Production of lactic acid and fructose from media with cane sugar using mutant of Lactobacillus delbrueckii NCIM 2365

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1 Letters in Applied Microbiology ISSN ORIGINAL ARTICLE Production of lactic acid and fructose from media with cane sugar using mutant of Lactobacillus delbrueckii NCIM 2365 S.S. Patil, S.R. Kadam, S.S. Patil, K.B. Bastawde, J.M. Khire and D.V. Gokhale NCIM Resource Centre, National Chemical Laboratory, Pune, Maharashtra, India Keywords fructose accumulation, hydrolyzed cane sugar, lactic acid production, Lactobacillus delbrueckii, mutant. Correspondence D.V. Gokhale, NCIM, National Chemical Laboratory, Pune 411 8, Maharashtra, India. 25/128: received 27 October 25, revised and accepted 25 January 26 doi:1.1111/j x x Abstract Aims: To examine the potential of Lactobacillus delbrueckii mutant, Uc-3 to produce lactic acid and fructose from sucrose-based media. Methods and Results: The mutant of L. delbrueckii NCIM 2365 was cultivated in shake flask containing hydrolysed cane sugar (sucrose)-based medium. The lactic acid yield and volumetric productivity with hydrolysed cane concentration up to 2 g l )1 were in the range of 92 97% of the theoretical value and between 2Æ7 and 3Æ8 gl )1 h )1, respectively. The fructose fraction of the syrup produced was more than 95% when the total initial sugar concentration in the medium was higher (15 2 g l )1 ). There are no unwanted byproducts detected in the fermentation broth. Conclusions: We demonstrated that L. delbrueckii mutant Uc-3 was able to utilize glucose preferentially to produce lactic acid and fructose from hydrolysed cane sugar in batch fermentation process. Significance and Impact of the Study: These findings will be useful in the production of lactic acid and high fructose syrups using media with high concentrations of sucrose-based raw materials. This approach can lead to modification of the traditional fermentation processes to obtain value-added byproducts, attaining better process economics. Introduction Lactic acid and its derivatives have been widely used in food, pharmaceutical, cosmetic and industrial applications (VickRoy 1985). It has also been receiving great attention as a feedstock for manufacture of polylactic acid (PLA), a biodegradable polymer used as environmental-friendly biodegradable plastic. PLA could be a good substitute for synthetic plastic derived from petroleum feedstock. Being highly reactive due to the presence of carboxylic and hydroxyl groups, lactic acid can undergo a variety of chemical conversions into potentially useful chemicals, such as propylene oxide, propylene glycol, acrylic acid, 2,3-pentanedione and lactate esters (Datta et al. 1995; Varadarajan and Miller 1999). Lactic acid is produced commercially either by chemical synthesis or by microbial fermentation. Approximately 9% of the total lactic acid produced worldwide is by bacterial fermentation and the rest is produced synthetically by the hydrolysis of lactonitrile. The chemical synthesis of lactic acid always results in racemic mixture of lactic acid, which is a major disadvantage. Fermentative production of lactic acid offers the advantages in both utilization of renewable carbohydrates and production of optically pure l- or d-lactic acid depending on the strain selected. The physical properties of PLA depend on the isomeric composition of lactic acid (Amass et al. 1998; Lunt 1998). Therefore, pure l- or d-lactic acid is polymerized to a high crystal polymer suitable for fibre and oriented film production and is expected to be useful in the production of ligand crystal as well (Ohara and Yahata 1996). The fructose is considered to be the sweetest sugar found in nature. It is the constituent of invert sugar and high fructose corn syrup (HFCS) produced commercially in large quantities. These products contain fructose and glucose in nearly equimolar concentrations. HFCS containing 42%, 55% and 9% fructose are produced Journal compilation ª 26 The Society for Applied Microbiology, Letters in Applied Microbiology 43 (26)

2 Coproduction of lactic acid and fructose S.S. Patil et al. commercially. The use of currently available syrups (42% and 55%) for the production of pure fructose requires a substantial reduction in their glucose content. The existing industrial methods use costly chromatographic techniques to produce HFCS containing 9% fructose from 42% HFCS. Selective conversion of glucose from sucrose media to products, such as ethanol or lactic acid, which are easily separated from fructose could be an alternative to get high fructose syrups with fructose content more than 9%. This approach also leads to modification of the fermentation processes to obtain value-added byproducts, attaining better process economics. The selective