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A new method for preparing raw material for biodiesel production
Process Biochemistry 41 (2006) 1699–1702 www.elsevier.com/locate/procbio
Short communication
A new method for preparing raw material for biodiesel production Feiyan Xue a, Xu Zhang a, Hui Luo b, Tianwei Tan a,* a Beijing Key Lab of Bioprocess, College of Biology Science and Technology, Beijing University of Chemical Technology, Beijing 100029, People’s Republic of China b Department of Environmental Engineering, University of Science and Technology Beijing, Beijing 100083, People’s Republic of China
Received 19 January 2006; received in revised form 28 February 2006; accepted 6 March 2006
Abstract Biodiesel has become more attractive recently because of its environmental benefits and the fact that it is made from renewable resources. The cost of biodiesel, however, is the bottleneck for its commercialization. In this paper the monosodium glutamate wastewater after diluted was well treated as a cheap fermentation broth for Rhodotorula glutinis to biosynthesize lipid as the raw material for the production of biodiesel. The optimum conditions were: the initial COD of inlet water was 10,000 mg/L, pH was 5.5, and the inoculum concentration of 12%. The flask treatment result indicated that the COD degradation ratio and the lipid content could reach (85.51 1.00%) and (9.04 0.11%), respectively. And the transesterification of the crude lipid obtained indicated that methyl ester yield was (92.54 2.00%). # 2006 Elsevier Ltd. All rights reserved. Keywords: Biodiesel; Lipid; Rhodotorula glutinis; Monosodium glutamate (MSG) wastewater; COD degradation ratio; Lipid content
1. Introduction Biodiesel is well known as an alternative diesel fuel. The raw materials for biodiesel production now mainly include biological sources such as vegetable seed oil, soybean oil and some recovered animal fats [1,2]. Biodiesel is a biodegradable, nontoxic, and clean renewable fuel with properties similar to conventional diesel. It is produced from renewable resources, and has low emission profiles. So it is environmentally beneficial. However the cost of biodiesel is high due to the high cost of raw material (about 70–75% of the total cost). So biodiesel is still not commonly used in daily life mostly due to the high production cost involved, though this fuel has been developed for about three decades [3–5]. A cheaper raw material for biodiesel production could be a solution. Microbial oils, otherwise referred to as single cell oils (SCO) produced by various microorganisms, are now believed as a potential feedstock for biodiesel production due to their specific characteristics such as they are not affected neither by seasons nor by climates, they own high lipid content, can be produced from a wide variety of sources with short period of production,
* Corresponding author. Tel.: +86 10 64416691; fax: +86 10 64715443. E-mail addresses: [email protected], [email protected] (T. Tan). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.03.002
especially from the residues with abundant nutrition, and so on [6,7]. The microbial oils, however, are mainly used as commercial sources of arachidonic acid (ARA) and docosahexaenoic acid (DHA) [8–10]. The research on microbial oils was focused on these polyunsaturated fatty acids (PUFAs) and related report that whether it could be used for biodiesel production was few. In addition, microbial oils must be absolutely safe if used as dietary supplements while microbial oils needed not when used for biodiesel production. Monosodium glutamate is one of the most popular products for its unique characteristic in the eastern Asia countries. Adding glutamate to foods increases their umami quality, their acceptability and their consumption [11]. However a large quantity of organic wastewater was produced in the course of monosodium glutamate production which caused serious pollution due to its high content of COD (>40,000 mg/L) and low pH (about 2.0). And monosodium glutamate wastewater in China now is usually treated by submerged culture Bacilus thuringeinsis and Candida tropicalis for bio-pesticide and mycelium protein as animal food additive production, respectively [12–14]. But till now, reports about utilizing monosodium glutamate wastewater for the production of microbial oils have not been published yet. In this work, it is intended to produce a cheaper raw material from an organic wastewater through microorganism fermentation. The raw material will be used for the biodiesel production.
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F. Xue et al. / Process Biochemistry 41 (2006) 1699–1702
2. Materials and methods 2.1. Strains Four microorganisms, Rhodotorula glutinis, Candida utilis, Saccharomyces cerevisiae (two strains) were studied in this work. The components of the seed medium were listed as following (%, g/dl): R. glutinis: glucose (4); yeast extract (0.6); urea (0.3); KH2PO4 (0.7); Na2SO4 (0.2). S. cerevisiae (two strains): glucose (2); yeast extract (1); peptone (1). C. utilis: glucose (1); malt extract (0.3); yeast extract (0.3); peptone (0.5).
