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Известия Тульского государственного университета Естественные науки. 2013. Вып. 1. С. 251-259 Биология
УДК 631.461:663.18:665.4
Degradation of asphaltenes by individual oil-utilizing aerobic bacterial strains *
A. N. Shkidchenko, L. I. Akhmetov, A. B. Gafarov
Аннотация. The possibility of biodegradation of asphaltenes at a room temperature by single aerobic strains Microbacterium liquefaciens Ash-10, Pseudomonas putida Ash-4, Rhodococcus erythropolis Sh-3 and Bacillus sp. 2, isolated from soil with chronic petroleum pollution has been shown. All strains possess high oil-utilizing activity and the ability to grow on agar media containing polycondensed hydrocarbons, black
oil, alcohol-benzene resins, benzene resins as sole sources of carbon and energy. The strains M. liquefaciens Ash-10, R. erythropolis Sh-3, P. putida Ash-4 and Bacillus sp. 2 assimilate 1.7 - 4.3% (w/v) of asphaltenes in a liquid medium in 6 days of incubation. Thus, this is the first time revealed that single cultures of aerobic bacteria can utilize pure asphaltenes.
Ключевые слова: asphaltenes, crude oil, biodegradation, bacteria.
Introduction
Widespread oil and oil products pollution of environment takes the size of ecological disaster. Despite numerous studies, the cardinal decision of the problem of oil-contaminated areas has not yet been found [1-3].
Crude oil is a mixture of several hundred chemical compounds, most of which are liquid hydrocarbons. Hydrocarbons composition is mainly represented by paraffin (30-50%) and naphthenic hydrocarbons (25-65%), and, to less extent, aromatic compounds (10-20%) [4]. Resin-asphaltene fraction of oil is an object of special interest since asphaltenes can clog soil pores, inducing water and oxygen deficiency and decreasing availability of light oil fractions [5].
The main role in the biodegradation of crude oil and its components belongs to microorganisms. Currently, more than 20 genera of bacteria and more than 10 ones of fungi are described which can assimilate oil components as a sole carbon and energy source [6, 7].
However, heterocyclic hydrocarbons with a high condensation degree, which include the resin-asphaltene fraction of oil, are characterized by the greatest
* The study has been funded by the Russian Foundation for Basic Research, grant 12-04-31390-mol_a, and the Federal State Contract 14.515.11.0027.
resistance to biochemical attack and exposed to microbial catabolism partially [8]. Asphaltenes biodegradation in nature is provided by mixed populations of microorganisms, which demonstrate cooperation and commensalism [9, 10]. Resins and asphaltenes are destroyed by the co-metabolism mechanism characteristic for many stable xenobiotics in natural environments. Pineda-Flores et al. [11] have shown that a microbial consortium in a bioreactor at room temperature is able to degrade asphaltenes.
In the most of studies on oil hydrocarbons biodegradation, total removal is shown as percentage, and it does not exceed 20-50% of the oil introduced [6, 7]. As a part of various oils asphaltenes are from 6 up to 11%. It is difficult to register whether destruction of asphaltenes is performed by microbial populations therefore researchers use indirect methods for detection such as CO2 evolution measurement and biomass growth [12].
The aim of this work was to assess the possibility of biodegradation of pure asphaltenes by individual strains of oil-degrading microorganisms.
1. Materials and methods
1. Bacterial strains and cultivation. Next oil-degrading strains from the Collection of Laboratory of Plasmid Biology, IBPM RAS, were studied, as follows: Microbacterium liquefaciens Ash-10, Pseudomonas putida Ash-4, and Rhodococcus erythropolis Sh-3, isolated from oil-polluted soils of the Caspian Sea [13], and Bacillus sp. 2, isolated from oil sludge pits of the Tyumen region.
Cultivation of bacteria was carried in a synthetic medium with the following composition (g/l): NH4C1 — 2.5; CaCl2 6H2O — 0.01; MnCl2 4H2O — 0.02; Na2HPO4 — 10.0; KH2PO4 — 1.0; MgSO4 7H2O — 0.2; FeSO4 7H2O — 0.001; NaCl — 5.0.
When checking the strains for the ability to utilize a range of substrates the agar medium (10 g/l) with the composition mentioned above was used. Crude oil, black oil, alcohol-benzene resins, benzene resins (2% w/w) were added into the hot agar medium that was then exposed to ultrasonic treatment by MSE-150B disintegrator (MSE, Great Britain) before preparing Petri dishes with agar. Powder of naphthalene was placed to the cover of Petri dishes. Anthracene, pyrene, acenaphthene and phenanthrene were dissolved in 1 ml of acetone and added into the hot agar medium up to a concentration of 5 g/l. Plating bacteria was realized after acetone being evaporated.
