Bioresource Technology 187 (2015) 167–172
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Production of D-lactic acid from hardwood pulp by mechanical milling followed by simultaneous saccharification and fermentation using metabolically engineered Lactobacillus plantarum Shinji Hama a, Shino Mizuno a,1, Maki Kihara a, Tsutomu Tanaka b, Chiaki Ogino b, Hideo Noda a, Akihiko Kondo b,⇑ a b
Bio-energy Corporation, Research and Development Laboratory, 2-9-7 Minaminanamatsu, Amagasaki 660-0053, Japan Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan
h i g h l i g h t s Process for high-titer D-lactic acid production from cellulosic feedstocks. Simultaneous saccharification and fermentation is suggested to improve the process. To improve saccharification of high-load pulp, high-impact pulverization is applied. A combined process for increasing the lactic acid titer above 100 g/L.
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Article history: Received 3 February 2015 Received in revised form 22 March 2015 Accepted 23 March 2015 Available online 28 March 2015 Keywords: D-Lactic acid Kraft pulp Pretreatment Ball milling
a b s t r a c t This study focused on the process development for the D-lactic acid production from cellulosic feedstocks using the Lactobacillus plantarum mutant, genetically modified to produce optically pure D-lactic acid from both glucose and xylose. The simultaneous saccharification and fermentation (SSF) using delignified hardwood pulp (5–15% loads) resulted in the lactic acid titers of 55.2–84.6 g/L after 72 h and increased productivities of 1.77–2.61 g/L/h. To facilitate the enzymatic saccharification of high-load pulp at a fermentation temperature, short-term (610 min) pulverization of pulp was conducted, leading to a significantly improved saccharification with the suppressed formation of formic acid by-product. The short-term milling followed by SSF resulted in a lactic acid titer of 102.3 g/L, an optical purity of 99.2%, and a yield of 0.879 g/g-sugars without fed-batch process control. Therefore, the process presented here shows promise for the production of high-titer D-lactic acid using the L. plantarum mutant. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Lactic acid, an important chemical used in a wide range of industrial applications, has attracted considerable attention as a raw material for the synthesis of biopolymers (e.g., polylactic acid). A polymer blend of poly L-lactic acid and poly D-lactic acid leads to the stereocomplex formation, which exhibits a melting temperature higher than that of the respective single polymers (Tsuji, 2005). This improvement of polymeric characteristics is believed to offer a broad application of the currently available poly L-lactic acid. Therefore, the fermentative production of polymer-grade ⇑ Corresponding author. Tel./fax: +81 78 803 6196. E-mail address: [email protected] (A. Kondo). Present address: TechnoPro R&D Corporation, 5-5-2 Minatojima-minamimachi, Chuoku, Kobe 650-0047, Japan. 1
http://dx.doi.org/10.1016/j.biortech.2015.03.106 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
D-lactic
acid has become a considerably significant research area (Tashiro et al., 2011; Tsuge et al., 2014). Cellulosic feedstocks consisting of cellulose and hemicellulose are abundant renewable resources, thus providing a sustainable alternative to existing petrochemical refineries for the production of industrially important chemicals including lactic acid. Glucose is a constitutional unit of cellulosic polysaccharides and is available for microbial lactic acid fermentation. In most cases, xylose is the most abundant sugar in hemicellulose, although this monosaccharide is not readily available as a nutritional resource for microorganisms (Jeffries, 1983). For efficient lactic acid production from cellulosic feedstocks, considerable efforts have been directed towards the development or isolation of lactic-acid-producing microorganisms that can assimilate xylose as well as glucose (Abdel-Rahman et al., 2013; Patel et al., 2006; Wang et al., 2015).
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Okano et al. (2009) successfully demonstrated homo-D-lactic acid production by xylose fermentation with disruption of phosphoketolase genes (xpk1 and xpk2) and by introduction of the xylose-assimilating operon (xylAB) and transketolase gene (tkt) in an ldhL1-deficient Lactobacillus plantarum strain. Moreover, to enhance xylose assimilation, the L. plantarum DldhL1::PxylABDxpk1::tkt-Dxpk2::PxylAB mutant was developed, in which the integration of two copies of xylAB into the genome resulted in the production of 41.0 g/L lactic acid with a yield of 0.88 g/g xylose after a 42-h cultivation (Yoshida et al., 2011). These findings show promise for the high-yield conversion of sugars potentially present in cellulosic biomass. What remains unclarified is, however, the applicability of the L. plantarum mutant to the high-titer lactic acid production, generally regarded as above 100 g/L and important for decreasing the overall purification costs (Zhao et al., 2013). Therefore, this study focused on the process development for high-titer D-lactic acid production from cellulosic feedstocks using the L. plantarum mutant. Thus far, two fermentation processes using cellulosic feedstocks have been proposed: separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). SHF, in which the activities of cellulase and microorganisms are inhibited by a high glucose concentration, requires a long overall process time. In contrast, the fermentable sugars produced during SSF can be converted rapidly into target compounds, thereby minimizing end-product inhibition and decreasing process time (Alfani et al., 2000). However, there are also disadvantages of SSF over SHF; the optimum temperature in enzymatic saccharification is often higher than that in fermentation (Olofsson et al., 2008). Increasing enzymatic saccharification at a fermentation temperature is thus considered a technical barrier to realizing a high-titer lactic acid production from cellulosic feedstocks. To facilitate enzymatic saccharification of cellulosic feedstocks, mechanical milling has been proposed. This is one of the pretreatment methods that crucially affect overall process efficiency in terms of cellulose digestibility, fermentation toxicity, and waste treatment. Although mechanical milling has one remaining issue, namely, the need for a very high energy to obtain cellulose powder suitable for enzymatic saccharification, high-impact pulverization of cellulosic biomass has been investigated extensively, in which different grinding tools including ball mill (Karthik et al., 2012), cog-ring mill (Takahashi et al., 2014), and disk mill (Hideno et al., 2009) are used. Pulverization of cellulosic biomass by such tools produces fine particles with a mean diameter in the range of 20–50 lm and a reduced crystallinity of cellulose (Takahashi et al., 2014). In this study, hardwood pulp recovered from biomass fractionation was used as a cellulosic feedstock, as it is widely applied in the paper industry and contains considerable amounts of hemicellulose. This paper first presents the successful conversion of cellulose and hemicellulose into lactic acid via simultaneous saccharification and fermentation of pulp using the L. plantarum mutant. Moreover, to facilitate the enzymatic saccharification of high-load pulp at a fermentation temperature, shortterm pulverization was applied to the pretreatment of pulp, resulting in the high-titer D-lactic acid production without a fed-batch operation.