2.2. Culture conditions The four yeasts, R. glutinis, C. utilis, S. cerevisiae (two strains) were kept in the plate medium for three days at 30 8C to get their activation and then inoculated into the culture medium. Under the culturing conditions, the fermentation went on 24 h at 28 8C with the rotation speed of 140 r/min. Then microorganisms had been further cultured for 5 days in the flask culture medium with monosodium glutamate wastewater which was diluted and pH adjusted. The monosodium glutamate wastewater with an initial COD of 43,210 mg/L and an initial pH 2.0–2.5 was provided by Hongmei Co., Ltd. in China.
2.3. Analytical methods Flasks were periodically taken out from the incubator and cells were collected by centrifugation (4000 rpm 10 min) and washed twice with distilled water. After that the cell was fragmented by sonication (the condition was: 3 s 3 s 90 times 500 W, working time gap time working times output power) and dried at 60–80 8C in a weighing bottle, which had been weighed, until constant weight to measure biomass (biomass = [weight of dry cell and weighing bottle weight of net weighing bottle]/volume of analyzed zymotic fluid). Then the crude lipid in the extraction solvent from the dried cell fragments was obtained with the Soxhlet extraction method [15]. At last the lipid components were analyzed by the gas-chromatograph (GC-2010, made in Shimadzu, Japan). The condition of GC analysis was listed as following: flame ionization detector (FID) 350 8C; OB-1ht, 30 m (length) 0.25 cm (inner diameter) 0.1 mm (thickness); PIV sample entrance (33 cm/s); diffluent ratio1:5; carrier gas N2. COD was tested by COD rapid determinator (made in Twt, Germany) including a heating reactor and a photoelectric colorimeter. And the value was determined by OD605 nm value through the working curve that can be expressed as: y ¼ 2110:3x 30:519
(1)
3.1. The result of wastewater treatment by the four strains
Fig. 1. Results of four different yeasts grown in the monosodium glutamate wastewater. The fermentation system was that slant seed inoculated into the 100 ml flask of wastewater zymotic fluid with a dilution ratio 5. And the culturing conditions were: temperature 30 8C, rotational speed 140 r/min, and pH 6.5, 10% of inoculum concentration.
Because there was a lot of unused organic components and nutrimental elements, furthermore most of TOC in the MGS wastewater can be used as carbon source [14], Four yeasts were studied to grow in MGS wastewater in the experiment. And the results of the wastewater treatment including biomass, total lipid content (%, lipid weight/dry cell weight) and COD degradation were compared for the four microorganisms. Biomass obtained from the four yeasts treatment systems was monitored everyday from the second day (Fig. 1). At the first 2 days of fermentation, the biomass increased very slowly that maybe the reason that the microorganisms were not used to growing in the new and disadvantageous environment compared with normal culturing medium. All of the yeasts’
biomass got to maximum at the 5th day then trended decline. The reason may be that not enough compositions in the wastewater were available for use. In addition, lipid content and COD degradation had the same trends afterward. The intracellular substance was presumed to be consumed or dissolved out of the cell and caused the lipid content and COD degradation decreased. All of the three parameters, biomass, lipid content and COD degradation of the wastewater treated by R. glutinis, were the highest (Fig. 1), which means that remarkable growth ability in the wastewater was the reason that it had been chosen as the destination strain for the monosodium glutamate wastewater treatment.
3. Results and discussion
F. Xue et al. / Process Biochemistry 41 (2006) 1699–1702
1701
Fig. 2. Effect of pH on wastewater treatment. Fermentation system was that slant seed inoculated into the 50 ml flask of wastewater zymotic fluid. And the culturing conditions were: temperature 30 8C, COD content of inlet water10,000 mg/L, and rotational speed 140 r/min, 10% of inoculum concentration.
Fig. 3. Effect of inoculum concentration. Fermentation system was that slant seed firstly inoculated into the 50 ml flask of basal culture medium (glucose: 4%; (NH4)2SO4: 0.2%; KH2PO4: 0.7%; NaSO4%: 0.2%; MgSO47H2O: 0.15%; yeast extract: 0.15%), then the fluid seed was transferred into the wastewater zymotic fluid with COD content 10,000 mg/L. And the culturing conditions were: temperature 30 8C, rotational speed 140 r/min, pH 5.5.
