Soil protection using bacteriaAbstract
At present, the restoration of oil-contaminated lands in arid regions is becoming increasingly relevant and represents one of the key research priorities for scientists in the southern region of Kazakhstan. Addressing the challenges posed by anthropogenic and technogenic impacts on natural ecosystems is an important scientific direction in the region. In this context, a number of researchers are actively engaged in soil microbiology studies aimed at developing innovative approaches and methods that facilitate the successful rehabilitation of disturbed or degraded natural resources. Microbial remediation is a promising technology for the treatment of environmental pollution caused by oil and petroleum products, based on the metabolic potential of microorganisms. Aim of the study: To investigate the remediation process and assess the effect of different petroleum hydrocarbons on the distribution of hydrocarbon-oxidizing and nitrogen-fixing microorganisms. The distribution of hydrocarbon-oxidizing and nitrogen-fixing microorganisms depends on the level and type of soil contamination with petroleum hydrocarbons. Soil contamination with heavy (dark) petroleum fractions leads to a decrease in the abundance of all analyzed groups of microorganisms, whereas the introduction of light petroleum fractions causes a sharp increase in microbial population. It was also established that a bioremediation method using a microbial consortium composed of hydrocarbon-oxidizing and nitrogen-fixing microorganisms, combined with the additional application of 1% mannitol, enhances the efficiency of oil-contaminated soil remediation.
Keywords:
phytoremediation; bacteria; oil; petroleum products; soil
Resumo
Atualmente, a recuperação de solos contaminados por petróleo em regiões áridas está se tornando cada vez mais relevante e representa uma das principais prioridades de pesquisa para cientistas na região sul do Cazaquistão. Enfrentar os desafios impostos pelos impactos antropogênicos e tecnogênicos nos ecossistemas naturais é uma importante linha de pesquisa científica na região. Nesse contexto, diversos pesquisadores estão ativamente envolvidos em estudos de microbiologia do solo, com o objetivo de desenvolver abordagens e métodos inovadores que facilitem a reabilitação bem-sucedida de recursos naturais perturbados ou degradados. A remediação microbiana é uma tecnologia promissora para o tratamento da poluição ambiental causada por petróleo e derivados, baseada no potencial metabólico de microrganismos. Objetivo do estudo: Investigar o processo de remediação e avaliar o efeito de diferentes hidrocarbonetos de petróleo na distribuição de microrganismos oxidantes de hidrocarbonetos e fixadores de nitrogênio. A distribuição de microrganismos oxidantes de hidrocarbonetos e fixadores de nitrogênio depende do nível e do tipo de contaminação do solo por hidrocarbonetos de petróleo. A contaminação do solo com frações pesadas (escuras) do petróleo leva a uma diminuição na abundância de todos os grupos de microrganismos analisados, enquanto a introdução de frações leves do petróleo provoca um aumento acentuado na população microbiana. Foi também estabelecido que um método de biorremediação utilizando um consórcio microbiano composto por microrganismos oxidantes de hidrocarbonetos e fixadores de nitrogênio, combinado com a aplicação adicional de 1% de manitol, aumenta a eficiência da remediação de solos contaminados com óleo.
Palavras-chave:
fitorremediação; bactérias; petróleo; derivados de petróleo; solo
1. Introduction
Soil is traditionally regarded as a unique natural component that regulates interactions between the biosphere, hydrosphere, and atmosphere of the Earth. The soil cover significantly influences and largely determines many processes occurring within the biosphere. In the environment, it performs a buffering function, acting as an absorber, decomposer, and neutralizer of various pollutants (Sozina and Danilov, 2023; Vasilyeva et al., 2020; Babaev, 2019).
Anthropogenic transformations and disturbances of the soil cover may result in its inability to perform essential ecological functions, thereby disrupting the functioning of the biosphere as a whole. Therefore, studying the biological and biochemical condition of soils and their changes under human impact is of particular importance (Tumanyan et al., 2017; Yesentaeva et al., 2024; Zhuniszhan et al., 2024; Tatykhanova et al., 2024).
