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DOI 10.1002/star.201100006

rke 2011, 63, 632636 Starch/Sta

RESEARCH ARTICLE

Production of L() lactic acid from sweet sorghum, date palm, and golden syrup as alternative carbon sources
Anil K. Yadav1, Nirichan K. Bipinraj2, Ambalal B. Chaudhari1 and Ramanlal M. Kothari2
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School of Life Sciences, North Maharashtra University, Jalgaon, India Rajiv Gandhi Institute of IT and BT, Bharati Vidyapeeth Deemed University, Pune, India

Lactic acid production has been carried out using (a) Lactobacillus delbruckii, (b) optimal production medium comprised of 10% carbon source derived from sweet sorghum (SS) (Sorghum bicolor)/golden syrup (GS; molasses after glucose crystallization)/date palm (DP) juice (Phoenix dactylifera L.), 1% yeast extract, 0.6% sodium acetate, 0.5% KH2PO4, and 0.5% MgSO4 7H2O, (c) in batch mode, (d) at 45 18C, 150 rpm, anaerobic condition, and pH 5.5 0.1, and (e) sterilized CaCO3 powder at regular intervals for neutralization to promote optimal utilization of sugar. The lactic acid (LA) productivity was higher from the use of GS and less from SS as well as DP juice. Decolorization of carbon sources prior to use in fermentation gave LA, meeting food grade specications. Keywords: Date palm juice / Golden syrup / L() Lactic acid / Lactobacillus delbruckii / Sweet sorghum juice

Received: January 6, 2011 Revised: April 18, 2011 Accepted: April 21, 2011

Introduction

Lactic acid (LA) is considered as a specialty chemical due to its use in various applications in food, non-food [1], and bioplastic industry [2, 3]. For this purpose, LA is produced by microbial fermentation [4], which has shown that production of D-or L-forms or their mixture depends on the microbial species used and source of carbon employed [1]. While most of the studies on LA fermentation are focused on using rened carbohydrates such as glucose [5, 6] or starch hydrolysate [7], it has been found that the cost of production medium alone accounts for 6080% of the total production cost, thus making it imperative to use economical agricultural residues/by-products/produce like sugarcane juice/molasses, sugar beet juice, etc. [1]. However,

their use has met with (a) variable conversion rates of carbon source into LA, (b) difculties in post-fermentation processing, (c) problems in the yield and quality of LA, (d) high cost of marketable grade LA, and (e) pollution problems as a result of additional steps in the separation of different isoforms. To address these problems, it was conceived that use of relatively superior quality agri-products such as juice of sweet sorghum (SS; Sorghum bicolor) and date palm (Phoenix dactylifera) available economically or industrial by-products such as golden syrup (GS; molasses after recrystallization of two crops of D-glucose) available in plenty and economically could be explored for the production of LA, which is theme of the present article.

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Correspondence: Professor Ramanlal M. Kothari, Rajiv Gandhi Institute of IT and BT, Bharati Vidyapeeth Deemed University, Pune-411046, India E-mail: rmkothari1942@gmail.com Fax: 91-20-24365713 Abbreviations: DP, date palm; GS, golden syrup; LA, lactic acid; RS, reducing sugars; SS, sweet sorghum

Materials and methods

2.1 Culture
Lactobacillus delbruckii was obtained from National Collection of Industrial Microbes (NCIM), NCL, Pune. It was grown in Man Rogosa Sharpe (MRS)-agar medium, sub-cultured regularly as per NCIM instructions and preserved at 48C. www.starch-journal.com

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633 ortho-phosphoric acid). While 0 and 24 h samples were diluted 25-fold and 48, 72, and 96 h samples were diluted 50-fold. They were subsequently ltered through 0.2 mm Millipore assembly and the ltrate used for HPLC analysis using Hamilton microsyringe. LA was determined by HPLC (Perkin Elmer, series 200), (i) using SUPELCOGELTM C-610 column (30 cm 7.8 mm) lled with H form of sulfonated polystyrene divinyl benzene resin, (ii) by injecting 10 mL of claried fermented liquor at 308C, (iii) mobile phase of 0.1% orthophosphoric acid at a ow rate of 0.5 mL/min, (iv) UV detector at 210 nm, and (v) monitoring detection time for LA at 17th minute (as judged from standard solution). All the solutions used in HPLC were ltered through 2 mm Millipore lter. The area obtained under each peak was computed to give concentration of LA [1].

2.2 Inoculum
L. delbruckii was grown in inoculum medium comprised of 5.5% de MRS nutrients and 24 h old culture (1.4 108 cells/mL) at 10% v/v of the total volume of production medium served as an inoculum.

