Effects of washing with water on enzymatic saccharification andD -lactic acid production from steam-exploded sugarcane bagasse

• lactic acid production from steam-exploded sugarcane bagasse
• lactic acid from unutilized sugarcane bagasse using steam explosion pretreatment. The optimal steam pressure for a steaming time of 5 min was determined. The steam- exploded sugarcane bagasse was hydrolyzed using cellu- lase (Meicelase) and then the hydrolyzate was subjected to fermentation substrate. By enzymatic saccharification using Meicelase, the highest recovery of glucose from raw bagasse, 73.7 %, was obtained at a steam pressure of 20 atm. For extracted residue with water after steam explosion, the glucose recovery increased up to 94.9 % at a steam pressure of 20 atm. These results showed that washing with water is effective in removing enzymatic reaction inhibitors. After steam pretreatment (steam pres- sure of 20 atm),
• lactic acid was produced by Lactoba- cillus delbrueckii NBRC 3534 from the enzymatic hydrolyzate of steam-exploded bagasse and washed resi- due. The conversion rate of

• lactic acid obtained from the glucose concentration was 66.6 % for the hydrolyzate of steam-exploded bagasse without washing with water and 90.0 % for that derived from the extracted residue with water after steam explosion. These results also demon- strated that the hydrolyzate of steam-exploded bagasse (without washing with water) contains fermentation inhibitors and washing with water can remove them. Keywords Sugarcane bagasse Steam explosion
• Lactic acid

  Lactic acid is widely used in the food, pharmaceutical, and chemical industries. In particular, lactic acid has been used as a raw material for manufacturing poly(lactic acid),

  explosion is a hydrothermal pretreatment method for plant biomass that uses high-pressure and high-temperature steam without the addition of any chemicals. And this method involves chemical effects such as auto hydrolysis, defibria- tion, and delignification. Compared to the generally used chemical treatments, it has several potential advantages: it eliminates the use of toxic substances, such as strong acids or alkalis, chemical tolerance equipment, waste solution processing, and preliminary feedstock size reduction. On the other hand, degradation products from sugarcane bagasse, which are inhibitors for enzymatic saccharification and fer- mentation, are generated. Therefore, the removal process with chemical reagents such as alkali is necessary [

   ] methods. Steam

  ical

  hydrothermal-chem-

  Over the years, various pretreatment methods for the sugarcane bagasse have been developed, including physical

  The main components of cellulosic biomass, sugarcane bagasse, are cellulose, hemicellulose, and lignin. Because cellulose is surrounded by a hard network of lignin, it is necessary to decompose it using an environmentally friendly pretreatment method prior to its conversion into glucose using an enzymatic method.

  Introduction Sugarcane bagasse, a byproduct of the sugar industry, is an abundant source of lignocellulose. Although it is used as fuel for boilers, a large quantity of this material is accu- mulated in sugar processing plants, which leads to envi- ronmental problems. Advanced utilization of this material as a carbon resource holds promise for the bioproduction of useful chemicals.

  Enzymatic saccharification

  D

  D

  D

  D

  This study investigated the production of

  Springer 2012 Abstract

  Chizuru Sasaki ^• Ryosuke Okumura ^• Ai Asakawa ^• Chikako Asada ^• Yoshitoshi Nakamura Received: 1 December 2011 / Accepted: 28 May 2012 / Published online: 27 June 2012 Ó

  Effects of washing with water on enzymatic saccharification and D

  O R I G I N A L A R T I C L E

  C. Sasaki (&) R. Okumura A. Asakawa C. Asada Y. Nakamura Department of Life System, Institute of Technology and Science, The University of Tokushima, 2-1 Minamijosanjima-cho, Tokushima 770-8506, Japan e-mail: csasaki@bio.tokushima-u.ac.jp DOI 10.1007/s10163-012-0064-y

• lactic acid production strategies using unutilized cellulosic plant material and starchy biomass, such as paper sludge, water wastes, agro wastes, and beer production byproducts
• type and
• type lactic acids form stable stereocomplexes that have thermal properties different from those of the original polylactic acid made from only
• type or
• type lactic acid
• lactic acid from unutilized plant biomass

• lactic acid by Lactobacillus delbrueckii were investigated. In the process, the removal method of hydrolyzate and fermentation inhibitors with water extraction was investigated, and the final amount of production of

  Water soluble material Residue 2 ( Holocellulose and Klason-lignin )

  Components analysis of bagasse and steam-exploded bagasse Components analysis of untreated bagasse and steam- exploded bagasse was conducted by Wayman’s method

   . The amount of

  components, i.e., water-soluble material (WSM), metha- nol-soluble material, acid-soluble lignin, holocellulose (cellulose and hemicellulose), and Klason lignin, and vol- atile materials were determined as follows.

