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ФИЗИКО-ХИМИЧЕСКАЯ БИОЛОГИЯ
УДК 631.417:581.142
THE CHANGE OF SOIL HUMIC SUBSTANCE (HS) AND HEAVY CARBON ISOTOPE (513С) IN SOIL AND SOIL MICROORGANISMS OVER IN VITRO BIOREMEDIATION OF OIL-SPILLED SOIL
L.I. Akhmetov, A.N. Shkidchenko, A.A. Andreev
Bioremediation of oil-contaminated soil by treatment with a consortium of active oil-degrading microorganisms- did not lead to complete biodegradation of crude oil, regardless of the duration of the experiment (up to 400 days) and the periodic additional application of the biopreparation. As the biodegradation of crude oil in the soil increased the content of hu-mic substances (GS), while the content of soluble GS increased by a factor of two, and the content of insoluble GS increased by a factor of three. Using mass spectrometry, the fractionation of carbon isotopes of oil hydrocarbons by microorganisms was established. Heavy isotopes (S!313C) are included into the biomass of microorganisms, and light ones (S!2C) are released in the form of CO2. The isotopic composition of the treated soil increased from -29.94 to -29.18%o, which confirms the effect of fractionation of carbon isotopes by microorganisms and, possibly, causes incomplete biodegradation of oil components in the soil.
Key words: soil, humic substance, carbon isotope, microorganisms, crude oil, degradation.
Introduction
One of the most hazardous environmental pollutants, due to its properties and extent of use, is crude oil (Robertson and Hansen, 2015). A ton of crude oil pollutes 12 km2 of the water surface, and one liter of oil deprives oxygen of 40 thousand liters of water. When the oil concentration in the water is 0.1-0.01 mg/l, fry die in a few days (O'Shaughnessy et al. 2018). Of the transported 2 billion tons of oil , 0.1 to 0.5 % goes to the ocean legal due to flushing tankers (https://www. statista.com/statistics/264013/transport-volume-of-crude-oil-in-seaborne-trade/). According to (Clark, 2001; EEA, 2007, p. 232), about 4.63 million tons of oil products per year fall into the sea as a result of transport losses (0.163 million tons due to operating losses of tankers and 0.162 million tons from tanker accidents) and 0.18 million tons from drilling rigs.
Emergency oil spills are systematically occurring in Russia, due to both deterioration of equipment and non-compliance with technical discipline. If we estimate oil losses at 1.5 % of production, they will exceed 5 million tons annually. The territories in which oil is extracted and refined, and over which oil pipelines run, amount to hundreds of thousands of square meters.
Spilled oil is sorbed by the soil and localized mainly in its upper horizon (Matveeva, Lipatov, 2015; Seredina et al., 2017; Rahman et al., 2010; Oluremi et al., 2015). Low boiling oil fractions, as a rule, evaporate, especially in the
summer (up to 15 % per year). Heavy oils and especially heavy fractions (for example, asphaltenes) practically do not erode and rather slowly penetrate deep into the soil.
To date, the use of consortia consisting of oil-degrading microorganisms (bacteria and yeast) has become an alternative to physicochemical and mechanical methods for eliminating oil contaminants from the soil, but complete (100 % or close to this) utilization of oil hydrocarbons in the soil is not achieved (Filonov et al., 2012; Delegan et al., 2016; Korshunova et al., 2016 and others).
The objective of the study was to determine the cause of the incomplete destruction of oil hydrocarbons in contaminated soil, as well as to assess the relationship of this reason with the potential return of soils to turnover.
Materials and methods
Objects of study. We used strains of psychrotrophic oil-degrading microorganisms from the collection of the Laboratory of Plasmid Biology, IBPM RAS, pre-selected in a three-stage flow culture system and identified as Microbacterium liquiefaciens 6, Pseudomonas sp. 22 and Rhodococcus erythropolis 21 [Shkidchenko and Akhmetov, 2013].
