Mannitol production from glycerol by resting cells of Candida magnoliae Asiya Khan, Amey Bhide, Ramchandra Gadre Chemical Engineering and Process Development Division National Chemical Laboratory Pune 411008 India
Running Title: Mannitol production by C. magnoliae
*Correspondence to Dr. R. V. Gadre, Chemical Engineering and Process Development Division National Chemical Laboratory, Pune, 411008, India E-mail:
[email protected] Telephone: +91 20 25902348 Fax: +91 20 25902612
Published in Bioresource Technology, 2009, 100:4911-4913
ABSTRACT
Production of mannitol from glycerol by resting cells of Candida magnoliae under aerobic condition was investigated. The resting cells were suspended in aqueous solution of glycerol in Erlenmeyer flasks and incubated on rotary shaker. The samples were analyzed by ion exclusion-HPLC equipped with refractive index and UV detector. The resting cells of C. magnoliae produced mannitol from fructose, sucrose and glycerol but not from glucose. Addition of yeast extract and/or potassium phosphate to the glycerol solution adversely affected its conversion to mannitol. The conversion of glycerol to mannitol was dependent on oxygen availability. Using resting cells, the yield of mannitol was as high as 45%. This is probably the first report of conversion of glycerol to mannitol by osmophilic yeast.
KEY WORDS
Candida magnoliae, resting cells, mannitol production, glycerol
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1. INTRODUCTION
Mannitol is widely distributed in nature and is present in small quantities in many fruits, vegetables and mushrooms. It exhibits low chemical reactivity, low hygroscopicity, has a sweet taste but a very low caloric value and therefore is used in several food and pharmaceutical preparations (Soetaert et al.1999). Mannitol is produced currently by highpressure catalytic hydrogenation of fructose and glucose mixture. Although mannitol production by fermentation using fungi, yeast, bacteria and also by enzyme reactions has been investigated in past, major improvement in the yield and productivity have been achieved only recently. Song et al. (2002) isolated osmophilic yeast, Candida magnoliae that produced 209 g l-1 mannitol using a mixture of fructose and glucose with a yield of 83%. Lee et al. (2003) achieved 213 g l-1 mannitol in fed-batch fermentation of C. magnoliae using glucose: fructose mixture at a ratio of 1: 20. Candida magnoliae also is reported to produce glycerol (Sahoo and Agarwal, 2002), erythritol (Koh et al. 2003) and xylitol (Tada et al. 2004), using different substrates and fermentation conditions. Glycerol is available as a cheap raw material generated in biodiesel production in several countries (Johnson and Taconi, 2007). Value addition and utilization of this glycerol is being investigated by its conversion to 1,3-propanediol, succinic acid, ethanol and lipids by fermentation (Zhao et al. 2006; Easterling et al. 2008). Production of citric acid and erythritol from glycerol using mutants of Yarrowia lipolytica has been studied recently (Rymowicz et al. 2008; Rymowicz et al. 2009). We report here production of mannitol from glycerol using resting cells of Candida magnoliae.
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2. MATERIALS AND METHODS 2.1. Microorganism and media Candida magnoliae (NCIM 3470) was obtained from National Collection of Industrial Microorganisms, National Chemical Laboratory, Pune, India and maintained on malt extract glucose yeast extract peptone (MGYP) agar. Media ingredients were purchased from HiMedia, Mumbai, India. 2.2. Analysis Optical density (OD600) was measured with a spectrophotometer and dry cell weight (DCW) was determined at 103 °C to constant weight. A ratio of 0.32 g l-1 DCW per 1 OD600 was established. Concentrations of glucose, fructose, mannitol, erythritol, glycerol and other cometabolites were determined using ion exclusion high performance liquid chromatograph equipped with an Aminex HPX-87H, 300 X 7.6 mm column (Bio-Rad) at 50°C. Mobile phase used was 0.01 N H2SO4 at 0.5 ml/min. Carbohydrates and polyols were detected by refractive index detector, Shodex RI-71. The quantification was done by external standard technique. A UV-VIS detector was used at 214 nm simultaneously, so as to eliminate possibility of coeluting, UV-detectable compounds. 2.3. Growth of C. magnoliae on different carbon sources The growth medium contained (g l-1) yeast extract 10, KH2PO4 5, MgSO4 0.25, pH 6.5 and one of the following carbon sources; fructose, glucose, glycerol or sucrose at 250 g l-1. A fresh slant was used for inoculation of 25 ml media in 250 ml Erlenmeyer flasks and the flasks were incubated at 28 oC, at 220 rpm, for 48 h. At 24 h interval, samples were withdrawn and analyzed for OD600, DCW, pH, residual substrate and the end products by ion exclusionHPLC described above.
