An economical approach for D-lactic acid production utilizing unpolished rice from aging paddy as major nutrient source Zhengdong Lu, Mingbo Lu, Feng He, Longjiang Yu * Institute of Resource Biology and Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, PR China article info Article history: Received 5 July 2008 Received in revised form 9 October 2008 Accepted 12 October 2008 Available online 21 November 2008 Keywords: D -Lactic acid Unpolished rice Aging paddy Wheat bran powder Production-cost abstract In order to reduce the raw material cost of D-lactic acid fermentation, the unpolished rice from aging paddy was used as major nutrient source in this study. The unpolished rice saccharificate, wheat bran powder and yeast extract were employed as carbon source, nitrogen source and growth factors, respec- tively. Response surface methodology (RSM) was applied to optimize the dosages of medium composi- tions. As a result, when the fermentation was carried out under the optimal conditions for wheat bran powder (29.10 g/l) and yeast extract (2.50 g/l), the D-lactic acid yield reached 731.50 g/kg unpolished rice with a volumetric production rate of 1.50 g/(l h). In comparison with fresh corn and polished rice, the D- lactic acid yield increased by 5.79% and 8.71%, and the raw material cost decreased by 65% and 52%, respectively, when the unpolished rice was used as a major nutrient source. These results might provide a reference for the industrial production of D-lactic acid. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Lactic acid (LA), a useful organic acid, is widely used in the food, pharmaceutical, leather and textile industries. Its most promising application is being used as a major raw material for the manufac- ture of poly(lactic acid) (PLA). As a kind of biodegradable polymer, PLA might become a potential environmentally friendly substitute of non-biodegradable plastics derived from petrochemicals (Aker- berg and Zacchi, 2000). There are three types of lactic acid: opti- cally active L -lactic acid, D -lactic acid and racemic DL -lactic acid. Recently, it was reported that an equimolar blend of poly( L -lactic acid) and poly( D -lactic acid) generated a racemic crystal called ste- reo-complex poly(lactic acid) which was more heat-resistant than the poly( L -lactic acid) homo-polymer due to the high melting tem- perature (Sawai et al., 2007). This finding made D -lactic acid more and more important. D -Lactic acid was primarily produced from a variety of feed- stocks by fermentation using lactic acid bacteria (Hofvendahl and Hahn-Hägerdal, 2000; Fukushima et al., 2004). A major concern in D -lactic acid fermentation was to reduce the cost of raw materi- als which accounted for more than 34% of total production-cost (Akerberg and Zacchi, 2000). Utilization of cheap carbon sources was considered as an effective approach. Though many starchy materials from agriculture such as corn, rice and rice starch were used as carbon sources in many studies, the media costs were still high in relation to synthetic media (Fukushima et al., 2004; Lee, 2007). The utilization of cellulosic wastes such as cardboard and corn cobs as substrates for lactic acid fermentation by simulta- neous saccharification and fermentation (SSF) was considered a promising approach (Rivas et al., 2004). However, there were many technical problems, for instance, the enzymes of cellulose hydroly- sis were inhibited by the intermediate product (cellobiose), and the lactic acid biosynthesis was inhibited by the final product (lactic acid). Many investigations had been carried out to relieve the inhi- bitions, for example, in situ product removal technology was ap- plied during the SSF process, which need large electric energy or high-level equipment (Li et al., 2004; Tanaka et al., 2006; Romani et al., 2008). Therefore, for the industrial production of D -lactic acid, it was quite necessary to provide cheap carbon sources which could be easily utilized by lactic acid bacteria, and to obtain the optimal conditions of fermentation with higher yield and produc- tion rate. Unpolished rice is a type of rice that had paddy hull removed during the processing but not the bran layer. Besides abundant starch, unpolished