  Five grams of dry untreated or steam-exploded bagasse was added to 100 mL of distilled water and extracted for 24 h at room temperature. The solid (Residue 1) and liquid materials were separated by filtration, the filtrate was recovered from the liquid, then concentrated, dried, and weighed (WSM). Residue 1 was extracted at 80 °C for 24 h in a Soxhlet extractor using 150 mL of methanol to dissolve the methanol-soluble material. After concentration and drying of the extract, the methanol-soluble material was weighed. Residue 2 from the methanol extraction consisted of holocellulose and Klason lignin, a high molecular weight lignin. This residue (1 g) was added to 15 mL of 72 % (w/v) sulfuric acid and kept at room tem- perature for 4 h. After that, the content was placed in a 100-mL conical flask, washed with 560 mL of distilled water, and then autoclaved for 1 h. After the insoluble material was washed with distilled water, it was heat-dried at 105 °C to a constant weight and weighed (Klason lignin). The weight of holocellulose was calculated by subtracting the weight of Klason lignin from 1 g of Residue 2. The volatile materials, i.e., degradation products from sugar [organic acids, i.e., acetic acid and formic acid, furfural, and 5-hydroxymethyl furfural (HMF)] analysis was carried out using the HPLC system with a refractive index detector with a Bio-Rad HPX-87H column at a

• lactic acid from sugarcane bagasse using steam explosion. For this purpose, optimal steam explosion pretreatment conditions for sugarcane bagasse followed by enzymatic saccharifica- tion and microbial conversion of hydrolyzates derived from sugarcane bagasse to

  Residue 1 ( Holocelluloseand lignin ) Untreated or Steam exploded bagasse

  Water extraction Dry Volatile material Dry and Weigh Weigh

  Methanol soluble material Klason-lignin Acid soluble lignin, Holocellulose Methanol extraction Acidic treatment with sulfuric acid

  Dry and Weigh Colorimetric method Glucose equivalent Residue 1 ( Holocelluloseand lignin ) Untreated or Steam exploded bagasse

  Water soluble material Residue 2 ( Holocellulose and Klason-lignin )

  Methanol soluble material Klason-lignin Acid soluble lignin, Holocellulose Methanol extraction Acidic treatment with sulfuric acid

  Water extraction Dry Dry Volatile material Dry and Weigh Weigh Weigh

  Dry and Weigh Colorimetric method Glucose equivalent

  Steam explosion Pretreatment of the bagasse was conducted in a steam explosion apparatus (Japan Chemical Engineering and Machinery Co. Ltd., Osaka, Japan). The reactor was charged with 100 g (dry matter) of feedstock per batch. Saturated steam from the boiler was then allowed to enter the reactor to heat the bagasse at a controlled pressure of 15, 20, 25, 30, or 35 atm. Each pressure was maintained for 5 min and then the reactor was suddenly depressurized. The exploded bagasse was recovered in a cyclone and cooled to room temperature.

  Materials and methods Sugarcane bagasse The raw sugarcane bagasse from Kyuyo Sugar Co. Ltd.

  (Okinawa, Japan) used in these experiments was cut into 1–3-cm-sized chips. The chemical composition (based on dry material) of bagasse was 45.1 % a-cellulose, 22.3 % Klason lignin, 27.9 % hemicellulose, and 4.7 % other.

  D -lactic acid was evaluated.

  D

  D

  The main objective of this study is to evaluate an envi- ronmentally friendly method for the production of

   ].

  D

  However, few studies have focused on strategies for pro- ducing

   ].