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All the experiments were done in triplicate. 2.4. Preparation of resting cells Erlenmeyer flasks of 250 ml capacity containing 25 ml growth medium composed of (g l-1) yeast extract 10, KH2PO4 5, MgSO4 0.25 and 100 glycerol were inoculated and incubated, at 28 oC, on a rotary shaker, for 48 h. The broth was aseptically centrifuged at 10000 rpm, at 5 o
C, for 10 min. The cell pellet was suspended in sterile physiological saline and the cells were
recovered by centrifugation. This procedure was repeated once more to get clean resting cells. Fresh cells were prepared for each experiment. 2.5. Effect of individual medium components on mannitol production Equal quantity of resting cells of C. magnoliae prepared as above was suspended in sterile 20 ml glycerol solution (100 g l-1) in separate flasks. To the different flasks, sterile solutions of yeast extract, KH2PO4, and MgSO4.7H2O were added individually or in different combinations, at the concentrations equal to that in the growth medium. Control flasks contained only glycerol solution. Volumes of the reaction mixtures were adjusted to 25 ml by addition of sterile distilled water. The flasks were incubated at 28 °C, on rotary shaker at 220 rpm, for 96 h. Samples were withdrawn periodically and analyzed as above. 2.6. Viability staining One ml cell suspension was mixed with 1 ml of methylene blue solution (10 mg ml-1) and held at 30 oC for 10 min. The cells were observed microscopically and classified as blue and colourless, representing dead and alive cells, respectively.
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2.7. Polyol production by resting cells from different carbohydrates and glycerol Resting cells (3 g) were suspended in 25 ml sterile solutions of glucose, fructose, sucrose and glycerol (100 gl-1) individually, in 250 ml Erlenmeyer flasks and incubated on a rotary shaker. Samples were withdrawn at regular interval and analyzed as above. 2.8. Effect of yeast extract and potassium phosphate addition Resting cells (3 g) were suspended in 25 ml sterile glycerol solution (100 g l-1) in 250 ml Erlenmeyer flasks. Small quantities of sterile yeast extract stock solution were added to the experimental flasks to achieve yeast extract concentrations of 0, 0.1 and 0.2 g l-1 in different flasks. The flasks were then incubated on rotary shaker at 220 rpm, 28 °C, for 96 h. The same quantities of yeast extract solution were added to the flasks at every 24 h, during incubation. Samples were withdrawn at 24 h interval and analyzed as above. Similarly, effect of inorganic phosphate on mannitol production from glycerol by resting cells of C. magnoliae was investigated by addition of different quantities of sterile KH2PO4 solution to the sterile glycerol solution (100 g l-1) and incubating the flasks for 96 h. Samples were withdrawn and analyzed at regular interval.