rice also contains greater amounts of dietary fi- bers, proteins, vitamins and minerals than polished rice (Das et al., 2008). Unpolished rice manufactured from fresh paddy is a healthy food. In China, an agricultural country that produces paddy in large volumes, a considerable amount of aging paddy is rejected for use as foodstuff, due to its less tasty flavor in comparison to fresh pad- dy. This aging paddy is mostly used in the feedstuff industry, which does not bring equivalent profit (Heerink et al., 2007; Liang et al., 2008). Thus, this cheap farm product containing abundant nutri- ents was used as a major nutrient source for D -lactic acid produc- tion in this study. 0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.10.015 * Corresponding author. Tel./fax: +86 27 87792265. E-mail address: yulj@hust.edu.cn (L. Yu). Bioresource Technology 100 (2009) 2026–2031 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech The objectives of this study were as follows: (i) to produce D - lactic acid by Lactobacillus delbrueckii HG 106 utilizing unpolished rice from aging paddy, and obtain the unpolished rice dregs, a byproduct of D -lactic acid production; (ii) to screen out an inexpen- sive nitrogen source from agricultural waste, and optimize the dos- age of medium compositions by response surface methodology (RSM) and (iii) to compare fermentative effect using the different raw materials such as unpolished rice, fresh corn and polished rice. 2. Methods 2.1. Samples of unpolished rice, polished rice and corn The samples of aging paddy and fresh paddy were harvested from Hubei Province of China in August 2004 and July 2007, respectively. The unpolished rice was manufactured from the aging paddy by removing the paddy hull. The polished rice was manufac- tured from the fresh paddy by removing the paddy hull and bran layer. The sample of fresh corn was harvested from Hebei Province of China in October 2007. 2.2. Microorganism, culture media and culture conditions Lactobacillus delbrueckii HG 106, a highly optical purity D -lactic acid producing strain (content of D -form lactic acid is more than 97.5%) used in this study, was provided by Guangshui Chemical Company of Hubei Province of China. MRS medium was used as the culture medium of seed activation. The media used for screen- ing nitrogen sources contained 2 g/l of yeast extract and 20 g/l of nitrogen sources such as corn steep liquor, soybean meal powder, wheat bran powder, and the treated products. Here, the nitrogen sources were limited at a lower level, namely the nitrogen sources were not superfluous. The media used for studying the effect of yeast extract on D -lactic acid fermentation contained 20 g/l of wheat bran powder, 0, 2 and 4 g/l of yeast extract, respectively. Other fermentation media’s compositions were shown further. The initial reducing sugar concentrations of unpolished rice sac- charificate were controlled at 100 g/l in all fermentations. All the media were sterilized at 121 °C for 15 min. Flask experiments were carried out in 250 ml Erlenmeyer flasks containing 100 ml of med- ium at 45 °C, with the shaking speed 150 rpm. A 5 l fermentor (Bio- stat Er B. Braun) was employed for the ampliative fermentation with an initial culture volume of 3 l. The agitation speed and cul- ture temperature were controlled at 150 rpm and 45 °C, respec- tively. The culture pH of shaking flask and fermentor were automatically controlled by the addition of 5% (w/v) sterilized CaCO 3 before fermentations. Five percent (v/v) activated inocu- lums incubated in MRS medium were used in all fermentations. 2.3. Preparation of the hydrolysate of wheat bran and soybean meal The wheat bran and soybean meal were comminuted into pow- der and sieved through a screen with 0.074 mm aperture. Then, the powder was added into water in a ratio of 3:10 (w/w). After the ini- tial pH was regulated to 1.0 by the addition of 3 M H 2 SO 4 and hydrolyzed at 121 °C for 20 min, the slurry was cooled to room temperature and filtered (Gao et al., 2007). The filtrates were read- justed to pH 7.0 as nitrogen sources of D -lactic acid production. 