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Improving lactic acid productivity from wheat straw hydrolysates by membrane integrated repeated batch fermentation under non-sterilized conditions
Bioresource Technology 163 (2014) 160–166
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Improving lactic acid productivity from wheat straw hydrolysates by membrane integrated repeated batch fermentation under non-sterilized conditions Yuming Zhang a,b,c, Xiangrong Chen a, Benkun Qi a, Jianquan Luo d, Fei Shen a, Yi Su a, Rashid Khan a, Yinhua Wan a,⇑ a
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China University of Chinese Academy of Sciences, Beijing 100049, China c College of Life Sciences, Hebei University, Baoding 071002, China d Department of Chemical and Biochemical Engineering, Center for Bioprocess Engineering, Building 229, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark b
h i g h l i g h t s LA was produced from wheat straw hydrolysates under non-sterilized conditions. Membrane integrated repeated batch fermentation (MIRB) could increase LA productivity. With MIRB, the simultaneous fermentation of hexose and pentose sugars was realized. LA productivity of 2.35 g/L/h was obtained from wheat straw hydrolysates by MIRB.
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Article history: Received 20 February 2014 Received in revised form 9 April 2014 Accepted 10 April 2014 Available online 19 April 2014 Keywords: Lactic acid Membrane Repeated batch Cell recycle Wheat straw hydrolysates
a b s t r a c t Bacillus coagulans IPE22 was used to produce lactic acid (LA) from mixed sugar and wheat straw hydrolysates, respectively. All fermentations were conducted under non-sterilized conditions and sodium hydroxide was used as neutralizing agent to avoid the production of insoluble CaSO4. In order to eliminate the sequential utilization of mixed sugar and feedback inhibition during batch fermentation, membrane integrated repeated batch fermentation (MIRB) was used to improve LA productivity. With MIRB, a high cell density was obtained and the simultaneous fermentation of glucose, xylose and arabinose was successfully realized. The separation of LA from broth by membrane in batch fermentation also decreased feedback inhibition. MIRB was carried out using wheat straw hydrolysates (29.72 g/L glucose, 24.69 g/L xylose and 5.14 g/L arabinose) as carbon source, LA productivity was increased significantly from 1.01 g/L/h (batch 1) to 2.35 g/L/h (batch 6) by the repeated batch fermentation. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Lactic acid (LA) is an important commodity chemical for various applications in food, pharmaceutical and the cosmetic industry (Datta and Henry, 2006). It can also serve as a precursor for producing poly lactic acid, which is a promising biodegradable, biocompatible and environment-friendly alternative to plastics derived from petrochemicals (Lasprilla et al., 2012). LA can be produced in two ways: chemical synthesis and fermentation. The fermentation process is attractive because of its advantages of using renewable carbohydrates and producing optically pure LA ⇑ Corresponding author. Tel./fax: +86 10 62650673. E-mail address: [email protected] (Y. Wan). http://dx.doi.org/10.1016/j.biortech.2014.04.038 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
(John et al., 2009). Abundant and renewable lignocellulosic materials are regarded as suitable feedstocks for LA production (Abdel-Rahman et al., 2013a). Lignocellulose is composed of cellulose, hemicellulose and lignin, which can be hydrolyzed to a mixture of hexose and pentose sugars. Therefore, a strain capable of fermenting all the lignocellulose released sugars is essential for the economical production of LA (Kim et al., 2010a). Production of LA using lignocellulosic hydrolysates by Bacillus coagulans has drawn much attention due to its strong ability to ferment both hexose and pentose sugars (Ou et al., 2011; Ye et al., 2013a; Zhou et al., 2013). Moreover, the fermentation could be operated under non-sterilized conditions by virtue of the strain’s thermophilic feature (Ouyang et al., 2012b; Ye et al., 2013b; Zhao et al., 2010; Zhou et al., 2013). Non-sterilized fermentation is
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energy-efficient, cost-effective and labor-saving. Despite having these advantages, production of LA by B. coagulans still has several challenges remaining to be resolved. As reported by Walton et al. (2010) and Ou et al. (2011), some B. coagulans strains could not simultaneously ferment hexose and pentose, and these mixed sugar could only be sequentially metabolized due to carbon catabolite repression (CCR) (Kim et al., 2010a), which would decrease the efficiency of fermentation. In addition, feedback inhibition during the fermentation could limit the LA production (Ou et al., 2011). To solve these problems, method of genetic modification is undoubtedly the primary strategy to be considered, while optimization of fermentation strategy is also an alternative. It is well known that the repeated batch fermentation is a feasible method for improving LA productivity due to the reduction in fermentation time and the skip of inoculums preparation (Abdel-Rahman et al., 2013b; John et al., 2007). During the repeated batch fermentation, cells could be reused by centrifugation (Abdel-Rahman et al., 2013b; Kim et al., 2010b; Zhao et al., 2010) or cell immobilization (Rosenberg et al., 2005; Shi et al., 2012). Usually, cell reuse by centrifugation could not realize automatically continuous operation. And the immobilized cell bioreactor may be suffered from productivity loss due to the limited mass transfer and the accumulation of dead cells (Shi et al., 2012). Membrane techniques have many advantages in separation of cells from fermentation broth, such as energy-efficient, low damage to cells and easy to scale up for industrial production (Pal et al., 2009; Zhao et al., 2010). The strategy of integrating membrane module with fermentor, termed as membrane integrated repeated batch fermentation (MIRB), could efficiently achieve cell-recycled repeated batch. Thus, a lot of efforts have been made to improve the productivity of LA by MIRB (Kim et al., 2006; Oh et al., 2003; Wee et al., 2006) and most of the studies concerned the MIRB using single sugar as carbon source. To the best of our knowledge, there has been no report regarding LA production from sugar mixture of hexose and pentose using MIRB, especially from lignocellulosic hydrolysates. In conventional LA production by fermentation, calcium hydroxide or calcium carbonate is normally used to neutralize the produced LA, thus resulting in the production of calcium-LA. The salt of calcium lactate has to be acidified with H2SO4, thus resulting in the production of insoluble CaSO4 in LA extraction and purification steps. Gypsum poses serious environmental problem in waste treatment during large-scale LA production. Fermentation using sodium hydroxide as neutralizing agent could avoid the above problem (Qin et al., 2010). Another advantage of fermentation without calcium ion is that the potential of membrane fouling can be significantly decreased, and thus making the MIRB technology more practical in industrial LA production (Pal et al., 2009). Recently, a thermophilic LA producing bacterium, B. coagulans IPE22, was isolated and characterized in our lab, and this strain showed remarkable capability to ferment pentose, hexose and cellobiose and was highly resistant to inhibitors from lignocellulosic hydrolysates (Zhang et al., 2014). The objective of the present work is to evaluate the performance of MIRB in LA production from wheat straw hydrolysates by B. coagulans IPE22, aiming to eliminate the sequential utilization of mixed sugar and feedback inhibition for efficient LA production. All the fermentations were operated under non-sterilized conditions and sodium hydroxide was used as neutralizing agent to avoid the production of insoluble CaSO4. Firstly, the effect of initial concentration of sugar mixture on the production of LA was investigated. Then, MIRB was employed to produce LA from both mixed sugar and wheat straw hydrolysates. Fermentative parameters in terms of sugar consumption, cell mass accumulation and viable cells were monitored and the mechanisms were discussed.
