Microbial desulfurization of fuel oil - Springer Link

Microbial desulfurization of fuel oil 1

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XU Ping , LI Fuli , YU Jian , MA Cuiqing , 3 1 4 ZHONG Jianjiang , QU Yinbo & H. D. Blankespoor 1. State Key Lab of Microbial Technology, Shandong University, Jinan 250100, China; 2. Department of Chemical Engineering, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China; 3. State Key Lab of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China; 4. Biology Department of Hope College, Holland, MI 49423, USA Correspondence should be addressed to Xu Ping (e-mail: pingxu@sdu. edu.cn)

Abstract Culture conditions of desulfurization microbes were investigated with a bioreactor controlled by computer. Factors such as pH, choice of carbon source, optimal concentrations of carbon, nitrogen and sulfur sources were determined. The addition of carbon in a culture with a constant pH greatly improved the growth of Rhodococcus. Cells and cell debris from microbes rested using a sulfur-specific pathway were used to desulfurize diesel oil treated by hydrodesulfurization (acquired from the Research Institute of Fushun Petroleum with total sulfur level at 205 ȝg/mL). Strains 1awq, IG, X7B, ZT, ZCR, and a mixture of No. 5 and No. 6, were used in the biodesulfurization process. The reduction of total sulfur was between 10.6% and 90.3%. Keywords: dibenzothiophene (DBT), biodesulfurization (BDS), hydrodesulfurization (HDS), fuel oil.

With the development and use of automobile exhaust systems, and the emphasis on fuel quality, the regulations against sulfur levels in fuels are becoming more stringent. Combustion of fossil fuels leads to the release of toxic sulfur dioxides into the environment, contributing significantly to air pollution and being the principal cause of acid rain[1, 2]. As a result of concern over sulfur emissions, environment regulations require the use of fossil fuels with low-sulfur content, but worldwide reserves of such a low-sulfur crude are limited, so high-sulfur crude oil needs to undergo desulfurization[3]. Hydrodesulfurization (HDS), a chemical method, is a high-pressure (1ü20 MPa) and high-temperature (290ü450§) catalytic process that uses hydrogen gas to reduce the sulfur in petroleum fractions to hydrogen sulfide[4]. Regulations against sulfur levels in fossil fuels are becoming more stringent, and hydrodesulfurization units are expensive to construct and to operate. In addition, the same processes do not work on organosulfur. On the other hand, biocatalytic processes are cheap, operate under mild conditions, and are endowed with high selectivity. There has been a considerable effort to develop fossil fuel desulfurization using bioprocesses. This makes biodesulfurization (BDS) an alternative means in place of HDS for fossil fuels desulfuChinese Science Bulletin Vol. 47 No. 5

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rization[1]. Dibenzothiophene (DBT) is generally considered the model compound for sulfur heterocycles present in hydrodesulfurization-treated fuel[1,3]. Although numerous organisms have been found to remove sulfur from DBT via a hydrocarbon degradative pathway, this method involves the destruction of carbon-carbon bonds and thus results in an unacceptable reduction of fuel value[1,2]. However, a small number of genera, mainly Rhodococcus, Bacillus, Corynebacterium, and Arthrobacter, are known to remove sulfur from DBT via a sulfur-specific pathway, selectively cleaving sulfur from DBT without ring destruction and, therefore, preserve the high quality of the fuel. Recently, the desulfurization pathway with Rhodococcus erythropolis IGTS8 (formerly Rhodococcus rhodochrous IGTS8, ATCC53698)[5] has been characterized (fig. 1). The dszA, -B, and -C (formerly named sox), responsible for DBT desulfurization, transcribed via a single direction, are located in a single operon, controlled by a promoter individually on a 120-kb linear plasmid of strain IGTS8. Three proteins, DszA, -B, and -C have been encoded[6ü8]. These genes have been cloned and sequenced, and their products have been characterized[9ü12]. It has been reported that dsz clusters have been successfully cloned into several Pseudomonas strains. The recombinant strain was able to desulfurize DBT more efficiently than the native one, and, in addition, offered new alternatives for the development of a commercially viable desulfurization process[13]. Most conventional refining processes are performed at much higher temperatures, it was not economic for desulfurization with cooling the stock[14]. More recently, several bacterial strains have been isolated that are capable of degrading DBT and several methylated DBTs. The present study was initiated to determine if microbial desulfurization has the potential of being used commercially. 1