2. Methods 2.1. Materials L. plantarum NCIMB 8826 DldhL1::PxylAB-Dxpk1::tktDxpk2::PxylAB (Yoshida et al., 2011) was used for lactic acid fermentation. The strain was cultivated at 37 °C in 6% (v/v) fermented
barley extract (Sanwa Shurui, Co., Ltd., Oita, Japan; Furuta et al., 2009) supplemented with sugars. The highly delignified hardwood kraft pulp contained 82.7% (w/w) glucose and 10.4% (w/w) xylose, as determined by a procedure described previously (Hama et al., 2014). Prior to use, sheets of pulp were shredded into pieces of approximately 2 20 mm2 in size. Commercial cellulase, Cellic CTec2 (Novozymes, Bagsvaerd, Denmark) with a filter paper unit (FPU) of 117.7 per 1 ml (measured in our laboratory), was used for enzymatic saccharification. 2.2. Enzymatic saccharification and fermentation using shredded pulp SHF and SSF were applied to lactic acid fermentation using a 3-L bioreactor (Tokyo Rikakikai, Co., Ltd., Tokyo, Japan). For SHF, the shredded pulp was added to the reactor vessel to reach a pulp concentration of 5%, 10%, or 15% (w/v) in water, and the mixture was autoclaved at 121 °C for 15 min. Enzymatic saccharification was initiated by adding cellulase to the reaction mixture at a dosage of 20 FPU/g pulp. The reactor vessel at an agitation speed of 200 rpm was maintained at 50 °C for 72 h. After saccharification, the hydrolysates were centrifuged at 4720g for 1 min. The resulting supernatant was then transferred to the reactor vessel containing 60 ml of fermented barley extract, the mixture was adjusted to pH 5.5 with sodium hydroxide, and distilled water was added to reach a total volume of 950 ml. Subsequently, 50 ml of the inoculum (precultured in fermented barley extract supplemented with 0.5% xylose) was added into the reactor vessel, and incubated at 37 °C and 200 rpm for 72 h. The pH of the culture medium was maintained at 5.5 by the automatic addition of 20% (w/v) sodium hydroxide. For SSF, the mixture of 50 g of pulp (5% load) and 60 ml of fermented barley extract was added into the reactor vessel to reach a total volume of 1 L and then autoclaved at 121 °C for 15 min. Prior to inoculation, presaccharification was performed; cellulase at a dosage of 20 FPU/g pulp was added into the reactor vessel, which was maintained at 50 °C and 200 rpm for 3–6 h. To facilitate liquefaction of high-load pulp (10% and 15%, w/v), fedbatch operation was conducted; 50 g of pulp (autoclaved and dried at 70 °C prior to use) and 1000 FPU of cellulase were added at 3 h (10% load) and 6 h (15% load) after the start of presaccharification. Namely, the total presaccharification time was 3 h at both 5% and 10% loads or 6 h at 15% load. After maintaining the temperature of the mixture at approximately 37 °C, 50 ml of the inoculum was added into the reactor vessel and then anaerobically incubated at 37 °C for 72 h. The pH of the culture medium was maintained at 5.5 as described above. 2.3. Enzymatic saccharification and fermentation using pulverized pulp The pulverized pulp was prepared using Simoloyer CM01 (Zoz GmbH, Wenden, Germany), a horizontal high-energy attritor ball mill equipped with a double-walled stainless steel chamber and a stainless steel impeller (Karthik et al., 2012). The shredded pulp (approximately 20 g) was added into the mill chamber containing 400 ml of steel balls of 5 mm diameter. The rotational speed of the rotor that propels the mill balls was programmed as follows: mixing at 500 rpm for 30 s; 5–60 cycles of milling at 1000 rpm for 30 s followed by milling at 1500 rpm for 30 s. The milling was carried out for 5–60 min, and samples were collected at 5, 10, 30, and 60 min of milling. Enzymatic saccharification of the pulverized pulp was performed by adding cellulase (20 FPU/g pulp) into 50-ml polypropylene tubes (Corning Inc., NY, USA) containing 150 g/L pulp in water (pH 5.0), which was then incubated at 50 °C in a rotator (Thermo Block Rotator SN-06BN; Nissin, Tokyo, Japan). The culture medium with a total volume of 6 ml in a test tube contained a 2.0-ml (1.8-
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ml at 0 min pulverization) aliquot of the resulting sugar solutions, 0.6 ml of 10-fold concentrated MRS medium (100 g/L proteose peptone No. 3, 100 g/L beef extract, 50 g/L yeast extract, 10 g/L Tween 80, 20 g/L ammonium citrate, 50 g/L sodium acetate, 1 g/L MgSO4, 0.5 g/L MnSO4H2O, and 20 g/L K2HPO4), 0.15 g of calcium carbonate, and sterilized water. After inoculation, the tubes were incubated at 37 °C for 24 h. SSF of the pulverized pulp was performed anaerobically in a 200-ml glass vessel (flat-bottom separable flask, Sansyo Co., Ltd., Tokyo, Japan) containing 15 g of pulp (15% load) and 6 ml of fermented barley extract with a total volume of 100 ml. The glass vessel was autoclaved at 121 °C for 15 min and then maintained at 37 °C and a stirring rate of approximately 200 rpm, into which cellulase at a dosage of 20 FPU/g pulp and 5 ml of the inoculum (precultured in fermented barley extract supplemented with 0.4% glucose and 0.1% xylose) were added. The pH of the culture medium was maintained at 6.0 by the automatic addition of 20% (w/v) sodium hydroxide.