3.2. Effect of pH
for the cell growth, and inoculum concentration had no significant influence on the total lipid content. So we should chose 12% as the better inoculum concentration because of a better COD degradation ratio and the highest biomass in an overall analysis.
R. glutinis was found to be not suitable to grow in an acid condition (Fig. 2). Cell growth and the COD degradation ratio were both very low at pH 2.5, however, the two parameters was relatively stable between pH 4.5 and 7.0. And the three target parameters, COD degradation ratio, biomass (dry cell weight/ culture broth volume) and lipid content peak at pH 5.5. So pH 5.5 was chosen as the optimal value. Otherwise, the result of pH effect indicated COD degradation (around 40%) was much less than the result shown in Fig. 1 (around 75%). All the conditions were thought over to be same except the volume of the fermentation system. So maybe we could say small system fermentation, which was not easy to be aerated and influenced remarkably by water evaporation, was found not suitable to exact treat wastewater exactly. 3.3. Effect of the inoculum concentration The inoculum concentration had significant influence on the COD degradation ratio (Fig. 3). But when the inoculum concentration was more than 12%, the COD degradation ratio remained constant approximately at 85%. In addition, the result indicated that the inoculum concentration of 12% was the best
3.4. Effect of COD of the inlet MSG wastewater The effect of COD of the inlet water on the degradation ratio by R. glutinis was shown in Table 1. As the COD value of the inlet monosodium glutamate (MSG) wastewater decreased from 40,000 to 5000 mg/L, the result of COD degradation ratio increased to a maximum value of 85.51% at COD = 10,000 mg/L, at the same time, biomass reach the peak too. And the COD of the inlet MSG wastewater has little effect on lipid content. So the optimal COD value of the inlet (MSG) wastewater was 10,000 mg/L. The reason is that both biomass and COD were the highest and the total lipid content was not too low at this level. In addition, comparing Table 1 (inoculum was 10%) and Fig. 2 (inoculum was 12%), the data of COD degradation was significantly different (in this case at pH 5.5). This further proved the result that inoculum concentration had significant influence on the COD degradation (Fig. 3).
Table 1 Effect of the concentration of inlet water COD of inlet water (mg/L)
Biomass (g/L)
Total lipid content (%, w/w)
COD degradation ratio (%)
40000 20000 10000 5000
1.56 1.52 2.44 0.80
10.95 9.09 9.04 11.56
36.80 43.80 85.51 80.53
(0.06) (0.08) (0.03) (0.04)
(0.12) (0.14) (0.11) (0.12)
(1.20) (1.50) (1.00) (1.30)
The fermentation system was that slant seed inoculated into the 100 ml flask of wastewater zymotic fluid. And the culturing conditions were: temperature 30 8C, rotational speed 140 r/min, pH 5.5, 12% of inoculum concentration.
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(92.54 2.00%), showing the crude lipid fermented by R. glutinis in MSG wastewater was a promising raw material for biodiesel production. From environmental and economical viewpoints, the work will be an important one and the further study is in progress. For example, adding glucose into monosodium glutamate wastewater fermenting fluid was found be advantageous to both biomass and lipid content which could be reached 34.3 g/L and 12.97%, respectively. Acknowledgements The authors wish to express their thanks for the financial supports from the National Natural Science Foundation of China (No. 20325622, 20576013 and 50373003), ‘‘863’’ High-Tech Project (No. 2002AA217022), National ‘‘973’’ Project (No. 2003CB716002), Beijing National Science Foundation (No. 2032013), Open-project of Beijing Key Lab of Bioprocess (No.SYS100100421), Doctor Program of High Education (No. 20030010004) and National key Program (No.2004BA411B05). References
Fig. 4. Result of GC analysis. (a) Composition of raw lipid and (b) composition after transesterification. The quantity of sample entrance is 1 ml, and the condition of analysis was listed in Section 2. Transesterification system was: 30 mg lipid + 1 ml KOH–CH3OH (0.5 M). After saponification for 10 min at 60 8C in water, then adding 1 ml BF3–CH3OH (12.5%, v/v), reacting for 5 min at 80 8C. The methyl ester was then extracted by the solvent n-hexane.
