CC BY
3UDK 504.53.054
IRSTI 87.21.23
DOI 10.56525/ACYI2155
THE EFFECT OF BIOAUGMENTATION
OF PETROLEUM CONTAMINATED
SOIL BY MICROORGANISMS
IMMOBILIZED ON MINERAL CARRIERS
*KHOZHANEPESSOVA F.
Sh.Yessenov Caspian state university
of technology and engineering, Аktau, Kazakhstan
E-mail: fariza_eco@mail.ru
SERIKBAYEVA A.
Sh.Yessenov Caspian state university
of technology and engineering, Аktau, Kazakhstan
E-mail: akmaral.serikbayeva@yu.edu.kz
*Correspondence: fariza_eco@mail.ru
Abstract. Oil pollution may arise either accidentally or operationally whenever oil is produced, transported, stored and processed or used at sea or on land. Oil spills are a major menace to the environment as they severely damage the surrounding ecosystems. To improve the survival and retention of the bioremediation agents in the contaminated sites, bacterial cells must be immobilized. This work presents the results of studies on the oil-oxidizing activity of microorganisms-oil-degraders cells free and immobilized on mineral sorbents of cells, carried out in the field at the Karazhanbas field in the Mangistau region. The highest oil destruction activity was observed in microorganisms-oil destructors immobilized on expanded clay.
Keywords: Bacteria, biodegradation, crude oil, soil, immobilization.
Introduction
Oil produced in Western Kazakhstan is highly paraffinic, with a high content of mercaptan compounds, which negatively affects the oil spill on the physicochemical parameters of soils, forming powerful bituminous crusts in the soil profile [1]. The process of oil degradation in soil under natural conditions is a complex physicochemical and biochemical process, the direction and speed of which depend on the climate, soil properties and regimes, seasonal microflora activity, moisture, concentration and fractional composition of oil in the soil. The process of biodegradation in the soil proceeds slowly, over a long time, more than 20-25 years [2, 3]. Therefore, the management of hydrocarbon biodegradation processes should be aimed, first of all, at activating microbial communities and creating optimal conditions for their existence. The use of microorganisms-oil destructors immobilized on various sorbents of cells and the creation of stable destructors of oil, with guaranteed functional stability in the environment, allows us to expand the application of the microbiological method in eliminating hydrocarbon contaminants and to further increase efficiency and reduce soil cleaning time. Cells attached to the carrier have increased viability, resistance to the action of adverse environmental factors, increased catalytic and oil-oxidizing activity, due to the high concentration of microorganism cells [4]. And the carrier itself, thanks to the sorption capacity, allows rapid adsorption of the toxic substrate, preventing its migration to the underlying layers, improves the aeration of the medium and, thanks to the microorganisms immobilized on it, allows the carbon of petroleum hydrocarbons to be assimilated by biochemical transformation into compounds that are safe for humans and the environment [5].
In this regard, the aim of this work was to study the oil-oxidizing activity of free and immobilized cells of destructive microorganisms in arid conditions at the Karazhanbas field in the Mangistau region.
Materials and methods of research
A field experiment to test the Rhodococcus erythropolis Str1 and Rhodococcus ruber Str4 hydrocarbon-oxidizing microorganism strains immobilized on mineral carriers was established in a stationary area in Aktau, Mangistau area. For the experiment, heavily contaminated soil from the Karazhanbas field was used. The site was planned for 36 experimental plots, the area of which was 1x1 m2, the experience was laid down in accordance with randomization [6].
The control was oil-contaminated soil without introducing microorganisms and with the introduction of mineral carriers of zeolite or expanded clay into the oil-contaminated soil (control, control + z, control + exp.cl). Also, variants with suspension of free cells of strains of the microorganisms Rhodococcus erythropolis Str1 and Rhodococcus ruber Str4 (Str1, Str4), immobilized on zeolite (Str1+z, Str4 + z) and immobilized on expanded clay (Str1 + exp.cl, Str4+exp.c).