For model soil systems, gray forest soil was used (Table 1), sampled in the vicinity of Pushchino (Moscow Region, Russia).
Table 1
Soil properties
Water extract pH Ash, % Corganic , % Ntotal, % Ptotal, % Ca, % Mg, % Fe, % K, % SiO2, %
7,05 91,00 2,89 0,25 0,06 0,48 0,14 1,20 2,47 72,5
Crude oil (5 % v/w) was introduced into the soil in the form of a solution in chloroform and thoroughly homogenized. Chloroform from the soil was removed by evaporation in a fume hood at room temperature for one day with periodic soil mixing. Samples of the soil prepared in this way (1 kg each) were placed in plastic containers with a layer of 10-12 cm. The system was nonsterile, open, the introduced hydrocarbons were also not sterilized. All experiments were performed in triplicate at 6°C for 400 days with periodic sampling for analysis 30 days before the next introduction of microorganisms-destructors.
Soil moisture throughout the experiment was maintained at 60 % of the total moisture capacity. The soil was inoculated with microorganisms-oil destructors grown in a liquid nutrient medium [Ivanova et al., 2006].
Quantitative determination of oil hydrocarbons was carried out by the gravimetric method. To perform, 5 g of the soil sample was poured into 50 ml of
chloroform, the hydrocarbons were extracted by shaking for 30 min, and analyzed according to the described procedure [Shkidchenko et al., 2007].
The carbon isotopic composition was analyzed using a BREATMATplus mass spectrometer (Finnigan MAT GmbH, Germany), which has a two-channel system for introducing analyzed gases and two-collector detection of ion currents. The amount of carbon was measured in the initial substrate (hydrocarbons) and in the organic matter of the soil after extraction of hydrocarbons. Quantitatively, the carbon isotopic composition was expressed in relative units of 513C%o using the expression
where Rs, Rst. - prevalence ratios 13CO2, 12CO2 in the sample and the international standard PDB, respectively
As the analyzed gas, we used CO2 obtained by burning hydrocarbon substrates, as well as obtained by burning soil samples, from which these hydrocarbons were previously extracted.
The content of total carbon and HS in soil samples was determined by the Tyurin method in the modification of Ponomareva-Plotnikova [Ponomareva and Plotnikova, 1980].
Previously, during non-sterile flow-through cultivation of active oil-degrading microorganisms in a battery of three bioreactors (the first carbon source was diesel fuel, the second and third oil) it was shown that diesel fuel was consumed to a threshold concentration (chemostat), but the total oil consumption did not occur regardless cultivation duration and the number of microorganisms. The oil configuration changed and an emulsion formed instead of the oil film (Akhmetov et al., 2006), probably as a result of the synthesis of biosurfactants by microorganisms-oil destructors.
The interaction of oil components with soil is more complex. It was shown that immediately after the introduction of oil hydrocarbons into the soil (before interaction with microorganisms), the content of diesel fuel determined during extraction with chloroform decreases by 25 %, crude oil - by 16 % (Shkidchenko et al., 2007).
Pyrolysis-GC and pyrolysis-GC-MS methods applied to oil-contaminated soil and extracted hydrocarbons fully confirmed the quantitatively irreversible sorption of hydrocarbons by humic substances of the soil (Andrus et al., 2003). The reason for this is the ability of the aromatic framework of humic substances to bind organic compounds, which occurs by the mechanism of hydrophobic interactions. As the contribution of the aromatic framework of humic substances increases, their affinity for hydrophobic organic compounds increases. Binding of petroleum hydrocarbons with humic substances is equivalent to the process of their transfer from the free state and reducing the
Results and Discussion
availability of the products formed for microorganisms. Humic substances give the necessary nutrients to living organisms gradually, as they are consumed, thereby preserving the necessary supply of these elements. In practice, this means that humic substances are responsible for the life support of soil biota.
