Isolation and Characterization of Petroleum Hydrocarbon Degrading Bacteria from the Bohai Sea, China

Transcription

1 Advanced Materials Research Online: ISSN: , Vols , pp doi: / Trans Tech Publications, Switzerland Isolation and Characterization of Petroleum Hydrocarbon Degrading Bacteria from the Bohai Sea, China Ping Guo 1, a, Jianguo Lin 1,b, Binxia Cao 1,c and Na Ta 1, d 1 The College of Environmental Science and Engineering, Dalian Maritime University, China a guoping214@126.com, b dlhsdxhjxy@126.com, c caobx2025@163.com, d tahaa123@126.com Keywords: Biodegradation, Oil contamination, Biodegradability Abstract. Fourteen petroleum hydrocarbon degrading bacteria strains were isolated from oil-contaminated site. Isolated strains were able use diesel oil as sole carbon and energy source. Bacterial strain HD1 was selected due to the luxuriant growth on oil agar. The oil degradation rate of strain HD1 was analyzed using UV-spectrometry-based methods. The result showed that the rate of diesel oil degradation of 75% was observed after 14days of cultivation. Introduction Almost all human activities rely on petroleum as a major source of energy, but environmental contamination by oil and its derivatives has become a serious problem in the last century. Oil spills are a major menace to the environment because they severely damage the surrounding ecosystems[1]. Petroleum hydrocarbons are the primary constituents in oil, gasoline, diesel, and a variety of solvents and penetrating oils. Petroleum hydrocarbons can be divided into four classes: the saturates, the aromatics, the asphaltenes (phenols, fatty acids, ketones, esters, and porphyrins), and the resins (pyridines, quinolines, carbazoles, sulfoxides, and amides) [2]. The petroleum constituents of primary interest to human health have been the aromatic hydrocarbons (i.e., benzene, ethylbenzene, toluene, and xylenes), polynuclear aromatic hydrocarbons (PAHs), gasoline additives (e.g., MTBE, TBA), and combustion emissions from fuels (e.g., carbon monoxide, benzene, acetaldehyde, formaldehyde, diesel particulates). The technologies commonly used for the oil contaminated sites include mechanical, burying, evaporation, dispersion, and washing. However, these technologies are expensive and can lead to incomplete decomposition of contaminants. Bioremediation is the promising technology for the treatment of these contaminated sites since it is cost-effective and will lead to complete mineralization[3]. The process of bioremediation, defined as the use of microorganisms to detoxify or remove pollutants owing to their diverse metabolic capabilities is an evolving method for the removal and degradation of many environmental pollutants including the products of petroleum industry [4]. In addition, bioremediation technology is believed to be noninvasive and relatively cost-effective [5]. Biodegradation by natural populations of microorganisms represents one of the primary mechanisms by which petroleum and other hydrocarbon pollutants can be removed from the environment [6]and is cheaper than other remediation technologies [7]. An attempt for bioremediation of oil was first seriously carried out in the Exxon Valdez spill accident in It was shown at that time that indigenous bacteria were useful for oil degradation [8]. The intensity of biodegradation is influenced by a number of limiting factors, such as temperature, nutrients, oxygen, ph-value, composition, concentration and bioavailability of the contaminants. The composition and inherent biodegradability of the petroleum hydrocarbon pollutant is the first and foremost important consideration when the suitability of a remediation approach is to be assessed.temperature plays a significant role in controlling the nature and the extent of microbial hydrocarbon metabolism. Bioavailability and solubility of hydrophobic substances with low solubility, such as some aliphatic and polyaromatic hydrocarbons, are temperature-dependent. The decreased volatilization and solubility of some hydrocarbons at low temperature affects toxicity. A All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of Trans Tech Publications, (ID: , Pennsylvania State University, University Park, USA-11/05/16,21:04:56)

