Production of l - and d -lactic acid from waste Curcuma longa biomass through simultaneous saccharification and cofermentation

Accepted Manuscript Production of L- and D-lactic acid from waste Curcuma longa biomass through simultaneous saccharification and cofermentation Cuong Mai Nguyen, Jin-Seog Kim, Thanh Ngoc Nguyen, Kim Seul Ki, Gyung Ja Choi, Yong Ho Choi, Kyoung Soo Jang, Jin-Cheol Kim PII: DOI: Reference:

S0960-8524(13)01093-6 http://dx.doi.org/10.1016/j.biortech.2013.07.035 BITE 12080

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

9 May 2013 3 July 2013 9 July 2013

Please cite this article as: Nguyen, C.M., Kim, J-S., Nguyen, T.N., Ki, K.S., Choi, G.J., Choi, Y.H., Jang, K.S., Kim, J-C., Production of L- and D-lactic acid from waste Curcuma longa biomass through simultaneous saccharification and cofermentation, Bioresource Technology (2013), doi: http://dx.doi.org/10.1016/j.biortech.2013.07.035

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Production of L- and D-lactic acid from waste Curcuma longa biomass through simultaneous saccharification and cofermentation

Cuong Mai Nguyena,b, Jin-Seog Kimb, Thanh Ngoc Nguyenc, Kim Seul Kib, Gyung Ja Choib, Yong Ho Choib, Kyoung Soo Jangb, Jin-Cheol Kima,b,*

a

Department of Green Chemistry and Environmental Biotechnology, University of Science

and Technology, 217, Gajungro, Yuseong-gu, Daejeon 305-333, Republic of Korea b

Research Center for Biobased Chemistry, Division of Convergence Chemistry, Korea

Research Institute of Chemical Technology, Daejeon 305-600, Republic of Korea c

Department of Phytochemistry, Vietnam Institute of Industrial Chemistry, No.2 Pham Ngu

Lao Street, Hanoi, Vietnam

*

Corresponding author. Tel.: +82 42 8607436; Fax: +82 42 8614913

E-mail address: [email protected]

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ABSTRACT

Simultaneous saccharification and cofermentation (SSCF) of Curcuma longa waste biomass obtained after turmeric extraction to L- and D-lactic acid by Lactobacillus coryniformis and Lactobacillus paracasei, respectively, was investigated. This is a rich, starchy, agro-industrial waste with potential for use in industrial applications. After optimizing the fermentation of the biomass by adjusting nitrogen sources, enzyme compositions, nitrogen concentrations, and raw material concentrations, the SSCF process was conducted in a 7-l jar fermentor at 140 g dried material/l. The maximum lactic acid concentration, average productivity, reducing sugar conversion and lactic acid yield were 97.13 g/l, 2.7 g/l/h, 95.99% and 69.38 g/100 g dried material for L-lactic acid production, respectively and 91.61 g/l, 2.08 g/l/h, 90.53% and 65.43 g/100 g dried material for D-lactic acid production, respectively. The simple and efficient process described in this study could be utilized by C. longa residue-based lactic acid industries without requiring the alteration of plant equipment.

Keywords: Curcuma longa, L-lactic acid, D-lactic acid, Lactobacillus paracasei, Lactobacillus coryniformis, Simultaneous saccharification and cofermentation.

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1. Introduction

Lactic acid, an important organic acid, is one of the top 30 potential building-block chemicals obtained from biomass. It is widely used in the food, pharmaceutical, cosmetic and other chemical industries. Recently, polylactic acid, a biodegradable and biocompatible chemical obtained from renewable material has garnered worldwide attention due to its characteristics and potential applications (Wee et al., 2006; Werpy and Petersen, 2004). Abrupt decreases in petrochemical resources and the environmental pollution caused by the petrochemical industry have led excessive production of lactic acid from fermentation, producing polylactic acid from a high optical purity of lactic acid, which is not obtained from synthetic processes. The pure polymers of poly(L-lactic acid) (PLLA) and poly(Dlactic acid) (PDLA) are relatively heat sensitive, while stereocomplexes of polylactic acid produced by blending PLLA and PDLA have a melting point approximately 50℃ higher than their respective pure polymers and are more biodegradable. The ratio of L- and Dlactic acid influences the properties and the degradability of the resulting poly-lactic acid (Datta and Henry, 2006; John et al., 2007; John et al., 2009; Wang et al., 2011). Inexpensive, renewable materials, including starch, agro-industrial residues or other lignocellulosic materials (such as green microalga and wood), are necessary for the feasible economic production of lactic acid, because insdustrial uses, such as the production of polymers usually require large quantities of lactic acid at a relatively low cost (Moldes et al., 2001; Nguyen et al., 2012a,b; Wee et al., 2006). Agro-industrial residues are produced at a rate of approximately 3.5 billion tons worldwide annually, and they are used as specific 3

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carbohydrate feedstock. Though agro-industrial residues are rich in carbohydrates, their utilization is limited due to their low protein content and poor digestibility (John et al., 2007) When the carbohydrates exist as polysaccharides, they must be hydrolyzed by inorganic acids or enzymatic catalysis to release fermentative sugar. The well-known disadvantages of enzymatic catalysis such as inhibition of the enzymes involved in the hydrolysis step by glucose or lactic acid and the high cost of the enzymes present drawbacks for industrial use. However, the use of simultaneous saccharification and fermentation (SSCF) can prevent enzyme inhibition (Abdel-Rahman et al., 2011; John et al., 2009). In addition, the development of modern biotechnology allows the enzymes for the hydrolysis of biomass to be manufactured at a substantially reduced cost. Thus, hydrolytic enzymes have gained traction in industry for liquefaction and saccharification (Schafer et al., 2007). To digest the lignocellulosic material, a pretreatment step is required to disrupt the carbohydrate-lignin complex and thus allow the hydrolytic enzyme to gain access to the carbohydrate. In biomass, lignin is considered to be difficult to use as a fermentation substrate because it makes the biomass resistant to chemical and biological degradation (Abdel-Rahman et al., 2011). Tumeric is a rhizomatous, herbaceous plant botanically known as Curcuma longa. The tuberous rhizomes (or underground stems) of turmeric have been used since antiquity as condiments, a dye and an aromatic stimulant in several medicines. It is also a very important spice in India and Southeast Asia. India accounts for 78% of the world’s turmeric production and boasts 60% of the world export share. Approximately 900,000 tons of fresh turmeric was produced in India in 2007. Curcuminoids, polyphenolic pigments present in 4

