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Ethanol production by continuous fermentation of d -(+)-cellobiose, d -(+)-xylose and sugarcane bagasse hydrolysate using the thermoanaerobe Caloramator boliviensis
Bioresource Technology 103 (2012) 186–191
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Ethanol production by continuous fermentation of D-(+)-cellobiose, D-(+)-xylose and sugarcane bagasse hydrolysate using the thermoanaerobe Caloramator boliviensis Carla F. Crespo, Malik Badshah, Maria T. Alvarez 1, Bo Mattiasson ⇑ Department of Biotechnology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden
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Article history: Received 27 June 2011 Received in revised form 19 September 2011 Accepted 6 October 2011 Available online 14 October 2011 Keywords: Pentose fermentation Lignocellulose hydrolysate Caloramator Bioethanol Continuous fermentation
a b s t r a c t The recently isolated anaerobic bacterium Caloramator boliviensis with an optimum growth temperature of 60 °C can efficiently convert hexoses and pentoses into ethanol. When fermentations of pure sugars and a pentose-rich sugarcane bagasse hydrolysate were carried out in a packed bed reactor with immobilized cells of C. boliviensis, more than 98% of substrates were converted. Ethanol yields of 0.40–0.46 g/g of sugar were obtained when sugarcane bagasse hydrolysate was fermented. These features reveal interesting properties of C. boliviensis in producing ethanol from a renewable feedstock. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Technology for production of ethanol from lignocellulosic feedstock is expected to become mature in the next 5–10 years and partly replace first generation ethanol. The importance of lignocellulose-based ethanol stems from the expected possibility to use assumed inexpensive feedstock, avoid direct and indirect competition with supply of human food and animal feed and to reduce environmental risks such as pollution and soil degradation which are generally associated to biofuels of the first generation (Gnansounou and Dauriat, 2010). Dry lignocellulosic feedstock generally consists of 35–50% cellulose, 20–35% hemicellulose and 10–25% lignin. Conversion of lignocellulose to higher value products requires fractionation of the material into its main components. Lignocellulosic biomass, such as sugarcane bagasse (SCB), which is a sugar production residue, allows the possible integration of second and first generation bioethanol (Ojeda et al., 2011). The efficient utilization of SCB which contains 40–45% cellulose, 30–35% hemicelluloses and 20–30% lignin (Peng et al., 2009) offers
⇑ Corresponding author. Tel.: +46 46 2228264; fax: +46 46 2224713. E-mail addresses: [email protected] (C.F. Crespo), Malik.Badshah@bio tek.lu.se (M. Badshah), [email protected] (M.T. Alvarez), Bo. [email protected] (B. Mattiasson). 1 Present address: Instituto de Investigaciones Fármaco Bioquímicas, Facultad de Ciencias Farmacéuticas y Bioquímicas, Universidad Mayor de San Andrés, P.O. Box 3239, La Paz, Bolivia. 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.10.020
an opportunity to reduce the cost of ethanol production provided that suitable fermentative organisms are available. While hexosefermenting organisms, such as Saccharomyces cerevisiae, are common, pentose-fermenting organisms are rare. A number of genetically engineered ethanol-producing strains capable of metabolizing xylose and other pentose sugars into ethanol have been developed (Martín et al., 2007; Yao and Mikkelsen, 2010; Zaldivar et al., 2001), but a common problem with these organisms is their sensitivity to inhibitors present in undetoxified hydrolysates (Dien et al., 2003). Thus, two important requirements for an efficient ethanol-producing microorganism are to ferment a variety of sugars (pentoses and hexoses) and to tolerate stress conditions (Zaldivar et al., 2001). Some anaerobic thermophilic bacteria are potential microorganisms for the production of ethanol due to their capability to metabolize a wide spectrum of sugars found in lignocellulose. Additionally, several advantages are associated with the production of ethanol at high temperatures, e.g. high bioconversion rates, reduced risk of contamination, and facilitated product recovery. However, bioconversion of lignocellulosic hydrolysates by thermoanaerobes has been studied only to a limited extent and mostly in batch fermentations (Georgieva and Ahring, 2007). For economic reasons, continuous fermentations are sometimes used for commercial production of ethanol from corn and sugarcane, since higher volumetric productivities can be achieved and time for emptying, cleaning and filling vessels and labor work can be minimized. In addition, continuous processing could be beneficial in
C.F. Crespo et al. / Bioresource Technology 103 (2012) 186–191
adapting fermentative organisms to inhibitors generated during biomass pretreatment (Brethauer and Wyman, 2010). Higher cell densities in a continuous than in a batch fermentation system could enable higher productivities and yields (Taniguchi et al., 1983). In a previous study, the thermoanaerobe Caloramator boliviensis strain 45BT was isolated, characterized and found to be efficient in fermenting a wide spectrum of substrates and producing ethanol in batch fermentations (Crespo et al., 2011). Therefore, the main objective of the present study was to evaluate the ability of the organism to Ferment D-(+)-cellobiose, D-(+)-xylose and pentose-rich SCB hydrolysate using immobilized C. boliviensis in an anaerobic packed bed reactor operated at 60 °C. Hexose and pentose utilization under continuous operational mode and the effect of substrate loading rate (LR) and hydraulic retention time (HRT) were studied. 2. Methods 2.1. Microorganism and cultivation C. boliviensis strain 45BT (T = Type strain) (Crespo et al., 2011) was maintained in serum bottles under anaerobic conditions at 4 °C. Inocula were prepared by cultivating C. boliviensis (2%, v/v) in 100-mL serum vials containing 70 mL of anaerobic mineral medium (AMM) as described by Angelidaki et al. (1990) with the following modifications: NH4Cl was omitted and instead 1 g/L of urea was supplemented as nitrogen source. The trace element solution was replaced with 1 mL of (g/L of deionized water): FeCl24H2O, 1.50; H3BO3, 0.06; CoCl26H2O, 0.12; MnCl24H2O, 0.10; Na2MoO42H2O, 0.02; NiCl26H2O, 0.02; ZnCl2, 0.07; CuCl22H2O, 0.01; Na2SeO3, 0.003; NaOH, 0.5 and HCl 25%, 6.5 mL. The medium was amended with 1 g/L of yeast extract. To ensure anaerobic conditions before autoclaving (121 °C for 20 min), the medium was prepared under an O2-free N2 stream using the modified Hungate technique (Ljungdahl and Wiegel, 1986). After autoclaving and prior to inoculation, the medium was reduced using a filter-sterilized anaerobic solution of Na2S9H2O to a final concentration of 0.5 g/L, and supplemented with a filter-sterilized solution of D-(+)-cellobiose (5 g/L) and a vitamin solution (DSMZ medium No. 141, German Collection of Microorganisms and Cell Cultures) (10 mL/L). The pH of the medium was adjusted to 7.0 ± 0.2 with H2SO4 (0.5 M). For continuous fermentation, AMM was used as synthetic medium. A sterile stock solution (100 g/L) of D-(+)-cellobiose was added to final concentrations of 5.0 ± 0.1 g/L and 7.5 ± 0.2 g/L (operational conditions 1 and 2, respectively; Table 1). Subsequently, cellobiose as a substrate was replaced by D-(+)-xylose to final concentrations of 5.0 ± 0.2 g/L and 7.5 ± 0.4 g/L (operational conditions 3–5; Table 1). AMM was amended with SCB hydrolysate to final concentrations of 5.5 ± 0.1 g/L and 8.9 g/L of total sugars (operational conditions 6 and 7, respectively). The pH of the medium was adjusted to 7.0 ± 0.5 with 0.5 M H2SO4 or 1 M NaOH. The pentose-rich SCB hydrolysate was prepared by SO2-catalyzed steam pretreatment according to Carrasco et al. (2010) and supplied by the Department of Chemical Engineering, Lund University. The pentose-rich hydrolysate with low concentration of byproducts (e.g. HMF, furfural, etc.) was used to evaluate xylose to ethanol conversion. The initial SCB hydrolysate composition was (in g/L): cellobiose, 1.5; glucose, 1.5; xylose, 22.2; mannose, (traces); arabinose, (traces); acetic acid, 4.1; 5-(Hydroxymethyl)furfural (HMF), 0.1 and furfural, 0.5. 2.2. Reactor operation The fermentation set-up (Fig. 1) consisted of a water-jacketed glass column (25 cm height and 5.5 cm inner diameter), packed