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2. Methods 2.1. Microorganisms and medium B. coagulans IPE22 was used in this study. A modified De Man-Rogosa-Sharpe (mMRS) medium was used for seed culture and fermentation. Medium of mMRS contained 10 g/L peptone, 10 g/L beef extract, 5 g/L yeast extract, 2 g/L dipotassium phosphate, 0.2 g/L magnesium sulfate heptahydrate, 0.05 g/L manganese sulfate tetrahydrate and different carbon sources. The type and concentration of carbon sources varied in different fermentation experiments. 2.2. Preparation of wheat straw hydrolysates Wheat straw was cleaned, chopped, and then pretreated by 2% (w/v) sulfuric acid at a 10% (w/v) loading. The mixture was treated at 121 °C for 90 min, and the obtained slurry was filtrated to achieve liquid and solid fractions. Both liquid and solid fractions were collected and defined as dilute acid hydrolysates and water insoluble solids (WIS), respectively. The WIS (containing 59.96% cellulose) was hydrolyzed by commercial cellulase (Sunson Group Ningxia Enzyme Preparation Plant, China) at a 10% (w/v) solid loading. Enzyme loading was 20 FPU (filter paper activity units)/g cellulose. The enzymatic hydrolysis was carried out in a 10 L jar fermentor (GUJS-10, Zhenjiang Dongfang Bioengineering Equipment Company, China) at 50 °C, pH 5.0 and 200 rpm. Enzymatic hydrolysates were obtained by filtration with filter paper (No. 43, Whatman, UK). Finally, dilute acid hydrolysates and enzymatic hydrolysates were mixed and concentrated by vacuum evaporation at 45 °C. The wheat straw hydrolysates after concentration normally contained 59.55 g/L mixed sugar (29.72 g/L glucose, 24.69 g/L xylose and 5.14 g/L arabinose). 2.3. Batch fermentations To prepare seed culture, the strain of B. coagulans IPE22 was grown on agar mMRS plate for 2 days at 45 °C. The cells were transferred to liquid mMRS media containing 10 g/L glucose and cultured for 6 h at 50 °C. This culture was used to provide 5% (v/ v) inocula for fermentation. The optimal fermentation condition for B. coagulans IPE22 to produce LA was 52 °C and pH 6.0, as described by Zhang et al. (2014). So, batch fermentations were carried out in fermentor at 52 °C, pH 6.0 and 100 rpm. Sodium hydroxide solution of 400 g/L was automatically added to maintain the pH value by a peristaltic pump. Samples were taken with specific time intervals to determine cell mass, residual sugars and products. 2.4. Membrane integrated repeated batch fermentation (MIRB) Repeated batch fermentation was performed in a membrane integrated bioreactor, the schematic diagram of which was shown in Fig. 1. The fermentor was coupled with a membrane module. Fermentation broth was transferred to the membrane module by a diaphragm pump. A polyacrylonitrile (PAN) ultrafiltration membrane with a nominal molecular weight cut-off of 20 kDa was used in the experiments. The effective area of the used ultrafiltration membrane was 0.18 m2. Fermentation was carried out in the fermentor with a working volume of 8 L, and the culture condition was same with that of batch fermentation (Section 2.3). When the sugars in the culture broth were depleted, the integrated membrane module was started to filter fermentation broth. Eighty percent (v/v) of the culture broth (6.4 L) was removed as the permeate liquid. Then 1.6 L broth with concentrated cells was obtained, and
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Fig. 1. Schematic diagram of fermentor coupled with a membrane module.
0.8 L of the obtained broth was used as seed culture to generate the next batch fermentation with 7.2 L fresh medium. So the volume of the second batch fermentation was also 8 L, which was equal to the initial fermentation. Subsequent batch fermentations were performed in the same manner as described above. All fermentations were conducted under non-sterilized conditions, and medium was used directly without sterilization. 2.5. Analytical methods Cell growth was measured by spectrophotometer (UV757, Shanghai Precision & Scientific Instrument, China) at a wavelength of 620 nm and the value was transformed into cell mass (g/L) according to a calibration curve. While in the measurement of cell growth in fermentation with the dilute acid hydrolysate, given the factor that impurities present in the hydrolysate could interfere with spectrophotometric measurement, the samples were centrifuged at 10,000 rpm for 10 min and the resulting cells were washed three times with distilled water and then dried to constant weight at 60 °C for 24 h. The number of viable cells was determined in terms of colony forming units (CFU). The liquid samples of the cultivations and the wheat straw hydrolysates were analyzed by HPLC (Shimadzu Corp., Kyoto, Japan), equipped with UV/Vis-detector (SPD-20A, Shimadzu Corp., Kyoto, Japan) and refractive index (RI) detector (RID-10A, Shimadzu Corp., Kyoto, Japan). The concentration of substrates, products and growth inhibitors in lignocellulosic hydrolysates were analyzed using an Aminex HPX-87H column (Bio-Rad, Richmond, CA, USA) at 50 °C with 0.6 mL/min eluent of 0.49 g/L sulfuric acid. 3. Results and discussion 3.1. Effect of mixed sugar concentration on LA production As reported previously, B. coagulans IPE22 could ferment many lignocellulose-related sugars (e.g., glucose, xylose, arabinose, mannose, galactose and cellobiose) to LA with high yields (Zhang et al., 2014). To further evaluate the fermentation performance of B. coagulans IPE22, experiments on different concentrations of mixed sugar were conducted. The ratio of the single sugar (glucose, xylose and arabinose) in the mixed sugar was the same as that in the obtained wheat straw hydrolysates (Section 2.2). So, the proportion of glucose, xylose and arabinose was 49.91% (w/v), 41.46%
(w/v) and 8.63% (w/v), respectively. The fermentations were performed under non-sterilized conditions with different concentrations of mixed sugar (40, 60, 80 and 100 g/L). Time course profiles for LA production under different mixed sugar concentrations were described in Fig. 2. In the present work, all the fermentations were successfully conducted under non-sterilized conditions and contamination was not observed. During the fermentations of 40, 60, 80 and 100 g/L mixed sugar, the obtained LA yields were 0.96, 0.93, 0.92 and 0.91 g/g, respectively. The result showed that B. coagulans IPE22 could homo-ferment the mixed sugar to LA. When B. coagulans IPE22 fermented 40 g/L mixed sugar (Fig. 2A), there was 5 h lag phase before metabolizing glucose. After 8 h fermentation, glucose declined to 4.36 g/L in the broth. Then xylose and arabinose was fermented to LA simultaneously. A maximum LA titer of 37.66 g/L was achieved after 21 h fermentation, with a yield of 0.96 g/g. The similar sugars utilization pattern was observed from the fermentation of the mixed sugar with other concentrations. The result illustrated that B. coagulans IPE22 was a typical carbon catabolite repression positive (CCR-positive) strain. During the fermentation of 40 g/L mixed sugar (Fig. 2A), the cell mass accumulation was parallel with the production of LA. However, the growth curves showed decline in the late stage of the fermentations when B. coagulans IPE22 fermenting 60, 80 and 100 g/L mixed sugar. For the fermentation of 60 g/L mixed sugar (Fig. 2B), a maximum cell mass of 4.34 g/L was obtained at the 20th hour and then the cell mass slightly declined to 4.18 g/L at the end of the fermentation (24 h). For the fermentation of 80 g/L mixed sugar (Fig. 2C), the cell mass reached its maximum value of 7.18 g/L at the 18th hour, and then declined gradually to 5.71 g/L at the end of the fermentation (34 h). The similar tendency of cell growth was also observed in the fermentation of 100 g/L mixed sugar (Fig. 2D). It is worth mentioning that the higher concentration of mixed sugar was used, the more significant decline of the cell mass was observed in the late stage of the fermentation. Interestingly, the turning points of the growth curves always occurred when the produced LA exceeded 50 g/L. Thus, the decline of cell mass could be resulted from feedback inhibition. In comparison to other LA producer (Lactobacillus), strain of B. coagulans was more sensitive to the product of LA (Ou et al., 2011; Patel et al., 2006). In the present work, LA productivities from 40, 60, 80 and 100 g/ L mixed sugar were 1.79, 2.25, 2.21 and 1.82 g/L/h, respectively. The highest LA productivity was attained from 60 g/L mixed sugar. Further increase of the mixed sugar concentration would cause
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severe feedback inhibition to cell growth and lead to lower productivity. Moreover, B. coagulans IPE22 metabolized hexose and pentose sugars sequentially, which also limited the bioconversion efficiency during fermenting mixed sugar, similar results were also reported by Walton et al. (2010) in LA fermentation from hemicellulose extracts by B. coagulans MXL-9. Compared with industrial production, the LA titer and productivity from mixed sugar by B. coagulans IPE22 were low. Therefore, in order to make the fermentation commercially-attractive, it is necessary to further improve fermentation efficiency. 3.2. Membrane integrated repeated batch fermentation (MIRB) of mixed sugar
Fig. 2. Time course profiles for LA production with different mixed sugar concentrations. Fermentations were performed in mMRS medium using mixed sugar of glucose, xylose and arabinose. Fermentation conditions: 52 °C, pH 6.0 and 100 rpm. Concentrations of the mixed sugar were 40 g/L (A), 60 g/L (B), 80 g/L (C) and 100 g/L (D), respectively.