  D

  L

  L

   ]. Recently, it was discovered that D

  L

  one of the most promising biodegradable polymers. For this reason, many researchers have studied

  Fig. 1 Component analysis method of untreated bagasse and steam- exploded bagasse

  D

  1.0 g/L yeast extract. Later, the cells were harvested by centrifugation (2000g), rinsed thoroughly with sterile dis- tilled water, centrifuged again, and then resuspended in sterile distilled water.

  production of sugars and further degraded materials, the amount of holocellulose decreased (steam pressures of 15 and 35 atm resulted in 53.3 and 39.0 %, respectively). The amount of methanol-soluble material increased with steam pressure. Steam pressures of 15 and 30 atm resulted in 7.9 and 23.7 %, respectively, and it contained low molecular weight lignin [

  With increased

  therefore, mainly the degradation of hemicellulose occur- red. Above 20 atm, the amount of WSM decreased owing to the degradation of pentose, i.e., xylose or arabinose sugars derived from hemicellulose, which changed to insoluble materials, such as furfural [

   ];

  of WSM (17.8 %), which contains sugars derived from holocellulose, was obtained at a steam pressure of 15 atm. In general, hemicellulose in the holocellulose hydrolyzes under less severe conditions than cellulose [

   . The highest amount

  5.0), and 10 % (v/v) preculture solution. The nutrient medium and buffer were autoclaved at 121 °C for 20 min. The hydrolyzate was added after sterilizing with a 0.22-lm pore size filter. Cultivation was carried out in a 300-mL flask with 100 mL of the medium using a static incubator at 37 °C. Aliquots of the samples were collected and assayed for determining ethanol and residual glucose concentra- tions. All fermentative experiments were performed in duplicate and the average results are shown. Determination of residual glucose and produced

2 SO

  D

• lactic acid Residual glucose and produced
• lactic acid concentrations in the fermentation medium were determined by the mutarotase GOD method (Glucose C-II test, Wako Pure Chemicals Co., Ltd., Japan) and an enzymatic UV test with

  D

  D

• lactate dehy- drogenase (

  D

  D

• LDH) (enzymatic fluid
• lactic acid, R-Biop- harm GmbH, Darmstadt, Germany), respectively. Results and discussion Composition analysis of steam-exploded bagasse To break down the lignin network that surrounds cellulose and increase the accessibility of the enzyme to cellulose, steam explosion was used to treat the bagasse. Table

  7H

  2 O, and

  )

  temperature of 50 °C. The mobile phase was 5.0 mM H

  4 , at a flow rate of 0.6 mL/min.

  Each reducing sugar component in WSM and water- insoluble residue from steam-exploded bagasse was deter- mined using a sulfuric acid hydrolyzate of bagasse according to the Technical Association of the Pulp and Paper Industry Standard. Following that, each hydrolyzate was analyzed by the reducing sugar analysis system (Prominence series with post-column derivatization system using arginine, Shimadzu Co., Ltd.) with a fluorescence detector (RF-20Axs)

   ]. All

  analytical determinations were performed in duplicate and the average results are shown. Enzymatic hydrolysis Steam-exploded bagasse was enzymatically hydrolyzed using Meicelase (produced by Trichoderma viride) pro- vided by Meiji Seika Co., Ltd. Enzymatic hydrolysis was performed using a 0.05-M sodium phosphoric acid buffer (pH 4.5) at 50 °C on a rotary shaker at 140 rpm for 48 h. The substrate, i.e., steam-exploded bagasse or water- extracted residue after steam explosion, concentration and enzyme loading were 100 g/L and 50 FPU/g substrate, respectively. The supernatant was centrifuged to remove solid waste and analyzed for glucose. All enzymatic hydrolysis experiments were performed in duplicate and the average results are shown. Microorganism and inoculum cultivation Lactobacillus delbrueckii NBRC 3534, a homofermenta- tive D -lactic acid producer, was used to produce D -lactic acid from the hydrolyzate of steam-exploded bagasse. The microorganisms were subcultured every 4 weeks. The strain was precultured in 100 mL of medium in a 300-mL flask at 30 °C for 24 h using a static incubator at 60 rpm. The preculture media comprised 0.1 g/L (NH

  4

  2 SO

  shows the chemical composition of untreated bagasse and steam-exploded bagasse (steam pressures of 15, 20, 25, 30, and 35 atm; steaming time 5 min) separated by the extraction method illustrated in Fig.