2% (w/w) of crude oil or 500 mg of asphaltenes per liter were added into the liquid medium as a sole source of carbon and energy. Cultivation of microorganisms was carried out in Erlenmeyer flasks on a rotary shaker at 180 revolutions per minute and 240C. Experiments were in triplicate.
2. Quantification. The number of microorganisms was counted by direct plating on Petri dishes with oil (2% w/w) and agar medium of the composition mentioned above and subsequent quantification after 7 days of incubation at
240C. The culture purity was inspected visually, using standard light phase-contrast microscopy.
3. Isolation of asphaltene fraction. Black oil was used to obtain asphaltenes. 25 g of black oil was added into 1000 ml of petroleum ether and stayed in a dark place for two days until complete precipitation of asphaltenes. Asphaltene precipitate (4 g, or 15.2% w/w) was filtered on a filter paper, washed with pentane until the filtrate bleaching and dried in a vacuum desiccator.
4. Determination of oil hydrocarbons. Determination of residual oil and asphaltenes after cultivation of microorganisms was performed by IR-spectrometer AN-2 («Heftechimavtomatika-SPb», Russia), with intensity of operational IR-beam with a frequency maximum of v = 2930 cm-1 and a basic IR-signal with a frequency of v = 3300 cm-1.
J.l. Preparation of the sample for measuring. Sulfuric acid was added to the sample glass (2 ml of acid per 100 ml sample) and then the sample was moved to extractor. The glass after the sample was washed by 10 ml of CCl4 and the washing solvent was added to extractor as well. Additionally 20 ml of CCl4 was added to extractor. Extractor was shaken for 4 min. After that the emulsion was stayed for 10 min. After the emulsion stratification the lower layer was transferred to 100 ml glass. The extract was dried with water-free sodium sulfate in 30 min (more than 5 g sodium sulfate per 30 ml of extract) till its clarification and the extract was carefully removed to 50 ml glass.
Analyzed culture broth was transferred to a glass 1 and the culture broth volume was measured.
J.2. Chromatographic column preparation. A level of glass fiber (about 1 cm in depths), previously washed by CCl4 and dried, was placed into the bottom part of the column. 6 g of Al2O3 was placed into the column and a glass fiber level (0,5 cm) was again added. Al2O3 was single used.
J.3. Extraction of oil products in the column and measurement procedure. 8 ml of CCl4 was added into a column to moisten. Since CCl4 was absorbed by Al2O3 the extract (see 1) was added triple in series of 10 ml every for the liquid level to cover Al2O3. Since the sample goes through the column additionally 5 ml of CCL4 was added (the CCl4 volume was previously used for washing the glass 1 (see 4.1) ). Eluate was collected into 50 ml glass, with the first 4 ml eluate being rejected. The eluate volume was fixed. Eluate was placed into a cuvette. Then measurement, using AN-2, was performed.
J.J. Calculation. Oil products concentration in the culture broth was calculated next: C Y k
^ Cmeas Yek
where Cmeas is oil products concentration measured (ml/l), Ve — the eluate volume (l), Vcb — the culture broth volume (l), k — eluate dilution coefficient.
4.5. Estimation of the fractional composition of residual oil was carried out by a chromatographic method [14] with the use of microcolumns filled with aluminum oxide.
2. Results and discussion
It was observed in the process of studying the growth and activity of oil-degrading microbial strains isolated from oil-contaminated soil of the Tyumen region and the Caspian Sea coast that a decrease in the content fraction of asphaltenes occurred along with assimilation of light oil fractions [13]. In a number of hydrocarbon extracts from the oil-polluted soil from the coast of the Caspian Sea the presence of asphaltenes was observed, while the asphaltenes content was much lower in other samples than in the original oil. Considering the duration of oil pollution (oil extraction is performed in the region over 100 years), we can assume that the change in the asphaltenes quantity is an effect of hydrocarbons microbial degradation. However, the change in the fraction of residual hydrocarbons in petropolluted soils after prolonged contact with oil-degrading microorganisms can not be a direct evidence of assimilation of certain petroleum fractions such as asphaltenes, since the data show not absolute but relative content of each fraction in a hydrocarbons pool.
Four oil-degrading strains, R. erythropolis Sh-3, M. liquefaciens Ash-10, P. putida Ash-4, and Bacillus sp. 2 were selected to study biodegradation of asphaltenes.
