Evaluation of the Effectiveness of the Biopreparation in Combination with the Polymer γ-PGA for the Biodegradation of Petroleum Contaminants in Soil

Biodegradation is a method of effectively removing petroleum hydrocarbons from the natural environment. This research focuses on the biodegradation of aliphatic hydrocarbons, monoaromatic hydrocarbons such as benzene, toluene, ethylbenzene, and all three xylene isomers (BTEX) and polycyclic aromatic hydrocarbons (PAHs) as a result of soil inoculation with a biopreparation A1 based on autochthonous microorganisms and a biopreparation A1 with the addition of γ-PGA. The research used biopreparation A1 made of the following strains: Dietzia sp. IN133, Gordonia sp. IN138 Mycolicibacterium frederiksbergense IN53, Rhodococcus erythropolis IN119, Rhodococcus sp. IN136 and Pseudomonas sp. IN132. The experiments were carried out in laboratory conditions (microbiological tests, respirometric tests, and in semi-technical conditions (ex-situ prism method). The biodegradation efficiency was assessed on the basis of respirometric tests, chromatographic analyses and toxicological tests. As a result of inoculation of AB soil with the biopreparation A1 within 6 months, a reduction of total petroleum hydrocarbons (TPH) (66.03%), BTEX (80.08%) and PAHs (38.86%) was achieved and its toxicity was reduced. Inoculation of AB soil with the biopreparation A1 with the addition of γ-PGA reduced the concentration of TPH, BTEX and PAHs by 79.21%, 90.19%, and 51.18%, respectively, and reduced its toxicity. The conducted research has shown that the addition of γ-PGA affects the efficiency of the biodegradation process of petroleum pollutants, increasing the degree of TPH biodegradation by 13.18%, BTEX by 10.11% and PAHs by 12.32% compared to pure biopreparation A1.

Effect of light-converting coatings on the growth of Sarepta mustard (Brassica juncea L.) plants colonized by associative microorganisms

The effect of colonization by beneficial associative microorganisms Pseudomonas putida KT 2442 and Rhodococcus erythropolis X5 on the growth of Sarepta mustard (Brassica juncea L.) under a covering light-converting material containing organic photoluminophore, in vitro and in vivo, was investigated. The combined use of microbial colonization and photoluminophore coating led to stimulation of plant growth much stronger (2.4 times more) than separately only photoluminophoric coating (1.3 times) or colonization (2.1 times). These data indicate that when covering materials with photoluminophores are used in agrobiotechnologies, luminescent red light (610-730 nm) induces an increase in biochemical processes not only in plants, but also in microorganisms that supply plants with growth regulators and other useful metabolites. The data obtained are relevant for further study of the photobiological mechanisms of interactions between the plant-microorganism system in agrobiotechnologies.

Degradation of Rhodococcus erythropolis SY095 modified with functional magnetic Fe 3 O 4 nanoparticles

Alkali-surfactant-polymer flooding technology is widely employed to extract crude oil to enhance its production. The bacterial strain Rhodococcus erythropolis SY095 has shown high degradation activity of alkane of crude oil. In the past, many treatment strategies have been implemented to reduce oil concentration in wastewater. Previous studies mainly focused on the extracellular products of Erythrococcus rather than its degradation properties. In the current study, we designed an immobilization method to modify the surface of R. erythropolis SY095 with functional Fe 3 O 4 nanoparticles (NPs) for biodegradation of crude oil and separation of the immobilized bacteria after degradation. We characterize the synthesized NPs through various methods, including scanning electron microscope energy-dispersive spectrometer, Fourier transform infrared spectroscopy, X-ray diffraction (XRD) and a vibrating sample magnetometer. We found that the size of the synthesized NPs was approximately 100 nm. Our results showed that R. erythropolis SY095 was successfully coated with functional magnetic NPs (MNPs) that could be easily separated from the solution via the application of an external magnetic field. The coated cells had a high tolerance for heavy metals. Our findings demonstrated that the immobilization of MNPs to bacterial surfaces is a promising approach for the degradation of crude oil.