In a long-term model experiment on the biodegradation of crude oil in the soil at 6°C, complete utilization was not observed even after 400 days: from the initial introduced 5 % of oil (v/w) at the end of the experiment, 2.85 % of the soil remained (Fig. 1).
1e+10
1e+9 -
HS soluble I I HS insoluble
microbial number crude oil content
(D
E 1e+8
1e+7
1e+6
100
200 300
t, days
400
4,6
- 4,4
- 4,2
- 4,0
- 3,8
£
I" 3,6 ~
<D
h 3,4 с
о о
о
- 3,2
- 3,0
- 2,8 2,6
500
Fig. 1. Dynamics of the number of heterotrophs (dot line), the amount of residual oil (solid line) and humic substances (diagram) in the soil during
the model experiment
Additional inoculation of the soil with active strains-oil destructors every 30 days also did not lead to an increase in the degree of biodegradation of oil. The initial number of heterotrophic microorganisms was created by additional inoculation of oil-destroying microorganisms at the beginning of the 30-day cycle. At the same time, the content of oil destructors did not exceed 50 % of the total number of heterotrophs. A gradual decrease in the number of heterotrophs with a decrease in the content of petroleum products in the soil occured due to limiting the growth of oil destructors when they change the content of available carbon sources in the soil (Fig. 2).
In the model soil, oil components of various degrees of condensation affected the humic complex of the soil as follows (Table 2). In the case of fuel
oil, the value of the HS1 fraction is reduced by 50 %, the HS2 fraction remains unchanged, the HS3 is reduced by 50 %, and the content of humin (a non-extractable source of soil carbon) increases five times. In the case of crude oil, the fraction of HS 1 is reduced by 30 %, the fraction HS2 reduced by 100 %, HS3 decreases by 30 %, the humin content increases by 4.75 times in the case of diesel fuel HS2 fraction is reduced by 95 %, HS3 - by 40 %, the humin content grows 3.5 times. Previously, it was shown that light fractions of crude oil inhibit the development of biota in the soil (Shkidchenko, 2016).
Fig. 2. The survival of heterotrophic microorganisms in the soil before and after reinoculation (after 30 day) of oil-degrading bacteria
In general, during the process of oil biodegradation, the content of determined humic substances in the soil increases in the case of soluble humates and gradually decreases for insoluble ones, and the total content of humates in the soil grows several times.
Blocking of the soluble fraction of the humic complex of the soil by light oil fractions causes the need of additional amendment of nitrogen and phosphorus sources simultaneously with biopreparations for bioremediation of oil-polluted soil. It is well known that organic substances entering the soil are decomposed by heterotrophic microflora, forming microbial biomass and carbon dioxide, and the residues of their vital activity form the humus complex of the soil.
Previously, it was found that carbon dioxide, formed as a result of the life of microorganisms (biogenic), inhibits their growth, and it is not used by heterotrophic microorganisms (Shkidchenko, 1976). Interestingly, the isotopic composition of the biomass of microorganisms-oil destructors does not depend on the degree of condensation of oil hydrocarbons. It is shown that such microorganisms fractionate the carbon of petroleum products and heavier carbon isotopes are included in the biomass; the formed abiogenic carbon dioxide contains lighter carbon isotopes (Zyakun et al., 1986).
Table 2
Influence of crude oil hydrocarbons on humic complex of the soil
Humic substances content, % CNon-extгacted, % CTotal, %
HS1 fraction HS2 fraction HS3 fraction s
Control 0,33 0,16 0,17 0,66 0,94 1,60
Black oil 5 % 0,17 0,17 0,08 0,42 4,80 5,22
Crude oil 5 % 0,22 0 0,11 0,33 5,02 5,35
Diesel fuel 5 % 0,23 0,03 0,10 0,36 2,79 3,15
Crude oil contains more than 400 chemical compounds, but the biomass of microorganisms has a constant isotopic composition. So, when microorganisms are grown in oil, diesel fuel or fuel oil, the isotopic composition of 513C biomass is from -30.24 to -30.26%o with different isotopic composition of the substrate, which is an additional proof of the carbon isotopes fractionation (Table 3).