The possibility of the strains to grow on agar media containing a number of oil components as carbon and energy sources was tested (Table 1). All strains had the ability to utilize benzene resins, alcohol-benzene resins and asphaltenes, i.e. fractions containing polycondensed hydrocarbons. The strains R. erythropolis Sh-3 and P. putida Ash-4 were capable of growing on naphthalene. All the studied microorganisms formed colonies on the agar medium with crude oil and black oil.
Oil-degrading activity of the examined microbial strains was relatively high, in 6 days of incubation the strains assimilated from 32 up to 42% of the oil added (Table 2). The use of asphaltenes as the sole source of carbon resulted in a sharp decrease of degradation degree (ten-fold) and achieved some percents only (1.7-4.3%).
It should be noted that the increase of microbial biomass in the medium containing asphaltenes was insignificant and did not exceed one order of magnitude. A cause for the low biodegradation of asphaltenes by microorganisms in the liquid medium could be the low water solubility, since a supplemented portion of asphaltene fraction in every flask was crystalline, and a surface contact with microbial cells could be insufficient. To test this hypothesis, we increased the period of incubation of microorganisms in the medium with asphaltenes up to 15 and 30 days (Table 3). In this case the initial concentration of microorganisms was created as close and relevant to those in previous experiments.
After 15 days of incubation, biological degradation of asphaltenes by P. putida Ash-4 increased up to 4.3%. At the same time, the number of bacterial cells for 15 dayss decreased slightly compared with baseline, but then exceeded the previous value by 3.6 times by the 30-th day of cultivation. The strain M. liquefaciens
Table 1
The ability of oil-degrading strains to grow on the solid agar with different ___________components of crude oil as carbon and energy sources__________________
Substrate
Strain Crude Oil Black Oil Naphthalene Anthracene Pyrene Acenaphthene Phenanthrene Benzene resins Alcohol-benzene resins
P. putida Ash-4 + + + + + + + + +
M. liquefaciens Ash-10 + + - + + + + + +
R. erythropolis Sh-3 + + + + - + + + +
Bacillus sp. 2 + + - + + + + + +
Table 2
Biodegradation of crude oil and asphaltenes in batch culture by single strains ___________________________(6 days of incubation)______________________________
Strains Asphaltenes Crude oil
Cell number/ml Degradation level, % Degradation level, %
initial final
P. putida Ash-4 l.lxlO8 3.2xl09 1.7±0.2 42.2±5.3
M. liquefaciens Ash-10 3.2x10s 1.2xl09 3.2±0.3 32.5±4.8
R. erythropolis Sh-3 О X 1.2xl09 4.3±0.5 33.7±6.4
Bacillus sp. 2 l.lxlO8 3.5xl09 3.6±0.4 39.5±5.2
Ash-10 did not demonstrate the increase of asphaltenes degradation degree by 15 day of incubation, however, to the end of the experiment it reached 3.8% of the amount introduced. The strain number stayed at a close level.
Increasing the incubation time for the strains R. erythropolis Sh-3 and Bacillus sp. 2 up to 15 and 30 days in the medium with asphaltenes was not accompanied by an increase in the degree of asphaltenes biodegradation.
When studying the degradation products of the asphaltene substrate in the culture broth, after precipitation of asphaltene residues a small quantity of hydrocarbons of hexane, benzene and alcohol-benzene fractions, not present in the medium previously, was detected (Table 4). Possibly, the content of revealed hydrocarbons in the medium was at the threshold concentration values for these substances [15] and the mentioned compounds might be physiologically inaccessible for the microorganisms studied.
Table 3
Biodegradation of asphaltenes in batch culture in the extended incubation
Strains 0 day 15 days 30 days
Cell number /ml Cell number /ml Degradat ion level, % Cell number /ml Degradat ion level, %
P. putida Ash-4 2.5xl08 1.1x10s 4.3±0.2 8.9x10s 4,7±0.3
M. liquefaciens Ash-10 1.3xl08 5.7x10s 3.4±0.1 1.0x10s 3.8±0.2
R. erythropolis Sh-3 8.7xl07 1.7xl09 4.3±0.3 7.9x10s 4.3±0.3
Bacillus sp. 2 7.4xl07 1.6xl09 3.7±0.2 2.4xl07 3.7±0.2
Table 4
The content of residual hydrocarbons after 30 days of bacterial incubation with _________________________asphaltenes in batch culture________________________
Strains Hydrocarbon fractions
Hexane fraction Benzene fraction Alcohol-benzene fraction
weight, g % weight, g % weight, g %
P. putida Ash-4 0.0012 4.4 0.0086 30.0 0.0133 46.3
M. liquefaciens Ash-10 0.0013 5.2 0.0087 34.8 0.0132 52.8
R. erythropolis Sh-3 0.0021 9.7 0.0077 34.8 0.0113 51.1
Bacillus sp. 2 0.0027 11.4 0.0092 38.8 0.0120 50.6
These hydrocarbon fractions proved to be products of the partial biodegradation of asphaltenes by microorganisms, they were evidently absent in the medium before the experiment. This can be an additional confirmation of the ability of bacterial strains to metabolize asphaltenes individually.