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Fig. 1. Time course of enzymatic saccharification of pulp. Shredded pulp at a concentration in the range of 5–15% was treated with cellulase at 50 °C. The concentrations of glucose (closed symbols) and xylose (open symbols) are shown.
2.4. Analytical methods Samples were obtained at specified intervals. Glucose and xylose concentrations were determined using a BF-5 biosensor (Oji Scientific Instruments, Hyogo, Japan) equipped with the corresponding enzyme electrodes. Lactic acid, acetic acid, and formic acid concentrations were determined by a high-performance liquid chromatography (HPLC) using a GL-C-610H-S column (300 mm 7.8 mm I.D.; Hitachi High-Technologies Co., Tokyo, Japan) maintained at 40 °C. The mobile phase of 3 mM perchloric acid was allowed to flow at a rate of 0.5 ml/min, and samples were applied using an injection volume of 10 ll. The absorbance of the products was measured at 440 nm using a UV–VIS detector by a bromothymol blue (BTB) postcolumn method in accordance with the supplier’s instructions (Hitachi High-Technologies Co.). The data and error bars in the figures show averages and standard deviations of triplicate experiments, respectively. The optical purity of lactic acid was determined by HPLC using an MCI GEL CRS15 W (50 mm 4.6 mm I.D.; Mitsubishi Chemical Co., Tokyo, Japan) column maintained at 35 °C. The mobile phase of 2 mM copper(II) sulfate was allowed to flow at a rate of 0.4 ml/min, and samples were applied using an injection volume of 10 ll. The absorbance of the separated D- or L-lactic acid was measured at 254 nm using a UV–VIS detector (Hitachi HighTechnologies Co.). Optical purity was defined as follows: optical purity (%) = 100 (D-lactic acid concentration L-lactic acid concentration)/(D-lactic acid concentration + L-lactic acid concentration). 3. Results and discussion 3.1. Lactic acid production from shredded pulp by SHF The production of lactic acid using the L. plantarum mutant and cellulosic feedstocks was investigated. To produce mixed sugars of glucose and xylose for fermentation, enzymatic saccharification was carried out at 50 °C and at various pulp loads of 5–15%. Fig. 1 shows the time courses of the concentrations of glucose and xylose released from the shredded pulp. The glucose concentration increased rapidly until 24 h, after which it increased slowly up to 40.6 g/L (5% load), 65.3 g/L (10% load), and 90.8 g/L (15% load) at 72 h. The increase in xylose concentration was also observed; the xylose concentrations at 72 h reached 8.6 g/L (5% load), 12.6 g/L (10% load), and 17.0 g/L (15% load), which are almost one-fifth of the glucose concentration at the respective load. Because the pulp used in this study contained 82.7% glucose and
10.4% xylose, the concentration ratios of glucose to xylose in the reaction mixtures were lower than those present in the pulp. This suggests that, in the enzymatic saccharification of shredded pulp, cellulose remains undigested to a greater extent than hemicellulose. The L. plantarum mutant was added to the sugar solution obtained at pulp loads of 5–15%. As can be seen in Fig. 2, the cells produced lactic acid in accordance with the consumption of both glucose and xylose. The initial concentrations of glucose and xylose at 5% load were 34.5 g/L and 6.36 g/L, respectively, from which the lactic acid concentration reached 41.0 g/L after 24 h. The use of the sugar solution at 10% load increased the lactic acid concentration to 63.0 g/L after 48 h and finally decreased the concentrations of glucose and xylose. At 15% load, however, considerable amounts of glucose and xylose remained even after 72 h, and the lactic acid concentration was comparable to that at 10% load. At all the pulp loads, the lactic acid productivities at 24 h were relatively low (1.67–1.83 g/L/h). Because the rates of bacterial growth and fermentation at a high sugar concentration decrease owing to the imbalance in osmotic pressure (John et al., 2009), SSF was suggested to improve the process in terms of the total residence time in the reactor and lactic acid concentration in the medium. 3.2. Lactic acid production from shredded pulp by SSF To avoid the inhibitory effect of high sugar concentration, SSF was applied to lactic acid production. Fig. 3 shows the time courses of SSF at 37 °C and pulp loads of 5–15%, in which the feedstocks were subjected to the presaccharification at 50 °C (see Section 2.2.). Although the mixing and handling of high-solid slurries are often problematic (Modenbach and Nokes, 2013), the presaccharification enabled the liquefaction of the insoluble pulp by degrading large fibers, so that inoculation and pH maintenance were achieved for lactic acid fermentation. The cells consumed sugars rapidly until 19 h and produced lactic acid at concentrations of 55.2 g/L (5% load), 81.6 g/L (10% load), and 84.6 g/L (15% load) after 72 h. The lactic acid productivities at 24 h also increased to 1.77–2.61 g/L/h. Under all the conditions examined, excessive accumulation of sugars was avoided, suggesting that the sugars released via enzymatic saccharification were rapidly converted to lactic acid during the course of SSF. However, the final lactic acid concentration at 15% load (84.6 g/L) was comparable to that at 10% load (81.6 g/L), indicating the low yield of available sugars in the medium at 15% pulp load. Given the significantly low
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concentrations of sugars in the fermentation broth, the hydrolysis of pulp appears to be a rate-limiting step for the lactic acid production at 15% pulp load. Although working enzymatic conversion of cellulosic feedstocks at high solid loads is effective in increasing the product concentration, the decrease in yield offsets the advantages of such a process. Of the several factors proposed to explain the decrease in yield, the inhibition of enzyme adsorption onto an insoluble substrate is one of the major factors determining the yield in high solids enzymatic hydrolysis of cellulose (Kristensen et al., 2009). Moreover, a previous study suggested the significant temperature dependence of the rate of cellulase adsorption/desorption during the enzymatic saccharification of cellulose (Hama et al., 2014). Increasing the enzymatic saccharification at a high solid load and a relatively low temperature is, therefore, necessary for the high-titer lactic acid production. 3.3. Effect of pulverization on enzymatic saccharification and fermentation of pulp
Fig. 2. Time course of lactic acid fermentation using pulp hydrolysates. Enzymatic hydrolysates of shredded pulp at a concentration in the range of 5–15% were subjected to lactic acid fermentation at 37 °C. The vertical axes show the concentrations of lactic acid (a) and sugars (b) in the fermentation broth. (b) The concentrations of glucose (closed symbols) and xylose (open symbols) are shown.
