Project objectives

1. Chemical and physicochemical analysis of soil (content of biogenic elements, moisture, temperature, pH, salinity) and the contaminant (petroleum hydrocarbons) to assess the conditions for the functioning of the soil microbiome of the Aral Sea region. Soil analysis will be carried out using spectral, chromatographic, gravimetric, and titrimetric methods.

2. Metagenomic studies of the taxonomic, metabolic, and genetic diversity of the native soil microbiome of the Aral Sea region depending on the season (time of year) to assess its biotechnological potential. Soil samples will be collected at different times of the year. The taxonomic, metabolic, and genetic diversity of the native soil microbiome will be evaluated using bioinformatic analysis of metagenomic sequencing data of soil DNA samples.

3. Metagenomic studies of the taxonomic, metabolic, and genetic diversity of the microbiome of oil-contaminated desert soil to assess the impact of oil pollution on the structure and succession of the soil microbiome of the Aral Sea region. Soil samples will be collected before and after oil contamination. The effect of oil pollution on the taxonomic, metabolic, and genetic diversity of the desert soil microbiome will be evaluated using bioinformatic analysis of metagenomic sequencing data of soil DNA samples.

4. Isolation of individual cultures of hydrocarbon-degrading strains as potential biotechnological agents and components of new biopreparations for the remediation of contaminated soils of the Aral Sea region, analysis of their genomes, and assessment of the possibility of expanding their degradative potential (through catabolic plasmids). Pure cultures of hydrocarbon-degrading strains will be isolated using enrichment cultivation and direct plating on selective media. Based on the results of whole-genome sequencing analysis of the most active strains, catabolic genes will be identified and their chromosomal or plasmid localization will be determined. The possibility of enhancing the catabolic capabilities of the most effective hydrocarbon-degrading strains will be investigated by introducing plasmids responsible for the biodegradation of various pollutants.

5. Investigation of biostimulation processes of indigenous microorganisms in soils of the Aral Sea region through the application of mineral nutrient amendments. The effectiveness of biostimulation of hydrocarbon-degrading indigenous microorganisms will be evaluated based on the reduction in total hydrocarbon content after the addition of biogenic elements in the form of phosphorus and nitrogen mineral fertilizers.

6. Investigation of bioaugmentation processes in soils of the Aral Sea region through the introduction of active indigenous hydrocarbon-degrading microorganisms isolated from the soils of the Aral Sea region, or through the application of a previously developed biopreparation (Patent of the Republic of Kazakhstan No. 33715). The effectiveness of bioaugmentation will be assessed based on the reduction in total hydrocarbon content after the introduction of active indigenous hydrocarbon-degrading microorganisms or an effective consortium of oil-degrading strains included in the previously developed biopreparation.

7. Development of a bioremediation strategy for oil-contaminated soils of the Aral Sea region based on the results of evaluating the efficiency of natural attenuation, biostimulation, and bioaugmentation processes. Based on data on the reduction of total hydrocarbon content obtained from field model experiments under conditions of natural attenuation, as well as through the application of biostimulation and bioaugmentation approaches, a strategy for the bioremediation of oil-contaminated soils of the Aral Sea region will be developed.

Expected results

During the first year of the project, an assessment of the taxonomic and metabolic diversity of the native microbiome and the impact of oil contamination on the native soil microbiome will be carried out.

As a result of the first year, data on physicochemical parameters (salinity, moisture, temperature, pH) and the chemical composition of the soil (including the presence and content of biogenic elements (N, P, K)) before and after oil application will be obtained. Data on the chemical composition of oil by fractions and on the oil content in soil during the experiment (0, 14, 30, 60, 180 days) will also be obtained.

Microbiomes of soil samples before and after oil contamination will be sequenced. The results of microbiome analysis will be obtained in the form of raw reads, taxonomically classified data, as well as contigs. Binning will be performed from the contigs, and metagenome-assembled genomes (MAGs) will be reconstructed. Dominant microbiome fractions (taxa) will be identified.

The possibility of isolating and cultivating indigenous hydrocarbon-degrading soil microorganisms under laboratory conditions will be investigated using enrichment cultivation of soil samples in mineral media with hydrocarbon sources of carbon and energy. Data will be obtained on the ratio of the total number of cultivable microorganisms to the number of hydrocarbon-degrading microorganisms, as well as on the impact of oil contamination on these parameters. Pure cultures of the most active hydrocarbon-degrading microorganisms will be isolated for further study.