2.9. Effect of resting cell concentration Different quantities of the resting cells were weighed aseptically and suspended in 25 ml sterile glycerol solution (100 g l-1) in 250 ml Erlenmeyer flasks and incubated at 220 rpm, 28 °C for 96 h. Samples were removed and analyzed as above. 2.10. Effect of different medium volumes in shake flasks Equal quantities (3 g) of fresh resting cell mass were aseptically transferred to different 250 ml Erlenmeyer flasks containing different volumes of glycerol solution (100 g l-1) ranging
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between 12.5 and 100 ml. All the flasks were incubated at 220 rpm, 28 °C for 96 h. Samples were withdrawn and analyzed as above. 3. RESULTS The strain of C. magnoliae used in the current investigation showed carbohydrate utilization pattern similar to the type strain (NCYC 2620). It could utilize fructose, galactose, glucose, glycerol, mannose, mannitol, raffinose, ribose and sucrose for growth. It did not utilize arabinose, cellobiose, maltose, melezitose, melibiose, rhamnose, salicin, trehalose and xylose. The ion exclusion chromatography used in the present investigation was well suited for polyols as well as sugars analysis. The sequence of elution was (min) glucose (11.30), fructose (12.35), mannitol (12.76), erythritol (14.65) and glycerol (16.87). In the complete medium, C. magnoliae grew well at 250 g l-1 initial substrate concentration and produced a mixture of erythritol and mannitol in the medium containing glucose and sucrose but produced only mannitol when fructose and glycerol were used (Table 1). The resting cells could not consume glucose effectively and only 32 g l-1 glucose was utilized in 96 h. The yield of mannitol was also low, just 10% based on the glucose utilized. Fructose was completely consumed by the resting cells of C. magnoliae within 96 h and resulted in 44.5 g l-1 mannitol production. Most interestingly, glycerol was completely consumed within 96 h and resulted in highest mannitol production (51 g l-1). In contrast to the earlier experiment where growing cultures produced mixtures of erythritol and mannitol, the resting cells produced only mannitol irrespective of the carbon source used. In the experiment when the resting cells were used in solution of glycerol with addition of different components of the growth medium, it was observed that the mannitol production was maximal when only glycerol was present. During experiment with periodic addition of yeast extract on glycerol
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conversion to mannitol it was observed that the addition of yeast extract at every 24 h interval to the glycerol solution drastically decreased the mannitol production by resting cells of C. magnoliae. The lesser production was not an effect of increased cell mass as could be seen from equal OD600 in all the flasks. Similarly, addition of 5 g l-1 potassium di-hydrogen phosphate in the glycerol solution decreased the mannitol production from 31 g l-1 to 13 g l-1. A small amount of erythritol was also produced in the presence of phosphate, which was otherwise not observed. Increase in resting cell mass from 3 g to 20 g resulted in decrease in glycerol uptake from 100 g l-1 to 62 g l-1 in 96 h and also decreased the mannitol concentration from 51 g l-1 to mere 3 g l-1. The yield of mannitol also decreased from 51% to a poor 4.8% on the basis of glycerol utilized. In shake flask experiments, the medium volume is known to influence the cell growth or metabolism because of differences in the oxygen transfer rates (Mantzouridou et al. 2005). In the present investigation, the medium volume in 250 ml Erlenmeyer flasks strongly influenced conversion of glycerol to mannitol by resting cells. Use of 12.5 ml medium in 250 ml Erlenmeyer flasks had the highest mannitol production rate as seen from Figure 1. In case of 50 ml and more volume, the rate of conversion was considerably low and decreased further with increase in volume of glycerol solution used. Methylene blue staining of resting cells was a suitable technique for viability determination and the cells remained viable till consumption of all the glycerol in the reaction mixture.
4. DISCUSSION
Candida magnoliae resting cells produced mannitol from glycerol in aqueous solution under aerobic condition without any other nutrient. Most of the chemical and biological processes
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investigated earlier use fructose or fructose and glucose mixture as carbon source. The investigations by Baek et al. (2003) while working with Candid magnoliae had showed that C. magnoliae did not produce mannitol from glycerol although fructose, sucrose and glucose were converted to mannitol. The strain of C. magnoliae used in the present investigation is probably different, because it produced mannitol from glycerol while growing as well as in resting condition, not observed by any investigator earlier. In the present investigation, we studied production of mannitol from glycerol, which has to follow gluconeogenesis pathway. Unlike mannitol production from fructose, conversion of glycerol to mannitol does not need NADPH2 and there is no need for any co-metabolite. Mannitol yield of about 50% based on glycerol could be achieved in the present investigation while rest of the glycerol was presumably converted to carbon dioxide. In the investigation by Lee et al. (2003), production of mannitol was a two-phase process. In the present investigation, we have used resting cells of C. magnoliae for mannitol production by physically separating the growth and production phase from each other. Resting cells of C. magnoliae did not produce any other metabolites detectable by UV-VIS or RI detector and resulted in a clean aqueous solution of mannitol from which recovery of mannitol would be relatively easy. The increased generation of glycerol as a by-product of bio-diesel production has resulted in availability of large stocks of crude and dilute glycerol solutions in the world. Although currently the crude petroleum has become cheaper, it is unlikely that the bio-diesel production will be discontinued. However, to sustain, the bio-diesel industry will require value addition and economic utilization of glycerol generated as its by-product. Mannitol production from glycerol using resting cells of C. magnoliae would be one of such possibilities.
