2.4. Conventional continuous culture with and without ph control

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1 Continuous butanol fermentation from xylose with high cell density by cell recycling system Jin Zheng a, Yukihiro Tashiro b, Tsuyoshi Yoshida a, Ming Gao a, Qunhui Wang c, Kenji Sonomoto a,d, a Laboratory of Microbial Technology, Division of Applied Molecular Microbiology and Biomass Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Hakozaki, Higashi-ku, Fukuoka , Japan b Laboratory of Soil Microbiology, Division of Applied Molecular Microbiology and Biomass Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Hakozaki, Higashi-ku, Fukuoka , Japan c Department of Environmental Engineering, University of Science and Technology Beijing, Beijing , China d Laboratory of Functional Food Design, Department of Functional Metabolic Design, Bio-Architecture Center, Kyushu University, Hakozaki, Higashi-ku, Fukuoka , Japan highlights " ph was a significant factor in continuous fermentation of butanol. " Butanol productivity were increased dramatically by cell recycling. " Maximum butanol productivity was obtained at a dilution rate of 0.78 h 1. article info abstract Article history: Received 23 September 2012 Received in revised form 12 November 2012 Accepted 17 November 2012 Available online 28 November 2012 Keywords: Xylose Butanol fermentation Continuous culture Cell recycling Clostridium saccharoperbutylacetonicum N1-4 A continuous butanol production system with high-density Clostridium saccharoperbutylacetonicum N1-4 generated by cell recycling was established to examine the characteristics of butanol fermentation from xylose. In continuous culture without cell recycling, cell washout was avoided by maintaining ph > 5.6 at a dilution rate of 0.26 h 1, indicating ph control was critical to this experiment. Subsequently, continuous culture with cell recycling increased cell concentration to 17.4 g L 1, which increased butanol productivity to 1.20 g L 1 h 1 at a dilution rate of 0.26 h 1 from g L 1 h 1 without cell recycling. The effect of dilution rates on butanol production was also investigated in continuous culture with cell recycling. Maximum butanol productivity (3.32 g L 1 h 1 ) was observed at a dilution rate of 0.78 h 1, approximately 6-fold higher than observed in continuous culture without cell recycling (0.529 g L 1 h 1 ). Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction The world s rapidly diminishing petroleum reserves and increasing environmental concerns over the impact of petroleum fuel emissions, has made the search for alternative biofuel sources more important (Van Hecke et al., 2012). Butanol is a valuable biofuel, as it possesses many favourable physical properties, Corresponding author at: Laboratory of Microbial Technology, Division of Applied Molecular Microbiology and Biomass Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Hakozaki, Higashi-ku, Fukuoka , Japan. Tel./fax: addresses: zhengjin2811@gmail.com (J. Zheng), tashiro@agr.kyushu-u. ac.jp (Y. Tashiro), jubeat.knit.tsuyoshi@gmail.com (T. Yoshida), gaoming402@ gmail.com (M. Gao), wangqh59@163.com (Q. Wang), sonomoto@agr.kyushu-u.ac.jp (K. Sonomoto). including higher energy content, higher boiling points, and a reduced need to modify combustion engines as compared to ethanol (Tran et al., 2010). Acetone butanol ethanol (ABE) fermentation from renewable resources has been paid much attention. Agricultural residues, which consist mainly of cellulose and hemicellulose, are the most abundant renewable resource, and have great potential for butanol fermentation (Jang et al., 2012). Various carbohydrates such as glucose, fructose, mannose, sucrose, lactose, and starch can be consumed by butanol-producing strains (Kumar and Gayen, 2011). However, few studies have focused on butanol fermentation with xylose as the sole carbon source. In previous study (Shinto et al., 2008), Clostridium saccharoperbutylacetonicum N1-4 gave a higher yield of butanol from xylose (0.62 C-mol/C-mol) than from glucose (0.53 C-mol/C-mol) in batch culture, suggesting xylose is a useful substrate for ABE fermentation, although a detailed mechanism Reproduced from Bioresource Technology 129: (2013). 76

