International Journal of Integrative Biology A journal for biology beyond borders ISSN

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1 Regular International Journal of Integrative Biology A journal for biology beyond borders ISSN Investigation on the ability of soil bacterial isolates to degrade petroleum Hanan I Malkawi 1,*, Mohammad Y Jahmani 1, Emad H Hussein 1, Fuad A AL-Horani 2, Taghleb M Al-Deeb 1 1 Department of Biological Sciences, Yarmouk University, Irbid, Jordan 2 Marine Science Station, Aqaba, Jordan Submitted: 24 Aug. 2009; Accepted: 2 Oct Abstract In an effort to develop active bacterial consortia that could be used in bioremediation of crude oil and/or from contaminated systems, 34 bacterial isolates were isolated from contaminated soil at the Jordanian Oil Refinery in Zarqa. Bacterial isolates were tested individually and as consortia for their potential ability for the degradation of individual (toluene, benzene, naphthalene, anthracene, pentane and heptane) and varying concentrations of mixed in minimal media (mixture (1) pentane, heptane and benzene. mixture (2) toluene, anthracene and naphthalene. mixture (3) naphthalene, toluene and heptane). Bacterial isolates showed variable abilities to grow on individual with concentration ranging from 200ppm to 1000ppm. Bacterial growth (CFU/ml) was used to monitor the biodegradation potential of the bacterial consortia. Bacterial consortia (termed MJ1-MJ5) showed variable abilities to grow on different hydrocarbon mixtures as well as on crude oil. They were also tested for their potential ability to degrade and crude oil in soil boxes, monitoring the evolution of CO 2 from the soil boxes. The cumulative amount of CO 2 released during the degradation of in the soil boxes were approximately 22% to 46.2% of the calculated amount of CO 2. Keywords: biodegradation,, bacterial isolates. INTRODUCTION Petroleum is a complex mixture of and other organic compounds including some organometallo constituents. Petroleum is recovered from different reservoirs that vary widely in their composition and physical properties. The petroleumderived can be divided into four classes as follows: the saturated, the aromatics, the asphaltenes (phenols, fatty acids, ketones, esters, and porphyrins), and the resins (pyridines, quinolines, carbazoles, sulfoxides, and amides) (Colwell et al., 1977). These compounds are of great environmental and human health concern due to their potential trophic biomagnification and the presence of some low molecular weight compounds that are acutely toxic (Darville et al., 1984; Natsuko et al., 2006) and high molecular weight compounds that have mutagenic, * Corresponding author: Hanan I. Malkawi, Ph.D. Director of Foreign Projects Management Unit Director of UNESCO Chair for Desert Studies Yarmouk University, Irbid-Jordan hanan@yu.edu.jo; hananmalkawi@gmail.com teratogenic, and potential carcinogenic effects (Mortelmans et al., 1986; Maria, 2007). The accidental and deliberate crude oil spills have been, and still continue to be, a significant source of environmental pollution, and pose a serious environmental problem due to the possibility of air, water and soil contamination (Trindade et al., 2005), which results in serious human health and ecological risks (Vila et al., 2001). Biodegradation of by natural population of microorganisms represents one of the primary mechanisms by which petroleum and other hydrocarbon pollutants are eliminated from the environment (Rahman et al., 2003). These microorganisms are involved in degrading considerable number of oil hydrocarbon pollutants including; aliphatic and aromatic compounds (Vila et al., 2001; Rahman et al., 2003; Sanger et al., 1984). Bacteria are the most common microbiota involved in oil degradation (Rahman et al., 2002), since they contain different degradative enzymes that are involved International Journal of Integrative Biology IJIB, 2009, Vol. 7, No. 2, 93