3.5. Composition of lipid Lipid extracted from the cell of R. glutinis which was fermented in the Monosodium Glutamate wastewater was analyzed by gas-chromatograph (GC) analysis. The result indicated that the composition of lipid fermented in monosodium glutamate wastewater was similar to that of vegetable seed oil and soybean oil. Fatty acid of C16 and C18 and triglyceride (TG) dominated the crude lipid compounds as shown Fig. 4(a). And the transesterification reactions were carried out further and methyl ester was analyzed. The methyl ester contents in the reaction mixture were quantified by GC and the result as shown Fig. 4(b) indicated that the crude lipid from R. glutinis fermentation in the MSG wastewater can be used for biodiesel production because of a high methyl ester yield (92.54 2.00%) obtained. 4. Conclusion A new method for preparing raw material for biodiesel production was developed to utilize high strength MSG wastewater. The system removed about 85% COD and 10% of crude lipid was obtained, respectively. And the transesterification reaction indicated that methyl ester yield was
[1] Ana V, Enoch YP. Lipase-catalyzed production of biodiesel fuel from vegetable oils contained in waste activated bleaching earth. Process Biochem 2003;38:1077–82. [2] Han HW, Cao WL, Zhang JC. Preparation of biodiesel from soybean oil using supercritical methanol and CO2 as co-solvent. Process Biochem 2005;40:3148–51. [3] Ma F, Hanna MA. Biodiesel production: a review. Biores Technol 1999;70:1–15. [4] Allen CAW, Watts KC, Ackman RG, Pegg MJ. Predicting the viscosity of biodiesel fuels from their fatty acid ester composition. Fuel 1999;78: 1319–26. [5] Leunc DYC. Development of a clean biodiesel fuel in HongKong using recycled oil. Water Air Soil Pollut 2001;130:277–82. [6] Papanikolaou S, Komaitis M, Aggelis G. Single cell oil (SCO) production by Mortierella isabellina grown on high-sugar content media. Biores Technol 2004;95:287–91. [7] Shi AH, Zhou B. The research on option of lipid fermentation by Rhodotorula glutinis GLR513. Chinese J Biotech 2003;24:48–51. [8] Eroshin VK, Satroutdinov AD, Dedyukhina EG, Chistyakova TI. Arachidonic acid production by Mortierella alpina with growth-coupled lipid synthesis. Process Biochem 2000;35:1171–5. [9] Wibert GJ, Burns RA, Diersenschade DA, Kelly CM. Evaluation of single cell sources of docosahexaenoic acid and arachidonic acid. Food Chem Toxicol 1997;35:967–74. [10] Harel M, Koven W, Lein I, Bar Y, Behrens P, Stubblefield J, et al. Advanced DHA, EPA and ArA enrichment materials for marine aquaculture using single cell heterotrophs. Aquaculture 2002;213:347–62. [11] John P. Effects of added glutamate on liking for novel food flavors. Appetite 2004;42:143–50. [12] Yang JZ, Wen G, Zhang HX, Wu FL. Study on fermentation properties of Bacillus thuringiensis in the glutamate wastewater. Techniq Equip Environ Pollut Control 2000;1:35–40. [13] Yang JZ, Zhang SP, Zhang HX. Production of microbial protein feed from monosodium glutamate wastewater by fermentation. Techniq Equip Environ Pollut Control 2002;3:21–4. [14] Yang QX, Yang M, Zhang SJ, Lv WZ. Treatment of wastewater from a monosodium glutamate manufacturing plant using successive yeast and activated sludge systems. Process Biochem 2005;40:2483–8. [15] Manirakiza P, Covaci A, Schepens P. Comparative study on total lipid determination using Soxhlet, Roese–Gottlieb, Bligh and Dyer, and modified Bligh and Dyer extraction methods. J Food Compos Anal 2001;14: 93–100.