2 for the increased yield involved in metabolic pathways was not well known. The traditional batch fermentation process for butanol production suffers from 2 major problems. The first one is low butanol productivity, ranging only from 0.1 to 0.3 g L 1 h 1, which means large fermentors and long periods of the operation are required (Zhang et al., 2009). The second problem is severe product inhibition, which hinders commercial development (Green, 2011). Processes that can be operated in continuous mode provide several advantages over batch processes (Survase et al., 2011). Increased productivity was also achieved by increasing cell concentration by cell recycling or immobilization (Afschar et al., 1985; Qureshi et al., 2000). Compared to fermentors with immobilized cells, cell-recycling fermentors are advantageous due to broth homogeneity, which facilitates diffusion and total recycling of microorganisms (Ferras et al., 1986). To date, there is no report on highly productive continuous fermentation for butanol production using xylose as the substrate. In addition, the characteristics of clostridial fermentation with cell recycling for producing butanol from xylose are poorly understood. The objective of this study was to establish a continuous butanol production system with high cell density of the N1-4 strain, xylose as the sole carbon source, and cell recycling. In comparison to conventional continuous culture with free cells, the cell-recycling fermentor achieved a high dry cell weight (DCW) of ca. 18 g L 1. The dilution rate in continuous culture with cell recycling was investigated, and a maximum butanol productivity of 3.32 g L 1 h 1 was obtained at a dilution rate of 0.78 h Methods 2.1. Strain C. saccharoperbutylacetonicum N1-4 ATCC was used for the investigation (Tashiro et al., 2004). It was kept at 4 C as a suspension of spores in potato glucose (PG) medium (Lee et al., 1995). To germinate the spores, 1 ml of spore suspension was transferred aseptically to 9 ml of PG medium (10%, v v 1 ), heat-shocked for 1 min in boiling water, then incubated at 30 C for h before pre-culture (Lee et al., 1995) Culture medium Tryptone yeast extract acetate (TYA) and tryptone yeast extract (TY) media were used in this experiment. The TYA medium contained the following ingredients per litre of distilled water: 20 g or 50 g xylose, 2 g yeast extract (Difco Laboratories, Detroit, MI, USA), 6 g tryptone (Difco Laboratories), 3 g CH 3 COONH 4, 0.3 g MgSO 4 7H 2 O, 0.5 g KH 2 PO 4, and 10 mg FeSO 4 7H 2 O. TY medium was TYA medium with substitution of 2.57 g L 1 (NH 4 ) 2 SO 4 for 3 g L 1 CH 3 COONH 4 (Oshiro et al., 2010). Xylose (20 g L 1 )in TYA medium and 50 g L 1 xylose in TY medium were used for pre-culture, and for main culture and feeding medium, respectively. Antifoam Y-30 emulsion (0.2 ml L 1 ; Sigma Aldrich Inc., Louis, MO, USA) was added to the feeding medium. In all experiments, the initial ph was adjusted to 6.5 (Baba et al., 2012; Oshiro et al., 2010; Shinto et al., 2008; Tashiro et al., 2004, 2005) by 2.5 M KOH prior to sterilization. The medium was sterilized at 115 C for 15 min Hollow fibre module for cell recycling In continuous butanol production with cell recycling, a hollow fibre module (MICROZA PMP-102; Asahi Kasei, Tokyo, Japan) was used to transfer cells without broth to the fermentor. The filtration area of this module is 0.17 m 2. Diameter and pore diameter of every fibre are 0.7 mm and 0.25 lm, respectively. Before use, the module was sterilized with 70% ethanol for >24 h, and then washed with sterilized distilled water (Baba et al., 2012). After the experiment, the module was washed with sterilized distilled water, and stored in 1 M NaOH Conventional continuous culture with and without ph control The N1-4 strain was pre-cultured anaerobically and statically for 15 h at 30 C in a flask with a 100-mL total working volume that included 10% inoculum. After pre-culture, the main culture was performed in a 1-L jar fermentor with a 0.4-L working volume that included 10% pre-cultured broth. Water at 30 C was circulated through the fermentor to control temperature with a circulating water bath. Following inoculation, the broth was sparged