Effect of Temperature on Biodegradation of Crude Oil

Energy Sources, 27:233–244, 2005 Copyright © Taylor & Francis Inc. ISSN: 0090-8312 print/1521-0510 online DOI: 10.1080/00908310490448299

Effect of Temperature on Biodegradation of Crude Oil ABDULRAZAG Y. ZEKRI OMAR CHAALAL United Arab Emirates University Chemical and Petroleum Engineering Department Al Ain, United Arab Emirates An active strain of anaerobic thermophilic bacteria was isolated from the environment of the United Arab Emirates. This project studied the effect of temperature, salinity and oil concentration on biodegradation of crude oil. Oil weight loss, microbial growth and the changes of the crude oil asphaltene concentration are used to evaluate the oil degradation by this strain. A series of batch experiments was performed to study the effects of bacteria on the degradation of crude oil. The effects of oil concentration, bacteria concentration, temperature and salinity on the biodegradation were investigated. The temperatures of the studied systems were varied between 35 and 75◦ C and the salt concentrations were varied between 0 and 10%. Oil concentrations were ranged from 5 to 50% by volume. Experimental work showed the bacteria employed in this project were capable of surviving the harsh environment and degrading the crude oil at various conditions. Increasing the temperature increases the rate of oil degradation by bacteria. Increasing the oil concentration in general decreases the rate of bacteria oil degradation. Salinity plays a major role on the acceleration of biodegradation process of crude oil. An optimum salinity should be determined for every studied system. The finding of this project could be used in either the treatment of oil spill or in-situ stimulation of heavy oil wells. Keywords asphaltene, biodegradation, thermophilic bacteria

Oil spills are one of the major concerns of the up- and downstream oil industry. The main concern of environmental groups and/or authorities is the danger to the marine life, caused by offshore oilspills. In recent years oil spills have demonstrated that the physico-chemical properties of spilled oil, in addition to the area environment and weather conditions, are all key factors that determine the effect of the oil spill. In the Arabian Gulf area, governments and oil companies operating in the region recognized the threat of a major oil spill in the late 1960’s. Brown and James (1985) reported the first oil spill in the region when a large oil slick invaded the north and west coasts of Bahrain on Aug. 25, 1980. The most major oil spill reported in the Arabian Gulf area was during the Received May 15, 2001; accepted August 31, 2003. This article was written as part of a research grant provided by the Abu Dhabi National Oil Company (ADNOC). We thank Ibrahim El-Magrabi for performing the experimental work and performing the image analyzer tests. Address correspondence to A. Y. Zekri, UAE University, P.O. Box 17555, Al Ain, UAE. E-mail: [email protected]

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Gulf war in 1991. This spill damaged the marine life of the whole Gulf area. Therefore, it is essential for those in the area to study and develop a technique to mitigate and combat oil spills to be ready in case of future accidents. Many techniques used by the oil industry to combat marine oil spills consist of mechanical and/or chemical methods in addition to biotreatment. Recently Boben and Yanting (1996) suggested utilizing in-situ burning as the primary means of response in the event of a major oil spill. Studies on the ability of microorganisms to degrade hydrocarbons of various structures which exist in crude oil started in the mid 1960’s (Kallio, 1996). Bartha and Atlas (1997) indicated that different species of bacteria and fungi are capable of degrading hydrocarbon. They listed 20 genera of bacteria, 14 genera of fungi and one algae genus that are capable of degrading hydrocarbon. These microorganisms exist in different environments: salt water, fresh water and soil. Biological oxidation of any organic matter is basically the utilization of the substance as a food source. The organic matter is converted by bacteria into energy and metabolic end products such as surfactant, gases and water. The growth of the bacteria is dependent on the chemical nature of the organic matter. Robichaux and Myrick (1992) stated that the growth process is a function of the surface area: as the interfacial area increases, the growth rate increases. Thus, dispersing oil through a water phase increases both the availability of the food (hydrocarbon) and the surface area. Therefore, using bacteria capable of producing surfactant in-situ substantially improves the degradation process. Seitinger and Peball (1994), El-Sayed et al. (1995), TjonJoePin et al. (1997), Nour and Thompson (1997) and Al-magrabi et al. (1998) investigated the possibility of using bacteria to degrade hydrocarbons. A substantial amount of research has been conducted in the area of mesophilic and thermophilic bacteria, in leaching and other forms of mineral extraction (Bryant et al., 1988). The study of thermophilic bacteria in the area of biodegradation is a rare phenomenon. Several acidophlic bacteria have been identified that can survive at temperatures higher than the one preferred by mesophiles. Norris and Hart (1989) identified iron-sulfur-oxidation eubacteria that grow at 60◦ C that can be considered moderately thermophilic (with an optimum temperature of 50◦ C). At higher temperatures, there are strains of sulfurous that can readily oxidize mineral sulfides. Morphologically strains belonging to the genus Acidiamus are active at temperature of at least 85◦ C. The isolation of thermophilic bacteria is similar to the isolation of other microorganisms except it requires high incubation temperature. The use of thermophilic bacteria in the treatment of oil spills received little attention in the past. However, if thermophilic bacteria can survive in the presence of high salinity and oil contaminants, their usefulness can be extended to biomediation of marine oil spills in hot climate areas like the Middle East. Only recently, Al-Maghrabi et al. (1998) and Zekri et al. (1999) have introduced a thermophilic strain of bacteria that can survive in a saline environment in the presence of hydrocarbon, making them useful tools for bioremediation of marine oil spills or stimulation of heavy oil wells.