Oil is considered a valuable energy resource, accounting for approximately 30-35% of global energy consumption. At the same time, the oil extraction and refining industries are among the leading sectors in terms of waste generation. Technological processes involved in oil production, petroleum product manufacturing, transportation, and storage are inevitably associated with losses, sometimes significant. The most severe and hazardous consequences occur during well blowouts, pipeline ruptures, and loss of integrity in transportation systems and oil storage facilities. Such incidents create environmentally dangerous situations, leading to soil degradation, atmospheric pollution, oil infiltration into water bodies, and ultimately adverse effects on human health (Yessentayeva et al., 2024; Gussenov et al., 2023).
A characteristic feature of oil, as a complex mixture of various hydrocarbons, is that when it enters soil or aquatic environments, it undergoes transformation: its hydrocarbon components are subject to partial chemical oxidation and microbial degradation. The study of oil behavior in natural geosystems, aimed at identifying patterns of its transformation under the influence of microbial communities at different trophic levels, is a relevant scientific problem. Such research can serve as a theoretical basis for organizing monitoring systems for oil pollution and for the effective restoration of oil-contaminated biocenoses (Nurzhanova et al., 2020; Mikolasch et al., 2020).
Natural self-purification of environmental objects from oil contamination is a slow process; therefore, the issue of reclaiming oil-contaminated soils, particularly through remediation, is of exceptional importance. A promising technology for cleaning oil-contaminated soils involves the introduction of various microbial consortia characterized by enhanced capacity for biodegradation of specific hydrocarbon components of oil and petroleum products (Mikolasch et al., 2015; Nurzhanova et al., 2025).
Heavy fractions of oil (fuel oil, resins, asphaltenes) although they have less direct toxicity, are highly stable in the soil and cause long-term violations of its physico-chemical and biological properties (hydrophobization, cementation, impaired air exchange). Their priority is determined by their ability to cause irreversible changes in the soil ecosystem.
Compounds capable of they accumulate in trophic chains according to the "soil – plant – animal – human" scheme. The most pronounced bioaccumulation ability is distinguished by individual polycyclic aromatic hydrocarbons and heavy metals, which are often found in the composition of oil in the formof impurities (Goncharova et al., 2021; Ibragimova et al., 2025).
2. Objective of the Study
to assess the effect of different petroleum hydrocarbons on the distribution of hydrocarbon-oxidizing and nitrogen-fixing microorganisms.
3. Materials and Methods
3.1. Soil samples
Laboratory studies were conducted using soils collected from the industrial area and the commercial raw materials workshop (CRM) of PetroKazakhstan Oil Products (PKOP). The soils represent typical medium loamy sierozems with varying levels of petroleum contamination. The main sources of oil and petroleum product pollution are associated with transportation and processing operations, particularly in the areas of tank farms, valve control units, and railway loading racks. Oil refining waste in the form of oil sludge accumulates in sludge storage facilities.
3.2. Oil and petroleum products
The objects of the study included Kumkol crude oil, diesel fuel, fuel oil, oil sludge, and various gasoline grades (AI-80, AI-85, AI-90, AI-96).
3.3. Microorganisms
Hydrocarbon-oxidizing and nitrogen-fixing microorganisms were isolated from oil-contaminated soils collected from the industrial territory of PKOP.
Soil samples for petroleum product content analysis were collected once a month in the presence of representatives of the Environmental Protection Department and a laboratory assistant from the Central Plant Laboratory of PKOP.
Experimental studies aimed at identifying patterns in the distribution of hydrocarbon-oxidizing microflora in oil-contaminated sierozem soils of the Turkistan region were carried out under laboratory conditions using samples from several local sites within the industrial area of PKOP, an enterprise engaged in the processing of Kumkol crude oil.
At the end of the experiment, soil phytotoxicity was assessed in all treatment variants using a biotest with oat and alfalfa seeds. The seeds were предварительно tested for germination capacity. Vegetation was carried out for 14 days. Soil moisture was maintained at 60% of total water-holding capacity by covering the containers with polyethylene film after the initial watering. Soil phytotoxicity was determined based on differences in seed germination energy and morphological parameters of seedlings between contaminated and control soil variants.