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with coated plastic carriers 0.7 1 cm in size (AnoxKaldnes, Sweden). Polyethylenimine of medium molecular weight (1%) was utilized for coating the plastic carriers as described by Senthuran et al. (1997). The packed glass column was connected to a water jacketed vessel at both ends. The vessel was equipped with a stirring device, a pH electrode connected to a control unit, a port for gas release and a port for pumping in fresh medium. The influent in the glass column entered from the bottom of the reactor. Recirculation of medium between both units was achieved by a peristaltic pump to ensure an up-flow velocity of 1 m/h. The reactor was maintained at 60 °C for continuous fermentation, and the pH was maintained at 7.0 ± 0.5 by automatic addition of 1 M NaOH to the water jacketed vessel, the pH adjusted medium was recirculated into the glass column reactor. The total working volume of the system was 680 mL. The entire reactor system, including the plastic carriers and tubings was autoclaved at 120 °C for 20 min. Before operation, the reactor system was flushed with O2-free N2 gas for 15 min in order to maintain anaerobic conditions. The reactor system was filled with AMM supplemented with 5.0 ± 0.1 g/L of D-(+)-cellobiose as sole carbon source. The reactor was started in batch operational mode by inoculating 100 mL of a cell suspension of C. boliviensis with an optical density (OD620) of 1.0 ± 0.3 to achieve cell enrichment and immobilization. The batch operational mode was maintained for 5 d and successively changed into seven other operational conditions (Table 1). Thus, the system was shifted to continuous operational mode by feeding D-(+)-cellobiose containing medium at a low LR and extended HRT to avoid cell detachment and wash out. Subsequently, a second condition was set to achieve higher fermentation rate of D-(+)-cellobiose. A third condition was applied by replacing D-(+)-cellobiose with D-(+)-xylose. Conversion of xylose at a higher rate was evaluated in a fourth operational condition. Since instability in the reactor performance was observed in the last stage, a fifth condition was applied by slightly increasing the HRT and decreasing the LR (Table 1). Consecutively, and after achieving xylose adaptation, the continuous fermentation of the pentose-rich SCB hydrolysate was evaluated. The sixth operational condition was set at constant HRT and slightly increased LR. The concentration of fermentable substrates in the hydrolysate-containing medium was (in g/L): cellobiose, 0.3; xylose, 4.9; glucose, 0.3 and acetate, 0.9. As the last operational condition, the HRT was as in the previous operational mode, the LR was increased (Table 1), and the concentration of fermentable substrates in the hydrolysate-containing medium was (in g/L): cellobiose, 0.5; xylose, 7.9; glucose, 0.5 and acetate, 1.5. During all experiments, the operational conditions (HRT and LR) were modified after steady state had been achieved. The system was considered to be in steady state when ethanol production was constant, and/or varied no more than 10% of the ethanol titer (standard deviation no larger than 0.02 g/L), for at least three retention times. Operating time for each operating condition was as detailed in Table 1. Liquid and gas samples were collected at regular time intervals and analyzed in duplicate for quantification of residual substrate and fermentation products. 2.3. Analytical methods Samples containing D-(+)-glucose, D-(+)-xylose, D-(+)-cellobiose and fermentation products (ethanol, acetate, succinate, formate, propionate, butyrate and lactate) were acidified with 20% H2SO4 (10 lL/mL of sample) and filtered through a 0.45 lm polypropylene membrane. Thereafter, samples were analyzed by high pressure liquid chromatography (JASCO Corporation, Tokyo, Japan) on an HPX-87H ion-exchange column (Biorad Laboratories Inc.,
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Table 1 Operational conditions for continuous fermentation of cellobiose, xylose and SCB hydrolysate by C. boliviensis. Operational conditions
Type of substrate
1 2 3
D-(+)-Xylose
5.0 ± 0.2
34
0.15
288
4
D-(+)-Xylose
7.5 ± 0.4
21
0.36
456
5
D-(+)-Xylose Xylose, cellobiose and glucose from SCB hydrolysate Xylose, cellobiose and glucose from SCB hydrolysate
5.0 ± 0.4
23
0.21
432
5.5 ± 0.1 8.9 ± 0.0
23 23
0.23 0.38
132 120
6 7
Substrate concentration (g/L)
Hydraulic retention time (h)
Loading rate (g/L h)
Operating time (h)
D-(+)-Cellobiose
5.0 ± 0.1
162
0.03
408
D-(+)-Cellobiose
7.5 ± 0.2
53
0.14
480
Gas collector pH controller
Outlet Sampling port
Gas collector
Sampling port Alkali
Medium
Fig. 1. Diagram of reactor set-up for ethanol production by immobilized C. boliviensis.
other solvents (Weimer and Zeikus, 1977; Zeikus et al., 1981), and only a few studies have been carried out with pretreated lignocellulosic feedstock (Ahring et al., 1996; Klinke et al., 2001; Sommer et al., 2004; Georgieva and Ahring, 2007; Georgieva et al., 2008). Microorganisms with high ethanol yields do not necessarily ferment pretreated lignocellulosic material because of the presence of inhibitory compounds. Evaluating the fermentation of lignocellulosic hydrolysates is crucial since this type of feedstock is envisioned for commercial ethanol production (Zaldivar et al., 2005), the capacity of C. boliviensis sp. nov., to ferment hexoses, pentoses and a pentose-rich SCB hydrolysate in a continuous process was evaluated. The reactor performance in terms of production of ethanol and other products, residual substrate, and type of substrate (hexoses
Hercules, CA, USA) at 55 °C using 5 mM sulfuric acid as eluent with a flow rate of 0.6 mL/min and detected with a refractive index detector (Erc Inc., Saitama, Japan). Carbohydrates, furfural and hydroxymethyl furfural were quantified as described by Sluiter et al. (2008). Determination of carbon dioxide was performed by gas chromatography as described by Parawira et al. (2008).
3. Results and discussion Thermophilic Clostridia growing optimally at 60–65 °C have been recognized for several decades as interesting candidates for processes involving conversions of pure sugars to ethanol and 5
1 Soluble products (g/L)
4
2 Succinic acid (g/L) Acetic acid (g/L) Ethanol (g/L)
3
4
Lactic acid (g/L) Propionic acid (g/L)
5
6
Formic acid (g/L) Butyric acid (g/L)
3
2
1
0 0
15
30
45
60
75
Time (days) Fig. 2. Product profile of C. boliviensis during continuous fermentation.
90
7
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C.F. Crespo et al. / Bioresource Technology 103 (2012) 186–191 Table 2 Summary of fermentation parameters at seven operational conditions of continuous fermentation of cellobiose, xylose or SCB hydrolysate by C. boliviensis. Operational condition
EtOH (g/L)
Ace (g/L)
Form (g/L)
Prop (g/L)
Succ (g/L)
Buty (g/L)
Lac (g/L)
Y CO2 a (mol/mol)
CCelb (%)
CXylc (%)
CGlud (%)
CSe (%)
YEtOHf (g/g)
TYEtOHg (%)
CEh (%)
CRi (%)
1 2 3 4 5 6 7
1.7 ± 0.2 2.9 ± 0.1 1.9 ± 0.1 2.9 ± 0.2 1.7 ± 0.1 2.2 ± 0.1 4.1 ± 0.1
1.2 ± 0.1 2.1 ± 0.2 1.3 ± 0.1 1.6 ± 0.2 1.3 ± 0.1 2.4l ± 0.2 3.9m ± 0.2
1.0 ± 0.1 1.4 ± 0.3 1.1 ± 0.1 2.0 ± 0.3 1.3 ± 0.1 1.3 ± 0.3 0.5 ± 0.0
ND 0.2 ± 0.0 ND ND ND ND 0.3 ± 0.2
0.2 ± 0.1 ND ND 0.1 ± 0.1 ND ND ND
0.1 ± 0.1 ND ND ND ND ND ND
ND 0.1 ± 0.1 ND ND ND ND ND
1.0 0.9 0.4 0.3 0.4 0.3 0.3
100 98.7 0 0 0 100 100
0 0 100 98.7 100 100 100
0 0 0 0 0 100 100
100 98.7 100 98.7 100 100 100
0.34 0.39 0.38 0.39 0.34 0.40 0.46
67 77 75 77 67 78 90
67 76 75 76 67 78 90
98.9 102.5 100 98 97.6 98.7j 97.8k
ND: not detected. a Yield based on the production of CO2 per mol of hexose or pentose unit converted. b Consumed cellobiose. c Consumed xylose. d Consumed glucose. e Total substrate consumed. f Ethanol yield based on sugars consumed. g Ethanol yield given as a percentage of theoretical possible yield of 0.51 g/g. Theoretical yields are assumed according to stoichiometrical balances, for hexoses: C6H12O6 ? 2 CH3CH2OH + 2 CO2 and for pentoses: C5H10O5 ? 1.67 CH3CH2OH + 1.67 CO2 (Zhao et al., 2010). h Conversion efficiency calculated by dividing the ethanol yield based on sugar concentrations present in the influent by theoretical possible yield of 0.51 g/g. i Carbon recovery percentage. j Mannose (0.6 10 3 mol) and arabinose (2 10 3 mol) were considered to estimate carbon recovery percentage. k Mannose (0.7 10 3 mol) and arabinose (2 10 3 mol) were considered to estimate carbon recovery percentage but are not shown in the stoichiometric balance due to their minor amounts. l Acetate production was 1.5 g/L and influent concentration was 0.9 g/L. m Acetate production was 2.4 g/L and influent concentration was 1.5 g/L.