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turmeric, comprise 3 to 6% of C. longa. The natural curcuminoids in tumeric have been used as folk medicines due to their antioxidant, anti-inflammatory properties, anticarcinogenic effects and hypoglycemic effects in humans (Cherubino and Alves 2005). Turmeric residue is a by-product of the curcuminoid production process with high polysaccharide content consisting mainly of starch. In the isolation of curcuminoids, oleoresin is extracted by an organic solvent such as acetone, ethyl acetate, chloroform, methanol or ethanol. The total extract yields 15.45-21.55% (g extract/g material) depending on the solvent, material and isolation conditions, and the waste residue is 78.45-84.55% (w/w) (Revathy et al., 2011). C. longa contains up to 60-70% carbohydrate, 2-7% fiber, 37% mineral matter and 6-8% protein, which remain in the residue (Balakrishman, 2007). Therefore, this turmeric residue is an attractive rich carbon and nitrogen source for lactic acid production that would not require a pretreatment step. However, lactic acid production from C. longa residue has been not yet reported. This study examines the production of L- and D-lactic acid from waste C. longa, an agro-industrial residue, through the SSCF process. The main objectives were (i) to demonstrate the feasibility of L- and D-lactic acid production by SSCF using strains L. paracasei LA104 and L. coryniformis ATCC 25600 from C. longa residue as carbon and nitrogen sources, respectively, and (ii) to optimize the enzyme component, nitrogen source and substrate concentration for lactic acid production through SSCF.

2. Material and Methods

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2.1. Raw material and media

The waste Curcuma longa (WCL) was collected from curcuminoid extraction pilot with capacity of approximately 1,200 ton raw material/year at Department of Phytochemistry, Vietnam Institute of Industrial Chemistry, Hanoi, Vietnam. A total of 1000 kg turmeric powder was extracted with ethyl acetate for 8 h using a reflux apparatus. The extract was concentrated, crystallized and purified to obtain curcuminoids. The organic solvent was removed from the C. longa residue by water vapor at 2.5 bar, and then the residue was dried in the sun for 2 days. The WCL was stored in plastic bags at 4℃ until use. L. paracasei LA104 and L. coryniformis ATCC 25600 are homofermentative L-lactic acid and D-lactic acid producing strains, respectively (Nguyen et al., 2012a,b). The seed cultures were prepared by inoculating these strains in 200 ml of culture broth containing 20 g glucose, 10 g yeast extract (YE), 10 g peptone (Pep), 0.2 g MgSO4, 1.5 g KH2PO4, 1.5 g K2HPO4, 1.5 g sodium acetate, 0.05 g MnSO4.H2O and 1 g Tween 80 (per liter) for strain LA104 and 20 g glucose, 10 g YE, 10 g Pep, 0.05 g MnSO4, 2 g K2HPO4, 5 g sodium acetate, 0.2 g MgSO4, 2 g triammonium citrate and 1 g Tween 80 for strain ATCC 25600 in 500-ml Erlenmeyer flasks. These flasks were incubated at 150 rpm for 24 h at 37℃ for strain LA1 and at 34℃ for strain ATCC 25600.

2.2. Enzymes

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Celluclast Conc BG (CC100133) was purchased from Novozymes (Bagsvaerd, Denmark) in solid form and had an activity of 385 FPU g-1, and 1 g powder was dissolved in 5 ml 1 M citrate buffer (pH 4.8) (64 FPU/ml; E1). Cellobiase (C6105: E2) from Aspergillus niger was purchased from Sigma–Aldrich Co., (St. Louis, MO. USA) and its βglucosidase activity was 311 U/ml. Aspergillus oryzae α-amylase Fungamyl 800 L (1,4-αD-glucan

glucanohydrolase, EC 3.2.1.1: E3), which is produced by Novozymes A/S, was

purchased from Sigma-Aldrich Co. (product number A-8220) in liquid form with an activity of 800 FAU/g. Amyloglucosidase (A7095: E4) from A. niger, which is produced by Novozymes, was purchased from Sigma-Aldrich Co. (product number A7095) in liquid form with an activity of 300 units/ml. S1, S2, S3, S4, S5, S6, S7, S8, S9 and S10 were mixtures containing the four enzymes at ratios of 2/2/2/2, 1/1/2/2, 2/0/2/2, 0/2/2/2, 2/2/1/2, 1/1/1/2, 2/2/2/1, 1/1/1/1, 0/0/2/2, and 2/2/1/1 (v/v/v/v) of E1/E2/E3/E4, respectively; the numbers indicate the amounts of the enzymes as percentages of the dried biomass material (v/w).

2.3. Effect of enzyme mixture loading on lactic acid production

Ten different enzyme mixtures, S1–S10, were tested to select the optimum enzyme compositions and amounts for the production of L- and D-lactic acid from WCL through SSCF using strains LA104 and ATCC 25600. The material (100 g/l) was incorporated into medium supplemented with 10 g YE/l, 10 g Pep/l, 40 g CaCO3/l and the same mineral salts described in section 2.1. After autoclaving at 121℃ for 21 min, the medium was added to 7

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the enzyme mixture and seed culture (10%) and incubated at 37℃ for strain LA104 or 34℃ for strain ATCC 25600 and 220 rpm under anaerobic conditions. Fifty milliliter Erlenmeyer flasks containing 30 ml medium in a vinyl anaerobic chamber (Coy Laboratory Products Inc., Michigan, United States) were used. Samples were taken after 24, 48 and 72 h and immediately immersed in a boiling water bath for 5 min. This experiment was repeated twice with two replicates. To determine the optimum substrate concentration, 90, 100, 110, 120, 130, 140, 150 and 160 g/l were added to the medium. CaCO3 was added at 40% of material (w/w) to control the pH. The S10 enzyme mixture was used for this experiment. Unless otherwise stated, the culture conditions were the same as those described in section 2.1, without glucose.