2.4. Preparation of the unpolished rice saccharificate and rice dregs The unpolished rice was comminuted into powder and sieved through a screen with 0.074 mm aperture. Then, the rice powder was mixed with tap water in a ratio of 1:2 (w/w). CaCl 2 was added into the slurry with a final concentration of 0.1 M. After the pH of the rice slurry was adjusted to 6.0, the a -amylase (20,000 U/ml, Wuxi Jieneng Bioengineering Company, China) was added into the rice slurry with a dosage of 12 U/g unpolished rice. The starch liquefaction was carried out at 95 °C for 60 min. When the starch liquefaction was completed, the solution was cooled to below 60 °C. After the pH of this hydrolysate was readjusted to 4.5, the amyloglucosidase (100,000 U/ml, Wuxi Jieneng Bioengineering Company, China) was added into the hydrolysate with a dosage of 120 U/g unpolished rice. The saccharification was carried out at 60 °C for 32 h. When the unpolished rice hydrolysate was en- tirely transformed into saccharificate, the mixture was heated at 100 °C for 10 min to make the enzymes inactivated. After the mix- ture was cooled to room temperature, the rice dregs were removed from the mixture by centrifugation at 8000g for 10 min (Yun et al., 2004). Finally, the pH of the unpolished rice saccharificate was ad- justed to 7.0 and the reducing sugar concentration was adjusted to 100 g/l. This unpolished rice saccharificate was used as the carbon source for D -lactic acid fermentation. 2.5. Optimization of the dosage of wheat bran powder and yeast extract Response surface methodology (RSM) and central composite design (CCD) were applied to optimize the dosage of fermentation medium compositions (Chauhan et al., 2006; John et al., 2007). The software Design-Expert 7.0.0 (Stat-Ease Inc., USA) was used for experimental design, data analysis and quadratic model building. For statistical calculation, independent variables were coded as x i ¼ðX i À X 0 Þ= D X i ð1Þ where x i represents the coded values for X i (i = 1, 2, 3, 4, etc.), X i is the experimental value of variable, X 0 is the mid-point of X i , and D X i is the step change in X i . For predicting the optimal point, a second-order polynomial equation was fitted to correlate the relationship between variables and response. The equation is y ¼ b 0 þ X k i¼1 b i x i þ X k i¼1 b ii x 2 i þ X k i X k j b ij x i x j ð2Þ where y is predicted response, x i and x j (i 6 j) are coded variables, b 0 , b i , b ii , b ij are regression coefficients calculated from the experimen- tal data by second-order multiple regression, and k is the number of factors. The experimental data were statistically analyzed using the Fischer’s statistical test for analysis of variance (ANOVA). The fitted polynomial equation was then expressed in the form of three- dimensional surface plots to illustrate the relationship between the responses and the experimental levels of each of the variables utilized in this study. 2.6. Analytical methods The samples of unpolished rice, saccharificate and dregs were analyzed after pre-treatment (Das et al., 2008). The amount of reducing sugar was determined by the 3,5 dinitro salicylic acid method (Miller, 1959). The amino acids were measured by amino acid analyzer (Beckman-6300) referring to GB/T 5009.124-2003 of China. The amount of vitamins was determined by HPLC system using a C18 column with guard column holder (Kromasil 5 l m 250  4.6 mm, Agilent 1200) (Das et al., 2008). The color of fer- mentation broth was measured by spectrophotometer at an absor- bency of 420 nm (OD 420 ). Unit price (RMB/kg) of raw material used in this study was present price in China. Incubation period was de- fined as the time (h) that the concentration of D -lactic acid reached the maximum during the process of the fermentation. Z. Lu et al. / Bioresource Technology 100 (2009) 2026–2031 2027 The fermentation broths were sampled once every 2 h during the process of the fermentation. The samples were heated immedi- ately at 100 °C for 10 min to make lactic acid bacteria inactivated and treated with 1 M H 2 SO 4 to release D -lactic acid which was formed as calcium lactate with the buffering agent, CaCO 3 . Then, the treated samples were centrifuged at 8000g for 10 min and the supernatants were filtered through a 0.45 l m cellulose acetate filter. The filtrates were diluted to