or pentoses), under seven operational conditions is summarized in Fig. 2 and Table 2. Ethanol was the main product, and its yield ranged from 0.34 to 0.39 (gram of ethanol per gram of sugar consumed) when cellobiose (operational conditions 1 and 2) or xylose (operational conditions 3 and 4) were fermented. However, when the HRT was the shortest (21 h) and the LR was the highest (0.36 g/L h) (fourth operational condition), a slight instability in the reactor performance was perceived since xylose was detected in the effluent. Despite of this occurrence, the ethanol concentration in the effluent remained constant. Ethanol yields under conditions 2–4 were stable and were the highest when fermenting pure sugars. The ethanol yield achieved in the first operational condition was 67% with respect to the assumed theoretical possible yield of
0.51 g ethanol/g sugar consumed. However, higher values were achieved under other operational conditions when cellobiose or xylose were utilized as substrate, yielding 77%, 75% and 77% under conditions 2–4, respectively (Table 2). These results show that C. boliviensis, is among the few species of wild type bacteria, fungi and yeast that can naturally convert hexoses (cellobiose) and pentoses (xylose) into ethanol with high yields in a continuous fermentation process. Even though high yields were achieved with low initial sugar concentrations (5 ± 0.2 g/L and 7.5 ± 0.4 g/L), it is extremely important to evaluate the fermentation process with higher influent sugar concentrations (>10% of total sugars) since ethanol titer as well as yield are the main considerations for commercial fermentations. A comparison of ethanol yields of
Table 3 Comparison of ethanol yields of thermophilic bacterial strains and engineered Saccharomyces, Zymomonas, Escherichia coli and naturally occurring yeast strains. Organism
Genotype
Fermentation substrate
Fermentation mode
YEtOHa (g/g)
Titer (g/L)
Ref.
Thermoanaerobacter mathranii BG1L1 Thermoanaerobacter mathranii BG1L1 Thermoanaerobacter saccharolyticum ALK2 Geobacillus thermoglucosidasius TM242
Dldh
Mixed sugars
Continuous
0.39–0.42
14.4
Dldh
Wet exploded wheat straw Xylose
Continuous
0.39–0.42
3.9
Continuous
0.46
37
Georgieva et al. (2008) Georgieva and Ahring (2007) Shaw et al. (2008)
Zymomonas mobilis CP4:pZB5
Batch Batch Batch Batch
0.42 0.35 0.47 0.48
14.4 12.3 14.8 23
Saccharomyces cerevisiae 1400
Carrying genes for xylose metabolism from E. coli XR, XD, XK
Glucose Xylose Cellobiose Xylose
Batch
0.46
47.9
E. coli FBR5 Caloramator boliviensis
Dldh, Dpfl Wild type
Pichia stipitis PXF58
UV induced mutant
Glucose and Xylose mix Xylose Cellobiose Xylose SCB hydrolysate Glucose
Continuous Continuous Continuous Continuous Batch
0.44 0.32–0.34 0.38–0.39 0.40–0.46 0.78
22 2.4 2.9 4.1 47
E. coli KO11 E. coli LY168
Pfl, PEToperon, Dfrd DPEToperon, DadhE, DackA, DldhA + pflB, rrIE, PET operon Wild type
Xylose Xylose Xylose
Batch Batch Batch
0.76 0.51 0.48
43 45.5 43.2
Glucose
Continuous
0.36
97
Saccharomyces cerevisiae a
Dldh, Dpta, Dak Dldh, pdhupregulated, Dpfl
Ethanol yield based on sugar consumed.
Cripps et al. (2009)
Lawford and Rousseau (1999) Krishnan et al. (1999) Martin et al. (2006) This study
Watanabe et al. (2011) Jarboe et al. (2007)
Nguyen and Shieh (1992)
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10
0.6
8 7
0.4
6 5
0.3 Yield (g EtOH/g sugar) pH Residual sugar (g/L)
0.2 0.1
4 3 2
Residual sugar (g/L), pH
Yield (g EtOH/g sugar)
9 0.5
1 0
0.0 0
15
30
45 60 Time (days)
75
90
Fig. 3. Residual substrate, ethanol production and pH profiles during fermentations with immobilized C. boliviensis.
C. boliviensis with those of other wild type and engineered strains was made as shown in Table 3. While C. boliviensis yielded less ethanol than recombinant strains (Zymomonas CP4:pZB5, S. cerevisiae 1400, E. coli FBR5, KO11 and LY168, and Thermoananerobacter BG1L1) from xylose, it produced comparable ethanol yields from SCB hydrolysate as substrate. It is likely that optimization of fermentation conditions and strain improvement efforts will result in higher yields with C. boliviensis. Higher and stable ethanol yields of 0.40–0.46 g ethanol/g of sugar consumed (corresponding to 78–90% of theoretical yield) were achieved under conditions 6 and 7, respectively (Table 2). Diluted SCB hydrolysate with 1.5 ± 0.1 g/L acetate had no apparent inhibitory effect on C. boliviensis fermentation since the highest ethanol production yields were achieved when fermenting the pentose-rich hydrolysate. Complete or almost complete substrate utilization was accomplished under all conditions regardless of whether D-(+)-cellobiose, D-(+)-xylose or mixed sugars from SCB hydrolysate were fermented (Fig. 2 and Table 2). Total substrate consumption varied from 98% to 100%. Therefore, no residual substrate was detected in the effluent stream except at some stages of the fermentation when the LR was increased by more than twofold (second and fourth operational conditions). The overall sugar to ethanol conversion efficiency ranked from 67% to 90% for all operational conditions. The high sugar to ethanol conversion achieved in this study is evidence for the catabolic efficiency of the anaerobes which possess a higher end-product-to-cell ratio compared to aerobic microorganisms. It is important to mention that the sugar to ethanol conversion achieved when xylose or cellobiose were utilized as substrates varied from 67% to 75%, (Table 2) and the highest conversion was achieved when mixed sugars of the SCB hydrolysate were fermented (78–90%; Table 2). Under all fermentation conditions, ethanol along with acetate and formate and, temporally, traces of propionate, succinate, lactate or butyrate, were produced (Table 2 and Fig. 2). Acetate was the main by-product. During the fermentation of SCB hydrolysate, acetate concentrations (0.9–1.5 g/L) in the feed stream resulted in rather high concentration of acetate (2.4–3.9 g/L) in the effluent. While acetic acid, released during pretreatment of lignocellulose can affect the overall cell physiology, viability, ethanol yield and productivity (Zaldivar et al., 2001), inhibition by acetic acid was not apparent in the current study with C. boliviensis. A rather low carbon dioxide production of 0.28–0.45 mol per mol of D-(+)-xylose or pentose-rich hydrolysate fermented was observed, while 0.91–1.00 mol of carbon dioxide were produced per mol of hexose unit when D-(+)-cellobiose was fermented (Table 2). The carbon recovery percentage was estimated for all operational conditions (Table 2) without including biomass due to the
impossibility to quantify immobilized cells without affecting the continuous feature of the system. Biofilm formation varied according to the environmental and operational conditions and the cell population was not completely homogeneous along the whole matrix, a large number of cells detached, settled and accumulated in the bottom of the reactor. Under all operational conditions the carbon recovery varied from 95% to 100% and, besides biomass, no by-products other than those included in the carbon balance were formed. It is important to mention that some carbon content was supplied by the yeast extract and vitamins added to the fermentation medium, and most of it was likely converted into biomass. The reactor performance deteriorated at some stages of the continuous fermentation presumably due to pH changes. Although the pH of the system was set to be maintained at 7.0 ± 0.5, the pH increased to around 8.0 due to decalibration of the pH probe. While at pH 8 or slightly higher no inhibition was observed, an increase to pH 9.5 caused a reduction to 0.1 g/g in the ethanol yield (Fig. 3). After pH readjustment, the reactor performance recovered due to the robustness of C. boliviensis.
4. Conclusion In this study, the recent isolated thermoanaerobe C. boliviensis was evaluated to ferment cellobiose, xylose and a pentose-rich SCB hydrolysate, and to produce ethanol under continuous operational mode. Ethanol yields achieved were comparable to those of other widely studied ethanol-producing microorganisms. The fermentation of diluted SCB hydrolysate yielded the highest ethanol production. Acetate, carbon dioxide, formate, and temporally, traces of propionate, succinate, lactate or butyrate, were by-products of fermentation. The continuous fermentation by C. boliviensis reveals the possibility to produce ethanol from renewable lignocellulosic feedstock. Fermentation of higher sugar concentrations, and optimization in continuous systems are our future goals.
