- Email: [email protected]
Simultaneous utilization of galactose and glucose by Saccharomyces cerevisiae mutant strain for ethanol production
Renewable Energy 65 (2014) 213e218
Contents lists available at ScienceDirect
Renewable Energy journal homepage: www.elsevier.com/locate/renene
Simultaneous utilization of galactose and glucose by Saccharomyces cerevisiae mutant strain for ethanol production Jeong-Hoon Park a, c, Sang-Hyoun Kim b, Hee-Deung Park c, Jun Seok Kim d, Jeong-Jun Yoon a, * a
Green Materials Technology Center, Korea Institute of Industrial Technology (KITECH), Chungnam 330-825, South Korea Department of Environmental Engineering, Daegu University, Gyeongbuk 712-714, South Korea School of Civil, Environmental and Architectural Engineering, Korea University, Seoul 136-714, South Korea d Department of Chemical Engineering, Kyonggi University, Gyeonggi 443-760, South Korea b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 February 2013 Accepted 9 September 2013 Available online 13 October 2013
Red algal biomass is a promising alternative feedstock for bioethanol production, due to several advantages including high carbohydrate content, growth rate, ethanol yield, and CO2 fixation ability. However, it has been known that most yeast strains can not utilize galactose, the major sugar of red algae, as efficiently it can utilize glucose. The authors report a novel ethanogenic strain capable of fermenting galactose, Saccharomyces cerevisiae. This mutant yeast strain exhibited exceptional fermentative performance on galactose and a mixture of galactose and glucose. At 120 g/L of initial galactose concentration, ethanol concentration reached 6.9% (v/v) within 36 h with 88.3% of theoretical ethanol yield (0.51 g ethanol/g galactose). The ethanol concentration and yield were higher than that for glucose at the same initial concentration. In a mixed sugar (galactose þ glucose) condition, the existence of glucose retarded galactose utilization however, 120 g/L of the mixed sugar was completely consumed within 60 h at any galactose concentration. The critical inhibitory levels of formic acid, levulinic acid and 5hydroxymethylfurfural (5-HMF) on ethanol fermentation were 0.5, 2.0, and 10.0 g/L; respectively. From this result, the ethanol fermentation efficiency of the novel S. cerevisiae strain using the galactose base of red algae was superior to the fermentation efficiency when using the wild type strain, and the novel strain was found to have resistance to the major inhibitors generated during the saccharification process. Ó 2013 Published by Elsevier Ltd.
Keywords: Saccharomyces cerevisiae Galactose Simultaneous utilization Algal biomass Gelidium amansii Bioethanol
1. Introduction Due to the shortage of fossil fuels and climate change, the importance of the bioenergy is increasing. Economical and sustainable production of biofuel must be established to substitute petroleum oil which is predicted to be exhausted within 40 years. Bioethanol, a representative biofuel, is produced by fermentation of sugar base (sugarcane, sugar beet, etc.), starch base (potato, sweet potato, corn, etc.) or cellulosic biomass (thinning logs, wood wastes, waste paper, etc.). When a sugary or starchy biomass is used as feedstock, ethanol can be produced in a relatively simple and affordable way. However, the use of sugary or starchy biomass inevitably leads to the controversy of “fuel vs
* Corresponding author. Tel.: þ82 41 589 8445; fax: þ82 41 589 8580. E-mail address: [email protected] (J.-J. Yoon). 0960-1481/$ e see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.renene.2013.09.010
food” [1]. Cellulosic biomass can be sustainably supplied without competition with food [2,3]. However, the lignin-containing structure of cellulosic biomass needs a harsh pretreatment/hydrolysis process, which not only increases the fermentation cost but also generates high levels of toxic compounds including 5hydroxymethylfurfural (5-HMF) [4]. Macroalgal biomass has several attributes as a potential feedstock for bioethanol [5]. Macroalgae can grow faster with higher CO2 fixation ability than land plants [6,7]. They can be cultivated on vast tracts of sea by sunlight without any nitrogenbased fertilizer [8]. Furthermore, the macroalgal biomass normally does not contain lignin, and thus sugars can be obtained through milder hydrolysis compared to cellulosic biomass [9]. Macroalgae are generally categorized into three phyla, red algae (Rhodophyta), green algae (Chlorophyta and Charophyta), and brown algae (Phaeophyceae). Some species of red algae belonging to the genera Gelidium, Gracilaria, and Euchema are known for
214
J.-H. Park et al. / Renewable Energy 65 (2014) 213e218
high carbohydrate content, implying great potential as feedstock for bioethanol. The yeasts such as Saccharomyces cerevisiae, and Brettanomyces custersii, and bacteria Zymomonas mobilis convert glucose into ethanol in an anaerobic state without an external electron acceptor. Since ethanol fermentation based on glucose has a long history, the strain and fermentation technology is well established. Red algal biomass is, however, composed of not only glucose but also galactose. For example, the ratio of galactose to glucose in Gelidium amansii is 61 to 39 [10]. Galactose is known to have lower ethanol productivity and yield than glucose with normal ethanogenic yeast strains [11e13]. Galactose must be converted to galactose-1phosphate, glucose-1-phosphate, and glucose-6-phosphate prior to glycolysis. The yeast and pathway used to convert galactose and glucose into glucose-6-phosphate [16e18]. Galactose has an opposite hydroxyle group at the 4-carbon compared to glucose, and this characteristic causes a big difference in early catabolism when the two are converted to glucose-6-phosphate [16]. Galactose conversion to glucose-1-phosphate is carried out via UDP (uridine diphosphate)-galactose, a saccharide-nucleotide derivative generated when galactose-1-phosphate is substituted with glucose-1phosphate from UDP-glucose. The UDP-galactose is converted to UDP-glucose through a reaction that includes C-4 acidification by NADþ and C-4 reduction by NADH through UDP-glucose4epimerase.Therefore, additional enzymes and reaction paths such as galactokinase, galactose-1-phosphateuridylyl transferase and phosphoglucomutase are required. Furthermore, the enzyme production is tightly repressed by the existence of glucose in a normal eukaryotic cell. Therefore, ethanol production from red algal biomass requires a stable and high-yielding strain enabling simultaneous utilization of galactose and glucose. In this manuscript, the authors report a galactose-utilizing mutant yeast strain, S. cerevisiae. The main goals of this study were i) to examine galactose fermentation of the mutant strain at highly concentrated sugar concentrations, which would reduce distillation costs by bioethanol, ii) to investigate the feasibility of the strain for bioethanol production from a galactose and glucose mixture, and iii) to characterize the inhibitory effects of hydrolysis byproducts of red algal biomass on the mixed sugar fermentation.
Table 1 Initial sugars concentrations at the ethanol fermentation of galactose and glucose mixture. Ratio (Gal.:Glu.)
Galactose (g/L)
Glucose (g/L)
Total sugar (g/L)
10:0 8:2 6:4 4:6 8:2 0:10
120 96 72 48 24 0
0 24 48 72 96 120
120 120 120 120 120 120
30 C for 24 h. 40 mL of the culture was centrifuged, transferred to another 200 mL culture medium (yeast extract 10 g/L, soy peptone 20 g/L, galactose 120 g/L) and cultured at 150 rpm and 30 C for 16 h. 2.3. Ethanol fermentation A predetermined amount of galactose and/or glucose (Table 1) with 2 g of yeast extract, and 4 g of soy peptone was added to a 500 mL medium bottle. The bottle was inoculated with 16 mL of the pre-culture, and then filled to 200 mL with distilled water. Subsequently, the headspace was purged with N2 gas for 3 min. The bottle was sealed with a screw cap with a septum, then agitated at 150 rpm and 30 C.