2.4. Effect of nitrogen source and soybean meal loading on lactic acid production

The optimal nitrogen source was selected by adding YE, Pep, soybean meal (SM), YE+Pep (1:1), corn steep liquor (CSL), beef extract, casein hydrolysate, ammonium sulfate, urea, malt sprouts, and skim milk at a 0.198% quantity of nitrogen. Media containing 140 g/l dried material, 56 g CaCO3/l and mineral salts (as described above) were used. The effect of SM dosage on LA production from WCL was determined at concentrations of 0, 5, 10, 15, 20, 25 and 30 g SM/l in medium supplemented with mineral salts as described in section 2.1. The pH was controlled by the addition of 56 g CaCO3/l. The effect of mineral salts was evaluated at 10 g of SM/l for strain LA104 and 15 g of SM/l 8

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for strain ATCC 25600. The S10 enzyme mixture and pre-culture were added to the fermentation medium at 6% (v/w) and 10% (v/v), respectively and then the medium was incubated at 37℃ and 220 rpm for strain LA104 or 34℃ and 220 rpm for strain ATCC 25600 for 72 h under anaerobic conditions. This experiment was repeated twice with two replicates.

2.5. SSCF of lactic acid production from Curcuma longa biomass in a jar fermentor

On the basis of the results from the optimization experiments, a medium containing 140 g WCL/l, 56 g CaCO3/l, 179.2 FPU/l cellulase, 870.8 U/l cellobiase, 1120 FAU/l αamylase and 420 U/l amyloglucosidase supplemented with 10 g SM/l or 15 g SM/l was chosen for batch fermentation by strains LA104 and ATCC 25600, respectively. The SSCF medium consisted of 353.54 g WCL (moisture: 20.8%) in 1726 ml of distilled water supplemented with 20 g SM for strain LA104 or 30 g SM for strain ATCC 25600. The medium was autoclaved at 121℃ for 21 min and then combined with 16.8 ml of the S10 enzyme mixture and inoculated with a 10% seed culture. The fermentation process was conducted in a 7-l jar fermentor with a working volume of 2 l. The temperature and agitation speed were maintained at 37℃ for LA104 or 34℃ for ATCC 25600 and 200 rpm. The pH of the culture was maintained at 6.0 by automatic addition of 28% (w/v) NH4OH. Nitrogen gas was purged into the reactor to keep the level of dissolved oxygen lower than 0.5 ppm.

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2.6. Analytical methods

Cell growth was monitored in terms of the colony-forming units per milliliter (cfu/ml) of broth culture by using the dilution spread plate method on MRS agar plates (diluted 10 -5 to 10-8). The samples were harvested at various time intervals, heated at 100℃ for 10 min, diluted 10-fold, and then centrifuged at 5720 × g and 37℃ for 20 min using a Gyrozen 1730MR temperature-controlled microcentrifuge. The supernatant was acidified by the addition of an equal volume of 5 M H2SO4, and free acid was liberated and analyzed. The levels of reducing sugars (RSs), glucose, and D- and L-lactic acid in the culture broths as well as the yield and optical purity were estimated and calculated using methods that have been previously described (Nguyen et al., 2012a). The WCL was analyzed as described in the Korea Food Standard Codex method.

3. Results and Discussion 3.1. Material composition

From 1000 kg of C. longa, 850 kg of waste residue was obtained after curcuminoid extraction process. The moisture content of WCL was 20.8% and contained 11% protein, 9.1% ash, 0.1% lipid and 76.5% carbohydrate including 50% starch, 0.1% fructose, 0.1% glucose, and 0.6% sucrose (w/w). Only a small amount of oleoresin (ethanol-toluene soluble residue: 2.58%) remained in the WCL, indicating that most of the curcuminoids and essential oils were extracted in the curcuminoid-isolation process. Additionally, significant 10

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contents of phosphorous, iron, potassium, thiamine, riboflavin, niacin, and ascorbic acid were observed in C. longa, and these compounds are necessary for the growth of lactic acid-producing bacteria (Balakrishman, 2007). In contrast to the results observed for C. longa containing oleoresin, the WCL did not show any effect on the growth of strains LA104 and ATCC 25600 (data not shown). These results support this residue as an interesting alternative substrate for lactic acid production by biotechnological synthesis.

3.2. Effect of enzyme mixture loading on lactic acid production

Different enzyme mixtures (S1-S10) were investigated at a concentration of 100 g dried material/l. As shown in Fig. 1, out of these enzyme mixtures tested, S1, S5, S7 and S10 reached high yields, RS conversions and final lactic acid concentrations. No significant differences were observed among them. When S1, S5, S7 and S10 were loaded, the yields, RS conversions and the lactic acid concentrations from strains LA104 and ATCC 25600 were approximately 67%, 93%, 67 g/l and 63%, 88%, 63 g/l after 72 h, respectively. In all of the treatments with less than 2% of E1 or E2, the lactic acid concentrations, RS conversions and yields were reduced for both strains. The RS conversion and yield for strain LA104 ranged from 83.6% to 93.23% and from 60.45% to 67.39%, respectively. The D-lactic

acid production by strain ATCC 25600 was also affected by the enzyme mixture

(Fig. 1). RS conversion, yield and lactic acid ranged from 79.17-87.93%, 57.23-63.56% and 57.23-63.56 g l-1, respectively. In both cases, the lactic acid concentration was the lowest when the SSCF was conducted using S9. After considering price and production of 11

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lactic acid, the S10 enzyme mixture was selected as the most effective (in terms of enzyme composition and concentrations) for the SSCF process using the two strains. At this enzyme dosage, the RS conversion, lactic acid and yield reached 93.23%, 67.39 g/l and 67.39% for strain LA104 and 87.18%, 63.02 g/l and 63.02% for strain ATCC 25600. Compared to the production of L-lactic acid from Hydrodictyon reticulatum, by LA104, the amount of amyloglucosidase decreased by 50%, although the amount of cellobiase increasing by 100% compared to that from D-lactic acid production from H. reticulatum by ATCC 25600 (Nguyen et al., 2012a,b).

3.3. Effect of Curcuma longa concentration on lactic acid production

The effect of WCL concentration on lactic acid production through SSCF was tested using S10 enzyme mixture at concentrations between 90-160 g/l. The results (Fig. 2A, B) show that the final lactic acid concentration increased with WCL levels, reaching maximum values of 101.53 g/l for strain LA104 and 97.87 g/l for strain ATCC 25600. At concentrations lower than 150 g/l, both strains showed the similar RS conversions and yields of approximately 92% and 67% for strain LA104 and 87% and 63% for ATCC 25600, respectively. However, at concentrations of 150 and 160 g/l, the lactic acid production and yield displayed opposite trends; the lactic acid concentration increased with increasing amount of substrate, but the RS conversion and yield decreased from 91.97% to 87.79% and from 66.48% to 63.46% for strain LA104, and from 87.32% to 84.63% and from 63.11% to 61.17% for strain ATCC25600, respectively. These decreases may be due 12

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to the inhibitory action of free lactic acid and calcium lactate on the activity of degrading enzymes and/or an increase in substrate viscosity. When the amount of substrate in the medium increases, the amount of fermentable sugars released by the enzymes also increases. However, it has been reported that the saccharification process becomes inefficient when the amount of substrate loaded is higher than 10% (w/w) of the material due to an increase in the substrate viscosity (Iyer and Lee, 1999; Rosgaard et al., 2007). These results suggest that the optimal substrate concentration was 140 g/l.