Acknowledgements This research has been supported by the Swedish International Development Cooperation Agency (SIDA) and the Swedish Agency for Research Cooperation with Developing Countries (SAREC). Malik Badshah was supported by a fellowship granted by Higher Education Commission of Pakistan. This study was part of the EU Interreg. Program ECOMOBILITY. Dietliend Adlecreutz helped initially to set up the HPLC analysis.
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To Cristhian Carrasco and Gunnar Liden from the Department of Chemical Engineering at Lund University to provide the sugarcane bagasse hydrolysate. To AnoxKaldnes to kindly provide plastic carriers. References Ahring, B.K., Jensen, K., Nielsen, P., Bjerre, A.B., Schmidt, A.S., 1996. Pretreatment of wheat straw and conversion of xylose and xylan to ethanol by thermophilic anaerobic bacteria. Bioresour. Technol. 58 (2), 107–113. Angelidaki, I., Petersen, S.P., Ahring, B.K., 1990. Effects of lipids on thermophilic anaerobic digestion and reduction of lipid inhibition upon addition of bentonite. Appl. Microbiol. Biotechnol. 33 (4), 469–472. Brethauer, S., Wyman, C.E., 2010. Review: continuous hydrolysis and fermentation for cellulosic ethanol production. Bioresour. Technol. 101 (13), 4862–4874. Carrasco, C., Baudel, H.M., Sendelius, J., Modig, T., Roslander, C., Galbe, M., HahnHägerdal, B., Zacchi, G., Lidén, G., 2010. SO2-catalyzed steam pretreatment and fermentation of enzymatically hydrolyzed sugarcane bagasse. Enzyme Microb. Technol. 46 (2), 64–73. Crespo, C., Pozzo, T., Nordberg-Karlsson, E., Alvarez, M.T., Mattiasson, B., 2011. Caloramator boliviensis sp. nov., a novel thermophilic, ethanol-producing bacterium isolated from a hot spring in Bolivia. Int. J. Syst. Evol. Microbiol. doi:10.1099/ijs.0.032664-0. Cripps, R.E., Eley, K., Leak, D.J., Rudd, B., Taylor, M., Todd, M., Boakes, S., Martin, S., Atkinson, T., 2009. Metabolic engineering of Geobacillus thermoglucosidasius for high yield ethanol production. Metab. Eng. 11 (6), 398–408. Dien, B.S., Cotta, M.A., Jeffries, T.W., 2003. Bacteria engineered for fuel ethanol production: current status. Appl. Microbiol. Biotechnol. 63 (3), 258–266. Georgieva, T., Ahring, B., 2007. Evaluation of continuous ethanol fermentation of dilute-acid corn stover hydrolysate using thermophilic anaerobic bacterium Thermoanaerobacter BG1L1. Appl. Microbiol. Biotechnol. 77, 61–68. Georgieva, T.I., Mikkelsen, M.J., Ahring, B.K., 2008. Ethanol production from wetexploded wheat straw hydrolysate by thermophilic anaerobic bacterium Thermoanaerobacter BG1L1 in a continuous immobilized reactor. Appl. Biochem. Biotechnol. 145 (1–3), 99–110. Gnansounou, E., Dauriat, A., 2010. Techno-economic analysis of lignocellulosic ethanol: a review. Bioresour. Technol. 101 (13), 4980–4991. Klinke, H.B., Thomsen, A.B., Ahring, B.K., 2001. Potential inhibitors from wet oxidation of wheat straw and their effect on growth and ethanol production by Thermoanaerobacter mathranii. Appl. Microbiol. Biotechnol. 57 (5–6), 631–638. Krishnan, M., Ho, N., Tsao, G., 1999. Fermentation kinetics of ethanol production from glucose and xylose by recombinant Saccharomyces 1400 (pLNH33). Appl. Biochem. Biotechnol. 78, 373–388. Jarboe, L., Grabar, T., Yomano, L., Shanmugan, K., Ingram, L., 2007. Development of ethanologenic bacteria. In: Olsson, L. (Ed.), Biofuels, vol. 108. Springer, Berlin/ Heidelberg, pp. 237–261. Lawford, H., Rousseau, J., 1999. The effect of glucose on high-level xylose fermentations by recombinant Zymomonas in batch and fed-batch fermentations. Appl. Biochem. Biotechnol. 77, 235–249. Ljungdahl, L., Wiegel, J., 1986. Anaerobic fermentations. In: Demain, A.L., Solomon, N.A. (Eds.), Manual of Industrial Microbiology and Biotechnology. American Society of Microbiology, pp. 84–96. Martín, C., Marcet, M., Almazán, O., Jönsson, L.J., 2007. Adaptation of a recombinant xylose-utilizing Saccharomyces cerevisiae strain to a sugarcane bagasse
191
hydrolysate with high content of fermentation inhibitors. Bioresour. Technol. 98 (9), 1767–1773. Martin, G.J.O., Bin Zhou, A.K., Pamment, N.B., 2006. Performance and stability of ethanologenic Escherichia coli strain FBR5 during continuous culture on xylose and glucose. J. Ind. Microbiol. Biotechnol. 33 (10), 834–844. Nguyen, V.T., Shieh, W.K., 1992. Continuous ethanol fermentation using immobilized yeast in a fluidized bed reactor. J. Chem. Technol. Biotechnol. 55 (4), 339–346. Ojeda, K., Ávila, O., Suárez, J., Kafarov, V., 2011. Evaluation of technological alternatives for process integration of sugarcane bagasse for sustainable biofuels production––Part 1. Chem. Eng. Res. Des. 89 (3), 270–279. Parawira, W., Read, J.S., Mattiasson, B., Björnsson, L., 2008. Energy production from agricultural residues: high methane yields in pilot-scale two-stage anaerobic digestion. Biomass Bioenergy 32, 44–50. Peng, F., Ren, J.-L., Xu, F., Bian, J., Peng, P., Sun, R.-C., 2009. Comparative study of hemicelluloses obtained by graded ethanol precipitation from sugarcane bagasse. J. Agric. Food Chem. 57 (14), 6305–6317. Senthuran, A., Senthuran, V., Mattiasson, B., Kaul, R., 1997. Lactic acid fermentation in a recycle batch reactor using immobilized Lactobacillus casei. Biotechnol. Bioeng. 55 (6), 841–853. Shaw, A.J., Podkaminer, K.K., Desai, S.G., Bardsley, J.S., Rogers, S.R., Thorne, P.G., Hogsett, D.A., Lynd, L.R., 2008. Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield. Proc. Nat. Acad. Sci. USA 105 (37), 13769–13774. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., 2008. Determination of sugars, byproducts, and degradation products in liquid fraction process samples. NREL/TP-510-42623. National Renewable Energy Laboratory, Midwest Research Institute, Golden, CO. Sommer, P., Georgieva, T., Ahring, B.K., 2004. Potential for using thermophilic anaerobic bacteria for bioethanol production from hemicellulose. Biochem. Soc. Trans. 32, 283–289. Taniguchi, M., Wakamiya, K., Tsuchiya, M., Matsuno, R., Kamikubo, T., 1983. Continuous ethanol production by cell-holding culture of yeasts. Appl. Microbiol. Biotechnol. 18 (4), 201–206. Watanabe, T., Watanabe, I., Yamamoto, M., Ando, A., Nakamura, T., 2011. A UVinduced mutant of Pichia stipitis with increased ethanol production from xylose and selection of a spontaneous mutant with increased ethanol tolerance. Bioresour. Technol. 102 (2), 1844–1848. Weimer, P., Zeikus, J., 1977. Fermentation of cellulose and cellobiose by Clostridium thermocellum in the absence of Methanobacterium thermoautotrophicum. Appl. Environ. Microbiol. 33 (2), 289–297. Yao, S., Mikkelsen, M.J., 2010. Metabolic engineering to improve ethanol production in Thermoanaerobacter mathranii. Appl. Microbiol. Biotechnol. 88, 199–208. Zaldivar, J., Nielsen, J., Olsson, L., 2001. Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration. Appl. Microbiol. Biotechnol. 56, 17–34. Zaldivar, J., Roca, C., Le Foll, C., Hahn-Hägerdal, B., Olsson, L., 2005. Ethanolic fermentation of acid pre-treated starch industry effluents by recombinant Saccharomyces cerevisiae strains. Bioresour. Technol. 96 (15), 1670–1676. Zeikus, J., Ben-Bassat, A., Ng, T., Lamed, R., 1981. Thermophilic ethanol fermentations. Basic Life Sci. 18, 441–461. Zhao, C., Karakashev, D., Lu, W., Wang, H., Angelidaki, I., 2010. Xylose fermentation to biofuels (hydrogen and ethanol) by extreme thermophilic (70 °C) mixed culture. Int. J. Hydrogen Energy 35, 3415–3422.