2. Materials and methods 2.1. Strains A wild type S. cerevisiae ATCC2341 was irradiated with light of 245 nm at 1200 erg/mm2. The treated culture was spread over an agar plate (galactose 2% (w/v), yeast extract 1% (w/v), peptone 2% (w/v), agar 1.5% (w/v)) and cultivated for 3 days at 30 C. Subsequently, colonies were sub-cultured to isolate the artificial mutant strain of S. cerevisiae. Also, to confirm genetic stability, the subculture was continuously carried out until the 20th generation, with galactose fermenting ability checked for each generation. The S. cerevisiae mutant strain cells are round to ovoid, 3e10 by 3e20 mm in size. They reproduce by budding aerobically or anaerobically. The optimal growth temperature and pH were 27e 30 C and 4.8e5.5, respectively. 2.2. Pre-culture S. cerevisiae mutant strain was inoculated on a YPD agar plate (yeast extract 10 g/L, bacto peptone 20 g/L, glucose 20 g/L, agar 20 g/ L) and cultivated at 30 C for 48 h. A single colony was transferred to 200 mL YSPG medium (yeast extract 10 g/L, soy peptone 20 g/L, galactose 20 g/L) and then cultured aerobically at 150 rpm and
Fig. 1. Ethanol production (b) for various galactose concentrations (a) in a batch culture by S. cerevisiae mutant strain. Symbols: 50 g/L galactose (closed circles), 75 g/L galactose (open circles), 100 g/L galactose (closed inverted triangles), 120 g/L galactose (open triangles).
J.-H. Park et al. / Renewable Energy 65 (2014) 213e218
The substrate utilization rate obtained from the inhibitor concentration curve was fitted to a noncompetitive inhibition model (Eq. (4)).
Table 2 The chemical composition of Gelidium amansii. Component
Concentration (%)
Carbohydrate Cellulose (glucose) Agar (Galactose) (3,6-Anhydrogalactose) Protein Lipid Ash Others
67.3 14.9 52.4 23.1 29.3 15.6 0.0 5.7 11.4
215
I n k ¼ k0 1 * I
(4)
Where k ¼ the substrate utilization rate at inhibitor concentration of I (g/L/h), k0 ¼ the substrate utilization rate without an added inhibitor (g/L/h), I ¼ the inhibitor concentration (g/L), I* ¼ the lethal
2.4. Inhibition effect The effects of hydrolysis byproducts from red algal biomass on the mixed sugar fermentation were examined at 100 g/L and 20 g/L of initial galactose and glucose, respectively. The concentrations of 5-HMF (Across, USA), levulinic acid (SigmaeAldrich, USA), and formic acid (Samchun, Korea) range from 5 to 12 g/L, 1e8 g/L, and 0.1e1.0 g/L, respectively. 2.5. Analysis All the liquid samples were filtered through a 0.45 mm membrane filter prior to liquid chromatographic analysis. 5-HMF, levulinic acid, formic acid, and ethanol were analyzed by high performance liquid chromatography (HPLC, YL9100 series, Younglin, Korea) using a refractive index detector, and a 300 mm 7.8 mm Aminex HPX-87H ion exclusion column (BioRad, USA) at 60 C with 5 mM H2SO4 with the mobile phase at a flow rate of 0.5 mL/min. Galactose, glucose, and 3.6anhyrogalactose (3.6-AHG) were analyzed by Bio-LC (ICS-3000, Dionex, USA), with a 250 mm 4 mm CarboPac PA1 column (Dionex, USA) at 30 C with 16 mM and 2 M NaOH as the mobile phases at a rate of 1.0 mL/min. Ethanol yield and percent yield (Eqs.(1) and (2))were calculated using the equation below:
½EtOHmax ½Sugarini
(1)
YEtOH 100 0:51
(2)
YEtOH ¼
Y%T ¼
where, YEtOH ¼ ethanol yield (g/g), [EtOH]max ¼ maximum ethanol concentration achieved during fermentation (g/L), [Sugar]ini ¼ total initial sugar concentration at the onset of fermentation (g/L), Y% T ¼ percent yield (%), and 0.51 theoretical maximum ethanol yield per galactose (g/g). 2.6. Assay The substrate utilization curve of each condition was fitted to a modified Gompertz equation (Eq. (3)), which provides a suitable model for describing the substrate utilization in a lab scale batch test [10].
R S ¼ S0 1 exp exp M e ðl tÞ þ 1 P
(3)
Where S ¼ the cumulative substrate consumption, S0 ¼ the initial sugar concentration (g/L), RM ¼ the substrate utilization rate (g/L/h), l ¼ the lag-phase time (h), t ¼ time (h) and e ¼ exp (1).
Fig. 2. Effect of glucose percentage (b) in mixed sugar on galactose utilization rate (a) and ethanol production (c) by S. cerevisiae mutant strain. Symbols: 120 g/L galactose (closed circles), 96 g/L galactose and 24 g/L glucose (open circles), galactose 72 g/L and glucose 48 g/L (closed inverted triangles), galactose 48 g/L and glucose 72 g/L (open triangle), galactose 24 g/L and glucose 96 g/L (closed squares), glucose 120 g/L (open squares).
216
J.-H. Park et al. / Renewable Energy 65 (2014) 213e218
reported that the existence of glucose at the early stage of yeast growth repressed the catabolism of other sugars such as xylose. It is assumed that the cell of S. cerevisiae grown on glucose needs the synthesis of enzymes required for galactose catabolism. Nevertheless, the glucose repression was overcome in a short period. The
Fig. 3. Effect of galactose and glucose ratio on the ethanol production rate and sugar utilization rate by S. cerevisiae mutant strain. Relative galactose utilization rate by glucose percentage of mixed sugar (closed circled) and estimated (line).
inhibition concentration beyond which the reaction can’t proceed (g/L), and n ¼ constant. 3. Results 3.1. Galactose fermentation Galactose ranging from 50 to 120 g/L was completely utilized within 36 h (Fig. 1). The maximum ethanol concentration was 54.0 g/L at 120 g/L of initial galactose. Based on the theoretical maximum ethanol yield (0.51 g ethanol/g galactose) (refer to the Eq. (5) below), the percent yield was 88%.
