1 Introduction (Courtesy:

Transcription

1 1 Introduction Petroleum, a natural and non-renewable source of energy, is considered the lifeblood of many other industries and of the industrialized civilization as well. The world consumption of oil, at 30 billion barrels per year, is supplied by the petroleum industry. This industry can be broadly divided into 2 categories: the upstream producers, including exploration, development and production of crude oil and natural gas and the downstream transporters including transport, refining and retailing. A problem that has plagued both these sectors from the beginning is that of paraffin deposition. When crude oil is transported, the temperature of the bulk fluid drops and the solution becomes supersaturated with respect to the long chain alkanes (Kané et al, 2003), the paraffins. Paraffins precipitate when the temperature of the crude oil falls below its cloud point (wax appearance temperature) (Seth and Towler, 2004). Paraffin-wax deposition in well bores and production tubing blocks the production lines (Figure 1.1) causing reduced flow and eventual shut downs for the treatment of the deposit. This paraffin deposition problem costs the petroleum industry billions of dollars annually in terms of cost of treatment, reduced production, wells shut-in, inefficient use of production capacity, choking of flow lines, premature abandoning and increased manpower (Towler and Rebbapragada, 2004). Figure 1.1: A cross section of an oil conduit showing the deposition of paraffins that leads to pipeline blockage (Courtesy:

2 2 The paraffin deposition problem is currently being combatted by mechanical treatments such as scraping (or pigging), thermal treatments like hot oiling, chemical treatments involving the use of solvents or dispersants or crystal modifiers and electromagnetic treatments. While most of these remedies are in use, they have certain shortcomings. Mechanical scraping is a temporary solution and causes losses due to production downtime. Chemicals and solvents are hazardous and only partially dissolve paraffins and thus the deposition recurs in other areas. Additionally, all these remedial options are expensive. A microbial approach to mitigate the problem of paraffin deposition is a nonhazardous and economically viable approach. The first aim of this research was therefore to obtain microbes that could survive at the high temperatures and degrade paraffinic deposits into shorter carbon chains to improve the flow of crude oil in oil well tubing and transport. Paraffin wax obtained from crude oil has several uses including candle making, coating of surfaces, food additives etc. Refining of petroleum wax employed the use of acids such as sulfuric acid for removal of impurities and improvement of the paraffinic wax. However, the residual sulfuric acid, which remained after refining, found its way into the oily sludge making it highly toxic and difficult to degrade. Oily sludge itself is carcinogenic and immunotoxic in nature (Mishra et al, 2001)). It is composed to a mixture of alkanes, polycyclic aromatic hydrocarbons and asphaltenes. However, when the oily sludge is acidic in nature, hydrocarbon degrading bacterial populations are unable to survive. Such a problem has occurred at Digboi refinery of Assam state in north-eastern India. More than 50,000 tonnes of acidic oily sludge is dumped inside the Digboi refinery premises (Figure 1.2), which is a major source of environmental pollution. Various physico-chemical treatments were attempted but were met with limited success. Bioremediation was then attempted as an environmentally benign and economically feasible option but the treatment of such a huge quantity of toxic waste was in itself a major bioremediation challenge. The second part of this study, therefore, involved the isolation, characterization and identification of microbes that could effectively degrade the acidic oily sludge/paraffinic waste of Digboi refinery.

3 3 Figure 1.2: The acidic oily sludge dumping site inside the refinery premises at Digboi, Assam, North-East India. (Courtesy: Indian Oil Corporation Ltd.)