Based on the above results, MIRB was employed to ferment 60 g/L mixed sugar of glucose, xylose and arabinose. The proportion of each single sugar in the mixture was same with that in wheat straw hydrolysates (see Section 2.2). The profile and the parameters of the MIRB were shown in Fig. 3 and Table 1. In the first batch, there was about 5 h lag phase and then B. coagulans IPE22 fermented the mixed sugar sequentially. After 24 h fermentation, the maximum titer of LA was attained. Then, the repeated batch cultures were performed using the method described in Section 2.4. Since the cells from batch 1 were reused to inoculate the next batch. The lag phase was almost eliminated in batch 2. The biocatalysts metabolized carbon source immediately and the fermentation time was reduced from 24 h (batch 1) to 20 h (batch 2). In batch 3, no lag phase was observed. Moreover, the assimilation of both hexose (glucose) and pentose (xylose and arabinose) was promoted by the cells surviving from batch 2. The phenomenon of simultaneously consuming glucose, xylose and arabinose was observed. Consequently, the fermentation time of batch 3 was only 18 h. The simultaneous utilization of glucose, xylose and arabinose was also observed in the following repeated culture cycles of batch 4 to batch 6. As described in Table 1, the fermentation time was reduced significantly from 24 h (for batch 1) to 17 h (for batch 4), and the time of subsequent batches remained 17 h. With the proceeding of MIRB, a continuous increase of cell mass was found from the batch 1 to batch 5, and then remained constant in batch 6. As showed in Table 1, the number of maximum viable cells increased continuously from batch 1 to batch 4. In the first batch, viable cells of 29 105 CFU/mL were detected. While, a 5.52-folds increase of viable cells (160 105 CFU/mL) was detected in batch 4. Subsequently, a reduction of viable cells appeared in batch 5. While the fermentation efficiency was not influenced, the productivity of LA was 3.30 g/L/h in batch 5. The result indicated that the existing viable cells in batch 5 were enough to sustain fermentation. In batch 6, viable cells of 125 105 CFU/mL were detected and a LA productivity of 3.31 g/L/h was achieved. The possible reason for decreased viable cells in batch 5 could be the limitation of nutrients in broth. This explanation was supported by the previous literature (Zhao et al., 2010). Although a slight rise and fall in viable cells was observed from batches 4 to 6, the LA productivity maintained constant at a relative high value. As a result, productivity of LA was improved from 2.26 g/L/h (batch 1) to 3.31 g/L/h (batch 6). Moreover, in each cycle of the repeated batch fermentation, the yield of LA from total mixed sugar was kept at the range of 0.93–0.96 g/g (Table 1). By application of MIRB, the productivity of LA was increased. The possible reason could be the synergistic effect of cell reuse and microorganism acclimation. The improved cell mass and viable cells enhanced the fermentation of mixed sugar. During the process of MIRB, the cells of the strain IPE22 were reused and resulted in the elimination of lag phase during the subsequent batch culture. In addition, it was speculated that the cells preferring to utilize pentose were reproducing and surviving in the late stage of
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Fig. 3. Membrane integrated repeated batch fermentation of mixed sugar. Fermentation was performed in mMRS medium using 60 g/L mixed sugar (30 g/L glucose, 25 g/L xylose and 5 g/L arabinose). Fermentation conditions: 52 °C, pH 6.0 and 100 rpm. Table 1 Parameters of membrane integrated repeated batch fermentation (MIRB) of mixed sugar. Batch number
Fermentation time (h)
Produced LA (g/L)
Yielda (g/g)
Productivity (g/L/h)
Maximum cell mass (g/L)
Maximum viable cellsb (105 CFU/mL)
1 2 3 4 5 6
24 20 18 17 17 17
54.14 54.93 54.92 55.68 56.03 56.27
0.93 0.94 0.94 0.96 0.95 0.96
2.26 2.75 3.08 3.28 3.30 3.31
4.36 5.35 7.37 8.08 8.86 8.81
29 ± 2 45 ± 6 118 ± 11 160 ± 12 108 ± 8 125 ± 10
Fermentation was performed in mMRS medium using 60 g/L mixed sugar (30 g/L glucose, 25 g/L xylose and 5 g/L arabinose). Fermentation conditions: 52 °C, pH 6.0 and 100 rpm. a The yields were calculated based on the produced LA (g)/consumed mixed sugar (g). b The values were the means and standard deviations of three independent experiments.
each batch without glucose, and the reuse of these cells resulted in an evolution in strain IPE22, which enhanced the strain’s capability of utilizing pentose. Govindaswamy and Vane (2010) also concluded that xylose utilization rate could be promoted by the biomass generated from the previous fermentor when multi-stage continous fermentaion was performed for ethanol production from glucose–xylose mixtures. Thus, the simultaneous utilization of hexose and pentose sugars was realized. By means of MIRB, the disadvantage of the CCR-positive strain B. coagulans IPE22 for fermentation mixed sugar was alleviated and the fermentation efficiency was improved significantly. 3.3. Membrane integrated repeated batch fermentation (MIRB) of wheat straw hydrolysates Wheat is the world’s most widely grown crop, wheat straw is an abundant agricultural residue with low commercial value (Talebnia et al., 2010). In the present work, wheat straw was used
to produce LA. The obtained wheat straw hydrolysates from Section 2.2 contained about 59.55 g/L mixed sugar (29.72 g/L glucose, 24.69 g/L xylose and 5.14 g/L arabinose) and some fermentation inhibitors (4.01 g/L acetate, 0.08 g/L formate, 0.05 g/L furfural, 0.08 g/L 5-HMF and 40.06 g/L sulfate). MIRB was performed using the hydrolysates to produce LA under non-sterilized conditions (Fig. 4 and Table 2). In batch 1 (Fig. 4), fermentation exhibited a lag phase of 18 h, which was longer than that of the previous mixed sugar fermentation (see Fig. 3). The prolonged lag phase could be caused by the fermentation inhibitors in the hydrolysates. Then, B. coagulans IPE22 fermented hexose (glucose) and pentose (xylose and arabinose) sequentially. After 54 h fermentation, a maximum LA titer of 54.55 g/L was obtained. When the MIRB was conducted, the lag phase of batch 2, batch 3 and batch 4 decreased to 8, 4 and 3 h, respectively. So, the fermentation time decreased from 54 h (batch 1) to 27 h (batch 4). In batch 5 and batch 6, lag phase was totally eliminated and the simultaneous fermentation of hexose
Fig. 4. Membrane integrated repeated batch fermentation of wheat straw hydrolysates. Fermentation was performed in mMRS medium using wheat straw hydrolysates containing 59.55 g/L mixed sugar (29.72 g/L glucose, 24.69 g/L xylose and 5.14 g/L arabinose). Fermentation conditions: 52 °C, pH 6.0 and 100 rpm.