Short communication
A new method for preparing raw material for biodiesel production Feiyan Xue a, Xu Zhang a, Hui Luo b, Tianwei Tan a,* a Beijing Key Lab of Bioprocess, College of Biology Science and Technology, Beijing University of Chemical Technology, Beijing 100029, People’s Republic of China b Department of Environmental Engineering, University of Science and Technology Beijing, Beijing 100083, People’s Republic of China
Received 19 January 2006; received in revised form 28 February 2006; accepted 6 March 2006
Abstract Biodiesel has become more attractive recently because of its environmental benefits and the fact that it is made from renewable resources. The cost of biodiesel, however, is the bottleneck for its commercialization. In this paper the monosodium glutamate wastewater after diluted was well treated as a cheap fermentation broth for Rhodotorula glutinis to biosynthesize lipid as the raw material for the production of biodiesel. The optimum conditions were: the initial COD of inlet water was 10,000 mg/L, pH was 5.5, and the inoculum concentration of 12%. The flask treatment result indicated that the COD degradation ratio and the lipid content could reach (85.51 1.00%) and (9.04 0.11%), respectively. And the transesterification of the crude lipid obtained indicated that methyl ester yield was (92.54 2.00%). # 2006 Elsevier Ltd. All rights reserved. Keywords: Biodiesel; Lipid; Rhodotorula glutinis; Monosodium glutamate (MSG) wastewater; COD degradation ratio; Lipid content
1. Introduction Biodiesel is well known as an alternative diesel fuel. The raw materials for biodiesel production now mainly include biological sources such as vegetable seed oil, soybean oil and some recovered animal fats [1,2]. Biodiesel is a biodegradable, nontoxic, and clean renewable fuel with properties similar to conventional diesel. It is produced from renewable resources, and has low emission profiles. So it is environmentally beneficial. However the cost of biodiesel is high due to the high cost of raw material (about 70–75% of the total cost). So biodiesel is still not commonly used in daily life mostly due to the high production cost involved, though this fuel has been developed for about three decades [3–5]. A cheaper raw material for biodiesel production could be a solution. Microbial oils, otherwise referred to as single cell oils (SCO) produced by various microorganisms, are now believed as a potential feedstock for biodiesel production due to their specific characteristics such as they are not affected neither by seasons nor by climates, they own high lipid content, can be produced from a wide variety of sources with short period of production,
* Corresponding author. Tel.: +86 10 64416691; fax: +86 10 64715443. E-mail addresses: [email protected], [email protected] (T. Tan). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.03.002
especially from the residues with abundant nutrition, and so on [6,7]. The microbial oils, however, are mainly used as commercial sources of arachidonic acid (ARA) and docosahexaenoic acid (DHA) [8–10]. The research on microbial oils was focused on these polyunsaturated fatty acids (PUFAs) and related report that whether it could be used for biodiesel production was few. In addition, microbial oils must be absolutely safe if used as dietary supplements while microbial oils needed not when used for biodiesel production. Monosodium glutamate is one of the most popular products for its unique characteristic in the eastern Asia countries. Adding glutamate to foods increases their umami quality, their acceptability and their consumption [11]. However a large quantity of organic wastewater was produced in the course of monosodium glutamate production which caused serious pollution due to its high content of COD (>40,000 mg/L) and low pH (about 2.0). And monosodium glutamate wastewater in China now is usually treated by submerged culture Bacilus thuringeinsis and Candida tropicalis for bio-pesticide and mycelium protein as animal food additive production, respectively [12–14]. But till now, reports about utilizing monosodium glutamate wastewater for the production of microbial oils have not been published yet. In this work, it is intended to produce a cheaper raw material from an organic wastewater through microorganism fermentation. The raw material will be used for the biodiesel production.
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F. Xue et al. / Process Biochemistry 41 (2006) 1699–1702
2. Materials and methods 2.1. Strains Four microorganisms, Rhodotorula glutinis, Candida utilis, Saccharomyces cerevisiae (two strains) were studied in this work. The components of the seed medium were listed as following (%, g/dl): R. glutinis: glucose (4); yeast extract (0.6); urea (0.3); KH2PO4 (0.7); Na2SO4 (0.2). S. cerevisiae (two strains): glucose (2); yeast extract (1); peptone (1). C. utilis: glucose (1); malt extract (0.3); yeast extract (0.3); peptone (0.5).
2.2. Culture conditions The four yeasts, R. glutinis, C. utilis, S. cerevisiae (two strains) were kept in the plate medium for three days at 30 8C to get their activation and then inoculated into the culture medium. Under the culturing conditions, the fermentation went on 24 h at 28 8C with the rotation speed of 140 r/min. Then microorganisms had been further cultured for 5 days in the flask culture medium with monosodium glutamate wastewater which was diluted and pH adjusted. The monosodium glutamate wastewater with an initial COD of 43,210 mg/L and an initial pH 2.0–2.5 was provided by Hongmei Co., Ltd. in China.