C6 H12 O6 /2CH3 CH2 OH þ 2CO2
(5)
At 100 g/L of initial galactose, the percent yield in this study was 92.7%. It was significantly higher than 52.4%, the percent yield using a previously reported mutant strain at the same galactose level [14]. A 50 g/L galactose complete utilization was observed by 20 h with ethanol concentration at approximately 25 g/L. At 75 g/L galactose less than 5 g/L remained with 33 g/L ethanol after at 20 h. These results show a doubling of ethanol yield with a almost doubling of the initial galactose concentration (75 g/L as opposed to 40 g/L) compared to previous results by Jin’s group [15]. The S. cerevisiae mutant strain is a potential strain for ethanol fermentation of galactose. 3.2. Fermentation of galactose and glucose mixture As red algal biomass is composed of galactose and glucose (Table 2), the fermentation of the sugars mixture was examined using the S. cerevisiae mutant strain. Glucose at 120 g/L was completely consumed within 12 h. The glucose utilization rate was not affected by the existence of galactose (Fig. 2(b)). On the other hand, the existence of glucose decreased the galactose utilization rate compared to the utilization rate observed in the galactose-only conditions (Fig. 3). When galactose was the sole substrate at 50 and 75 g/L, they were completely utilized within 20 h as mentioned in 3.1. On the other hand, the complete utilization of galactose at 48 and 72 g/L with the existence of glucose took 48e60 h as shown in Fig. 2(a) and (b). Moreover, even after glucose was completely consumed, the galactose utilization rate could not be recovered to the level of the galactose-only conditions. Bisson et al. (1993)
Fig. 4. Effect of byproducts on ethanol fermentation. Symbol: (a) Control e Gal (closed circles), Formic acid 0.1 g/L e Gal (closed inverted triangle), Formic acid 0.3 g/L e Gal (closed squares), Formic acid 0.5 g/L e Gal (closed diamonds), Formic acid 0.5 g/L e Gal (closed diamonds), Formic acid 0.7 g/L e Gal (closed triangles), Formic acid 1.0 g/L e Gal (closed hexagons), Control e Glu (open circles), Formic acid 0.1 g/L e Glu (open inverted triangle), Formic acid 0.3 g/L e Glu (open squares), Formic acid 0.5 g/L e Glu (open diamonds), Formic acid 0.7 g/L e Glu (open triangles), Formic acid 1.0 g/L e Glu (open hexagons) Control e EtOH (line cross), Formic acid 0.1 g/L e EtOH (dotted cross), Formic acid 0.3 g/L e EtOH (short dash cross), Formic acid 0.5 g/L e EtOH (double dotted dash cross), Formic acid 0.7 g/L e EtOH (long dash cross), Formic acid 1.0 g/L e EtOH (single dotted dash cross), (b) Control e Gal (closed circles), 5-HMF 5 g/L e Gal (closed inverted triangles), 5-HMF 7.5 g/L e Gal (closed squares), 5-HMF 10 g/L e Gal (closed diamonds), 5-HMF 12 g/L e Gal (closed triangles), Control e Glu (open circles), 5-HMF 5 g/L e Glu (open inverted triangles), 5-HMF 7.5 g/L e Glu (open squares), 5HMF 10 g/L e Glu (open diamonds), 5-HMF 12 g/L e Glu (open triangles), Control e EtOH (line cross), 5-HMF 5 g/L e EtOH (dotted cross), 5-HMF 7.5 g/L e EtOH (short dash cross), 5-HMF 10 g/L e EtOH (double dotted cross), 5-HMF 12 g/L e EtOH (long dash cross), (c) Control e Gal (closed circles), Lev. 1 g/L e Gal (closed inverted triangles), Lev. 2 g/L e Gal (closed squares), Lev. 4 g/L e Gal (closed diamonds), Lev. 8 g/L e Gal (closed triangles), Control e Glu (open circles), Lev. 1 g/L e Glu (open inverted triangles), Lev. 2 g/L e Glu (open squares), Lev. 4 g/L e Glu (open diamonds), Lev. 8 g/L e Glu (open triangles), Control e EtOH (line cross), Lev. 1 g/L e EtOH (dotted cross), Lev. 2 g/L e EtOH (short dash cross), Lev. 4 g/L e EtOH (double dotted cross), Lev. 8 g/L e EtOH (long dash cross).
J.-H. Park et al. / Renewable Energy 65 (2014) 213e218
217
Fig. 5. Effect of Formic acid (a), 5-HMF (b) and Levulinic acid (c) on galactose utilization rate by S. cerevisiae mutant strain. Symbols: relative galactose utilization rate by formic acid concentrations (closed circles), relative galactose utilization rate by 5-HMF concentrations (closed squared), relative galactose utilization rate by levulinic acid concentrations (closed inverted triangles).
substrate utilization was completed within 60 h in all the examined conditions. The final ethanol concentration and yield were in the range of 5.8e6.6%(v/v) and 70.8e84.2%, indicating that the sugar mixture ratios had little influence on the ethanol yield. The yields and the utilization rates were superior to the reported values at 120 g/L of glucose as the sole substrate (90% by 80 h incubation) [19]. It implied that mutant S. cerevisiae mutant strain was an efficient yeast strain for ethanol fermentation of galactose and glucose mixture. 3.3. Inhibition by hydrolysis byproducts There are four potential inhibitors in ethanol fermentation of red algal biomass. Firstly, ethanol itself acts as the product inhibitor [20]. Secondly, high substrate concentration would cause substrate inhibition. The third is glucose repression, the inhibition of the catabolism for other sugars by glucose. The last is an inhibitory byproduct generated during the hydrolysis of red algal biomass. The product inhibition, the substrate inhibition, and the glucose repression were mentioned in Sections 3.1 and 3.2. The novel strain, S. cerevisiae, was not practically affected by ethanol and galactose levels up to 55 g/L and 120 g/L, respectively. Glucose repression retarded galactose utilization. However, 120 g/L of the mixed sugar was completely consumed within 60 h at any galactose content. In this part, the effects of the inhibitory byproducts are discussed. In a physicochemical hydrolysis process such as dilute-acid hydrolysis, sugar is converted to 5-HMF, which is further degraded to levulinic acid and formic acid [21,]. Dilute-acid hydrolysis of red algal biomass generates 5-HMF, formic acid, and levulinic acid, although the byproducts yields are less than those of lignocellulose [5]. In this work, the authors examined the effects of 5-HMF, formic acid, and levulinic acid on the ethanol fermentation of a galactose and glucose mixture. The initial sugar concentration was 120 g/L (galactose 100 g/L þ glucose 20 g/L). The inhibition was more pronounced on galactose utilization than glucose utilization. Fig. 4 shows the effect of the byproducts on galactose utilization with the assay using the noncompetitive inhibition model (Fig. 5) (Eq. (2)). Formic acid significantly inhibited the ethanol fermentation from galactose. At a formic acid concentration over 1 g/L, galactose could not be consumed at all. The galactose utilization rate dropped to 50% of the control at a formic acid concentration of 0.43 g/L (Fig. 5(a)). 5-HMF (Fig. 5(b)) and levulinic (Fig. 5(c)) acid showed much milder inhibitory effects. The 50% maximal inhibitory concentrations (IC50) of 5-HMF and levulinic acid were 10.43 and 12.09 g/L, respectively (Table 3). This result would be useful as criteria for controlling byproduct levels in both hydrolysis and fermentation unit processes.