To facilitate enzymatic saccharification of high-load pulp by mechanical processing, the pulverized pulp was used as a feedstock. Because short-term pulverization was sufficient for improving the enzymatic saccharification [Supplementary data (Fig. S1)], the effect of pulverization time on lactic acid fermentation was also investigated. Fig. 4 shows the concentrations of sugars (Fig. 4a) and lactic acid (Fig. 4b) in the fermentation broth at 0 h and 24 h. There were significant differences in sugar consumption and lactic acid production between 10 min and 30 min of pulverization. Further investigations showed the significantly increased concentrations of formic acid in the sugar solutions. The concentrations of formic and acetic acids in the sugar solutions at 10 min of pulverization were 0.19 g/L and 0.07 g/L, respectively. At 30 min of pulverization, however, they reached 0.42 g/L and 0.10 g/L [Supplementary data (Fig. S2)]. The extended time of pulverization increased the formic acid concentration more significantly than the acetic acid concentration. Thus, the presence of formic acid, a strong inhibitor of microbial growth (Hasunuma and Kondo, 2012), is likely one of the major reasons for the fermentation inhibition observed in this study. In addition, formic acid is considered a degradation compound from the pulverized pulp, as shown by a recently proposed mechanism as follows. At elevated temperatures, glucose in cellulose can be thermally dehydrated to 5-(hydroxymethyl)-2-furaldehyde (HMF) and then degraded to formic acid or levulinic acid (Rasmussen et al., 2014). Although the pulverization was conducted in a water-jacketed chamber, the extended time might inevitably increase the surface temperature of the cellulose powder, resulting in the formation of the formic acid by-product. 3.4. High-titer D-lactic acid production from pulverized pulp by SSF
Fig. 3. Lactic acid production from shredded pulp by SSF. Shredded pulp at a concentration in the range of 5–15% was subjected to lactic acid fermentation at 37 °C. The vertical axis shows the concentrations of lactic acid (d, N, j), glucose (s, 4, h) and xylose (e, , +), whereas the horizontal axis shows the fermentation time after inoculation. Prior to inoculation, presaccharification and fed-batch operation were conducted (see Section 2.2).
On the basis of the above findings, SSF of the pulverized pulp (10 min) was performed. The short-term pulverization facilitated the liquefaction of pulp even at 15% load, thus requiring no presaccharification and fed-batch operation during SSF. As can be seen in Fig. 5, the lactic acid concentration increased rapidly with the maximum lactic acid productivity of 2.29 g/L/h, reaching the final lactic acid concentration of 102.3 g/L. Given the dilution of the fermentation broth by the neutralization with sodium hydroxide, the amount of lactic acid produced per liter of the initial culture medium was 122.8 g, which results from 139.7 g of sugars present in the feedstocks (i.e., a lactic acid yield of 0.879 g/g-sugars). The optical purity of the lactic acid produced was 99.2%, although the feedstocks contained a small amount of L-lactic acid originating from
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Fig. 4. Effect of pulp pulverization on lactic acid fermentation. (a) Sugar (glucose and xylose) consumption and (b) lactic acid production at 0 h and 24 h were investigated.
100 g/L and thus expand the practical utility of the L. plantarum mutant. 4. Conclusions The conversion of hardwood pulp into optically pure D-lactic acid has been demonstrated using the L. plantarum mutant. Although SSF is suggested to improve the process in terms of lactic acid productivity and sugar release, enzymatic saccharification of high-load pulp at a fermentation temperature needs to be further improved for the production of high-titer D-lactic acid. Therefore, to expand the practical utility of the L. plantarum mutant, the combined process employing short-term milling and SSF is useful for increasing the lactic acid titer above 100 g/L.
Fig. 5. Lactic acid production from pulverized pulp by SSF. Substrate loading: 15% pulp pulverized for 10 min. The vertical axis shows the concentrations of lactic acid (d), glucose (s), and xylose (e) in the fermentation broth.
the fermented barley extract (Furuta et al., 2009). In a previous study (Tsuge et al., 2014), the maximum lactic acid titer of 82.6 g/L was obtained using the L. plantarum mutant and mixed sugars of glucose and xylose (used as monosaccharides). In this study using cellulosic feedstocks, high sugar concentrations and low saccharification efficiency are suggested to underlie the difficulties in the further increase in lactic acid titer. Therefore, the combined process employing short-term milling and SSF can be a useful approach to increasing the lactic acid titer above
Acknowledgements This work was financially supported by the New Energy and Industrial Technology Development Organization (NEDO), Japan. We also thank Professor Tadahisa Iwata (The University of Tokyo, Japan) for providing hardwood pulp samples. Simoloyer CM01 was installed by Grants-in-Aid from the Network of Centers of Carbon Dioxide Resource Studies in Plants: NC-CARP project and the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. This collaborative study was supported by Special Coordination Funds for Promoting Science and Technology, Creation of Innovative Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe) from MEXT, Japan.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Production of D-lactic acid from hardwood pulp by mechanical milling followed by simultaneous saccharification and fermentation using metabolically engineered Lactobacillus plantarum Shinji Hama a, Shino Mizuno a,1, Maki Kihara a, Tsutomu Tanaka b, Chiaki Ogino b, Hideo Noda a, Akihiko Kondo b,⇑ a b
Bio-energy Corporation, Research and Development Laboratory, 2-9-7 Minaminanamatsu, Amagasaki 660-0053, Japan Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan
h i g h l i g h t s Process for high-titer D-lactic acid production from cellulosic feedstocks. Simultaneous saccharification and fermentation is suggested to improve the process. To improve saccharification of high-load pulp, high-impact pulverization is applied. A combined process for increasing the lactic acid titer above 100 g/L.