2.3. Analytical methods Flasks were periodically taken out from the incubator and cells were collected by centrifugation (4000 rpm 10 min) and washed twice with distilled water. After that the cell was fragmented by sonication (the condition was: 3 s 3 s 90 times 500 W, working time gap time working times output power) and dried at 60–80 8C in a weighing bottle, which had been weighed, until constant weight to measure biomass (biomass = [weight of dry cell and weighing bottle weight of net weighing bottle]/volume of analyzed zymotic fluid). Then the crude lipid in the extraction solvent from the dried cell fragments was obtained with the Soxhlet extraction method [15]. At last the lipid components were analyzed by the gas-chromatograph (GC-2010, made in Shimadzu, Japan). The condition of GC analysis was listed as following: flame ionization detector (FID) 350 8C; OB-1ht, 30 m (length) 0.25 cm (inner diameter) 0.1 mm (thickness); PIV sample entrance (33 cm/s); diffluent ratio1:5; carrier gas N2. COD was tested by COD rapid determinator (made in Twt, Germany) including a heating reactor and a photoelectric colorimeter. And the value was determined by OD605 nm value through the working curve that can be expressed as: y ¼ 2110:3x 30:519
(1)
3.1. The result of wastewater treatment by the four strains
Fig. 1. Results of four different yeasts grown in the monosodium glutamate wastewater. The fermentation system was that slant seed inoculated into the 100 ml flask of wastewater zymotic fluid with a dilution ratio 5. And the culturing conditions were: temperature 30 8C, rotational speed 140 r/min, and pH 6.5, 10% of inoculum concentration.
Because there was a lot of unused organic components and nutrimental elements, furthermore most of TOC in the MGS wastewater can be used as carbon source [14], Four yeasts were studied to grow in MGS wastewater in the experiment. And the results of the wastewater treatment including biomass, total lipid content (%, lipid weight/dry cell weight) and COD degradation were compared for the four microorganisms. Biomass obtained from the four yeasts treatment systems was monitored everyday from the second day (Fig. 1). At the first 2 days of fermentation, the biomass increased very slowly that maybe the reason that the microorganisms were not used to growing in the new and disadvantageous environment compared with normal culturing medium. All of the yeasts’
biomass got to maximum at the 5th day then trended decline. The reason may be that not enough compositions in the wastewater were available for use. In addition, lipid content and COD degradation had the same trends afterward. The intracellular substance was presumed to be consumed or dissolved out of the cell and caused the lipid content and COD degradation decreased. All of the three parameters, biomass, lipid content and COD degradation of the wastewater treated by R. glutinis, were the highest (Fig. 1), which means that remarkable growth ability in the wastewater was the reason that it had been chosen as the destination strain for the monosodium glutamate wastewater treatment.
3. Results and discussion
F. Xue et al. / Process Biochemistry 41 (2006) 1699–1702
1701
Fig. 2. Effect of pH on wastewater treatment. Fermentation system was that slant seed inoculated into the 50 ml flask of wastewater zymotic fluid. And the culturing conditions were: temperature 30 8C, COD content of inlet water10,000 mg/L, and rotational speed 140 r/min, 10% of inoculum concentration.
Fig. 3. Effect of inoculum concentration. Fermentation system was that slant seed firstly inoculated into the 50 ml flask of basal culture medium (glucose: 4%; (NH4)2SO4: 0.2%; KH2PO4: 0.7%; NaSO4%: 0.2%; MgSO47H2O: 0.15%; yeast extract: 0.15%), then the fluid seed was transferred into the wastewater zymotic fluid with COD content 10,000 mg/L. And the culturing conditions were: temperature 30 8C, rotational speed 140 r/min, pH 5.5.
3.2. Effect of pH
for the cell growth, and inoculum concentration had no significant influence on the total lipid content. So we should chose 12% as the better inoculum concentration because of a better COD degradation ratio and the highest biomass in an overall analysis.
