Assessing the efficiency and microbial diversity of H2S-removing biotrickling filters at various pH conditions.

This study aimed to investigate the operation of three parallel biotrickling filters (BTFs) in removing H2S at different pH conditions (haloalkaliphilic, neutrophilic, and acidophilic) and their associated microbial population in the biodesulfurization process. BTF columns were inoculated with enriched inoculum and experiments were performed by gradually reducing Empty Bed Retention Time (EBRT) and increasing inlet concentration in which the maximum removal efficiency and maximum elimination capacity in EBRT 60 s reached their maximum level in haloalkaline condition (91% and 179.5 g S-H2S m-3 h-1). For visualizing the attached microbial biofilms on pall rings, Scanning Electron Microscopy (SEM) was used and microbial community structure analysis by NGS showed that the most abundant phyla in haBTF, nBTF, and aBTF belong to Gammaproteobacteria, Betaproteobacteria, and Acidithiobacillia, respectively. Shannon and Simpson indexes evaluation showed a lower diversity of bacteria in the aBTF reactor than that of nBTF and haBTF and beta analysis indicated a different composition of bacteria in haBTF compared to the other two filters. These results indicated that the proper performance of BTF under haloalkaliphilic conditions is the most effective way for H2S removal from air pollutants of different industries.

Elucidated potential of immobilized Janibacter sp. for saline wastewater phenol removal.

Phenolic compounds are commonly found in industrial effluents and can be hazardous to organisms even at low concentrations. Over the years, researchers have demonstrated that bioremediation is a cost-effective and environmentally friendly alternative to physicochemical approaches used to remove phenol. The aim of this study was to investigate the removal of phenol from saline wastewaters by a halotolerant strain of the genus Janibacter. For this purpose, bacterial cells were immobilized on different supports, from which mica and zeolite were ultimately chosen due to their higher removal efficiency. The wet weight of immobilized cells per 1 g of mica and zeolite was 0.51 and 0.48 g, respectively. Free cells consumed 100 mg/L of phenol in 88 h, while immobilized cells used it in 40 h. Immobilized cells revealed a higher thermostability and could operate over a wider pH range and salinity. Unlike free cells, immobilized cells could remove 700 mg/L of phenol and could be reused for at least nine cycles. Interestingly the phenol removal efficiency of zeolite-immobilized cells remained unchanged after 4 months of storage at 4 and - 20 °C, which could be of great advantage for industrial applications. Complete destruction of phenol was observed through the meta pathway comprising phenol hydroxylase and catechol 2,3-dioxygenase enzymes. KEY POINTS: • Mica- and zeolite-immobilized cells were able to consume high concentrations of phenol. • Cells immobilized on mica and zeolite had considerable operational and storage stability. • Immobilized cells could be a good candidate for phenol removal in saline environments.

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.

Biodegradation of high molecular weight hydrocarbons under saline condition by halotolerant Bacillus subtilis and its mixed cultures with Pseudomonas species.

Biodegradation of high-molecular-weight petroleum hydrocarbons in saline conditions appears to be complicated and requires further investigation. This study used heavy crude oil to enrich petroleum-degrading bacteria from oil-contaminated saline soils. Strain HG 01, with 100% sequence similarity to Bacillus subtilis, grew at a wide range of salinities and degraded 55.5 and 77.2% of 500 mg/l pyrene and 500 mg/l tetracosane, respectively, at 5% w/v NaCl. Additionally, a mixed-culture of HG 01 with Pseudomonas putida and Pseudomonas aeruginosa, named TMC, increased the yield of pyrene, and tetracosane degradation by about 20%. Replacing minimal medium with treated seawater (C/N/P adjusted to 100/10/1) enabled TMC to degrade more than 99% of pyrene and tetracosane, but TMC had lesser degradation in untreated seawater than in minimal medium. Also, the degradation kinetics of pyrene and tetracosane were fitted to a first-order model. Compared to B. subtilis, TMC increased pyrene and tetracosane's removal rate constant (K1) from 0.063 and 0.110 per day to 0.123 and 0.246 per day. TMC also increased the maximum specific growth rate of B. subtilis, P. putida, and P. aeruginosa, respectively, 45% higher in pyrene, 24.5% in tetracosane, and 123.4% and 95.4% higher in pyrene and tetracosane.

Evaluation of pyrene and tetracosane degradation by mixed-cultures of fungi and bacteria.