a r t i c l e
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Article history: Received 3 February 2015 Received in revised form 22 March 2015 Accepted 23 March 2015 Available online 28 March 2015 Keywords: D-Lactic acid Kraft pulp Pretreatment Ball milling
a b s t r a c t This study focused on the process development for the D-lactic acid production from cellulosic feedstocks using the Lactobacillus plantarum mutant, genetically modified to produce optically pure D-lactic acid from both glucose and xylose. The simultaneous saccharification and fermentation (SSF) using delignified hardwood pulp (5–15% loads) resulted in the lactic acid titers of 55.2–84.6 g/L after 72 h and increased productivities of 1.77–2.61 g/L/h. To facilitate the enzymatic saccharification of high-load pulp at a fermentation temperature, short-term (610 min) pulverization of pulp was conducted, leading to a significantly improved saccharification with the suppressed formation of formic acid by-product. The short-term milling followed by SSF resulted in a lactic acid titer of 102.3 g/L, an optical purity of 99.2%, and a yield of 0.879 g/g-sugars without fed-batch process control. Therefore, the process presented here shows promise for the production of high-titer D-lactic acid using the L. plantarum mutant. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Lactic acid, an important chemical used in a wide range of industrial applications, has attracted considerable attention as a raw material for the synthesis of biopolymers (e.g., polylactic acid). A polymer blend of poly L-lactic acid and poly D-lactic acid leads to the stereocomplex formation, which exhibits a melting temperature higher than that of the respective single polymers (Tsuji, 2005). This improvement of polymeric characteristics is believed to offer a broad application of the currently available poly L-lactic acid. Therefore, the fermentative production of polymer-grade ⇑ Corresponding author. Tel./fax: +81 78 803 6196. E-mail address: [email protected] (A. Kondo). Present address: TechnoPro R&D Corporation, 5-5-2 Minatojima-minamimachi, Chuoku, Kobe 650-0047, Japan. 1
http://dx.doi.org/10.1016/j.biortech.2015.03.106 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
D-lactic
acid has become a considerably significant research area (Tashiro et al., 2011; Tsuge et al., 2014). Cellulosic feedstocks consisting of cellulose and hemicellulose are abundant renewable resources, thus providing a sustainable alternative to existing petrochemical refineries for the production of industrially important chemicals including lactic acid. Glucose is a constitutional unit of cellulosic polysaccharides and is available for microbial lactic acid fermentation. In most cases, xylose is the most abundant sugar in hemicellulose, although this monosaccharide is not readily available as a nutritional resource for microorganisms (Jeffries, 1983). For efficient lactic acid production from cellulosic feedstocks, considerable efforts have been directed towards the development or isolation of lactic-acid-producing microorganisms that can assimilate xylose as well as glucose (Abdel-Rahman et al., 2013; Patel et al., 2006; Wang et al., 2015).
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Okano et al. (2009) successfully demonstrated homo-D-lactic acid production by xylose fermentation with disruption of phosphoketolase genes (xpk1 and xpk2) and by introduction of the xylose-assimilating operon (xylAB) and transketolase gene (tkt) in an ldhL1-deficient Lactobacillus plantarum strain. Moreover, to enhance xylose assimilation, the L. plantarum DldhL1::PxylABDxpk1::tkt-Dxpk2::PxylAB mutant was developed, in which the integration of two copies of xylAB into the genome resulted in the production of 41.0 g/L lactic acid with a yield of 0.88 g/g xylose after a 42-h cultivation (Yoshida et al., 2011). These findings show promise for the high-yield conversion of sugars potentially present in cellulosic biomass. What remains unclarified is, however, the applicability of the L. plantarum mutant to the high-titer lactic acid production, generally regarded as above 100 g/L and important for decreasing the overall purification costs (Zhao et al., 2013). Therefore, this study focused on the process development for high-titer D-lactic acid production from cellulosic feedstocks using the L. plantarum mutant. Thus far, two fermentation processes using cellulosic feedstocks have been proposed: separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). SHF, in which the activities of cellulase and microorganisms are inhibited by a high glucose concentration, requires a long overall process time. In contrast, the fermentable sugars produced during SSF can be converted rapidly into target compounds, thereby minimizing end-product inhibition and decreasing process time (Alfani et al., 2000). However, there are also disadvantages of SSF over SHF; the optimum temperature in enzymatic saccharification is often higher than that in fermentation (Olofsson et al., 2008). Increasing enzymatic saccharification at a fermentation temperature is thus considered a technical barrier to realizing a high-titer lactic acid production from cellulosic feedstocks. To facilitate enzymatic saccharification of cellulosic feedstocks, mechanical milling has been proposed. This is one of the pretreatment methods that crucially affect overall process efficiency in terms of cellulose digestibility, fermentation toxicity, and waste treatment. Although mechanical milling has one remaining issue, namely, the need for a very high energy to obtain cellulose powder suitable for enzymatic saccharification, high-impact pulverization of cellulosic biomass has been investigated extensively, in which different grinding tools including ball mill (Karthik et al., 2012), cog-ring mill (Takahashi et al., 2014), and disk mill (Hideno et al., 2009) are used. Pulverization of cellulosic biomass by such tools produces fine particles with a mean diameter in the range of 20–50 lm and a reduced crystallinity of cellulose (Takahashi et al., 2014). In this study, hardwood pulp recovered from biomass fractionation was used as a cellulosic feedstock, as it is widely applied in the paper industry and contains considerable amounts of hemicellulose. This paper first presents the successful conversion of cellulose and hemicellulose into lactic acid via simultaneous saccharification and fermentation of pulp using the L. plantarum mutant. Moreover, to facilitate the enzymatic saccharification of high-load pulp at a fermentation temperature, shortterm pulverization was applied to the pretreatment of pulp, resulting in the high-titer D-lactic acid production without a fed-batch operation.