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Y. Zhang et al. / Bioresource Technology 163 (2014) 160–166 Table 2 Parameters of membrane integrated repeated batch fermentation (MIRB) of wheat straw hydrolysates. Batch number
Fermentation time (h)
LA titer (g/L)
Yielda (g/g)
Productivity (g/L/h)
Maximum cell mass (g/L)
Maximum viable cellsb (105 CFU/mL)
1 2 3 4 5 6
54 39 30 27 24 24
54.55 55.13 55.29 55.61 55.45 56.45
0.94 0.94 0.94 0.95 0.95 0.96
1.01 1.41 1.84 2.06 2.31 2.35
4.93 7.66 8.53 9.47 9.33 9.48
51 ± 4 73 ± 10 108 ± 5 167 ± 9 162 ± 7 165 ± 11
Fermentation was performed in mMRS medium using wheat straw hydrolysates containing 59.55 g/L mixed sugar (29.72 g/L glucose, 24.69 g/L xylose and 5.14 g/L arabinose). Fermentation conditions: 52 °C, pH 6.0 and 100 rpm. a The yields were calculated based on the produced LA (g)/consumed mixed sugar (g). b The values were the means and standard deviations of three independent experiments.
and pentose sugars occurred. The fermentation time for batch 5 and batch 6 was the same, both were 24 h. As the repeated batch proceeded from batch 1 to batch 4, a gradual increase of cell mass and viable cells was observed, as shown in Table 2. From batch 4 to batch 6, cell mass and viable cells remained constant. By virtue of MIRB, fermentation efficiency of wheat straw hydrolysates was accelerated significantly. The LA productivity of 2.35 g/L/h was obtained in batch 6, a significant increase in comparison to batch 1. Moreover, the LA yield of each repeated batch cycles was more or less constant, ranging from 0.94 to 0.96 g/g. Comparing Table 1 with Table 2, LA productivity from hydrolysates was lower than the MIRB fermentation of mixed sugar, which could be ascribed to the presence of inhibitors in hydrolysates. Even so, the improvement of LA productivity by MIRB was prominent. It is worth mentioning that both cell mass and number of viable cell in the wheat straw hydrolysates fermentation were higher than those from the MIRB fermentation of mixed sugar. The reason was deduced to the function of extra nutrients in wheat straw hydrolysates. During the pretreatment of biomass, some nutrients (such as amino acids) could release from lignocellulosic materials and that was favorable to cell growth (Lau and Dale, 2009). As a whole, the acquired high cell density by MIRB favored the consumption of both hexose and pentose sugars in the hydrolysates. The simultaneous fermentation of glucose, xylose and arabinose was successfully realized. In addition, the cells were reused during the MIRB. The cells may be adapted to the hydrolysates, so high fermentation efficiency could be attained. Many microorganisms had been employed to produce LA from lignocellulosic hydrolysates containing mixed sugar, including Rhizopus oryzae (Bai et al., 2008), Lactobacillus brevis (Guo et al., 2010), Lactobacillus pentosus (Bustos et al., 2005, 2007; Moldes et al., 2006), B. coagulans (Bischoff et al., 2010; Ouyang et al., 2012a) and Bacillus sp. 17C5 (Patel et al., 2004). The highest LA productivity of 3.10 g/L/h was achieved by L. pentosus from vine shoots hydrolysates in a continuous fermentation process (Bustos et al., 2007). The continuous fermentation usually led to incomplete utilization of the carbon sources. So, the subsequent separation of LA from residual sugars increased the costs of production. B. coagulans IPE22 was suitable to produce LA from mixed sugar in lignocellulosic hydrolysates due to its high yield of LA. Moreover, thermophilic B. coagulans IPE22 has robust property to resist contamination. The non-sterilized fermentation greatly simplified the operation and reduced the costs of production. It is well known that in conventional LA production process, calcium hydroxide or calcium carbonate is added during fermentation to neutralize lactic acid and maintain the pH around 5–6. A large amount of gypsum is produced in the subsequent LA extraction and purification steps, posing serious economic and environmental problems in terms of waste treatment. While in the present study, sodium hydroxide was selected as neutralizing agent to maintain pH, so the above problem could be avoided, as reported by Qin et al. (2010). Besides,
fermentation without calcium ion could reduce membrane fouling, as a result, the MIRB technology could be more practical in industrial LA production (Pal et al., 2009). In this work, by means of MIRB, the highest LA productivity of 2.35 g/L/h was obtained from wheat straw hydrolysates. The result indicated that MIRB was an efficient way to produce LA from lignocellulosic hydrolysates. Furthermore, MIRB is also expected to be applicable for other biotechnological processes based on the conversion of lignocellulosic hydrolysates containing sugar mixtures.
4. Conclusions B. coagulans IPE22 could homo-ferment mixed sugar of glucose, xylose and arabinose to LA under non-sterilized conditions. By application of MIRB strategy, the CCR feature of the strain and the feedback inhibition during fermentation were greatly mitigated because of the strain acclimation, cell reuse and product removal during fermentation. With MIRB, LA productivity was increased to 2.35 g/L/h (batch 6) from 1.01 g/L/h (batch 1) when wheat straw hydrolysates was used as carbon source. MIRB fermentation under non-sterilized conditions leads to great savings in terms of both time and labor, making it easy to scale up for industrial production. Acknowledgements The authors would like to thank the National Natural Science Foundation of China for the financial support (Grant No. 21176239) and the CAS/SAFEA International Partnership Program for Creative Research Teams. References Abdel-Rahman, M.A., Tashiro, Y., Sonomoto, K., 2013a. Recent advances in lactic acid production by microbial fermentation processes. Biotechnol. Adv. 31, 877–902. Abdel-Rahman, M.A., Tashiro, Y., Zendo, T., Sonomoto, K., 2013b. Improved lactic acid productivity by an open repeated batch fermentation system using Enterococcus mundtii QU 25. RSC Adv. 3, 8437–8445. Bai, D.M., Li, S.Z., Liu, Z.L., Cui, Z.F., 2008. Enhanced L-(+)-lactic acid production by an adapted strain of Rhizopus oryzae using corncob hydrolysate. Appl. Biochem. Biotechnol. 144, 79–85. Bischoff, K.M., Liu, S.Q., Hughes, S.R., Rich, J.O., 2010. Fermentation of corn fiber hydrolysate to lactic acid by the moderate thermophile Bacillus coagulans. Biotechnol. Lett. 32, 823–828. Bustos, G., de la Torre, N., Moldes, A.B., Cruz, J.M., Dominguez, J.M., 2007. Revalorization of hemicellulosic trimming vine shoots hydrolyzates trough continuous production of lactic acid and biosurfactants by L. pentosus. J. Food Eng. 78, 405–412. Bustos, G., Moldes, A.B., Cruz, J.M., Dominguez, J.M., 2005. Influence of the metabolism pathway on lactic acid production from hemicellulosic trimming vine shoots hydrolyzates using Lactobacillus pentosus. Biotechnol. Progr. 21, 793–798. Datta, R., Henry, M., 2006. Lactic acid: recent advances in products, processes and technologies – a review. J. Chem. Technol. Biotechnol. 81, 1119–1129.
