Abstract
One of the serious problems in the oil industry is precipitation and deposition of asphaltenes in the different oil production stages including formation, wellbore, production tubing, flow lines, and separation units. This phenomenon causes a dramatic increase in the cost of oil production, processing, and transferring. Thus, it seems to be very necessary to use the removing methods for precipitated asphaltenes in different crude oil production and transferring stages. In this study, the ability of microorganisms for biodegradation of precipitated asphaltenes was investigated. For this purpose, four bacterial consortiums were isolated from oil-contaminated soil, crude oil, reservoir water, and oil sludge samples of an oil field located in the southwest of Iran. Based on the results of the designed experiments, by using response surface methodology (RSM) and central composite design, the bacterial consortiums were cultured in the flasks. Three levels of temperatures, salinity, pH, and initial asphaltene concentration as the substrate were considered as the parameters of culture medium and incubated growth mediums for 60 days. The maximum asphaltene biodegradation was 46.41% caused by the crude oil consortium including Staphylococcus saprophyticus sp. and Bacillus cereus sp. at 45 °C, salinity 160 g·L–1, pH 6.5, and 25 g·L–1 initial asphaltene concentration. Also, it was observed that the negative or positive impacts of culture media conditions such as temperature and salinity on asphaltene degradation depended on the type of the available bacterial consortium. The carbon–hydrogen–nitrogen–sulfur analysis showed that carbon, hydrogen, nitrogen, and in some cases, the sulfur in biodegraded samples are less than in control samples. Moreover, Fourier transform infrared analysis indicated that the alkyne groups were less resistant to biodegradation and were eliminated thoroughly after 2 months of incubation. In addition, alkane components were partially removed in treated asphaltene fraction. The parameters of culture medium were optimized by RSM, and besides, their effects on the performance of bacteria in the asphaltene biodegradation process were discussed. The validity of some available kinetic models to describe the behavior of the studied bacteria consortium was investigated, and it was observed that Tessier, Moser, and Contois models accurately predict the values of asphaltenes and biomass concentration at 30, 45, and 60 °C, respectively.
1. Introduction
Asphaltenes are considered as a fraction of crude oil that is insoluble in normal alkanes, e.g., pentane, heptane, and decane, but can be dissolved in aromatic solvents, e.g., toluene, benzene, and xylenes, at room temperature.1−5 X-ray diffraction analysis demonstrated the asphaltene structure as flat plates of aromatics systems that are interconnected by sulfur, ether, aliphatic chains, and/or naphthenic rings.6,7
Asphaltene precipitation from reservoir fluids in all stages of oil production and transportation causes a serious problem and increases the cost of production.8−12 Because of these problems, it seems to be mandatory to find and employ asphaltene precipitation removal methods such as chemical methods, physical techniques, thermal methods, and microbial methods.13−16 Recently, biological processes as one of the affordable and environment-friendly methods have encouraged researchers to use them in removing precipitated asphaltenes. Hydrocarbon biodegradation is a very important mechanism that causes the elimination of crude oil and other pollutant hydrocarbons from ecosphere.17
The limitations of microorganisms’ culture medium are one of the significant controlling factors in the biodegradation of oil pollution in the environment.18,19 The hydrophobic nature of the hydrocarbon components of the petroleum makes them connect with soil components and causes reducing the oil components elimination or degradation. The microorganisms’ effects on hydrocarbons and the possibility of hydrocarbons biodegradation are different. Resistance downtrend of hydrocarbons in the biodegradation process between linear alkanes and asphaltenes is as follows:
Asphaltenes > resin > polycyclic aromatics > cyclic alkanes > small aromatics > branched alkanes > linear alkanes.
The alteration of the crude oil structure due to biodegradation processes and the effect of microorganisms on its chemical and physical properties have been studied significantly.20−24 In comparison, there is not enough studies on the asphaltene biodegradation and the alteration of asphaltene structure in the biodegradation process. Pineda et al. inoculated microbial consortium including Corynebacterium sp., Bacillus sp., Brevibacillus sp., and Staphylococcus sp. to the medium containing mineral salts and asphaltene precipitation. The asphaltene precipitation as the sole source of energy and carbon was added to the mineral medium and asphaltenes biodegradability evaluated using the ISO 9439 method after 13 days of incubation.25 On the other hand, it was confirmed that just a part of asphaltenes and bitumen is affected by enzymes and microbial activity.10 Also, it was proven that asphaltene bio catalytic reformation can be occurred by some hemeproteins such as chloroperoxidase, cytochrome C peroxidase, and lignin peroxidase from Caldariomyces fumago, Bacillus megaterium, and Escherichia coli and remove the nickel and vanadium from petro porphyrins and asphaltenes.10,26,27 Moreover, the possibility of isolating some species from the Dorood oil field and Bangestan reservoir in Iran which can utilize asphaltene precipitation as the sole source of carbon and energy at 28 and 40 °C under shaking and static conditions was studied.28,29 Aditiawati and Kamarisima30 evaluated the performance of four isolated bacteria from a sludge oil sample from Balikpapan on precipitated asphaltene. Bacillus sp. and Lysinibacillus fusiformis have the best ability to degrade asphaltene, and percentage of their asphaltene biodegradation was 50 and 55%, respectively.30 Moreover, Asadollahi et al.,31 identified Bacillus cereus as a bio surfactant-producing bacterium in the oil-contaminated soil and oily sludge samples around the Kermanshah Oil Refining Company which could degrade 40% of asphaltenes after 60 days at 28 °C.31 Iraji and Ayatollahi32 studied on the effect of a surfactant on the microorganism adhesion and cell surface hydrophobicity of Enterobacter cloacae, E. cloacae, and Pseudomonas aeruginosa and evaluated the performance of these bacteria in the asphaltene biodegradation at 40 °C. They observed that cell hydrophobicity alteration leads to more asphaltene biodegradability up to 49%.32
The asphaltene precipitation phenomenon in oil equipment is a serious challenge in the production facilities in the Iranian southwest oilfields,33,34 and in the previous work, it has been shown that some isolated bacteria from the Bangestan reservoir located in the one of the Iranian southwest oilfields could degrade asphaltene precipitation and use it as a sole source of carbon and energy.10
The main goal of this study was an investigation on the precipitated asphaltene biodegradation process by isolated bacterial consortia, from oil contaminated soil, crude oil, reservoir water, and oil sludge samples in the Bangestan reservoir of an Iranian southwest oil field. Furthermore, the effects of cultural medium factors including temperature, salinity, pH, and substrate concentration on the performance of identified microorganisms were evaluated. The values of these parameters have been attempted to be close to operating conditions.