conversion of glucose from glucose/fructose mixtures has been carried out using Mucor sp. M15 (Ueng et al. 1982), Zymomonas mobilis (Doelle 1989; Doelle and Doelle 1991; Kirk and Doelle 1994) were used for the production of fructose from sucrose media. The production of enriched fructose syrups and ethanol from sugar beet molasses (Atiyeh and Duvnjak 22) and sucrose-based media (Atiyeh and Duvnjak 21a,b) using Saccharomyces cerevisiae ATCC was also studied. The preliminary studies carried out by di Luccio et al. (22) on the economic analysis of the process for the co-production of ethanol and fructose represents a very promising alternative to improve the ethanol process economics. The present work reports on the co-production of lactic acid and fructose from hydrolysed cane sugar using mutant of Lactobacillus delbrueckii NCIM Fructose has been investigated to be a byproduct of the selective fermentation that might improve the process economics of lactic acid production. The proposed work is based on the preferential utilization of glucose from acid hydrolysed sucrose media, yielding lactic acid and fructose as products. Materials and methods Micro-organism Lactobacillus delbrueckii Uc-3 mutant used in this study was obtained by mutation (Kadam et al. 26). It was maintained in liquid MRS medium (Hi-Media, Mumbai, India) supplemented with Æ1% CaCO 3 which was used as stock culture for preparation of inoculum. Growth and production media The growth medium consisted of (w/v), 1 g l )1 hydrolysed cane sugar, 1 g l )1 yeast extract and 5 g l )1 CaCO 3. The basic production medium was the same as the growth medium. The ingredients of the media were separately added after sterilization at 121 C for 2 min. The ph of both growth and fermentation media was adjusted to 7Æ prior to sterilization. The cane sugar was hydrolysed by adding 1 ml of 2% H 2 SO 4 in 1 ml of sugar solution. The acidified sugar solution was heated in boiling water bath for 2 min. The ph of the hydrolysed cane sugar was adjusted to 7Æ. The hydrolysed cane sugar (1 g) contained glucose (5 g) and fructose (5 g) as determined by high-performance liquid chromatography (HPLC). Batch fermentation in shake flask Inoculum was prepared by transferring the culture (2 ml) pregrown in liquid MRS medium to 1 ml of the growth medium in 25-ml screw cap conical flask. The flask was incubated at 42 C with shaking at 15 rev min )1 for 24 h. Batch fermentation experiments were carried out in 25-ml screw cap conical flasks containing 1 ml fermentation medium. Flasks were inoculated with inoculum culture (5%) and incubated in shaker incubator at 42 C with shaking at 15 rev min )1. The culture samples harvested at various time intervals were centrifuged at 6 g for 2 min to separate the cells. The supernatant was analysed for sugars and lactic acid and for determining the ph of the fermentation broth. The supernatant was acidified by adding equal volume of 1 mol l )1 HCl, when free acid is liberated. Analytical methods Samples were removed aseptically at various time intervals to determine biomass, lactic acid, glucose and fructose. The biomass concentration was determined by drying the samples at 8 C for 24 h in preweighed glass dishes. Glucose, fructose and lactic acid were determined using a HPLCsystem (Dionex India Limited, Mumbai, India) equipped with UV- or RI-detectors. An ion exclusion column (Aminex HPX-87H; Bio-Rad, Hercules, CA, USA) was used at a temperature of 38 C with 8 mmol l )1 H 2 SO 4 as a mobile phase at a flow rate of Æ6 ml min )1. An injection volume of the sample was 5 ll. Results Fermentation pattern of mutant, Uc-3 in hydrolysed cane sugar medium Kinetics of growth, lactic acid production and fructose accumulation by mutant Uc-3 in a fermentation medium containing 19 g l )1 of hydrolysed sucrose is shown in Fig. 1. The results of this fermentation showed that this mutant was able to preferentially convert glucose to lactic acid and biomass. In the beginning of the process, the 54 Journal compilation ª 26 The Society for Applied Microbiology, Letters in Applied Microbiology 43 (26) 53 57