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Ethanol production by continuous fermentation of D-(+)-cellobiose, D-(+)-xylose and sugarcane bagasse hydrolysate using the thermoanaerobe Caloramator boliviensis Carla F. Crespo, Malik Badshah, Maria T. Alvarez 1, Bo Mattiasson ⇑ Department of Biotechnology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden
a r t i c l e
i n f o
Article history: Received 27 June 2011 Received in revised form 19 September 2011 Accepted 6 October 2011 Available online 14 October 2011 Keywords: Pentose fermentation Lignocellulose hydrolysate Caloramator Bioethanol Continuous fermentation
a b s t r a c t The recently isolated anaerobic bacterium Caloramator boliviensis with an optimum growth temperature of 60 °C can efficiently convert hexoses and pentoses into ethanol. When fermentations of pure sugars and a pentose-rich sugarcane bagasse hydrolysate were carried out in a packed bed reactor with immobilized cells of C. boliviensis, more than 98% of substrates were converted. Ethanol yields of 0.40–0.46 g/g of sugar were obtained when sugarcane bagasse hydrolysate was fermented. These features reveal interesting properties of C. boliviensis in producing ethanol from a renewable feedstock. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Technology for production of ethanol from lignocellulosic feedstock is expected to become mature in the next 5–10 years and partly replace first generation ethanol. The importance of lignocellulose-based ethanol stems from the expected possibility to use assumed inexpensive feedstock, avoid direct and indirect competition with supply of human food and animal feed and to reduce environmental risks such as pollution and soil degradation which are generally associated to biofuels of the first generation (Gnansounou and Dauriat, 2010). Dry lignocellulosic feedstock generally consists of 35–50% cellulose, 20–35% hemicellulose and 10–25% lignin. Conversion of lignocellulose to higher value products requires fractionation of the material into its main components. Lignocellulosic biomass, such as sugarcane bagasse (SCB), which is a sugar production residue, allows the possible integration of second and first generation bioethanol (Ojeda et al., 2011). The efficient utilization of SCB which contains 40–45% cellulose, 30–35% hemicelluloses and 20–30% lignin (Peng et al., 2009) offers
⇑ Corresponding author. Tel.: +46 46 2228264; fax: +46 46 2224713. E-mail addresses: [email protected] (C.F. Crespo), Malik.Badshah@bio tek.lu.se (M. Badshah), [email protected] (M.T. Alvarez), Bo. [email protected] (B. Mattiasson). 1 Present address: Instituto de Investigaciones Fármaco Bioquímicas, Facultad de Ciencias Farmacéuticas y Bioquímicas, Universidad Mayor de San Andrés, P.O. Box 3239, La Paz, Bolivia. 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.10.020
an opportunity to reduce the cost of ethanol production provided that suitable fermentative organisms are available. While hexosefermenting organisms, such as Saccharomyces cerevisiae, are common, pentose-fermenting organisms are rare. A number of genetically engineered ethanol-producing strains capable of metabolizing xylose and other pentose sugars into ethanol have been developed (Martín et al., 2007; Yao and Mikkelsen, 2010; Zaldivar et al., 2001), but a common problem with these organisms is their sensitivity to inhibitors present in undetoxified hydrolysates (Dien et al., 2003). Thus, two important requirements for an efficient ethanol-producing microorganism are to ferment a variety of sugars (pentoses and hexoses) and to tolerate stress conditions (Zaldivar et al., 2001). Some anaerobic thermophilic bacteria are potential microorganisms for the production of ethanol due to their capability to metabolize a wide spectrum of sugars found in lignocellulose. Additionally, several advantages are associated with the production of ethanol at high temperatures, e.g. high bioconversion rates, reduced risk of contamination, and facilitated product recovery. However, bioconversion of lignocellulosic hydrolysates by thermoanaerobes has been studied only to a limited extent and mostly in batch fermentations (Georgieva and Ahring, 2007). For economic reasons, continuous fermentations are sometimes used for commercial production of ethanol from corn and sugarcane, since higher volumetric productivities can be achieved and time for emptying, cleaning and filling vessels and labor work can be minimized. In addition, continuous processing could be beneficial in
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adapting fermentative organisms to inhibitors generated during biomass pretreatment (Brethauer and Wyman, 2010). Higher cell densities in a continuous than in a batch fermentation system could enable higher productivities and yields (Taniguchi et al., 1983). In a previous study, the thermoanaerobe Caloramator boliviensis strain 45BT was isolated, characterized and found to be efficient in fermenting a wide spectrum of substrates and producing ethanol in batch fermentations (Crespo et al., 2011). Therefore, the main objective of the present study was to evaluate the ability of the organism to Ferment D-(+)-cellobiose, D-(+)-xylose and pentose-rich SCB hydrolysate using immobilized C. boliviensis in an anaerobic packed bed reactor operated at 60 °C. Hexose and pentose utilization under continuous operational mode and the effect of substrate loading rate (LR) and hydraulic retention time (HRT) were studied. 2. Methods 2.1. Microorganism and cultivation C. boliviensis strain 45BT (T = Type strain) (Crespo et al., 2011) was maintained in serum bottles under anaerobic conditions at 4 °C. Inocula were prepared by cultivating C. boliviensis (2%, v/v) in 100-mL serum vials containing 70 mL of anaerobic mineral medium (AMM) as described by Angelidaki et al. (1990) with the following modifications: NH4Cl was omitted and instead 1 g/L of urea was supplemented as nitrogen source. The trace element solution was replaced with 1 mL of (g/L of deionized water): FeCl24H2O, 1.50; H3BO3, 0.06; CoCl26H2O, 0.12; MnCl24H2O, 0.10; Na2MoO42H2O, 0.02; NiCl26H2O, 0.02; ZnCl2, 0.07; CuCl22H2O, 0.01; Na2SeO3, 0.003; NaOH, 0.5 and HCl 25%, 6.5 mL. The medium was amended with 1 g/L of yeast extract. To ensure anaerobic conditions before autoclaving (121 °C for 20 min), the medium was prepared under an O2-free N2 stream using the modified Hungate technique (Ljungdahl and Wiegel, 1986). After autoclaving and prior to inoculation, the medium was reduced using a filter-sterilized anaerobic solution of Na2S9H2O to a final concentration of 0.5 g/L, and supplemented with a filter-sterilized solution of D-(+)-cellobiose (5 g/L) and a vitamin solution (DSMZ medium No. 141, German Collection of Microorganisms and Cell Cultures) (10 mL/L). The pH of the medium was adjusted to 7.0 ± 0.2 with H2SO4 (0.5 M). For continuous fermentation, AMM was used as synthetic medium. A sterile stock solution (100 g/L) of D-(+)-cellobiose was added to final concentrations of 5.0 ± 0.1 g/L and 7.5 ± 0.2 g/L (operational conditions 1 and 2, respectively; Table 1). Subsequently, cellobiose as a substrate was replaced by D-(+)-xylose to final concentrations of 5.0 ± 0.2 g/L and 7.5 ± 0.4 g/L (operational conditions 3–5; Table 1). AMM was amended with SCB hydrolysate to final concentrations of 5.5 ± 0.1 g/L and 8.9 g/L of total sugars (operational conditions 6 and 7, respectively). The pH of the medium was adjusted to 7.0 ± 0.5 with 0.5 M H2SO4 or 1 M NaOH. The pentose-rich SCB hydrolysate was prepared by SO2-catalyzed steam pretreatment according to Carrasco et al. (2010) and supplied by the Department of Chemical Engineering, Lund University. The pentose-rich hydrolysate with low concentration of byproducts (e.g. HMF, furfural, etc.) was used to evaluate xylose to ethanol conversion. The initial SCB hydrolysate composition was (in g/L): cellobiose, 1.5; glucose, 1.5; xylose, 22.2; mannose, (traces); arabinose, (traces); acetic acid, 4.1; 5-(Hydroxymethyl)furfural (HMF), 0.1 and furfural, 0.5. 2.2. Reactor operation The fermentation set-up (Fig. 1) consisted of a water-jacketed glass column (25 cm height and 5.5 cm inner diameter), packed