The bacterial consortium’s performance in asphaltenes biodegradation process at 30, 45, and 60 °C with salinity 0, 80, and 160 g·L–1; pH 5.5, 6.5, and 7.5; and initial asphaltenes concentration as substrate 15, 25, and 35 g·L–1 for two months was assessed. In addition, the culture conditions of each available bacterial consortium were optimized through the response surface methodology (RSM). The alteration of asphaltene molecules during biodegradation was investigated using spectroscopy and elemental analysis. Besides, the validation of some available kinetic models to predict the concentration of asphaltenes and microorganisms’ dry weight at pH 6.5, salinity 80 g·L–1, initial asphaltene concentration 25 g·L–1, and temperature 30, 45, and 60 °C have been studied.
2. Results and Discussion
2.1. Identification of Microorganisms
Based on the results of 16S rRNA sequence analysis and morphological and biochemical tests which are listed in Table 2, isolated species from oil-contained samples were identified. The isolated species from the same oil polluted environment was categorized in a consortium. The Soil consortium which was obtained from a polluted soil sample in the burning pit includes Bacillus cereus YSH-1 and Microbacterium paraoxydans YSH-2. Three strains including E. cloacae YSH-3, Bacillus cereus YSH-4, and Stenotrophomonas maltophilia YSH-5 were extracted from the reservoir water sample and placed in Reservoir water consortium. Staphylococcus saprophyticus YSH-6 and Bacillus cereus YSH-7 strains as isolated strains from the crude oil sample were categorized as the Crude oil consortium. Moreover, Sludge consortium includes E. cloacae YSH-8, and Bacillus cereus YSH-9 strains were identified in the oil sludge sample.10
Table 2. Experimental Results of Asphaltene Biodegradation and Dry Weight of Crude Oil and Sludge Consortium.
| response
Crude oil consortium |
response
Sludge consortium |
|||||||
|---|---|---|---|---|---|---|---|---|
| run no. | temperature (°C) | salinity (g·L–1) | pH | asphaltene concentration (g·L–1) | biodegradation (%) | dry weight (g·L–1) | biodegradation (%) | dry weight (g·L–1) |
| 1 | 30.00 | 0.00 | 5.50 | 15.00 | 4.93 | 2.6 | 8.55 | 1.88 |
| 2 | 30.00 | 0.00 | 7.50 | 15.00 | 8.98 | 2.2 | 3.05 | 1.13 |
| 3 | 30.00 | 160.00 | 5.50 | 15.00 | 16.5 | 4.6 | 39.37 | 3.2 |
| 4 | 30.00 | 160.00 | 7.50 | 15.00 | 24.02 | 5.30 | 6.01 | 2.23 |
| 5 | 30.00 | 80.00 | 6.50 | 25.00 | 15.48 | 5.2 | 19 | 1.6 |
| 6 | 30.00 | 0.00 | 5.50 | 35.00 | 4.88 | 1.16 | 4.92 | 3.05 |
| 7 | 30.00 | 0.00 | 7.50 | 35.00 | 1.7 | 1.4 | 11.65 | 1.37 |
| 8 | 30.00 | 160.00 | 5.50 | 35.00 | 15.92 | 3.54 | 33.07 | 3.94 |
| 9 | 30.00 | 160.00 | 7.50 | 35.00 | 12.88 | 1.5 | 12.36 | 4.18 |
| 10 | 45.00 | 80.00 | 6.50 | 15.00 | 10.02 | 3.16 | 1.6 | 1.4 |
| 11 | 45.00 | 0.00 | 6.50 | 25.00 | 21.55 | 5.55 | 8.75 | 3.38 |
| 12 | 45.00 | 80.00 | 5.50 | 25.00 | 4.54 | 1.33 | 15.42 | 4.4 |
| 13 | 45.00 | 80.00 | 6.50 | 25.00 | 13.38 | 1.88 | 11.34 | 5.32 |
| 14 | 45.00 | 80.00 | 6.50 | 25.00 | 13.45 | 2.24 | 10.25 | 5.45 |
| 15 | 45.00 | 80.00 | 6.50 | 25.00 | 14.01 | 1.11 | 11.11 | 4.61 |
| 16 | 45.00 | 80.00 | 6.50 | 25.00 | 13.98 | 1.55 | 11.55 | 4.32 |
| 17 | 45.00 | 80.00 | 6.50 | 25.00 | 13.88 | 1.22 | 10.44 | 5.23 |
| 18 | 45.00 | 80.00 | 6.50 | 25.00 | 13.88 | 3.66 | 11.34 | 1.23 |
| 19 | 45.00 | 80.00 | 7.50 | 25.00 | 4.72 | 5.83 | 10.42 | 2.77 |
| 20 | 45.00 | 160.00 | 6.50 | 25.00 | 46.71 | 8.2 | 15.67 | 4.8 |
| 21 | 45.00 | 80.00 | 6.50 | 35.00 | 7.35 | 1.81 | 8.03 | 1.25 |
| 22 | 60.00 | 0.00 | 5.50 | 15.00 | 4.062 | 2.64 | 5.79 | 1.38 |
| 23 | 60.00 | 0.00 | 7.50 | 15.00 | 15.51 | 8.16 | 16.56 | 1.45 |
| 24 | 60.00 | 160.00 | 5.50 | 15.00 | 5.22 | 4.76 | 18.93 | 2.25 |
| 25 | 60.00 | 160.00 | 7.50 | 15.00 | 20.82 | 1.15 | 3.56 | 0.625 |
| 26 | 60.00 | 80.00 | 6.50 | 25.00 | 14.78 | 8.54 | 39.72 | 9.45 |
| 27 | 60.00 | 0.00 | 5.50 | 35.00 | 9.12 | 4.77 | 12.26 | 4.25 |
| 28 | 60.00 | 0.00 | 7.50 | 35.00 | 11.41 | 2.12 | 35.58 | 5.22 |
| 29 | 60.00 | 160.00 | 5.50 | 35.00 | 7.16 | 0.78 | 23.075 | 4.33 |
| 30 | 60.00 | 160.00 | 7.50 | 35.00 | 12.37 | 4.5 | 19.015 | 2.66 |
It was reported that benzene and other polyaromatic hydrocarbons can be degraded by Bacillus sp. and also, branched alkanes, phenol, naphthalene, acetone, and resin were degraded by Pseudomonas sp.35,36
Therefore, there is the probability of bacterial isolation from oil-contaminated samples that can degrade asphaltenes and as can be observed in some conditions of culture mediums, in the comparison of pure bacteria culture, the consortium of them can provide the maximum asphaltene biodegradation.25,28,29
2.2. Asphaltene Biodegradation Results
The effect of four bacterial consortiums and one mixed culture of all available strains on the asphaltene degradation in the different culture mediums was evaluated. The experimental results of asphaltenes degradation percent (% w/w) and biomass dry weight after two months of incubation are presented in Tables 1–3. The uncertainty in the samples’ weight was ±0.0001 g. It can be seen that in the different growth medium, a specific bacterial consortium had various performances and in similar conditions, the ability of consortiums to degrade asphaltenes was dissimilar. After 60 days, the highest asphaltene biodegradation by the Crude oil consortium was 46.71% at 45 °C, 160 g·L–1 salinity, pH equal to 6.5 and 25 g·L–1 as initial asphaltene concentration. In addition, 39.72, 18.90, 40.35, and 26.03% were the maximum asphaltene biodegradation using sludge, soil, reservoir water, and all-isolated strain consortiums, respectively. Also, it was determined that biodegradation ability of a specific bacterial consortium can be higher or lower than the all-strain consortium. Soil and Crude oil consortiums had the maximum and minimum performances at the lowest temperature, salinity, pH and asphaltene concentration, respectively. Furthermore, at the maximum temperature, salinity, pH and asphaltene concentration, the best and worst efficiencies were observed for Sludge and Soil consortiums, respectively.