Statistical analysis of the results included calculation of the arithmetic mean and standard deviation at a confidence level of 0.95 > P > 0.80. All determinations were performed in three- and fivefold replicates. Data processing was carried out using a personal computer (IBM Pentium) with the application software package Microsoft Excel.
4. Results and Discussion
Microorganisms are highly sensitive to environmental changes of various origins, which determines the high mobility and dynamic nature of microbiological indicators. The processes of chemical and biological transformation of hydrocarbons in soil are influenced by numerous factors, including the concentration and composition of oil contamination, the qualitative and quantitative composition of soil microflora, the mineral and organic composition of the soil, as well as temperature, moisture, aeration, and pH.
The results of the study demonstrated that the distribution of hydrocarbon-oxidizing microorganisms across soil horizons is heterogeneous and correlates with the molecular weight of the contaminating petroleum products (Figure 1).
Based on the obtained data, it was determined that in soils contaminated with crude oil, fuel oil, and sludge-like wastes (heavy fractions) containing asphaltenes and maltenes, the highest number of hydrocarbon-oxidizing microorganisms (HOM) was concentrated in the 10-20 cm and 20-30 cm horizons. Analysis of petroleum product content in soil samples collected from different horizons showed that the highest concentrations were detected at depths of 10-20 cm and below.
In contrast, in areas contaminated with diesel fuel (primarily containing maltenes) and various gasoline grades (light fractions), both the petroleum product content and microbial counts were highest in the 0-10 cm horizon. The highest microbial titers were recorded in soil samples contaminated with different gasoline grades.
Heterotrophic bacteria in soil carry out the full range of organic matter decomposition reactions and serve as indicators of the soil’s functional state. Several authors have reported that petroleum contamination may stimulate bacterial activity within a certain concentration range of the pollutant, whereas exceeding a critical contamination level may inhibit the activity of the bacterial community.
Our studies showed that bacterial cultures isolated from contaminated soils were represented by the generaMicrococcus,Bacillus, andPseudomonas. The obtained results were consistent with previously published data. It was established that the highest hydrocarbon-oxidizing activity was exhibited by hydrocarbon-oxidizing microorganisms in association with nitrogen-fixing microorganisms isolated from sludge-like wastes. These microorganisms actively utilized hexadecane, benzene, naphthalene, diesel fuel, crude oil, and fuel oil.
As a result, 12 microbial cultures capable of actively using petroleum products as the sole carbon source were selected. Three strains belonging to the genusMicrococcuswere tested for non-pathogenicity and non-allergenicity to humans and warm-blooded animals at the South Kazakhstan Regional Branch of the Republican Veterinary Laboratory. These strains were identified as pathogenic and therefore excluded from further study.
The remaining eight cultures, confirmed to be non-pathogenic and non-allergenic, were classified according to their taxonomic characteristics as follows: B1Ag16G, B1Ag8G, and B1Ag6G – genusMicrococcus; G311/1 – genusBacillus; GR11, GR21, GR35, and GR149 – genusAzotobacter.
Microbial growth on nutrient media containing the above-mentioned hydrocarbons was evaluated using a four-point scale: 0 – no growth; 1 – weak growth; 2 – moderate growth; 3 – intensive growth.
Strain B1Ag6G grew intensively on hexadecane, moderately on benzene, naphthalene, and diesel fuel, and showed no growth on fuel oil. Strain G 311/1 grew intensively on hexadecane, weakly on benzene and naphthalene, moderately on diesel fuel, and did not grow on fuel oil.
Strain GR21 exhibited weak growth on mineral media containing hexadecane, benzene, and diesel fuel; weak growth was also observed on media with fuel oil, naphthalene, and crude oil. Strain GR35 grew intensively on hexadecane, moderately on benzene, naphthalene, and crude oil, weakly on diesel fuel, and did not grow on fuel oil. Strain GR149 demonstrated intensive growth on hexadecane and weak growth on benzene, naphthalene, diesel fuel, crude oil, and fuel oil.