Materials and methods

( ν ) Chemicals. DBT, methylated DBTs, and 2-hydroxybiphenyl (HBP) were of the highest quality available, purchased from Sigma; all the other commercially available chemicals were of analytical grade. Diesel oil samples treated by HDS, containing 205 ȝg/mL or so of total sulfur, were supplied by the Fushun Research Institute of Petroleum. (ξ) Bacterial strains, medium and cultivation. R. erythropolis IGTS8 (ATCC53698), a well characterized bacterium cleaving C-S bond of DBT via ‘4S’ pathway (fig. 1), was used as a control. Strains 1awq, X7B, ZT, No. 5, No. 6, and ZCR, with similar ability to desulfurize DBT via ‘4S’ pathway, all isolated in this work, were used to desulfurize fuel oil with IGTS8. Strains 1awq and X7B 365

NOTES were identified in morphological and biochemical characterization as belonging to genuses Rhodococcus and Bacillus, respectively. The strains were cultured in basic medium with glycerol as carbon source and 0.2 mmol/L DBT or dimetylsulfoxide as the sulfur source. Basal salt medium (BSM) was a sulfur free medium and contained 5 g of glycerol, 0.5 g of KH2PO4, 4 g of K2HPO4, 1 g of NH4Cl, 2 mL of 1% CaCl2, 2 mL of 10% MgCl2, 200 µL of 1% FeCl3, and 200 µL of 5% NaCl. All strains were grown at 30§ with vertical shaking at 150 rotations/min. The strains were harvested after mid-log phase with centrifugation at 5000 r/min and then stored at a low temperature for future use. Cell growth was measured by monitoring the optical density at 620 nm (OD620) by a model 721 spectrophotometer with distilled water as control. Cells were harvested by centrifugation and washed twice by pH 7.0 phosphate buffer, and placed overnight in an oven at 105§.

flask (10%, by volume) into a 1.5-L culture medium. The temperature was kept at 30§; the ventilation ratio was controlled at 1 VVM. (π) Assay of HBP. Desulfurization activity was monitored by using Gibb’s reagent (2,6-dichloroquinone4-chloromide; Sigma) as follows[14]: after centrifugation, 1 mL of supernant of the culture, mixed with 4 mL buffer (pH 9.0), was adjusted to pH 9.0. Then 60 µL of Gibb’s reagent (1 mg/mL in ethanol solution) was added, and the reaction mixture was shaked at 30§ for 30 min untill the color reaction was fully achieved. The absorbance of the reaction mixture was determined at 610 nm (model 721 spectrophotometer; Shanghai No. 3 Analysis Instrument Manufactory) and converted to the concentration on an HBP-generated standard curve. (ρ) Desulfurization of fuel oil. Desulfuriztion of fuel oil was proceeded as follows: cultivation of the seeds of desulfurization microorganisms»scale-up cultivation »cells harvest by centrifugation»washing 2k3 times with a physiological salt solution»resuspension in 120 mL of 20 mmol/L phosphate buffer»mixing aliquot cell debris by ultrasonic waves»adding an appropriate volume of oil and shaking at 30§ for 24 h»oil phase separation. The reaction conditions were as follows: bacterial corcentration of 15 g dry cell per liter, a 3„1 water-to-oil ratio, 20 mmol/L phosphate buffer, and a pH of 7.5. Sample oil was then added; after desulfurization, the oil was separated by centrifugation at 10000 r/min for 5 min[14]. (ς) Estimation of total sulfur in fuel oil. After the above treatment, total sulfur was measured at the Research Institute of Qilu Petrochemical Co. To do this, 3 mL of samples were placed in a WK-2B microcomputer integrated dynamic microcoulomb analysis apparatus (Jiangsu Electroanalysis Instrument Manufactory, China). 2