Table 1. Experimental Results of Asphaltene Biodegradation and Dry Weight of Soil and Reservoir Water Consortiums.
| response
Soil consortium |
response
Reservoir water consortium |
|||||||
|---|---|---|---|---|---|---|---|---|
| run no. | temperature (°C) | salinity (g·L–1) | pH | asphaltene concentration (g·L–1) | biodegradation (%) | dry weight (g·L–1) | biodegradation (%) | dry weight (g·L–1) |
| 1 | 30.00 | 0.00 | 5.50 | 15.00 | 17.56 | 13.33 | 14.28 | 1.16 |
| 2 | 30.00 | 0.00 | 7.50 | 15.00 | 9.19 | 3.63 | 40.35 | 12.55 |
| 3 | 30.00 | 160.00 | 5.50 | 15.00 | 11.56 | 2.55 | 4.10 | 4.30 |
| 4 | 30.00 | 160.00 | 7.50 | 15.00 | 1.18 | 8.8 | 8.12 | 3.66 |
| 5 | 30.00 | 80.00 | 6.50 | 25.00 | 3.87 | 4.8 | 21.07 | 5.53 |
| 6 | 30.00 | 0.00 | 5.50 | 35.00 | 14.65 | 1.2 | 8.30 | 1.6 |
| 7 | 30.00 | 0.00 | 7.50 | 35.00 | 6.48 | 1.6 | 28.03 | 1.92 |
| 8 | 30.00 | 160.00 | 5.50 | 35.00 | 15.02 | 5.94 | 12.67 | 1.14 |
| 9 | 30.00 | 160.00 | 7.50 | 35.00 | 4.85 | 2.27 | 21.39 | 6.72 |
| 10 | 45.00 | 80.00 | 6.50 | 15.00 | 5.06 | 1.21 | 6.07 | 2.00 |
| 11 | 45.00 | 0.00 | 6.50 | 25.00 | 5.76 | 7.55 | 12.5 | 11.66 |
| 12 | 45.00 | 80.00 | 5.50 | 25.00 | 22.0 | 1.27 | 8.54 | 3.83 |
| 13 | 45.00 | 80.00 | 6.50 | 25.00 | 6.9 | 4.76 | 5.54 | 11.8 |
| 14 | 45.00 | 80.00 | 6.50 | 25.00 | 7.1 | 3.8 | 5.85 | 12.4 |
| 15 | 45.00 | 80.00 | 6.50 | 25.00 | 6.88 | 5.46 | 6.76 | 13.55 |
| 16 | 45.00 | 80.00 | 6.50 | 25.00 | 6.95 | 4.22 | 6.37 | 12.88 |
| 17 | 45.00 | 80.00 | 6.50 | 25.00 | 6.85 | 3.77 | 5.07 | 10.75 |
| 18 | 45.00 | 80.00 | 6.50 | 25.00 | 7.15 | 4.66 | 5.79 | 11.33 |
| 19 | 45.00 | 80.00 | 7.50 | 25.00 | 12.28 | 7.36 | 6.87 | 2.8 |
| 20 | 45.00 | 160.00 | 6.50 | 25.00 | 4.13 | 9.43 | 7.82 | 6.00 |
| 21 | 45.00 | 80.00 | 6.50 | 35.00 | 4.83 | 2.60 | 13.08 | 3.81 |
| 22 | 60.00 | 0.00 | 5.50 | 15.00 | 18.56 | 3.41 | 13.04 | 2.00 |
| 23 | 60.00 | 0.00 | 7.50 | 15.00 | 9.31 | 1.26 | 8.30 | 0.6 |
| 24 | 60.00 | 160.00 | 5.50 | 15.00 | 16.93 | 6.20 | 11.46 | 2.27 |
| 25 | 60.00 | 160.00 | 7.50 | 15.00 | 5.68 | 4.28 | 4.48 | 0.76 |
| 26 | 60.00 | 80.00 | 6.50 | 25.00 | 6.019 | 5.94 | 12.67 | 2.45 |
| 27 | 60.00 | 0.00 | 5.50 | 35.00 | 14.44 | 3.33 | 27.25 | 5.5 |
| 28 | 60.00 | 0.00 | 7.50 | 35.00 | 5.39 | 4.43 | 4.10 | 0.88 |
| 29 | 60.00 | 160.00 | 5.50 | 35.00 | 19.19 | 1.22 | 7.23 | 1.52 |
| 30 | 60.00 | 160.00 | 7.50 | 35.00 | 8.13 | 5.25 | 15.37 | 2.33 |
Table 3. Experimental Results of Asphaltene Biodegradation and Dry Weight of All Isolated Strains.