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5. CONCLUSIONS Production of mannitol from glycerol by resting cells of Candida magnoliae was investigated in the present study. The conversion was higher with high oxygen availability while it was adversely affected by excessive quantity of resting cells, addition of potassium phosphate and even small quantities of yeast extract. The decrease in the mannitol production because of yeast extract addition was presumably due to a change in the metabolism and not because of oxygen limitation caused by increased cell mass. Mannitol was the only metabolite produced from glycerol by resting cells of C. magnoliae with yield of mannitol as high as 50%. This is probably the first report of mannitol production from glycerol.
ACKNOWLEDGEMENT The authors are thankful to Department of Biotechnology, Government of India for financial support under Research Grant Number BT/PR9118/PID/06/386/2007.
REFERENCES Baek, H., Song, K.-H., Park, S.-M., Kim, S.-Y., Hyun, H.-H., 2003. Role of glucose in the bioconversion of fructose into mannitol by Candida magnoliae. Biotechnol. Lett. 25, 761765. Easterling, E.R., Todd French, W., Hernandez, R., Licha, M. 2009. The effect of glycerol as a sole source and secondary substrate on the growth and fatty acid composition of Rhodotorula glutinis. Bioresource Technol. 100, 356-361. Johnson, D.T., Taconi, K.A. 2007. The glycerin glut: Options for the value added conversion of crude glycerol from biodiesel production. Environ. Progress. 26, 338-348.
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Koh, E.-S., Lee, T.-H., Lee, D.-Y., Kim, H.-J., Ryu. Y.-W., Seo, J.-H., 2003. Scale up of erythritol production by an osmophilic mutant of Candida magnoliae. Biotechnol. Lett. 25, 2103-2105. Lee, J.-K., Song, J.-Y., Kim, S.-Y., 2003. Controlling substrate concentration in fed batch Candida magnoliae culture increases mannitol production. Biotechnol. Progress 19, 768-775. Mantzouridou, F., Roukas, T., Achatz, B. 2005. Effect of oxygen transfer rate on β-carotene production from synthetic medium by Blakeslea trispora in shake flask culture. Enzyme Microbial Technol. 37, 687-694. Rymowicz, W., Rywinska, A., Gladkowski, W., 2008. Simultaneous production of erythritol from crude glycerol by Yarrowia lipolytica Wratislavia K1. Chemical Papers 62, 239-246. Rymowicz, W., Rywinska, A., Marcinkiewicz, M., 2009. High yield production of erythritol from raw glycerol in fed-batch cultures of Yarrowia lipolytica. Biotechnol. Lett. 31, 377-380. Sahoo D.K., Agarwal G.P. 2002. Effect of oxygen transfer on glycerol biosynthesis by an osmophilic yeast Candida magnoliae I2B. Biotechnol. Bioengg. 78, 545-555. Soetaert, W., Vanhooren, P.T.,Vandamme, E.J., 1999. Production of mannitol by fermentation. Methods in Biotechnol. 10. Humana Press, Totowa, New Jersey, USA, pp. 261275 Song K.-H., Lee, J -K., Song. J.-Y., Hong S.-G., Baek, H., Kim, S.-Y., Hyun, H.-H., 2002. Production of mannitol by a novel strain of Candida magnoliae. Biotechnol. Lett. 24, 9-12. Tada, K., Horiuchi, J.-I., Kanno, T., Kobayashi, M. 2004. Microbial xylitol production fromcorn cobs using Candida magnoliae. J. Biosci. Bioengg. 98, 228-230. Zhao Y.-N, Chen, G., Yao S.-J. 2006. Microbial production of 1-3 propanediol from glycerol by encapsulated Klebsiella pneumoniae. Biochem. Engg. J. 32, 93-99.
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Asiya Khan Figure 1 35
Mannitol Conc (g l-1)
30 25 12.5 25
20
50 75
15
100
10 5 0 0
50
100
150
Medium Volume (ml)
Asiya.Khan et al. Table 1 Growth and polyol production by C. magnoliae in different carbon sources.
Carbon
pH at the
source
end
Sugar/ glycerol
Erythritol
Mannitol
utilized
produced
produced
g l-1
g l-1
g l-1
g l-1
DCW
Glucose
2.3
15.0
154
23.4
7.2
Fructose
3.3
7.4
250
0
39
Sucrose
2.2
19.5
219
8
15
Glycerol
4.1
16.6
171
0
20
12