Table 3
Isotopic compositions of oil hydrocarbons and biomass of degrading
microorganisms (Sl3C%o)
Oil products
Black oil Crude oil Diesel fuel
Hydrocarbon isotopes -30,1 -30,17 -30,37
Biomass isotopes -30,24 -30,24 -30,26
The isotopic composition of the source soil 513C is -27.04 %. After applying 5 % of crude oil, it and decreases to -29.94 %, and as a result of bioremediation for 400 days, it increases to -29.18 %, which may be evidence of
the activity of degrading microorganisms. As a result of bioremediation of oil-contaminated laboratory soil, the isotopic composition of hydrocarbons extracted from this soil changed (before inoculation of microorganisms it was -29.94 %, after - -29.18 %).
It is possible that the change in the carbon isotope composition of oil components is the reason for the decrease in the activity of microorganisms degrading oil and embedding of oil hydrocarbon residues into the humic complex of the soil. Assessment of the impact of biodegradation of petroleum products on the agricultural value of the restored soil is possible after large-scale field research.
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Akhmetov Lenar Imametdinovich, candidate of biological sciences, research scientist, akhmetovscience'arambler.ru, Russia, Pushchino, G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms RAS,
Shkidchenko Alexander Nikolaevich, candidate of biological sciences, senior research scientist, shkidchenkoaigmail. com, Russia, Pushchino, G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms RAS,
Andreev Alexey Alexeevich, candidate of biological sciences, senior research scientist, alandreev'amail. ru, Russia, Pushchino, Institute for Cell Biophysics RAS
ИЗМЕНЕНИЕ ГУМИНОВОГО ВЕЩЕСТВА И ТЯЖЕЛОГО
ИЗОТОПА УГЛЕРОДА (513С) В ПОЧВЕ И ПОЧВЕННЫХ МИКРООРГАНИЗМАХ ПРИ IN VITRO БИОРЕМЕДИАЦИИ НЕФТЕЗАГРЯЗНЁННОЙ ПОЧВЫ
Л.И. Ахметов, А.Н. Шкидченко, А.А. Андреев
Биоремедиация нефтезагрязнённой почвы путём обработки ассоциацией активных микроорганизмов-нефтедеструкторов не привела к полной биодеградации нефти независимо от времени эксперимента (до 400 сут) периодического дополнительного внесения биопрепарата. По мере биодеградации нефти в почве росло содержание гуминовых веществ (ГВ), при этом содержание растворимых ГВ увеличивалось в два раза, а содержание нерастворимых ГВ - в три раза. С использованием масс-спектрометрии установлено фракционирование изотопов углерода углеводородов нефти микроорганизмами. Тяжелые изотопы ($3С) включаются в состав биомассы микроорганизмов, а лёгкие ($2С) удаляются в виде CO2. Изотопный состав обрабатываемой почвы вырос с -29,94 до -29,18%о, что подтверждает эффект фракционирования изотопов углерода микроорганизмами и, возможно, является причиной неполной биодеструкции компонентов нефти в почве.
Ключевые слова: почва, гуминовые вещества, изотоп углерода, микроорганизмы, нефть, деградация
Ахметов Ленар Имаметдинович, канд. биол. наук, науч. сотр., akhmetovscience'arambler.rii, Россия, Пущино, Институт биохимии и физиологии микроорганизмов им. Г.К. Скрябина РАН,
Шкидченко Александр Николаевич, канд. биол. наук, стар. науч. сотр., shkidchenko'gmail. com, Россия, Пущино, Институт биохимии и физиологии микроорганизмов им. Г.К. Скрябина РАН,
Андреев Алексей Алексеевич, канд. биол. наук, старш. науч. сотр., alandreev'a mail. ru, Россия, Пущино, Институт биофизики клетки РАН
