Evaluation of Galactose Adapted Yeasts for Bioethanol Fermentation from Kappaphycus alvarezii Hydrolyzates

Abstract

Bioethanol was produced from Kappaphycus alvarezii seaweed biomass using separate hydrolysis and fermentation (SHF). Pretreatment was evaluated for 60 min at 121℃ using 12% (w/v) biomass slurry with 364 mM H₂SO₄. Enzymatic saccharification was then carried out at 45℃ for 48 h using Celluclast 1.5 L. Ethanol fermentation with 12% (w/v) K. alvarezii hydrolyzate was performed using the yeasts Saccharomyces cerevisiae KCTC1126, Kluyveromyces marxianus KCTC7150, and Candida lusitaniae ATCC42720 with or without prior adaptation to high concentrations of galactose. When non-adapted S. cerevisiae, K. marxianus, and C. lusitaniae were used, 11.5 g/l, 6.7 g/l, and 6.0 g/l of ethanol were produced, respectively. When adapted S. cerevisiae, K. marxianus, and C. lusitaniae were used, 15.8 g/l, 11.6 g/l, and 13.4 g/l of ethanol were obtained, respectively. The highest ethanol concentration was 15.8 g/l, with Y_EtOH = 0.43 and Y_T% = 84.3%, which was obtained using adapted S. cerevisiae.

Introduction

Macroalgae is considered as a biomass feedstock for bioenergy resources [7]. Macroalgae are composed of carbohydrate-containing cell walls and various constitutive polysaccharides, which can be transformed from sugar to ethanol [8]. Moreover, macroalgae do not represent competition to food crops, and can grow more rapidly than land-based biomass [5]. Seaweed can be classified into macroalgae and microalgae. Macroalgae are typically red, brown, or green algae, which are composed of agar and carrageenan (from red seaweeds), alginate (from brown seaweeds), and cellulose (from green seaweeds). Kappaphycus alvarezii (Eucheuma cottonii) is a red seaweed [16], and is one of the largest tropical carrageenophytes and one of the most abundant aquatic feedstocks. In this work, K. alvarezii was used for ethanol fermentation because of its abundance and high D-type galactose concentration [17].

Bioethanol production from macroalgae biomass requires three major processes: thermal hydrolysis using acids, saccharification using enzymes, and fermentation using yeasts. Many previous works on bioconversion of seaweed biomass to ethanol via the acid hydrolysis and enzymatic saccharification processes have been preported. Ra et al. [22] studied ethanol fermentation from Gracilaria verrrucosa. Cho et al. [4] has also reported about ethanol production from red seaweed Gelidium amansii.

In this work, K. alvarezii slurry was used for the production of ethanol. Thermal acid hydrolysis of K. alvarezii slurry was carried out to obtain a high concentration galactose. Enzymatic treatment for the saccharification was carried out to obtain glucose from cellulose using Celluclast 1.5 L, Viscozyme L, and/or Spirizyme.

Saccharomyces cerevisiae produces a high yield coefficient of ethanol near the theoretical yield coefficient of YEtOH = 0.51 (51%) from glucose [14]. Saccharomyces cerevisiae and Kluyveromyces marxianus are recognized as being safe for fermented food at high temperatures. They had a high growth rate and similar fermentation efficiency at 30℃ [2,18]. Candida lusitaniae has also been reported to have high initial concentrations of ethanol production [6] and high ethanol conversion [25]. Therefore, in this study, fermentation was performed using S. cerevisiae, K. marxianus, or C. lusitaniae to determine the optimal yeast strain for ethanol production.

 

Materials and Methods

Raw Materials and Composition Analysis

Saccharomyces cerevisiae KCTC 1126 and Kluyveromyces marxianus KCTC 7150 were obtained from the Korean Collection for Type Cultures (KCTC), Biological Resource Center (Korea). Candida lusitaniae ATCC 42720 was obtained from the American Type Culture Collection (ATCC). K. alvarezii was produced in Indonesia and was obtained from Biolsystems Co. Ltd. (Korea).