2.3 Sources of carbon

SS (Praj SSV-84, Mumbai) seeds procured from the market were cultivated and harvested after 110 days [8]. Its stem juice extracted through mechanical crusher was treated with 20% slurry of milk of lime at pH 7.5, using 2.5 ppm Magnaock (occulating agent) at 958C for 30 min, ltered, and used for fermentation. Date palm (DP; P. dactylifera L.) fruits procured from the open market were cleaned, seeds taken out, biomass soaked in water (1:1 w/w) for softening for 8 h at ambient temperature, and juice prepared using kitchen blender. It was decolorized using 0.5% bone charcoal at 808C for 15 min, ltered under vacuum, and colorless ltrate was used for fermentation. GS, the molasses after crystallization of second crop of D-glucose, collected from M/S Universal Starch Chem. & Allied Industries, Dondaicha, Maharashtra, was per se used for fermentation. Ingredients used for preparing the production medium were procured from the open market, Mumbai.

Results and discussion

Efforts were focused to reduce the cost of carbon source against glucose or sucrose used presently [7].

3.1 Sweet sorghum

SS is a dryland coarse cereal grown in India (on 9.5 m ha of land) and around the world (on 45.8 m ha of land) for food, animal feed, and ber. The crop is (3.04.0 m tall), and relatively photoperiod sensitive, generally having low grain yield with higher stalk yield (7075% of fresh biomass). Sugarcane (i) is presently used bioenergy crop, (ii) has poor ability to adapt to drought/salinealkaline conditions, (iii) is water guzzling crop, (iv) has evapotranspiration rate of 1275 mm, and (v) needs 360 450 days for harvesting rendering in a single crop in 1216 months. In contrast, SS (i) is new generation bioenergy crop, (ii) has excellent ability to adapt to drought and salinealkaline conditions, permitting use of such lands/crops for cultivation, (iii) has 35% requirement of water vis-a-vis sugarcane on comparable time scale, (iv) has evapo-transpiration rate of 508 mm, (v) needs 110120 days for harvesting, rendering a feasibility of three crops in 12 months, and (vi) requires 22% less water than that required by maize, another energy crop used abundantly in USA. Thus, agronomically, SS use makes an eco-friendly, pragmatic, and cost-effective choice.

2.4 Typical fermentation protocol

Batch fermentation was carried out in 2 L pre-sterilized optimized medium in a lab fermenter (Navin Process Systems, Pune) containing 10% carbon source, 1% yeast extract, 0.6% sodium acetate, 0.5% KH2 PO4, and 0.5% MgSO4 7H2O at 45 18C, pH 5.5 0.1, 150 rpm, and without aeration [8]. The LA formed during fermentation was neutralized by the addition of sterilized 2% w/v slurry of CaCO3 at regular intervals to minimize the inhibition of LA production. Using this protocol, LA production was carried out as a function of above-mentioned carbon sources at least 5 times and repeated 15 times using GS for statistically meaningful work.

2.5 Analytical methods

Reducing sugars (RS) were estimated by DNSA reaction [9]. Cell growth was monitored using Neubauers chamber [10].

3.2 Date palm 2.5.1 Sample preparation for lactic acid estimation
The fermented liquor was centrifuged (3000 rpm, 5 min, 428C) and diluted suitably using mobile phase (0.1% 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DP is a widely distributed horticultural crop in the tropical and sub-tropical climatic zones in the world. It has three main types of cultivars according to the moisture content of fruits [11]. www.starch-journal.com

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3.3 Golden syrup

It is a by-product of glucose industry obtained after the recovery of crystallized second crop of D-glucose by basket centrifugation. The centrifugate is concentrated to attain 80% solids (w/w) so that it is stable at ambient temperature. It was explored for value addition as attempted earlier for the production of sorbitol (12).