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Pal, P., Sikder, J., Roy, S., Giorno, L., 2009. Process intensification in lactic acid production: a review of membrane based processes. Chem. Eng. Process. 48, 1549–1559. Patel, M.A., Ou, M.S., Harbrucker, R., Aldrich, H.C., Buszko, M.L., Ingram, L.O., Shanmugam, K.T., 2006. Isolation and characterization of acid-tolerant, thermophilic bacteria for effective fermentation of biomass-derived sugars to lactic acid. Appl. Environ. Microbiol. 72, 3228–3235. Patel, M., Ou, M., Ingram, L.O., Shanmugam, K.T., 2004. Fermentation of sugar cane bagasse hemicellulose hydrolysate to L(+)-lactic acid by a thermotolerant acidophilic Bacillus sp.. Biotechnol. Lett. 26, 865–868. Qin, J.Y., Wang, X.W., Zheng, Z.J., Ma, C.Q., Tang, H.Z., Xu, P., 2010. Production of Llactic acid by a thermophilic Bacillus mutant using sodium hydroxide as neutralizing agent. Bioresour. Technol. 101, 7570–7576. Rosenberg, M., Rebros, M., Kristofikova, L., Malatova, K., 2005. High temperature lactic acid production by Bacillus coagulans immobilized in LentiKats. Biotechnol. Lett. 27, 1943–1947. Shi, Z.M., Wei, P.L., Zhu, X.C., Cai, J., Huang, L., Xu, Z.N., 2012. Efficient production of Llactic acid from hydrolysate of Jerusalem artichoke with immobilized cells of Lactococcus lactis in fibrous bed bioreactors. Enzyme Microb. Technol. 51, 263–268. Talebnia, F., Karakashev, D., Angelidaki, I., 2010. Production of bioethanol from wheat straw: an overview on pretreatment, hydrolysis and fermentation. Bioresour. Technol. 101, 4744–4753. Walton, S.L., Bischoff, K.M., van Heiningen, A.R.P., van Walsum, G.P., 2010. Production of lactic acid from hemicellulose extracts by Bacillus coagulans MXL-9. J. Ind. Microbiol. Biotechnol. 37, 823–830. Wee, Y.J., Yun, J.S., Kim, D., Ryu, H.W., 2006. Batch and repeated batch production of L(+)-lactic acid by Enterococcus faecalis RKY1 using wood hydrolyzate and corn steep liquor. J. Ind. Microbiol. Biotechnol. 33, 431–435. Ye, L.D., Bin Hudari, M.S., Zhou, X.D., Zhang, D.X., Li, Z., Wu, J.C., 2013b. Conversion of acid hydrolysate of oil palm empty fruit bunch to L-lactic acid by newly isolated Bacillus coagulans JI12. Appl. Microbiol. Biotechnol. 97, 4831–4838. Ye, L., Zhou, X., Hudari, M.S.B., Li, Z., Wu, J.C., 2013a. Highly efficient production of Llactic acid from xylose by newly isolated Bacillus coagulans C106. Bioresour. Technol. 132, 38–44. Zhang, Y., Chen, X., Luo, J., Qi, B., Wan, Y., 2014. An efficient process for lactic acid production from wheat straw by a newly isolated Bacillus coagulans strain IPE22. Bioresour. Technol. 158, 396–399. Zhao, B., Wang, L.M., Ma, C.Q., Yang, C.Y., Xu, P., Ma, Y.H., 2010. Repeated open fermentative production of optically pure L-lactic acid using a thermophilic Bacillus sp. strain. Bioresour. Technol. 101, 6494–6498. Zhou, X., Ye, L., Wu, J., 2013. Efficient production of L-lactic acid by newly isolated thermophilic Bacillus coagulans WCP10-4 with high glucose tolerance. Appl. Microbiol. Biotechnol. 97, 4309–4314.
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Improving lactic acid productivity from wheat straw hydrolysates by membrane integrated repeated batch fermentation under non-sterilized conditions Yuming Zhang a,b,c, Xiangrong Chen a, Benkun Qi a, Jianquan Luo d, Fei Shen a, Yi Su a, Rashid Khan a, Yinhua Wan a,⇑ a
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China University of Chinese Academy of Sciences, Beijing 100049, China c College of Life Sciences, Hebei University, Baoding 071002, China d Department of Chemical and Biochemical Engineering, Center for Bioprocess Engineering, Building 229, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark b
h i g h l i g h t s LA was produced from wheat straw hydrolysates under non-sterilized conditions. Membrane integrated repeated batch fermentation (MIRB) could increase LA productivity. With MIRB, the simultaneous fermentation of hexose and pentose sugars was realized. LA productivity of 2.35 g/L/h was obtained from wheat straw hydrolysates by MIRB.
a r t i c l e
i n f o
Article history: Received 20 February 2014 Received in revised form 9 April 2014 Accepted 10 April 2014 Available online 19 April 2014 Keywords: Lactic acid Membrane Repeated batch Cell recycle Wheat straw hydrolysates
a b s t r a c t Bacillus coagulans IPE22 was used to produce lactic acid (LA) from mixed sugar and wheat straw hydrolysates, respectively. All fermentations were conducted under non-sterilized conditions and sodium hydroxide was used as neutralizing agent to avoid the production of insoluble CaSO4. In order to eliminate the sequential utilization of mixed sugar and feedback inhibition during batch fermentation, membrane integrated repeated batch fermentation (MIRB) was used to improve LA productivity. With MIRB, a high cell density was obtained and the simultaneous fermentation of glucose, xylose and arabinose was successfully realized. The separation of LA from broth by membrane in batch fermentation also decreased feedback inhibition. MIRB was carried out using wheat straw hydrolysates (29.72 g/L glucose, 24.69 g/L xylose and 5.14 g/L arabinose) as carbon source, LA productivity was increased significantly from 1.01 g/L/h (batch 1) to 2.35 g/L/h (batch 6) by the repeated batch fermentation. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Lactic acid (LA) is an important commodity chemical for various applications in food, pharmaceutical and the cosmetic industry (Datta and Henry, 2006). It can also serve as a precursor for producing poly lactic acid, which is a promising biodegradable, biocompatible and environment-friendly alternative to plastics derived from petrochemicals (Lasprilla et al., 2012). LA can be produced in two ways: chemical synthesis and fermentation. The fermentation process is attractive because of its advantages of using renewable carbohydrates and producing optically pure LA ⇑ Corresponding author. Tel./fax: +86 10 62650673. E-mail address: [email protected] (Y. Wan). http://dx.doi.org/10.1016/j.biortech.2014.04.038 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
(John et al., 2009). Abundant and renewable lignocellulosic materials are regarded as suitable feedstocks for LA production (Abdel-Rahman et al., 2013a). Lignocellulose is composed of cellulose, hemicellulose and lignin, which can be hydrolyzed to a mixture of hexose and pentose sugars. Therefore, a strain capable of fermenting all the lignocellulose released sugars is essential for the economical production of LA (Kim et al., 2010a). Production of LA using lignocellulosic hydrolysates by Bacillus coagulans has drawn much attention due to its strong ability to ferment both hexose and pentose sugars (Ou et al., 2011; Ye et al., 2013a; Zhou et al., 2013). Moreover, the fermentation could be operated under non-sterilized conditions by virtue of the strain’s thermophilic feature (Ouyang et al., 2012b; Ye et al., 2013b; Zhao et al., 2010; Zhou et al., 2013). Non-sterilized fermentation is
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energy-efficient, cost-effective and labor-saving. Despite having these advantages, production of LA by B. coagulans still has several challenges remaining to be resolved. As reported by Walton et al. (2010) and Ou et al. (2011), some B. coagulans strains could not simultaneously ferment hexose and pentose, and these mixed sugar could only be sequentially metabolized due to carbon catabolite repression (CCR) (Kim et al., 2010a), which would decrease the efficiency of fermentation. In addition, feedback inhibition during the fermentation could limit the LA production (Ou et al., 2011). To solve these problems, method of genetic modification is undoubtedly the primary strategy to be considered, while optimization of fermentation strategy is also an alternative. It is well known that the repeated batch fermentation is a feasible method for improving LA productivity due to the reduction in fermentation time and the skip of inoculums preparation (Abdel-Rahman et al., 2013b; John et al., 2007). During the repeated batch fermentation, cells could be reused by centrifugation (Abdel-Rahman et al., 2013b; Kim et al., 2010b; Zhao et al., 2010) or cell immobilization (Rosenberg et al., 2005; Shi et al., 2012). Usually, cell reuse by centrifugation could not realize automatically continuous operation. And the immobilized cell bioreactor may be suffered from productivity loss due to the limited mass transfer and the accumulation of dead cells (Shi et al., 2012). Membrane techniques have many advantages in separation of cells from fermentation broth, such as energy-efficient, low damage to cells and easy to scale up for industrial production (Pal et al., 2009; Zhao et al., 2010). The strategy of integrating membrane module with fermentor, termed as membrane integrated repeated batch fermentation (MIRB), could efficiently achieve cell-recycled repeated batch. Thus, a lot of efforts have been made to improve the productivity of LA by MIRB (Kim et al., 2006; Oh et al., 2003; Wee et al., 2006) and most of the studies concerned the MIRB using single sugar as carbon source. To the best of our knowledge, there has been no report regarding LA production from sugar mixture of hexose and pentose using MIRB, especially from lignocellulosic hydrolysates. In conventional LA production by fermentation, calcium hydroxide or calcium carbonate is normally used to neutralize the produced LA, thus resulting in the production of calcium-LA. The salt of calcium lactate has to be acidified with H2SO4, thus resulting in the production of insoluble CaSO4 in LA extraction and purification steps. Gypsum poses serious environmental problem in waste treatment during large-scale LA production. Fermentation using sodium hydroxide as neutralizing agent could avoid the above problem (Qin et al., 2010). Another advantage of fermentation without calcium ion is that the potential of membrane fouling can be significantly decreased, and thus making the MIRB technology more practical in industrial LA production (Pal et al., 2009). Recently, a thermophilic LA producing bacterium, B. coagulans IPE22, was isolated and characterized in our lab, and this strain showed remarkable capability to ferment pentose, hexose and cellobiose and was highly resistant to inhibitors from lignocellulosic hydrolysates (Zhang et al., 2014). The objective of the present work is to evaluate the performance of MIRB in LA production from wheat straw hydrolysates by B. coagulans IPE22, aiming to eliminate the sequential utilization of mixed sugar and feedback inhibition for efficient LA production. All the fermentations were operated under non-sterilized conditions and sodium hydroxide was used as neutralizing agent to avoid the production of insoluble CaSO4. Firstly, the effect of initial concentration of sugar mixture on the production of LA was investigated. Then, MIRB was employed to produce LA from both mixed sugar and wheat straw hydrolysates. Fermentative parameters in terms of sugar consumption, cell mass accumulation and viable cells were monitored and the mechanisms were discussed.