2 Advanced Materials Research Vols temperature decrease also results in a decrease in diffusion rates of organic compounds and in an increase in viscosity, which affects the degree of distribution[9,10]. Nutrients are very important ingredients for successful biodegradation of hydrocarbon pollutants. The effects of nutrient application on hydrocarbon degradation have been well studied. Nitrogen and phosphorus are often the dominant limiting factors in biodegradation of hydrocarbons [11,12]. Atlas [13] reported that when a major oil spill occurred in marine and freshwater environments, the supply of carbon was significantly increased and the availability of nitrogen and phosphorus generally became the limiting factor for oil degradation. In marine environments, it was found to be more pronounced due to low levels of nitrogen and phosphorous in seawater [14].Use of poultry manure as organic fertilizer in contaminated soil was also reported [15], and biodegradation was found to be enhanced in the presence of poultry manure alone. Therefore, additions of nutrients were necessary to enhance the biodegradation of oil pollutant. Compared to microbial products, very few nutrient additives have been developed and marketed specifically as commercial bioremediation agents for oil spill cleanup. It is probably because common fertilizers are inexpensive, readily available, and have been shown effective if used properly. However, due to the limitations of common fertilizers (e.g., being rapidly washed out due to tide and wave action), several organic nutrient products, such as oleophilic nutrient products, have recently been evaluated and marketed as bioremediation agents. Four agents, namely, Inipol EAP22, Oil Spill Eater II (OSE II), BIOREN 1, and BIOREN 2, listed on the National Oil and Hazardous Substances Pollution Contingency Plan(NCP )Product Schedule have also been put into this category[3]. There are the two main approaches to oil spill bioremediation: (a) bioaugmentation, in which known oil degrading bacteria are added to supplement the existing microbial population, and (b) biostimulation, in which the growth of indigenous oil degraders is stimulated by the addition of nutrients or other growth-limiting cosubstrates[3]. For bioaugmentation, bacteria are the most active agents, and they work as primary degraders of spilled oil in environment [16,17]. Several bacteria are even known to feed exclusively on hydrocarbons [18]. Floodgate [14] listed 25 genera of hydrocarbon degrading bacteria and 25 genera of hydrocarbon degrading fungi which were isolated from marine environment. The main goal of this study was the isolation of hydrocarbon degrading bacteria from oil contaminated Dalian Port, China. Materials and methods Sampling. Oil contaminated seawater samples were collected from Dalian Port, China. The samples were obtained from surface seawater and the in-situ temperature was about 15 C. Samples were collected in pre-sterilized brown bottles. The bottles were kept at 4 C during transportation back to the laboratory and stored at 4 C refrigeratory to further processing. Enrichment and culturing techniques. The medium used was MMC liquid medium. The medium was prepared by weighing mineral salts with the following composition in gl -1 : 24NaCl, 2.0 K 2 HPO 4, 1.0NH 4 NO 3, 4.0 NH 4 Cl, 0.7 KCl and 3.0Na 2 HPO 4 into conical flask of 1l capacity ml distilled water was added into the flask and the ph adjusted to 7.4. Sterilization was carried out by autoclaving at 121 C for 15 min. Diesel oil was used as the sole carbon and energy source to enrich petroleum hydrocarbon degrading bacteria. About 5mL samples of seawater were transferred into 250mL conical flasks containing 100mL MMC liquid medium; Sterilization was carried out by autoclaving at 121 C for 15 min. Diesel oil was then added at a concentration of 1% (v/v).the flask was incubated at 20 C on an orbital shaker at 150 rpm, and 1mL aliquots were transferred weekly to fresh MMC medium and incubated under the same conditions. The concentration of diesel oil in the MMC medium was increased weekly by 1% up to 3% (v/v). All enrichment experiments were performed in triplicate. Isolation and purification techniques. The medium used for isolation was 2216E agar plates. The medium was prepared by weighing nutrients with the following composition in gl -1 :5.0 peptone,

3 730 Advances in Environmental Technologies III 1.0 yeast extract powder, 0.1Fe 3 (PO 4 ) 4 and 20 agar into conical flask of 1l capacity ml aged seawater was added into the flask and the ph adjusted to 7.4. Sterilization was carried out by autoclaving at 121 C for 15 min. Diesel oil was added into the pre-sterilized 2216 E agar culture media at a concentration of 1% (v/v). The media was poured into culture dish while hot and allowed to cool and solidify before inoculation. To isolate bacterial strains, samples of the enrichment cultures were serially diluted and aliquot (0.l ml) was streaked on sterile oil agar using sterilized wire loop for the isolation of oil degrading bacteria. The plates were incubated at temperature20 C for 5 days. The microbial cultivations were done in triplicates. After incubation, morphologically distinct colonies were re-isolated by transfer to fresh agar plates at least three times to obtain pure cultures. Pure cultures were subsequently observed for basic morphological characteristics, such as colony color, size, and texture. The best petroleum hydrocarbon degrader, bacterial strain HD1, was selected for further analysis due to the luxuriant growth on oil agar. Pure cultures were stored at -21 C in 2216E liquid culture medium containing 30% (v/v) glycerol solution. Growth rate. Bacterial isolates were grown at 20 C for 1 week on rotator shaker at 150 rpm. The growth of the isolates was routinely assessed indirectly by measuring the turbidity (OD600nm) using a UV-visible spectrophotometer. petroleum hydrocarbon degradation. Bacterial strains were grown in 250ml conical flasks containing 100mL MMC liquid medium supplemented with 1ml of diesel oil. Blank flasks were also set up containing 100ml medium and 1ml of diesel oil but without added bacteria strain. After a 14 day incubation experiment, at 20 C on an orbital shaker at 150 rpm, residual compounds were extracted from the culture media (100ml) three times with 20 ml of petroleum ether. The extracts were combined and detected the optical density using a UV-visible spectrophotometer at a wave-length of 227nm. Results and discussion Isolation. Fourteen bacterial strains were isolated from enrichment cultures. One isolated strain(hd1) that showed the luxuriant growth on oil agar was selected from among the 14 isolates. The morphology of HD1 colonies was white, convex and wet. Growth rate. The levels of HD1 growth were analyzed using spectrometry-based methods. The result showed that the amount of HD1 was reached the highest value after 48 hours incubation, then slowly decreased with time. Degradation rate. 75% of the diesel oil was degraded after 14 days, which indicated the selected bacterial strain HD1 could be used in bioremediation of oil contaminated seawater Conclusion Fourteen petroleum hydrocarbon degrading bacteria strains were isolated from oil-contaminated Dalian Port, China. One high biodegradability strain HD1 was selected among these bacteria strains. Further studies are field studies, this is because laboratory studies cannot always simulate complicated real world conditions such as spatial heterogeneity, biological interactions, climatic effects, and nutrient mass transport limitations. Therefore, field studies and applications are the ultimate tests or the most convincing demonstration of the effectiveness of petroleum hydrocarbon degrading bacteria strains.

