4.32 for the control column samples, while it was reduced to 2.73 and 2.00 in the natural and biostimulation columns, respectively. Therefore, the present study provides a considerable insight into the impact of ORCs on the groundwater microbial community during the bioremediation process.

Biodegradation of long chain alkanes in halophilic conditions by Alcanivorax sp. strain Est-02 isolated from saline soil.

In this study, through a multistep enrichment and isolation procedure, a halophilic bacterial strain was isolated from unpolluted saline soil, which was able to effectively and preferentially degrade long chain alkanes (especially tetracosane and octacosane). The strain was identified by 16S rRNA gene sequence as an Alcanivorax sp. The growth of strain Est-02 was optimized at the presence of tetracosane in different NaCl concentrations, temperatures, and pH. The consumption of different heavy alkanes was also investigated. Optimal culture conditions of the strain were determined to be as follows: 10% NaCl, temperature 25-35 °C and pH 7. Alcanivorax sp. strain Est-02 was able to use a wide range of aliphatic substrates ranging from C14 to C28 with clear tendency to utilize heavy chain hydrocarbons of C24 and C28. During growth on a mixture of alkanes (C14-C28), the strain consumed 60% and 65% of tetracosane and octacosane, respectively, while only about 40% of the lower chain alkanes were degraded. This unique ability of the strain Est-02 in efficient and selective biodegradation of long chain hydrocarbons could be further exploited for remediation of wax and heavy oil contaminated soils or upgrading of heavy crude oils. Comparison of the sequence of alkane hydroxylase gene (alkB) of strain Est-02 with previously reported sequences for Alcanivorax spp. and other hydrocarbon degraders, showed a remarkable phylogenetic distance between the sequence alkB of Est-02 and other alkane-degrading bacteria.

Bioremediation of benzene-contaminated groundwater by calcium peroxide (CaO2) nanoparticles: Continuous-flow and biodiversity studies.

Calcium peroxide (CaO2) nanoparticles have been extensively applied in treatment of contaminated groundwater through bioremediation or modified Fenton (MF) processes. In the present study utilization of CaO2 in bioremediation and MF (CaO2+FeSO4) reaction is investigated for benzene (50 mg/L) removal in continuous flow sand-packed columns. The results indicated that MF produced OH radicals markedly increased benzene remediation at first 30 days (up to 93%). But, OH generation rate was gradually declined when the pH was increased and finally 75% of initial benzene removed after 100d. In bioremediation column, because of supplying adequate oxygen by CaO2, the number of planktonic bacteria logarithmically increased to more than 5 × 106 CFU/mL (two orders of magnitude) and consequently 100% benzene removal was achieved by the end of experiment. Scanning electron microscopy analysis visualized the attached biofilm growth on sand surfaces in CaO2 injected columns indicating their key role in the remediation process. The impact of each process on the microbial biodiversity of groundwater was investigated by next generation sequencing (NGS) of the 16S rRNA gene. The alpha and beta analysis indicated that microbial diversity is decreased by CaO2 injection while benzene-degrading species such as Silanimonas, Arthrobacter and Pseudomonas spp. were dominated in remediation column.

Application of encapsulated magnesium peroxide (MgO2) nanoparticles in permeable reactive barrier (PRB) for naphthalene and toluene bioremediation from groundwater.

One of the challenges in the petroleum hydrocarbon contaminated groundwater remediation by oxygen releasing compounds (ORCs) is to identify the remediation mechanism and determine the impact of ORCs on the environment and the intrinsic groundwater microorganisms. In this research, the application of encapsulated magnesium peroxide (MgO2) nanoparticles in the permeable reactive barrier (PRB) for bioremediation of the groundwater contaminated by toluene and naphthalene was studied in the continuous flow sand-packed plexiglass columns within 50 d experiments. For the biodiversity studies, next generation sequencing (NGS) of the 16S rRNA gene was applied. The results showed that naphthalene was metabolized (within 20 days) faster than toluene (after 30 days) by microorganisms of the aqueous phase. By comparing the contaminant removal in the biotic (which resulted in the complete contaminant removal) and abiotic (around 32% removal for naphthalene and 36% for toluene after 50 d) conditions, the significant role of microorganisms on the decontamination process was proved. Furthermore, the attached microbial communities on the porous media were visualized by scanning electron microscopy (SEM). Microbial community structure analysis by NGS technique revealed that the microbial species which were able to degrade toluene and naphthalene such as P. putida and P. mendocina respectively were stimulated by addition of MgO2 nanoparticles. The presented study resulted in a momentous insight into the application of MgO2 nanoparticles in the hydrocarbon compounds removal from groundwater.