  4

  , 10 g/L glucose, 0.1 g/L KH

  2 PO

  4

  , 0.1 g/L MgSO

  4

  

• Lactic acid fermentation using bagasse hydrolyzate at optimal conditions The nutrient medium for
• lactic acid fermentation con- tained hydrolyzate derived from steam-exploded bagasse (the initial glucose concentration was adjusted at 20 g/L, the hydrolyzate was freeze-dried and then dissolved with water to adjust the concentration of the quantity of 20 g/L), yeast extract 0.1 g/L, ammonium sulfate 0.1 g/L, KH

  2 O 0.01 g/L, 0.05 M acetate buffer (pH

  7H

  4

  0.1 g/L, MgSO

  2 PO drying under 105 °C, which was contained in the WSM. From the steam-exploded bagasse, furfural, HMF, acetic acid, and formic acid were generated. Acetic acid is liberated from acetyl groups in the hemicellulose fraction; furfural and HMF are products of pentose and hexose degradation, respectively. Moreover, formic acid is formed from furfural and HMF at elevated temperatures

  D

   ]. Furthermore, the maximum reduc-

  tion of Klason lignin (reduction rate 19.8 %) was observed at a steam pressure of 20 atm. At higher pressures, a gradual increase was observed owing to the combination of lignin with low molecular weight lignin and WSM

   ].

  Figure

   shows the volatile materials volatiled during

  4

   ].

  17.8

  0.4

  0.4 Holocellulose

  72.6

  53.3

  46.2

  44.6

  41.7

  39.0 Klason lignin

  22.2

  20.1

  21.0

  0.4

  24.5

  28.0 Total 100 100 100 100 100 100

  Steam pressure

  12

  10

  8

  6

  4

  2 Furfural HMF Acetic acid Formic acid g / 100 g-Steam exploded bagasse

  15 atm 20 atm 25 atm 30 atm 35 atm Furfural HMF Acetic acid Formic acid Furfural HMF Acetic acid Formic acid

  0.4

  0.9

  As can be observed, by increasing the pressure, i.e., tem- perature, the amount of these products was increased. Generally, these compounds are known to the inhibitors for fermentation [

  Table 1 Chemical composition (%) of untreated and steam-exploded bagasse at different steam pressures (steaming time 5 min) Components Untreated bagasse Steam pressure (atm) [steaming temperature] 15 [198 °C] 20 [213 °C] 25 [224 °C] 30 [234 °C] 35 [243 °C] Water-soluble material

  therefore, it is necessary to remove these products.

  Sugar composition analysis of steam-exploded bagasse It is necessary to analyze the sugar composition of pre- treated material to investigate the relationship between sugar and the conditions of steam explosion. Table

   shows

  the sugar composition in WSM and water-insoluble material (WISM) of steam-exploded bagasse at different steam pressures with a steaming time of 5 min. It was clear that untreated bagasse contained 0.494 and 0.26 g of fer- mentable sugars, i.e., glucose (converted into lactic acid or ethanol) and xylose (mainly converted into xylitol) in 1 g of dry bagasse, respectively. After steam explosion, the amounts of glucose in WISM were 0.485, 0.471, and 0.472 g in 1 g of dry untreated bagasse at steam pressures of 15, 20, and 25 atm, respectively. These correspond to 98.2, 95.3, and 95.5 % recovery of glucose (glucose in the untreated bagasse, i.e., 0.494 g of glucose in 1 g of dried untreated bagasse). At steam pressures of 30 and 35 atm, the amount of glucose gradually decreased. The xylose in WISM decreased dramatically with the increase in steam pressure. The amount was 0.098 g in 1 g of untreated bagasse at a steam pressure of 15 atm, which corresponds to a recovery rate of 37.7 % from the initial amount of xylose.

  In contrast, the amount of xylose in WSM increased (0.146 g in 1 g of dry bagasse). This result shows that the decomposition and solubilization of hemicellulose (mainly xylose) in water is caused by the increase of the treatment temperature (pressure). The amount of xylose in WSM was maximum (0.15 g in 1 g of dried untreated bagasse) at a steam pressure of 20 atm and then decreased with an increase in steam pressure. These results indicate that the decomposition of xylose started at around 210 °C [

  

  Enzymatic saccharification of steam-exploded bagasse The effect of steam explosion conditions on the enzymatic saccharification of bagasse was studied using a substrate concentration of 100 g/L and the results are presented in Fig.