4. Discussion Recently, red algal biomass is a candidate of one of the promising alternative energy sources to substitute fossil fuels. Although marine biomass has a myriad of advantages as a feedstock for ethanol production, challenges in the successful ethanol fermentation process include the generation of high amounts of fermentable sugars and low amounts of inhibitors from saccharification process as well as the discovery or modification of new strains utilizing galactose conversion to ethanol. Because galactose is main monomer sugar of fermentable sugar in G. amansii Galactose is also known to have lower ethanol productivity and yield than glucose with normal ethanogenic yeast strains [11e13]. In this study, the authors investigated the feasibility of bioethanol production from red algae, G. amansii, by S. cerevisiae mutant strain according to this study goal. (i) To examine galactose fermentation of the mutant strain at high sugar concentrations, which would reduce the overall distillation costs of bioethanol. This study investigated the effect of utilizing galactose rate at different concentrations. Fig. 1 shows the characteristics of galactose utilization. From these result, at 100 g/L of initial galactose, the percent yield in this study was 92.7%. It was significantly higher than 52.4%, the percent yield using a previously reported mutant strain at the same galactose level [14] as well as initial galactose utilizing rate higher than in other previous study. In a same time (20 h) at 75 g/L galactose less than 5 g/L remained with 33 g/L ethanol yield after at 20 h. These results show a doubling of ethanol yield in the same reaction time with an almost doubling of the initial galactose concentration (75 g/L as opposed to 40 g/L) compared to previous results by Jin’s group [15]. (ii) To investigate the feasibility of the strain for bioethanol production is a galactose and glucose mixture. Although galactose is the major sugar, is also present glucose in G. amansii. The existence of glucose at the initial stage of yeast growth
Table 3 10%, 50%, and 90% maximal inhibitory concentrations (IC10, IC50, and IC90, respectively) of formic acid, levulinic acid, and 5-HMF on ethanol fermentation of galactose.
Formic acid Levulinic acid 5-HMF
IC10
IC50
IC90
0.08 0.36 3.15
0.43 2.09 10.43
0.85 5.07 12.08
218
J.-H. Park et al. / Renewable Energy 65 (2014) 213e218
repressed the catabolism of other sugars such as xylose [22]. Fig. 2 shows the characteristics of the mixed-sugar solution. Within 60 h at any galactose content 120 g/L of the mixed sugar was completed consumed. The yields and the utilization rates were superior to the reported values at 120 g/L of glucose as the sole substrate (90% by 80 h incubation) [19]. (iii) To characterize the inhibitory effects of hydrolysis byproducts of red algal biomass on mixed sugar fermentation. Sugar is converted to 5-HMF, which is further degraded to levulinic acid and formic acid from saccharification [21]. To find the critical concentration for ethanol fermentation by the mutant strain, we investigated the effect of utilizing mixed-sugar with different inhibitors and concentrations. Fig. 5 shows the characteristics of ethanol fermentation by inhibitors. The critical inhibitory concentration of Formic acid, Levulinic acid and 5-HMF on ethanol fermentation was 0.5, 2.0 and 10.0 g/L, respectively. From this result, byproducts were kept under critical levels for successful ethanol fermentation In conclusion, S. cerevisiae a mutant strain, showed notable ethanol fermentation performance on galactose and a mixture of galactose and glucose. The effects of substrate concentration, composition, and the potential inhibitors on ethanol fermentation using the strain were quantified. The results could be useful for bioethanol production using sustainable and renewable biomass such as red algae. Acknowledgments This work was supported by a grant (JA-13-0002) from Korea Institute of Industrial Technology, Republic of Korea. References [1] Panagiotou G, Olsson L. Effect of compounds released during pretreatment of wheat straw on microbial growth and enzymatic hydrolysis rates. Biotechnol Bioeng 2007;96:250e8. [2] Tomas Pejo E, Oliva JM, Ballesteros M, Olsson L. Comparison of SHF and SSF processes from steam exploded wheat straw for ethanol production by xylose fermenting and robust glucose fermenting Saccharomyces cerevisiae strains. Biotechnol Bioeng 2008;100:1122e31.
[3] Jorgensen H, Vibe Pedersen J, Larsen J, Felby C. Liquefaction of lignocellulose at high solids concentrations. Biotechnol Bioeng 2007;96:862e70. [4] Mosier N, Wyman C, Dale B, Elander R, Lee Y, Holtzapple M, et al. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour Technol 2005;96:673e86. [5] Kim C, Ryu HJ, Kim SH, Yoon JJ, Kim HS, Kim YJ. Acidity tunable ionic liquids as catalysts for conversion of agar into mixed sugars. Bull Korean Chem Soc Notes 2010;31:511e4. [6] Luning K, Pang S. Mass cultivation of seaweeds: current aspects and approaches. J Appl Phycol 2003;15:115e9. [7] Packer M. Algal capture of carbon dioxide; biomass generation as a tool for greenhouse gas mitigation with reference to New Zealand energy strategy and policy. Energy Policy 2009;37:3428e37. [8] Buck BH, Buchholz CM. The offshore-ring: a new system design for the open ocean aquaculture of macroalgae. J Appl Phycol 2004;16:355e68. [9] Jol CN, Neiss TG, Penninkhof B, Rudolph B, De Ruiter GA. A novel highperformance anion-exchange chromatographic method for the analysis of carrageenans and agars containing 3, 6-anhydrogalactose. Anal Biochem 1999;268:213e22. [10] Park JH, Yoon JJ, Park HD, Kim YJ, Lim DJ, Kim SH. Feasibility of biohydrogen production from Gelidium amansii. Int J Hydrog Energy 2011;36:13997e4003. [11] Knutsen S, Myslabodski D, Larsen B, Usov A. A modified system of nomenclature for red algal galactans. Bot Mar 1994;37:163e70. [12] Ostergaard S, Roca C, Rønnow B, Nielsen J, Olsson L. Physiological studies in aerobic batch cultivations of Saccharomyces cerevisiae strains harboring the MEL1 gene. Biotechnol Bioeng 2000;68:252e9. [13] Alexander M, Jeffries T. Respiratory efficiency and metabolite partitioning as regulatory phenomena in yeasts. Enzym Microb Technol 1990;12:2e19. [14] Keating JD, Robinson J, Bothast RJ, Saddler JN, Mansfield SD. Characterization of a unique ethanologenic yeast capable of fermenting galactose. Enzym Microb Technol 2004;35:242e53. [15] Lee KS, Hong ME, Jung SC, Ha SJ, Yu BJ, Koo HM, et al. Improved galactose fermentation of Saccharomyces cerevisiae through inverse metabolic engineering. Biotechnol Bioeng 2011;108:621e31. [16] Ostergaard S, Olsson L, Nielsen J. In vivo dynamics of galactose metabolism in Saccharomyces cerevisiae: metabolic fluxes and metabolite levels. Biotechnol Bioeng 2001;73:412e25. [17] Ross KL, Davis CN, Fridovich-Keil JL. Differential roles of the Leloir pathway enzymes and metabolites in defining galactose sensitivity in yeast. Mol Genet Metab 2004;83:103e16. [18] Holden HM, Rayment I, Thoden JB. Structure and function of enzymes of the Leloir pathway for galactose metabolism. J Biol Chem 2003;278:43885e8. [19] Blondin B, Ratomahenina R, Arnaud A, Galzy P. A study of cellobiose fermentation by a Dekkera strain. Biotechnol Bioeng 1982;24:2031e7. [20] Ha SJ, Galazka JM, Rin Kim S, Choi JH, Yang X, Seo JH, et al. Engineered Saccharomyces cerevisiae capable of simultaneous cellobiose and xylose fermentation. Proc Natl Acad Sci 2011;108:504e9. [21] Larsson S, Palmqvist E, Hahn-Hagerdal B, Tengborg C, Stenberg K, Zacchi G, et al. The generation of fermentation inhibitors during dilute acid hydrolysis of softwood. Enzym Microb Technol 1999;24:151e9. [22] Bisson LF, Coons DM, Kruckeberg AL, Lewis DA. Yeast sugar transporters. Crit Rev Biochem Mol Biol 1993;28:259e308.