Application of magnesium peroxide (MgO2) nanoparticles for toluene remediation from groundwater: batch and column studies.

In the present study, magnesium peroxide (MgO2) nanoparticles were synthesized by electro-deposition process and characterized by X-ray diffraction (XRD) and field emission scanning electron microscopy (FESEM). The batch experiments were conducted to evaluate the MgO2 half-life (600 mg/L) in groundwater under various temperatures (4, 15, and 30 °C) and initial pH (3, 7, and 12). The effect of Fe2+ ions (enhanced oxidation) on the toluene remediation by MgO2 was also investigated. Nanoparticles were injected to sand-packed continuous-flow columns, and toluene removal (50 ppm) was studied within 50 days at 15 °C. The results indicated that the half-life of MgO2 at pH 3 and 12 were 5 and 15 days, respectively, in comparison to 10 days at the initial pH 7 and 15 °C. The nanoparticles showed 20 and 7.5 days half-life at 4 and 30 °C temperatures, respectively. Injection of Fe2+ ions indicated an impressive effect on toluene removal by MgO2, and the contaminant was completely removed after 5 and 10 days, in the batch and column experiments, respectively. Confocal laser scanning microscope (CLSM) analysis indicated that the attached biofilm had a significant role in the decontamination of groundwater. Comparison of bioremediation and enhanced oxidation resulted in a considerable insight into the application of magnesium peroxide in groundwater remediation. Graphical abstract ᅟ.

Naphthalene remediation form groundwater by calcium peroxide (CaO2) nanoparticles in permeable reactive barrier (PRB).

This study investigated the applicability of synthesized calcium peroxide (CaO2) nanoparticles for naphthalene bioremediation by permeable reactive barrier (PRB) from groundwater. According to the batch experiments the application of 400 mg/L of CaO2 nanoparticles was the optimum concentration for naphthalene (20 mg/L) bioremediation. Furthermore, the effect of environmental conditions on the stability of nanoparticles showed the tremendous impacts of the initial pH and temperature on the stability and oxygen releasing potential of CaO2. Therefore, raising the initial pH from 3 to 12 elevated the dissolved oxygen from 4 to 13.6 mg/L and the stability of nanoparticles was significantly improved around 70 d. Moreover, by increasing the temperature from 4 to 30 °C, the stability of CaO2 declined from 120 to 30 d. The continuous-flow experiments revealed that the naphthalene-contaminated groundwater was completely bio-remediated in the presence of CaO2 nanoparticles and microorganisms from the effluent of the column within 50 d. While, the natural remediation of the contaminant resulted in 19.7% removal at the end of the experiments (350 d). Additionally, the attached biofilm on the surface of the PRB zone was studied by scanning electron microscopy (SEM) which showed the higher biofilm formation on the pumice surfaces in the bioremediation column in comparison to the natural remediation column. The physic-chemical characteristics of the effluents from each column was also analyzed and indicated no negative impact of the bioremediation process on the groundwater. Consequently, the present paper provides a comprehensive study on the application of the CaO2 nanoparticles in PAH-contaminated groundwater treatment.

Bioremediation of oil-based drill cuttings by a halophilic consortium isolated from oil-contaminated saline soil.