The present study was conducted to compare the efficiency of different microbial mixed-cultures consists of fifteen oil-degrading microorganisms with different combinations. The investigation was targeted toward the removal of 500 mg/l pyrene and 1% w/v tetracosane, as single compounds or mixture. Sequential Fungal-Bacterial Mixed-Culture (SMC) in which bacteria added one week after fungi, recorded 60.76% and 73.48% degradation for pyrene and tetracosane; about 10% more than Traditional Fungal-Bacterial Mixed-Culture (TMC). Co-degradation of pollutants resulted in 24.65% more pyrene degradation and 6.41% less tetracosane degradation. The non-specified external enzymes of fungi are responsible for initial attacks on hydrocarbons. Delayed addition of bacteria and co-contamination would result in higher growth of fungi which increases pyrene degradation. The addition of Rhamnolipid potently increased the extent of pyrene and tetracosane degradation by approximately 16% and 23% and showed twice better performance than Tween-80 in 20 times less concentration. The results indicated the importance of having sufficient knowledge on the characteristics of the contaminated site and its contaminants as well as oil-degrading species. Gaining this knowledge and using it properly, such as the later addition of bacteria (new method of mixed-cultures inoculation) to the contaminated culture, can serve as a promising approach.

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.

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.

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.

Evaluation of heavy petroleum degradation using bacterial-fungal mixed cultures.

The use of potent microbial mixed cultures is a promising method for the bioremediation of recalcitrant compounds. In this study, eight molds, three yeasts, and four bacterial isolates were screened from an aged oil-polluted area. An oil degradation assay with various combinations including Bacterial Mixed Culture (BMC), Fungal Mixed Culture (FMC), Fungal-Bacterial Mixed Culture (TMC), and Sequential Fungal-Bacterial Mixed Culture (SMC) was investigated. The results indicated that the SMC culture had the highest yield of degradation (65.96%) in comparison with the degradation yields of TMC, FMC and BMC, which were 59.04%, 56.64%, and 47.56%, respectively. The degradation of saturates, aromatics, resins, and asphaltenes in the crude oil found using the Iatroscan system were, as follows: 64.21%, and 67.63% for aromatics, 72.90%, and 73.59% for saturates, and 53.88% and 58.25% for resins with respect to the TMC and SMC cultures as the superior mixed cultures. The growth rates of yeasts, molds, and bacteria in the TMC and SMC cultures were compared for further evaluation of the role of each microorganism in the degradation. Our findings support the use of mixed cultures in the bioremediation of recalcitrant petroleum pollution.

The ability of Silybum marianum to phytoremediate cadmium and/or diesel oil from the soil.

Phytoremediation is a new ecological and cost-effective technology applied for cleaning heavy metals and total petroleum hydrocarbon contaminated (TPH-contaminated) soils. This study was conducted to evaluate the potential of milk thistle (Silybum marianum) to phytoremediate cadmium (Cd (II)) from contaminated soils. To this end, the investigators applied a completely randomized design with the factorial arrangement and four replications. The results indicated that all the evaluated parameters of S. Marianum, including shoot and root fresh and dry weight, as well as shoot and root Cd, were significantly influenced by Cd (II) concentration and diesel oil (DO). The Cd-contaminated soil showed minor declining effects on the produced plant biomass, whereas the DO-contaminated soil had more inhibitory effects. Moreover, the soil contaminated with both Cd and DO led to adverse effects on the plant biomass. The shoot and root Cd concentration had an increasing trend in the presence of DO as the bioconcentration factor (BCF) by 1.740 (+90.78%), 1.410 (+36.89%), 2.050 (+31.41%), 1.68 (+32.28%), and 1.371 (+22.41%) compared to the soil without DO at Cd (II) concentrations of 20, 40, 60, 80, and 100 mg/kg, respectively. Biological accumulation coefficient also showed the same trend as the BCF. In all the treatments, the translocation factor was >1. Therefore, it was demonstrated that milk thistle had high potential for transferring Cd from root to shoot and reducing its concentration in the soil. Moreover, the study revealed that milk thistle had high potential for absorbing Cd in the soil contaminated with Cd and DO.

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.

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.

Construction of a horseradish peroxidase resistant toward hydrogen peroxide by saturation mutagenesis.

Horseradish peroxidase (HRP) with a variety of potential biotechnological applications is still isolated from the horseradish root as a mixture of different isoenzymes with different biochemical properties. There is an increasing demand for preparations of high amounts of pure enzyme but its recombinant production is limited because of the lack of glycosylation in Escherichia coli and different glycosylation patterns in yeasts which affects its stability parameters. The goal of this study was to increase the stability of non-glycosylated enzyme, which is produced in E. coli, toward hydrogen peroxide via mutagenesis. Asparagine 268, one of the N-glycosylation sites of the enzyme, has been mutated via saturation mutagenesis using the megaprimer method. Modification and miniaturization of previously described protocols enabled screening of a library propagated in E. coli XJb (DE3). The library of mutants was screened for stability toward hydrogen peroxide with azinobis (ethylbenzthiazoline sulfonate) as a reducing substrate. Asn268Gly mutant, the top variant from the screening, exhibited 18-fold increased stability toward hydrogen peroxide and twice improved thermal stability compared with the recombinant HRP. Moreover, the substitution led to 2.5-fold improvement in the catalytic efficiency with phenol/4-aminoantipyrine. Constructed mutant represents a stable biocatalyst, which may find use in medical diagnostics, biosensing, and bioprocesses.

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.