2. Methods 2.1. Materials L. plantarum NCIMB 8826 DldhL1::PxylAB-Dxpk1::tktDxpk2::PxylAB (Yoshida et al., 2011) was used for lactic acid fermentation. The strain was cultivated at 37 °C in 6% (v/v) fermented
barley extract (Sanwa Shurui, Co., Ltd., Oita, Japan; Furuta et al., 2009) supplemented with sugars. The highly delignified hardwood kraft pulp contained 82.7% (w/w) glucose and 10.4% (w/w) xylose, as determined by a procedure described previously (Hama et al., 2014). Prior to use, sheets of pulp were shredded into pieces of approximately 2 20 mm2 in size. Commercial cellulase, Cellic CTec2 (Novozymes, Bagsvaerd, Denmark) with a filter paper unit (FPU) of 117.7 per 1 ml (measured in our laboratory), was used for enzymatic saccharification. 2.2. Enzymatic saccharification and fermentation using shredded pulp SHF and SSF were applied to lactic acid fermentation using a 3-L bioreactor (Tokyo Rikakikai, Co., Ltd., Tokyo, Japan). For SHF, the shredded pulp was added to the reactor vessel to reach a pulp concentration of 5%, 10%, or 15% (w/v) in water, and the mixture was autoclaved at 121 °C for 15 min. Enzymatic saccharification was initiated by adding cellulase to the reaction mixture at a dosage of 20 FPU/g pulp. The reactor vessel at an agitation speed of 200 rpm was maintained at 50 °C for 72 h. After saccharification, the hydrolysates were centrifuged at 4720g for 1 min. The resulting supernatant was then transferred to the reactor vessel containing 60 ml of fermented barley extract, the mixture was adjusted to pH 5.5 with sodium hydroxide, and distilled water was added to reach a total volume of 950 ml. Subsequently, 50 ml of the inoculum (precultured in fermented barley extract supplemented with 0.5% xylose) was added into the reactor vessel, and incubated at 37 °C and 200 rpm for 72 h. The pH of the culture medium was maintained at 5.5 by the automatic addition of 20% (w/v) sodium hydroxide. For SSF, the mixture of 50 g of pulp (5% load) and 60 ml of fermented barley extract was added into the reactor vessel to reach a total volume of 1 L and then autoclaved at 121 °C for 15 min. Prior to inoculation, presaccharification was performed; cellulase at a dosage of 20 FPU/g pulp was added into the reactor vessel, which was maintained at 50 °C and 200 rpm for 3–6 h. To facilitate liquefaction of high-load pulp (10% and 15%, w/v), fedbatch operation was conducted; 50 g of pulp (autoclaved and dried at 70 °C prior to use) and 1000 FPU of cellulase were added at 3 h (10% load) and 6 h (15% load) after the start of presaccharification. Namely, the total presaccharification time was 3 h at both 5% and 10% loads or 6 h at 15% load. After maintaining the temperature of the mixture at approximately 37 °C, 50 ml of the inoculum was added into the reactor vessel and then anaerobically incubated at 37 °C for 72 h. The pH of the culture medium was maintained at 5.5 as described above. 2.3. Enzymatic saccharification and fermentation using pulverized pulp The pulverized pulp was prepared using Simoloyer CM01 (Zoz GmbH, Wenden, Germany), a horizontal high-energy attritor ball mill equipped with a double-walled stainless steel chamber and a stainless steel impeller (Karthik et al., 2012). The shredded pulp (approximately 20 g) was added into the mill chamber containing 400 ml of steel balls of 5 mm diameter. The rotational speed of the rotor that propels the mill balls was programmed as follows: mixing at 500 rpm for 30 s; 5–60 cycles of milling at 1000 rpm for 30 s followed by milling at 1500 rpm for 30 s. The milling was carried out for 5–60 min, and samples were collected at 5, 10, 30, and 60 min of milling. Enzymatic saccharification of the pulverized pulp was performed by adding cellulase (20 FPU/g pulp) into 50-ml polypropylene tubes (Corning Inc., NY, USA) containing 150 g/L pulp in water (pH 5.0), which was then incubated at 50 °C in a rotator (Thermo Block Rotator SN-06BN; Nissin, Tokyo, Japan). The culture medium with a total volume of 6 ml in a test tube contained a 2.0-ml (1.8-
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ml at 0 min pulverization) aliquot of the resulting sugar solutions, 0.6 ml of 10-fold concentrated MRS medium (100 g/L proteose peptone No. 3, 100 g/L beef extract, 50 g/L yeast extract, 10 g/L Tween 80, 20 g/L ammonium citrate, 50 g/L sodium acetate, 1 g/L MgSO4, 0.5 g/L MnSO4H2O, and 20 g/L K2HPO4), 0.15 g of calcium carbonate, and sterilized water. After inoculation, the tubes were incubated at 37 °C for 24 h. SSF of the pulverized pulp was performed anaerobically in a 200-ml glass vessel (flat-bottom separable flask, Sansyo Co., Ltd., Tokyo, Japan) containing 15 g of pulp (15% load) and 6 ml of fermented barley extract with a total volume of 100 ml. The glass vessel was autoclaved at 121 °C for 15 min and then maintained at 37 °C and a stirring rate of approximately 200 rpm, into which cellulase at a dosage of 20 FPU/g pulp and 5 ml of the inoculum (precultured in fermented barley extract supplemented with 0.4% glucose and 0.1% xylose) were added. The pH of the culture medium was maintained at 6.0 by the automatic addition of 20% (w/v) sodium hydroxide.