| response
all-strain consortium |
||||||
|---|---|---|---|---|---|---|
| run no. | temperature (°C) | salinity (g·L–1) | pH | asphaltene concentration (g·L–1) | biodegradation (%) | dry weight (g·L–1) |
| 1 | 30.00 | 0.00 | 5.50 | 15.00 | 12.7 | 1.66 |
| 2 | 30.00 | 0.00 | 7.50 | 15.00 | 18.6 | 2.23 |
| 3 | 30.00 | 160.00 | 5.50 | 15.00 | 6.76 | 3.5 |
| 4 | 30.00 | 160.00 | 7.50 | 15.00 | 7.5 | 1.83 |
| 5 | 30.00 | 80.00 | 6.50 | 25.00 | 14.8 | 8.33 |
| 6 | 30.00 | 0.00 | 5.50 | 35.00 | 15.97 | 1.81 |
| 7 | 30.00 | 0.00 | 7.50 | 35.00 | 18.67 | 2.22 |
| 8 | 30.00 | 160.00 | 5.50 | 35.00 | 10.85 | 1.45 |
| 9 | 30.00 | 160.00 | 7.50 | 35.00 | 8.3 | 1.8 |
| 10 | 45.00 | 80.00 | 6.50 | 15.00 | 16.4 | 2.09 |
| 11 | 45.00 | 0.00 | 6.50 | 25.00 | 16.4 | 5.69 |
| 12 | 45.00 | 80.00 | 5.50 | 25.00 | 4.73 | 1.0 |
| 13 | 45.00 | 80.00 | 6.50 | 25.00 | 13.2 | 4.45 |
| 14 | 45.00 | 80.00 | 6.50 | 25.00 | 13.45 | 4.68 |
| 15 | 45.00 | 80.00 | 6.50 | 25.00 | 12.85 | 4.37 |
| 16 | 45.00 | 80.00 | 6.50 | 25.00 | 13.11 | 5.12 |
| 17 | 45.00 | 80.00 | 6.50 | 25.00 | 13.25 | 5.02 |
| 18 | 45.00 | 80.00 | 6.50 | 25.00 | 13.08 | 4.8 |
| 19 | 45.00 | 80.00 | 7.50 | 25.00 | 5.86 | 4.33 |
| 20 | 45.00 | 160.00 | 6.50 | 25.00 | 12.96 | 8.09 |
| 21 | 45.00 | 80.00 | 6.50 | 35.00 | 26.03 | 7.63 |
| 22 | 60.00 | 0.00 | 5.50 | 15.00 | 6.24 | 2.44 |
| 23 | 60.00 | 0.00 | 7.50 | 15.00 | 11.05 | 3.54 |
| 24 | 60.00 | 160.00 | 5.50 | 15.00 | 9.74 | 5.2 |
| 25 | 60.00 | 160.00 | 7.50 | 15.00 | 9.3 | 1.32 |
| 26 | 60.00 | 80.00 | 6.50 | 25.00 | 12.2 | 2.45 |
| 27 | 60.00 | 0.00 | 5.50 | 35.00 | 8.98 | 4.68 |
| 28 | 60.00 | 0.00 | 7.50 | 35.00 | 10.51 | 3.5 |
| 29 | 60.00 | 160.00 | 5.50 | 35.00 | 13.3 | 4.6 |
| 30 | 60.00 | 160.00 | 7.50 | 35.00 | 9.57 | 2.94 |
In a previous work, it was observed that Bacillus cereus YSH-9 which was isolated from oil sludge had maximum efficiency in the biodegradation of asphaltene precipitation. Maximum biodegradation was achieved using YSH-1, YSH-2, YSH-4, YSH-5, YSH-6, and YSH-8 strains as 41.5, 38.1, 41.51, 30.64, 30.65, and 33.33%, respectively.10 A comparison between the performance of pure isolates and consortia showed that microorganisms in this consortium did not have synergy activity in all growth mediums and in some cases, biodegradation potential of the bacterial consortium can be higher than pure bacteria.
It was reported that compared with pure bacteria, a mixed culture of Pseudomonas sp., Bacillus licheniformis sp., Bacillus lentus sp., Bacillus cereus sp., and Bacillus firmus sp. had the highest reported degradation result, 48%, and obtained at 28 °C.28 In addition, the best consortiums’ performance was achieved by an isolated bacterial consortium from a soil sample from the Shiraz refinery including P. aeruginosa and Pseudomonas fluorescens at 40 °C and 35 g·L–1 of asphaltenes concentration under shaking and static conditions. The highest observed precipitated asphaltene degradation was 51.5 and 32% under shaking and static conditions, respectively.29
The contribution of the assimilation or dissimilation metabolisms in asphaltene biodegradation would be evaluated by measuring the biomass dry weight. In other words, the values of biomass dry weight explained which one of them is more sensible. In assimilation, carbon was absorbed and causes an increase in microorganisms’ dry weight by increasing the substrate utilization. In the dissimilation metabolism, carbon may be released as CO2 and lead to lack of microorganisms’ dry weight with increases in substrate consumption.10 For example, investigation on asphaltene biodegradation and biomass dry weight results of Soil consortium performance in three different growth mediums (run number 1, 20, and 25) shows that despite the rise in asphaltene consumption in growth medium 25 compared with 20, the biomass dry weight decreases. It seems that in medium 25, dissimilation metabolism is more effective than medium 20. On the other hand, such as asphaltene biodegradation percent, the amount of dry weight of biomass in medium 1 is higher than medium 20. It can be concluded that in medium 1, assimilation metabolism is more operative and compared with medium 20, and more carbon is absorbed by bacterial members of the Soil consortium.
2.3. Influence of Independent Variables on the Asphaltene Biodegradation
The effect of temperature, salinity, pH, and asphaltene concentration on the asphaltene biodegradation as independent variables can be illustrated by perturbed figures. These figures are derived from RSM and include some curves that each presents the influence of one of the factors (in this study: growth medium condition) on the response (in this study: precipitated asphaltene biodegradation) qualitatively and can be observed in Figures 1–5.
Figure 1.
Effect of culture medium variables [(A) temperature, (B) salinity, (C) pH, and (D) initial asphaltene concentration] on asphaltene biodegradation with the Soil consortium.
Figure 5.