Contents lists available at ScienceDirect
Renewable Energy journal homepage: www.elsevier.com/locate/renene
Simultaneous utilization of galactose and glucose by Saccharomyces cerevisiae mutant strain for ethanol production Jeong-Hoon Park a, c, Sang-Hyoun Kim b, Hee-Deung Park c, Jun Seok Kim d, Jeong-Jun Yoon a, * a
Green Materials Technology Center, Korea Institute of Industrial Technology (KITECH), Chungnam 330-825, South Korea Department of Environmental Engineering, Daegu University, Gyeongbuk 712-714, South Korea School of Civil, Environmental and Architectural Engineering, Korea University, Seoul 136-714, South Korea d Department of Chemical Engineering, Kyonggi University, Gyeonggi 443-760, South Korea b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 February 2013 Accepted 9 September 2013 Available online 13 October 2013
Red algal biomass is a promising alternative feedstock for bioethanol production, due to several advantages including high carbohydrate content, growth rate, ethanol yield, and CO2 fixation ability. However, it has been known that most yeast strains can not utilize galactose, the major sugar of red algae, as efficiently it can utilize glucose. The authors report a novel ethanogenic strain capable of fermenting galactose, Saccharomyces cerevisiae. This mutant yeast strain exhibited exceptional fermentative performance on galactose and a mixture of galactose and glucose. At 120 g/L of initial galactose concentration, ethanol concentration reached 6.9% (v/v) within 36 h with 88.3% of theoretical ethanol yield (0.51 g ethanol/g galactose). The ethanol concentration and yield were higher than that for glucose at the same initial concentration. In a mixed sugar (galactose þ glucose) condition, the existence of glucose retarded galactose utilization however, 120 g/L of the mixed sugar was completely consumed within 60 h at any galactose concentration. The critical inhibitory levels of formic acid, levulinic acid and 5hydroxymethylfurfural (5-HMF) on ethanol fermentation were 0.5, 2.0, and 10.0 g/L; respectively. From this result, the ethanol fermentation efficiency of the novel S. cerevisiae strain using the galactose base of red algae was superior to the fermentation efficiency when using the wild type strain, and the novel strain was found to have resistance to the major inhibitors generated during the saccharification process. Ó 2013 Published by Elsevier Ltd.
Keywords: Saccharomyces cerevisiae Galactose Simultaneous utilization Algal biomass Gelidium amansii Bioethanol
1. Introduction Due to the shortage of fossil fuels and climate change, the importance of the bioenergy is increasing. Economical and sustainable production of biofuel must be established to substitute petroleum oil which is predicted to be exhausted within 40 years. Bioethanol, a representative biofuel, is produced by fermentation of sugar base (sugarcane, sugar beet, etc.), starch base (potato, sweet potato, corn, etc.) or cellulosic biomass (thinning logs, wood wastes, waste paper, etc.). When a sugary or starchy biomass is used as feedstock, ethanol can be produced in a relatively simple and affordable way. However, the use of sugary or starchy biomass inevitably leads to the controversy of “fuel vs
* Corresponding author. Tel.: þ82 41 589 8445; fax: þ82 41 589 8580. E-mail address: [email protected] (J.-J. Yoon). 0960-1481/$ e see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.renene.2013.09.010
food” [1]. Cellulosic biomass can be sustainably supplied without competition with food [2,3]. However, the lignin-containing structure of cellulosic biomass needs a harsh pretreatment/hydrolysis process, which not only increases the fermentation cost but also generates high levels of toxic compounds including 5hydroxymethylfurfural (5-HMF) [4]. Macroalgal biomass has several attributes as a potential feedstock for bioethanol [5]. Macroalgae can grow faster with higher CO2 fixation ability than land plants [6,7]. They can be cultivated on vast tracts of sea by sunlight without any nitrogenbased fertilizer [8]. Furthermore, the macroalgal biomass normally does not contain lignin, and thus sugars can be obtained through milder hydrolysis compared to cellulosic biomass [9]. Macroalgae are generally categorized into three phyla, red algae (Rhodophyta), green algae (Chlorophyta and Charophyta), and brown algae (Phaeophyceae). Some species of red algae belonging to the genera Gelidium, Gracilaria, and Euchema are known for
214
J.-H. Park et al. / Renewable Energy 65 (2014) 213e218
high carbohydrate content, implying great potential as feedstock for bioethanol. The yeasts such as Saccharomyces cerevisiae, and Brettanomyces custersii, and bacteria Zymomonas mobilis convert glucose into ethanol in an anaerobic state without an external electron acceptor. Since ethanol fermentation based on glucose has a long history, the strain and fermentation technology is well established. Red algal biomass is, however, composed of not only glucose but also galactose. For example, the ratio of galactose to glucose in Gelidium amansii is 61 to 39 [10]. Galactose is known to have lower ethanol productivity and yield than glucose with normal ethanogenic yeast strains [11e13]. Galactose must be converted to galactose-1phosphate, glucose-1-phosphate, and glucose-6-phosphate prior to glycolysis. The yeast and pathway used to convert galactose and glucose into glucose-6-phosphate [16e18]. Galactose has an opposite hydroxyle group at the 4-carbon compared to glucose, and this characteristic causes a big difference in early catabolism when the two are converted to glucose-6-phosphate [16]. Galactose conversion to glucose-1-phosphate is carried out via UDP (uridine diphosphate)-galactose, a saccharide-nucleotide derivative generated when galactose-1-phosphate is substituted with glucose-1phosphate from UDP-glucose. The UDP-galactose is converted to UDP-glucose through a reaction that includes C-4 acidification by NADþ and C-4 reduction by NADH through UDP-glucose4epimerase.Therefore, additional enzymes and reaction paths such as galactokinase, galactose-1-phosphateuridylyl transferase and phosphoglucomutase are required. Furthermore, the enzyme production is tightly repressed by the existence of glucose in a normal eukaryotic cell. Therefore, ethanol production from red algal biomass requires a stable and high-yielding strain enabling simultaneous utilization of galactose and glucose. In this manuscript, the authors report a galactose-utilizing mutant yeast strain, S. cerevisiae. The main goals of this study were i) to examine galactose fermentation of the mutant strain at highly concentrated sugar concentrations, which would reduce distillation costs by bioethanol, ii) to investigate the feasibility of the strain for bioethanol production from a galactose and glucose mixture, and iii) to characterize the inhibitory effects of hydrolysis byproducts of red algal biomass on the mixed sugar fermentation.
Table 1 Initial sugars concentrations at the ethanol fermentation of galactose and glucose mixture. Ratio (Gal.:Glu.)
Galactose (g/L)
Glucose (g/L)
Total sugar (g/L)
10:0 8:2 6:4 4:6 8:2 0:10
120 96 72 48 24 0
0 24 48 72 96 120
120 120 120 120 120 120
30 C for 24 h. 40 mL of the culture was centrifuged, transferred to another 200 mL culture medium (yeast extract 10 g/L, soy peptone 20 g/L, galactose 120 g/L) and cultured at 150 rpm and 30 C for 16 h. 2.3. Ethanol fermentation A predetermined amount of galactose and/or glucose (Table 1) with 2 g of yeast extract, and 4 g of soy peptone was added to a 500 mL medium bottle. The bottle was inoculated with 16 mL of the pre-culture, and then filled to 200 mL with distilled water. Subsequently, the headspace was purged with N2 gas for 3 min. The bottle was sealed with a screw cap with a septum, then agitated at 150 rpm and 30 C.