3.4 Production of lactic acid as a function of alternate carbon sources

Fermentation of LA from substrates like juice of SS, DP , and GS was explored due to their abundant, easy, and economic availability (Table 1). It was observed that L. delbruckii strain is efcient in converting 10.0% substrate concentration from the juice of DP , SS, and GS into LA (Fig. 1). Accumulation of LA proceeded with an increase in the viable cell mass, consequently causing a delay in the LA production between 12 and 24 h (Table 2). It was probably due to presence of natural inhibitory compounds such as organic acids, aldehydes, phenolic compounds, and heavy metals in SS juice and DP juice [13]. Although SS and DP juice are nutritious and refreshing, containing natural sugars, proteins, amino acids, vitamins, and minerals that are suitable for the growth of L. delbruckii (Table 3), lower yields observed initially in SS and DP juice were probably due to presence of inhibitory compounds. After 24 h, however, LA concentration increased rapidly, indicating that when L. delbruckii was presumably adapted to the inhibitory compounds in the juice of SS and DP , it could efciently utilize the fermentable sugars in these juices. The rate of LA accumulation and sugar utilization is shown in Fig. 1. Table 2 has summarized the results of conversion of GS, SS, and DP juice into LA by L. delbruckii as a function of duration of fermentation. Similarly, productivity of LA as a function of these sources is depicted in Fig. 2. Table 1. Profiles of availability and cost of alternate carbon sources Availability Cost Concentration of (MT/year) (US$/T) lactic acid (%) 154.0 166.0 142.0 88.5 48.6 92.0

Figure 1. Utilization of total reducing sugars in golden syrup, sweet sorghum, and date palm juice as a function of lactate production.

3.5 Golden syrup as superior substrate

Few studies on LA fermentation from sugarcane juice have been published. Timbuntan et al. [14] have reported the production of L-LA from sugarcane juice by a newly isolated Lactobacillus sp. To our knowledge, present study is the rst report on LA fermentation from three carbon sources reported presently. LA yield was considerably high in GS than in the juice of SS and DP (Table 2 and Fig. 2). This could be attributed to the high % of glucose found in GS compared to those in SS and DP (Table 3). All fermentation experiments showed high optical purity of LA using these substrates as source of carbon [1, 8].

3.6 Economics of use of alternative carbon sources

LA production with GS was found to be more as compared to SS and DP juice. GS (also known as Hydrol in trade circles) is commercially available in large quantities (1.6 million tons, 12% RS) with reproducibility at US $555 44 per ton (80% solids w/w; Table 1), while LA is sold commercially at US $2780 per ton or calcium lactate at US $1660 per ton. Thus, presently GS forms a yet cheaper alternative to BISS (below Indian standard sugar) [4]. Use of GS in LA production promotes its value-addition, rendering dextrose industry robust to absorb shocks of nancial uncertainty faced from time to time. Its ready availability enables industry in its alternate applications [12]. In contrast dependence on an agri-products, like SS, whose commercial viability in large measure depends upon the availability of water for irrigation in all seasons for undertaking three crop cycles per annum. www.starch-journal.com

Carbon source

Sweet sorghuma) 10.8 Date palm 0.15 Golden syrup 1.60

MT, million tons. a) Need at least one pre-treatment which adds to direct cost by 10% and pollution control cost by another 5%. The availability and corresponding cost is in terms of 12% w/v reducing sugars used for conversion to lactic acid by fermentation.

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 rke 2011, 63, 632636 Starch/Sta Table 2. Profiles of lactic acid production as a function of fermentation duration and carbon source LA productivity (g/L/h)

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Duration (h) Golden syrup 0 24 48 72 96 Sweet sorghum juice 0 24 48 72 96 Date palm juice 0 24 48 72 96

pH 6.81 5.50 5.50 5.53 5.48 6.74 5.85 5.53 5.56 5.45 6.85 5.83 5.51 5.57 5.59

% RS 10.51 8.45 5.22 2.16 0.26 10.48 8.67 5.31 2.45 0.54 10.57 9.95 8.95 7.18 5.17

% LA 00 1.84 4.80 7.65 9.20 00 1.63 4.27 7.13 8.85 00 0.56 1.67 3.04 4.86

Cell count 109 0.001 0.3 2.0 3.0 3.0 0.001 0.03 0.4 0.5 0.5 0.001 0.03 0.03 0.04 0.04

Nil 0.7675 (SD 0.0095) 1.235 (SD 0.0159) 1.187 (SD 0.0125) 0.647 (SD 0.0095) Nil 0.675 (SD 0.01) 1.10 (SD 0.0) 1.195 (SD 0.0129) 0.715 (SD 0.0129) Nil 0.23(SD 0.00816) 0.465 (SD 0.00577) 0.557 (SD 0.0095) 0.767 (SD 0.0189)

LA, lactic acid; RS, reducing sugars. Table 3. Comparative analysis of composition of golden syrup, sweet sorghum, and date palm juice Golden syrup [12] 72.0 60 3 51 3 0.1 2 0.2 % 28 12 10 Sweet sorghum juice 13.0 12 2 1.3 0.3 0.2 0.5 0.1 303.8 344.0 116.0 Date palm juice [14, 15] 79.9 70 2 16 1.5 3.9 1.6 0.2 0.6 0.45 0.55