with filtered oxygen-free nitrogen gas to maintain a strict anaerobic condition. Before the continuous culture, the batch culture was performed statically for 36 h, when the solventogenesis phase occurred, and then, continuous culture was initiated at an agitation rate of rpm. Dilution rates were set at 0.14 h 1, 0.20 h 1, and 0.26 h 1. During all experiments, dilution rates were changed after steady state had been achieved. The system was considered to be in steady state when butanol production was constant, and/or varied no more than 10% of the butanol titer, for at least three retention times. In total, samples from the fermentor at steady state were withdrawn 4 times at every dilution rate. In the ph control experiment, the device for ph control consisted of a ph-metre, a peristaltic pump, a vessel with KOH solution, and a ph controller (PHC-2201; Able, Tokyo, Japan). Broth ph was controlled automatically by the ph controller with 2.5 M KOH to be > Continuous culture with cell recycling A schematic diagram for continuous culture production is shown in Fig. 1. Before continuous culture, refresh culture and pre-culture were performed, and then the N1-4 strain was cultivated in batch mode for 36 h in a 5-L jar fermentor with a 4-L working volume. After 36 h cultivation, the broth was gradually transferred into a 1-L jar fermentor. During transfer, the concentration of cells in the 1-L jar fermentor was achieved by recirculation through the hollow fibre module, and the permeate from the module was collected spontaneously. Finally, 4 L of the broth in the 5-L jar fermentor was concentrated to 0.4 L in a 1-L jar fermentor. After cell concentration, continuous culture was initiated with feeding TY medium at an agitation rate of rpm with cell recycling. Broth ph was maintained >5.6 by the ph controller with 10 M KOH. In continuous culture by cell recycling, the inflow rate of the feeding medium was balanced to the same rate of outflow of permeated solution from the module by the pump. Samples were collected at regular intervals Analytical methods The collected samples were centrifuged at 17,800 g for 10 min. The supernatant was used for measurements of solvents (ABE), xylose, and organic acids. Cell density was estimated by measuring the optical density (OD) of the suspension at 562 nm in a spectrophotometer (V-530; JASCO, Tokyo, Japan); an OD of 1.0 was equivalent to g DCW per litre (Oshiro et al., 2010). The concentration of xylose was determined by high-performance liquid chromatography (US HPLC-1210; JASCO, Tokyo, Japan) using an RSpak KC-811 column (Shodex, Tokyo, Japan) at 40 C. The mobile phase was 0.1% HClO 4 at a flow rate of 1.0 ml min 1. The concentrations of acids and solvents were determined by gas 77

3 ON OFF ON OFF 8 Fig. 1. Fermentation setup for the continuous culture with cell recycling: (1) 1-L jar fermentor; (2) 5-L jar fermentor; (3) hollow fibre module; (4) ph controller; (5) ph electrode; (6) agitator for 1-L jar fermentor; (7) agitator for 5-L jar fermentor; (8) magnetic stirrer; (9) pump; (10) permeate reservoir; (11) potassium hydroxide solution reservoir; (12) fresh medium reservoir. chromatography (6890A; Agilent Technologies, Palo Alto, CA, USA) as described previously (Tashiro et al., 2005). Kinetic parameters were expressed using the following definitions: Dilution rate (h 1 ) was defined as the feed flow rate (ml h 1 ) of the feeding medium divided by the working fermentor volume (ml). Butanol productivity (g L 1 h 1 ) was calculated as butanol produced (g L 1 ) multiplied by the dilution rate (h 1 ). 3. Results and discussion 3.1. Effect of ph control and dilution rate on butanol production from xylose by conventional continuous culture Various carbohydrates can be consumed by clostridia (Kumar and Gayen, 2011). D-Xylose is the second most abundant sugar in lignocellulose after glucose (Yanase et al., 2007), and batch culture using xylose as the substrate has been reported for butanol production by N1-4 (Shinto et al., 2008). Therefore, conventional continuous culture using xylose without cell recycling was carried out and the effect of dilution rates and ph on butanol production was also investigated. Conventional continuous