2. Materials and methods 2.1. Strains A wild type S. cerevisiae ATCC2341 was irradiated with light of 245 nm at 1200 erg/mm2. The treated culture was spread over an agar plate (galactose 2% (w/v), yeast extract 1% (w/v), peptone 2% (w/v), agar 1.5% (w/v)) and cultivated for 3 days at 30 C. Subsequently, colonies were sub-cultured to isolate the artificial mutant strain of S. cerevisiae. Also, to confirm genetic stability, the subculture was continuously carried out until the 20th generation, with galactose fermenting ability checked for each generation. The S. cerevisiae mutant strain cells are round to ovoid, 3e10 by 3e20 mm in size. They reproduce by budding aerobically or anaerobically. The optimal growth temperature and pH were 27e 30 C and 4.8e5.5, respectively. 2.2. Pre-culture S. cerevisiae mutant strain was inoculated on a YPD agar plate (yeast extract 10 g/L, bacto peptone 20 g/L, glucose 20 g/L, agar 20 g/ L) and cultivated at 30 C for 48 h. A single colony was transferred to 200 mL YSPG medium (yeast extract 10 g/L, soy peptone 20 g/L, galactose 20 g/L) and then cultured aerobically at 150 rpm and
Fig. 1. Ethanol production (b) for various galactose concentrations (a) in a batch culture by S. cerevisiae mutant strain. Symbols: 50 g/L galactose (closed circles), 75 g/L galactose (open circles), 100 g/L galactose (closed inverted triangles), 120 g/L galactose (open triangles).
J.-H. Park et al. / Renewable Energy 65 (2014) 213e218
The substrate utilization rate obtained from the inhibitor concentration curve was fitted to a noncompetitive inhibition model (Eq. (4)).
Table 2 The chemical composition of Gelidium amansii. Component
Concentration (%)
Carbohydrate Cellulose (glucose) Agar (Galactose) (3,6-Anhydrogalactose) Protein Lipid Ash Others
67.3 14.9 52.4 23.1 29.3 15.6 0.0 5.7 11.4
215
I n k ¼ k0 1 * I
(4)
Where k ¼ the substrate utilization rate at inhibitor concentration of I (g/L/h), k0 ¼ the substrate utilization rate without an added inhibitor (g/L/h), I ¼ the inhibitor concentration (g/L), I* ¼ the lethal
2.4. Inhibition effect The effects of hydrolysis byproducts from red algal biomass on the mixed sugar fermentation were examined at 100 g/L and 20 g/L of initial galactose and glucose, respectively. The concentrations of 5-HMF (Across, USA), levulinic acid (SigmaeAldrich, USA), and formic acid (Samchun, Korea) range from 5 to 12 g/L, 1e8 g/L, and 0.1e1.0 g/L, respectively. 2.5. Analysis All the liquid samples were filtered through a 0.45 mm membrane filter prior to liquid chromatographic analysis. 5-HMF, levulinic acid, formic acid, and ethanol were analyzed by high performance liquid chromatography (HPLC, YL9100 series, Younglin, Korea) using a refractive index detector, and a 300 mm 7.8 mm Aminex HPX-87H ion exclusion column (BioRad, USA) at 60 C with 5 mM H2SO4 with the mobile phase at a flow rate of 0.5 mL/min. Galactose, glucose, and 3.6anhyrogalactose (3.6-AHG) were analyzed by Bio-LC (ICS-3000, Dionex, USA), with a 250 mm 4 mm CarboPac PA1 column (Dionex, USA) at 30 C with 16 mM and 2 M NaOH as the mobile phases at a rate of 1.0 mL/min. Ethanol yield and percent yield (Eqs.(1) and (2))were calculated using the equation below:
½EtOHmax ½Sugarini
(1)
YEtOH 100 0:51
(2)
YEtOH ¼
Y%T ¼
where, YEtOH ¼ ethanol yield (g/g), [EtOH]max ¼ maximum ethanol concentration achieved during fermentation (g/L), [Sugar]ini ¼ total initial sugar concentration at the onset of fermentation (g/L), Y% T ¼ percent yield (%), and 0.51 theoretical maximum ethanol yield per galactose (g/g). 2.6. Assay The substrate utilization curve of each condition was fitted to a modified Gompertz equation (Eq. (3)), which provides a suitable model for describing the substrate utilization in a lab scale batch test [10].
R S ¼ S0 1 exp exp M e ðl tÞ þ 1 P
(3)
Where S ¼ the cumulative substrate consumption, S0 ¼ the initial sugar concentration (g/L), RM ¼ the substrate utilization rate (g/L/h), l ¼ the lag-phase time (h), t ¼ time (h) and e ¼ exp (1).
Fig. 2. Effect of glucose percentage (b) in mixed sugar on galactose utilization rate (a) and ethanol production (c) by S. cerevisiae mutant strain. Symbols: 120 g/L galactose (closed circles), 96 g/L galactose and 24 g/L glucose (open circles), galactose 72 g/L and glucose 48 g/L (closed inverted triangles), galactose 48 g/L and glucose 72 g/L (open triangle), galactose 24 g/L and glucose 96 g/L (closed squares), glucose 120 g/L (open squares).
216
J.-H. Park et al. / Renewable Energy 65 (2014) 213e218
reported that the existence of glucose at the early stage of yeast growth repressed the catabolism of other sugars such as xylose. It is assumed that the cell of S. cerevisiae grown on glucose needs the synthesis of enzymes required for galactose catabolism. Nevertheless, the glucose repression was overcome in a short period. The
Fig. 3. Effect of galactose and glucose ratio on the ethanol production rate and sugar utilization rate by S. cerevisiae mutant strain. Relative galactose utilization rate by glucose percentage of mixed sugar (closed circled) and estimated (line).
inhibition concentration beyond which the reaction can’t proceed (g/L), and n ¼ constant. 3. Results 3.1. Galactose fermentation Galactose ranging from 50 to 120 g/L was completely utilized within 36 h (Fig. 1). The maximum ethanol concentration was 54.0 g/L at 120 g/L of initial galactose. Based on the theoretical maximum ethanol yield (0.51 g ethanol/g galactose) (refer to the Eq. (5) below), the percent yield was 88%.