Material and Apparatus Bacteria Two strains of bacteria, both belonging to the Bacillus family, were isolated from local hot water streams. These bacteria were the only kind that survived the harsh temperature and salinity conditions prevailing in the UAE environment.

Effect of Temperature on Biodegradation of Oil

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Crude Oil and Water A sample of crude oil obtained from one of UAE local oil reservoirs was used through out the study. The standard was asphaltenic-base crude with an initial asphaltene content of 1.24% by weight. The oil was heated for a period of one week at a temperature of 80◦ C. Distilled water was used in this study, to which a specified amount of sodium chloride was added to prepare the specified salinity. Medium The growth medium was prepared in 120-ml sterilized light weight plastic bottles. The plastic bottles are capable of withstanding temperature up to 200◦ C. Each bottle contained a specified amount of saline water and crude oil. The water salinities were varied from 0 to 10% by weight. The following water to oil-ratios were used in this project: 5, 10, and 50% by volume. The National Oil Company (ADNOC) made the crude oil available. The medium was contiousely agitated to insure a complete mixing of the system. In addition to that, the culture medium was exposed to different temperatures conditions using controlled temperature bath (see Figure 1). Computer Image Analyzer A computer image analyzer was used to measure concentration of bacteria in the culture. Several methods are available to determine the growth of bacteria (Seeley et al., 1991). The simplest one is by microscopic enumeration. The basic system consists of a high resolution video camera mounted on an optical microscope, an image processor, a Pentium PC, a high resolution image monitor, and high resolution text monitor. The image is visulized with the video camera through a microscope lens. The signal from the video camera is in analog form and must be digitized so that the computer is able to store the image in the library. Therefore, the siginal has to be processed by an analog to digital converter. However, the signal has to be coverted into its analog form in order for the image to be produced in the monitor. Once the binary images are produced from an accepted microphography, a feature count is performed. This simply means that the desired bacteria plane is selected and the feature count option is activated. Bioreactor This reactor was used to growth the bacteria required for this project. It consists of a very successful air curtain driven fluidized bed reactor. Compressed air is injected

Figure 1. Schematic drawing of oil degradation setup.

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into the reactor through a series of perforations in a transverse tube in order to create fluid circulation with an air curtain. Complete details of the bioreactor is presented by Al-maghrabi et al. (1998). Interfacial Tension Apparatus The spinning drop model 500 interfacial tensiometer was used in this project. The apparatus includes a variable temperature air bath so that reservoir temperatures can be emulated. Weighting Scale Apparatus The Mettler PE 300 weighting scale model was used in this research to measure the weight loss of oil as function of time. The model has a three digital accuracy.