Effect of culture medium variables on [(A) temperature, (B) salinity, (C) pH, and (D) initial asphaltene concentration] asphaltene biodegradation with the All-strain consortium.
Figure 2.
Effect of culture medium variables [(A) temperature, (B) salinity, (C) pH, and (D) initial asphaltene concentration] on asphaltene biodegradation with the Reservoir water consortium.
Figure 3.
Effect of culture medium variables [(A) temperature, (B) salinity, (C) pH, and (D) initial asphaltene concentration] on asphaltene biodegradation with the Crude oil consortium.
Figure 4.
Effect of culture medium variables [(A) temperature, (B) salinity, (C) pH, and (D) initial asphaltene concentration] on asphaltene biodegradation with the Sludge consortium.
The negative and positive slopes of curves point to that the factors have negative and positive effects on consortium performance, respectively. The zero slope means that increasing or decreasing the values of these parameters does not affect the bacterial consortium activity. As can be observed the influence of temperature, salinity, pH, and substrate concentration on asphaltene degradation depended on the type of the available bacterial consortium. For instance, before the intermediate (center coded) quantities, pH and initial asphaltene concentration had a positive and after that had a negative effect on Crude oil consortium performance. Moreover, in comparison with pH and initial asphaltene concentration, salinity had a reverse effect and temperature did not significantly effect on the biodegradation process by this mixed culture. As can be observed in each perturbed figure, the effect of two independent variables on asphaltene biodegradation are more significant. For example, salinity and pH influences on the Crude oil consortium performance and temperature and initial asphaltene concentration effect on Sludge consortium activity are more remarkable. The concurrent effect of two variables which had the most significant influence on bacteria consortium performance have been illustrated by 3D surface images in Figures 6–10. In these figures, the influences of temperature and pH for the Soil consortium, temperature and salinity for the Reservoir water consortium, salinity and pH for the Crude oil consortium, temperature and initial asphaltene concentration for the Sludge consortium, and pH and initial asphaltene concentration for the all-strain consortium have been studied, respectively. Two other factors for each bacterial consortium have been kept constant in their mid-range.
Figure 6.
Response surface of the asphaltene biodegradation percentage using the Soil consortium on pH and temperature in a salinity of 80 g·L–1 and an initial asphaltene concentration of 25 g·L–1.
Figure 10.
Response surface of asphaltene biodegradation percentage using the All-strain consortium on initial asphaltene concentration and pH at 45 °C and a salinity of 80 g·L–1.
Figure 6 shows that in a salinity of 80 g·L–1 and asphaltene concentration of 25 g·L–1 when increasing the temperature and pH simultaneously, asphaltene degradation by the Soil consortium was initially reduced and then increased. Also, at the highest pH value, increase in the temperature had a positive influence on the Soil consortium activity and more precipitated asphaltene degraded. At the maximum temperature, pH growth before 6.5 led to degradation decrease and after that increase. In addition, asphaltene biodegradation was more sensitive to pH and the effect of the pH alteration on it is more remarkable. Also, maximum biodegradation was obtained in the lowest and highest pH and temperature, respectively.
The concurrent effects of salinity and temperature on asphaltene biodegradation using the Reservoir water consortium in pH 6.5 and an initial asphaltene concentration of 25 g·L–1 have been demonstrated in Figure 7. It can be observed that the growth values of salinity and temperature before intermediate values caused in the performance of the Reservoir water consortium drop and then increase. It is notable that biodegradation in the lowest values of salinity and temperature rather their maximum values occurs more.
Figure 7.
Response surface of the asphaltene biodegradation percentage using the Reservoir water consortium on salinity and temperature in pH = 6.5 and an initial asphaltene concentration of 25 g·L–1.
The 3D surface figure of the asphaltene biodegradation percent using the Crude oil consortium (Figure 8) demonstrates that at 45 °C and an initial substrate concentration of 25 g·L–1, simultaneous increase in salinity and pH before their mid-range led to degradation reduce and after that increase. Furthermore, maximum biodegradation occurred in the highest value of salinity and the mean value of pH.
Figure 8.
Response surface of the asphaltene biodegradation percentage using the Crude oil consortium on pH and salinity at a temperature of 45 °C and an initial asphaltene concentration of 25 g·L–1.
Figure 9 shows that in salinity 80 g·L–1 and pH 6.5, at the same time, the increase in the initial asphaltene concentration and temperature before and after mean values caused to decrease and increase asphaltene biodegradation by the Sludge consortium, respectively. Also, it is detected that maximum biodegradation has been obtained at maximum temperature and mid-range of asphaltene concentration.
Figure 9.
Response surface of the asphaltene biodegradation percentage using the Sludge consortium on initial asphaltene concentration and temperature in a salinity of 80 g·L–1 and pH = 6.5.
In Figure 10, it is revealed that at 45 °C and a salinity of 80 g·L–1, simultaneous increase in asphaltene concentration and pH led to improve the All-strain consortium performance. However, the graph illustrates that the rate of asphaltene biodegradation growth in the higher values of asphaltene concentration and pH was lower than the smaller amounts of their value.
Environmental factors such as temperature, pH, salinity, and so forth, as extrinsic factors can directly affect bacterial growth and metabolism. These factors can disturb membrane fluidity of the bacterial cell and with regard to this fact that many physiological processes in bacterial cell are dependent on membrane integrity, and the disturbing agent that can affect membrane fluidity will reduce bacterial metabolism performance and lead to a reduction in substrate consumption and bacterial growth. Asphaltene concentration cannot be a limiting factor except in very high concentrations that disturbs the carbon to nitrogen ratio and leads to the reduction of bacterial growth and multiplication.
In a previous study, it was determined that at 40 °C, salinity and pH affect asphaltene biodegradation more significantly than asphaltene concentration and also increasing in the salinity had an unfavorable effect on bacteria consortium’s performance including Pseudomonas, Bacillus licheniformis, Bacillus lentus, Bacillus cereus, and Bacillus firmus in asphaltene biodegradation.28
2.4. Elemental Analysis
The results of asphaltene elemental analyses of untrained and trained asphaltene precipitation have been listed in Table 4.