cultures were performed at dilution rates of 0.14 h 1, 0.20 h 1, and 0.26 h 1. As shown in Table 1, maximum xylose consumption of 6.46 g L 1 was obtained without ph control at the dilution rate of 0.20 h 1, resulting in butanol concentration and DCW of 1.36 g L 1 and 1.25 g L 1, respectively. Meanwhile, the maximum butanol productivity of g L 1 h 1 was observed at a dilution rate of 0.20 h 1. When increasing dilution rate from 0.20 h 1 to 0.26 h 1, DCW drastically fell to g L 1, as did butanol concentration, which decreased to g L 1.It was speculated that cell washout might occur at the dilution rate of 0.26 h 1. It is noteworthy that the ph in fermentation broth without ph control fell to approximately 4.6 from the 6.5 of the feeding medium. Previous work showed the optimum ph to stimulate N1-4 growth and butanol production was approximately 5.6 in batch culture (Table 1). Therefore, ph was maintained above 5.6 in subsequent conventional continuous cultures. Continuous culture with ph control sustained the ph of the broth at approximately 5.6 during fermentation at all dilution rates. As expected, the ph-controlled continuous cultures exhibited higher butanol concentration, DCW, and xylose consumption than the ph-uncontrolled continuous cultures at each dilution rate (Table 1). In particular, the maximum respective values of 2.63 g L 1, 2.78 g L 1, and 15.1 g L 1 were obtained at a dilution rate of 0.20 h 1 with ph control, approximately 2-fold higher than without ph control at the same dilution rate (Table 1). In contrast, the maximum butanol productivity increased from g L 1 h 1 in ph-uncontrolled culture at a dilution rate of 0.20 h 1 to g L 1 h 1 in ph-controlled culture at a dilution rate of 0.26 h 1. Increasing the dilution rate from 0.20 h 1 to 0.26 h 1 still maintained DCW above 2.2 g L 1. Thus, cell washout could be avoided by ph control even at a dilution rate of 0.26 h 1 in conventional continuous culture. In addition, under ph control, butanol productivity increased with increasing dilution rate from 0.14 h 1 Table 1 Kinetic parameters of butanol production by conventional continuous culture using xylose with and without ph control. D (h 1 ) ph DCW (g L 1 ) C xylose a (g L 1 ) C butanol b (g L 1 ) P butanol c (g L 1 h 1 ) Without ph control (Batch) 4.83 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± With ph control (Batch) 6.12 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Conventional continuous culture was conducted in a 1-L jar fermentor of 0.4 L working volume at 30 C, feeding with TY medium containing 50 g L 1 xylose. In the phcontrolled continuous culture, ph was maintained above 5.6 with 2.5 M KOH. The dilution rate (D) was set at 0.14 h 1, 0.20 h 1, or 0.26 h 1. a Xylose consumption. b Butanol concentration. c Butanol productivity. 78

4 to 0.26 h 1. It was concluded that ph control in the broth was a significant factor for cell growth and butanol production in continuous culture using xylose; therefore, ph control was conducted in subsequent experiments Continuous culture with cell recycling using xylose as carbon source The hollow fibre module can retain cells in the fermentor; thus, integration of the module can provide high cell density by cell recycling while maintaining continuous culture at higher dilution rates (Qureshi and Ezeji, 2008). Most reports have used glucose as the carbon source for butanol production in continuous culture by cell recycling (Afschar et al., 1985; Ferras et al., 1986; Pierrot et al., 1986). Therefore, it was aimed to establish a bioreactor with high butanol productivity from xylose by cell recycling and to obtain a better understanding of the characteristics of fermentation. Initially, the dilution rate was set at 0.26 h 1 with ph maintained at >5.6 as compared with the continuous culture without cell recycling. Cell recycling allowed the overall DCW to be maintained at 17.4 g L 1 during fermentation (Fig. 2 and Table 2); this was 8-fold higher than in conventional continuous culture (2.26 g L 1, Table 1), but much lower than culture cell recycling with glucose as the substrate (>100 g L 1 )(Tashiro et al., 2005), because the N1-4 strain should prefer glucose for its growth to xylose. In