2 in the metabolism of oil (Kok et al., 1989; Kurkela et al., 1988; Van Beilen et al., 2004). Jordan refines the imported crude petroleum at the Oil Refinery in Zarqa, which represents a source of contamination for soil, air, and water in the surrounding areas. Few studies have been done in Jordan on the availability of microorganisms that have biodegradation ability (Saadoun, 2002). Also single study has been done at the molecular level on the microbial biodegradation ability (Al-Deeb et al., 2009). However, no studies were done in the past to examine consortia of bacterial isolates to test their ability in degrading and the clean up of oil spills in different contaminated sites. In a previous study in our lab., we have isolated and characterized 34 oil-degrading bacterial isolates from oil contaminated soil collected from the Oil Refinery in Zarqa (Al-Deeb et al., 2009), which showed high oil degradation ability. The purpose of this study was to examine the potential of 34 bacterial strains and their consortia to degrade pure or mixed and/or crude oil. MATERIALS AND METHODS Bacterial Isolates Thirty-four bacterial isolates were previously isolated and identified using biochemical methods (Al-Deeb et al., 2009) from oil-contaminated soil samples collected from 21 locations at the Jordanian Oil Refinery in Zarqa. These 34 isolates were isolated according to their degradation ability for crude oil; all these isolates were used in this study (Table 1). Preparation of bacterial isolates for the biodegradation study Bacterial isolates were revived from glycerol stocks and sub-cultured on: Nutrient agar, tryptic soy broth (TSB), tryptic soy agar (TSA), and Stanier's minimal broth medium (Stanier et al., 1966). All these media were supplemented with 400ppm crude oil, respectively. different concentrations of individual Individual bacterial isolates were cultured on Stanier's minimal media (Stanier et al., 1966) supplemented with different concentrations (100ppm, 200ppm, 400ppm, 800ppm and 1000ppm) of various types of (toluene, benzene, naphthalene, anthracene, pentane and heptane). Each bacterial isolate was cultured on each of the above mentioned at all concentrations separately. The resulting bacterial colonies were counted and repeatedly sub-cultured on the same media to confirm their ability to degrade. different concentrations of mixed Individual isolates were plated on Stanier's minimal media supplemented with different concentrations (100 ppm, 200ppm, 400ppm, 800ppm and 1000ppm) of hydrocarbon mixtures as follows: Mixture (I) pentane, heptane, and benzene. Mixture (II) toluene, anthracene, and naphthalene, Mixture (III) contained naphthalene, toluene, and heptane. The resulting bacterial colonies were repeatedly sub-cultured on the same media to confirm their oil-degrading ability. Growth of bacterial consortia on minimal media containing different concentrations of mixed Thirty four bacterial isolates were grouped into five consortia (termed; MJ1, MJ2, MJ3, MJ4 and MJ5) (Table 2 [Supplementary data]). Each bacterial consortium was grown on Stanier's minimal broth containing the following : naphthalene, toluene, and heptane at 800ppm each, and incubated for two weeks at 28 C, with shaking at 160rpm. After that, each bacterial consortium was grown on the same medium at 1000ppm, 1500ppm, 2000ppm and 2500ppm of the same using the same culture conditions. isolates in minimal media containing crude oil Each bacterial isolate was grown on Stanier's minimal broth containing 1000ppm crude oil for 6 weeks at 28 C, with shaking at 160rpm. Growth of bacterial consortia in minimal media containing crude oil Each bacterial consortium was grown on Stanier's minimal broth containing different concentrations (1000 ppm, 1500ppm, 2000ppm, 2500ppm and 3000ppm) of crude oil for two weeks at 28 C, with shaking at 160rpm. Biodegradation of and crude oil in the soil box system The biodegradation ability of each bacterial consortium for or crude oil was tested by the addition of each bacterial consortium to a soil box system. The International Journal of Integrative Biology IJIB, 2009, Vol. 7, No. 2, 94