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Fig. 1. Time course of enzymatic saccharification of pulp. Shredded pulp at a concentration in the range of 5–15% was treated with cellulase at 50 °C. The concentrations of glucose (closed symbols) and xylose (open symbols) are shown.
2.4. Analytical methods Samples were obtained at specified intervals. Glucose and xylose concentrations were determined using a BF-5 biosensor (Oji Scientific Instruments, Hyogo, Japan) equipped with the corresponding enzyme electrodes. Lactic acid, acetic acid, and formic acid concentrations were determined by a high-performance liquid chromatography (HPLC) using a GL-C-610H-S column (300 mm 7.8 mm I.D.; Hitachi High-Technologies Co., Tokyo, Japan) maintained at 40 °C. The mobile phase of 3 mM perchloric acid was allowed to flow at a rate of 0.5 ml/min, and samples were applied using an injection volume of 10 ll. The absorbance of the products was measured at 440 nm using a UV–VIS detector by a bromothymol blue (BTB) postcolumn method in accordance with the supplier’s instructions (Hitachi High-Technologies Co.). The data and error bars in the figures show averages and standard deviations of triplicate experiments, respectively. The optical purity of lactic acid was determined by HPLC using an MCI GEL CRS15 W (50 mm 4.6 mm I.D.; Mitsubishi Chemical Co., Tokyo, Japan) column maintained at 35 °C. The mobile phase of 2 mM copper(II) sulfate was allowed to flow at a rate of 0.4 ml/min, and samples were applied using an injection volume of 10 ll. The absorbance of the separated D- or L-lactic acid was measured at 254 nm using a UV–VIS detector (Hitachi HighTechnologies Co.). Optical purity was defined as follows: optical purity (%) = 100 (D-lactic acid concentration L-lactic acid concentration)/(D-lactic acid concentration + L-lactic acid concentration). 3. Results and discussion 3.1. Lactic acid production from shredded pulp by SHF The production of lactic acid using the L. plantarum mutant and cellulosic feedstocks was investigated. To produce mixed sugars of glucose and xylose for fermentation, enzymatic saccharification was carried out at 50 °C and at various pulp loads of 5–15%. Fig. 1 shows the time courses of the concentrations of glucose and xylose released from the shredded pulp. The glucose concentration increased rapidly until 24 h, after which it increased slowly up to 40.6 g/L (5% load), 65.3 g/L (10% load), and 90.8 g/L (15% load) at 72 h. The increase in xylose concentration was also observed; the xylose concentrations at 72 h reached 8.6 g/L (5% load), 12.6 g/L (10% load), and 17.0 g/L (15% load), which are almost one-fifth of the glucose concentration at the respective load. Because the pulp used in this study contained 82.7% glucose and
10.4% xylose, the concentration ratios of glucose to xylose in the reaction mixtures were lower than those present in the pulp. This suggests that, in the enzymatic saccharification of shredded pulp, cellulose remains undigested to a greater extent than hemicellulose. The L. plantarum mutant was added to the sugar solution obtained at pulp loads of 5–15%. As can be seen in Fig. 2, the cells produced lactic acid in accordance with the consumption of both glucose and xylose. The initial concentrations of glucose and xylose at 5% load were 34.5 g/L and 6.36 g/L, respectively, from which the lactic acid concentration reached 41.0 g/L after 24 h. The use of the sugar solution at 10% load increased the lactic acid concentration to 63.0 g/L after 48 h and finally decreased the concentrations of glucose and xylose. At 15% load, however, considerable amounts of glucose and xylose remained even after 72 h, and the lactic acid concentration was comparable to that at 10% load. At all the pulp loads, the lactic acid productivities at 24 h were relatively low (1.67–1.83 g/L/h). Because the rates of bacterial growth and fermentation at a high sugar concentration decrease owing to the imbalance in osmotic pressure (John et al., 2009), SSF was suggested to improve the process in terms of the total residence time in the reactor and lactic acid concentration in the medium. 3.2. Lactic acid production from shredded pulp by SSF To avoid the inhibitory effect of high sugar concentration, SSF was applied to lactic acid production. Fig. 3 shows the time courses of SSF at 37 °C and pulp loads of 5–15%, in which the feedstocks were subjected to the presaccharification at 50 °C (see Section 2.2.). Although the mixing and handling of high-solid slurries are often problematic (Modenbach and Nokes, 2013), the presaccharification enabled the liquefaction of the insoluble pulp by degrading large fibers, so that inoculation and pH maintenance were achieved for lactic acid fermentation. The cells consumed sugars rapidly until 19 h and produced lactic acid at concentrations of 55.2 g/L (5% load), 81.6 g/L (10% load), and 84.6 g/L (15% load) after 72 h. The lactic acid productivities at 24 h also increased to 1.77–2.61 g/L/h. Under all the conditions examined, excessive accumulation of sugars was avoided, suggesting that the sugars released via enzymatic saccharification were rapidly converted to lactic acid during the course of SSF. However, the final lactic acid concentration at 15% load (84.6 g/L) was comparable to that at 10% load (81.6 g/L), indicating the low yield of available sugars in the medium at 15% pulp load. Given the significantly low
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concentrations of sugars in the fermentation broth, the hydrolysis of pulp appears to be a rate-limiting step for the lactic acid production at 15% pulp load. Although working enzymatic conversion of cellulosic feedstocks at high solid loads is effective in increasing the product concentration, the decrease in yield offsets the advantages of such a process. Of the several factors proposed to explain the decrease in yield, the inhibition of enzyme adsorption onto an insoluble substrate is one of the major factors determining the yield in high solids enzymatic hydrolysis of cellulose (Kristensen et al., 2009). Moreover, a previous study suggested the significant temperature dependence of the rate of cellulase adsorption/desorption during the enzymatic saccharification of cellulose (Hama et al., 2014). Increasing the enzymatic saccharification at a high solid load and a relatively low temperature is, therefore, necessary for the high-titer lactic acid production. 3.3. Effect of pulverization on enzymatic saccharification and fermentation of pulp
Fig. 2. Time course of lactic acid fermentation using pulp hydrolysates. Enzymatic hydrolysates of shredded pulp at a concentration in the range of 5–15% were subjected to lactic acid fermentation at 37 °C. The vertical axes show the concentrations of lactic acid (a) and sugars (b) in the fermentation broth. (b) The concentrations of glucose (closed symbols) and xylose (open symbols) are shown.