3.4. Effect of nitrogen source on lactic acid production

In order to replace YE with another inexpensive nutrient source, we tested, the effects of eleven nitrogen sources on L- and D-lactic acid production through SSCF. Each nitrogen source was supplemented in an amount that was equivalent to a nitrogen concentration of 10 g YE/l + 10 g Pep/l (corresponding to 0.198% nitrogen). The lactic acid production, RS conversions and yields are presented in Table 1. The strain preferred organic nitrogen sources for L-lactic acid production. Furthermore, lactic acid production was markedly influenced by the type of nitrogen source used. YE, Pep, SM, YE+Pep, beef extract and casein hydrolysate increased lactic acid production compared to the other nitrogen sources. In the sample supplemented with SM, the lactic acid concentration, RS conversion and yield reached 92.59 g/l, 91.50% and 66.13%, respectively. On the other hand, when CSL, ammonium sulfate, urea, malt sprouts and skim milk were used as the supplement, the lactic acid productions was decreased in comparison to the sample without the 13

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supplemented nitrogen source. This result may be due to the inhibitory action of these nitrogen sources on strain LA104, due to inadequate amino acids or the lower amounts of vitamins in these resources (Altaf et al., 2005). For D-lactic acid production by strain ATCC 25600, the highest concentrations of lactic acid (approximately 90 g/l) were obtained when the nitrogen sources were YE, Pep, YE+Pep and beef extract, whereas a concentration of 87.35 g lactic acid/l was obtained with SM treatment (Table 1). In contrast to strain LA104, no significant effect was observed between casein hydrolysate and the unsupplemented control. The treatment with other nitrogen sources strongly decreased the lactic acid concentration down to 32.75 g lactic acid/l. These results indicated that most of the nitrogen required could be provided by WCL. Without a nitrogen source, the lactic acid concentrations, RS conversions and yields were 84.44 g/l, 83.84%, and 60.31% for strain LA104 and 73.42 g/l, 72.55% and 52.44% for strain ATCC 25600, respectively. The nitrogen or vitamin deficiency can be overcome by the addition of SM, YE, Pep, etc. Soybean meal, a cheap solid residue that is a food industry by-product was observed to contribute to high lactic acid production in the absence of Pep and YE compared to other sources. In an economic analysis, Sikder et al. (2012) reported that the two largest components of the operating costs are the yeast extract (87%) and carbon source (6%); the total annual costs of the fermentation step are high and the major contributor is the cost of the supplemental yeast extract. Therefore, using SM as an inexpensive nitrogen source may reduce operating costs.

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3.5. Effect of soybean meal loading on lactic acid production

Seven different concentrations of soybean meal (0-30 g/l) were assayed using the S10 enzyme mixture. As shown in Table 2, the lactic acid concentrations increased with SM levels. Concentrations lower than 10 g SM/l produced low L-lactic acid contents, yields and RS conversions. When the concentration of SM was greater than 10 g/l, there was no significant increase in lactic acid production. Under conditions with an optimum nitrogen source (10 g SM/l), WCL concentration and enzyme mixture, mineral salts did not affect Llactic acid, demonstrating that WCL could provide all of the required mineral salts for the growth of strain LA104. In the absence of mineral salts, the concentration of L-lactic acid, yield and RS conversion were 92.73 g/l, 66.24% and 91.64%, respectively, at a concentration of 10 g SM/l. For D-lactic acid production by L. coryniformis ATCC 25600, the lactic acid concentration, yield and RS conversion were associated with the SM dosage; lactic acid, yield and RS conversion increased as the nitrogen source concentration increased. However, these parameters were not significantly affected at concentration higher than 15 g SM/l. Therefore, the optimal concentration of SM for D-lactic acid production was 15 g/l. Under these conditions, there is no significant difference in the SSCF of D-lactic acid from WCL between mineral salt supplemented and unsupplemented groups: 88.67 and 89.06 g/l were produced from 140 g WCL/l, respectively.

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In the comparison between lactic acid production from WCL and other materials using SM as an inexpensive nitrogen source, our processes required low concentrations of SM. Zhou et al. (1996) used L. pentosus B-227 to produce lactic acid from double sugar municipal solid waste hydrolysate supplemented with 2.8% SM, and they obtained a lactic acid yield of 85% and 77.1 g/l lactic acid after 72 h. In a study by Wang et al. (2011), a lactic acid concentration of 50 g/l was obtained from 90 g/l initial glucose using Sporolactobacillus sp. strain CASD in a medium that contained 42 g SM/l as a nitrogen source. Li et al. (2006) used acid hydrolysate of soybean meal (AHSM)-supplemented with corn steep liquor at a concentration of 24.3 g AHSM/l for L-lactic acid production from glucose by L. casei LA-04-1 and they obtained 110 g/l lactic acid with a productivity of 1.15 g/l/h.

3.6. SSCF of lactic acid production from Curcuma longa biomass in a jar fermentor

Production of D- and L-lactic acid from WCL was performed in a 7-l jar fermentor through SSCF using L. paracasei LA104 and L. coryniformis ATCC 25600. CaCO3 is exchanged by NH4OH to avoid excessive solid waste (calcium salts) to be treated. Fig. 3 and 4 present the profiles of lactic acid production by LA104 and ATCC 25600, respectively. These results indicate that the two strains have different biochemical characteristics with regard to fermentation. In general, the strain LA104 showed a higher capacity for lactic acid production under optimal SSCF conditions.