the required concentration for D - lactic acid and reducing sugar analysis. The D -lactic acid concentra- tion (g/l) was estimated by colorimetric assay method (Taylor, 1996). For the calculation of D -lactic acid yield, the following equa- tion was introduced: W ¼ ðL 1 À L 0 ÞY S 1 À S 0 ð3Þ where W is the D -lactic acid yield (g/kg unpolished rice), L 1 is the to- tal D -lactic acid concentration (g/l), L 0 is the D -lactic acid concentra- tion (g/l) transformed from the reducing sugar of the wheat bran powder, Y is the reducing sugar yield (g/kg) of the unpolished rice, S 1 is the total reducing sugar concentration (g/l) and S 0 is the reduc- ing sugar concentration (g/l) released from the wheat bran powder. All experimental values were means of three replicates ± stan- dard deviation, (p < 0.05). 3. Results and discussion 3.1. Results of preparation of unpolished rice saccharificate and dregs Reducing sugar was the primary composition of the unpolished rice saccharificate. The yields of reducing sugar and rice dregs reached 810.26 ± 24.21 g/kg unpolished rice and 185.67 ± 5.87 g/ kg unpolished rice, respectively. The unpolished rice was rich in essential amino acids and B-vitamins (Table 1). It was found that the contents of amino acids and B-vitamins of the unpolished rice were generally more than that of the corn. It was reported that amino acids and B-vitamins were essential to the growth of lactic acid bacteria (Nancib et al., 2005). However, the contents of various nutrients of the rice dregs were more than that of the saccharifi- cate. It was probably because that many nutrients were enwrapped in the rice cellulose. The unpolished rice was proved nutritionally better than the polished rice. Mechanical milling and removal of bran layers resulted in a large loss of amino acids, vitamins, pro- teins, and mineral elements (Das et al., 2008). The rice dregs could be processed into rice proteins and amino acids which had been used as additives in the health food and feedstuff industry and showing good development prospect (Chandi and Sogi, 2007). As a byproduct of the D -lactic acid production, the added value of unpolished rice dregs was estimated as 10% approximately. 3.2. Screening of inexpensive and light-colored nitrogen source Although the highest D -lactic acid concentration was achieved, the incubation period was longer than the others, and the unit price was the highest, when yeast extract was used as a nitrogen source. Thus, yeast extract was an uneconomical material when it was used in a large quantity in D -lactic acid production (Table 2). Lower OD 420 implies lower cost of the fermentation broth decoloration. Obviously, though the D -lactic acid concentration and production rate were not the highest, the OD 420 of the fermen- tation broth and unit price of raw material were the lowest when the wheat bran powder was used as a nitrogen source, which ac- corded with the aims of this study. Meanwhile, the effect of different treated products of the wheat bran and soybean meal on D -lactic acid production was investi- gated. It was discovered that the crude wheat bran and crude soy- bean meal were difficult to be used as nitrogen sources for D -lactic acid fermentations. This was probably because that the nutrients were enwrapped in cellulose and were difficult to be utilized by lactic acid bacteria. Although the highest production rate was ob- tained, the D -lactic acid concentration was low and the unit price Table 1 Content of some essential amino acids (g/100 g) and B-vitamins (mg/100 g) in the corn, unpolished rice, saccharificate and dregs. Nutrient components Corn Unpolished rice Unpolished rice saccharificate Unpolished rice dregs Leucine 1.02 ± 0.25 0.72 ± 0.09 0.17 ± 0.03 0.52 ± 0.08 Isoleucine 0.22 ± 0.03 0.34 ± 0.04 0.11 ± 0.03 0.21 ± 0.03 Methionine 0.18 ± 0.02 0.21 ± 0.03 0.12 ± 0.03 0.78 ± 0.08 Phenylalanine 0.37 ± 0.04 0.42 ± 0.06 0.12 ± 0.03 0.25 ± 0.04 Valine 0.41 ± 0.06 0.52 ± 0.06 0.15 ± 0.04 0.35 ± 0.05 Threonine 0.28 ± 0.04 0.35 ± 0.04 0.11 ± 0.03 0.22 ± 0.04 Lysine 0.32 ± 0.05 0.38 ± 0.04 0.12 ± 0.03 0.23 ± 0.04 Histidine 0.23 ± 0.03 0.21 ± 0.03 0.11 ± 0.03 0.07 ± 0.01 Niacin 1.36 ± 0.15 1.65 ± 0.13 0.71 ± 0.08 0.94 ± 0.09 Pyridoxine 0.98 ± 0.12 1.35 ± 0.23 0.58 ± 0.06 0.75 ± 0.08 Thiamine 0.12 ± 0.03 0.18 ± 0.03 0.06 ± 0.01 0.09 ± 0.01 Riboflavin 0.04 ± 0.01 0.06 ± 0.01 0.02 ± 0.01 0.03 ± 0.01 Table 2 Comparison of D-lactic acid fermentations with different nitrogen sources and the treated products. Compositions of the media (g/l): reducing sugar of the unpolished rice saccharificate, 100; yeast extract, 2 and different nitrogen sources, 20. Nitrogen sources Evaluated items D -Lactic acid concentration (g/l) Incubation periods (h) OD 420 of fermentation broth Unit price of raw material (RMB/kg) Yeast extract 80.24 ± 2.56 70 0.41 ± 0.05 12.1 Corn steep liquor 74.70 ± 2.48 58 1.75 ± 0.09 4.5 Soybean meal powder 78.35 ± 2.36 56 0.39 ± 0.04 4.7 Wheat bran powder 76.55 ± 2.28 56 0.38 ± 0.04 1.6 Soybean meal hydrolysate 73.68 ± 2.46 52 0.47 ± 0.05 5.3 Wheat bran hydrolysate 71.17 ± 2.31 52 0.45 ± 0.04 2.1 Crude soybean meal 46.58 ± 1.63 66 0.39 ± 0.05 4.4 Crude wheat bran 42.65 ± 1.35 68 0.38 ± 0.05 1.4 2028 Z. Lu et al. / Bioresource Technology 100 (2009) 2026–2031 was high when soybean meal hydrolysate was used as a nitrogen source. It was also found that D -lactic acid concentration were slightly lower than that of wheat bran powder and soybean meal powder when the hydrolysate of wheat bran and soybean meal were used as nitrogen sources. The reason might be that important nutritional components were destroyed or removed inadvertently in the severe acid-hydrolysis and separation steps for its produc- tion (Kwon et al., 2000; Gao et al., 2007). On the contrary, acid- hydrolysis process aggravated the color of fermentation broth and increased the production-costs. As a result, the wheat bran powder, an inexpensive and light- colored agricultural waste was screened out and used as the nitro- gen source for D -lactic acid production, through comprehensive analysis and comparison. After incubated 60 h, 76.55 ± 2.28 g/l of D -lactic acid concentra- tion was obtained when 20 g/l of wheat bran powder was used as the major nitrogen source, while the sugar still maintained at the high concentration of 16.20 ± 1.20 g/l, which indicated that the maximum D -lactic acid concentration could not be obtained only when the unpolished rice saccharificate and wheat bran powder were employed as nutrients. Therefore, it was possible that some other nutrients, especially growth factors must be added into the fermentation medium to increase the yield of D -lactic acid and transforming ratio of sugar into acid. 3.3. Supplement of yeast extract provided necessary growth factors Yeast extract was demonstrated to be an excellent nutrient for most lactic acid bacteria according to previous reports (Nancib et al., 2005). The D -lactic acid concentration increased significantly with increasing initial yeast extract concentration, but in a non- proportional manner (Fig. 1). As it was expected, D -lactic acid con- centration increased greatly when a small quantity of yeast extract was supplied. The final D -lactic acid concentration was only 42.30 ± 2.50 g/l because of the lack of yeast extract in the medium, which implied that the yeast extract was essential for D -lactic acid fermentation by Lactobacillus delbrueckii HG 106 even with a low dosage. The importance of yeast extract for lactic acid bacteria was re- ported by some researchers. Some researchers speculated that the main contribution of yeast extract in the medium was supply- ing purine, pyrimidine and B-vitamins for lactic acid bacteria (Nancib et al., 2005). Yeast extract was too expensive (accounted for about 38% of to- tal medium cost) to hinder its use in large quantities in lactic acid production (Altaf et al., 2007). If yeast extract was replaced by other cheap raw materials, there was either decrease in lactic acid production or increase in incubation period (Altaf et al., 2006; Gao et al., 2007). Therefore, the supplementation of small amount of yeast extract was necessary to avoid a limitation of essential growth factors for Lactobacillus delbrueckii HG 106. However, it was also necessary to optimize the dosage of yeast extract in the fermentation medium to reduce the material cost. 3.4. Optimization of the dosage of wheat bran powder and yeast extract by RSM The wheat bran