To facilitate enzymatic saccharification of high-load pulp by mechanical processing, the pulverized pulp was used as a feedstock. Because short-term pulverization was sufficient for improving the enzymatic saccharification [Supplementary data (Fig. S1)], the effect of pulverization time on lactic acid fermentation was also investigated. Fig. 4 shows the concentrations of sugars (Fig. 4a) and lactic acid (Fig. 4b) in the fermentation broth at 0 h and 24 h. There were significant differences in sugar consumption and lactic acid production between 10 min and 30 min of pulverization. Further investigations showed the significantly increased concentrations of formic acid in the sugar solutions. The concentrations of formic and acetic acids in the sugar solutions at 10 min of pulverization were 0.19 g/L and 0.07 g/L, respectively. At 30 min of pulverization, however, they reached 0.42 g/L and 0.10 g/L [Supplementary data (Fig. S2)]. The extended time of pulverization increased the formic acid concentration more significantly than the acetic acid concentration. Thus, the presence of formic acid, a strong inhibitor of microbial growth (Hasunuma and Kondo, 2012), is likely one of the major reasons for the fermentation inhibition observed in this study. In addition, formic acid is considered a degradation compound from the pulverized pulp, as shown by a recently proposed mechanism as follows. At elevated temperatures, glucose in cellulose can be thermally dehydrated to 5-(hydroxymethyl)-2-furaldehyde (HMF) and then degraded to formic acid or levulinic acid (Rasmussen et al., 2014). Although the pulverization was conducted in a water-jacketed chamber, the extended time might inevitably increase the surface temperature of the cellulose powder, resulting in the formation of the formic acid by-product. 3.4. High-titer D-lactic acid production from pulverized pulp by SSF
Fig. 3. Lactic acid production from shredded pulp by SSF. Shredded pulp at a concentration in the range of 5–15% was subjected to lactic acid fermentation at 37 °C. The vertical axis shows the concentrations of lactic acid (d, N, j), glucose (s, 4, h) and xylose (e, , +), whereas the horizontal axis shows the fermentation time after inoculation. Prior to inoculation, presaccharification and fed-batch operation were conducted (see Section 2.2).
On the basis of the above findings, SSF of the pulverized pulp (10 min) was performed. The short-term pulverization facilitated the liquefaction of pulp even at 15% load, thus requiring no presaccharification and fed-batch operation during SSF. As can be seen in Fig. 5, the lactic acid concentration increased rapidly with the maximum lactic acid productivity of 2.29 g/L/h, reaching the final lactic acid concentration of 102.3 g/L. Given the dilution of the fermentation broth by the neutralization with sodium hydroxide, the amount of lactic acid produced per liter of the initial culture medium was 122.8 g, which results from 139.7 g of sugars present in the feedstocks (i.e., a lactic acid yield of 0.879 g/g-sugars). The optical purity of the lactic acid produced was 99.2%, although the feedstocks contained a small amount of L-lactic acid originating from
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Fig. 4. Effect of pulp pulverization on lactic acid fermentation. (a) Sugar (glucose and xylose) consumption and (b) lactic acid production at 0 h and 24 h were investigated.
100 g/L and thus expand the practical utility of the L. plantarum mutant. 4. Conclusions The conversion of hardwood pulp into optically pure D-lactic acid has been demonstrated using the L. plantarum mutant. Although SSF is suggested to improve the process in terms of lactic acid productivity and sugar release, enzymatic saccharification of high-load pulp at a fermentation temperature needs to be further improved for the production of high-titer D-lactic acid. Therefore, to expand the practical utility of the L. plantarum mutant, the combined process employing short-term milling and SSF is useful for increasing the lactic acid titer above 100 g/L.
Fig. 5. Lactic acid production from pulverized pulp by SSF. Substrate loading: 15% pulp pulverized for 10 min. The vertical axis shows the concentrations of lactic acid (d), glucose (s), and xylose (e) in the fermentation broth.
the fermented barley extract (Furuta et al., 2009). In a previous study (Tsuge et al., 2014), the maximum lactic acid titer of 82.6 g/L was obtained using the L. plantarum mutant and mixed sugars of glucose and xylose (used as monosaccharides). In this study using cellulosic feedstocks, high sugar concentrations and low saccharification efficiency are suggested to underlie the difficulties in the further increase in lactic acid titer. Therefore, the combined process employing short-term milling and SSF can be a useful approach to increasing the lactic acid titer above
Acknowledgements This work was financially supported by the New Energy and Industrial Technology Development Organization (NEDO), Japan. We also thank Professor Tadahisa Iwata (The University of Tokyo, Japan) for providing hardwood pulp samples. Simoloyer CM01 was installed by Grants-in-Aid from the Network of Centers of Carbon Dioxide Resource Studies in Plants: NC-CARP project and the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. This collaborative study was supported by Special Coordination Funds for Promoting Science and Technology, Creation of Innovative Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe) from MEXT, Japan.
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