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YflK 577.15
ISOLATION OF SOIL BACTERIA FOR BIOREMEDIATION OF HYDROCARBON CONTAMINATION
Ilyina A.*, Castillo Sanchez M.I., Villarreal Sanchez J. A.*, Ramirez Esquivel G., Candelas Ramirez J.
(Corporacion Mexicana de Investigacion en Materiales S.A. de C. V (COMIMSA); Blvd. Oceania 190, Frac. Saltillo-400, C.P. 25290, Saltillo, Coah., Mexico Tel: 52-844-411-32-00 (Ext. 1145); *Universidad Autonoma de Coahuila, Facultad de Ciencias Quimicas, Departamento de Biotecnologia; Blvd. V Carranza e Ing. J. Cardenas V., C.P. 25280, Saltillo, Coahuila, Mexico, Fax: 52-844-415-95-34, E-mail: anna_ilina@hotmail.com)
Bacterial strains were isolated from sites impacted by spill of petroleum hydrocarbons for the development a product (COBE-10) applicable in soil bioremediation. Initially, 82 bacterial strains were isolated in selective agars (agar diesel, combustion oil and petroleum). After monitoring absorbance change of mineral media (containing petroleum as sole carbon source) inoculated by isolated strains, 30 strains were selected. The strains were evaluated for their potential to degrade the hydrocarbons of petroleum in soil, artificially contaminated under laboratory conditions. Based on DTPH/day, finally 6 strains were selected. Seven carrier materials were tested to select suitable vehicle for final formulation of COBE-10. Ex -situ evaluation of the developed product (COBE-10) was performed in field with soils contaminated with diesel and refinery wastes. The field test showed a high efficiency of biopreparation COBE-10.
Life in our planet is sustained in a fragile biological balance; microorganisms play an important role on nutritional chains, that are an important part of this biological balance [1]. Adapting several abilities, microorganisms have become an important influence on the ecological systems, making them necessary for superior organisms life in this planet. Ability of microorganisms to transform and degrade many types of pollutants in different matrixes (soil, water, sediments and air) has been widely recognized during the last decades [2, 3].
Soil contamination with hydrocarbons causes extensive damage of local ecosystems since accumulation of pollutants in animals and plants tissues, may cause progeny's death or mutation [4]. In Mexico, an endless number of contaminated sites exists as a result of more than 60 years of oil petroleum activity; in recent years this problem has motivated researches to recover these contaminated sites [2].
Microorganisms survive in contaminated habitat because they are metabolically capable of utilizing its resources and can occupy a suitable niche. Contaminants are often potential energy sources for microorganisms [1]. Bioremediation, a process that exploits the catalytic abilities of living organisms to enhance the rate or extent of pollutant destruction, is an important tool in attempts to mitigate environmental contamination [3, 5]. Bioremediation achieves contaminant decomposition or immobilization by exploiting the existing metabolic potential in microorganisms with cata-bolic functions derived through selection, or by the introduction of genes encoding such functions. The effectiveness of bioremediation is often a function of the extent to
which a microbial population or consortium can be enriched and maintained in environment. When few or no indigenous degradative microorganisms exist in a contaminated area and practically does not allow time for the natural enrichment of suitable population, inoculation may be a convenient option [5].
The goal of the present work is to isolate from hydrocarbon contaminated soils the bacterial strains to assess their potential for bioremediation and develop a bioproduct useful for soil inoculation.
MATERIALS AND METHODS
Soils contaminated by different petroleum hydrocarbons were collected from 3 different places (Table 1), by simple soil sampling method [6] at different depths (minor of 3 m). Microorganism isolation was carried out using selective mediums (agars containing petroleum, diesel and heavy fractions of refined petroleum) as unique carbon source. The selective medium used for isolation contained 15 g of agar-agar, 0.5 g of (NH4)2SO4, 0.2 g of Na2HPO4, 1L of sterilized water and 10 ml of carbon source (petroleum hydrocarbons). After dilution of soil samples (10-210-12), selective agar media were inoculated and incubated at 25 °C. For the bacterial strain selection, the following rules were considered: colonies of microorganisms grown within 48-72 hr period and colonies with the bigger size at the end of incubation period (12 days).