Procedure The crude oil used in this project was initially subjected to heating at temperature of 80◦ C for a period of one week to get rid of the volatile components that might evaporate during the experimental work. Plank tests were performed to measure the weight loss due to the evaporation process for the oil and water systems under various temperatures, salinity and water-oil ratio conditions. The evaporation effects were measured in all cases to be less than 0.05%, and these factors have been corrected in all of the experimental results. A second test involved mixing of bacteria in a batch system under various water-oil ratios and various temperatures. Weights of the bottles were taken as a function of time in order to determine the degradation of oil. Initially, weight losses were determined under three different oil concentrations, namely 5, 10, and 15% by volume. In the second patch of experiments, the following temperatures were used: 35, 50 and 75◦ C. The third patch of experimental work consisted of the following four different salinity conditions: 0, 3, 6, and 10% by weight. Keeping in mind that other variables were kept constant during the study of the effects of a specific parameter (salinity, temperature and oil concentration) on the biodegradation of crude oil. The same initial bacteria concentration (30 ∗ 103 cells/ ml) was used in all of the experimental work. To confirm the degradation of the crude oil by the thermophilic bacteria, two additional experiments were conducted. In the first set, 50% by volume crude oil was added to a brine solution. The brine solution consisted of 5% by weight NaCl and 5 ml of indigenous bacteria solution (30 ∗ 103 cells/ ml) to form a 100 ml mixture. The mixture was placed in a special glass flask, as shown in Figure 2. The system was subjected to continue agitation at 50◦ C for 30 days. Asphaltine content of the system was measured as function of time using IP 143 method. In this method, a quantity of the sample is dissolved in n-heptane and the insoluble material, consisting of asphaltenes separated under hot reflux with normal n-heptane, and the asphaltenes are isolated by extraction with toluene. The previous experimental set up was used to study bacteria growth in the oil and water phases as function of time. A mixture consisted of 50 ml of oil and 50 ml of bacteria solution was agitated continuously and bacteria count in both oil and water phases were taken as function of time. The experiment was conducted for 150 hours at constant temperature of 50◦ C and 5% salinity.


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Figure 2. Oil-bacteria mixing and sampling system.

Results and Discussion Figure 3 shows bacteria growth curves at two different temperatures of 50◦ C and 80◦ C. The most rapid growth was observed at 80◦ C. Note both cases used 50% crude oil, 5% salinity and 30 ∗ 103 cell/ml bacteria solution. Figure 3 indicates that the growth rate of bacteria improved drastically at higher temperature. At 80◦ C bacteria count jumped from initial value of 30 ∗ 103 cell/ml to 185 ∗ 103 cell/ml after 24 hours of contact time. In both cases the bacteria count dropped initially for a very short period of time followed by a rapid increase. The drop of initial bacteria count during initial short period of time is due to the adaptation of the bacteria to the new environment. This period of time is normally called adaptation time. The growth rate of the bacteria used in this project is function of temperature. The growth process of the bacteria was drastically accelerated at high temperature of 80◦ C. Therefore, these bacteria could be used for degradation of crude oil at elevated temperatures. Effect of Oil Concentration Figure 4 shows the weight loss of oil as function of time for three different oil concentrations: 5, 10, and 15%. The salinity and temperature were kept constant at 5% and

Figure 3. Effect of temperature on bacteria growth.

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Figure 4. Effect of oil concentration.

50◦ C, respectively, for all tested systems. During the first two hours of mixing time, no significant difference in oil degradation between 5 and 10% oil concentration systems was observed. Increasing mixing time tends in general to increase the difference in oil degradation between the three systems. The experiment was conducted for 25 hours. At that point, a maximum difference in the weight loss of oil between the three different crude oil concentrations was observed. Bacteria were able to degrade the crude oil efficiently at 5% crude oil, where about 9.5% of the crude oil had disappeared in a reasonable period of time (25 hours). At relatively high crude oil concentration (15%), the extent of degradation by bacteria decreased, where only 1% of the oil had disappeared in 25 hours of incubation time. However, using crude oil concentration of 10% resulted in a 3.6% loss of crude oil in the same span of time. The same phenomenon was observed by El-Sayed et al. (1995). At high oil concentration, the oil components may reduce ability of the bacteria to degrade crude oil due to toxic effect on the viability of the cells. High cell density may overcome such problem of substance toxicity to some extent. Effect of Temperature In order to study the effect of temperature on the biodegradation of crude oil, experiments were conducted at 35, 50, and 75◦ C. The following salinity, oil concentration and initial bacteria concentration were used in all runs: 5% NaCl, 50% oil and 30 ∗ 103 cells/ml respectively. Figure 5 shows the percent loss of oil as function of time for the three

Figure 5. Effect of temperature on biodegradation.