The biomass of 2 active strains of microorganisms of the genus Rhodococcus with a cell titer of 3-5 × 109 CFU / g was accumulated. The experiment was laid in 3 replicates, in the following variants: with the introduction of only free or immobilized cells of hydrocarbon-oxidizing microorganisms immobilized on zeolite and expanded clay. The soil before and after inoculation with its microorganisms was thoroughly loosened and moistened.
To determine the change in oil content in the soil, soil sampling was carried out before laying, in the middle and at the end of the field experiment. Soil sampling was carried out according to established methods for the selection and preparation of soil samples for microbiological and chemical analysis [7].
The oil content in the soil was determined by the gravimetric method after extraction with chloroform [8].
The dynamics of the number of hydrocarbon-oxidizing microorganisms (HOM) was determined in soil samples by the method of limiting dilutions followed by seeding on a Voroshilov-Dianova agar medium; the Karazhanbas oil was used as the sole source of carbon and energy [7].
Statistical processing was performed using the Excel application package.
Research results
Determination of the oil content in the soil of the experimental section of the Karazhanbas deposit before inoculation with its microorganisms showed a high degree of contamination. The oil content in the soil ranged from 58.2 to 69.2 g / kg of soil, while the presence of oil in uncontaminated background soil was not established.
Soil inoculation was carried out by a suspension with free cells of the hydrocarbon-oxidizing microorganisms Rhodococcus erythropolis Str1 and Rhodococcus ruber Str4, and immobilized on zeolite and expanded clay. Then the soil in the plots was loosened and moistened. The change in the oil content in the soil of the field experiment was determined by the gravimetric method after 30 and 60 days; the changes in the total microbial number (TMN) and the number of hydrocarbon-oxidizing microorganisms (HOM), and the change in the activity of the soil enzyme catalase were also monitored.
During the experiment, a decrease in the content of oil hydrocarbons was observed in the soil of the control plots, which can be explained by the activity of the soil microbial community and the partial evaporation of oil fractions. So after 30 days in the soil of the control variant, the destruction of oil was 3.3%, after 60 days - 7.2%. In the soil in areas with only zeolite or expanded clay added, after 30 days the destruction of oil was 6.1% and 9%, respectively, on the 60th day - 10.9% and 12.9%, respectively (Figure 1).
Figure 1 - Oil destruction in the soil of a field experiment
In the soil of the experimental plots with the suspension of free cells of the microorganisms Rhodococcus erythropolis Str1 and Rhodococcus ruber Str4, the destruction of oil for 30 days was 48.2% and 43.4%, and after 60 days it was 62.7% and 58.6%, respectively.
Whereas, upon inoculation of soil with cells of strains of microorganisms Rhodococcus erythropolis Str1 and Rhodococcus ruber Str4, immobilized onto zeolite, oil degradation was observed at 60 and 57.7% on day 30 and 75.8 and 71.8%, respectively, on day 60. A high percentage of oil destruction was noted in the variants when these strains were immobilized on expanded clay introduced into the soil. So for 30 days, the destruction of oil by microbial strains immobilized on expanded clay was 64.9 and 61.1%, and after 60 days, 80.2 and 76.8%, respectively.
According to the results obtained after 60 days, it was noted that the cells of the hydrocarbon-oxidizing microorganisms Rhodococcus erythropolis Str1 and Rhodococcus ruber Str4, immobilized to expanded clay cells had the highest oil-oxidizing activity, and the decrease in the concentration of hydrocarbons in the soil was 5.4 and 4.3 times lower, respectively. In the embodiment, when introducing strains of microorganisms immobilized onto a zeolite, a decrease in oil content was observed in comparison with the initial indicator by 4.1 and 3.5 times, then introduced in a free state reduced the oil content by 2.6 and 2.4 times, respectively.