Oil-based drill cuttings are hazardous wastes containing complex hydrocarbons, heavy metals, and brine. Their remediation is a crucial step before release to the environment. In this work, we enriched a halophilic consortium, from oil-polluted saline soil, which is capable of degrading diesel as the main pollutant of oil-based drill cuttings. The degradation ability of the consortium was evaluated in microcosms using two different diluting agents (fine sand and biologically active soil). During the bioremediation process, the bacterial community dynamics of the microcosms was surveyed using PCR amplification of a fragment of 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE). The diesel degradation rates were monitored by total petroleum hydrocarbon (TPH) measurement and the total count of heterotrophic and diesel-degrading bacteria. After 3 months, the microcosm containing fine sand and drill cuttings with the ratio of 1:1 (initial TPH of 36,000 mg/kg) showed the highest TPH removal (40%) and its dominant bacterial isolates belonged to the genera Dietzia, Arthrobacter, and Halomonas. DGGE results also confirmed the role of these genera in drill cuttings remediation. DGGE analysis of the bacterial diversity showed that Propionibacterium, Salinimicrobium, Marinobacter, and Dietzia are dominant in active soil microcosm; whereas Bacillus, Salinibacillus, and Marinobacter are abundant in sand microcosm. Our results suggest that the bioaugmentation strategy would be more successful if the diluting agent does not contain a complex microbial community.

Investigation of transient forms of sulfur during biological treatment of spent caustic.

In the present study, the production of various transient forms of sulfur during biological oxidation of sulfidic spent caustics under haloalkaline conditions in a stirred tank bioreactor is investigated. Also, the effects of abiotic aeration (chemical oxidation), dissolved oxygen (DO) concentration and sodium concentration on forms of sulfur during biological treatment are demonstrated. Thioalkalivibrio versutus strain was used for sulfide oxidation in spent caustic (SC). The aeration had an important effect on sulfide oxidation and its final products. At DO concentrations above 2 mg l-1, majority of sulfide was oxidized to sulfate. Maximum sulfide removal efficiency (%R) and yield of sulfate production [Formula: see text] was obtained in Na+ concentration ranging from 0.6 to 2 M. Abiotic aeration, which is the most important factor of production of thiosulfate, resulted in the formation of an undesired product-polysulfide. However, abiotic aeration can be used as a pretreatment to biological treatment. In the bioreactor the removal efficiency was obtained as 82.7% and various forms of sulfur such as polysulfide, biosulfur, thiosulfate and sulfate was observed during biological treatment of SC.

Prauserella oleivorans sp. nov., a halophilic and thermotolerant crude-oil-degrading actinobacterium isolated from an oil-contaminated mud pit.

A crude-oil-degrading, Gram-stain-positive actinobacterial strain, RIPIT, was isolated from a soil sample collected from an oil-contaminated mud pit in Khangiran oil and gas field, in the north-east of Iran. RIPIT was strictly aerobic, catalase- and oxidase-positive. The strain grew with 0-12.5 % (w/v) NaCl (optimum 3-5 %), at 25-55 °C (optimum 45 °C) and at pH 6.0-9.5 (optimum pH 7.0). The results of 16S rRNA gene sequence comparative analysis indicated that RIPIT represents a member of the genus Prauserella, with high phylogenetic similarity to Prauserella coralliicola SCSIO 11529T (97.5 %), Prauserella endophytica SP28S-3T (97.5 %) and Prauserella marina MS498T (97.2 %). DNA-DNA relatedness values between the novel strain and P. coralliicola DSM 45821T, P. endophytica DSM 46655T and P. marina DSM 45268T were 28 , 19 and 23 %, respectively. The cell wall peptidoglycan of RIPIT contained meso-diaminopimelic acid as the diamino acid and the whole-cell sugars are galactose and arabinose. The polar lipids pattern contained phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol and two unknown phospholipids. Its cellular fatty acids pattern consisted of C17 : 1ω6c, iso-C16 : 0 and summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH), and the major respiratory quinone was MK-9(H4). The G+C content of the genomic DNA was 69 mol%. On the basis of polyphasic taxonomic data we propose that RIPIT represents a novel species of the genus Prauserella, for which the name Prauserella oleivorans sp. nov. is proposed. The type strain of Prauserellaoleivorans is RIPIT (=IBRC-M 10906T=LMG 28389T).

Benzene-contaminated groundwater remediation using calcium peroxide nanoparticles: synthesis and process optimization.