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Figure 6. The effect of time on AH degradation.

temperatures. It shows that the highest degradation was achieved at 75◦ C, where 11% of the employed crude disappeared after 10 hours. This is a result of optimum bacteria growth around this temperature, as reported previously and shown in Figure 3. The effect of oil evaporation due to high temperature has been taken into account. Bacteria were still capable of degrading crude oil at temperature of 50◦ C. Around 2.0% of the crude oil degraded after 10 hours of mixing time. Therefore, increasing temperature increases and accelerates the growth of bacteria that resulted in increasing the degradation process of the crude oil at high temperature. Effect of Salinity Figures 6 and 7 show the effect of salinity on the biodegradation of crude oil. The following bacteria concentration, temperature and oil concentration were used in all runs: 30 ∗ 103 cells/ml, 50◦ C and 50% oil, respectively. Initially mixtures contained 3 and 6% salinity were tested for one month. At salinity of 6%, around 93% of the crude oil disappeared after 4 weeks. At relatively lower salt concentration (3% salt), 74% of the crude oil disappeared after 4 weeks. Further experimental work was conducted using 0 and 10% salinity in addition to the previously used salt concentrations. The results are shown in Figure 7 and indicate that increasing salinity tends to increase the biodegradation process up to a salinity of around 6%; after that we observed the reverse trend. Al-Maghrabi et al. (1998) concluded that high salinity decreased the bacteria

Figure 7. Effect of salinity on oil biodegradation.

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Figure 8. Formation of middle phase micro-emulsion.

growth rate, keeping in mind that they investigated only two salinity systems of 0 and 10%. Our study indicates that there is an optimum salinity for the degradation of the crude oil by the thermophilic bacteria. The optimum salinity is around 6% for the studied systems. To verify the previous conclusion, five 100 ml mixtures of bacteria, oil and NaCl were prepared. These mixtures contained: 50% oil, 1 ml bacteria solution (30 ∗ 103 cells/ml) and the following different salinities: 0, 1000, 7000, 10000, and 100 ∗ 103 ppm. The systems were mixed very well and kept without any further agitation for one month. As shown in Figure 8, a middle phase microemulsion was developed in the case of 7% salinity. A middle phase microemulsion is associated with lowering of the interfacial tension and optimum salinity. Therefore, we concluded that the 7% salinity is the optimum salinity for the studied systems. Lin and Townsley (1970) reported that lignosulfates increase the rate of fermentation of hydrocarbon in water. They attributed the increased rate of fermentation to the large surface area and the efficiency of cell-to-cell contact provided by the emulsifier. The growth process and, consequently, the degradation process are related to surface area; as the interfacial area increases the degradation rate increases. Thus, dispersing oil through a water phase (microemulsion) increases both the availability of the organic matter and the surface area. As a result of that, the rate of biodegradation also increases. Robichaux and Myrick (1992) suggested increasing the rate of biological destruction of hydrocarbon by emulsifying the oil with a suitable chemical agent. Mechanism of Biodegradation To confirm the oil degradation phenomena by bacteria established by weighting technique additional experimental work was conducted by mixing bacteria solution and oil at room temperature and measuring the asphaltene content of the oil phase and bacteria count in both oil and water phases as function of time. The hypothesis is that if the asphaltene content of the oil decreases at the same time bacteria growth increases, then asphaltene is one of the oil components that bacteria is consuming; this confirms the crude oil degradation by bacteria. Two systems were employed in this phase of the work. The first system contains 50% oil concentration, 5% salinity and 1 ml of 30 ∗ 103 cells/ml bacteria solution. The total mixture volume was 500 ml. Figure 9 shows the reduction of asphaltene


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Figure 9. Aspheltene content vs. time.

content of the oil phase as function of time. The asphaltene content dropped from 1.24% to 0.05% after 28 days. The second system consisted of 30% oil concentration, 5% salinity and 1 ml of 30 ∗ 103 cells/ml. Bacteria were used to evaluate the bacteria count in both oil and water phases simultaneously as function of time. Figure 10 shows the bacteria count in both oil phase and water phases as function of time. The plot indicates that bacteria quickly transferred into the oil phase and a rapid growth of bacteria was observed in the oil phase. Figure 10 also shows that the growth rate of bacteria in the water phase is quite low. Therefore, it is clear that the media that contains the food attracted bacteria. One interesting observation is that growth curves have a similar area under them, leading one to believe that the growth behavior of the bacteria in the oil and the water phases are the same and the only difference is the rate of growth and the amount of bacteria exist in the system. Two microphotographs were taken in order to visualize the transfer of bacteria into the oil phase. Figure 11 shows a microphotograph that exhibits the existence of both round-shaped and rod like shape bacteria before mixing bacteria solution with the oil. The round shaped bacteria are very active in degrading the crude oil and production of a surfactant. The rod shaped bacteria was observed to die at a temperature of 45◦ C. After mixing bacteria and oil and agitation for one hour, a second microphotograph was obtained. Figure 12 clearly shows the spread of bacteria into the oil phase, which confirms the partition of bacteria into the oil phase phenomena.