3 S.S. Patil et al. Coproduction of lactic acid and fructose Biomass (g l 1 ), ph Time (h) biomass was produced by consuming preferentially 7% glucose within 24 h where only 15% fructose was consumed. The glucose consumption rate was faster than that of fructose, which resulted in the accumulation of fructose and lactic acid in the fermentation broth. The biomass increased from Æ3 gl )1 to 5Æ7 gl )1, which represents a biomass yield of Æ42 g g )1 of glucose and fructose consumed. Fructose and lactic acid yields after 36 h of fermentation were 7 and 95% of the theoretical values respectively. It was also noticed that approx. 25% of the fructose was used for lactic acid production. Effects of hydrolysed cane sugar concentration 12 1 Figure 1 Kinetics of growth of Lactobacillus delbrueckii mutant Uc-3 and lactic acid production and fructose accumulation in synthetic medium containing 19 g l )1 of hydrolysed cane sugar: (h) biomass; ( ) glucose; (s) ph; (d) fructose; ( ) lactic acid. The results of the effects of sugar concentration on the production of lactic acid and fructose are summarized in Table 1. No significant difference in total biomass obtained was observed when the concentration of sugar increased from 5 to 2 g l )1. The media containing 25 g l )1 of sugar gave lower biomass. However, the biomass yield in the media decreased from Æ14 to 8 6 Glucose, fructose, lactic acid (g l 1 ) Biomass (g l 1 ) Time (h) 12 1 Figure 2 Kinetics of fermentation of synthetic media containing hydrolysed cane sugar 5 g l )1 (+, open symbols) and 25 g l )1 (x, closed symbols) by Lactobacillus delbrueckii mutant UC-3: (+, x) biomass production; (n, ) glucose consumption; (h, ) fructose consumption and (s, d) lactic acid production. Æ35 g g )1 of glucose and fructose consumed. This decrease in biomass yield was expected because of the growth inhibition by a high substrate concentration. There was not much difference in lactic acid yield and productivity up to 2 g l )1 sugar concentration. However, these values are much affected at 25 g l )1 concentration of sugar indicating the greater growth inhibition due to osmotic effects at high sugar concentration. The more accumulation of fructose in the media was observed when hydrolysed sucrose concentration was increased from 5 to 25 g l )1. The fermentation of medium containing high sugar concentration of 2 g l )1 yielded 12 g l )1 of lactic acid, 7 g l )1 fructose and 4 g l )1 of glucose. The kinetics of biomass production, glucose consumption, fructose accumulation and lactic acid production by L. delbrueckii mutant Uc-3 in fermentation media with various sugar concentrations are shown in Fig. 2. Higher concentration (25 g l )1 ) of hydrolysed cane sugar resulted in significant initial decrease in biomass production Glucose, fructose, lactic acid (g l 1 ) Table 1 Biomass, lactic acid production, fructose and glucose consumption, lactic acid and biomass yields, lactic acid productivities for media with different hydrolysed cane sugar concentrations using Lactobacillus delbrueckii mutant Uc-3 Total sugar Biomass Lactic acid Fructose Glucose Lactic acid yield (%) Biomass yield (g g )1 ) Lactic acid productivity (g l )1 h )1 ) 5 5Æ2 46 (12 h)* 92Æ Æ14 3Æ8 8 5Æ7 75 (24 h)* 2Æ8 97Æ1 Æ73 3Æ1 1 5Æ6 77 (24 h)* 15Æ4 2Æ 93Æ2 Æ67 3Æ2 15 5Æ5 92 (3 h)* 5Æ 3Æ5 95Æ3 Æ57 3Æ 2 5Æ7 12 (36 h)* 7Æ6 4Æ6 95Æ6 Æ45 3Æ3 25 4Æ7 112 (48 h)* 9Æ3 28Æ 84Æ8 Æ35 2Æ3 *, The numbers in parentheses show time in hours when the samples were analysed. The values calculated in the table are the average of three independent experiments with 4 7% variations. Journal compilation ª 26 The Society for Applied Microbiology, Letters in Applied Microbiology 43 (26)

4 Coproduction of lactic acid and fructose S.S. Patil et al. indicating the growth inhibition due to high substrate concentration. Glucose was used at a higher rate from the very beginning of the fermentation for biomass production than that of fructose leading to accumulation of fructose in the broth. Hydrolysed cane sugar at 25 g l )1 concentration resulted in production of lactic acid with lower yields and also accumulation of glucose in addition to fructose even after 48 h of fermentation. Discussion The results obtained in the present study showed that L. delbrueckii mutant Uc-3 was able to preferentially utilize glucose rapidly in media containing hydrolysed cane sugar. It utilized fructose at a much lower rate than that of glucose leading to accumulation of fructose and lactic acid in the fermented broth. The mutant was isolated by exposing the cells of L. delbrueckii NCIM 2365 (parent strain) to UV-irradiation (Kadam et al. 26). The parent strain showed slow growth in a medium containing higher (2 g l )1 ) hydrolysed cane sugar, which resulted in low productivity of lactic acid (Kadam et al. 26). The parent strain also exhibited the characteristics of preferential glucose utilization. The mutant Uc-3 gave very high lactic acid yields even at 2 g l )1 hydrolysed cane sugar concentration than the parent strain and hence it was selected for this study. There are many reports on co-production of fructose and ethanol from sucrose by fructose nonutilizing mutants of S. cerevisiae (Atiyeh and Duvnjak 21a,b, 22) and Z. mobilis (Doelle and Doelle 1991; Kirk and Doelle 1994). These mutants were capable of utilizing glucose selectively from media with glucose/fructose mixtures or sucrose and producing fructose and ethanol. Majority of the micro-organisms used in these processes produced