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After the first 4 h cultivation period in SSCF, the lag phase was barely detectable for both strains, indicating that they were in the lag phase for less than 4 h. In this period, the cfu of the two strains rapidly increased (Fig. 3B, 4B). At approximately 4 h, the glucose and RS build up were maximal, and no further increase was detected. This result indicated that the saccharification rate was higher than the fermentation rate. After 20 h, the RS remained steady at a low level, approximately 4 g/l for strain LA104 and 5 g/l for strain ATCC 25600, and the glucose concentrations were nearly zero. Most of lactic acid was produced within 36 h by LA104 and 44 h by ATCC 25600 (Fig. 3A, 4A). After the first 4 h, enzymatic hydrolysis was the rate limiting process in SSCF. The RS and glucose released by enzyme hydrolysis were immediately converted to lactic acid. These results were also observed by Marques et al. (2008). Generally, lactic acid at high concentrations affects enzymatic saccharification. The enzymatic hydrolysis rate gradually decreases as fermentation proceeds and lactic acid concentrations slowly reach maximum levels. The rate-limiting step in the present study did not shift from hydrolysis to fermentation during the SSCF processes, even though the lactic acid concentrations were higher than those observed by other researchers (Iyer and Lee, 1999). It seems to be due to the rapid and complete hydrolysis of the WCL cellulose (within 36 h by LA104 and 44 h by ATCC 25600). The RS and glucose concentrations remaining in the cultures of LA104 and ATCC 25600 were nearly identical during the SSCF processes. In the bacterial stationary phase (after 8 h), lactic acid production by LA104 was faster than that of ATCC 25600 due to the characteristics of the strains. These differences were also observed in lactic acid production 17

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from Hydrodictyon reticulatum through SSCF by LA104 and ATCC 25600 (Nguyen et al., 2012a,b). As shown in Fig. 3A and 4A, the glucose and RS levels in the medium from both cultures were almost the same as each other during the initial 20 h of cultivation. The lactic acid concentration from the SSCF process of strain LA104 reached a maximal level of approximately 97 g/l at 36 h and then steadily maintained that level until 60 h. In the case of strain ATCC 25600, the lactic acid accumulated more slowly, reaching a maximum concentration of approximately 91 g/l at 44 h. The lower lactic acid concentration obtained with ATCC 25600 compared to LA104 may be due to different substrate availabilities between the two strains and/or their different effects on enzyme activities. During the SSCF processes, small amounts of lactic acid were produced during the initial 4 h (approximately 6 g/l for both strains). After initial delay, lactic acid was produced with maximum productivities of 4.46 g/l/h for strain LA104 and 4.27 g/l/h for strain ATCC 25600. The final lactic acid concentration, RS conversion and yield produced by strain LA104 were 97.13 g/l, 95.99% and 69.38%, respectively. Most of the RS was converted to lactic acid after 36 h with an average productivity of 2.70 g/l/h and an optical purity of 95.17%. During the fermentation, the optical purity increased from 89.24% to 96.74% and then gradually decreased to 91.08% (Fig. 3B). Low initial optical purity is likely due to the D-lactic acid present in initial medium. The decreasing optical purity is likely due to the conversion of L-lactic acid into D-lactic acid by the lactate racemization (Goffin et al., 2005). When SSCF was conducted with ATCC 25600, the lactic acid concentration was 91.61 g/l with productivity of 2.08 g/l/h and a yield of 65.43% after 44 h. Unlike the results obtained with LA104, the optical purity of D-lactic acid increased from 18

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90.41 to over 99.5% and then remained steady during the rest of the fermentation process. In comparison to SSCF conducted in a shake flask, the lactic acid production in the jar fermentor produced higher lactic acid concentration, RS conversion and yield. This difference may be due to the effect of agitation, which is an important factor for adequate mixing, mass transfer and heat transfer in enzymatic hydrolysis and neutralization processes. The agitation not only assists the mass transfer between the different phases presented in the culture but also maintains homogeneous chemical and physical conditions in the culture (Mussatto et al., 2008; Palmqvist et al., 2011). The economics of the production of lactic acid and its derivatives depends on many factors. Among these, the cost of raw materials is very significant (John et al., 2007). Therefore, the production of lactic acid from various low-cost and renewable biomasses, including by-products or agricultural residues with high lactic acid concentration, yield and productivity has recently garnered considerable interest (Ouyang et al., 2013, Ye et al., 2013). Table 3 shows the lactic acid production from renewable biomass through SSCF, based on the results of this study and those from the literature. Lactic acid concentration in our study were higher than those that have been previously observed, with exception of production through SSCF-fed batch or semi-continuous modes (Budhavaram and Fan, 2009; Moldes et al., 2001; Shen and Xia, 2006), and from wheat bran treated by protease (John et al., 2006b). This difference may be due to high starch content in our material. Moreover, the productivity of L-lactic acid in this study was the highest among those using cellulosic materials. It is clear that SSCF performed on waste C. longa using L. paracasei LA104 and L. coryniformis ATCC 25600 can produce high concentrations of L- and D19

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lactic acid with high productivities, which will reduce processing costs. In addition, Karp et al. (2011) achieved a concentrion of 138 g lactic acid/l with productivity rate of 0.863 g/l/h and product yield from sugar of 0.849 g/g by combination of soybean vinasse and soybean molasses using strain L. agilis LPB 56 in a pilot plant. Modern biotechnology permits the enzymes for the hydrolysis of biomass to fermentative sugars to be manufactured at substantially reduced costs. In additionally, several hydrolytic enzymes such as cellulase, cellobiase, α-amylase and amyloglucosidase, have been used extensively in industry for liquefaction and saccharification (Schafer et al., 2007). Thus, waste C. longa biomass is a potential raw material for the production of both L- and D-lactic acid through SSCF in a simple medium supplemented with the inexpensive nitrogen source SM, and this method boasts a high lactic acid concentration, productivity, and yield.

4. Conclusions

The present study highlights the production of L- and D-lactic acid with a high lactic acid concentration and productivity from WCL obtained from curcuminoid production. This procedure is performed in a simple medium supplemented with an inexpensive nitrogen source (SM) through SSCF. Because WCL is a non-grain and waste material, it does not compete with grain as an agricultural crop, and its use has no impact on the food chain of humans and animals. This economical L- and D-lactic acid production process using a renewable biomass has enormous potential for industrial applications.