Component analyzed Total solids (%) Total reducing sugar (%) Glucose (%) Total protein (%) Total ash (%) Magnesium (ppm) Calcium (ppm) Phosphorus (ppm)

Availability of DP juice on commercial scale in India does not appear to be feasible in the present scenario as fresh or dried DP is wholly consumed as a table fruit. DP and SS juice add a step of purication and decolorization for the removal of inhibitory chemicals, both of which render their use logistically difcult, whereas, GS does not require any purication for the production of LA. Secondly, its use for producing LA in preference to food carries a public outrage as perceived in India in the use of soybean oil in USA for biodiesel production instead of exporting in large measure as an edible oil.

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Figure 2. Productivity of lactic acid (g/L/h) from SS, GS, and DP . SS, sweet sorghum; GS, golden syrup; DP , date palm; RS, reducing sugars; LA, Lactic acid. 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Conclusions

The above results have demonstrated that conversion of GS into LA/calcium lactate of high purity is cost-efciently www.starch-journal.com

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[6] Ishida, N., Saitoh, S., Ohnishi, T., Tokuhiro, K., et al., Metabolic engineering of Saccharomyces cerevisiae for efcient production of pure L-()-lactic acid. Appl. Biochem. Biotechnol. 2006, 129132, 795807. [7] Tanaka, T., Hoshima, M., Tanabe, S., Sakai, K., et al., Production of D-lactic acid from defatted rice bran by simultaneous saccharication and fermentation. Bioresour. Technol. 2006, 97, 211217. [8] Yadav, A. K., Studies on fermentative production and applications of lactates, Ph.D. Thesis, North Maharashtra University, Jalgaon (India) 2011. [9] Miller, G. L., Use of dinitrosalicylic acid reagent for determination of reducing sugars. Anal. Chem. 1959, 31, 426428. [10] Rodrigues, A., Pina Vaz, C., Freitas da Fonseca, A., Martinez-de-Oliveira, J., Evaluating the concentration of a Candida albicans suspension. Infect. Dis. Obstet. Gynecol. 1993, 1, 134136. [11] Emna, B. S., Maher, T., Raouf, T., Mohamed, H., Lot, A., Common date palm in Tunisia: Chemical composition of pulp and pits. Int. J. Food Sci. Technol. 2008, 43, 20332037. [12] Nehete, P . N., Shah, N. K., Kothari, R. M., Recycling of mother liquor of sorbose and glucose for hexitol production. Resour. Conserv. Recycling 1991, 5, 8187. [13] Roukas, T., Pretreatment of beet molasses to increase pullulan production. Process Biochem. 1998, 33, 805810. [14] Timbuntan, W., Sriroth, K., Tokiwa, Y., Lactic acid production from sugarcane juice by a newly isolated Lactobacillus sp. Biotechnol. Lett. 2006, 28, 811814. [15] Chaira, N., Ferchichi, A., Mrabet, A., Sghairoun, M., Chemical composition of the esh and the pit of date palm fruit and radical scavenging activity of their extracts. Pak. J. Biosci. 2007, 10, 22022207.

feasible, while use of SS and DP juice by L. delbruckii have potential in future. Authors are grateful to Dr. V L Maheshwari, DirectorSchool of Life Sciences for providing experimental facilities and Smt. Irmagard Velagapudi M. Rao, Managing Director- KCP Sugar & Ind. Corp. Ltd., Vuyyuru (A.P.) for nancially supporting the project. The authors have declared no conict of interest.

5 References
[1] Yadav, A. K., Chaudhari, A. B., Kothari, R. M., Bioconversion of renewable resources into lactic acid: An industrial overview. Crit. Rev. Biotechnol. 2011, 31, 19. [2] Tokiwa, Y., Jarerat, A., Biodegradation of poly(L-lactide). Biotechnol. Lett. 2004, 26, 771777. [3] Tokiwas, Y., Calabia, B. P ., Biodegradability and biodegradation of poly(lactide). Appl. Microbiol. Biotechnol. 2006, 72, 244251. [4] Yadav, A. K., Chaudhari, A. B., Kothari, R. M., Cost-effective fermentative production of calcium lactate using BISS (Below Indian Standard Sugar) and Spirulina hydrolysate. Indian J. Biotechnol. 2009, 8, 418424. [5] Yadav, A. K., Chaudhari, A. B., Kothari, R. M., Enhanced viability of Bacillus coagulans after spray drying with calcium lactate, storage and re-hydration. Indian J. Chem. Technol. 2009, 16, 519522.

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