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with coated plastic carriers 0.7 1 cm in size (AnoxKaldnes, Sweden). Polyethylenimine of medium molecular weight (1%) was utilized for coating the plastic carriers as described by Senthuran et al. (1997). The packed glass column was connected to a water jacketed vessel at both ends. The vessel was equipped with a stirring device, a pH electrode connected to a control unit, a port for gas release and a port for pumping in fresh medium. The influent in the glass column entered from the bottom of the reactor. Recirculation of medium between both units was achieved by a peristaltic pump to ensure an up-flow velocity of 1 m/h. The reactor was maintained at 60 °C for continuous fermentation, and the pH was maintained at 7.0 ± 0.5 by automatic addition of 1 M NaOH to the water jacketed vessel, the pH adjusted medium was recirculated into the glass column reactor. The total working volume of the system was 680 mL. The entire reactor system, including the plastic carriers and tubings was autoclaved at 120 °C for 20 min. Before operation, the reactor system was flushed with O2-free N2 gas for 15 min in order to maintain anaerobic conditions. The reactor system was filled with AMM supplemented with 5.0 ± 0.1 g/L of D-(+)-cellobiose as sole carbon source. The reactor was started in batch operational mode by inoculating 100 mL of a cell suspension of C. boliviensis with an optical density (OD620) of 1.0 ± 0.3 to achieve cell enrichment and immobilization. The batch operational mode was maintained for 5 d and successively changed into seven other operational conditions (Table 1). Thus, the system was shifted to continuous operational mode by feeding D-(+)-cellobiose containing medium at a low LR and extended HRT to avoid cell detachment and wash out. Subsequently, a second condition was set to achieve higher fermentation rate of D-(+)-cellobiose. A third condition was applied by replacing D-(+)-cellobiose with D-(+)-xylose. Conversion of xylose at a higher rate was evaluated in a fourth operational condition. Since instability in the reactor performance was observed in the last stage, a fifth condition was applied by slightly increasing the HRT and decreasing the LR (Table 1). Consecutively, and after achieving xylose adaptation, the continuous fermentation of the pentose-rich SCB hydrolysate was evaluated. The sixth operational condition was set at constant HRT and slightly increased LR. The concentration of fermentable substrates in the hydrolysate-containing medium was (in g/L): cellobiose, 0.3; xylose, 4.9; glucose, 0.3 and acetate, 0.9. As the last operational condition, the HRT was as in the previous operational mode, the LR was increased (Table 1), and the concentration of fermentable substrates in the hydrolysate-containing medium was (in g/L): cellobiose, 0.5; xylose, 7.9; glucose, 0.5 and acetate, 1.5. During all experiments, the operational conditions (HRT and LR) were modified after steady state had been achieved. The system was considered to be in steady state when ethanol production was constant, and/or varied no more than 10% of the ethanol titer (standard deviation no larger than 0.02 g/L), for at least three retention times. Operating time for each operating condition was as detailed in Table 1. Liquid and gas samples were collected at regular time intervals and analyzed in duplicate for quantification of residual substrate and fermentation products. 2.3. Analytical methods Samples containing D-(+)-glucose, D-(+)-xylose, D-(+)-cellobiose and fermentation products (ethanol, acetate, succinate, formate, propionate, butyrate and lactate) were acidified with 20% H2SO4 (10 lL/mL of sample) and filtered through a 0.45 lm polypropylene membrane. Thereafter, samples were analyzed by high pressure liquid chromatography (JASCO Corporation, Tokyo, Japan) on an HPX-87H ion-exchange column (Biorad Laboratories Inc.,
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Table 1 Operational conditions for continuous fermentation of cellobiose, xylose and SCB hydrolysate by C. boliviensis. Operational conditions
Type of substrate
1 2 3
D-(+)-Xylose
5.0 ± 0.2
34
0.15
288
4
D-(+)-Xylose
7.5 ± 0.4
21
0.36
456
5
D-(+)-Xylose Xylose, cellobiose and glucose from SCB hydrolysate Xylose, cellobiose and glucose from SCB hydrolysate
5.0 ± 0.4
23
0.21
432
5.5 ± 0.1 8.9 ± 0.0
23 23
0.23 0.38
132 120
6 7
Substrate concentration (g/L)
Hydraulic retention time (h)
Loading rate (g/L h)
Operating time (h)
D-(+)-Cellobiose
5.0 ± 0.1
162
0.03
408
D-(+)-Cellobiose
7.5 ± 0.2
53
0.14
480
Gas collector pH controller
Outlet Sampling port
Gas collector
Sampling port Alkali
Medium
Fig. 1. Diagram of reactor set-up for ethanol production by immobilized C. boliviensis.
other solvents (Weimer and Zeikus, 1977; Zeikus et al., 1981), and only a few studies have been carried out with pretreated lignocellulosic feedstock (Ahring et al., 1996; Klinke et al., 2001; Sommer et al., 2004; Georgieva and Ahring, 2007; Georgieva et al., 2008). Microorganisms with high ethanol yields do not necessarily ferment pretreated lignocellulosic material because of the presence of inhibitory compounds. Evaluating the fermentation of lignocellulosic hydrolysates is crucial since this type of feedstock is envisioned for commercial ethanol production (Zaldivar et al., 2005), the capacity of C. boliviensis sp. nov., to ferment hexoses, pentoses and a pentose-rich SCB hydrolysate in a continuous process was evaluated. The reactor performance in terms of production of ethanol and other products, residual substrate, and type of substrate (hexoses
Hercules, CA, USA) at 55 °C using 5 mM sulfuric acid as eluent with a flow rate of 0.6 mL/min and detected with a refractive index detector (Erc Inc., Saitama, Japan). Carbohydrates, furfural and hydroxymethyl furfural were quantified as described by Sluiter et al. (2008). Determination of carbon dioxide was performed by gas chromatography as described by Parawira et al. (2008).
3. Results and discussion Thermophilic Clostridia growing optimally at 60–65 °C have been recognized for several decades as interesting candidates for processes involving conversions of pure sugars to ethanol and 5
1 Soluble products (g/L)
4
2 Succinic acid (g/L) Acetic acid (g/L) Ethanol (g/L)
3
4
Lactic acid (g/L) Propionic acid (g/L)
5
6
Formic acid (g/L) Butyric acid (g/L)
3
2
1
0 0
15
30
45
60
75
Time (days) Fig. 2. Product profile of C. boliviensis during continuous fermentation.
90
7
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C.F. Crespo et al. / Bioresource Technology 103 (2012) 186–191 Table 2 Summary of fermentation parameters at seven operational conditions of continuous fermentation of cellobiose, xylose or SCB hydrolysate by C. boliviensis. Operational condition
EtOH (g/L)
Ace (g/L)
Form (g/L)
Prop (g/L)
Succ (g/L)
Buty (g/L)
Lac (g/L)
Y CO2 a (mol/mol)
CCelb (%)
CXylc (%)
CGlud (%)
CSe (%)
YEtOHf (g/g)
TYEtOHg (%)
CEh (%)
CRi (%)
1 2 3 4 5 6 7
1.7 ± 0.2 2.9 ± 0.1 1.9 ± 0.1 2.9 ± 0.2 1.7 ± 0.1 2.2 ± 0.1 4.1 ± 0.1
1.2 ± 0.1 2.1 ± 0.2 1.3 ± 0.1 1.6 ± 0.2 1.3 ± 0.1 2.4l ± 0.2 3.9m ± 0.2
1.0 ± 0.1 1.4 ± 0.3 1.1 ± 0.1 2.0 ± 0.3 1.3 ± 0.1 1.3 ± 0.3 0.5 ± 0.0
ND 0.2 ± 0.0 ND ND ND ND 0.3 ± 0.2
0.2 ± 0.1 ND ND 0.1 ± 0.1 ND ND ND
0.1 ± 0.1 ND ND ND ND ND ND
ND 0.1 ± 0.1 ND ND ND ND ND
1.0 0.9 0.4 0.3 0.4 0.3 0.3
100 98.7 0 0 0 100 100
0 0 100 98.7 100 100 100
0 0 0 0 0 100 100
100 98.7 100 98.7 100 100 100
0.34 0.39 0.38 0.39 0.34 0.40 0.46
67 77 75 77 67 78 90
67 76 75 76 67 78 90
98.9 102.5 100 98 97.6 98.7j 97.8k
ND: not detected. a Yield based on the production of CO2 per mol of hexose or pentose unit converted. b Consumed cellobiose. c Consumed xylose. d Consumed glucose. e Total substrate consumed. f Ethanol yield based on sugars consumed. g Ethanol yield given as a percentage of theoretical possible yield of 0.51 g/g. Theoretical yields are assumed according to stoichiometrical balances, for hexoses: C6H12O6 ? 2 CH3CH2OH + 2 CO2 and for pentoses: C5H10O5 ? 1.67 CH3CH2OH + 1.67 CO2 (Zhao et al., 2010). h Conversion efficiency calculated by dividing the ethanol yield based on sugar concentrations present in the influent by theoretical possible yield of 0.51 g/g. i Carbon recovery percentage. j Mannose (0.6 10 3 mol) and arabinose (2 10 3 mol) were considered to estimate carbon recovery percentage. k Mannose (0.7 10 3 mol) and arabinose (2 10 3 mol) were considered to estimate carbon recovery percentage but are not shown in the stoichiometric balance due to their minor amounts. l Acetate production was 1.5 g/L and influent concentration was 0.9 g/L. m Acetate production was 2.4 g/L and influent concentration was 1.5 g/L.
