Effect of Temperature on Biodegradation of Crude Oil - Envis

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

Effect of Temperature on Biodegradation ofCrude Oil

ABDULRAZAG Y. ZEKRIOMAR CHAALAL

United Arab Emirates UniversityChemical and Petroleum Engineering DepartmentAl Ain, United Arab Emirates

An active strain of anaerobic thermophilic bacteria was isolated from the environmentof the United Arab Emirates. This project studied the effect of temperature, salinity andoil concentration on biodegradation of crude oil. Oil weight loss, microbial growthand the changes of the crude oil asphaltene concentration are used to evaluate the oildegradation by this strain. A series of batch experiments was performed to study theeffects of bacteria on the degradation of crude oil. The effects of oil concentration,bacteria concentration, temperature and salinity on the biodegradation were inves-tigated. The temperatures of the studied systems were varied between 35 and 75◦Cand the salt concentrations were varied between 0 and 10%. Oil concentrations wereranged from 5 to 50% by volume. Experimental work showed the bacteria employedin this project were capable of surviving the harsh environment and degrading thecrude oil at various conditions. Increasing the temperature increases the rate of oildegradation by bacteria. Increasing the oil concentration in general decreases therate of bacteria oil degradation. Salinity plays a major role on the acceleration ofbiodegradation process of crude oil. An optimum salinity should be determined forevery studied system. The finding of this project could be used in either the treatmentof 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. Themain concern of environmental groups and/or authorities is the danger to the marinelife, caused by offshore oilspills. In recent years oil spills have demonstrated that thephysico-chemical properties of spilled oil, in addition to the area environment and weatherconditions, are all key factors that determine the effect of the oil spill. In the ArabianGulf area, governments and oil companies operating in the region recognized the threatof a major oil spill in the late 1960’s. Brown and James (1985) reported the first oil spillin the region when a large oil slick invaded the north and west coasts of Bahrain onAug. 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 andperforming the image analyzer tests.

Address correspondence to A. Y. Zekri, UAE University, P.O. Box 17555, Al Ain, UAE.E-mail: [email protected]

233

234 A. Y. Zekri and O. Chaalal

Gulf war in 1991. This spill damaged the marine life of the whole Gulf area. Therefore, itis essential for those in the area to study and develop a technique to mitigate and combatoil spills to be ready in case of future accidents.

Many techniques used by the oil industry to combat marine oil spills consist ofmechanical and/or chemical methods in addition to biotreatment. Recently Boben andYanting (1996) suggested utilizing in-situ burning as the primary means of response inthe event of a major oil spill. Studies on the ability of microorganisms to degrade hydro-carbons 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 arecapable of degrading hydrocarbon. They listed 20 genera of bacteria, 14 genera of fungiand one algae genus that are capable of degrading hydrocarbon. These microorganismsexist in different environments: salt water, fresh water and soil. Biological oxidation ofany organic matter is basically the utilization of the substance as a food source. Theorganic matter is converted by bacteria into energy and metabolic end products such assurfactant, gases and water. The growth of the bacteria is dependent on the chemicalnature of the organic matter. Robichaux and Myrick (1992) stated that the growth pro-cess is a function of the surface area: as the interfacial area increases, the growth rateincreases. Thus, dispersing oil through a water phase increases both the availability of thefood (hydrocarbon) and the surface area. Therefore, using bacteria capable of producingsurfactant 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 degradehydrocarbons.

A substantial amount of research has been conducted in the area of mesophilicand thermophilic bacteria, in leaching and other forms of mineral extraction (Bryantet al., 1988). The study of thermophilic bacteria in the area of biodegradation is a rarephenomenon. Several acidophlic bacteria have been identified that can survive at tem-peratures higher than the one preferred by mesophiles. Norris and Hart (1989) identifiediron-sulfur-oxidation eubacteria that grow at 60◦C that can be considered moderatelythermophilic (with an optimum temperature of 50◦C). At higher temperatures, there arestrains of sulfurous that can readily oxidize mineral sulfides. Morphologically strains be-longing to the genus Acidiamus are active at temperature of at least 85◦C. The isolationof thermophilic bacteria is similar to the isolation of other microorganisms except it re-quires high incubation temperature. The use of thermophilic bacteria in the treatment ofoil spills received little attention in the past. However, if thermophilic bacteria can survivein the presence of high salinity and oil contaminants, their usefulness can be extendedto biomediation of marine oil spills in hot climate areas like the Middle East. Only re-cently, Al-Maghrabi et al. (1998) and Zekri et al. (1999) have introduced a thermophilicstrain 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 heavyoil wells.

Material and Apparatus

Bacteria

Two strains of bacteria, both belonging to the Bacillus family, were isolated from localhot water streams. These bacteria were the only kind that survived the harsh temperatureand salinity conditions prevailing in the UAE environment.