3 soil used was sandy with a ph of 6.5. The soil box was designed in a way that ensures continuous supply with oxygen and the release of carbon dioxide was collected in an alkaline trap. Each bacterial consortium was added to two soil box systems. In the first one, bacterial consortium was tested for their potential to degrade the (naphthalene, toluene, and heptane) in which each hydrocarbon was added at 2000 ppm at the beginning of the experiment. Hydrocarbons were added every five days starting with 3000ppm until it reached a concentration of 5000ppm. In the second soil box system, each bacterial consortium was tested for its potential to degrade crude oil, which was added at ppm. Two control systems were carried out for both and crude oil (control 1: no or crude oil in the system, control 2: no bacterial consortium in the System). Systems were monitored for 30days by measuring the amount of CO 2 that was trapped in the alkaline solutions and compared with the theoretical amount that should be released from complete degradation of the that were added to the soil box system. Measurement of CO 2 released The released CO 2 gas was trapped in an alkaline solution of 4N NaOH. This solution was replaced and analyzed by titration to determine the amount (gm) of released CO 2 (APHA, 1998). Table 1: List of the oil degrading bacterial isolates (Al-Deeb & Malkawi, 2009). Bacterial isolates Bacterial isolates Species codes codes Species TDJ1 Alcaligens denitrificans TDJ18 P. acidovorans TDJ2 P. aeruginosa TDJ19 Micrococcus roseus TDJ3 Bacillus megaterium TDJ20 P. stutzeri TDJ4 P. mallei TDJ21 P. alcaligenes TDJ5 A. calcoaceticus TDJ22 P. cepacia TDJ6 P. putida TDJ23 P. acidovorans TDJ7 P. maltophilia TDJ24 P. stutzeri TDJ8 Alcaligens denitrificans TDJ25 P. vesicularis TDJ9 Moraxella sp. TDJ26 P. fluorescens TDJ10 Comamonas sp. TDJ27 P. stutzeri TDJ11 Micrococcus luteus TDJ28 Moraxella sp. TDJ12 P. mallei TDJ29 P. mallei TDJ13 P. oleovorans TDJ30 A. lowffii TDJ14 P. acidovorans TDJ31 Bordetella sp. TDJ15 Micrococcus varians TDJ32 Bordetella sp. TDJ16 Micrococcus lylae TDJ33 Bordetella sp. TDJ17 A. lowffii TDJ34 P. oleovorans Table 2: The mixed bacterial isolates used in each consortium. Consortium Selected isolates MJ1 TDJ9, TDJ28, TDJ2, TDJ6, TDJ11, TDJ18, TDJ23, TDJ27. MJ2 TDJ1, TDJ16, TDJ30, TDJ3, TDJ7, TDJ13, TDJ20, TDJ24, TDJ29. MJ3 TDJ12, TDJ19, TDJ32, TDJ4, TDJ8, TDJ15, TDJ21, TDJ25, TDJ33. MJ4 TDJ14, TDJ31, TDJ34, TDJ5, TDJ10, TDJ17, TDJ22, TDJ26. MJ5 TDJ31, TDJ9, TDJ16, TDJ30, TDJ28, TDJ19. concentrations (100ppm, 200ppm, 400ppm, 800ppm and 1000ppm). Selected bacterial isolates have shown the ability to grow on those at the concentrations used. These include: Bacillus megaterium (TDJ3), Pseudomonas mallei (TDJ4), Pseudomonas maltophilia (TDJ7), Moraxella sp. (TDJ9), Pseudomonas oleovorans (TDJ13), Micrococus lylae (TDJ16), Acinetobacter lowffii (TDJ17), Micrococcus roseus (TDJ19), Pseudomonas cepacia (TDJ22), Pseudomonas stutzeri (TDJ27), Moraxella sp. (TDJ28), Acinetobacter lowffii (TDJ30), Bordetella sp. (TDJ31) and Bordetella sp. (TDJ33) (Fig. 1 [Supplementary data]). RESULTS different concentrations of individual The ability of each single bacterial isolate was tested to grow on single individual including toluene, benzene, naphthalene, anthracene, pentane and heptane as a sole carbon source at varying different concentrations of mixed The ability of each of the selected bacterial isolate to grow on mixed (Mixture (I) pentane, heptane, and benzene, Mixture (II) toluene, anthracene, and naphthalene and Mixture (III) contained naphthalene, toluene, and heptane) as a sole carbon source at the various concentrations used, which International Journal of Integrative Biology IJIB, 2009, Vol. 7, No. 2, 95