4 Advanced Materials Research Vols References [1] I.M.Head, D.M.Jones, and W.F.Roling: Marine microorganisms make a meal of oil. Nat. Rev. Microbiol. Vol.4 (2006), pp [2] R. R. Colwell, J. D. Walker, and J. J. Cooney: Ecological aspects of microbial degradation of petroleum in the marine environment. Critical Reviews in Microbiology, vol. 5, no. 4(1977), pp [3] D.Nilanjana, and C.Preethy: Microbial Degradation of Petroleum Hydrocarbon Contaminants: An overview. Biotechnology Research International. vol.(2011),doi: /2011/ [4] J. I.Medina-Bellver, P.Mar ın, and A. Delgado et al:evidence for in situ crude oil biodegradation after the Prestige oil spill. Environmental Microbiology.vol. 7, no. 6(2005), pp [5] T. M. April, J. M. Foght, and R. S. Currah: Hydrocarbon degrading filamentous fungi isolated from flare pit soils in northern and western Canada. Canadian Journal of Microbiology. vol. 46, no. 1(2000), pp [6] W. Ulrici: Contaminant soil areas, different countries and contaminant monitoring of contaminants, in: Environmental Process II. Soil Decontamination Biotechnology, edited by H. J. Rehm and G. Reed, Eds., vol. 11(2000), pp [7] J. G. Leahy and R. R. Colwell: Microbial degradation of hydrocarbons in the environment. Microbiological Reviews. vol. 54, no. 3(1990), pp [8] R.P.J.Swannell, K.Lee, M.McDonagh: Field evaluations of marine oil spill bioremediation. Microbiological Reviews 60(1996), pp [9] L.G.Whyte, J.Hawari, E.Zhou, L.Bourbonnière, W.E. Inniss, and C.W.Greer: Biodegradation of variable-chain-lenght alkanes at low temperatures by a psychrotrophic Rhodococcus sp. Applied and Environmental Microbiology vol. 64(1998), pp [10] F.Rojo: Degradation of alkanes by bacteria. Environmental Microbiology vol. 11(2009), pp [11]D.W.S.Westlake, A.M.Jobson and F.D.Cook,1978. In situ degradation of oil in a soil of the boreal region of the Northwest Territories. Can.J. Microbiol. 24: [12]J.D.Van Hamme, A.Pera, M.Agnolucci, and M.M.Valdrighi,1997.Humic acids stimulate growth and activity of in vitro tested axenic cultures of soil autotrophic nitrifying bacteria.biol.fertil.soils 24: [13] R.M.Atlas: Effects of hydrocarbons on micro-organisms and biodegradation in Arctic ecosystems, in: Petroleum Effects in the Arctic Environment, edited by F. R. Engelhardt, Ed., pp.63 99, Elsevier, London, UK, [14] G. Floodgate: The fate of petroleum in marine ecosystems, in: Petroleum Microbiology, edited by R. M. Atlas, Ed., pp ,Macmillion, New York, NY, USA, [15] J. C. Okolo, E. N. Amadi, and C. T. I. Odu: Effects of soil treatments containing poultry manure on crude oil degradation in a sandy loam soil. Applied Ecology and Environmental Research, vol. 3, no. 1(2005), pp [16] K. S. M. Rahman, T. J. Rahman, Y. Kourkoutas, I. Petsas, R. Marchant, and I. M. Banat: Enhanced bioremediation of n-alkane in petroleum sludge using bacterial consortium amended with rhamnolipid and micronutrients. Bioresource Technology. vol. 90, no. 2(2003), pp [17] R. J. W. Brooijmans, M. I. Pastink, and R. J. Siezen,: Hydrocarbon-degrading bacteria: the oil-spill clean-up crew. Microbial Biotechnology, vol. 2, no. 6(2009), pp [18] M. M. Yakimov, K. N. Timmis, and P. N. Golyshin: Obligate oil-degrading marine bacteria.current Opinion in Biotechnology, vol. 18, no. 3(2007), pp

5 Advances in Environmental Technologies III / Isolation and Characterization of Petroleum Hydrocarbon Degrading Bacteria from the Bohai Sea, China /