20

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simultaneous saccharification and co-fermentation using Lactobacillus coryniformis subsp. Torquens. Biotechnol. Lett. 34, 2235-2240. 22. Ouyang, J. Ma, R., Zheng, Z., Cai, C., Zhang, M., Jiang, T., 2013. Open fermentative production of L-lactic acid by Bacillus sp. strain NL01 using lignocellulosic hydrolyzates as low-cost raw material. Bioresour. Technol. 135, 475-480. 23. Palmqvist, B., Wiman, M., Liden, G., 2011. Effect of mixing on enzymatic hydrolysis of steam-pretreated spruce: a quantitative analysis of conversion and power consumption. Biotechnol. Biofuels 4:10. 24. Revathy, S., Elumalai, S., Benny, M., Antony, B., 2011 Isolation, purification and identification of curcuminoids from turmeric (Curcuma longa L.) by column chromatography. J. Exper. Sci. 2 (7), 21-25. 25. Romani, A., Yanez, R., Garrote, G., Alonso, J.L., 2008. SSF production of lactic acid from cellulosic biosludges. Bioresour. Technol. 99, 4247-4254. 26. Rosgaard, L., Andric, P., Dam-Johansen, K., Pedersen, S., Meyer, A.S., 2007. Effects of substrate loading on enzymatic hydrolysis and viscosity of pretreated barley straw, Appl. Biochem. Biotechnol. 143, 27-40. 27. Schafer, T., Borchert, T.W., Nielsen, V.S., Skagerlind, P., Gibson, K., Wenger, K., Hatzack, F., Nilsson, L.D., Salmon, S., Pedersen, S., Heldt-Hansen, H.P., Poulsen, P.B., Lund, H., Oxenbøll, K.M., Wu, G.F., Pedersen, H.H., Xu, H., 2007. Industrial enzymes. Adv. Biochem. Eng. Biotechnol. 105, 59-131. 28. Shen, X., Xia, L., 2006. Lactic acid production from cellulosic waste by immobilized cells of Lactobacillus delbrueckii. World J. Microbiol. Biotechnol. 22, 1109-1114. 24

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29. Sikder, J., Roy, M., Dey, P., Pala, P., 2012. Techno-economic analysis of a membraneintegrated bioreactor system for production of lactic acid from sugarcane juice. Biochem. Eng. J. 63, 81-87. 30. Wang, L., Zhao, B., Li, F., Xu, K., Ma, C., Tao, F., Li, Q., Xu, P., 2011. Highly efficient production of D-lactate by Sporolactobacillus sp. CASD with simultaneous enzymatic hydrolysis of peanut meal. Appl. Microbiol. Biotechnol. 89, 1009-1017. 31. Watanabe, M., Makino, M., Kaku, N., Koyama, M., Nakamura, K., Sasano, K., 2013. Fermentative L-(+)-lactic acid production from non-sterilized rice washing drainage containing rice bran by a newly isolated lactic acid bacterial without any additions of nutrients. J. Biosci. Bioeng. 115(4), 449-452. 32. Wee, Y.J., Kim, J.N., Ryu, H.W., 2006. Biotechnological production of lactic acid and its recent applications. Food Technol. Biotechnol. 44, 163-172. 33. Werpy, T., Petersen, G., 2004. Top value added chemicals from biomass. US DOE 1, 1-67. 34. Ye, L., Zhou, X., Hudari, M.S.B., Li, Z., Wu, J.C., 2013. Highly efficient production of L-lactic

acid from xylose by newly isolated Bacillus coagulans C106. Bioresour.

Technol. 132, 38-44. 35. Zhou, S.D., McCaskey, T.A., Broder, J., 1995. Evaluation of nitrogen supplement for bioconversion of municipal solid waste for lactic acid. Appl. Biochem. Biotechnol. 57– 58, 517-527.

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Table 1 Effects of nitrogen sources on L- and D-lactic acid production from waste Curcuma longa after 72 h Lactobacillus paracasei LA104 Nitrogen source

Lactobacillus coryniformis ATCC 25600

Glucose

RS

Lactic acid

RS conversion Yield

Glucose

RS

Lactic acid

RS conversion Yield

(g/l)

(g/l)

(g/l)

(%)

(g/l)

(g/l)

(g/l)

(%)

(%)

(%)