Nano-size calcium peroxide (nCaO2) is an appropriate oxygen source which can meet the needs of in situ chemical oxidation (ISCO) for contaminant remediation from groundwater. In the present study, an easy to handle procedure for synthesis of CaO2 nanoparticles has been investigated. Modeling and optimization of synthesis process was performed by application of response surface methodology (RSM) and central composite rotatable design (CCRD) method. Synthesized nanoparticles were characterized by XRD and FESEM techniques. The optimal synthesis conditions were found to be 5:1, 570 rpm and 10 °C for H2O2:CaSO2 ratio, mixing rate and reaction temperature, respectively. Predicted values showed to be in good agreement with experimental results (R 2 values were 0.915 and 0.965 for CaO2 weight and nanoparticle size, respectively). To study the efficiency of synthesized nanoparticles for benzene removal from groundwater, batch experiments were applied in biotic and abiotic (chemical removal) conditions by 100, 200, 400, and 800 mg/L of nanoparticles within 70 days. Results indicated that application of 400 mg/L of CaO2 in biotic condition was able to remediate benzene completely from groundwater after 60 days. Furthermore, comparison of biotic and abiotic experiments showed a great potential of microbial stimulation using CaO2 nanoparticles in benzene remediation from groundwater.

Pseudorhodoplanes sinuspersici gen. nov., sp. nov., isolated from oil-contaminated soil.

Strain RIPI 110T was isolated from a soil sample collected from an oil-contaminated site on Siri Island, Persian Gulf, Iran. Cells of the novel isolate were Gram-stain-negative, facultatively anaerobic, non-motile and rod-shaped. Cells divided asymmetrically by budding and formed rosette-like clusters. The optimum pH and temperature for growth were pH 7 and 30 °C, while the strain was able to grow at pH 5.5-8 and 15-35 °C. Strain RIPI 110T utilized only complex carbon sources and pyruvate as the sole carbon source and could not grow under photoautotrophic conditions. The highest 16S rRNA gene sequence similarities, 93.9, 93.9 and 93.5 %, were obtained with Variibacter gotjawalensis GJW-30T, Rhodoplanes roseus 941T and Rhodoplanes elegans AS130T, respectively. The major cellular fatty acids were summed feature 8 (C18 : 1ω7c/ω6c), C16 : 0 and C19 : 0 cyclo ω8c. Polar lipid analyses revealed that strain RIPI 110T contained phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, an unknown aminophospholipid and four unknown phospholipids. Ubiquinone-10 was the predominant quinone component. The DNA G+C content was 59.4 mol%. On the basis of the 16S rRNA gene sequence analysis, in combination with chemotaxonomic and physiological data, the novel isolate could not be classified in any recognized genera. Strain RIPI 110T is thus considered to represent a novel species of a new genus within the order Rhizobiales, for which the name Pseudorhodoplanes sinuspersici gen. nov., sp. nov. is proposed. The type strain of the type species is RIPI 110T ( = IBRC-M 10770T = CECT 8374T).

Biodegradation of polycyclic aromatic hydrocarbons by a halophilic microbial consortium.

In this study we investigated the phenanthrene degradation by a halophilic consortium obtained from a saline soil sample. This consortium, named Qphe, could efficiently utilize phenanthrene in a wide range of NaCl concentrations, from 1% to 17% (w/v). Since none of the purified isolates could degrade phenanthrene, serial dilutions were performed and resulted in a simple polycyclic aromatic hydrocarbon (PAH)-degrading culture named Qphe-SubIV which was shown to contain one culturable Halomonas strain and one unculturable strain belonging to the genus Marinobacter. Qphe-SubIV was shown to grow on phenanthrene at salinities as high as 15% NaCl (w/v) and similarly to Qphe, at the optimal NaCl concentration of 5% (w/v), could degrade more than 90% of the amended phenanthrene in 6 days. The comparison of the substrate range of the two consortiums showed that the simplified culture had lost the ability to degrade chrysene but still could grow on other polyaromatic substrates utilized by Qphe. Metabolite analysis by HPLC and GC-MS showed that 2-hydroxy 1-naphthoic acid and 2-naphthol were among the major metabolites accumulated in the Qphe-SubIV culture media, indicating that an initial dioxygenation step might proceed at C1 and C2 positions. By investigating the growth ability on various substrates along with the detection of catechol dioxygenase gene, it was postulated that the uncultured Marinobacter strain had the central role in phenanthrene degradation and the Halomonas strain played an auxiliary role in the culture by utilizing phenanthrene metabolites whose accumulation in the media could be toxic.