Evaluation of P450 monooxygenase activity in lyophilized recombinant E. coli cells compared to resting cells

Cytochromes P450 catalyze oxidation of chemically diverse compounds and thus offer great potential for biocatalysis. Due to the complexity of these enzymes, their dependency of nicotinamide cofactors and redox partner proteins, recombinant microbial whole cells appear most appropriate for effective P450-mediated biocatalysis. However, some drawbacks exist that require individual solutions also when P450 whole-cell catalysts are used. Herein, we compared wet resting cells and lyophilized cells of recombinant E. coli regarding P450-catalyzed oxidation and found out that lyophilized cells are well-appropriate as P450-biocatalysts. E. coli harboring CYP105D from Streptomyces platensis DSM 40041 was used as model enzyme and testosterone as model substrate. Conversion was first enhanced by optimized handling of resting cells. Co-expression of the alcohol dehydrogenase from Rhodococcus erythropolis for cofactor regeneration did not affect P450 activity of wet resting cells (46% conversion) but was crucial to obtain sufficient P450 activity with lyophilized cells reaching a conversion of 72% under the same conditions. The use of recombinant lyophilized E. coli cells for P450 mediated oxidations is a promising starting point towards broader application of these enzymes.

Establishing and Optimizing a Bacterial Consortia for Effective Biodegradation of Petroleum Contaminants: Advancing Classical Microbiology via Experimental and Mathematical Approach

In classical microbiology, developing a high-efficiency bacterial consortium is a great challenge for faster biodegradation of petroleum contaminants. In this study, a systematic experimental and mathematical procedure was adopted to establish a bacterial consortium for the effective biodegradation of heavy oil constituents. A total of 27 bacterial consortia were established as per orthogonal experiments, using 8 petroleum-degrading bacterial strains. These bacteria were closer phylogenetic relatives of Brevundimonas sp. Tibet-IX23 (Y1), Bacillus firmus YHSA15, B. cereus MTCC 9817, B. aquimaris AT8 (Y2, Y6 and Y7), Pseudomonas alcaligenes NBRC (Y3), Microbacterium oxydans CV8.4 (Y4), Rhodococcus erythropolis SBUG 2052 (Y5), and Planococcus sp. Tibet-IX21 (Y8), and were used in different combinations. Partial correlation analysis and a general linear model hereafter were applied to investigate interspecific relationships among different strains and consortia. The Y1 bacterial species showed a remarkable synergy, whereas Y3, Y4, and Y6 displayed a strong antagonism in all consortia. Inoculation ratios of different strains significantly influenced biodegradation. An optimal consortium was constructed with Y1, Y2, Y5, Y7, and Y8, which revealed maximum degradation of 11.238 mg/mL OD600 for oil contaminants. This study provides a line of evidence that a functional consortium can be established by mathematical models for improved bioremediation of petroleum-contaminated environment.

Cloning, Amplified Expression and Bioinformatics Analysis of a Putative Nucleobase Cation Symporter-1 (NCS-1) Protein From Rhodococcus erythropolis

The Rhodococcus erythropolis gene DYC18_RS18060 (1437 bp) putatively codes for a secondary transporter of the Nucleobase Cation Symporter-1 (NCS-1) protein family (478 amino acids). The DYC18_RS18060 gene was successfully cloned from R. erythropolis genomic DNA with addition of EcoRI and PstI restriction sites at the 5′ and 3′ ends, respectively, using PCR technology. The amplified gene was introduced into IPTG-inducible plasmid pTTQ18 immediately upstream of the sequence coding for a His6-tag. The construct was transformed into Escherichia coli BL21(DE3), then amplified expression of the DYC18_RS18060-His6 protein was achieved with detection by SDS-PAGE and western blotting. Computational methods predicted that DYC18_RS18060 has a molecular weight of 51.1 kDa and isoelectric point of 6.58. The protein was predicted to be hydrophobic in nature (aliphatic index 113.24, grand average of hydropathicity 0.728) and to form twelve transmembrane spanning α-helices with both N- and C-terminal ends at the cytoplasmic side of the membrane. Whilst database sequence similarity searches and phylogenetic analysis suggested that the substrate of DYC18_RS18060 could be cytosine, this was not certain based on comparisons of residues involved in substrate binding in experimentally characterised NCS-1 proteins. This study has laid foundations for further structural and functional studies of DYC18_RS18060 and other NCS-1 proteins. Copyright(c) The Authors