Large Scale Bioremediation of Petroleum Hydrocarbon Contaminated Waste at Indian Oil Refineries: Case Studies
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Research Article ISSN 2250-0480 Vol 2/Issue 4/Oct-Dec 2012 LARGE SCALE BIOREMEDIATION OF PETROLEUM HYDROCARBON CONTAMINATED WASTE AT INDIAN OIL REFINERIES: CASE STUDIES Ajoy Kumar Mandal 1*, Priyangshu Manab Sarma 1, C Paul Jeyaseelan 1, Veeranna A Channashettar 1, Bina Singh 1, Banwari Lal1* and Jayati Datta 2 1 The Energy and Resources Institute (TERI), Habitat Place, Lodhi Road, New Delhi, India. Phone: +91-11-2468 2100, Fax: +91 11 2468 2145, 2 Bengal Engineering and Science University, Shibpur, PO: Botanic Garden, Dist: Howrah, West Bengal, India – 711103. India. ABSTRACT The petroleum industry effluents, oily sludge and oil spills cause a serious threat to the environment as their constituents are toxic, mutagenic and carcinogenic. Safe disposal of these wastes is serious problem. None of the available conventional disposal methods are environment friendly. Biological methods have been well reviewed and acknowledged for remediation of petroleum hydrocarbon contaminated waste (oily waste). An indigenous microbial consortium was developed by assemble of four species of bacteria, isolated from various oil contaminated sites of India, which could biodegrade different fractions of total petroleum hydrocarbon (TPH) of the oily waste to environment friendly end products. The said consortium was applied on field scale at different oil refineries in India and successfully bioremediated 48,914 tons of different types of oily waste. In 44 field case studies of different batch size of ex situ bioremediation process, the initial TPH content varying from 83.50 to 531.30 gm/kg of oily waste, has been biodegraded to < 10 gm/kg of oily waste in major cases in 2 – 12 months. In one refinery due to coastal climate, the bioremediation time was > 20 months. The bioremediated soil was non-toxic and natural vegetation was found to be grown on the same. Bioremediation technology has helped various oil industries for the management of their hazardous oily wastes in environment friendly manner. KEYWORDS: Bioremediation, Biodegradation, Oily waste, Microbial consortium, Total Petroleum Hydrocarbon. INTRODUCTION Petroleum refineries unavoidably generate (United States Environmental Protection Agency) enormous quantity of waste oily sludge, in terms of and OECD (Organization for Economic Co- tank bottom and effluent treatment plant (ETP) oily operation and Development) countries designated sludge, and oil contaminated soil in their day to day oily wastes as hazardous wastes (Zhu et al., 2001; refining process. These waste oily sludge and oil Ministry of Environment and Forest, Government contaminated soil (termed as “oily waste”) are of India, 2000). The hazardous oily waste is stored in sludge pits inside the refinery premises composed of total petroleum hydrocarbons (TPH), and due to stringent regulatory norms, the disposal water, and sediments (Dibble et al., 1979). The TPH of these oily waste in an environment friendly constitutes a complex mixture of alkane; aromatic; manner is a serious problem. India, US EPA nitrogen, sulfur, and oxygen containing compounds L - 114 Life Science Environmental Science Research Article ISSN 2250-0480 Vol 2/Issue 4/Oct-Dec 2012 (NSO); and asphaltene fractions (Bhattacharya et enhance oil biodegradation on contaminated al., 2003). Oil contamination has severe impacts in shorelines (Prince, 1993 and Swannell et al., 1996). the plant and animal ecosystem including human The success of bioremediation depends on having health (Mandal et al., 2007; EPA, undated). Crude the appropriate microorganisms in place under oil exposure may cause damage to lungs, liver, suitable environmental conditions and composition kidneys, intestines and other internal organs. of the contaminant. Although extensive laboratory Polycyclic aromatic hydrocarbons (PAH) may lead research has been conducted on oil bioremediation, to cancer, Inhalation leads to headache, nausea, only limited numbers of pilot-scale and field trials dizziness, respiratory irritation, BTEX (Benzene, with small quantity of oily sludge, which may Toluene, Eethyl benzene & Xylene) cause provide the most convincing demonstrations of this mutations, cancers, birth defects, nervous disorders, technology, have been carried out in India and and liver disease, depression, irregular heartbeats abroad. (Raghavan et. al., 1999; Mishra et. al., etc. (Gomer et al., 1980; Knafla et al., 2006; Zhang 2001; B K Gogoi et. al., 2003, Ouyang et. al., 2005, et al., 1992; Carpenter et al., 1977; Lee et al., 2006; Chikere et. al., 2009 and Liu et. al., 2009). The Chen et al., 2008; Lewis et al.,2008 and Rice et al., present paper describes our experience on field case 2007). Oil contaminated soil