In the next step, the test tubes containing liquid mineral medium with 3% v/v of petroleum as sole carbon source were inoculated by isolated strains. Four test tubes per strain, were shaken at 250 rpm for three weeks at 25-
27°C. The absorbance change (turbidity) of the mineral medium was measured in HACH spectrophotometer at 540 nm once a week. Absorbance change was the evaluation criteria for microorganism adaptation in the used media. In some cases degradative activity caused changes in petroleum during the incubation period, these changes did not allow microbial growth kinetics evaluation.
For the pathogenicity evaluation of the selected strains, some microbiological tests were performed [1]. Gram positive microorganisms were cultivated in S-110 agar and manitol. Gram negatives were inocluated in ENDO agar, Salmonella-Shigella agar, EMB-agar, eosin-blue of methylene and Mc Conkey agar [1].
The final microorganisms selection was performed using the soil fertilized with a 200:1 hydrocarbon-nitrogen ratio, 800:1 hydrocarbon-phosphate ratio, using (NH4)2SO4 and Na2HPO4.7H2O, respectively [7]. Then, the soil was sterilized and petroleum was atomized into the soil until it reached 3% w/w concentration. Soil was distributed in petri plates, inoculated with a dilution of 2 ml of the original strain in 10 ml of sterilized water and incubated for 15 days at 25-27°C. Three commercial products (A, B and C) were used as positive controls. Their activity was compared against the isolated strain's activity. Uncontaminated soil was used as blank. Further, a soil sample with the same petroleum content but without any strain was used as
T a b l e 1
Sampling place features
Sample indentification Features
A Samples from a contaminated soil with refinery wastes. Two years of contamination. Sampling depth 0.15 m. (SITE 2).
B Samples from a site contaminated with petroleum. 5 years of contamination. Sampling depth 1.00 m. (SITE 1).
C Samples from site contaminated for 1 year by gasoline from a damaged pipeline. Sampling depth 0.15 m. (SITE 3).
D Samples from a contaminated soil with refinery wastes. Two years of contamination. Sampling depth 1.00 m. (SITE 2).
E Samples from site contaminated for 1 year by gasoline from a damaged pipeline. Sampling depth 1.00 m. (SITE 3).
F Samples from a site contaminated with petroleum. 5 years of contamination. Sampling depth 2.00 m. (SITE 1).
G Samples from a contaminated soil with refinery wastes. Two years of contamination. Sampling depth 2.00 m. (SITE 2).
T a b l e 2
Primary isolation results
Samples keys Agar-Petroleum Agar-Diesel Agar- Combustion oil Number of isolated strains per sample
A 4 4 1 9
B 5 5 3 13
C 6 4 0 10
D 3 4 2 9
E 3 5 3 11
F 4 11 4 19
G 2 5 4 11
Isolated strains per medium 27 38 17 82
experimental control. After 15 days total petroleum hydrocarbons (TPH) were measured by EPA 418.1 method [8].
To select the carriers for microorganisms, several materials were tested: perlita, medium and bulky vermiculite, TBK, PGX, sand and brand. The commercial materials were selected according to their price, availability in the market and physical and chemical properties (apparent density, real density, porous fraction, pH, humidity and ability to absorb water or oil) [9, 10].
Using the isolated microorganisms and selected materials the bioproduct COBE-10 was formulated. The bioreme-diation test was performed at field [11]. One cubic meter of soil from an area freshly contaminated with diesel and refinery wastes, was treated with: a) water; b) COBE-10 and c) commercial product A. The samples were used to form mounds on a high density polyethylene liner to avoid leaching. Total petroleum hydrocarbons (TPH) were measured by EPA 418.1 method [8].
RESULTS AND DISCUSSION
Initially, 82 bacterial strains were isolated from 7 samples of soil originating from three different places contaminated with petroleum hydrocarbons on Northwest of Mexico (Table 1).