To control the effect of introduced microorganism strains on the soil condition, soil samples were taken from all sections of the field experiment in order to determine changes in TMN and the number of HOM. In the soil of the field experiment, before introducing the strains of Rhodococcus erythropolis Str1 and Rhodococcus ruber Str4, the TMN of the soil was 1.3 × 103 CFU / g of soil and the abundance was 1.2 × 102 CFU / g of soil. The analysis of soil samples from the field experiment after 30 and 60 days for TMN and the number of HOM showed an increase in the number of microorganisms.
When only mineral sorbents were introduced into the soil after 30 and 60 days of the experiment, an increase in the number of TMN and HOM by 1 order compared to the initial value was observed. In the variants with suspension of free cells of the hydrocarbon-oxidizing microorganisms Rhodococcus erythropolis Str1 and Rhodococcus ruber Str4, after 30 days, an increase in TMN and HOM by 1 order was observed, after 60 days, TMN was increased by 3 orders of magnitude, HOM - by 2 orders of magnitude compared to the initial values. Then, after 30 days, in the variants with the introduction of microorganisms immobilized to zeolite and expanded clay, an increase in both the TMN and the number of HOM by 1 order compared with the initial indices was observed, and after 60 days, an increase in both TMN and the number of HOM was noted by 3 orders of magnitude.
In the control variant, without the introduction of microorganisms, an increase in the number of HOMs was noted by 1.2 times, and in the experimental plots with the introduction of microbial cells immobilized onto mineral sorbents, the number of HOMs increased by 3 times (Table 1).
Table 1 - Microbiological analysis of the soil of a field experiment, CFU/g of soil
Experiments options Total microbial number (TMN) hydrocarbon-oxidizing microorganisms (HOM)
30 days 60 days 30 days 60 days
Background value (8,00±0,12)×103 (2,75±0,30)×103 (2,23±0,16)×102 (1,62±0,32)×103
Control (1,05±0,24)×103 (2,75±0,30)×103 (1,32±0,23)×102 (1,93±0,43)×102
Control+zeolite (1,65±0,24)×103 (2,09±0,19)×104 (2,62±0,12)×102 (1,03±0,37)×103
Control+expan ded clay (2,63±0,22)×103 (4,24±0,28)×104 (4,73±0,22)×102 (1,59±0,22)×103
Free cells of microorganisms
Rhodococcus erythropolis Str1 (2,73±0,16)×104 (3,14±0,42)×106 (8,42±0,20)×103 (2,57±0,47)×104
Rhodococcus ruber Str4 (2,27±0,21)×104 (2,21±0,24)×106 6,36±0,28)×103 (1,97±0,20)×104
Immobilized Cells on Zeolite
Rhodococcus erythropolis Str1 (3,83±0,22)×104 (2,57±0,84)×106 (7,16±0,44)×103 (2,75±0,47)×105
Rhodococcus ruber Str4 2,45±0,13)×104 (1,60±0,27)×106 (5,98±0,36)×103 (2,14±0,17)×105
Immobilized cells for expanded clay
Rhodococcus erythropolis Str1 (5,17±0,26)×104 (3,63±0,42)×106 (8,25±0,14)×103 (3,68±0,33)×105
Rhodococcus ruber Str4 (4,62±0,63)×104 (2,84±0,27)×106 (7,39±0,17)×103 (3,04±0,51)×105
Conclusion
The determination of TMN and HOM at the experimental sites during soil cleaning using free and immobilized cells of the strains of hydrocarbon-oxidizing microorganisms Rhodococcus erythropolis Str1 and Rhodococcus ruber Str4 after 60 days of the experiment showed an increase in TMN by 3 orders of magnitude, and the number of HOMs when introducing free cells by 2 orders of magnitude, in variant with the introduction of strains of microorganisms immobilized on mineral carriers by 3 orders of magnitude.