Table 4. Elemental Analysis of Untrained Asphaltene Precipitation (Control Sample) and Biodegraded Asphaltene after 60 days Incubation with Bacterial Consortiums of Bacterial Isolates at 45 °C, 80 g·L–1 Salinity, pH Equal to 6.5 and 25 g·L–1 Initial Asphaltene.
| consortium | % carbon | % hydrogen | % nitrogen | % sulfur | carbon/hydrogen | nitrogen/carbon | sulfur/carbon |
|---|---|---|---|---|---|---|---|
| Control sample | 80.65 | 9.68 | 2.65 | 5.24 | 8.33 | 0.032 | 0.064 |
| Soil consortium | 66.78 | 1.09 | 0.05 | 25.40 | 61.26 | 0.00075 | 0.3803 |
| Reservoir water consortium | 70.52 | N/A | 0.43 | 9.96 | N/A | 0.0060 | 0.141 |
| Crude oil consortium | 55.39 | 0.40 | 0.17 | 1.56 | 138.47 | 0.0030 | 0.0281 |
| Sludge consortium | 58.31 | 0.01 | 0.14 | 5.38 | 5831 | 0.0024 | 0.0922 |
| All-strain consortium | 79.20 | 1.90 | 1.54 | 3.49 | 41.68 | 0.0194 | 0.0440 |
It can be observed that following biodegradation, carbon, hydrogen, and nitrogen contents of asphaltenes precipitation were decreased. Also, only the Crude oil consortium and All-strain consortium were able to decline the weight fraction of sulfur in asphaltene molecules. Increasing in the C/H mass ratio in asphaltene samples after the biodegradation pointed to the fact that consortiums have broken asphaltene molecules, consumed the elemental carbon and released hydrogen.
It was revealed that some heterocyclic aromatic organic nitrogen components such as pyrrolic and pyridinic are relatively resistant to biodegradation while less resistive compounds are degraded, the nitrogen to the carbon ratio in asphaltene molecules will increase.37
2.5. Fourier Transform Infrared Analysis
In Figures 11 and 12, the results of Fourier transform infrared (FT-IR) analysis on (1) control and (2) biodegraded asphaltenes precipitation after two months’ incubation by the Reservoir water and All-strains consortium have been illustrated. The peaks at 3429.58, 2921, and 1611 cm–1 in the control sample correspond to stretch for sp C–H in alkyne groups, aldehyde, and alkene groups, respectively.38,39 It was detected that the alkyne groups had the least resistance to biodegradation in the asphaltene structure and were disappeared during the biodegradation process by each consortium. It was observed that after 2 month incubation, the aldehyde group has not been degraded significantly and as compared to alkyne and alkene compounds, it was more resistant to biodegradation. Furthermore, some relative peaks at almost 1350–1450 cm–1 belonged to bend −CH3 in alkane components which were partially eliminated in treated asphaltenes.10,38 It was identified that in slight or moderate stages of biodegradation, the components such as n-fatty acids and linear aliphatic alcohol which bonded to the asphaltene core by ester and hydrogen bonds were affected. On the other hand, because of the strong covalent bonds such as C–C and ether bonds, the linear alkyl group which was attached by them was degraded at heavy—severe stages.10,40 FT-IR analysis of other samples gave similar results.
Figure 11.
FT-IR spectra of natural asphaltenes (1) and degraded asphaltenes after 60 days of incubation (2) at 45 °C, salinity = 80 g·L–1, pH = 6.5 and initial asphaltene concentration = 25 g·L–1 using the Reservoir water consortium.
Figure 12.
FT-IR spectra of natural asphaltenes (1) and degraded asphaltenes after 60 days of incubation (2) at 45 °C, salinity = 80 g·L–1, pH = 6.5 and initial asphaltene concentration = 25 g·L–1 using the All-strain consortium.
2.6. Optimization of Asphaltene Biodegradation
To optimize the variables of the asphaltene biodegradation process, independent factors including temperature (Tem), salinity (Sal), pH (pH), and initial asphaltenes concentration (Asp) have been applied as input data in quadric models as below
 |
1 |
where Y, α0, and αi (i = 1–14) are biodegraded asphaltenes, intercept and coefficients of the model, respectively, and have been listed in Table 5. The high value of R2 as the coefficient of determination pointed to the adequate fit of this equation for each system over the given experimental domain. The analysis of variance (ANOVA) of the selected model has been presented in Table 6. It can be seen that the proposed quadric model has been significant for each sample. For example, the model F-value of 11485.87 and values of “Prob > F” less than 0.0001 for the Soil consortium indicates that the model is significant. The optimum value of factors predicted the percentage of biodegradation and desirability and is given in Table 7.
Table 5. Values of Intercept, Coefficients, and the Coefficient of Determination of Models.
| factor | ||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| strain | Intercept | Tem | Sal | pH | Asp | Tem × Sal | Tem × pH | Tem × Asp | Sal × pH | Sal × Asp | pH × Asp | Tem2 | Sal2 | pH2 | Asp2 | R2 |
| Soil consortium | 6.96 | 1.07 | –0.82 | –4.86 | –0.11 | 1.09 | –0.22 | –0.30 | –0.50 | 1.59 | 0.050 | –2.01 | –2.00 | 10.19 | –2.00 | 0.89 |
| Reservoir water consortium | –87.06 | –1.163 | –0.187 | 40.08 | –0.236 | 0.001 | –0.35 | 0.005 | –0.003 | 0.0028 | –0.030 | 0.033 | 0.0001 | –1.645 | 0.002 | 0.71 |
| Crude oil consortium | 15.21 | –0.27 | 4.41 | 2.23 | –1.52 | –2.71 | 1.82 | 0.84 | 0.67 | –0.74 | –2.33 | –1.53 | 17.47 | –12.03 | –7.97 | 0.90 |
| Sludge consortium | 12.47 | 2.03 | 3.55 | –2.40 | 3.14 | –4.27 | 4.22 | 2.50 | –6.80 | –0.68 | 3.05 | 15.43 | –1.72 | –1.01 | –9.12 | 0.93 |
| All-strain consortium | –355.0 | +0.16 | –0.025 | 122.9 | –2.736 | 0.001 | –0.01 | –0.001 | –0.015 | 0.0002 | –0.076 | –0.003 | 0.0000 | –9.127 | +0.06 | 0.94 |
Table 6. Analysis of variance (ANOVA).