  

  a. Steam pressures of 15, 20, 25, 30, and 35 atm for a steaming time of 5 min were investigated. The maximum amount of glucose produced was observed at a steam pressure of 20 atm and steaming time of 5 min: 364 mg/g dry steam-exploded bagasse, which corresponds to a 73.7 % glucose yield from untreated bagasse. For steam pressures of 25, 30, and 35 atm, the glucose yield decreased more than that for a steam pressure of 15 atm. Figure

   b shows the enzymatic saccharification of washed

  1.9

  1.8

  17.8

  13.6

  11.2

  9.7

  10.0 Methanol-soluble material

  1.5

  7.9

  22.0

  22.8

  23.7

  22.6 Acid-soluble lignin

  Fig. 2 Composition of the volatile materials resulting from steam explosion at different pressures (steaming time 5 min)

  Table 2 Sugar composition of water soluble material (WSM) and water insoluble material (WISM) of steam exploded bagasse at different steam pressures Sugar Untreated bagasse 15 atm (198 °C) 20 atm (213 °C) 25 atm (224 °C) _{a a a a a a a} WISM WSM WISM WSM WISM WSM g/g % g/g % g/g % g/g % g/g % g/g % g/g %

• _{-4 -4} Arabinose 0.028

  3.55 0.005 0.81 0.010 6.33 0.002 0.41 0.007

  4.1

  0.05

  1.93 2.0 9 10 5.0 9 10

  Galactose 0.010 1.28 0.005 0.83 0.004 2.53 0.004 0.74 0.005 2.92 0.004 0.85 0.001

  3.81 Glucose 0.494 61.8 0.485 81.5 0.002 1.26 0.471 91.8 0.007 4.09 0.472 _{-4 -4} 96.7 0.014

  46.3 Mannose 0.007 0.85 0.001 0.25 0.002 1.26 0.001 0.20 0.002

  1.17

  0.15

  2.77 7.0 9 10 8.0 9 10

  Xylose 0.260 32.5 0.098 16.6 0.146 88.6 0.035 6.8 0.150 87.7 0.010 2.05 0.014

  45.2 30 atm (234 °C) 35 atm (243 °C) WISM WSM WISM WSM _{a a a a} g/g % g/g % g/g % g/g %

• _{-4 -4 -4 -4}

  0.047

  1.03

  0.04

  0.35 2.0 9 10 2.0 9 10 1.0 9 10 1.0 9 10 _{-4 -4} 0.003

  0.58

  2.53

  0.05 – – 5.0 9 10 2.0 9 10

  0.451 98.3 0.016 77.3 0.427 99.4 0.018

  90.9 ^{-4 -4 -4}

  0.05 2.67 –

• – 0.004 _a

  2.3 2.0 9 10 6.0 9 10 4.0 9 10

  0.88 0.003 16.4 0.002 0.39 0.001

  6.6 Based on untreated bagasse Fig. 3 Enzymatic saccharification of steam-

  A B

500 500

  exploded bagasse (a) and water- insoluble residue after steam

  

400 400

  explosion (b). Open circles steam pressure 15 atm, filled squares 20 atm, open squares

  300 300

  25 atm, filled triangles 30 atm, and open triangles 35 atm, at a

  200 200

  steaming time of 5 min

  

100 100

  10

  20

  

30

  40

  50

  10

  20

  30

  40

  50 mg-glucose/g-dry steam exploded bagasse

  Time (h) Time (h) mg-glucose/g-dry steam exploded bagasse

  steam-exploded bagasse, i.e., WISM, after steam explosion Palmqvist et al. [

  reported that organic acids, i.e., acetic

  as a substrate (substrate concentration is 50 g/L). Under all acid and fran derivatives, are well-known inhibitors for conditions, the amount of glucose produced increased. yeasts and bacteria, but have little effect on cellulases. Kim Similarly, higher glucose concentration was achieved at et al. [

  also demonstrated that 13 g/L acetic acid and

  steam pressures of 20 and 25 atm. The value was 469 mg furfurals at 4 g/L, which is larger than of our concentration, in 1 g of steam-exploded dry bagasse, corresponding to a were not inhibitory for enzymatic saccharification using 94.9 % glucose yield from untreated bagasse. These results cellulase. On the other hand, simple phenolic compounds, indicated that, if water-soluble inhibitors for saccharifica- such as vanillin, caffeine acid, and catechol [

  were

  tion were washed with water, the enzyme could access reported as cellulase inhibitors. Other researchers reported cellulose in the treated bagasse. Dunlop [

   ].

  

   Consequently, the removal of phenolic compounds with

  reported that, during high-temperature or acid pretreatment water resulted in an increase of glucose recovery. From the of wood, water-soluble inhibitors such as sugar- and lignin- viewpoint of low energy input, this study indicated that the derived materials, organic acids, fran compounds, and optimal conditions for the steam explosion of bagasse was phenol compounds, are formed. Delgenes et al.