or pentoses), under seven operational conditions is summarized in Fig. 2 and Table 2. Ethanol was the main product, and its yield ranged from 0.34 to 0.39 (gram of ethanol per gram of sugar consumed) when cellobiose (operational conditions 1 and 2) or xylose (operational conditions 3 and 4) were fermented. However, when the HRT was the shortest (21 h) and the LR was the highest (0.36 g/L h) (fourth operational condition), a slight instability in the reactor performance was perceived since xylose was detected in the effluent. Despite of this occurrence, the ethanol concentration in the effluent remained constant. Ethanol yields under conditions 2–4 were stable and were the highest when fermenting pure sugars. The ethanol yield achieved in the first operational condition was 67% with respect to the assumed theoretical possible yield of
0.51 g ethanol/g sugar consumed. However, higher values were achieved under other operational conditions when cellobiose or xylose were utilized as substrate, yielding 77%, 75% and 77% under conditions 2–4, respectively (Table 2). These results show that C. boliviensis, is among the few species of wild type bacteria, fungi and yeast that can naturally convert hexoses (cellobiose) and pentoses (xylose) into ethanol with high yields in a continuous fermentation process. Even though high yields were achieved with low initial sugar concentrations (5 ± 0.2 g/L and 7.5 ± 0.4 g/L), it is extremely important to evaluate the fermentation process with higher influent sugar concentrations (>10% of total sugars) since ethanol titer as well as yield are the main considerations for commercial fermentations. A comparison of ethanol yields of
Table 3 Comparison of ethanol yields of thermophilic bacterial strains and engineered Saccharomyces, Zymomonas, Escherichia coli and naturally occurring yeast strains. Organism
Genotype
Fermentation substrate
Fermentation mode
YEtOHa (g/g)
Titer (g/L)
Ref.
Thermoanaerobacter mathranii BG1L1 Thermoanaerobacter mathranii BG1L1 Thermoanaerobacter saccharolyticum ALK2 Geobacillus thermoglucosidasius TM242
Dldh
Mixed sugars
Continuous
0.39–0.42
14.4
Dldh
Wet exploded wheat straw Xylose
Continuous
0.39–0.42
3.9
Continuous
0.46
37
Georgieva et al. (2008) Georgieva and Ahring (2007) Shaw et al. (2008)
Zymomonas mobilis CP4:pZB5
Batch Batch Batch Batch
0.42 0.35 0.47 0.48
14.4 12.3 14.8 23
Saccharomyces cerevisiae 1400
Carrying genes for xylose metabolism from E. coli XR, XD, XK
Glucose Xylose Cellobiose Xylose
Batch
0.46
47.9
E. coli FBR5 Caloramator boliviensis
Dldh, Dpfl Wild type
Pichia stipitis PXF58
UV induced mutant
Glucose and Xylose mix Xylose Cellobiose Xylose SCB hydrolysate Glucose
Continuous Continuous Continuous Continuous Batch
0.44 0.32–0.34 0.38–0.39 0.40–0.46 0.78
22 2.4 2.9 4.1 47
E. coli KO11 E. coli LY168
Pfl, PEToperon, Dfrd DPEToperon, DadhE, DackA, DldhA + pflB, rrIE, PET operon Wild type
Xylose Xylose Xylose
Batch Batch Batch
0.76 0.51 0.48
43 45.5 43.2
Glucose
Continuous
0.36
97
Saccharomyces cerevisiae a
Dldh, Dpta, Dak Dldh, pdhupregulated, Dpfl
Ethanol yield based on sugar consumed.
Cripps et al. (2009)
Lawford and Rousseau (1999) Krishnan et al. (1999) Martin et al. (2006) This study
Watanabe et al. (2011) Jarboe et al. (2007)
Nguyen and Shieh (1992)
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10
0.6
8 7
0.4
6 5
0.3 Yield (g EtOH/g sugar) pH Residual sugar (g/L)
0.2 0.1
4 3 2
Residual sugar (g/L), pH
Yield (g EtOH/g sugar)
9 0.5
1 0
0.0 0
15
30
45 60 Time (days)
75
90
Fig. 3. Residual substrate, ethanol production and pH profiles during fermentations with immobilized C. boliviensis.
C. boliviensis with those of other wild type and engineered strains was made as shown in Table 3. While C. boliviensis yielded less ethanol than recombinant strains (Zymomonas CP4:pZB5, S. cerevisiae 1400, E. coli FBR5, KO11 and LY168, and Thermoananerobacter BG1L1) from xylose, it produced comparable ethanol yields from SCB hydrolysate as substrate. It is likely that optimization of fermentation conditions and strain improvement efforts will result in higher yields with C. boliviensis. Higher and stable ethanol yields of 0.40–0.46 g ethanol/g of sugar consumed (corresponding to 78–90% of theoretical yield) were achieved under conditions 6 and 7, respectively (Table 2). Diluted SCB hydrolysate with 1.5 ± 0.1 g/L acetate had no apparent inhibitory effect on C. boliviensis fermentation since the highest ethanol production yields were achieved when fermenting the pentose-rich hydrolysate. Complete or almost complete substrate utilization was accomplished under all conditions regardless of whether D-(+)-cellobiose, D-(+)-xylose or mixed sugars from SCB hydrolysate were fermented (Fig. 2 and Table 2). Total substrate consumption varied from 98% to 100%. Therefore, no residual substrate was detected in the effluent stream except at some stages of the fermentation when the LR was increased by more than twofold (second and fourth operational conditions). The overall sugar to ethanol conversion efficiency ranked from 67% to 90% for all operational conditions. The high sugar to ethanol conversion achieved in this study is evidence for the catabolic efficiency of the anaerobes which possess a higher end-product-to-cell ratio compared to aerobic microorganisms. It is important to mention that the sugar to ethanol conversion achieved when xylose or cellobiose were utilized as substrates varied from 67% to 75%, (Table 2) and the highest conversion was achieved when mixed sugars of the SCB hydrolysate were fermented (78–90%; Table 2). Under all fermentation conditions, ethanol along with acetate and formate and, temporally, traces of propionate, succinate, lactate or butyrate, were produced (Table 2 and Fig. 2). Acetate was the main by-product. During the fermentation of SCB hydrolysate, acetate concentrations (0.9–1.5 g/L) in the feed stream resulted in rather high concentration of acetate (2.4–3.9 g/L) in the effluent. While acetic acid, released during pretreatment of lignocellulose can affect the overall cell physiology, viability, ethanol yield and productivity (Zaldivar et al., 2001), inhibition by acetic acid was not apparent in the current study with C. boliviensis. A rather low carbon dioxide production of 0.28–0.45 mol per mol of D-(+)-xylose or pentose-rich hydrolysate fermented was observed, while 0.91–1.00 mol of carbon dioxide were produced per mol of hexose unit when D-(+)-cellobiose was fermented (Table 2). The carbon recovery percentage was estimated for all operational conditions (Table 2) without including biomass due to the
impossibility to quantify immobilized cells without affecting the continuous feature of the system. Biofilm formation varied according to the environmental and operational conditions and the cell population was not completely homogeneous along the whole matrix, a large number of cells detached, settled and accumulated in the bottom of the reactor. Under all operational conditions the carbon recovery varied from 95% to 100% and, besides biomass, no by-products other than those included in the carbon balance were formed. It is important to mention that some carbon content was supplied by the yeast extract and vitamins added to the fermentation medium, and most of it was likely converted into biomass. The reactor performance deteriorated at some stages of the continuous fermentation presumably due to pH changes. Although the pH of the system was set to be maintained at 7.0 ± 0.5, the pH increased to around 8.0 due to decalibration of the pH probe. While at pH 8 or slightly higher no inhibition was observed, an increase to pH 9.5 caused a reduction to 0.1 g/g in the ethanol yield (Fig. 3). After pH readjustment, the reactor performance recovered due to the robustness of C. boliviensis.
4. Conclusion In this study, the recent isolated thermoanaerobe C. boliviensis was evaluated to ferment cellobiose, xylose and a pentose-rich SCB hydrolysate, and to produce ethanol under continuous operational mode. Ethanol yields achieved were comparable to those of other widely studied ethanol-producing microorganisms. The fermentation of diluted SCB hydrolysate yielded the highest ethanol production. Acetate, carbon dioxide, formate, and temporally, traces of propionate, succinate, lactate or butyrate, were by-products of fermentation. The continuous fermentation by C. boliviensis reveals the possibility to produce ethanol from renewable lignocellulosic feedstock. Fermentation of higher sugar concentrations, and optimization in continuous systems are our future goals.