| response | sum of squares | DOF | mean squares | F-value | P-value Prob > F | |
|---|---|---|---|---|---|---|
| % biodegradation Soil consortium | model | 845.45 | 14 | 60.39 | 11485.87 | <0.0001 |
| residual | 0.079 | 15 | 0.0058 | |||
| % biodegradation Reservoir water consortium | model | 1458.81 | 14 | 104.20 | 2.65 | 0.0357 |
| residual | 590.25 | 15 | 39.35 | |||
| % biodegradation Crude oil consortium | model | 1885.40 | 14 | 134.67 | 10.43 | <0.0001 |
| residual | 193.76 | 15 | 12.92 | |||
| % biodegradation Sludge consortium | model | 2871.22 | 14 | 205.09 | 16.30 | <0.0001 |
| residual | 188.74 | 15 | 18.73 | |||
| % biodegradation All-strain consortium | model | 532.19 | 14 | 38.01 | 17.45 | <0.0001 |
| residual | 32.67 | 15 | ||||
Table 7. Optimum Conditions of Asphaltene Biodegradation Predict Degradation and Desirability.
| parameter |
||||||
|---|---|---|---|---|---|---|
| optimum value | temperature (°C) | salinity (g·L–1) | pH | asphaltene concentration (g·L–1) | predict biodegradation (%) | desirability |
| Soil consortium | 49.49 | 76.26 | 5.50 | 26.72 | 22.0724 | 1.000 |
| Reservoir water consortium | 30.00 | 0.00 | 7.42 | 15.00 | 31.7818 | 0.764 |
| Crude oil consortium | 30.15 | 160.00 | 6.56 | 22.97 | 38.896 | 0.826 |
| Sludge consortium | 30.01 | 146.73 | 5.55 | 28.55 | 39.7995 | 1.000 |
| All-strain consortium | 30.21 | 0.67 | 6.39 | 34.94 | 26.4188 | 1.000 |
2.7. Kinetic Studies on Asphaltene Biodegradation
The ability of Monod, Contois, and Tessier models as kinetic models to predict the concentration of substrate and biomass dry weight of the Crude oil consortium has been evaluated. The asphaltene concentration and microorganism dry weight were measured every five days during 60 days of incubation. In general, the mass balance equations for asphaltenes and biomass are as follows
 |
2 |
 |
3 |
where x, μ, S, Yx/S, and t are the biomass concentration in liquid medium (g·L–1), the specific growth rate (day–1), the substrate concentrations in the liquid medium (g·L–1), the Biomass yield on asphaltenes (g/g) and time, respectively.29,41
The various equations have been proposed for the specific growth rate as a key role in kinetic models. Some of them are as follows
 |
4 |
 |
5 |
 |
6 |
where μm and KS are the maximum specific growth rate (day–1) and kinetic constant (g·L–1), respectively. The kinetic parameters and root-mean-square deviation (rmsd) of these models have been listed in Table 8.
Table 8. Kinetic Parameters and rmsd of Applied Models for the Crude Oil Consortium in Asphaltenes Degradation at pH = 6.5, Salinity = 80 g·L–1, Initial Asphaltene Concentration = 25 g·L–1 and Temperatures = 30, 45, and 60 °C.
| model | temperature (°C) | μm (day–1) | KS (g·L–1) | Yx/S | rmsd |
|---|---|---|---|---|---|
| Monod | 30 | 21.785 | 2513.421 | 2.740 | 0.898 |
| 45 | 8.563 | 1198.620 | 0.334 | 0.699 | |
| 60 | 161.871 | 19721.34 | 0.414 | 0.959 | |
| Contois | 30 | 5.231 | 1629.539 | 2.440 | 0.288 |
| 45 | 1.939 | 1578.588 | 0.439 | 0.146 | |
| 60 | 0.377 | 67.844 | 0.444 | 0.265 | |
| Tessier | 30 | 40.34 | 5436.43 | 0.325 | 1.68 |
| 45 | 33.491 | 4777.128 | 0.331 | 0.698 | |
| 60 | 42.101 | 5123.776 | 0.416 | 0.959 |
It was found that temperature has a significant effect on model precision, and at each temperature, the Contois model could predict the concentration of asphaltenes and microorganisms’ dry weight as a function of time with the lowest rmsd. But as can be seen in Figures 13–15 for 45 °C, in comparison with other models, the Contois model cannot show the lag, exponential, and stationary phase of microorganisms consistent with alteration concentration of asphaltenes by time correctly. Therefore, based on the results of rmsd and curve fitting diagrams, the Monod model is the best kinetics model to predict the behavior of systems at 30 °C and at 45 and 60 °C of both the Monod and Tessier models have almost the same precision.
Figure 13.
Correlation results of the Tessier model with experimental asphaltene biodegradation data using Crude oil consortium at 45 °C, pH = 6.5, salinity = 80 g·L–1 and initial asphaltene concentration = 25 g·L–1.
Figure 15.
Correlation results of the Contois model with experimental asphaltene biodegradation data using the Crude oil consortium at 45 °C, pH = 6.5, salinity = 80 g·L–1, and initial asphaltene concentration = 25 g·L–1.
Figure 14.
Correlation results of the Monod model with experimental asphaltene biodegradation data using the Crude oil consortium at 45 °C, pH = 6.5, salinity = 80 g·L–1, and initial asphaltenes concentration = 25 g·L–1.
It was revealed that in some growth medium conditions, the Tessier model was the best model, and unlike some other models such as Monod, Moser, and logistic models, it had the highest R2 for the description of asphaltene biodegradation using bacterial consortiums and Bacillus lentus.28,29
3. Conclusions
The performances of four bacterial consortiums which identified in the oil-contaminated soil, crude oil, reservoir water, and oil sludge on precipitated asphaltene biodegradation at three levels of temperatures, pH, salinity, and initial substrate concentrations during two months’ incubation were studied. The results showed that after the incubation period, the Crude oil consortium degraded the highest amount of asphaltene precipitation, 46.71%, at 45 °C, 160 g·L–1 salinity, 6.50 pH, and 25 g·L–1 initial asphaltene concentration. The share of assimilation and dissimilation metabolism was not same in all culture mediums and a more powerful effect of assimilation makes increase in the biomass dry weight concurrent with increasing the substrate utilization. The elemental analysis presented that the bacterial consortium broke asphaltene molecules into smaller segments and consumed the carbon, hydrogen, nitrogen, and in some cases, sulfur. The spectroscopy results indicated that alkyne groups and aldehyde groups in asphaltenes have the least and most resistances to biodegradation, respectively. Using the RSM, the statistical optimization was done and the optimum values of culture media parameters were obtained. The results of the kinetic study showed that at different temperatures, Tessier, Moser, and Contois models have the different precision to predict the concentration of precipitated asphaltene and microorganisms’ dry weight of the Crude oil consortium as a function of time and Monod and Tessier models could better correlate the experimental data.