Strain GR35, which intensively utilized hexadecane and showed moderate growth on other hydrocarbons, was further tested for its ability to grow on Voroshilova–Dianova medium containing benzo[a]pyrene, xylene, toluene, and oil sludge as hydrocarbon sources. After seven days, visual changes were observed in the variants containing benzo[a]pyrene, toluene, and xylene, including turbidity, changes in medium color, and disappearance of the oily film. In the variant containing oil sludge, the changes were insignificant.
Chemical analysis showed that the reduction in petroleum product concentration began on the third day. It was established that under aeration alone (as an abiotic factor), xylene content decreased by 5.0%, whereas the introduction of nitrogen-fixing microorganisms reduced petroleum product concentration by 52.0%.
The experiments assessing the growth capacity of different microbial groups on selective media supplemented with petroleum products demonstrated that, in addition to hydrocarbon-oxidizing microorganisms, strains ofAzotobacter chroococcum(GR11, GR21, GR35, GR149) showed a positive dynamic in reducing petroleum product concentrations, indicating their ability to utilize petroleum hydrocarbons as readily available carbon sources.
Morphological, physiological, and biochemical characteristics of the isolated strains were determined and are presented in Table 1.
Thus, hydrocarbon-oxidizing and nitrogen-fixing microorganisms isolated from oil-contaminated soils of the industrial zone of PetroKazakhstan Oil Products (PKOP) were identified as follows:Micrococcus varians B1Ag16G, Micrococcus luteus B1Ag8G, Micrococcus roseus B1Ag6G, Bacillus subtilis G311/1, and Azotobacter chroococcumstrains GR11, GR21, GR35, and GR149.
In laboratory conditions, the potential use of the following cultivated plants as phyto-test objects was studied: garden bean (Phaseolus sp.) and alfalfa. The choice of these crops was determined by their rapid germination and large seedling biomass.
It was found that plants respond more sharply to soil contamination by the light fractions of petroleum products. It was observed that, regardless of the gasoline brand, a soil content of up to 0.1% causes complete seed death.
Diesel fuel and crude oil exhibit less toxic effects. It was established that their concentration in soil up to 0.1% has a stimulating effect on plant development, with all experimental plants exceeding the control samples by 1.75 ± 0.5 cm in height and by 0.2 ± 0.05 g in biomass.
Analysis of soil phytotoxicity levels in the studied experimental variants showed that the control variant, where no agrotechnical measures were applied, had the lowest seed germination and the highest phytotoxicity levels. The lowest toxicity level was noted in the second variant, which received a minimal initial dose of mineral fertilizers (Table 2). The high toxicity in petroleum-contaminated soil may be due to the accumulation, in early stages of microbiological degradation, of large amounts of petroleum acids and other primary degradation products of oil, which have a high degree of toxicity to both plants and most microorganisms.
At the same time, the absence of visible chlorosis in plants in this variant is an indirect indicator that the soil, despite the minimal total amount of nitrogen fertilizers used in the experiment, contained a sufficient amount of nitrogen, possibly due to active nitrogen fixation processes.
Thus, the development of a reclamation scheme, and particularly the calculation of nitrogen fertilizer doses, should begin with an analysis of the activity level of nitrogen-fixing microflora in the specific petroleum-contaminated soil. In cases of high nitrogen fixation activity, applying high initial doses of mineral nitrogen fertilizers is inadvisable. Although such doses may initially reduce petroleum content in the soil, they can subsequently sharply suppress the rate of petroleum biodegradation and increase soil phytotoxicity, which may negatively affect the effectiveness of subsequent phytoremediation.
5. Conclusions
The distribution of hydrocarbon-oxidizing and nitrogen-fixing microorganisms depends on petroleum hydrocarbon contamination in the soil. Contamination with the dark fractions of petroleum products leads to a decrease in the population of all analyzed groups of microorganisms, whereas the introduction of light fractions into the soil triggers a sharp increase in microbial numbers.
It was also established that a bioremediation method for petroleum-contaminated soils using a microbial consortium composed of hydrocarbon-oxidizing and nitrogen-fixing microorganisms, along with the addition of 1% mannitol, enhances the degree of soil decontamination.
Data Availability Statement
Research data is only available upon request.
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Edited by
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Editor:
Takako Matsumura Tundisi