Figure 10. Bacteria counts in the oil-phase and water-phase.

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Figure 11. Microphotograph of bacteria solution prior to mixing with oil.

Figure 12. Microphotograph of partitioning bacteria into oil phase.

Figure 13. FT of AH crude for bacteria system and water system, salinity 10%.


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An additional benefit obtained from thermophilic bacteria is its ability to produce surfactant, which could be considered as emulsifying agent. The surfactant production by the thermophilic bacteria was established by measuring the interfacial tension between the thermophilic bacteria solution and the crude oil. Two systems were tested employing the crude oil: a bacteria- free solution and mixture contains bacteria. Results are shown in Figure 13. The results clearly demonstrate that bacteria are capable of reducing IFT by producing surfactant at relatively high temperatures of 45◦ C. A 70% reduction in IFT of the crude for the microbial rich solution was observed at a temperature of 60◦ C.

Conclusions Based on the experimental results, the following conclusions are obtained: 1. The theromophilic bacteria proved its ability to degrade crude oil. 2. Increasing the oil concentration reduces the extent of crude oil biodegradation for constant thermophilic bacteria concentration. 3. Salinity affects the rate and amount of biodegradation, an optimum salinity should be determined for the studied system. 4. Rate and amount of biodegradation is function of temperature, increasing temperature increases both the rate and amount of oil degradation by thermophilic bacteria.

References Al-maghrabi, I., O. Chaalal, and M. R. Islam. 1998. Thermophilic bacteria in uae environment can enhance biodegradation and mitigate wellbore problems. SPE 49545 presented at Abu Dhabi International Petroleum Exhibition Conference, Abu Dhabi, UAE. Oct. 11–14. Bartha, B., and R. M. Atlas. 1997. The microbiology of aquatic oil spills. Advance Applied Microbiology 22:225. Boben, M., and Y. Yanting. 1996. In-situ burning—One method of effective oil spill response in the South China Sea. International SPE 35894 Presented at the Conference on Health, Safety & Environment, New Orleans, Louisiana, June 9–12. Brown, D. J. S., and J. D. James. 1985. Major oil spill response coordination in combat of spills in Bahrain waters. JPT Jan.:131–136. Bryant, R. S. et al. 1988. State of the art of microbial EOR field technology. U.S. DOE Report No. NIPER-138. Bartlesville, OK. El-Sayed, A. H., A. M. Shebi, and M. A. Ramadan. 1995. Laboratory study of microbial cleaning of oil spills under Saudi environmental conditions. SPE 29789 presented at the Middle East Oil Show, Bahrain, March 18–19. Kallio, R. E. 1996. Microbial Transformation of Alkanes. International D. Periman edition, Fermentation Advances. New York: Academic Press. Inc., 635–636. Lin, D. L., and P. M. Townsley. 1970. Lignosulfates in petroleum fermentation. J. of Water Pollution Control Federation 2:68–70. Norris, P. N., and A. Hart. 1989. Acidophilic-mineral-oxidizing bacteria. The utilization of carbon dioxide with particular reference to autotrophy in sulfolobus. Microbiology of extreme environments and its potential for biotechnology. FEMS Symposium, Da Costa, MS. Nour, M. H., and I. Thompson. 1997. Biomediation of marine oil spills: The results of extensive lab research using mutagensis. SPE 37722 presented at the Middle East Oil Show, Bahrain. March 15–18.

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Robichaux, T. J., and N. H. Myrick. 1992. Chemical enhancement of the biodegradation of crude-oil pollutants. JPT Feb.:16–19. Seeley, H. W., Jr., P. J. Vandemark, and P. J. Lee. 1991. Microbes in Action. New York: W.H. Freeman and Company. Seitinger, P., and U. Peball. 1994. Oil spill remediation: An integrated approach for in-situ site clean-up. SPE 27159 presented at the Second International Conference on Health, Safety & Environment in Oil & Gas Exploration and Production, Jakarta, Jan. 25–27. TjonJoePin, M. R., H. D. Brannon, and B. B. Beall. 1997. Biotechnological treatment removes xanthan-base skin damage. SPE 38304 presented at the Western Regional Meeting, Long Beach, Calif., June 25–27. Zekri, A. Y., R. A. Al-Mehaideb, and O. Chaalal. 1999. Project of increasing oil recovery from UAE reservoirs using bacteria flooding. SPE 56827 presented at the Annual Technical Conference & Exhibition, Houston, TX, Oct. 3–6.