powder and yeast extract were used as nitrogen source and growth factors of D -lactic acid production based on above experiments. Central composite design (CCD) was applied to find their appropriate dosage and predict maximum D -lactic acid concentration. The variables and responses of D -lactic acid produc- tion were listed in Table 3. Analysis of variance for the quadratic model was calculated from the response obtained for 13 runs (Table 4). The ANOVA showed that The model F-value of 48.45 implied the model was significant. Values of ‘‘p > F” <0.05 indicated model terms were sig- nificant. In this case, X 1 , X 2 , X 1 X 2 , X 2 1 and X 2 2 were significant model terms which indicated that the change of concentrations of wheat bran powder and yeast extract influenced D -lactic acid production directly. The F-value of X 1 X 2 term is 20.68, which indicated that interaction of wheat bran powder and yeast extract was significant. By ANOVA, the coefficient of determination (R 2 ) of the regres- sion model was 0.9719, which implied that 97.19% of the variation in the response could be explained by the model. The R 2 of 0.9719 was in reasonable agreement with the adjusted R 2 of 0.9519, which indicated a good agreement between the experimental values and the predicted values of D -lactic acid production. The experimental results of the CCD were fitted with the coded second-order polynomial function for the estimation of D -lactic acid production: Y ¼ 88:64 þ 3:08X 1 þ 3:14X 2 À 2:02X 1 X 2 À 1:18X 2 1 À 1:41X 2 2 ð4Þ where Y is the response, D -lactic acid concentration, X 1 and X 2 are the concentrations of wheat bran powder and yeast extract, respectively. The effect of wheat bran powder, yeast extract and their interactions on D -lactic acid concentration was shown in Fig. 2. It was evident that D -lactic acid production increased with increasing wheat bran powder concentration. A similar trend was observed in yeast extract. However, the two variables showed a synergistic effect on D -lactic acid production, and fur- ther increase of D -lactic acid concentration could not be achieved when the two variables increased unceasingly. The surface plots of yield indicated that the D -lactic acid concentration could not exceed 92.00 g/l. Using the desirability function for point prediction of the Design Expert 7.0.0 software, the optimal conditions for wheat bran pow- der (29.10 g/l) and yeast extract (2.50 g/l) were obtained. The pre- dicted maximum response of D -lactic acid concentration was 90.10 g/l. Validation of the model was carried out in 250 ml Erlen- meyer flasks under the optimal conditions to confirm the pre- dicted response. As a result, the D -lactic acid concentration reached 91.20 ± 3.34 g/l which neared to the foregoing pre- dicted value of 90.10 g/l and also neared to the maximum D - lactic acid concentration of 91.10 g/l in CCD experiment (Table 3, run 4) while the dosages of raw materials were obviously decreased. Fig. 1. Effect of different initial concentrations of yeast extract on D -lactic acid production. Closed key: D -lactic acid concentration and open key: residual sugar. Z. Lu et al. / Bioresource Technology 100 (2009) 2026–2031 2029 3.5. Comparison of fermentations using unpolished rice, fresh corn and polished rice as major nutrient sources D -Lactic acid fermentations with different media were carried out in a 5 l fermentor under the aforementioned optimal condi- tions (Fig. 3). As a result, the D -lactic acid concentration reached 90.80 ± 3.20 g/l, and incubation period was <60 h when the unpol- ished rice saccharificate uncontaining rice dregs was used as a ma- jor nutrient source (Fig. 3a). There was no significant difference on D -lactic acid production when using unpolished rice saccharificate in which the unpolished rice dregs were contained or not. As a re- sult, the D -lactic acid yield reached 731.50 g /kg unpolished rice with a volumetric production rate of 1.50 g/(l Á h), when unpol- ished rice saccharificate was used as major nutrient source. The yield was higher about 5.79% and 8.71% than that of fresh corn and polished rice, respectively. The OD 420 of fermentation broth was lower than that of fresh corn saccharificate when unpolished