Yeast extract (YE) 0.60 ± 0.42 3.92 ± 0.01 93.97 ± 3.49 92.87 ± 3.45

67.12 ± 2.50 0.53 ± 0.04 4.89 ± 0.20 89.79 ± 1.39 88.74 ± 1.37

64.14 ± 0.99

Peptone

1.00 ± 0.28 4.07 ± 0.10 92.52 ± 1.32 91.43 ± 1.30

66.08 ± 0.94 2.94 ± 0.93 7.54 ± 0.99 88.86 ± 1.16 87.82 ± 1.14

63.47 ± 0.83

Soybean meal

1.20 ± 0.28 3.56 ± 0.02 92.59 ± 1.25 91.50 ± 1.24

66.13 ± 0.89 4.09 ± 0.89 7.80 ± 0.84 87.35 ± 1.76 86.32 ± 1.74

62.39 ± 1.26

YE+Peptone

1.00 ± 0.28 3.91 ± 0.05 93.95 ± 3.58 92.84 ± 3.54

67.10 ± 2.56 1.69 ± 1.41 6.10 ± 1.50 89.04 ± 0.44 87.99 ± 0.43

63.60 ± 0.31

Corn steep liquor

1.65 ± 0.07 3.67 ± 0.11 83.40 ± 0.36 82.42 ± 0.36

59.57 ± 0.26 50.48 ±1.45 57.67 ± 1.06 32.75 ± 0.01 32.36 ± 0.01

23.39 ± 0.01

Beef extract

1.10 ± 0.42 3.66 ± 0.00 92.43 ± 1.20 91.34 ± 1.19

66.02 ± 0.86 0.49 ± 0.02 4.24 ± 0.13 89.06 ± 0.83 88.01 ± 0.82

63.61 ± 0.59

Casein hydrolysate 1.15 ± 0.35 4.19 ± 0.13 92.87 ± 2.15 91.77 ± 2.13

66.33 ± 1.54 8.93 ± 2.65 14.60 ± 2.74 74.35 ± 2.61 73.48 ± 2.58

53.11 ± 1.86

Ammonium sulfate 1.45 ± 0.21 5.65 ± 0.17 76.98 ± 1.27 76.08 ± 1.25

54.99 ± 0.91 9.96 ± 1.18 19.14 ± 2.96 61.86 ± 2.95 61.13 ± 2.91

44.18 ± 2.10

Urea

1.55 ± 0.07 6.85 ± 0.26 81.35 ± 2.18 80.40 ± 2.15

58.11 ± 1.56 18.40 ± 0.57 29.41 ± 0.26 59.44 ± 1.18 58.74 ± 1.16

42.46 ± 0.84

Malt sprouts

1.76 ± 1.79 5.44 ± 1.22 76.86 ± 0.50 75.95 ± 0.49

54.90 ± 0.35 23.68 ± 0.40 36.40 ± 3.69 59.02 ± 0.92 58.32 ± 0.91

42.15 ± 0.66

Skim milk

1.45 ± 0.21 7.35 ± 0.86 77.51 ± 1.26 76.60 ± 1.25

55.37 ± 0.90 29.08 ± 0.04 36.44 ± 0.21 58.00 ± 0.93 57.32 ± 0.92

41.43 ± 0.66

Without nitrogen

1.90 ± 0.57 6.11 ± 1.24 84.44 ± 1.87 83.44 ± 1.85

60.31 ± 1.34 14.11 ± 3.69 22.18 ± 1.06 73.42 ± 3.44 72.55 ± 3.40

52.44 ± 2.45

The mineral salts containing 0.2 g MgSO4, 1.5 g KH2PO4, 1.5 g K2HPO4, 1.5 g sodium acetate, 0.05 g MnSO4.H2O and 1 g Tween 80 for strain LA104 and 0.05 g MnSO4, 2 g K2HPO4, 5 g sodium acetate, 0.2 g MgSO4, 2 g triammonium citrate and 1 g Tween 80 for strain ATCC 25600. The total amount of nitrogen was 0.198%. The fermentation was carried out at 140 g dried material/l, 56 g CaCO 3/l with working volume of 30 ml and 37℃ for strain LA104 or 34℃ for strain ATCC 25600 at 220 rpm.

26

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Table 2 Effects of soybean meal concentration on L- and D-lactic acid production from waste Curcuma longa (WCL) after 72 h Soybean meal (g/l)

Lactobacillus paracasei LA104

Lactobacillus coryniformis ATCC 25600

Glucose

RS

Lactic acid

RS conversion Yield

Glucose

RS

Lactic acid RS conversion Yield

(g/l)

(g/l)

(g/l)

(%)

(g/l)

(g/l)

(g/l)

(%)

(%)

(%)

0

1.30 ± 0.28 2.04 ± 0.38 75.85 ± 2.29 74.96 ± 2.27

54.18 ± 1.64 9.71 ± 0.12 14.98 ± 0.16 76.81 ± 0.28 75.91 ± 0.28

54.87 ± 0.20

5

0.70 ± 0.28 2.02 ± 0.20 88.04 ± 0.88 87.00 ± 0.87

62.88 ± 0.63 1.76 ± 0.53 3.07 ± 0.27

85.25 ± 0.87 84.25 ± 0.86

60.90 ± 0.62

10

0.95 ± 0.21 2.16 ± 0.29 92.73 ± 2.07 91.64 ± 2.04

66.23 ± 1.48 0.33 ± 0.01 2.29 ± 0.01

86.56 ± 1.80 85.54 ± 1.78

61.83 ± 1.29

15

0.70 ± 0.14 2.21 ± 0.07 92.82 ± 1.16 91.73 ± 1.15

66.30 ± 0.83 0.32 ± 0.04 2.42 ± 0.02

88.67 ± 2.03 87.62 ± 2.00

63.33 ± 1.45

20

0.80 ± 0.14 2.32 ± 0.17 92.93 ± 2.09 91.84 ± 2.06

66.38 ± 1.49 0.35 ± 0.00 2.55 ± 0.10

89.09 ± 2.39 88.04 ± 2.36

63.63 ± 1.71

25

0.65 ± 0.21 2.20 ± 0.00 93.11 ± 0.30 92.02 ± 0.29

66.51 ± 0.21 0.35 ± 0.01 2.60 ± 0.03

88.82 ± 1.51 87.78 ± 1.50

63.45 ± 1.08

30

0.75 ± 0.21 2.37 ± 0.10 92.84 ± 2.48 91.75 ± 2.45

66.32 ± 1.77 0.34 ± 0.00 2.63 ± 0.02

89.35 ± 1.72 88.30 ± 1.70

63.82 ± 1.23

10a

0.70 ± 0.28 1.76 ± 0.01 92.73 ± 1.72 91.64 ± 1.70

66.24 ± 1.23 -

-

-

15a

-b

-

-

-

-

-

0.30 ± 0.01 2.32 ± 0.05

-

89.06 ± 1.15 88.01 ± 1.14

63.61 ± 0.82

The mineral salts containing 0.2 g MgSO4, 1.5 g KH2PO4, 1.5 g K2HPO4, 1.5 g sodium acetate, 0.05 g MnSO4.H2O and 1 g Tween 80 for strain LA104 and 0.05 g MnSO4, 2 g K2HPO4, 5 g sodium acetate, 0.2 g MgSO4, 2 g triammonium citrate and 1 g Tween 80 for strain ATCC 25600. The fermentation was carried out at 140 g dried material/l, 56 g CaCO3/l with working volume of 30 ml and 37℃ for strain LA104 or 34℃ for strain ATCC 25600 at 220 rpm. a

Medium containing only the nitrogen source, 56 g CaCO3/l and 140 g WCL/l , without mineral sources.

b

Not tested

27

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Table 3 Comparison of recent data with the present work on SSCF lactic acid from agro-industrial residues and celullosic materials Lactic acid Productivity Yield References (g/l) (g/l/h) (%)d 37.1a 1.03 0.46 Nguyen et al. (2012a)

Material

Nitrogen source (g/l)

Microorganism

Process

Hydrodictyon reticulatum

5 g YE

L. paracasei LA104

Batch

Hydrodictyon reticulatum

3 g YE, 3 g Pep

L. coryniformis ATCC 25600 Batch

36.6b

1.02

0.46 Nguyen at al., (2012b)

Cellulosic biosludge

4 g YE, 8 g ME, 10 g Pep

L. rhamnosus CECT-288

42a

0.87

0.38 Romani et al. (2008)

0.96

0.77* Budhavaram and Fan, (2009)