C6 H12 O6 /2CH3 CH2 OH þ 2CO2
(5)
At 100 g/L of initial galactose, the percent yield in this study was 92.7%. It was significantly higher than 52.4%, the percent yield using a previously reported mutant strain at the same galactose level [14]. A 50 g/L galactose complete utilization was observed by 20 h with ethanol concentration at approximately 25 g/L. At 75 g/L galactose less than 5 g/L remained with 33 g/L ethanol after at 20 h. These results show a doubling of ethanol yield with a almost doubling of the initial galactose concentration (75 g/L as opposed to 40 g/L) compared to previous results by Jin’s group [15]. The S. cerevisiae mutant strain is a potential strain for ethanol fermentation of galactose. 3.2. Fermentation of galactose and glucose mixture As red algal biomass is composed of galactose and glucose (Table 2), the fermentation of the sugars mixture was examined using the S. cerevisiae mutant strain. Glucose at 120 g/L was completely consumed within 12 h. The glucose utilization rate was not affected by the existence of galactose (Fig. 2(b)). On the other hand, the existence of glucose decreased the galactose utilization rate compared to the utilization rate observed in the galactose-only conditions (Fig. 3). When galactose was the sole substrate at 50 and 75 g/L, they were completely utilized within 20 h as mentioned in 3.1. On the other hand, the complete utilization of galactose at 48 and 72 g/L with the existence of glucose took 48e60 h as shown in Fig. 2(a) and (b). Moreover, even after glucose was completely consumed, the galactose utilization rate could not be recovered to the level of the galactose-only conditions. Bisson et al. (1993)
Fig. 4. Effect of byproducts on ethanol fermentation. Symbol: (a) Control e Gal (closed circles), Formic acid 0.1 g/L e Gal (closed inverted triangle), Formic acid 0.3 g/L e Gal (closed squares), Formic acid 0.5 g/L e Gal (closed diamonds), Formic acid 0.5 g/L e Gal (closed diamonds), Formic acid 0.7 g/L e Gal (closed triangles), Formic acid 1.0 g/L e Gal (closed hexagons), Control e Glu (open circles), Formic acid 0.1 g/L e Glu (open inverted triangle), Formic acid 0.3 g/L e Glu (open squares), Formic acid 0.5 g/L e Glu (open diamonds), Formic acid 0.7 g/L e Glu (open triangles), Formic acid 1.0 g/L e Glu (open hexagons) Control e EtOH (line cross), Formic acid 0.1 g/L e EtOH (dotted cross), Formic acid 0.3 g/L e EtOH (short dash cross), Formic acid 0.5 g/L e EtOH (double dotted dash cross), Formic acid 0.7 g/L e EtOH (long dash cross), Formic acid 1.0 g/L e EtOH (single dotted dash cross), (b) Control e Gal (closed circles), 5-HMF 5 g/L e Gal (closed inverted triangles), 5-HMF 7.5 g/L e Gal (closed squares), 5-HMF 10 g/L e Gal (closed diamonds), 5-HMF 12 g/L e Gal (closed triangles), Control e Glu (open circles), 5-HMF 5 g/L e Glu (open inverted triangles), 5-HMF 7.5 g/L e Glu (open squares), 5HMF 10 g/L e Glu (open diamonds), 5-HMF 12 g/L e Glu (open triangles), Control e EtOH (line cross), 5-HMF 5 g/L e EtOH (dotted cross), 5-HMF 7.5 g/L e EtOH (short dash cross), 5-HMF 10 g/L e EtOH (double dotted cross), 5-HMF 12 g/L e EtOH (long dash cross), (c) Control e Gal (closed circles), Lev. 1 g/L e Gal (closed inverted triangles), Lev. 2 g/L e Gal (closed squares), Lev. 4 g/L e Gal (closed diamonds), Lev. 8 g/L e Gal (closed triangles), Control e Glu (open circles), Lev. 1 g/L e Glu (open inverted triangles), Lev. 2 g/L e Glu (open squares), Lev. 4 g/L e Glu (open diamonds), Lev. 8 g/L e Glu (open triangles), Control e EtOH (line cross), Lev. 1 g/L e EtOH (dotted cross), Lev. 2 g/L e EtOH (short dash cross), Lev. 4 g/L e EtOH (double dotted cross), Lev. 8 g/L e EtOH (long dash cross).
J.-H. Park et al. / Renewable Energy 65 (2014) 213e218
217
Fig. 5. Effect of Formic acid (a), 5-HMF (b) and Levulinic acid (c) on galactose utilization rate by S. cerevisiae mutant strain. Symbols: relative galactose utilization rate by formic acid concentrations (closed circles), relative galactose utilization rate by 5-HMF concentrations (closed squared), relative galactose utilization rate by levulinic acid concentrations (closed inverted triangles).
substrate utilization was completed within 60 h in all the examined conditions. The final ethanol concentration and yield were in the range of 5.8e6.6%(v/v) and 70.8e84.2%, indicating that the sugar mixture ratios had little influence on the ethanol yield. The yields and the utilization rates were superior to the reported values at 120 g/L of glucose as the sole substrate (90% by 80 h incubation) [19]. It implied that mutant S. cerevisiae mutant strain was an efficient yeast strain for ethanol fermentation of galactose and glucose mixture. 3.3. Inhibition by hydrolysis byproducts There are four potential inhibitors in ethanol fermentation of red algal biomass. Firstly, ethanol itself acts as the product inhibitor [20]. Secondly, high substrate concentration would cause substrate inhibition. The third is glucose repression, the inhibition of the catabolism for other sugars by glucose. The last is an inhibitory byproduct generated during the hydrolysis of red algal biomass. The product inhibition, the substrate inhibition, and the glucose repression were mentioned in Sections 3.1 and 3.2. The novel strain, S. cerevisiae, was not practically affected by ethanol and galactose levels up to 55 g/L and 120 g/L, respectively. Glucose repression retarded galactose utilization. However, 120 g/L of the mixed sugar was completely consumed within 60 h at any galactose content. In this part, the effects of the inhibitory byproducts are discussed. In a physicochemical hydrolysis process such as dilute-acid hydrolysis, sugar is converted to 5-HMF, which is further degraded to levulinic acid and formic acid [21,]. Dilute-acid hydrolysis of red algal biomass generates 5-HMF, formic acid, and levulinic acid, although the byproducts yields are less than those of lignocellulose [5]. In this work, the authors examined the effects of 5-HMF, formic acid, and levulinic acid on the ethanol fermentation of a galactose and glucose mixture. The initial sugar concentration was 120 g/L (galactose 100 g/L þ glucose 20 g/L). The inhibition was more pronounced on galactose utilization than glucose utilization. Fig. 4 shows the effect of the byproducts on galactose utilization with the assay using the noncompetitive inhibition model (Fig. 5) (Eq. (2)). Formic acid significantly inhibited the ethanol fermentation from galactose. At a formic acid concentration over 1 g/L, galactose could not be consumed at all. The galactose utilization rate dropped to 50% of the control at a formic acid concentration of 0.43 g/L (Fig. 5(a)). 5-HMF (Fig. 5(b)) and levulinic (Fig. 5(c)) acid showed much milder inhibitory effects. The 50% maximal inhibitory concentrations (IC50) of 5-HMF and levulinic acid were 10.43 and 12.09 g/L, respectively (Table 3). This result would be useful as criteria for controlling byproduct levels in both hydrolysis and fermentation unit processes.
