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Известия Тульского государственного университета Естественные науки. 2013. Вып. 3. С. 310-316
Биология =
УДК 631.461:663.18:665.4
Autoselection of a stable consortium formed by psychrophilic oil-degrading microorganisms *
A. N. Shkidchenko, L. I. Akhmetov
Аннотация. A stable consortium of psychrophylic oil-degrading microorganisms Microbacterium liquefaciens 6, Rhodococcus erythropo-lis 21 and Pseudomonas putida 22 has been obtained, using an open three-stage bioreactor system. The consortium created by autoselection under continuous cultivation conditions is up to 80 % more effective than the most active strain, and up to 48 % more active than the mixture of the five strains initially introduced into the system. The consortium is shown to be stable when cultivated in a non-sterile soil within 90 days at 6°C.
Ключевые слова: degrader microorganisms, crude oil hydrocarbons, chemostat cultivation, autoselection, bacterial consortium.
Introduction
Elimination of oil spills in the environment in a cold climate is a slow process, for 50 years or more [1]. The main reason for the low rates of the biological processes of the soil remediation in these conditions is the prevalence of low temperatures. Deep freezing and prolonged state of permafrost does not promote the biota distribution into the rocks [2]. Because of low microbial activity of native microorganisms the foreground recultivation method, due to Atlas [3], is introduction of hydrocarbon-oxidizing bacteria into oil-spilled sites [3].
Microbial preparations applied for disposing oil spills consist of two or more microbial species [4-7]. Crude oil is a complex multi-component substance formed by hundreds of chemical compounds, and its total destruction by one species of bacterium or fungi is impossible. Preparations consisting of some species of hydrocarbon-utilizing microorganisms have got a great pool of adaptation reactions to changes of environmental conditions and can oxidize a wider range of oil components [8].
* 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.
At present, however, there is not a typical method of creating laboratory consortia of oil-degrading microorganisms under laboratory conditions. As a rule, composing the consortium occurs by combining active strains destructing hydrocarbons [5, 6]. At the same time, criteria such as the hydrocarbons catabolic activity of strains, the growth at a wide range of temperatures and pH, the lack of antagonism among microbes, are most often applied. Considering the criteria mentioned does not guarantee development of high-effective consortium of oil degraders, with the consortium activity being not equal to a sum of activities of all the strains when single used [9].
The most important trait is the stability of introduced laboratory oil-degrading consortium in open systems since the introduction of a biopreparation into oil-spilled site results in acute drop of its number [10]. Recommendations for repeated introduction of biopreparations into oil spills during bioremediation period is a consequence of insufficient sustainability of applied consortium.
The aim of the study was the development of a consortium of psychrophylic oil-utilizing microorganisms stable both in liquid and solid-phase open systems.
1. Materials and methods
2.1. Bacterial strains. To create a stable microbial consortium of psychrophilic oil-degrading microorganisms five strains were chosen from the collection of the Laboratory of Plasmid Biology (IBPM RAS). All the strains were able to utilize oil hydrocarbons actively at a temperature range 4-6°C [11]: Microbacterium liq-uefaciens 6, Rhodococcus erythropolis 21, Pseudomonas putida 22, Pseudomonas sp. 4 and Pseudomonas sp. 9.
2.2. Cultivation of microorganisms. Continuous cultivation. To create the consortium a principle of flow chemostat was applied. The system included three serial bioreactors with the working volume 1.5 l for each one (flow rate D 0.07 h-1). Diesel fuel and crude oil were chosen as selective factors (Fig. 1). The system was non-sterile, open, air and components of synthetic medium were not being sterilized. The medium composition and identification of the strains used were described in [12]. Diesel fuel flew into the first reactor (1.5 ml/h), 20% oil water emulsion (1.5 ml/h) was added into the second reactor, final oxidation of hydrocarbon intermediates proceeded in the third one. The first reactor was inoculated with a mixture of five strains in equal ratio. Culture broth with microorganisms from the first reactor flew into two other reactors in series, sequentially.
Batch cultivation. Microorganisms were cultivated in Erlenmeyer’s flasks in liquid mineral medium with crude oil as a sole carbon and energy source in 10 days at 4-6° C.
Model open systems with oil-spilled soil. Crude oil (2 % v/w) was added into the grey forest soil and the soil was then homogenized. Soil samples of 1 kg were placed into plastic containers to reach a layer of 10-15 cm in depth. (NH4)2HPO4 served as the additional source of nitrogen and phosphorus. Microcosms were
Phc. 1. The scheme of three-stage flow system
watered up to 65 % of the soil water capacity, inoculated with microorganisms and incubated at 4-6 °C. All the experiments were in triplicate.
2.3. Oil-degrading activity was estimated by the ratio of utilized crude oil to the added oil volume. Residual content of hydrocarbons was assessed by IR-spectrometry, using AN-2 spectrophotometer («Neftekhimanalytika-SPb», Russia).
2.4. Counting the number of introduced strains. Plating onto agar mineral medium with crude oil (2 % v/v) after serial dilutions was realized. Oil was added into the hot agar medium [12] that was then exposed to ultrasonic treatment by MSE-150B disintegrator (MSE, Great Britain) before preparing Petri dishes with agar.