A fungi (Neosartorya fischeri) strain was first revealed [12] to grow, using asphaltenes as a sole carbon and energy source. Asphaltenes consumption was checked by the change of the content of CO2 expired and the laccase activity change as well. The strain utilized different fractions of asphaltenes (determined by a ratio of acetone/toluene solubility).
A thermophylic bacterium Thermus sp. was described [16] which degraded asphaltenes fraction in crude oils (oils were carbon and energy sources).
Asphaltenes were possibly not to be used by the microorganisms but were co-metabolized in a parallel with consumption of other oil fractions.
Asphaltenes are polycondensed macromolecular compounds that results in low level of biodegradation. We showed previously that the activity of assimilation of hydrocarbons by microorganisms decreased according to increasing degree of condensation [17, 18].
To test the hypothesis whether oil-degrading microorganisms were able to assimilate a part of asphaltenes only, the asphaltenes remaining after prolonged incubation of microorganisms were isolated from the culture broth and re-used in the experiments with the same microbial strains. No changes in the weight content of asphaltenes and no microbial growth was observed. The application of the mix of all the strains studied to degrade rest of asphaltenes demonstrated negative results.
Thus, aerobic bacterial strains were shown to utilize 3-4% of crystalline asphaltenes at a room temperature only while the fungi N. fischeri isolate could consume ~13% [12]. A distinction in the level of asphaltene consumption was possibly provided by different degree of the asphaltene substrate purification process. Recently the anaerobe termophilic strain Garciaella petrolearia was discovered that is able to utilize asphaltenes in single culture (about 40% of asphalt fraction from heavy oil) at a temperature of 500C. Its extremely high activity against to our bacterial strains may be provided by, first, higher availability of asphalt to G. petrolearia and, second, high activity of catabolic enzymes at high temperature.
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Shkidchenko Alexander (shkidchenko@gmail.com), PhD, senior scientist, laboratory of plasmid biology, Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences.
Akhmetov Lenar (akhmetovscience@rambler.ru), PhD, research scientist, laboratory of plasmid biology, Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences.
Gafarov Arslan (gafarov@ibpm pushchino.ru), research scientist, laboratory of plasmid biology, Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences.
Деградация асфальтенов монокультурами бактериальных аэробных штаммов — деструкторов нефти
А. Н. Шкидченко, Л. И. Ахметов, А. Б. Гафаров
Abstract. Показана возможность биодеструкции асфальтенов при комнатной температуре монокультурами аэробных штаммов Microbacterium liquefaciens Ash-10, Pseudomonas putida Ash-4, Rhodococcus erythropolis Sh-3 и Bacillus sp. 2, выделенными из почв с хроническим загрязнением углеводородами нефти. Все штаммы обладали высокой деструктивной активностью и способностью к росту на агаризованных средах, содержавших в качестве единственного источника углерода поликонденсированные углеводороды, мазут, спирто-бензольные смолы, бензольные смолы. Штаммы M. liquefaciens Ash-10, R. erythropolis Sh-3, P. putida Ash-4 и Bacillus sp. 2 ассимилировали в жидкой питательной среде от 1,7 до 4,3% внесенных асфальтенов в течение 6 сут инкубирования. Таким образом, впервые показано, что отдельные штаммы аэробных бактерий могут утилизировать асфальтены.
Keywords: асфальтены, нефть, биодеструкция, бактерии.
Шкидченко Александр Николаевич (shkidchenko@gmail.com), к.б.н., с.н.с., лаборатория биологии плазмид, Институт биохимии и физиологии микроорганизмов им. Г.К. Скрябина РАН.
Ахметов Ленар Имаметдинович (akhmetovscience@rambler.ru), к.б.н., н.с., лаборатория биологии плазмид, Институт биохимии и физиологии микроорганизмов им. Г.К. Скрябина РАН.
Гафаров Арслан Булатович (gafarov@ibpm pushchino.ru), н.с., лаборатория биологии плазмид, Институт биохимии и физиологии микроорганизмов им. Г.К. Скрябина РАН.
Поступила 20.01.2013