Isolation was carried out using the traditional microbiological technique with petri dishes containing selective agars with hydrocarbons (petroleum, diesel and heavy fraction of refined petroleum) as the sole source of carbon (Fig. 1). Results demonstrated (Table 2) that soil sample (F) which showed higher contamination age yielded more number of colonies [5]: 11 colonies grown in diesel media, 4 colonies in petroleum media and 4 colonies in combustion oil media (Table 2).
T a b l e 3
TPH values in soils contained petroleum measured on 15th day after inoculation with bacterial strains. (TPHInitial = 30,000 ppm; TPH control on 15 days = 25,280 ppm. 100% is equivalent to TPH value of control after 15 days of test).
KpRaR««J
Fig. 1. Test tubes with mineral media containing 3% v/v petroleum applied in Assay for selection of strains. Compare the FIRST TUBE (Control) with the other tubes inoculated with the bacterial strains. Solubility of petroleum in water can be observed in tubes inoculated with bacterial strains
T a b l e 4
Characteristics of tested carrier materials
Materials Apparent density g/cm3 Real density g/cm3 % porous space pH Oil abs. ml/gr Water abs. ml/gr % Humidity
Medium Vermiculite 0.12 2.79 95.49 5.71 7.00 5.00 0
Perlita 0.0б 0.80 91.75 8.57 10.00 5.00 0
TBK 0.13 1.30 89.39 4.00 б.00 4.30 28
PGX 0.14 2.71 93.00 5.58 5.00 4.00 20
Bulky Vermiculite 0.13 1.б3 91.72 7.53 7.00 5.00 10
Sand 1.58 1.37 27.01 8.41 0.70 0.25 0
Brand 0.40 1.50 72.80 б.71 3.00 2.50 14
T a b l e 5
Results obtained in the field test
Key Treatment identification % TPH Remaining in tested soil
Initial 4° Week 8° Week
1 CONTROL 100 50.б 55.7
2 C0BE-10 100 30.3 9.3
3 COMMERCIAL PRODUCT (A) 100 38.2 38.3
Based on the monitoring of absorbance change in the mineral medium (with 3% petroleum as sole source of carbon), 30 strains were selected. In the tubes with selected strains absorbance increase was related with the microbial growth as well as with the increase of water solubility of petroleum hydrocarbons as a result of transformation of petroleum due to the bacterial degrading activity. This phenomenon is demonstrated in Fig. 1. It can be observed, at the end of the experiment, that in the control tube, first of the presented tubes (first of the presented tubes), petroleum phase is located on top of culture media. While, in tubes inoculated with some isolated strains, hydrocarbons were mixed with the aqueous phase.
After tests for evaluating pathogenicity, 3 strains were excluded. Common biochemical tests were performed to identify the genus of isolated strains.
The strains were evaluated for their potential to degrade the hydrocarbons of petroleum in soil, artificially contaminated under laboratory conditions. Based on ATPH/day (Table 3), 6 strains were selected. Table 3 shows that the activities of the 6 selected strains were similar to the activity of commercial products (A, B and C) used in this assay as positive control. The genus of those strains were identified as Bacillus sp., Rhodococcus sp., Providencia sp. and Citrobacter sp.