4. Materials and Methods
4.1. Culture Medium Preparation
The content of broth culture medium was mineral salts as the nutrient, precipitated asphaltenes as the substrate and inoculated bacterial consortium. The composition of mineral salts medium used in this study was similar to the previous work, and asphaltene was extracted by the ASTM D3279-90 standard procedure.42 The members of consortiums were isolated from oil-contaminated soil in the burning pit, reservoir water, crude oil, and oil sludge samples from an Iranian southwest oil field and 16S rRNA gene sequencing analysis, and some biochemical tests were carried out to identify them.10,43,44 The utilized general bacterial primer sets were (5′CCGAATTCGTCGACAACAGAGTTTGATCCTGGCTCAG-3′) as a forward primer and (5′-CCCGGGATCCAAGCTTAAGGAGGTGATCCAGCC-3′) as a reverse primer.20 The polymerase chain reaction (PCR) was performed on a thermal cycler by using 25 μL reaction mixture. The reaction mixture contains 1, 12.5, 9.5, and 1 μL of each forward and reverse primers, master mix 2×, Milli-Q water and genome, respectively.16 The reaction mix was exposed to the denaturation process at 94 °C for 5 min; next, the amplification was performed with 25 cycles of 1 min at 94 °C, 40 s at 55 °C, 2 min at 72 °C chased by 1 cycle extension of 10 min at 72 °C.20 The analysis of PCR products was carried out by using 1% agarose gel. As mentioned, the precipitated asphaltene was added to culture medium as sole substrate. Therefore, the isolated strains were able to utilize asphaltene precipitation as a sole source of carbon and energy.
4.2. Asphaltene Biodegradation and Biomass Quantification
The experiments of asphaltene biodegradation were carried out in the flasks, containing 5 mL of inoculum, 50 mL of the broth growth medium in three pH levels 5.5, 6.5, and 7.5, and three levels of salinity 0, 80, and 160 g·L–1, with considering three initial concentrations of asphaltenes, 15, 25, and 35 g·L–1. The flasks were incubated at 30, 45, and 60 °C for two months. A pure colony of each bacterium on the latest solid culture was removed, added to 10 mL of Muller-Hinton broth, were incubated at 30 °C for 24 h and utilized as pure inoculum.10 Then, based on the oil-contaminated source of the bacterial consortiums, 2 mL of pure members of each consortium was inoculated to same fresh Muller–Hinton broth, and the culture mediums were incubated at 30 °C for 24 h. Sampling and analysis were performed at 5 days’ intervals during the experiments. Two samples were provided for each test, and the average result of two experiments was reported.10 To separate the trained asphaltene and quantify the percentage of biodegradation, the IP 143 method was applied.10,45 The free asphaltene medium was centrifuged at 5000 rpm for 10 min, the sediment was dried at 70 °C, and the constant weight was measured and reports as the dry weight of the biomass.28,29
4.3. Analytical Procedures
FT-IR spectroscopy (Spectrum GX model, US) was performed to determine the alteration of the asphaltene chemical structure through the biodegradation procedure. For this purpose, the samples were run as KBr pellets (Merck, Germany). The spectra were normally acquired at 4 cm–1 resolutions over the range of 400–4000 cm–1. Also, carbon–hydrogen–nitrogen–sulfur (CHNS) analysis was performed using the CHNSO analyser (ECS 4010 model, Italy) to evaluate the ability of bacterial consortiums in removing these elements from asphaltene precipitation at 45 °C, 80 g·L–1 salinity, pH equal to 6.5, and 25 g·L–1 of initial asphaltene concentration.
4.4. Asphaltene Biodegradation Optimization
Design of experiments and process variables optimization were done using RSM and central composition design by software design expert 7 (DX7). The process variables including temperature, salinity, pH, and initial asphaltene concentration were identified, and three levels were considered for each of them. For statistical evaluation, the lowest, middle, and highest of available independent factors were coded as −1, 0 and +1, respectively.46,47 Based on the number of desired levels and factors, 30 experiments, including six replicates at the center point to find the experimental error, were designed to determine the effect of various parameters on the precipitated asphaltene biodegradation process.
A quadratic polynomial empirical model was employed to predict the response as below
 |
7 |
where, Y, k, β0, βi, b, βii, βij, and ε represent response (asphaltenes biodegradation percent), number of variables, intercept, coefficients of the linear parameters, variables, coefficients of the quadratic parameters, coefficients of the interaction parameters and residual associated to the experiments, respectively.
4.5. Kinetic Studies
The kinetic study provides a theoretical framework for optimal design in biotechnologies based on the employment of outdoor activity of natural microbial populations in various aspects such as fermentation, enzyme catalysis, wastewater treatment, and soil bioremediation.41 In this study, the ability of some kinetic models such as Monod, Contois, and Tessier models to determine the kinetic coefficients of the Crude oil consortium at three temperatures 30, 45, and 60 °C, with a salinity of 80 g·L–1, pH equal to 6.5, and an initial asphaltene concentration of 25 g·L–1 was evaluated. The cell growth and asphaltene concentration was measured every 5 days for 60 days of the incubation period, and then, the experimental data were used as the input of these models.
Acknowledgments
Authors thank the National Iranian South Oil Company (NISOC) for supporting this research study.
Glossary
Nomenclature
- ANOVA
analysis of variance
- Asp
initial asphaltene concentration (g·L–1)
- DOF
degree of freedom
- k
number of variables
- KS
kinetic constant (g·L–1)
- pH
pH
- R2
coefficient of determination
- Tem
temperature (°C)
- Sal
salinity (g·L–1)
- S
substrate concentrations (g·L–1)
- t
time
- Y
response (biodegraded asphaltene)
- Yx/S
biomass yield on asphaltene (g/g)
- b
variable
- x
biomass concentration (g·L–1)
Greek letter
- α0
intercept
- αi
coefficients of the linear parameters
- αii
coefficients of the quadratic parameters
- αij
coefficients of the interaction parameters
- ε
residual associated to the experiments
- λ
parameter of the Moser model
- μ
the specific growth rate (day–1)
- μm
maximum specific growth rate (day–1)
The authors declare no competing financial interest.
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