4 included 100ppm, 200ppm, 400ppm, 800ppm and 1000ppm was tested. Several bacterial isolates were able to grow on all concentrations used. These include: Bacillus megaterium (TDJ3), Pseudomonas mallei (TDJ4), Pseudomonas maltophilia (TDJ7), Moraxella sp. (TDJ9), Pseudomonas oleovorans (TDJ13), Micrococus lylae (TDJ16), Acinetobacter lowffii (TDJ17), Micrococcus roseus (TDJ19), Pseudomonas cepacia (TDJ22), Pseudomonas stutzeri (TDJ27), Moraxella sp. (TDJ28), Acinetobacter lowffii (TDJ30), Bordetella sp. (TDJ31) and Bordetella sp. (TDJ33) (Fig. 2 [Supplementary data]). Growth of selected bacterial consortia on minimal media containing different concentrations of mixed The ability of the selected bacterial consortium to grow on mixed (naphthalene, toluene and heptane) as the sole carbon and energy source was tested at varying concentrations (800ppm, 1000ppm, 1500ppm, 2000ppm, 2500ppm and 3000ppm of each hydrocarbon). Bacterial growth (CFU/ml) was used as indicator for evaluating the biodegradation potential of bacterial consortia for mixed (Table 3 [Supplementary data]). The bacterial consortium MJ5 exhibited the highest bacterial count (3.5 x 10 8 ), followed by MJ2 (2.9 x 10 8 ), MJ3 (2.4 x 10 8 ) and MJ1 (2.0 x 10 8 ), while the bacterial consortium MJ4 exhibited the lowest growth (6.1 x 10 7 ) at 2500ppm. Growth of bacterial consortia in minimal media containing crude oil The ability of each bacterial consortium to grow on crude oil as the sole carbon source was tested at concentrations of 800ppm, 1000ppm, 1500ppm, 2000ppm, 2500ppm and 3000ppm. Bacterial growth (CFU/ml) was used as indicator for evaluating the biodegradation potential of bacterial consortia for crude oil (Table 4 [Supplementary data]). The bacterial consortium MJ5 exhibited the highest bacterial count (2.1 x ) followed by MJ2 (9.2 x ), MJ1 (2.3 x ) and MJ3 (1.7 x ) respectively, while MJ4 (7.2 x ) exhibited the lowest growth at 3000 ppm. Biodegradation of or crude oil in soil box system and the release of carbon dioxide During the biodegradation of or crude oil in the soil box system, the amount of carbon dioxide that was released during the utilization of or crude oil was used as indicator for evaluating the biodegradation potential of each bacterial consortium in the soil box. In case of hydrocarbon degradation, the total amount of (in grams) that was added to each soil box system was as follows: 1.915gm heptane, 2.427gm toluene, and 2.8gm naphthalene which should theoretically release 5.886gm, 8.113gm, and 9.612gm CO 2, respectively from the complete degradation of those. This theoretical figure was obtained by calculating the stoichiometry of each hydrocarbon according to the weight of the respective hydrocarbon used. The cumulative amount (gm) of CO 2 released during the biodegradation of in the soil boxes are shown in (Fig. 3). On the other hand, the cumulative amount (gm) of CO 2 released during the biodegradation of crude oil in the soil boxes are shown in (Fig. 4). CO2 evolved (gm) Time (days) MJ1 MJ3 MJ5 No bacteria Figure 3: The cumulative amount (gm) of carbon dioxide released from soil box systems during the utilization of by the bacterial consortia. CO2 evolved (gm) MJ2 MJ4 No Time (days) MJ1 MJ3 MJ5 No bacteria MJ2 MJ4 No Figure 4: The cumulative amount (gm) of carbon dioxide released from soil box systems during the utilization of crude oil by the bacterial consortia. Bacterial consortium MJ5 exhibited the highest CO 2 production (10.9gm), which represents 46.2% of the theoretical CO 2, followed by MJ2 (9.1gm) representing 38.5%, then MJ3 (8.2gm) representing 34.7%, and then MJ1 (7.8gm) representing 33%, while the MJ4 (5.2gm) exhibited the lowest CO 2 production which represents 22% of the theoretical CO 2 that should be released from complete degradation of the added (Fig. 3). Bacterial consortium MJ5 exhibited the highest CO 2 production (13.1gm) followed by MJ2 (11.3gm), then MJ1 (10.8gm), and then MJ3 (9.8gm), while MJ4 (6.1 gm) exhibited the lowest CO 2 production (Fig. 4). International Journal of Integrative Biology IJIB, 2009, Vol. 7, No. 2, 96