K. alvarezii was dried using sunlight, ground using a roller mill, and sieved with a 200-mesh sieve before pretreatment. The composition analysis of K. alvarezii was conducted according to the AOAC method [1] by the Feed and Foods Nutrition Research Center at Pukyong National University in Busan, Korea

Thermal Acid Hydrolysis Pretreatment

Thermal acid hydrolysis of 121℃ temperature was enough to obtain a high concentration of sugars from seaweed K. alvarezii [22,26]. Therefore, pretreatment was focused on the effects of the three factors such as slurry contents, H2SO4 concentration, and treatment time. The pretreatment was carried out using the weight/volume fraction of slurry contents ranging 8–16% (w/v) with 182 mM H2SO4 at 121℃ for 60 min. Then, the H2SO4 concentration was optimized. The pretreatment was carried out using the optimal condition of slurry content determined previously and H2SO4 concentrations ranging 182–564 mM at 121℃ for 60 min. The thermal hydrolysis time was also optimized. The pretreatment was carried out using the optimal slurry content and optimal H2SO4 concentration at 121℃ for the determination the thermal hydrolysis time ranging 30–150 min.

K. alvarezii slurry (100 ml working volume in a 250 ml flask) was heated to 121℃, and the hydrolyzates were neutralized to pH 5.0 using 10 M NaOH. The efficiency of thermal acid hydrolysis pretreatment [21] was calculated using Eq. (1) as follows:

where ΔSmono is the change in monosaccharide (g/l) during the thermal acid hydrolysis, and TC is the total carbohydrate content (g/l) of K. alvarezii.

Enzymatic Saccharification

The optimal conditions for the enzymatic saccharification of K. alvarezii were investigated after finding the optimal condition for thermal acid hydrolysis, using 12% slurry concentration. A 250 ml Erlenmeyer flask with 100 ml working volume was used for the enzymatic saccharification, and 16 U/ml Viscozyme L (121 fungal β-glucanase unit (FBG)/ml; Novozyme, Denmark), Celluclast 1.5 L (854 endo-glucanase unit (EGU)/ml; Novozyme), Spirizyme Fuel (862 amyloglucosidase unit (AUG)/ml; Novozyme), and 16 U/ml of their mixed enzymes (with 1:1 ratio of 2 mixed enzymes, 1:1:1 r atio o f 3 mixed enzymes) were u sed at pH 5.0, 45℃, 150 rpm for 48 h [22]. Viscozyme L contains endo-beta-glucanase that hydrolyzes (1,3)- or (1,4)-linkages in beta-ᴅ-glucans, with side activities of xylanase, cellulose, and hemicellulose. Celluclast 1.5 L contains cellulase hydrolyzing (1,4)-beta-ᴅ-glucosidic linkages in cellulose and other beta-ᴅ-glucans. Spirizyme Fuel contains amyloglucosidase hydrolyzing both 1,4- and 1,6-alpha linkages to produce glucose for fermentation using yeast. The activities of β-glucosidase and cellulase were determined according to Mandels et al. [15] and Kubicek et al. [13]. The efficiency of enzymatic saccharification [21] was calculated using Eq. (2) as follows:

where ΔSglu is the change in glucose concentration (g/l) when enzymatic saccharification was carried out, and C is the cellulose concentration (g/l) in K. alvarezii.

Fermentation

Seed culture and adaptation of yeasts. This adaptation study has been reported by Ra et al. [22]. The yeast can increase the metabolic activities of galactose utilization by culturing yeasts in a high-concentration galactose medium. The adaptation of high galactose concentration can reduce the glucose repression.

Adaptation of yeasts to various galactose concentrations was carried out. Optimal conditions for yeast growth were determined using yeast extract-peptone-galactose (YPG) medium containing 10 g/l of yeast extract and 20 g/l of peptone, as well as a systematically varied concentration of galactose (in the range 20–120 g/l). Saccharomyces cerevisiae KCTC 1126, Kluyveromyces marxianus KCTC 7150, and Candida lusitaniae ATCC 42720 were cultured on YPG agar plates containing 20 g/l galactose for 24 h. Each colony of yeasts was transferred to 30 ml of YPG medium containing 20 g/l of galactose, and was cultured at 30℃ and 150 rpm for 24 h. Ten milliliters of culture was then transferred to 100 ml of YPG with various galactose concentrations (20–120 g/l) in a 250 ml flask, and incubated in a shaking incubator at 30℃ and 150 rpm for 72 h [22]. Cultured yeast strains were sampled to determine the dry cell weight using the optical density (OD600).