During the second year of the project, the features of genetic organization and the degradative potential of indigenous strains – promising hydrocarbon degraders – will be investigated.

Data will be obtained on the spectrum of substrates utilized by the most active indigenous hydrocarbon-degrading strains (aliphatic, aromatic, and cyclic non-aromatic hydrocarbons), as well as on the limits of strain survival under different temperatures and salinity levels of the cultivation medium. A collection of the most promising hydrocarbon-degrading strains for bioremediation will be established.

Complete genome sequences of the strains selected as the most promising hydrocarbon degraders will be obtained. Genes and operons involved in hydrocarbon catabolism will be identified in the genomes. The localization of these genes and operons (chromosomal or plasmid) will be determined.

The possibility of transfer of mobile genetic elements (plasmids) containing genes and operons responsible for hydrocarbon catabolism will be investigated, both between representatives of the same genus and between different genera. It is expected that transconjugants will be obtained, their stability will be assessed, and their potential for application in petroleum hydrocarbon degradation will be evaluated.

During the third year of the project, a bioremediation strategy will be developed based on the efficiency of petroleum hydrocarbon biodegradation depending on the outcomes of different approaches tested in field experiments for the remediation of oil-contaminated soil (biostimulation/bioaugmentation).

Dry lyophilized preparations or biomass in the form of concentrated suspensions of each culture will be obtained and used in remediation experiments. The survival rate of the cultures and their purity after concentration and lyophilization will be assessed. The efficiency of petroleum biodegradation under biostimulation (application of mineral fertilizers) due to microbial degradation will be determined. The effect of biostimulation on changes in the native soil microbiome will also be evaluated.

The efficiency of petroleum biodegradation under bioaugmentation (application of mineral fertilizers and reintroduction of indigenous hydrocarbon-degrading microorganisms) as a result of microbial degradation will be determined. The effect of bioaugmentation on changes in the native soil microbiome will be evaluated.

The efficiency of petroleum biodegradation under bioaugmentation (application of mineral fertilizers and a previously developed consortium of hydrocarbon-degrading microorganisms) as a result of microbial degradation will be determined. The effect of bioaugmentation on changes in the native soil microbiome will be evaluated.

Soil microbiomes after field experiments on biostimulation, reintroduction of indigenous hydrocarbon degraders, and application of the previously developed microbial consortium will be sequenced; taxonomic classification and de novo metagenome assembly will be performed. Metabolic pathways of hydrocarbon catabolism will be identified in the metagenomes, and their representation will be evaluated based on sequencing reads.

Based on the obtained results, conclusions on the effectiveness of the remediation approaches will be drawn. The most effective strategy for the bioremediation of oil-contaminated soils of the Aral Sea region will be developed.

1) Based on the results of the conducted research, three articles will be published: one in Microorganisms (Q2, citation index 0.8), one in Frontiers in Microbiology (Q2, citation index 0.96), and one in PLOS One (Q2, citation index 0.91), or in other journals indexed in the Science Citation Index Expanded and ranked in Q1, Q2, or Q3 by impact factor in the Web of Science database or having a CiteScore percentile of at least 60 in the Scopus database, as well as one article in Proceedings of the National Academy of Sciences of the Republic of Kazakhstan. Series of Chemistry and Technology or in other journals recommended by the Committee for Quality Assurance in Science and Higher Education.

2) The publication of monographs, books, and/or book chapters in foreign and/or domestic publishing houses is not planned.

3) Submission of a patent application to the national patent office is planned.

4) The development of scientific, technical, and engineering documentation is not planned.

5) Dissemination of research results among potential users, the scientific community, and the general public will be carried out through participation in international scientific conferences, as well as through national and regional mass media and social networks.

6) Other measurable results in accordance with the requirements of the tender documentation and the specifics of the project. Additionally, the following are specified in this section:

1) Field of application and target consumers of each expected result:
The target consumers of the project results may include oil production and transportation companies, oil refining enterprises, emergency response services, and regulatory authorities. The results obtained during the implementation of this project may be applied in the development of technologies for the remediation of accidental oil and petroleum product spills at oil extraction sites, processing facilities, fuel storage depots, filling stations, airfields, testing grounds, as well as coastal areas contaminated with petroleum products.