4. Discussion Recently, red algal biomass is a candidate of one of the promising alternative energy sources to substitute fossil fuels. Although marine biomass has a myriad of advantages as a feedstock for ethanol production, challenges in the successful ethanol fermentation process include the generation of high amounts of fermentable sugars and low amounts of inhibitors from saccharification process as well as the discovery or modification of new strains utilizing galactose conversion to ethanol. Because galactose is main monomer sugar of fermentable sugar in G. amansii Galactose is also known to have lower ethanol productivity and yield than glucose with normal ethanogenic yeast strains [11e13]. In this study, the authors investigated the feasibility of bioethanol production from red algae, G. amansii, by S. cerevisiae mutant strain according to this study goal. (i) To examine galactose fermentation of the mutant strain at high sugar concentrations, which would reduce the overall distillation costs of bioethanol. This study investigated the effect of utilizing galactose rate at different concentrations. Fig. 1 shows the characteristics of galactose utilization. From these result, at 100 g/L of initial galactose, the percent yield in this study was 92.7%. It was significantly higher than 52.4%, the percent yield using a previously reported mutant strain at the same galactose level [14] as well as initial galactose utilizing rate higher than in other previous study. In a same time (20 h) at 75 g/L galactose less than 5 g/L remained with 33 g/L ethanol yield after at 20 h. These results show a doubling of ethanol yield in the same reaction time with an almost doubling of the initial galactose concentration (75 g/L as opposed to 40 g/L) compared to previous results by Jin’s group [15]. (ii) To investigate the feasibility of the strain for bioethanol production is a galactose and glucose mixture. Although galactose is the major sugar, is also present glucose in G. amansii. The existence of glucose at the initial stage of yeast growth
Table 3 10%, 50%, and 90% maximal inhibitory concentrations (IC10, IC50, and IC90, respectively) of formic acid, levulinic acid, and 5-HMF on ethanol fermentation of galactose.
Formic acid Levulinic acid 5-HMF
IC10
IC50
IC90
0.08 0.36 3.15
0.43 2.09 10.43
0.85 5.07 12.08
218
J.-H. Park et al. / Renewable Energy 65 (2014) 213e218
repressed the catabolism of other sugars such as xylose [22]. Fig. 2 shows the characteristics of the mixed-sugar solution. Within 60 h at any galactose content 120 g/L of the mixed sugar was completed consumed. The yields and the utilization rates were superior to the reported values at 120 g/L of glucose as the sole substrate (90% by 80 h incubation) [19]. (iii) To characterize the inhibitory effects of hydrolysis byproducts of red algal biomass on mixed sugar fermentation. Sugar is converted to 5-HMF, which is further degraded to levulinic acid and formic acid from saccharification [21]. To find the critical concentration for ethanol fermentation by the mutant strain, we investigated the effect of utilizing mixed-sugar with different inhibitors and concentrations. Fig. 5 shows the characteristics of ethanol fermentation by inhibitors. The critical inhibitory concentration of Formic acid, Levulinic acid and 5-HMF on ethanol fermentation was 0.5, 2.0 and 10.0 g/L, respectively. From this result, byproducts were kept under critical levels for successful ethanol fermentation In conclusion, S. cerevisiae a mutant strain, showed notable ethanol fermentation performance on galactose and a mixture of galactose and glucose. The effects of substrate concentration, composition, and the potential inhibitors on ethanol fermentation using the strain were quantified. The results could be useful for bioethanol production using sustainable and renewable biomass such as red algae. Acknowledgments This work was supported by a grant (JA-13-0002) from Korea Institute of Industrial Technology, Republic of Korea. References [1] Panagiotou G, Olsson L. Effect of compounds released during pretreatment of wheat straw on microbial growth and enzymatic hydrolysis rates. Biotechnol Bioeng 2007;96:250e8. [2] Tomas Pejo E, Oliva JM, Ballesteros M, Olsson L. Comparison of SHF and SSF processes from steam exploded wheat straw for ethanol production by xylose fermenting and robust glucose fermenting Saccharomyces cerevisiae strains. Biotechnol Bioeng 2008;100:1122e31.
[3] Jorgensen H, Vibe Pedersen J, Larsen J, Felby C. Liquefaction of lignocellulose at high solids concentrations. Biotechnol Bioeng 2007;96:862e70. [4] Mosier N, Wyman C, Dale B, Elander R, Lee Y, Holtzapple M, et al. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour Technol 2005;96:673e86. [5] Kim C, Ryu HJ, Kim SH, Yoon JJ, Kim HS, Kim YJ. Acidity tunable ionic liquids as catalysts for conversion of agar into mixed sugars. Bull Korean Chem Soc Notes 2010;31:511e4. [6] Luning K, Pang S. Mass cultivation of seaweeds: current aspects and approaches. J Appl Phycol 2003;15:115e9. [7] Packer M. Algal capture of carbon dioxide; biomass generation as a tool for greenhouse gas mitigation with reference to New Zealand energy strategy and policy. Energy Policy 2009;37:3428e37. [8] Buck BH, Buchholz CM. The offshore-ring: a new system design for the open ocean aquaculture of macroalgae. J Appl Phycol 2004;16:355e68. [9] Jol CN, Neiss TG, Penninkhof B, Rudolph B, De Ruiter GA. A novel highperformance anion-exchange chromatographic method for the analysis of carrageenans and agars containing 3, 6-anhydrogalactose. Anal Biochem 1999;268:213e22. [10] Park JH, Yoon JJ, Park HD, Kim YJ, Lim DJ, Kim SH. Feasibility of biohydrogen production from Gelidium amansii. Int J Hydrog Energy 2011;36:13997e4003. [11] Knutsen S, Myslabodski D, Larsen B, Usov A. A modified system of nomenclature for red algal galactans. Bot Mar 1994;37:163e70. [12] Ostergaard S, Roca C, Rønnow B, Nielsen J, Olsson L. Physiological studies in aerobic batch cultivations of Saccharomyces cerevisiae strains harboring the MEL1 gene. Biotechnol Bioeng 2000;68:252e9. [13] Alexander M, Jeffries T. Respiratory efficiency and metabolite partitioning as regulatory phenomena in yeasts. Enzym Microb Technol 1990;12:2e19. [14] Keating JD, Robinson J, Bothast RJ, Saddler JN, Mansfield SD. Characterization of a unique ethanologenic yeast capable of fermenting galactose. Enzym Microb Technol 2004;35:242e53. [15] Lee KS, Hong ME, Jung SC, Ha SJ, Yu BJ, Koo HM, et al. Improved galactose fermentation of Saccharomyces cerevisiae through inverse metabolic engineering. Biotechnol Bioeng 2011;108:621e31. [16] Ostergaard S, Olsson L, Nielsen J. In vivo dynamics of galactose metabolism in Saccharomyces cerevisiae: metabolic fluxes and metabolite levels. Biotechnol Bioeng 2001;73:412e25. [17] Ross KL, Davis CN, Fridovich-Keil JL. Differential roles of the Leloir pathway enzymes and metabolites in defining galactose sensitivity in yeast. Mol Genet Metab 2004;83:103e16. [18] Holden HM, Rayment I, Thoden JB. Structure and function of enzymes of the Leloir pathway for galactose metabolism. J Biol Chem 2003;278:43885e8. [19] Blondin B, Ratomahenina R, Arnaud A, Galzy P. A study of cellobiose fermentation by a Dekkera strain. Biotechnol Bioeng 1982;24:2031e7. [20] Ha SJ, Galazka JM, Rin Kim S, Choi JH, Yang X, Seo JH, et al. Engineered Saccharomyces cerevisiae capable of simultaneous cellobiose and xylose fermentation. Proc Natl Acad Sci 2011;108:504e9. [21] Larsson S, Palmqvist E, Hahn-Hagerdal B, Tengborg C, Stenberg K, Zacchi G, et al. The generation of fermentation inhibitors during dilute acid hydrolysis of softwood. Enzym Microb Technol 1999;24:151e9. [22] Bisson LF, Coons DM, Kruckeberg AL, Lewis DA. Yeast sugar transporters. Crit Rev Biochem Mol Biol 1993;28:259e308.