5 DISCUSSION Soil contaminations by petroleum, which results from accidental oil spills and various industrial operations are threatening the environment all over the world. Currently, there are many clean up methods available. However, bioremediation has gained approval because of its removal efficiency and cost effectiveness as compared to the other methods such as the incineration and land-farming (Lageman et al., 2005). The remediation of hazardous wastes including petroleum by non-biological processes is estimated to cost about US$750 billion over the next 30 years, whereas the cost of using bioremediation methods is about ten times less (Pimentel et al, 1997). A number of studies have shown that several bacterial genera are able to degrade oil components. Among those, the genus Pseudomonas is predominant (Radwan et al., 1995; Daane et al., 2001). Other genera include Acinetobacter, Micrococcus, Alcaligenes, Bacillus, Moraxella and Comamonas (Weissenfels, et al., 1990; Radwan et al., 1995; Ashoke et al., 1995; Zylstra et al., 1997; Briganti et al., 1997; Rockne, et al., 2000; Zilli et al., 2001; Rahman et al., 2002). In a previous study conducted at our Lab., fourteen bacterial isolates (out of 34 bacterial isolates obtained), most of them belonging to the genus Pseudomonas, were isolated from contaminated soils and demonstrated high efficiency in degrading oil components added to the culture media compared with the rest of the 34 isolates (Al-Deeb et al., 2009). Those isolates include; Bacillus megaterium (TDJ3), Pseudomonas mallei (TDJ4), Pseudomonas maltophilia (TDJ7), Moraxella sp. (TDJ9), Pseudomonas oleovorans (TDJ13), Micrococus lylae (TDJ16), Acinetobacter lowffii (TDJ17), Micrococcus roseus (TDJ19), Pseudomonas cepacia (TDJ22), Pseudomonas stutzeri (TDJ27), Moraxella sp. (TDJ28), Acinetobacter lowffii (TDJ30), Bordetella sp. (TDJ31) and Bordetella sp. (TDJ33). In this study, the 14 bacterial isolates were grown on Stanier's minimal media supplemented with varying concentrations of different types of including; toluene, benzene, naphthalene, anthracene, pentane and heptane. These bacterial isolates have shown the ability to grow on all added up to 1000ppm. Among the bacteria tested, Bordetella sp. (TDJ31) and Moraxella sp. (TDJ9), demonstrated the maximum growth on all hydrocarbon concentrations up to 1000ppm, while Bacillus megaterium (TDJ3) and Pseudomonas mallei (TDJ4), demonstrated the least growth on all hydrocarbon concentrations with the rest of the bacteria showing moderate growth. The ability of these bacterial isolates to live on minimal media containing different oil, suggests that these bacteria have the necessary enzymes that are capable of degrading these oil as also indicated by other studies (Kok et al., 1989; Kurkela et al., 1988). Efroymson (Efroymson et al., 1991), showed that there is an upper limit for the concentration of each hydrocarbon, which the different bacteria can tolerate after then it will be toxic to some bacterial species. This might reflect the differential growth of the 14 bacterial isolates on the different concentrations of in the current study. The ability of the bacterial consortia to grow on mixed (Naphthalene, Toluene and Heptane) as the sole carbon source was tested at varying concentrations. The data suggests that the bacterial consortia showed better hydrocarbon degradation behavior than the single isolates. Pure bacterial cultures may not have the metabolic capability to degrade certain compounds in a rate that is enough to meet treatment criteria. Therefore, consortia of mixed populations with overall broad enzymatic capacities may be required to achieve total degradation of the petroleum as indicated by Westlake (Westlake, 1982). The ability of the bacterial consortia to grow on mixed as the sole carbon source was tested at varying concentrations of the. The bacterial consortium MJ5 exhibited the highest growth, followed by MJ2, MJ3 and MJ1, respectively. MJ4 exhibited the lowest growth (Table 3). On the other hand, the ability of the bacterial consortia to grow on crude oil as the