2.5. A method of genomic fingerprints was applied due to [13] to confirm adequate definition of tested strains.
2.6. Statistical calculations of data were carried on, using a built-in analytical tool of MS Excel (MS Office 2003).
2. Results and discussion
3.1. Creation of the bacterial consortium. After the inoculate being introduced into the first bioreactor a batch cultivation in 48 h was carried on until full consumption of the 2 % w/w of diesel fuel added, with its threshold concentration being 5 mg/l medium. Common number of heterotrophic microorganisms reached 5.6 x109 cells/ml medium, number of oil degraders — 2.4 x109 cells/ml medium. The ratio of the introduced strains is presented in Figure 2A. The other 19 % oil destructors (so-called «indigenous») might germinate from non-sterile components of medium and/or the environment. After the continuous cultivation being started (flow rate D 0.07 h-1) the content of diesel fuel was at the threshold level, i.e. the process in the first reactor was chemostat by the carbon source.
There was no full consumption of supplemented crude oil in the second and third bioreactors, and the carbon source did not serve a limiting factor.
Рис. 2. The ratio of introduced strains during cultivation in three-stage flow system: A — initial ratio, B — final ratio, h — heterotrophic microorganisms, o — oil-degrading microorganisms
Continuous cultivation was carried on in 12 days. Analysis of the composition of the microbial population after continuous cultivation (Fig. 2B) showed the strains Pseudomonas sp. 4 and Pseudomonas sp. 9 appear to be non-competitive in open flow system and to be washed out by medium flow from bioreactors. The number of heterotrophic microbes during continuous cultivation in comparison with the batch one slightly decreased (from 9.6x109 to 5.4x 109 cells/ml medium), however, the ratio of oil degraders to total number of heterotrophs stayed stable at a level of 1:4. The content of non-identified oil-utilizing microorganisms totaled from 2 up to 6% of the oil degraders total number and diminished in the cultivation process.
To create the stable consortium plating of oil-oxidizing microorganisms from the third bioreactor was performed. Then the most active degrader strain R. ery-thropolis 21 [11], the initial mixture of 5 strains, and the autoselected consortium were cultivated separately in flasks with mineral medium and crude oil (2 % w/w) in 10 days to assess their efficiency (Fig. 3). The consortium proved to be almost twice more active (up to 80 % more) than the single strain mentioned, and up to ^50% more active than the initial mixture of the five strains (M. liquefaciens 6, R. erythropolis 21, P. putida 22, Pseudomonas sp. 4, and Pseudomonas sp. 9).
3.2. Testing of the consortium stability in open soil microcosms. Soil microcosms amended with M. liquefaciens 6, R. erythropolis 21, and P. putida 22 were incubated in 90 h at 6°C. All three strains were detected in soil, and they totaled
Рис. 3. Comparative oil-degrading activity of the strains studied (batch cultivation in flasks at 4-6 °C, 10 days)
90 % of oil degraders number, with non-identified indigenous microbes amounting 10 % (Fig. 4).
Рис. 4. The number change of consortial strains in open soil microcosms: A — initial ratio, B — final ratio, h — heterotrophic microorganisms, o — oil-degrading microorganisms
R. erythropolis 21 dominated. Oil degradation degree reached 38 %, with oil-utilizing microorganisms being 72 % of total content of heterotrophic microorganisms in soil samples.
Thus, the application of the method of continuous cultivation with selective pressure (low temperature and the carbon source) appeared to be fruitful in creating the stable consortium of oil-utilizing bacteria. The method seems to be promising when developing new microbial consortia to remove specific pollutants.
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Shkidchenko Alexander Nikolaevich (shkidchenko@gmail.com), PhD, Senior Scientist, Laboratory of Plasmid Biology, G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences.
Akhmetov Lenar Imametdinovich (akhmetovscience@rambler.ru), PhD, Research Scientist, Laboratory of Plasmid Biology, G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences.
Автоселекция стабильной ассоциации психрофильных микроорганизмов-нефтедеструкторов
А. Н. Шкидченко, Л. И. Ахметов
Abstract. С использованием открытой проточной трехступенчатой системы получен стабильный консорциум психрофильных микроорганизмов-нефтедеструкторов Microbacterium liquefaciens 6, Rhodococcus erythropolis 21 и Pseudomonas putida 22. Созданный путем автоселекции при непрерывном культивировании консорциум на 80 % эффективнее самого активного штамма и на 48 % активнее смеси пяти исходно интродуцированных в систему штаммов. Показано, что данный консорциум стабилен и при культивировании в нестерильной почве в течение 90 суток при 6 °С.
Keywords: микроорганизмы-деструкторы, углеводороды нефти,
хемостатное культивирование, автоселекция, бактериальный консорциум.
Шкидченко Александр Николаевич (shkidchenko@gmail.com), к.б.н., с.н.с., лаборатория биологии плазмид, Институт биохимии и физиологии микроорганизмов им. Г.К. Скрябина, Российская академия наук.
Ахметов Ленар Имаметдинович (akhmetovscience@rambler.ru), к.б.н., н.с., лаборатория биологии плазмид, Институт биохимии и физиологии микроорганизмов им. Г.К. Скрябина, Российская академия наук.
Поступила 23.09.2013