   ] and a steam pressure of 20 atm for a steaming time of 5 min. D

• Lactic acid production from bagasse Figure
• lactic acid produced decreased (conversion rate was 66.5 %) and no dramatic glucose consumption was observed. This phenomenon also showed the presence of fermentation inhibition materials in the steam-exploded bagasse, similar to the case of enzymatic saccharification. This study found that a simple method with low environmental loading, such as washing only with water, could remove the inhibitors for

• lactic acid production by Lactobacillus delbrueckii NBRC 3534 using available glucose, steam-exploded bagasse (steam pressure of 20 atm, steaming time 5 min), and water-insoluble residue after steam explosion (20 atm, 5 min) as substrates. When water-insoluble residue after steam explosion was used as the substrate,
• lactic acid pro- ducers and cellulase from steam-exploded bagasse. As a result, the final amounts of
• lactic acid production were compared between the method without water extraction and with water extraction after steam explosion of bagasse (Fig.
• lactic acid was produced from 100 g of bagasse using the process without water extrac- tion; however, 42.2 g of

• lactic acid production behavior similar to the results obtained using commercially available glucose was observed. In this case, the maximum amounts of

  D

• lactic acid produced were 16.5 g/L (available glucose) and 17.1 g/L (water-insoluble residue), at a 72-h cultivation time, which corresponds to 81.7 and 90.0 % of the conversion rate of the initial glucose concentration, respectively. On the other hand, when steam-exploded bagasse was used as the substrate, i.e., without washing with water after steam explosion treatment, the amount of
• lactic acid can be produced from 100 g of bagasse using the process with water extraction after steam explosion.(steam pressure of 20 atm and steaming time 5 min). Conclusions This study showed that high amounts of glucose could be produced from the enzymatic hydrolyzate of steam-explo- ded bagasse. In particular, washing the steam-exploded sample with water effectively removes the inhibitors that interrupt the reaction between cellulose and the enzyme (cellulase). Furthermore, it was found that a large amount of

  D

  Only 24.2 g of D

   shows the time courses of glucose consumption

  and

  D

  D

  D

  D

  D

25.0 Concentration (g/L)

• lactic acid could be produced from a hydrolyzate of water-extracted residue of bagasse after steam explosion using Lactobacillus delbrueckii NBRC 3534, while, only a small amount of

  60

80 Time (h)

• lactic acid was produced from a hydrolyzate of steam-exploded bagasse. Thus, washing with water is a simple and environmentally friendly method for removing inhibitors from the pretreated residue. This study will contribute to improving large-scale indus- trial methods for the production of
• lactic acid from unutilized plant materials.
• lactic acid produced from steam-exploded bagasse without and with washing with water in the process

  Conversion rate* ² ( 90.0 %)

  D-lactic acid (42.2 g)

  Steam exploded bagasse (100 g)

  Conversion rate* ¹ Conversion rate* ¹ ( 36.4 %)

  Glucose (36.4 g)

  Conversion rate* ² Conversion rate* ² ( 66.6 %)

  D-lactic acid (24.2 g)

  Without water extraction after steam explosion of bagasse With water extraction after steam explosion of bagasse

  Steam exploded bagasse (100 g)

  Conversion rate* ¹ Conversion rate* ¹ ( 46.9 %)

  Glucose (46.9 g)

  Conversion rate* ² Conversion rate* ² ( 90.0 %)

  D-lactic acid (42.2 g)

  Conversion rate* ¹ ( 46.9 %)

  Conversion rate* ² ( 66.6 %)

  D-lactic acid (24.2 g)

  40

  D

  D

  D

  10

  20

  30

  50

  Conversion rate* ¹ ( 36.4 %)

  70

  5.0

  10.0

  15.0

  20.0

  Fig. 4 Residual glucose concentration and ^D -lactic acid production profiles by Lactobacillus delbrueckii NBRC3534. Filled circles glucose with commercial glucose, open circles ^D -lactic acid with commercial glucose, filled squares glucose with steam-exploded bagasse, open squares ^D -lactic acid with steam-exploded bagasse, filled triangles glucose with water-insoluble residue after steam explosion, and open triangles ^D -lactic acid with water-insoluble residue after steam explosion

• 1: = Produced glucose (mg) / 1 g of dry steam exploded bagasse 100
• 2: = Produced D-lactic acid (g/l) / Initial glucose concentration (g/l) 100 Fig. 5 Comparison of the amounts of ^D

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  Acknowledgments A part of this study was funded by the Shiseido Female Researcher Science Grant and a Grant-in-Aid for Young Scientists (B) (no. 21750159) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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