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2. Methods 2.1. Microorganisms and medium B. coagulans IPE22 was used in this study. A modified De Man-Rogosa-Sharpe (mMRS) medium was used for seed culture and fermentation. Medium of mMRS contained 10 g/L peptone, 10 g/L beef extract, 5 g/L yeast extract, 2 g/L dipotassium phosphate, 0.2 g/L magnesium sulfate heptahydrate, 0.05 g/L manganese sulfate tetrahydrate and different carbon sources. The type and concentration of carbon sources varied in different fermentation experiments. 2.2. Preparation of wheat straw hydrolysates Wheat straw was cleaned, chopped, and then pretreated by 2% (w/v) sulfuric acid at a 10% (w/v) loading. The mixture was treated at 121 °C for 90 min, and the obtained slurry was filtrated to achieve liquid and solid fractions. Both liquid and solid fractions were collected and defined as dilute acid hydrolysates and water insoluble solids (WIS), respectively. The WIS (containing 59.96% cellulose) was hydrolyzed by commercial cellulase (Sunson Group Ningxia Enzyme Preparation Plant, China) at a 10% (w/v) solid loading. Enzyme loading was 20 FPU (filter paper activity units)/g cellulose. The enzymatic hydrolysis was carried out in a 10 L jar fermentor (GUJS-10, Zhenjiang Dongfang Bioengineering Equipment Company, China) at 50 °C, pH 5.0 and 200 rpm. Enzymatic hydrolysates were obtained by filtration with filter paper (No. 43, Whatman, UK). Finally, dilute acid hydrolysates and enzymatic hydrolysates were mixed and concentrated by vacuum evaporation at 45 °C. The wheat straw hydrolysates after concentration normally contained 59.55 g/L mixed sugar (29.72 g/L glucose, 24.69 g/L xylose and 5.14 g/L arabinose). 2.3. Batch fermentations To prepare seed culture, the strain of B. coagulans IPE22 was grown on agar mMRS plate for 2 days at 45 °C. The cells were transferred to liquid mMRS media containing 10 g/L glucose and cultured for 6 h at 50 °C. This culture was used to provide 5% (v/ v) inocula for fermentation. The optimal fermentation condition for B. coagulans IPE22 to produce LA was 52 °C and pH 6.0, as described by Zhang et al. (2014). So, batch fermentations were carried out in fermentor at 52 °C, pH 6.0 and 100 rpm. Sodium hydroxide solution of 400 g/L was automatically added to maintain the pH value by a peristaltic pump. Samples were taken with specific time intervals to determine cell mass, residual sugars and products. 2.4. Membrane integrated repeated batch fermentation (MIRB) Repeated batch fermentation was performed in a membrane integrated bioreactor, the schematic diagram of which was shown in Fig. 1. The fermentor was coupled with a membrane module. Fermentation broth was transferred to the membrane module by a diaphragm pump. A polyacrylonitrile (PAN) ultrafiltration membrane with a nominal molecular weight cut-off of 20 kDa was used in the experiments. The effective area of the used ultrafiltration membrane was 0.18 m2. Fermentation was carried out in the fermentor with a working volume of 8 L, and the culture condition was same with that of batch fermentation (Section 2.3). When the sugars in the culture broth were depleted, the integrated membrane module was started to filter fermentation broth. Eighty percent (v/v) of the culture broth (6.4 L) was removed as the permeate liquid. Then 1.6 L broth with concentrated cells was obtained, and
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Fig. 1. Schematic diagram of fermentor coupled with a membrane module.
0.8 L of the obtained broth was used as seed culture to generate the next batch fermentation with 7.2 L fresh medium. So the volume of the second batch fermentation was also 8 L, which was equal to the initial fermentation. Subsequent batch fermentations were performed in the same manner as described above. All fermentations were conducted under non-sterilized conditions, and medium was used directly without sterilization. 2.5. Analytical methods Cell growth was measured by spectrophotometer (UV757, Shanghai Precision & Scientific Instrument, China) at a wavelength of 620 nm and the value was transformed into cell mass (g/L) according to a calibration curve. While in the measurement of cell growth in fermentation with the dilute acid hydrolysate, given the factor that impurities present in the hydrolysate could interfere with spectrophotometric measurement, the samples were centrifuged at 10,000 rpm for 10 min and the resulting cells were washed three times with distilled water and then dried to constant weight at 60 °C for 24 h. The number of viable cells was determined in terms of colony forming units (CFU). The liquid samples of the cultivations and the wheat straw hydrolysates were analyzed by HPLC (Shimadzu Corp., Kyoto, Japan), equipped with UV/Vis-detector (SPD-20A, Shimadzu Corp., Kyoto, Japan) and refractive index (RI) detector (RID-10A, Shimadzu Corp., Kyoto, Japan). The concentration of substrates, products and growth inhibitors in lignocellulosic hydrolysates were analyzed using an Aminex HPX-87H column (Bio-Rad, Richmond, CA, USA) at 50 °C with 0.6 mL/min eluent of 0.49 g/L sulfuric acid. 3. Results and discussion 3.1. Effect of mixed sugar concentration on LA production As reported previously, B. coagulans IPE22 could ferment many lignocellulose-related sugars (e.g., glucose, xylose, arabinose, mannose, galactose and cellobiose) to LA with high yields (Zhang et al., 2014). To further evaluate the fermentation performance of B. coagulans IPE22, experiments on different concentrations of mixed sugar were conducted. The ratio of the single sugar (glucose, xylose and arabinose) in the mixed sugar was the same as that in the obtained wheat straw hydrolysates (Section 2.2). So, the proportion of glucose, xylose and arabinose was 49.91% (w/v), 41.46%
(w/v) and 8.63% (w/v), respectively. The fermentations were performed under non-sterilized conditions with different concentrations of mixed sugar (40, 60, 80 and 100 g/L). Time course profiles for LA production under different mixed sugar concentrations were described in Fig. 2. In the present work, all the fermentations were successfully conducted under non-sterilized conditions and contamination was not observed. During the fermentations of 40, 60, 80 and 100 g/L mixed sugar, the obtained LA yields were 0.96, 0.93, 0.92 and 0.91 g/g, respectively. The result showed that B. coagulans IPE22 could homo-ferment the mixed sugar to LA. When B. coagulans IPE22 fermented 40 g/L mixed sugar (Fig. 2A), there was 5 h lag phase before metabolizing glucose. After 8 h fermentation, glucose declined to 4.36 g/L in the broth. Then xylose and arabinose was fermented to LA simultaneously. A maximum LA titer of 37.66 g/L was achieved after 21 h fermentation, with a yield of 0.96 g/g. The similar sugars utilization pattern was observed from the fermentation of the mixed sugar with other concentrations. The result illustrated that B. coagulans IPE22 was a typical carbon catabolite repression positive (CCR-positive) strain. During the fermentation of 40 g/L mixed sugar (Fig. 2A), the cell mass accumulation was parallel with the production of LA. However, the growth curves showed decline in the late stage of the fermentations when B. coagulans IPE22 fermenting 60, 80 and 100 g/L mixed sugar. For the fermentation of 60 g/L mixed sugar (Fig. 2B), a maximum cell mass of 4.34 g/L was obtained at the 20th hour and then the cell mass slightly declined to 4.18 g/L at the end of the fermentation (24 h). For the fermentation of 80 g/L mixed sugar (Fig. 2C), the cell mass reached its maximum value of 7.18 g/L at the 18th hour, and then declined gradually to 5.71 g/L at the end of the fermentation (34 h). The similar tendency of cell growth was also observed in the fermentation of 100 g/L mixed sugar (Fig. 2D). It is worth mentioning that the higher concentration of mixed sugar was used, the more significant decline of the cell mass was observed in the late stage of the fermentation. Interestingly, the turning points of the growth curves always occurred when the produced LA exceeded 50 g/L. Thus, the decline of cell mass could be resulted from feedback inhibition. In comparison to other LA producer (Lactobacillus), strain of B. coagulans was more sensitive to the product of LA (Ou et al., 2011; Patel et al., 2006). In the present work, LA productivities from 40, 60, 80 and 100 g/ L mixed sugar were 1.79, 2.25, 2.21 and 1.82 g/L/h, respectively. The highest LA productivity was attained from 60 g/L mixed sugar. Further increase of the mixed sugar concentration would cause
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severe feedback inhibition to cell growth and lead to lower productivity. Moreover, B. coagulans IPE22 metabolized hexose and pentose sugars sequentially, which also limited the bioconversion efficiency during fermenting mixed sugar, similar results were also reported by Walton et al. (2010) in LA fermentation from hemicellulose extracts by B. coagulans MXL-9. Compared with industrial production, the LA titer and productivity from mixed sugar by B. coagulans IPE22 were low. Therefore, in order to make the fermentation commercially-attractive, it is necessary to further improve fermentation efficiency. 3.2. Membrane integrated repeated batch fermentation (MIRB) of mixed sugar
Fig. 2. Time course profiles for LA production with different mixed sugar concentrations. Fermentations were performed in mMRS medium using mixed sugar of glucose, xylose and arabinose. Fermentation conditions: 52 °C, pH 6.0 and 100 rpm. Concentrations of the mixed sugar were 40 g/L (A), 60 g/L (B), 80 g/L (C) and 100 g/L (D), respectively.