Ethanol fermentation. Fermentation was evaluated in 250 ml flasks with a working volume of 100 ml. Following pretreatment, neutralization to pH 5.0 and enzymatic saccharification were carried out. The following nutrients were added to the fermentation medium: 2.5 g/l of NH4Cl, 5 g/l o f K2HPO4, 0.25 g/l of MgSO4, and 2.5 g/l of yeast extract. Fermentation was performed with S. cerevisiae, K. marxianus, or C. lusitaniae with or without prior adaptation to high concentrations of galactose, at 30℃, 150 rpm, and for 144 h. The bioethanol yield coefficient was calculated using Eq. (3):

where [EtOH]max is the highest ethanol concentration achieved during fermentation (g/l) and [sugar]ini is the total initial sugar concentration at the start of fermentation (g/l). The definition of yield coefficient is generally accepted for the ethanol fermentation. The maximum yield coefficient is 0.51 [19] by the total conversion of 2 mol ethanol (MW = 46) from the hexose (MW = 180)[YEtOHmax=92/180=0.51]. The percent theoretical yield coefficient (Y%T) was calculated using Eq. (4)

Analytical Methods

Cell growth was determined based on the optical density at 600 nm (OD600), and was converted to the dry cell weight (dcw) using a standard curve. The glucose, galactose, 5-hydroxymethylfurfural (5-HMF), and ethanol concentrations were determined using HPLC (1100 Series; Agilent Technologies, USA) equipped with a RID. A Bio-Rad Aminex HPX-87H column (300 × 7.8 mm; Bio-Rad, USA) was used with filtered and degassed 5 mM H2SO4 as an eluent at a flow rate of 0.6 ml/min and a temperature of 65℃. The fermentation samples were centrifuged for 10 min at 14,240 ×g, and the supernatant was filtered using 0.2 μm filter paper prior to analysis.

 

Results and Discussion

Composition of K. alvarezii

K. alvarezii is a red alga with a particularly high carbohydrate content. The major carbohydrates are kappa-carrageenan consisting of galactose [11] and cellulose consisting of glucose [27]. The composition of K. alvarezii was analyzed by the AOAC method and found to contain 65.8% carbohydrate, 4.6% crude protein, 0.8% crude lipids, 22.9% crude ash, and 5.9% cellulose. The total carbohydrate content of the K. alvarezii used in this study was 71.7%, including cellulose.

Thermal Acid Hydrolysis Pretreatment

K. alvarezii was used for acid hydrolysis to produce sugars [17]. Thermal acid hydrolysis was performed to determine the effects of the following three factors: the concentration of K. alvarezii slurry, the H2SO4 concentration, and the treatment time. During thermal acid hydrolysis pretreatment, 5-HMF (regarded as a fermentation inhibitor) was generated from the degradation of 3,6-anhydrogalactose (AHG) owing to acid-labile character of AHG [7,15].

The slurry content was varied in the range 8–16% (w/v) and 182 mM H2SO4 at 121℃ for 60 min for thermal acid hydrolysis to determine the optimal slurry contents. Fig. 1A shows that the sugar concentration increased as the slurry content increased, and the monosaccharide concentration with a slurry content of 16% (w/v) was 30.6 g/l, giving an efficiency of Ep = 26.6%. However, increasing the slurry content over 12% during thermal acid hydrolysis pretreatment resulted in a decrease in Ep (from 30.3% to 26.6%). Levels of 5-HMF increased from 4.1 to 7.0 g/l with increasing slurry content from 8% to 16% (w/v). Wu et al. [26] also reported that during acid hydrolysis of red alga Pterocladiella capillacea, the 5-HMF increased with increasing slurry content; therefore, a slurry content of 12% (w/v) with Ep = 30.3% was selected for ethanol production.

Fig. 1.The effects of thermal acid hydrolysis pretreatment with various slurry contents, acid concentrations, and thermal hydrolysis time on the efficiency of pretreatments. (A) The K. alvarezii slurry content (8–16% (w/v), 182 mM H2SO4 at 121℃ for 60 min), (B) the H2SO4 concentration (182–546 mM, 12% (w/v) slurry content at 121℃ for 60 min), and (C) the thermal hydrolysis time (30–150 min, 12% (w/v) slurry content, 364 mM H2SO4 at 121℃). *indicates highest efficiency of pretreatment with given variables.