Fig. 1. Proposed pathway for DBT desulfurization by Rhodococcus rhodochrous IGTS8. DszC, DBT monooxygenase; DszA, DBT-sulfone monoxygenase; DszB, HBPS desulfinase.

(ο) Fermentation conditions. Fermentation apparatus was a Biostat. B2 automatic bioreactor made by B. Braun Co., Germany; the electrode was an Ingold electrode. Inocula were prepared by transferring cells from 366

Results and discussion

(ν) Effects of pH on the growth of Rodococcus sp. 1awq strain. Bacteria were grown in BSM containing 0.5 mmol/L DBT for 48 h. The effects of different pH values on the growth of Rhodococcus sp. 1awq are shown in fig. 2. The optimal pH value for the growth of strain 1awq is 7.0. (ξ) Determination of carbon source. The effects of several carbon sources, such as glycerol, glucose, acetate and citrate, on the growth of 1awq were investigated in BSM, supplemented with each of them at 0.2%. The cells were cultured at 30§ for 48 h with 10% (volume percent) seeds; their growth was monitored at 620 nm as shown in fig. 3. As illustrated in fig. 3, the best carbon source for the growth was glycerol, while citrate, glucose, and acetate Chinese Science Bulletin

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Fig. 2. Effects of pH on the growth of Rodococcus sp. 1awq strain.

Fig. 3. Effects of different carbon sources on the growth of Rodococcus sp. 1awq.

follow it. Of course, glycerol was determined to be the best carbon source. (ο) Determination of optimal concentration of carbon source. The 1awq strain was cultured in BSM, with different concentrations of glycerol as the carbon source. Samples were withdrawn at specific intervals and then measured by monitoring optical density at 620 nm. The optimal growth rate at the log phase was determined, as shown in fig. 4. Data shown in fig. 4 indicate that if the concentration of glycerol is over 0.5%, the cells do not grow well. (π) Determination of optimal concentration of the nitrogen source. The 1awq strain was cultured in BSM with different concentrations of NH4Cl as nitrogen source using 1 mmol/L Na2SO4 as the sulfur source and 0.4% glycerol as the carbon source. After 48 h, the cell growth was measured by monitoring OD620 nm. The results are illustrated in fig. 5. The figure shows that the optimal concentration of nitrogen source was 0.5%; above the level, the cells do not grow well. Chinese Science Bulletin Vol. 47 No. 5

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Fig. 4. Effects of concentrations of glycerol on the growth rate of Rodococcus sp. 1awq.

Fig. 5. Effects of different concentrations of nitrogen source on the growth of the 1awq strain of Rodococcus sp.

(ρ) Determination of the optimal concentration of the sulfur source. Sulfur accounts for 1% of the dry weight of the cell[15]; therefore, the sulfur level for cell growth is limited. The 1awq strain was cultured in BSM with different concentrations of Na2SO4; a mixture of 0.4% glycerol and 0.5% NH4Cl was added. Cell growth was measured by monitoring the OD at 620 nm (fig. 6). From the curve, it is seen that a sulfur level of 0.5 mmol/L will meet the need of cell growth. (ς) Feeding in fed-batch culture with constant pH value. On the basis of the results mentioned above, the optimal pH value for cell growth was 7.0; however, the pH value decreased as the experiment progressed because the NH4Cl was utilized during culturing; at the end of the experiment, the bacteria did not grow well. In subsequent cultures, the pH was controlled at 7.0. This was done by keeping the concentrations of carbon and nitrogen respectively at a level without inhibiting the growth (fig. 7). 367