unwanted byproducts, such as sorbitol and glycerol along with fructose and ethanol. Saha and Nakamura (23) have reported the production of mannitol from fructose by Lactobacillus intermedius NRRL B The bacterium produced mannitol, lactic acid and acetic acid from fructose. However, there are no reports in the literature on co-production of lactic acid and fructose from sucrose. The results obtained in the present study demonstrated that the L. delbrueckii mutant Uc-3 was able to produce lactic acid and fructose from media containing hydrolysed cane sugar. The lactic acid can be separated as solid calcium lactate at low temperatures leaving behind the syrup containing high fructose concentration (> 95%). Lactic acid can also be recovered from fermented broth by incorporating nanofiltration or electrodialysis in downstream processing rejecting the nonutilized sugars like fructose, which can be either recycled or converted into HFCS. This syrup can be used commercially for the production of pure and solid fructose. The use of currently available syrups (42% and 55% HFCS) requires a substantial reduction in their glucose content in order to produce pure fructose. Considering that India is producing 2 million metric tonnes of cane sugar, and that fructose market is expanding, the production of fructose as a byproduct of lactic acid fermentation may represent an alternative to increase the profitability of lactic acid production. Conclusions The present study showed that L. delbrueckii mutant Uc-3 was able to utilize preferentially glucose from media containing hydrolysed cane sugar. This resulted in the production of lactic acid and accumulation of fructose in the fermented broth. The lactic acid yield was more than 9%, which can be separated as calcium lactate. The syrups obtained after separation of lactic acid contained more than 9% fructose when media with 1 2 g l )1 of hydrolysed cane sugar was used for fermentation. Even at 25 g l )1 of hydrolysed cane sugar concentration, the produced syrup contained 75% fructose which is richer in fructose content than the ordinary and 55% HFCS. No other unwanted byproduct like acetic acid was detected in the fermented broth. Acknowledgements The authors acknowledge the financial support of NMI- TLI Division of Council of Scientific and Industrial Research, New Delhi, India. References Amass, W., Amass, A. and Tighe, B.A. (1998) Review of biodegradable polymers: uses, current developments in the synthesis and characterization of biodegradable polyesters, blends of biodegradable polymers and recent advances in biodegradation studies. Polym Int 47, Atiyeh, H. and Duvnjak, Z. (21a) Study of the production of fructose and ethanol from sucrose media by Saccharomyces cerevisiae. Appl Microbiol Biotechnol 57, Atiyeh, H. and Duvnjak, Z. (21b) Production of fructose and ethanol from media with high sucrose concentrations by a mutant of Saccharomyces cerevisiae. 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5 S.S. Patil et al. Coproduction of lactic acid and fructose and lactic acid derivatives. FEMS Microbiol Rev 16, Doelle, H.W. (1989) Conversion of sucrose to fructose and ethanol. US Patent 4,797,36. Doelle, M.B. and Doelle, H.W. (1991) High fructose formation from sugarcane syrup and molasses using Zymomonas mobilis mutants. Biotechnol Lett 13, Kadam, S.R., Patil, S.S., Bastawde, K.B., Khire, J.M. and Gokhale, D.V. (26) Strain improvement of Lactobacillus delbrueckii NCIM 2365 for lactic acid production. Process Biochem 41, Kirk, L.A. and Doelle, H.W. (1994) Simultaneous fructose and ethanol production from sucrose using Zymomonas mobilis 2864 co-immobilized with invertase. Biotechnol Lett 16, di Luccio, M., Borges, C.P. and Alves, T.L.M. (22) Economic analysis of ethanol and fructose production by selective fermentation coupled to pervaporation: effect of membrane costs on process economics. Desalination 147, Lunt, J. (1998) Large scale production, properties and commercial applications of polylactic acids polymers. Polym Degrad Stab 59, Ohara, H. and Yahata, M. (1996) l-lactic acid production by Bacillus sp. in anaerobic and aerobic culture. J Ferment Bioeng 81, Saha, B.C. and Nakamura, L.K. (23) Production of mannitol and lactic acid by fermentation with Lactobacillus intermedius NRRL B Biotechnol Bioeng 82, Ueng, P.P., McCracken, L.D., Gong, C.S. and Tsao, G.T. (1982) Fructose production from sucrose and high fructose syrup: a mycelial fungal system. Biotechnol Lett 4, Varadarajan, S. and Miller, D.J. (1999) Catalytic upgrading of fermentation-derived organic acids. Biotechnol Prog 15, VickRoy, T.B. (1985) Lactic acid. In Comprehensive Biotechnology, vol. 3, ed. Moo-Young, M. pp New York: Pergamon Press. Journal compilation ª 26 The Society for Applied Microbiology, Letters in Applied Microbiology 43 (26)