The H2SO4 concentration was varied in the range 182–546 mM and 12% (w/v) slurry content at 121℃ for 60 min. As shown in Fig. 1B, the galactose concentration increased slightly with increasing H2SO4 concentration. Redding et al. [23] reported that the release of high amounts of monosaccharide was resulted by high acid concentrations; however, high H2SO4 concentrations in the range 455–546 mM resulted in a lower value of Ep compared with 364 mM H2SO4. Increasing the H2SO4 concentration during thermal acid hydrolysis resulted in a decrease in HMF concentration due to the subsequent decomposition of HMF into inhibitory compounds such as levulinic acid and formic acid [12]. These results were consistent with monosaccharide conversion to 5-HMF and subsequent conversion to levulinic acid and formic acid. Therefore, 364 mM H2SO4 was considered as the optimal acid concentration, giving Ep = 35.6%.

The treatment time was varied in the range 30–150 min, with a slurry content of 12% (w/v) and an H2SO4 concentration of 364 mM at 121℃. Fig. 1C shows that the high monosaccharide concentration was 30.7 g/l with Ep = 35.7% at 60 min, and the 5-HMF concentration increased as the hydrolysis time increased. Ra et al. [22] reported that hydrolysis times of over 60 min decreased ethanol production owing to the high concentration of HMF. Therefore, 60 min was selected as the optimal hydrolysis time.

From these results, the optimal conditions for thermal acid pretreatment were as follows: 12% (w/v) slurry and 364 mM H2SO4 at 121℃ for 60 min. The HMF concentration increased as the slurry content increased. Thermal acid hydrolysis pretreatment with these optimal conditions produced 4.2 g/l of 5-HMF.

Enzymatic Saccharification

Enzymatic saccharification is the process of hydrolyzing the cellulose to form monosaccharides to facilitate the ethanol fermentation using yeasts [10]. The effects of single and mixed enzyme saccharifications on glucose release with 12% (w/v) K. alvarezii hydrolyzate are shown in Fig. 2. The glucose content after thermal acid hydrolysis was 5.6 g/l. For hydrolysis of the cellulose, 16 U/ml of Viscozyme L (121 FBG/ml), Celluclast 1.5L (854 EGU/ml), or Spirizyme Fuel (862 AUG/ml), as well as mixed enzymes (with 8 U/ml: 8 U/ml ratio of 2 mixed enzymes, 5.3 U/ml:5.3 U/ml: 5.3 U/ml ratio of 3 mixed enzymes), was used with 12% (w/v) of K. alvarezii slurry at pH 5.0, 45℃, and 150 rpm for 48 h [22]. The maximum glucose concentration was 11.5g /l, and was obtained using Celluclast 1.5 L, giving Es = 83.6%. Similar results have been reported in Pusawati et al. [20] that high reducing sugar production from the enzymatic saccharification process was obtained from K. alvarezii. Therefore, Celluclast 1.5L alone, consisting mainly of cellulase, was selected as the optimal enzyme treatment.

Fig. 2.The effects of single and mixed enzyme treatments on glucose release from K. alvarezii hydrolyzate with a 12% (w/v) slurry following thermal acid hydrolysis pretreatment. The conditions were as follows: pH 5.0, 45℃, and 48 h. The initial glucose concentration was 5.6 g/l (i.e., after thermal acid hydrolysis pretreatment).

Adaptation of Yeasts to Galactose

Primary observations showed that galactose consumption was inhibited by glucose uptake. The yeasts produce ethanol from glucose [9]. However, when ethanol production is performed from a mixture of galactose and glucose, galactose consumption is inhibited by glucose repression. The uptake of galactose by yeasts requires the Leloir pathway of enzymes, which convert galactose to glucose-6-phosphate [4]. These enzymes are encoded in the GAL gene family, and their expression is induced by growth in galactose, and repressed by growth in glucose. When glucose was present in the medium, the GAL gene family was repressed by the inactivation of the transcriptional activator [4,22]. Therefore, the adaptation of yeasts to high galactose concentration eliminates the glucose repression to galactose utilization [3].

The effects of galactose adaptation on cell growth of three different yeasts were evaluated using various galactose concentrations (20–120 g/l) in YPG media. In general, ethanol production is associated with yeast cell growth under anaerobic conditions; thus, the substrate yield coefficient (Yx/s) of various concentrations is related to the stoichiometrically obtained theoretical Yx/s. The estimation of theoretical growth and product yield coefficients for ethanol fermentation by yeast has been well established [24]. The ATP yield (Yx/ATP) in anaerobic fermentation is approximately 10.5g dcw/mol ATP. Ethanol production from hexose produces 2 ATP. Thus, the predicted growth yield coefficient is Yx/s = (10.5g dcw/mol ATP) × (2 ATP/mol)/(180 g glucose) = 0.117 g dcw/g glucose for the optimal ethanol fermentation. Therefore, in this study, we selected the galactose concentration showing similar Yx/s to theoretical Yx/s = 0.117 as the optimal adaptation concentration of yeasts in various galactose concentrations.