NOTES

Fig. 6. Effects of different concentrations of Na2SO4 on the growth of Rodococcus sp. (strain 1awq).

mmol/L DBT. HBP was assayed after 48 h of cultivation and the results are shown in fig. 8. HBP was evident for all the strains; however, strain 1awq had the highest amount. Measurement of X7B was performed at 45§. (τ) Growth of IG and X7B at different temperatures. Cells were cultured in BSM supplemented with 0.2 mmol/L DBT for 48 h and the effects of different temperatures on the growth of strains are shown in fig. 9. The figure shows that the optimal temperature for the X7B was 45§or so; on the contrary, IG did not grow over 40§Interestingly, X7B can grow well at a broad range of temperatures. (υ) Desulfurization of fuel oil. Desulfurization of hydrodesulfurized diesel oil was performed by using several bacteria strains, including 1awq, IG, X7B, mixture of No. 5 and No. 6, and ZCR, respectively, as shown in table 1. X7B, a mesophilic bacterium used for desulfurization at 45§, gets the best results because of reducing total sulfur from 205 to 19.8 ȝg/mL (90.3%).

Fig. 8.

The production of HBP with different strains.

Table 1 Summary of the results of the treatment of desulfurized diesel oil by various strains of bacteria Before After Sulfur Reduction treatment treatment eliminated Bacterium (%) 1 1 1 /Pg • mL /Pg • mL /Pg • mL a) 1awq 205 41.0 164.0 80.0 IGa) Fig. 7. Feeding in fed-batch culture with constant pH value (a) and the curve of monitoring parameters online (b).

Based on the data shown in fig. 7, it is obvious that cells grew faster when the culture was maintained at a constant pH. The best growth resulted in concentration of bateria over 14 mg/mL, but it is only 4 mg/mL with the flask fed-batch culture. (σ) Production of HBP using various strains. Bacteria were cultured in BSM, supplemented with 0.2 368

b)

205

63.3

141.7

69.1

205

19.8

185.2

90.3

ZTa)

205

169.1

35.9

17.5

a)

205

183.3

21.7

10.6

205

85.0

120.0

58.5

X7B

5,6

ZCR

a)

a) 30§; b) 45§.

Diesel oil with HDS treatment (205 ȝg/mL S) was obtained from the Petroleum Research Institute of Fushun. The sulfur compounds eliminated by HDS treatment inChinese Science Bulletin

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cluded most of the mercaptan and sulfidic components, the remains were primarily thiophenic sulfur compounds that are refractory to HDS. In the present work, a mesophilic bacterium was good for diesel oil desulfurization. Under higher temperature condition, the total sulfur was decreased from 205 to 19.8 ȝg/mL, showing a 90.3% reduction. Comparable sulfur reductions have been reported previously for desulfurization of diesel oils by a Gordona sp. using sulfur-specific pathway: this yielded a 70% reduction for a middle-distillate unit feed and 50% for light oil gas[16]. Rhodococcus sp. ECRD-1 reduced the sulfur content of a straight-run middle distillate by 30% and oxidized another 35% into oil-soluble products[17]. Rhodococcus erythropolis I-19, a genetic engineering bacterium, a product of Energy Biosystems Corp, USA, reduced an oxidized middle-distillate unit feed from 1850 to 615 ȝg/mL, a 67% reduction[18k20].

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14. Fig. 9. Growths of IG and X7B at different temperatures.

In Japan, a researcher isolated a strain that can desulfurize at 50ć; however, the reduction of sulfur was less[14]. X7B, isolated in our lab, can grow and desulfurize at 45ć with a better reduction. This may result in higher profits from the higher mass transfer rate at higher temperature[21]. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 29977011), and the Visiting Scholar Foundation for Key Laboratory in University, the Ministry of Education of China (State Key Lab of Bioreactor Engineering, East China University of Science and Technology; State Key Lab of Microbial Technology, Shandong University).

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