sole carbon source was tested at varying concentrations of the oil, where the consortium MJ5 exhibited the highest growth, followed by MJ2, MJ1 and MJ3, respectively. MJ4 exhibited the lowest growth (Table 4). As indicated herein, there is a difference in the arrangement of oil degradation ability with crude oil and purified between MJ1 and MJ3 consortia which indicated that MJ1 consortium is better than MJ3 in case of crude oil degradation because the crude oil has other than those used in the previous experiments so it can utilize it efficiently more than MJ3. Most of the published literature, on biodegradation, focused on the degradation of one or two pure chemicals at very low concentrations by pure cultures or a mixed culture of two or three organisms but not on the degradation of complex mixtures of organic pollutants by mixed cultures. The extent of hydrocarbon biodegradation demonstrated in this study was higher than that obtained in other studies. Limbert (Limbert et al., 1994), showed, 3 bacterial isolates were required to treat a mixture of compounds consisting of benzene, o-xylene, nitrobenzene, naphthalene, and other chemicals at extremely low concentrations of 15-60ppm and their isolates were able to degrade up to 60% of these chemicals, in 18hrs. Grant and colleagues (Grant et al., 2002) have shown how the concentration of a target chemical can influence the growth of International Journal of Integrative Biology IJIB, 2009, Vol. 7, No. 2, 97

6 bacteria. In their experiments, higher concentrations resulted in lower cell growth due to the increased toxicity of that chemical. Arcangeli et al., (1992) used very low concentrations of toluene, less than 1ppm to 6ppm, in their study. Heitkamp (Heitkamp et al., 1987) showed that it takes 17 to 31 days to degrade naphthalene effectively, when added to selected soil microcosms at levels of less than 1ppm. Speitel (Speitel et al., 1989) demonstrated the degradation of phenols (e.g. p-nitrophenol, 2, 4- dinitrophenol, and pentachlorophenol) at very low levels, i.e., 1-100ppb. In our study, we have used much higher concentration and still obtained effective degradation. In the current study, the ability of bacterial consortia to degrade mixed (naphthalene, toluene and heptane) or crude oil in the soil box was tested, the amount of carbon dioxide that was released during the utilization of or crude oil was used as indicator for evaluating the biodegradation potential of bacterial consortia in the soil box system. The cumulative amount of carbon dioxide released during the biodegradation of, was approximately 22% to 46.2% of the theoretical amount of carbon dioxide (Fig. 3). This rate of carbon dioxide production is close to the maximum value that can be reached by microbial degradation, while the cumulative amount of carbon dioxide released during the biodegradation of crude oil in the soil boxes indicated that the bacterial consortium MJ5 exhibited the highest carbon dioxide production than MJ2, MJ1, MJ3, while MJ4 exhibited the lowest carbon dioxide production (Fig. 4). This indicates that the bacterial consortia were able to use and crude oil as a sole carbon and energy source as reported by Walter (Walter et al., 1991). The differences between the actual and the theoretical amount of carbon dioxide might be likely due to high volatilizations rate of as indicated by Atlas (Atlas, 1977) and the amount of the biodegraded might be incorporated into cell mass and not released as carbon dioxide (Thomas et al., 1989). The current study showed that, four unique bacterial isolates (Moraxella sp. (TDJ9), Micrococcus lylae (TDJ16), Micrococcus roseus (TDJ19), Bordetella sp. (TDJ31) showed the highest biodegradation ability (compared to the rest of bacterial isolates) for all at high concentration up to 1000ppm, also two unique bacterial consortia (MJ5 and MJ2) showed high biodegradation ability for both and crude oil at high concentrations up to 3000 ppm. 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