Emulsification potential of a newly isolated biosurfactant-producing bacterium, Rhodococcus sp. strain TA6.

An indigenous biosurfactant producing bacterium, Rhodococcus sp. strain TA6 was isolated from Iranian oil contaminated soil using an efficient enrichment and screening method. During growth on sucrose and several hydrocarbon substrates as sole carbon source, the bacterium could produce biosurfactants. As a result of biosurfactant synthesis, the surface tension of the growth medium was reduced from 68mNm(-1) to values below 30mNm(-1). The biosurfactant was capable of forming stable emulsions with various hydrocarbons ranging from pentane to light motor oil. Preliminary chemical characterization revealed that the TA6 biosurfactant consisted of extracellular lipids and glycolipids. The biosurfactant was stable during exposure to high salinity (10% NaCl), elevated temperatures (120°C for 15min) and within a wide pH range (4.0-10.0). The culture broth was effective in recovering up to 70% of the residual oil from oil-saturated sand packs which indicates the potential value of the biosurfactant in enhanced oil recovery.

Genomic structure and promoter analysis of the dsz operon for dibenzothiophene biodesulfurization from Gordonia alkanivorans RIPI90A.

The bacterium Gordonia alkanivorans RIPI90A has been previously reported as dibenzothiophene-desulfurizing strain. The present study provides a complete investigation of the dsz operon including dsz promoter analysis from desulfurization competent strain belonging to the genus Gordonia. PCR was used to amplify the dszABC genes and adaptor ligation-based PCR-walking strategy used to isolate the dsz promoter. Unlike the dsz operon of Rhodococcus erythropolis, the operon of RIPI90A was located on chromosome. Despite the remarkably high homology between dsz genes of G. alkanivorans RIPI90A and R. erythropolis IGST8, promoter sequences of the strains were not very similar. The dsz promoter of G. alkanivorans RIPI90A shows only 52.5% homology to that of R. erythropolis IGTS8 and Gordonia nitida. Deletion analysis of the dsz promoter from RIPI90A using luciferase as a reporter gene revealed that the dsz promoter was located in regions from -156 to -50.

High-level expression of lipase in Escherichia coli and recovery of active recombinant enzyme through in vitro refolding.

Microbial lipases are widely used for biotechnological applications. In this study we have cloned and sequenced the lipase and lipase specific foldase genes of a Pseudomonas sp., which was isolated from Southern Iran. The lipase was composed of 936 bp which encoded 311 amino acids and the lipase specific foldase gene consisted of 1008 bp which encoded 336 amino acids. The low amount of recombinant lipase was expressed as an active enzyme in Escherichia coli harboring a plasmid with the clustered lipase and lipase specific foldase genes. To increase the enzyme expression level, the lipase and lipase specific foldase genes subcloned into two separate expression vectors. The lipase was expressed as inactive inclusion bodies under the control of the strong T7 promoter. Inclusion bodies were dissolved in 8M urea and 1mM dithiothreitol (DTT) and purified using Ni-nitrilotriacetic acid column. Subsequently, purified lipase diluted in 20mM phosphate buffer (pH 7) containing the lipase specific foldase which was expressed in another clone of E. coli. In the presence of foldase, it was possible to achieve active lipase with a specific activity of up to 240 IU/mg using a simple refolding procedure. Moreover, the effect of different concentrations of various additives was investigated on the refolding of denatured lipase. The best yield of 70 IU/ml with the specific activity of 3000 IU/mg were obtained after incubation of denatured enzyme in a refolding buffer containing lipase specific foldase (0.005 mg/ml), 1M NaCl and 10% glycerol at 4 degrees C.