2) Impact of the expected results on the development of the main scientific field and related areas of science and technology: The results of this project will make a significant contribution to the development of eco-biotechnology, environmental biotechnology for the protection of ecosystems from anthropogenic pollution, and the development of new biopreparations and technologies for the reclamation of oil-contaminated areas. The project outcomes may also be used in educational programs of universities and research institutions in the fields of Biology, Microbiology, Biotechnology, and Ecology, including lectures, practical training, and the planning of research and development activities in the field of land remediation.

3) Applicability and/or potential for commercialization of the obtained scientific results:
Patent applications for the most effective hydrocarbon-degrading strains or their consortia will be submitted in the Republic of Kazakhstan, enabling their commercialization and the development of original innovative biotechnologies, including the creation of new jobs.

4) Social, economic, environmental, scientific, technical, multiplicative, and/or other effects of the project results, with justification: The results obtained during the implementation of this project will significantly expand current understanding of the development of strategies for bioremediation and reclamation of soils contaminated with oil and petroleum products, and will enhance knowledge, based on metagenomic analysis, of the diversity and succession of soil microbiomes affected by anthropogenic impacts.

5) Other direct and indirect results of the project, including their qualitative and quantitative characteristics:
The project results will be published in high-impact international journals and presented at international and national conferences.

Results

Data on physicochemical parameters (salinity, moisture, temperature, pH) and the chemical composition of the soil (including the presence and content of biogenic elements (N, P, K)) before and after oil application were obtained. Data on the fractional chemical composition of oil and its content in soil during the experiment (0, 14, 30, 60, 180 days) were also obtained.

The detailed and quantitative composition of oil in soil samples (oil from the Akshabulak field was used) was determined by gas chromatography–mass spectrometry (Agilent 7890A/5975C, USA). The number of compounds identified in the oil extracted from contaminated soil samples, after removal of asphaltenes and resins, was 220.

The degree of oil degradation by the studied strains was investigated in a liquid mineral medium containing 2% oil at 24 °C and 5 °C for 10 days. Based on the determination of oil degradation by individual strains, four microbial consortia of effective hydrocarbon degraders were constructed. The degree of petroleum biodegradation by these consortia was determined using gravimetric and IR spectrophotometric methods and ranged from 30% to 70%.

Metagenomic sequencing of the full 16S ribosomal RNA (rRNA) gene has proven to be a powerful strategy for the taxonomic classification of bacteria. The soil metagenome sequencing experiment was conducted as follows. The bacterial 16S rRNA gene contains nine variable regions (V1–V9) separated by highly conserved sequences across different taxa. For bacterial identification, the 16S rRNA gene was first amplified by PCR using primers annealing to conserved regions and then sequenced. The sequencing data were processed by bioinformatic analysis, in which the variable regions were used to discriminate between bacterial taxa. In control (uncontaminated) samples, the presence of Proteobacteria, Acidobacteria, Actinobacteria, and Bacteroidetes was established.

It was found that during the first 15 days after oil application, the proportion of Actinobacteria and Proteobacteria in the microbiome increased, as these groups possess a diverse repertoire of genes involved in the catabolism of alkanes and polycyclic aromatic hydrocarbons (PAHs). The presence of Bacilli was also observed. The taxonomic diversity and community composition at day 60 of the experiment demonstrated a trend toward stabilization and a return of the microbiome to its initial state; this sample was similar in composition to the control. These findings are consistent with literature data indicating that indigenous soil microbiomes exhibit a microbial stress response during the first 2–3 months, after which the microbiome stabilizes to its original state.

In both uncontaminated and oil-contaminated soils under study, the total number of cultivable microorganisms during the 60-day period remained relatively low, amounting to approximately 2–4 × 10⁶ CFU/g of soil. The number of hydrocarbon-degrading microorganisms during the first 30 days was also low, reaching 2–4 × 10² CFU/g of soil (approximately 0.01% of the total cultivable microorganisms). However, by day 60, a slight increase in the number of hydrocarbon degraders was observed, reaching 4–5 × 10³ CFU/g of soil (approximately 0.1% of the total cultivable microorganisms).

Promising cultures of oil-oxidizing microorganisms were isolated, capable of growth on crude oil, diesel fuel, n-alkanes (hexadecane), polycyclic aromatic hydrocarbons (naphthalene), and monocyclic aromatic hydrocarbons (toluene, phenol).