Effect of Temperature on Biodegradation of Oil 235

Crude Oil and Water

A sample of crude oil obtained from one of UAE local oil reservoirs was used throughout the study. The standard was asphaltenic-base crude with an initial asphaltene contentof 1.24% by weight. The oil was heated for a period of one week at a temperature of80◦C. Distilled water was used in this study, to which a specified amount of sodiumchloride was added to prepare the specified salinity.

Medium

The growth medium was prepared in 120-ml sterilized light weight plastic bottles. Theplastic bottles are capable of withstanding temperature up to 200◦C. Each bottle containeda specified amount of saline water and crude oil. The water salinities were varied from0 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 conditionsusing controlled temperature bath (see Figure 1).

Computer Image Analyzer

A computer image analyzer was used to measure concentration of bacteria in the cul-ture. 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 ofa 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. Theimage is visulized with the video camera through a microscope lens. The signal fromthe video camera is in analog form and must be digitized so that the computer is ableto store the image in the library. Therefore, the siginal has to be processed by an analogto digital converter. However, the signal has to be coverted into its analog form in orderfor the image to be produced in the monitor. Once the binary images are produced froman accepted microphography, a feature count is performed. This simply means that thedesired 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 ofa very successful air curtain driven fluidized bed reactor. Compressed air is injected

Figure 1. Schematic drawing of oil degradation setup.


into the reactor through a series of perforations in a transverse tube in order to createfluid circulation with an air curtain. Complete details of the bioreactor is presented byAl-maghrabi et al. (1998).

Interfacial Tension Apparatus

The spinning drop model 500 interfacial tensiometer was used in this project. The ap-paratus includes a variable temperature air bath so that reservoir temperatures can beemulated.

Weighting Scale Apparatus

The Mettler PE 300 weighting scale model was used in this research to measure theweight 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 of80◦C for a period of one week to get rid of the volatile components that might evaporateduring the experimental work. Plank tests were performed to measure the weight lossdue 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 casesto be less than 0.05%, and these factors have been corrected in all of the experimentalresults.

A second test involved mixing of bacteria in a batch system under various water-oilratios and various temperatures. Weights of the bottles were taken as a function of time inorder to determine the degradation of oil. Initially, weight losses were determined underthree different oil concentrations, namely 5, 10, and 15% by volume. In the second patchof experiments, the following temperatures were used: 35, 50 and 75◦C. The third patchof 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 thestudy 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 addi-tional experiments were conducted. In the first set, 50% by volume crude oil was addedto a brine solution. The brine solution consisted of 5% by weight NaCl and 5 ml ofindigenous bacteria solution (30 ∗ 103 cells/ ml) to form a 100 ml mixture. The mixturewas placed in a special glass flask, as shown in Figure 2. The system was subjected tocontinue agitation at 50◦C for 30 days. Asphaltine content of the system was measuredas function of time using IP 143 method. In this method, a quantity of the sample isdissolved in n-heptane and the insoluble material, consisting of asphaltenes separatedunder hot reflux with normal n-heptane, and the asphaltenes are isolated by extractionwith toluene. The previous experimental set up was used to study bacteria growth in theoil and water phases as function of time. A mixture consisted of 50 ml of oil and 50 mlof bacteria solution was agitated continuously and bacteria count in both oil and waterphases were taken as function of time. The experiment was conducted for 150 hours atconstant temperature of 50◦C and 5% salinity.


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 highertemperature. At 80◦C bacteria count jumped from initial value of 30 ∗ 103 cell/ml to185∗103 cell/ml after 24 hours of contact time. In both cases the bacteria count droppedinitially for a very short period of time followed by a rapid increase. The drop of initialbacteria count during initial short period of time is due to the adaptation of the bacteria tothe new environment. This period of time is normally called adaptation time. The growthrate of the bacteria used in this project is function of temperature. The growth processof the bacteria was drastically accelerated at high temperature of 80◦C. Therefore, thesebacteria 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 con-centrations: 5, 10, and 15%. The salinity and temperature were kept constant at 5% and

Figure 3. Effect of temperature on bacteria growth.


Figure 4. Effect of oil concentration.

50◦C, respectively, for all tested systems. During the first two hours of mixing time, nosignificant difference in oil degradation between 5 and 10% oil concentration systemswas observed. Increasing mixing time tends in general to increase the difference in oildegradation between the three systems.

The experiment was conducted for 25 hours. At that point, a maximum difference inthe 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 relativelyhigh 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 samespan of time. The same phenomenon was observed by El-Sayed et al. (1995). At highoil concentration, the oil components may reduce ability of the bacteria to degrade crudeoil due to toxic effect on the viability of the cells. High cell density may overcome suchproblem of substance toxicity to some extent.