4 4 The current research was performed in two parts and the study was done under the following two sets of objectives: Part I 1. Collection of paraffinic crude oil from oil wells with a history of paraffin deposition 2. Characterisation of the paraffinic crude oil in terms of its alkane fingerprint 3. Enrichment and isolation of bacteria capable of degrading paraffinic wax at temperatures of 50 C to 70 CScreening the paraffin-wax degradation ability of the isolated bacterial strains 4. Selection of the most efficient paraffin-wax degrading strain (TERI NSM) for further studies and identification of the selected strain 5. Optimization of cultural conditions for enhanced paraffin degradation by the selected bacterial strain 6. Identification of the intermediate metabolites arising during the degradation of paraffinic alkanes by the selected bacterial strain Geobacillus kaustophilus TERI NSM 7. Detection of the genes involved in the oxidation of hydrocarbons 8. Testing the ability of G. kaustophilus TERI NSM to degrade the paraffinic crude oil collected from oil wells with a history of paraffin deposition problems and study of flow behaviour of paraffinic-crude oil after bacterial degradation 9. Mass scale cultivation of G. kaustophilus TERI NSM and injection into the oil well tubing to prevent paraffin-wax deposition in oil well tubing. Part II 1. Characterization of the hydrocarbon content of acidic oily sludge collected from Digboi refinery situated in north-eastern India. 2. Enrichment for microbes capable of degrading the petroleum hydrocarbons of acidic oily sludge generated by the oil refinery 3. Screening the efficiency of the isolated microbes to degrade the acidic oily sludge at low ph (ph 3) 4. Selection of the most efficient acidic oily sludge degrading strain

5 5 5. Characterization and identification of most efficient strain: Candida digboiensis TERI ASN6 6. Determination of the alkane and aromatic hydrocarbon oxidation pathway of C. digboiensis TERI ASN6 by identifying the intermediate metabolites arising during the degradation of hydrocarbons and detecting the genes involved in hydrocarbon oxidation 7. Mass scale cultivation of C. digboiensis TERI ASN6 and immobilization on a carrier material for bioremediation of the acidic oily sludge at Digboi refinery 8. Study the bioremediation of acidic oily sludge by the carrier based C. digboiensis TERI ASN6.

6 6 Thesis Outline This study was conducted in two parts. The first part involved the study of microbes that could selectively degrade the paraffinic (waxy) fractions of crude oil at high temperatures. The second part of the study involved the selection of microbes that could degrade the alkane and aromatic fractions of oily sludge under acidic conditions. This thesis is divided into the following chapters: Introduction This chapter explains the nature of the two problems that have been addressed in this study. The problem of paraffin deposition that has plagued the oil industry by hindering production of crude oil from oil wells and also the problem of the resultant acidic oily sludge that was generated as a result of processing of the paraffin wax of the paraffinic crude oil. Review of Literature The second chapter of this thesis briefly reviews the existing literature in the area of microbial hydrocarbon degradation. The degradation of hydrocarbons is described under the following heads: 1. The paraffin deposition problem 2. Mechanism of alkane (paraffin) degradation 3. Monoterminal oxidation a. Alkane hydroxylase b. Alcohol dehydrogenase c. Aldehyde dehydrogenase d. Rubredoxins 4. Diterminal oxidation 5. Subterminal oxidation 6. Genetics of alkane degradation

7 7 The bioremediation is reviewed under the following heads: 1. The acidic oily sludge generation and its problem 2. Factors affecting biodegradation a. Physical and Chemical factors b. Biological factors 3. Hydrocarbon degradation a. Aliphatic hydrocarbon degradation b. Aromatic hydrocarbon degradation c. Degradation of NSO containing compounds d. Degradation of Resins and Asphaltenes Materials and Methods This section mentions in detail the experimental procedures used in the current study. The experimental regime was designed to meet the objectives mentioned earlier. This chapter was bifurcated into two parts. The first part details the materials and methods employed to study paraffin degradation. The methodology employed to isolate, characterize and identify the paraffin degrading bacterial isolates and estimate their degradation capacity has been detailed. The mechanism for the detection of the hydrocarbon degradation pathway by the use of GC MS and degenerate primers has also been detailed. The implementation of the selected microbe to the oil wells has also been described. The second part details the characterization of the acidic oily sludge that was to be degraded as also the enrichment, isolation and characterization of the acidic oily sludge degrading microbes. The procedures for the estimation of degradation and the pathways of degradation of alkane and aromatic hydrocarbons have also been detailed. Additionally the detection of the genes involved in hydrocarbon oxidation and the field implementation and performance evaluation of the selected microbe has also been detailed. Results

8 8 The results section describes the findings of the experiments done under the current objectives. This chapter has also been divided into two parts to detail the results of both sets of objectives. Discussion In this section the results of the current study have been discussed and implications and inferences drawn. Summary This section summarizes the major findings of this study. The results of the both parts of this study have been summarized here. The last chapter mentions the patents and publications that have resulted from this study.