loose its fertility and studies on bioremediation of oily waste at various have impact on seed germination. (Yoshida et al., oil refineries in India using indigenously developed 2006 and Gong et al., 2001). Hence disposal of the microbial consortium. oily waste in an improper manner may cause a serious environmental problem (Yustle et al., 2000). MATERIALS AND METHODS Various conventional methods like land filling, incineration, air spurging, etc. have been applied 3.1 Isolation and Identification of Microbial Strains since early times for remediation of oily waste The crude oil contaminated soil samples were (Vidali, 2011 and Mandal et. al., 2007). It is collected from different oil refineries and oil observed that none of the conventional methods is exploration sites of India. The solvent extractable environment friendly solution (Sood et al., 2009). TPH in the crude oil contaminated soil samples was The common drawback is that they are not the estimated (Mishra et al. 2001a). For enrichment, 5 g permanent solution for the environmental pollution samples of soil were inoculated into 100 mL of and sometimes they are not cost effective (Mandal minimal salt medium (MSM) (Lal and Khanna 1996) et. al., 2007, and Ouyang et. al., 2005) containing steam sterilized crude oil (1%, w/v) as It is established that virtually all types of carbon source and incubated at 37°C on a rotary hydrocarbons are susceptible to microbial shaker (200 rpm) for 7 days. 5 mL of enriched culture degradation and hence the relevance of using the was reinoculated in fresh medium under similar biotechnological approach using the microbial conditions and five such cycles were repeated. After capability for bioremediation of the hazardous five cycles of enrichment, 1 mL of culture was waste is justified (Atlas, 1991; Head, 1998). diluted up to 108 fold, and 100µL of all dilutions were Bioremediation has emerged as one of the most plated on MSM agar plates with crude oil (1 % w/v). promising treatment options for oil contamination The bacterial colonies obtained were further purified (Bragg et al., 1994 and Prince et al., 1994). on the MSM agar plates (with crude oil 1 % w/v). Bioremediation has been applied as a cost effective, The isolates were routinely sub cultured and frozen ecologically friendly and efficient treatment stock cultures were stored in 25 % glycerol at -70°C. technology for the contamination of hydrocarbon Identification of the isolated bacterial strains polluted soils (Chikere et. al., 2009). was done by sequencing of the 16S rDNA gene with Bioremediation is a process that uses naturally the Microseq 16S DNA sequencing Kit TM (PE occurring microorganisms to transform harmful Applied Biosystems, Inc, USA) (Sarma et al, 2004). substances to nontoxic compounds (Lal et al.,1996 The sequences were analyzed with an ABI Prism 310 and Bartha et al., 1984). Laboratory studies and Genetic Analyzer (PE Applied Biosystems, Inc, field tests have shown that bioremediation can L - 115 Life Science Environmental Science Research Article ISSN 2250-0480 Vol 2/Issue 4/Oct-Dec 2012 USA) as per manufacturer's instructions. The (IOCL), Chennai Petroleum Corporation Limited sequences of 16S rDNA genes were subjected to (CPCL) and Mangalore Refinery and BLAST searches of the NCBI GenBank database for Petrochemicals Limited (MRPL). The type of identification. contamination included acidic oily sludge (only at Digboi refinery of IOCL) and non-acidic waste oily 3.2 Degradation of Crude Oil by Microbial Strains sludge (at other refineries) as described in Table- 1 Degradation of crude oil by the bacterial isolates was (Mandal et al., 2011, Lal et al., 2007 and Sarma et. monitored in 250 mL flasks in triplicates containing al., 2006). The bioremediation jobs were carried out 100 mL MSM with 1 % (w/v) of crude oil (steam ex situ in different batches, where a secured HDPE sterilized) as sole carbon source and incubation on a (high density poly ethylene) lined bioremediation rotary shaker (200 rpm) at 37°C. The isolates were site was prepared near the sludge storage pit inside grown previously in MSM for 24 h with 1 % (w/v) the refinery premises. The oily waste was excavated crude oil to a cell density of 108 cells mL-1 and were by using excavator and transported to the secured inoculated into the medium with 5 % (v/v) as bioremediation site using dumper / trailer where the inoculum. Uninoculated controls were kept to bioremediation process was executed. The monitor natural weathering of crude oil. Residual bioremediation sites were located at different crude oil was extracted from the cultures by using climatic zones spread all over India. In some places solvents (Sarma et al. 2004). For quantitative like Gujarat refinery, IOCL, the climate was semi- analysis, the residual crude oil was fractionated by arid with average maximum temperature