Batch

c

Paper sludge

10 g Tryptone, 5 g YE

B. coagulan strains 36D1

SCM

92.0

Waste sugarcane bagasse

8 g YE

L. delbrueckii mutant Uc-3

Batch

67a

0.93

0.83 Adsul et al. (2007)

Eucalyptus globules wood 5 g YE, 10 g Pep

L. delbrueckii (NRRL-B445) FB

108

0.94

0.65 Moldes et al. (2001)

Corn cob residue

5 ml WBH, 5 g YE

L. delbrueckii ZU-S2

Batch

55.6

0.927

0.56 Shen and Xia (2006)

Corn cob residue

5 ml WBH, 5 g YE

L. delbrueckii ZU-S2

FB

107.6

1.345

0.51 Shen and Xia (2006)

α-Cellulose

5 g YE

L. delbrueckii (NRRL-B445) FB

75

0.35

0.63 Iyer and Lee (1999)

a

1.40

0.54 John et al. (2006a)

Cassava bagasse

5 g YE, 5 g NH4Cl

L. casei NCIMB 3254

Bach

83.8

Lime-treated wheat straw

21 g YE

B. coagulan DSM 2314

FB

40.7a

0.74

0.26 Maas et al. (2008)

a

1.23

0.85** Watanabe et al. (2013)

2.3

0.62 John et al. (2006b)

2.70

0.69 This study

RWD and rice bran

Without nitrogen source

Protease treated wheat bran 1 g YE Waste Curcuma Longa

10 g SM

L. rhamnosus M-23

Batch

59

L. delbrueckii and L.casei

Batch

123a

L. paracasei LA104

Batch

97.13

a b

Waste Curcuma Longa 15 g SM L. coryniformis ATCC 25600 Batch 91.61 2.08 0.65 This study L: Lactobacillus; B: Bacillus; YE: yeast extract; Pep: peptone; SM: soybean meal; WBH: wheat bran hydrolysate; ME: meat extract; RWD: rice washing drainage; SSCF: simultanous saccharification and cofermentation; SCM: semi-continuous mode; FB: fed batch * Based on consumed glucose and xylose ** Base on sugar equivalent a L-lactic acid b D-lactic acid c DL-lactic acid d Lactic acid (g)/ initial material (g)

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Figure captions Figure 1 Effect of enzyme mixtures on the simultaneous saccharification and cofermentation of Land D-lactic acid from Curcuma longa by Lactobacillus paracasei LA104 and Lactobacillus coryniformis ATCC 25600. The method is described in section 2.3. RS: reducing sugar. (A) RS and lactic acid, (B) RS conversion (100 × g lactic acid/g RS) and yield (100 × g lactic acid/g material). Figure 2 Effect of substrate concentrations on the simultaneous saccharification and cofermentation of L- and D-lactic acid from Curcuma longa by Lactobacillus paracasei LA104 and Lactobacillus coryniformis ATCC 25600. The fermentation was carried out at 37℃ for strain LA104 and 34℃ for strain ATCC 25600, at 220 rpm using S10 enzyme mixture. The pH was controlled by using 40% CaCO3 (% dried material). RS: reducing sugar, (A) L. paracasei LA104: glucose, RS, lactic acid, RS conversion (100 × g lactic acid/g RS) and yield (100 × g lactic acid/g material), (B) L. coryniformis ATCC 25600: glucose, RS, lactic acid, RS conversion and yield. Figure 3 Simultaneous saccharification and cofermentation of lactic acid from Curcuma longa by Lactobacillus paracasei LA104 in a jar fermentor. The fermentation was carried out at 140 g dried material/l, 10 g soybean meal/l with working volume of 2l and 37℃, at 200 rpm. The pH was maintained at 6 using 28% NH4OH using S10 enzyme mixture. RS: reducing

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sugar, (A) glucose, lactic acid and RS, productivity, yield and RS conversion, (B) log10(cfu) and optical purity of L-lactic acid. Figure 4 Simultaneous saccharification and cofermentation of lactic acid from Curcuma longa by Lactobacillus coryniformis ATCC 25600 in a jar fermentor. The fermentation was carried out at 140 g dried material/l, 15 g soybean meal/l with working volume of 2l and 34℃, at 200 rpm. The pH was maintained at 6 using 28% NH4OH using S10 enzyme mixture. RS: reducing sugar, (A) glucose, lactic acid and RS, productivity, yield and RS conversion, (B) log10(cfu) and optical purity of D-lactic acid.

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Fig. 1

Lactic acid, RS remaining (g/l)

A

RS remaining-LA104

Lactic acid-LA104

RS remaining-ACTC 25600

Lactic acid-ACTC 25600

60

40

20

0 S1

100

RS conversion and yield (%)

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B

S2

S3

S4

S5

S6

S7

S8

S9

RS conversion-LA104

Yield-LA104

RS conversion-ATCC 25600

Yield-ATCC 25600

S10

85

70

55

40 S1

S2

S3

S4

S5 S6 Enzyme mixture

31

S7

S8

S9

S10

Fig. 2 Lactic acid

RS conversion

Glucose remaining

RS remaining

5

Yield

95

4

85

3

75

2

65

1

55

0

100

B

Lactic acid

RS conversion

Glucose remaining

RS remaining

Glucose, RS (g/l)

A

6

Yield

5

90

4 80 3 70

2 60

1

50

0 90

100

110

120 130 140 Substrate concentration (g/l)

32

150

160

Glucose, RS (g/l)

Lactic acid (g/l) RS conversion, yield (%)

105

Lactic acid (g/l); RS conversion, yield (%)

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Fig. 3

80

4

60

3

40

2

20

Glucose remaining

RS remaining

Lactic acid

RS conversion

Yield

Productivity

Productivity (g/l/h)

5

A

1

0

0

11

98

B

96

10

94 9

92 Log10(cfu)

Optical purity

8

90

7

88 0

10

20 30 40 Fermentation time (h)

33

50

60

Optical purity (%)

Glucose, lactic acid, RS (g/l); RS conversion, yield (%)

100

Log10(cfu)

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Fig. 4

80

4

60

3

40

2

20

0 11

Glucose remaining

RS remaining

Lactic acid

RS conversion

Yield

Productivity

Productivity (g/l/h)

5

A

1

0 100

B

98

10

96 9

Log10(cfu)

94

Optical purity

8

92

7

90 0

10

20

30

40

Fermentation time (h)

34

50

60

Optical purity (%)

Glucose, lactic acid, RS (g/l); RS conversion, yield (%)

100

Log10(cfu)

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1 2

3

Graphical abstract

Research highlights

> A method for L- and D-lactic acid production from waste Curcuma longa biomass > The method produces L-lactic acid at 97.13 g/l with productivity of 2.7 g/l/h > The method produces D-lactic acid at 91.61 97.13 g/l with productivity of 2.08 g/l/h > This economical lactic acid production process using a renewable biomass has potential for industrial applications.