Based on the above results, MIRB was employed to ferment 60 g/L mixed sugar of glucose, xylose and arabinose. The proportion of each single sugar in the mixture was same with that in wheat straw hydrolysates (see Section 2.2). The profile and the parameters of the MIRB were shown in Fig. 3 and Table 1. In the first batch, there was about 5 h lag phase and then B. coagulans IPE22 fermented the mixed sugar sequentially. After 24 h fermentation, the maximum titer of LA was attained. Then, the repeated batch cultures were performed using the method described in Section 2.4. Since the cells from batch 1 were reused to inoculate the next batch. The lag phase was almost eliminated in batch 2. The biocatalysts metabolized carbon source immediately and the fermentation time was reduced from 24 h (batch 1) to 20 h (batch 2). In batch 3, no lag phase was observed. Moreover, the assimilation of both hexose (glucose) and pentose (xylose and arabinose) was promoted by the cells surviving from batch 2. The phenomenon of simultaneously consuming glucose, xylose and arabinose was observed. Consequently, the fermentation time of batch 3 was only 18 h. The simultaneous utilization of glucose, xylose and arabinose was also observed in the following repeated culture cycles of batch 4 to batch 6. As described in Table 1, the fermentation time was reduced significantly from 24 h (for batch 1) to 17 h (for batch 4), and the time of subsequent batches remained 17 h. With the proceeding of MIRB, a continuous increase of cell mass was found from the batch 1 to batch 5, and then remained constant in batch 6. As showed in Table 1, the number of maximum viable cells increased continuously from batch 1 to batch 4. In the first batch, viable cells of 29 105 CFU/mL were detected. While, a 5.52-folds increase of viable cells (160 105 CFU/mL) was detected in batch 4. Subsequently, a reduction of viable cells appeared in batch 5. While the fermentation efficiency was not influenced, the productivity of LA was 3.30 g/L/h in batch 5. The result indicated that the existing viable cells in batch 5 were enough to sustain fermentation. In batch 6, viable cells of 125 105 CFU/mL were detected and a LA productivity of 3.31 g/L/h was achieved. The possible reason for decreased viable cells in batch 5 could be the limitation of nutrients in broth. This explanation was supported by the previous literature (Zhao et al., 2010). Although a slight rise and fall in viable cells was observed from batches 4 to 6, the LA productivity maintained constant at a relative high value. As a result, productivity of LA was improved from 2.26 g/L/h (batch 1) to 3.31 g/L/h (batch 6). Moreover, in each cycle of the repeated batch fermentation, the yield of LA from total mixed sugar was kept at the range of 0.93–0.96 g/g (Table 1). By application of MIRB, the productivity of LA was increased. The possible reason could be the synergistic effect of cell reuse and microorganism acclimation. The improved cell mass and viable cells enhanced the fermentation of mixed sugar. During the process of MIRB, the cells of the strain IPE22 were reused and resulted in the elimination of lag phase during the subsequent batch culture. In addition, it was speculated that the cells preferring to utilize pentose were reproducing and surviving in the late stage of
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Fig. 3. Membrane integrated repeated batch fermentation of mixed sugar. Fermentation was performed in mMRS medium using 60 g/L mixed sugar (30 g/L glucose, 25 g/L xylose and 5 g/L arabinose). Fermentation conditions: 52 °C, pH 6.0 and 100 rpm. Table 1 Parameters of membrane integrated repeated batch fermentation (MIRB) of mixed sugar. Batch number
Fermentation time (h)
Produced LA (g/L)
Yielda (g/g)
Productivity (g/L/h)
Maximum cell mass (g/L)
Maximum viable cellsb (105 CFU/mL)
1 2 3 4 5 6
24 20 18 17 17 17
54.14 54.93 54.92 55.68 56.03 56.27
0.93 0.94 0.94 0.96 0.95 0.96
2.26 2.75 3.08 3.28 3.30 3.31
4.36 5.35 7.37 8.08 8.86 8.81
29 ± 2 45 ± 6 118 ± 11 160 ± 12 108 ± 8 125 ± 10
Fermentation was performed in mMRS medium using 60 g/L mixed sugar (30 g/L glucose, 25 g/L xylose and 5 g/L arabinose). Fermentation conditions: 52 °C, pH 6.0 and 100 rpm. a The yields were calculated based on the produced LA (g)/consumed mixed sugar (g). b The values were the means and standard deviations of three independent experiments.
each batch without glucose, and the reuse of these cells resulted in an evolution in strain IPE22, which enhanced the strain’s capability of utilizing pentose. Govindaswamy and Vane (2010) also concluded that xylose utilization rate could be promoted by the biomass generated from the previous fermentor when multi-stage continous fermentaion was performed for ethanol production from glucose–xylose mixtures. Thus, the simultaneous utilization of hexose and pentose sugars was realized. By means of MIRB, the disadvantage of the CCR-positive strain B. coagulans IPE22 for fermentation mixed sugar was alleviated and the fermentation efficiency was improved significantly. 3.3. Membrane integrated repeated batch fermentation (MIRB) of wheat straw hydrolysates Wheat is the world’s most widely grown crop, wheat straw is an abundant agricultural residue with low commercial value (Talebnia et al., 2010). In the present work, wheat straw was used
to produce LA. The obtained wheat straw hydrolysates from Section 2.2 contained about 59.55 g/L mixed sugar (29.72 g/L glucose, 24.69 g/L xylose and 5.14 g/L arabinose) and some fermentation inhibitors (4.01 g/L acetate, 0.08 g/L formate, 0.05 g/L furfural, 0.08 g/L 5-HMF and 40.06 g/L sulfate). MIRB was performed using the hydrolysates to produce LA under non-sterilized conditions (Fig. 4 and Table 2). In batch 1 (Fig. 4), fermentation exhibited a lag phase of 18 h, which was longer than that of the previous mixed sugar fermentation (see Fig. 3). The prolonged lag phase could be caused by the fermentation inhibitors in the hydrolysates. Then, B. coagulans IPE22 fermented hexose (glucose) and pentose (xylose and arabinose) sequentially. After 54 h fermentation, a maximum LA titer of 54.55 g/L was obtained. When the MIRB was conducted, the lag phase of batch 2, batch 3 and batch 4 decreased to 8, 4 and 3 h, respectively. So, the fermentation time decreased from 54 h (batch 1) to 27 h (batch 4). In batch 5 and batch 6, lag phase was totally eliminated and the simultaneous fermentation of hexose
Fig. 4. Membrane integrated repeated batch fermentation of wheat straw hydrolysates. Fermentation was performed in mMRS medium using wheat straw hydrolysates containing 59.55 g/L mixed sugar (29.72 g/L glucose, 24.69 g/L xylose and 5.14 g/L arabinose). Fermentation conditions: 52 °C, pH 6.0 and 100 rpm.