Key TPH after 15 days Л TPH compared with control Л TPH?S per day % Hydrocarbon removing
Control 25.280 0 - 0
PROD, A 17.536 7.744 51б.25 30.63
PROD, B 18.142 7.138 475.89 28.24
PROD, C 18.612 б.бб8 444.50 26.37
C4-7C 17.604 7.б7б 511.75 30.37
F4-6C 19.150 б.130 408.б4 24.25
A4-8A 18.881 б.399 42б.57 25.31
F5-6A 19.083 б.197 413.12 24.51
F3-7C 19.957 5.323 354.84 21.05
B4-8F 19.285 5.995 399.б7 23.71
G3-6B 20.294 4.98б 332.42 19.72
D4-9A 21.571 3.709 247.24 14.67
D5-9A 20.630 4.б50 310.00 18.39
E5-7A 20.495 4.785 318.97 18.93
A3-11A 21.437 3.843 25б.21 15.20
F5-6B 20.428 4.852 323.45 19.19
F5-11A 20.630 4.б50 310.00 18.39
G5-7A 20.966 4.314 287.59 17.06
E4-9A 21.235 4.045 2б9.бб 16.00
F4-11B 21.773 3.507 233.79 13.87
B5-7B 22.513 2.7б7 184.47 10.95
E3-7A 21.437 3.843 25б.21 15.20
G4-7A 21.033 4.247 283.10 16.80
G5-6A 21.437 3.843 25б.21 15.20
F4-6B 20.832 4.448 29б.55 17.60
A3-6A 22.109 3.171 211.37 12.54
G4-6B 21.706 3.574 238.27 14.14
B5-8C 21.706 3.574 238.27 14.14
D3-9A 23.185 2.095 139.б4 8.29
C3-7C 23.454 1.82б 121.71 7.22
D3-8A 22.446 2.834 188.9б 11.21
2 ВМУ, химия, № 1
Seven carrier materials were tested to select suitable support and vehicle for final formulation of COBE-10 (Table 4). The actual and apparent densities are two important parameters related to such characteristics as weight and volume and defined the management of final bioproduct during its preparation and field application [10]. Another important parameter is the material's water and liquid oil absorption, since the final bioproduct will be in contact with these two types of liquids. Based on the properties of selected bacterias, materials with pH near to 7 are preferred. Table 4 shows results for the tests applied on carrier materials.
Using 3 selected carriers and the 6 isolated strains the bioproduct COBE-10 was formulated. Ex-situ evaluation of the developed product (COBE-10) was performed in the field with soils contaminated with diesel and refinery wastes [11]. The field test showed a high efficiency of biopreparation COBE-10 (Table 5). The analysis of data obtained in the control assay demonstrates that without
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treatment the contamination was diminished by 50%. This effect could be attributed to hydrocarbons volatilization by the periodic movement of soil as well as other physical factors [3, 5].
The Table 5 shows that after 8 weeks of resting, 91% of contamination was removed. Whereas, in the control, it was removed only 44% and 62% in treatment with a commercial bioproduct, commonly used in Mexico for biore-mediation.
Thus, using the bacterial strains isolated from the regional soils and the carriers selected for bacterial inoculation and transportation, the bioproduct C0BE-10 was developed. In the field test, COBE - 10 removed the hydrocarbon contaminants better than the commercial bioprod-uct. Based on the obtained results we conclude that C0BE-10 has good prospects for application in bioremedi-ation and is competitive with the common commercial products available in the market.
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Mexicana NMX-AA-015-1985.
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Поступила в редакцию 25.10.02
УДК 577.15
ПОЛУЧЕНИЕ ПРЕПАРАТА НА ОСНОВЕ БАКТЕРИЙ, ВЫДЕЛЕННЫХ ИЗ ПОЧВЫ, ДЛЯ БИОРЕМЕДИАЦИИ НЕФТЯНЫХ ЗАГРЯЗНЕНИЙ
А. Ильина*, М.И. Кастилю Санчес, Х.А. Винареаль Санчес*, Г. Рамирес Эскивель, Х. Кандэлас Рамирес
(Мексиканская корпорация по исследованию материалов (СОМ1М8Л); *кафедра биотехнологии, химический факультет; Университет штата Коауила, Мексика)
Получен микробиологический препарат (КОБЕ-10) на основе бактериальных штаммов, выделенных из почв, загрязненных нефтяными отходами. Он использован для биоремедиации почв. Использование селективных сред на основе агара и углеводородов нефти (агар-нефть, агар-дизельное топливо и т.д.) позволило выделить на начальном этапе работы 82 бактериальные культуры. На основании оценки способности роста культур в жидкой минеральной среде, содержащей нефть в качестве единственного источника углерода, были отобраны 30 штаммов. Количество штаммов было уменьшено до 6 после сравнения их активности в процессе деградации углеводородов нефти, добавленных к почве в лабораторных условиях. Разработанный микробиологический препарат был использован для биоремедиации почвы, загрязненной дизельным топливом и отходами нефтеперерабатывающей промышленности в полевых условиях. Полученные результаты показали, что КОБЕ-10 является эффективным биопрепаратом, пригодным для обезвреживания почвы, загрязненной углеводородами нефти.