Thus, field studies at the Karazhanbas deposits in the Mangistau region showed that the use of strains of hydrocarbon-oxidizing microorganisms Rhodococcus erythropolis Str1 and Rhodococcus ruber Str4 immobilized on mineral carriers accelerates the destruction of oil in the soil and, most effectively, their immobilization with oil at cer after 60 days, it reached 80.2 and 76.8%, respectively, which is 1.3 times more compared to free cells. The introduction of strains of microorganisms immobilized onto expanded clay into the soil also affected the increase in the number of microorganisms in the soil, so the number of TMN increased by 3 orders of magnitude, HOM by 2 orders of magnitude, an increase in the activity of the soil enzyme catalase by almost 6 times compared to the initial value.
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1Хожанепесова Фариза, 1Серикбаева Акмарал
1Ш. Есенов атындағы Каспий технологиялар және инжиниринг университеті,
Ақтау қ., Қазақстан
ҚҰРАМЫНДА МҰНАЙЫ БАР ТОПЫРАҚТЫ МИНЕРАЛДЫ ТАСЫМАЛДАУШЫЛАРДА ИММОБИЛИЗАЦИЯЛАНҒАН МИКРООРГАНИЗМДЕРМЕН БИОАГМЕНТАЦИЯЛАУДЫҢ ӘСЕРІ
Аңдатпа. Мұнайдың ластануы кездейсоқ немесе пайдалану нәтижесінде пайда болуы мүмкін, мұнай теңізде немесе құрлықта өндірілген, тасымалданған, сақталған және өңделген немесе пайдаланылған кезде. Мұнайдың төгілуі қоршаған ортаға үлкен қауіп төндіреді, өйткені олар қоршаған экожүйелерге айтарлықтай зиян келтіреді. Ластанған жерлерде биоремедиация агенттерінің өмір сүруін және сақталуын жақсарту үшін бактериялық жасушаларды иммобилизациялау керек. Бұл жұмыста микроорганизмдердің мұнай Қышқылдандыру белсенділігін зерттеу нәтижелері ұсынылған-Мұнай бұзушы жасушалар, Маңғыстау облысындағы Қаражанбас кен орнында дала жағдайында жүргізілген минералды сорбенттерде бос және иммобилизацияланған жасушалар. Ең жоғары мұнай өткізгіштік белсенділігі кеңейтілген сазға иммобилизацияланған микроағзаларда байқалды.
Түйінді сөздер: бактериялар, биодеградация, шикі мұнай, топырақ, иммобилизация.
1Хожанепесова Фариза, 1Серикбаева Акмарал
1Каспийский университет технологии и инжиниринга им. Ш. Есенова,
г. Актау, Казахстан
ЭФФЕКТ БИОАУГМЕНТАЦИИ ЗАГРЯЗНЕННОЙ НЕФТЬЮ ПОЧВЫ МИКРООРГАНИЗМАМИ, ИММОБИЛИЗОВАННЫМИ НА МИНЕРАЛЬНЫХ НОСИТЕЛЯХ
Аннотация. Загрязнение нефтью может возникать как случайно, так и в результате эксплуатации всякий раз, когда нефть добывается, транспортируется, хранится и перерабатывается или используется на море или на суше. Разливы нефти представляют серьезную угрозу для окружающей среды, поскольку они наносят серьезный ущерб окружающим экосистемам. Чтобы улучшить выживаемость и удержание агентов биоремедиации на загрязненных участках, бактериальные клетки должны быть иммобилизованы. В данной работе представлены результаты исследований нефтеокисляющей активности микроорганизмов-нефтеразрушающих клеток, свободных и иммобилизованных на минеральных сорбентах клеток, проведенных в полевых условиях на месторождении Каражанбас в Мангистауской области. Наибольшая нефтедеструктивная активность наблюдалась у микроорганизмов-нефтедеструкторов, иммобилизованных на керамзите.
Ключевые слова: Бактерии, биодеградация, сырая нефть, почва, иммобилизация.