R. glutinis was found to be not suitable to grow in an acid condition (Fig. 2). Cell growth and the COD degradation ratio were both very low at pH 2.5, however, the two parameters was relatively stable between pH 4.5 and 7.0. And the three target parameters, COD degradation ratio, biomass (dry cell weight/ culture broth volume) and lipid content peak at pH 5.5. So pH 5.5 was chosen as the optimal value. Otherwise, the result of pH effect indicated COD degradation (around 40%) was much less than the result shown in Fig. 1 (around 75%). All the conditions were thought over to be same except the volume of the fermentation system. So maybe we could say small system fermentation, which was not easy to be aerated and influenced remarkably by water evaporation, was found not suitable to exact treat wastewater exactly. 3.3. Effect of the inoculum concentration The inoculum concentration had significant influence on the COD degradation ratio (Fig. 3). But when the inoculum concentration was more than 12%, the COD degradation ratio remained constant approximately at 85%. In addition, the result indicated that the inoculum concentration of 12% was the best
3.4. Effect of COD of the inlet MSG wastewater The effect of COD of the inlet water on the degradation ratio by R. glutinis was shown in Table 1. As the COD value of the inlet monosodium glutamate (MSG) wastewater decreased from 40,000 to 5000 mg/L, the result of COD degradation ratio increased to a maximum value of 85.51% at COD = 10,000 mg/L, at the same time, biomass reach the peak too. And the COD of the inlet MSG wastewater has little effect on lipid content. So the optimal COD value of the inlet (MSG) wastewater was 10,000 mg/L. The reason is that both biomass and COD were the highest and the total lipid content was not too low at this level. In addition, comparing Table 1 (inoculum was 10%) and Fig. 2 (inoculum was 12%), the data of COD degradation was significantly different (in this case at pH 5.5). This further proved the result that inoculum concentration had significant influence on the COD degradation (Fig. 3).
Table 1 Effect of the concentration of inlet water COD of inlet water (mg/L)
Biomass (g/L)
Total lipid content (%, w/w)
COD degradation ratio (%)
40000 20000 10000 5000
1.56 1.52 2.44 0.80
10.95 9.09 9.04 11.56
36.80 43.80 85.51 80.53
(0.06) (0.08) (0.03) (0.04)
(0.12) (0.14) (0.11) (0.12)
(1.20) (1.50) (1.00) (1.30)
The fermentation system was that slant seed inoculated into the 100 ml flask of wastewater zymotic fluid. And the culturing conditions were: temperature 30 8C, rotational speed 140 r/min, pH 5.5, 12% of inoculum concentration.
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F. Xue et al. / Process Biochemistry 41 (2006) 1699–1702
(92.54 2.00%), showing the crude lipid fermented by R. glutinis in MSG wastewater was a promising raw material for biodiesel production. From environmental and economical viewpoints, the work will be an important one and the further study is in progress. For example, adding glucose into monosodium glutamate wastewater fermenting fluid was found be advantageous to both biomass and lipid content which could be reached 34.3 g/L and 12.97%, respectively. Acknowledgements The authors wish to express their thanks for the financial supports from the National Natural Science Foundation of China (No. 20325622, 20576013 and 50373003), ‘‘863’’ High-Tech Project (No. 2002AA217022), National ‘‘973’’ Project (No. 2003CB716002), Beijing National Science Foundation (No. 2032013), Open-project of Beijing Key Lab of Bioprocess (No.SYS100100421), Doctor Program of High Education (No. 20030010004) and National key Program (No.2004BA411B05). References
Fig. 4. Result of GC analysis. (a) Composition of raw lipid and (b) composition after transesterification. The quantity of sample entrance is 1 ml, and the condition of analysis was listed in Section 2. Transesterification system was: 30 mg lipid + 1 ml KOH–CH3OH (0.5 M). After saponification for 10 min at 60 8C in water, then adding 1 ml BF3–CH3OH (12.5%, v/v), reacting for 5 min at 80 8C. The methyl ester was then extracted by the solvent n-hexane.
3.5. Composition of lipid Lipid extracted from the cell of R. glutinis which was fermented in the Monosodium Glutamate wastewater was analyzed by gas-chromatograph (GC) analysis. The result indicated that the composition of lipid fermented in monosodium glutamate wastewater was similar to that of vegetable seed oil and soybean oil. Fatty acid of C16 and C18 and triglyceride (TG) dominated the crude lipid compounds as shown Fig. 4(a). And the transesterification reactions were carried out further and methyl ester was analyzed. The methyl ester contents in the reaction mixture were quantified by GC and the result as shown Fig. 4(b) indicated that the crude lipid from R. glutinis fermentation in the MSG wastewater can be used for biodiesel production because of a high methyl ester yield (92.54 2.00%) obtained. 4. Conclusion A new method for preparing raw material for biodiesel production was developed to utilize high strength MSG wastewater. The system removed about 85% COD and 10% of crude lipid was obtained, respectively. And the transesterification reaction indicated that methyl ester yield was
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