Acknowledgements This research has been supported by the Swedish International Development Cooperation Agency (SIDA) and the Swedish Agency for Research Cooperation with Developing Countries (SAREC). Malik Badshah was supported by a fellowship granted by Higher Education Commission of Pakistan. This study was part of the EU Interreg. Program ECOMOBILITY. Dietliend Adlecreutz helped initially to set up the HPLC analysis.
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To Cristhian Carrasco and Gunnar Liden from the Department of Chemical Engineering at Lund University to provide the sugarcane bagasse hydrolysate. To AnoxKaldnes to kindly provide plastic carriers. References Ahring, B.K., Jensen, K., Nielsen, P., Bjerre, A.B., Schmidt, A.S., 1996. Pretreatment of wheat straw and conversion of xylose and xylan to ethanol by thermophilic anaerobic bacteria. Bioresour. Technol. 58 (2), 107–113. Angelidaki, I., Petersen, S.P., Ahring, B.K., 1990. Effects of lipids on thermophilic anaerobic digestion and reduction of lipid inhibition upon addition of bentonite. Appl. Microbiol. Biotechnol. 33 (4), 469–472. Brethauer, S., Wyman, C.E., 2010. Review: continuous hydrolysis and fermentation for cellulosic ethanol production. Bioresour. Technol. 101 (13), 4862–4874. Carrasco, C., Baudel, H.M., Sendelius, J., Modig, T., Roslander, C., Galbe, M., HahnHägerdal, B., Zacchi, G., Lidén, G., 2010. SO2-catalyzed steam pretreatment and fermentation of enzymatically hydrolyzed sugarcane bagasse. Enzyme Microb. Technol. 46 (2), 64–73. Crespo, C., Pozzo, T., Nordberg-Karlsson, E., Alvarez, M.T., Mattiasson, B., 2011. Caloramator boliviensis sp. nov., a novel thermophilic, ethanol-producing bacterium isolated from a hot spring in Bolivia. Int. J. Syst. Evol. Microbiol. doi:10.1099/ijs.0.032664-0. Cripps, R.E., Eley, K., Leak, D.J., Rudd, B., Taylor, M., Todd, M., Boakes, S., Martin, S., Atkinson, T., 2009. Metabolic engineering of Geobacillus thermoglucosidasius for high yield ethanol production. Metab. Eng. 11 (6), 398–408. Dien, B.S., Cotta, M.A., Jeffries, T.W., 2003. Bacteria engineered for fuel ethanol production: current status. Appl. Microbiol. Biotechnol. 63 (3), 258–266. Georgieva, T., Ahring, B., 2007. Evaluation of continuous ethanol fermentation of dilute-acid corn stover hydrolysate using thermophilic anaerobic bacterium Thermoanaerobacter BG1L1. Appl. Microbiol. Biotechnol. 77, 61–68. Georgieva, T.I., Mikkelsen, M.J., Ahring, B.K., 2008. Ethanol production from wetexploded wheat straw hydrolysate by thermophilic anaerobic bacterium Thermoanaerobacter BG1L1 in a continuous immobilized reactor. Appl. Biochem. Biotechnol. 145 (1–3), 99–110. Gnansounou, E., Dauriat, A., 2010. Techno-economic analysis of lignocellulosic ethanol: a review. Bioresour. Technol. 101 (13), 4980–4991. Klinke, H.B., Thomsen, A.B., Ahring, B.K., 2001. Potential inhibitors from wet oxidation of wheat straw and their effect on growth and ethanol production by Thermoanaerobacter mathranii. Appl. Microbiol. Biotechnol. 57 (5–6), 631–638. Krishnan, M., Ho, N., Tsao, G., 1999. Fermentation kinetics of ethanol production from glucose and xylose by recombinant Saccharomyces 1400 (pLNH33). Appl. Biochem. Biotechnol. 78, 373–388. Jarboe, L., Grabar, T., Yomano, L., Shanmugan, K., Ingram, L., 2007. Development of ethanologenic bacteria. In: Olsson, L. (Ed.), Biofuels, vol. 108. Springer, Berlin/ Heidelberg, pp. 237–261. Lawford, H., Rousseau, J., 1999. The effect of glucose on high-level xylose fermentations by recombinant Zymomonas in batch and fed-batch fermentations. Appl. Biochem. Biotechnol. 77, 235–249. Ljungdahl, L., Wiegel, J., 1986. Anaerobic fermentations. In: Demain, A.L., Solomon, N.A. (Eds.), Manual of Industrial Microbiology and Biotechnology. American Society of Microbiology, pp. 84–96. Martín, C., Marcet, M., Almazán, O., Jönsson, L.J., 2007. Adaptation of a recombinant xylose-utilizing Saccharomyces cerevisiae strain to a sugarcane bagasse
191
hydrolysate with high content of fermentation inhibitors. Bioresour. Technol. 98 (9), 1767–1773. Martin, G.J.O., Bin Zhou, A.K., Pamment, N.B., 2006. Performance and stability of ethanologenic Escherichia coli strain FBR5 during continuous culture on xylose and glucose. J. Ind. Microbiol. Biotechnol. 33 (10), 834–844. Nguyen, V.T., Shieh, W.K., 1992. Continuous ethanol fermentation using immobilized yeast in a fluidized bed reactor. J. Chem. Technol. Biotechnol. 55 (4), 339–346. Ojeda, K., Ávila, O., Suárez, J., Kafarov, V., 2011. Evaluation of technological alternatives for process integration of sugarcane bagasse for sustainable biofuels production––Part 1. Chem. Eng. Res. Des. 89 (3), 270–279. Parawira, W., Read, J.S., Mattiasson, B., Björnsson, L., 2008. Energy production from agricultural residues: high methane yields in pilot-scale two-stage anaerobic digestion. Biomass Bioenergy 32, 44–50. Peng, F., Ren, J.-L., Xu, F., Bian, J., Peng, P., Sun, R.-C., 2009. Comparative study of hemicelluloses obtained by graded ethanol precipitation from sugarcane bagasse. J. Agric. Food Chem. 57 (14), 6305–6317. Senthuran, A., Senthuran, V., Mattiasson, B., Kaul, R., 1997. Lactic acid fermentation in a recycle batch reactor using immobilized Lactobacillus casei. Biotechnol. Bioeng. 55 (6), 841–853. Shaw, A.J., Podkaminer, K.K., Desai, S.G., Bardsley, J.S., Rogers, S.R., Thorne, P.G., Hogsett, D.A., Lynd, L.R., 2008. Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield. Proc. Nat. Acad. Sci. USA 105 (37), 13769–13774. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., 2008. Determination of sugars, byproducts, and degradation products in liquid fraction process samples. NREL/TP-510-42623. National Renewable Energy Laboratory, Midwest Research Institute, Golden, CO. Sommer, P., Georgieva, T., Ahring, B.K., 2004. Potential for using thermophilic anaerobic bacteria for bioethanol production from hemicellulose. Biochem. Soc. Trans. 32, 283–289. Taniguchi, M., Wakamiya, K., Tsuchiya, M., Matsuno, R., Kamikubo, T., 1983. Continuous ethanol production by cell-holding culture of yeasts. Appl. Microbiol. Biotechnol. 18 (4), 201–206. Watanabe, T., Watanabe, I., Yamamoto, M., Ando, A., Nakamura, T., 2011. A UVinduced mutant of Pichia stipitis with increased ethanol production from xylose and selection of a spontaneous mutant with increased ethanol tolerance. Bioresour. Technol. 102 (2), 1844–1848. Weimer, P., Zeikus, J., 1977. Fermentation of cellulose and cellobiose by Clostridium thermocellum in the absence of Methanobacterium thermoautotrophicum. Appl. Environ. Microbiol. 33 (2), 289–297. Yao, S., Mikkelsen, M.J., 2010. Metabolic engineering to improve ethanol production in Thermoanaerobacter mathranii. Appl. Microbiol. Biotechnol. 88, 199–208. Zaldivar, J., Nielsen, J., Olsson, L., 2001. Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration. Appl. Microbiol. Biotechnol. 56, 17–34. Zaldivar, J., Roca, C., Le Foll, C., Hahn-Hägerdal, B., Olsson, L., 2005. Ethanolic fermentation of acid pre-treated starch industry effluents by recombinant Saccharomyces cerevisiae strains. Bioresour. Technol. 96 (15), 1670–1676. Zeikus, J., Ben-Bassat, A., Ng, T., Lamed, R., 1981. Thermophilic ethanol fermentations. Basic Life Sci. 18, 441–461. Zhao, C., Karakashev, D., Lu, W., Wang, H., Angelidaki, I., 2010. Xylose fermentation to biofuels (hydrogen and ethanol) by extreme thermophilic (70 °C) mixed culture. Int. J. Hydrogen Energy 35, 3415–3422.