addition, overall xylose consumption and butanol concentration increased to 20.2 g L 1 and 4.68 g L 1 (Table 2) from 11.2 g L 1 and 2.03 g L 1 without cell recycling (Table 1). Approximately 2-fold higher butanol productivity of 1.22 g L 1 h 1 was obtained in continuous culture with cell recycling, compared to g L 1 h 1 in conventional culture. Fig. 2 shows the time course profiles of ABE production, acid production, and xylose concentration in the ph-controlled continuous culture by cell recycling at dilution rate of 0.26 h 1. A decrease in xylose concentration tended to be associated with increases in total production of butanol, acetone, acetic acid, and butyric acid, while ethanol concentration was invariably low (<0.3 g L 1 ) throughout fermentation. In addition, a slight fluctuation in DCW and concentrations of products and substrate appeared in continuous culture with cell recycling even after more than 3 retention times. Similar types of fluctuations were reported for Clostridium acetobutylicum ATCC 824 in continuous culture with cell recycling by ultrafiltration (Ferras et al., 1986). Furthermore, it seems that solvent production (i.e. butanol and acetone) correspond to acid production (i.e. acetic acid and butyric acid) adversely. The early period of continuous culture (50 70 h) was characterised by acidogenesis, because total acid production was higher than solvent production; solventogenesis was observed in the late period ( h of cultivation). Cells fermenting ABE are generally composed of a mixture of actively dividing cells (acidogenic), non-dividing cells (solventogenic), sporulated cells, and dead cells (Ezeji et al., 2007). Therefore, the oscillatory behaviour of solvent and acid production during continuous culture with cell recycling indicates that actively dividing and non-dividing cells were dominant at the early and late phases of the culture, respectively. Thus, continuous culture with cell recycling was an efficient method to increase DCW, which could improve butanol concentration and productivity from xylose Continuous culture with cell recycling using xylose at different dilution rates The effect of dilution rates on butanol productivity was investigated in ph-controlled (>5.6) continuous culture using xylose with cell recycling. Compared to the conventional continuous cultures (A) Solvents (g L -1 ) (B) DCW and xylose(g L -1 ) (C) Acids (g L -1 ) Time (h) Time (h) Time (h) Fig. 2. Time course of continuous culture with cell recycling using xylose at a dilution rate of 0.26 h 1. Batch culture was performed for 36 h; then, cells were concentrated for ca. 4 h. Continuous culture by cell recycling was performed in a 1-L jar fermentor with 0.4 L working volume at 30 C, feeding with TY medium containing 50 g L 1 xylose. ph was maintained above 5.6 with 10 M KOH. The dilution rate was set at 0.26 h 1. Profile (A) shows acetone, butanol, and ethanol; (B) shows DCW and xylose; (C) shows acetate and butyrate. d, butanol; N, acetone; j, ethanol;, DCW; D, xylose; s, acetate; e, butyrate. without cell recycling, the dilution rates could be increased from 0.26 h 1 to 0.52 h 1, 0.78 h 1, and 0.85 h 1 in continuous culture because cell washout was eliminated by cell recycling. Table 2 shows the overall values of butanol productivity, xylose utilization, butanol concentration, and ratio of butanol to total solvent using xylose at each dilution rate. With cell recycling, the butanol concentrations at all dilution rates were >3.69 g L 1 (Table 2), higher than without cell recycling (Table 1). As the dilution rate increased from 0.26 h 1 to 0.85 h 1, 79