rice saccharificate was used in the medium, which implied that the cost of decoloration would be lower in the downstream steps (Fig. 3b). Besides, the unit price of unpolished rice was nearly one half of that of the fresh corn and polished rice. It was estimated that the raw material cost of D -lactic acid production decreased by 65% and 52%, respectively, when the unpolished rice was used as major nutrient source. These values were related to increase in D - lactic acid yield. When the unpolished rice was used as a major nutrient, the D - lactic acid yield increased and material cost reduced, compared with that of in former researches using fresh corn and polished rice as carbon sources (Fukushima et al., 2004; Oh et al., 2005; Lee, 2007). Thus, in comparison with fresh corn, unpolished rice showed several advantages, because unpolished rice was rich in nutrition and showed little color, which influenced the yield and cost of D -lactic acid production directly. Table 3 Central composite design for optimization of two variables in mathematically predicted and experimental values for D-lactic acid production. Run X 1 Wheat bran powder (g/l) X 2 Yeast extract (g/l) Y: D -lactic acid (g/l) Experimental values Predicted values 1 10 1 78.3 77.8 2 30 1 88.3 88.0 3 10 3 89.2 88.1 4 30 3 91.1 90.3 5 5.86 2 81.1 81.9 6 34.14 2 90.1 90.6 7 20 0.59 81.1 81.4 8 20 3.14 89.2 90.3 9 a 20 2 88.3 88.6 10 a 20 2 89.6 88.6 11 a 20 2 88.3 88.6 12 a 20 2 88.5 88.6 13 a 20 2 88.5 88.6 a Centre point replicate runs: 9–13. Experimental values were means of three replicates. Table 4 Analysis of variance (ANOVA) for the fitted quadratic polynomial model. Source Degree of freedom Sum of square Mean square F- value p-Value p > F Model 5 192.16 38.43 48.45 <0.0001 X 1 1 75.82 75.82 95.58 <0.0001 X 2 1 79.10 79.10 99.72 <0.0001 X 1 X 2 1 16.40 16.40 20.68 0.0026 X 2 1 1 9.73 9.73 12.26 0.0100 X 2 2 1 13.78 13.78 17.37 0.0042 Residual 7 5.55 0.79 Lack of fit 3 4.36 1.45 4.88 0.08 Pure error 4 1.19 0.30 Cor total 12 197.71 C.V.% = 1.10 R 2 = 0.9719 Adj R 2 = 0.9519 Fig. 2. Response surface and contour plots showing the interaction of wheat bran powder and yeast extract on D -lactic acid production. Fig. 3. Comparison of D -lactic acid fermentations with different major nutrient sources. Major nutrient sources: A, unpolished rice saccharificate uncontaining rice dregs; B, unpolished rice saccharificate containing rice dregs; C, fresh corn saccharificate uncontaining corn dregs and D, fresh polished rice saccharificate uncontaining rice dregs. 2030 Z. Lu et al. / Bioresource Technology 100 (2009) 2026–2031 4. Conclusion The unpolished rice provided not only carbon source but also other important nutrients such as amino acids and B-vitamins. The wheat bran powder was an inexpensive and light-colored nitrogen source. The supplement of a small quantity of yeast ex- tract provided growth factors and increased the D -lactic acid yield significantly. Their optimal concentrations to be added were suc- cessfully established by applying a statistical experimental tool such as RSM. The D -lactic acid yield increased a lot and raw mate- rial cost decreased remarkably compared with that of fresh corn and polished rice. Thus, the unpolished rice from aging paddy was a cheap and excellent nutrient source for D -lactic acid production. Acknowledgements This work was supported by Grants from the National High Technology Research and Development Key Program of China (Pro- ject No. 2008AA10Z339) and the Key Sci-Tech Project of Hubei Province of China (Project No. 2006AA201C47). 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An economical approach for D-lactic acid production utilizing unpolished rice from aging paddy as major nutrient source Zhengdong Lu, Mingbo Lu, Feng He, Longjiang Yu * Institute. bran layer. Besides abundant starch, unpolished rice also contains greater amounts of dietary fi- bers, proteins, vitamins and minerals than polished rice (Das et al., 2008). Unpolished rice manufactured. produce D - lactic acid by Lactobacillus delbrueckii HG 106 utilizing unpolished rice from aging paddy, and obtain the unpolished rice dregs, a byproduct of D -lactic acid production; (ii) to screen out an inexpen- sive