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Y. Zhang et al. / Bioresource Technology 163 (2014) 160–166 Table 2 Parameters of membrane integrated repeated batch fermentation (MIRB) of wheat straw hydrolysates. Batch number
Fermentation time (h)
LA titer (g/L)
Yielda (g/g)
Productivity (g/L/h)
Maximum cell mass (g/L)
Maximum viable cellsb (105 CFU/mL)
1 2 3 4 5 6
54 39 30 27 24 24
54.55 55.13 55.29 55.61 55.45 56.45
0.94 0.94 0.94 0.95 0.95 0.96
1.01 1.41 1.84 2.06 2.31 2.35
4.93 7.66 8.53 9.47 9.33 9.48
51 ± 4 73 ± 10 108 ± 5 167 ± 9 162 ± 7 165 ± 11
Fermentation was performed in mMRS medium using wheat straw hydrolysates containing 59.55 g/L mixed sugar (29.72 g/L glucose, 24.69 g/L xylose and 5.14 g/L arabinose). Fermentation conditions: 52 °C, pH 6.0 and 100 rpm. a The yields were calculated based on the produced LA (g)/consumed mixed sugar (g). b The values were the means and standard deviations of three independent experiments.
and pentose sugars occurred. The fermentation time for batch 5 and batch 6 was the same, both were 24 h. As the repeated batch proceeded from batch 1 to batch 4, a gradual increase of cell mass and viable cells was observed, as shown in Table 2. From batch 4 to batch 6, cell mass and viable cells remained constant. By virtue of MIRB, fermentation efficiency of wheat straw hydrolysates was accelerated significantly. The LA productivity of 2.35 g/L/h was obtained in batch 6, a significant increase in comparison to batch 1. Moreover, the LA yield of each repeated batch cycles was more or less constant, ranging from 0.94 to 0.96 g/g. Comparing Table 1 with Table 2, LA productivity from hydrolysates was lower than the MIRB fermentation of mixed sugar, which could be ascribed to the presence of inhibitors in hydrolysates. Even so, the improvement of LA productivity by MIRB was prominent. It is worth mentioning that both cell mass and number of viable cell in the wheat straw hydrolysates fermentation were higher than those from the MIRB fermentation of mixed sugar. The reason was deduced to the function of extra nutrients in wheat straw hydrolysates. During the pretreatment of biomass, some nutrients (such as amino acids) could release from lignocellulosic materials and that was favorable to cell growth (Lau and Dale, 2009). As a whole, the acquired high cell density by MIRB favored the consumption of both hexose and pentose sugars in the hydrolysates. The simultaneous fermentation of glucose, xylose and arabinose was successfully realized. In addition, the cells were reused during the MIRB. The cells may be adapted to the hydrolysates, so high fermentation efficiency could be attained. Many microorganisms had been employed to produce LA from lignocellulosic hydrolysates containing mixed sugar, including Rhizopus oryzae (Bai et al., 2008), Lactobacillus brevis (Guo et al., 2010), Lactobacillus pentosus (Bustos et al., 2005, 2007; Moldes et al., 2006), B. coagulans (Bischoff et al., 2010; Ouyang et al., 2012a) and Bacillus sp. 17C5 (Patel et al., 2004). The highest LA productivity of 3.10 g/L/h was achieved by L. pentosus from vine shoots hydrolysates in a continuous fermentation process (Bustos et al., 2007). The continuous fermentation usually led to incomplete utilization of the carbon sources. So, the subsequent separation of LA from residual sugars increased the costs of production. B. coagulans IPE22 was suitable to produce LA from mixed sugar in lignocellulosic hydrolysates due to its high yield of LA. Moreover, thermophilic B. coagulans IPE22 has robust property to resist contamination. The non-sterilized fermentation greatly simplified the operation and reduced the costs of production. It is well known that in conventional LA production process, calcium hydroxide or calcium carbonate is added during fermentation to neutralize lactic acid and maintain the pH around 5–6. A large amount of gypsum is produced in the subsequent LA extraction and purification steps, posing serious economic and environmental problems in terms of waste treatment. While in the present study, sodium hydroxide was selected as neutralizing agent to maintain pH, so the above problem could be avoided, as reported by Qin et al. (2010). Besides,
fermentation without calcium ion could reduce membrane fouling, as a result, the MIRB technology could be more practical in industrial LA production (Pal et al., 2009). In this work, by means of MIRB, the highest LA productivity of 2.35 g/L/h was obtained from wheat straw hydrolysates. The result indicated that MIRB was an efficient way to produce LA from lignocellulosic hydrolysates. Furthermore, MIRB is also expected to be applicable for other biotechnological processes based on the conversion of lignocellulosic hydrolysates containing sugar mixtures.
4. Conclusions B. coagulans IPE22 could homo-ferment mixed sugar of glucose, xylose and arabinose to LA under non-sterilized conditions. By application of MIRB strategy, the CCR feature of the strain and the feedback inhibition during fermentation were greatly mitigated because of the strain acclimation, cell reuse and product removal during fermentation. With MIRB, LA productivity was increased to 2.35 g/L/h (batch 6) from 1.01 g/L/h (batch 1) when wheat straw hydrolysates was used as carbon source. MIRB fermentation under non-sterilized conditions leads to great savings in terms of both time and labor, making it easy to scale up for industrial production. Acknowledgements The authors would like to thank the National Natural Science Foundation of China for the financial support (Grant No. 21176239) and the CAS/SAFEA International Partnership Program for Creative Research Teams. References Abdel-Rahman, M.A., Tashiro, Y., Sonomoto, K., 2013a. Recent advances in lactic acid production by microbial fermentation processes. Biotechnol. Adv. 31, 877–902. Abdel-Rahman, M.A., Tashiro, Y., Zendo, T., Sonomoto, K., 2013b. Improved lactic acid productivity by an open repeated batch fermentation system using Enterococcus mundtii QU 25. RSC Adv. 3, 8437–8445. Bai, D.M., Li, S.Z., Liu, Z.L., Cui, Z.F., 2008. Enhanced L-(+)-lactic acid production by an adapted strain of Rhizopus oryzae using corncob hydrolysate. Appl. Biochem. Biotechnol. 144, 79–85. Bischoff, K.M., Liu, S.Q., Hughes, S.R., Rich, J.O., 2010. Fermentation of corn fiber hydrolysate to lactic acid by the moderate thermophile Bacillus coagulans. Biotechnol. Lett. 32, 823–828. Bustos, G., de la Torre, N., Moldes, A.B., Cruz, J.M., Dominguez, J.M., 2007. Revalorization of hemicellulosic trimming vine shoots hydrolyzates trough continuous production of lactic acid and biosurfactants by L. pentosus. J. Food Eng. 78, 405–412. Bustos, G., Moldes, A.B., Cruz, J.M., Dominguez, J.M., 2005. Influence of the metabolism pathway on lactic acid production from hemicellulosic trimming vine shoots hydrolyzates using Lactobacillus pentosus. Biotechnol. Progr. 21, 793–798. Datta, R., Henry, M., 2006. Lactic acid: recent advances in products, processes and technologies – a review. J. Chem. Technol. Biotechnol. 81, 1119–1129.
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