5 Table 2 Effect of dilution rates on butanol production from xylose in continuous culture by cell recycling. D (h 1 ) DCW (g L 1 ) C xylose a (g L 1 ) C butanol b (g L 1 ) R butanol c (g g 1 ) P butanol d (g L 1 h 1 ) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.59 Continuous culture by cell recycling was performed in a 1-L jar fermentor with 0.4 L working volume at 30 C, feeding with TY medium containing 50 g L 1 xylose. ph was maintained above 5.6 with 10 M KOH. Cells were recycled by a hollow fibre module after initiating continuous culture. The dilution rate (D) was set at 0.26h 1, 0.52 h 1, 0.78 h 1, or 0.85 h 1. a Xylose consumption. b Butanol concentration. c Butanol/total solvent. d Butanol productivity. Table 3 Comparison of butanol production by different continuous cultures using different carbon sources with free cells, cell recycling, or immobilization. Fermentation mode Strains Carbon source D a (h 1 ) C butanol b (g L 1 ) P butanol c (g L 1 h 1 ) Reference Free cells C. saccharoperbutylacetonicum N1-4 Xylose This study Free cells C. saccharoperbutylacetonicum N1-4 Glucose Tashiro et al., 2005 Free cells C. acetobutylicum ATCC 824 Glucose + glycerol Andrade and Vasconcelos, 2003 Free cells C. beijerinckii BA 101 Starch d 0.2 e Ezeji et al., 2005 Cell recycling C. saccharoperbutylacetonicum N1-4 Xylose This study Cell recycling C. saccharoperbutylacetonicum N1-4 Glucose d 7.34 Tashiro et al., 2005 Cell recycling C. acetobutylicum P262 Lactose Ennis and Maddox, 1989 Cell recycling C. acetobutylicum DSM 792 Starch d 3.3 e Afschar and Schaller, 1991 Immobilization C. acetobutylicum DSM 792 Glucose d e Survase et al., 2012 Immobilization C. saccharobutylicum NCP 262 Lactose Qureshi and Maddox, 1995 Immobilization C. saccharobutylicum NCP 262 Starch Badr et al., 2001 a b c d e Dilution rate. Butanol concentration. Butanol productivity. ABE concentration. ABE productivity. there was a slight decrease in butanol concentration from 4.68 g L 1 to 3.69 g L 1. At all dilution rates, DCW was maintained at approximately 18 g L 1, and xylose consumption did not significantly differ (>ca. 20 g L 1 ). On the other hand, increasing the dilution rate from 0.26 h 1 to 0.78 h 1 increased butanol productivity, while a further increase from 0.78 h 1 to 0.85 h 1 led to a slight decrease. Maximal butanol productivity of 3.32 g L 1 h 1 was obtained at a dilution rate of 0.78 h 1. Note that this value was approximately 6-fold higher than the g L 1 h 1 for maximum butanol productivity in conventional continuous culture without cell recycling (Table 1). Meanwhile, this value was 18-fold higher than maximum butanol productivity of g L 1 h 1 in batch culture with ph control (Table 1). That means a highly efficient continuous fermentation for butanol production from xylose was achieved. In addition, it is interesting to note that the ratio of butanol to total solvent increased from to with increasing dilution rates from 0.26 h 1 to 0.85 h 1. This result indicates that metabolic fluxes of butanol production were dependent on the dilution rate, although a detailed mechanism for the alteration involved in solvent production fluxes is not well known. The ratio of butanol to total solvent in this study was also higher than that (0.63) in continuous culture with glucose and immobilized C. acetobutylicum (Yen and Li, 2011). Therefore, this study has advantages because a higher butanol ratio leads to easier separation from the culture. Table 3 shows the results of different continuous culture systems for butanol production using various carbon sources (xylose, glucose, glycerol, starch, lactose). There has been no report on continuous butanol fermentation using xylose as the sole carbon source without and with high cell density by either cell recycling or immobilization. Compared with butanol productivity in continuous culture using glucose by free (1.24 g L 1 h 1 ) and highdensity (7.34 g L 1 h 1 ) N1-4 cells, the values (0.529 g L 1 h 1 and 3.32 g L 1 h 1 ) in continuous culture using xylose were low. Except for glucose, continuous culture with xylose and high cell density by cell recycling showed the highest butanol productivity (3.32 g L 1 h 1 ) among other carbon sources and systems published to date. Therefore, a highly productive culture system for butanol production using xylose has been established. 4. Conclusions A continuous butanol production system by cell recycling N1-4 using xylose as the sole carbon source was investigated. Compared with conventional continuous culture, a DCW of 17.4 g L 1 was obtained by cell recycling, which led to a 2-fold increase in xylose consumption, butanol concentration and productivity. Furthermore, the dilution rate in continuous culture with cell recycling was optimized. 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