Effect of Temperature

In order to study the effect of temperature on the biodegradation of crude oil, experimentswere conducted at 35, 50, and 75◦C. The following salinity, oil concentration and initialbacteria concentration were used in all runs: 5% NaCl, 50% oil and 30 ∗ 103 cells/mlrespectively. Figure 5 shows the percent loss of oil as function of time for the three

Figure 5. Effect of temperature on biodegradation.


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 bacteriagrowth around this temperature, as reported previously and shown in Figure 3. The effectof oil evaporation due to high temperature has been taken into account. Bacteria werestill capable of degrading crude oil at temperature of 50◦C. Around 2.0% of the crudeoil degraded after 10 hours of mixing time. Therefore, increasing temperature increasesand accelerates the growth of bacteria that resulted in increasing the degradation processof the crude oil at high temperature.

Effect of Salinity

Figures 6 and 7 show the effect of salinity on the biodegradation of crude oil. Thefollowing bacteria concentration, temperature and oil concentration were used in allruns: 30 ∗ 103 cells/ml, 50◦C and 50% oil, respectively. Initially mixtures contained3 and 6% salinity were tested for one month. At salinity of 6%, around 93% of the crudeoil disappeared after 4 weeks. At relatively lower salt concentration (3% salt), 74%of the crude oil disappeared after 4 weeks. Further experimental work was conductedusing 0 and 10% salinity in addition to the previously used salt concentrations. Theresults are shown in Figure 7 and indicate that increasing salinity tends to increase thebiodegradation process up to a salinity of around 6%; after that we observed the reversetrend. Al-Maghrabi et al. (1998) concluded that high salinity decreased the bacteria

Figure 7. Effect of salinity on oil biodegradation.


Figure 8. Formation of middle phase micro-emulsion.

growth rate, keeping in mind that they investigated only two salinity systems of 0 and10%. Our study indicates that there is an optimum salinity for the degradation of thecrude oil by the thermophilic bacteria. The optimum salinity is around 6% for the studiedsystems. To verify the previous conclusion, five 100 ml mixtures of bacteria, oil andNaCl 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 thecase of 7% salinity. A middle phase microemulsion is associated with lowering of theinterfacial tension and optimum salinity. Therefore, we concluded that the 7% salinityis the optimum salinity for the studied systems. Lin and Townsley (1970) reported thatlignosulfates increase the rate of fermentation of hydrocarbon in water. They attributedthe increased rate of fermentation to the large surface area and the efficiency of cell-to-cellcontact provided by the emulsifier. The growth process and, consequently, the degradationprocess are related to surface area; as the interfacial area increases the degradation rateincreases. Thus, dispersing oil through a water phase (microemulsion) increases both theavailability 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 emulsi-fying the oil with a suitable chemical agent.

Mechanism of Biodegradation

To confirm the oil degradation phenomena by bacteria established by weighting techniqueadditional experimental work was conducted by mixing bacteria solution and oil at roomtemperature and measuring the asphaltene content of the oil phase and bacteria count inboth oil and water phases as function of time. The hypothesis is that if the asphaltenecontent of the oil decreases at the same time bacteria growth increases, then asphalteneis one of the oil components that bacteria is consuming; this confirms the crude oildegradation by bacteria. Two systems were employed in this phase of the work. The firstsystem contains 50% oil concentration, 5% salinity and 1 ml of 30∗103 cells/ml bacteriasolution. The total mixture volume was 500 ml. Figure 9 shows the reduction of asphaltene


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 countin both oil and water phases simultaneously as function of time. Figure 10 shows thebacteria count in both oil phase and water phases as function of time. The plot indicatesthat bacteria quickly transferred into the oil phase and a rapid growth of bacteria wasobserved in the oil phase. Figure 10 also shows that the growth rate of bacteria in thewater phase is quite low. Therefore, it is clear that the media that contains the foodattracted 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 waterphases are the same and the only difference is the rate of growth and the amount ofbacteria exist in the system.

Two microphotographs were taken in order to visualize the transfer of bacteria intothe oil phase. Figure 11 shows a microphotograph that exhibits the existence of bothround-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 productionof 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 wasobtained. Figure 12 clearly shows the spread of bacteria into the oil phase, which confirmsthe partition of bacteria into the oil phase phenomena.

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


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%.


An additional benefit obtained from thermophilic bacteria is its ability to producesurfactant, which could be considered as emulsifying agent. The surfactant productionby the thermophilic bacteria was established by measuring the interfacial tension be-tween the thermophilic bacteria solution and the crude oil. Two systems were testedemploying the crude oil: a bacteria- free solution and mixture contains bacteria. Resultsare shown in Figure 13. The results clearly demonstrate that bacteria are capable ofreducing IFT by producing surfactant at relatively high temperatures of 45◦C. A 70% re-duction in IFT of the crude for the microbial rich solution was observed at a temperatureof 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 tem-

perature increases both the rate and amount of oil degradation by thermophilicbacteria.

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Effect of Temperature on Biodegradation of Crude Oil - Envis