As shown in Fig. 3A, the galactose concentration in the range of 20-40 g/l showed high Yx/s of 0.275 and 0.167 g dcw/g galactose, producing less amount of ethanol. These results indicated that a high value of Yx/s suggests that S. cerevisiae has a high uptake of galactose for cell growth. However, the Yx/s of 80 g/l and 120 g/l galactose in anaerobic fermentation obtained 0.108 and 0.074 g dcw/g galactose, respectively. The substrate yield coefficient of 80 g/l showed a similar value of Yx/s to the theoretical Yx/s of 0.117. Therefore, 80 g/l galactose was selected for adaptation of S. cerevisiae.

Fig. 3.The growth of cells with different galactose concentrations in the medium. (A) Saccharomyces cerevisiae, (B) Kluyveromyces marxianus, and (C) Candida lusitaniae.

The same results were obtained with K. marxianus and C. lusitaniae. As shown in Figs. 3B and 3C, galactose concentrations of 20, 40, 80, and 120 g/l showed Yx/s of 0.210, 0.123, 0.120, and 0.129 g dcw/g galactose with K. marxianus and Yx/s of 0.425, 0.268, 0.149, and 0.216 g dcw/g galactose with C. lusitaniae, respectively. Yx/s of 80 g/l galactose in anaerobic fermentation of K. marxianus and C. lusitaniae showed 0.120 and 0.149 g dcw/g galactose, similar to the theoretical Yx/s of 0.117, respectively. Therefore, 80 g/l galactose was also selected for the adaptation of K. marxianus and C. lusitaniae.

From these results, we concluded that adapted S. cerevisiae, K. marxianus, and C. lusitaniae grown in YPG media with a galactose concentration of 80 g/l could perform the optimal fermentations.

Ethanol Fermentation

Thermal acid hydrolysis pretreatment and enzymatic saccharification were evaluated with 12% (w/v) K. alvarezii slurry to produce the maximum amount of monosaccharide. The hydrolyzate was then used for fermentation. SHF was performed by adding the adapted or non-adapted S. cerevisiae, K. marxianus, and C. lusitaniae to galactose.

Fig. 4A shows the results of fermentation using non-adapted S. cerevisiae. The sugar concentration at the start of fermentation was 37.2 g/l. Glucose was the preferred substrate (as opposed to galactose). Glucose was consumed during the first 48 h, and then galactose was consumed until 144 h. However, the galactose was not totally consumed at 144 h, and 9.8 g/l of galactose remained. The ethanol concentration after 144 h of fermentation with non-adapted S. cerevisiae was 11.5 g/l, with YEtOH = 0.31 and Y%T = 60.8%.

Fig. 4.Ethanol production from K. alvarezii hydrolyzate via separate hydrolysis and fermentation with adapted or non-adapted Saccharomyces cerevisiae KCTC1126, Kluyveromyces marxianus KCTC7150, and Candida lusitaniae ATCC42720 to high concentration of galactose (80 g/l). (A) Non-adapted S. cerevisiae, (B) adapted S. cerevisiae, (C) non-adapted K. marxianus, (D) adapted K. marxianus, (E) non-adapted C. lusitaniae, and (F) adapted C. lusitaniae.

Fig. 4B shows the results of fermentation with adapted S. cerevisiae. The galactose concentration was 25.80 g/l, and the initial glucose concentration was 11.30 g/l. The glucose and galactose were consumed simultaneously. The glucose was consumed after 48 h, and after 144 h, 1.9 g/l of galactose remained. The final ethanol concentration was 15.8 g/l, with YEtOH = 0.43 and Y%T = 84.3%. Meinita et al. [17] reported that when using non-adapted S. cerevisiae for ethanol fermentation from K. alvarezii hydrolyzate, the ethanol yield coefficient was 0.21 g/g on the basis of galactose, which corresponds to 41% of the theoretical maximum yield coefficient of galactose. Therefore, the adaptation of S. cerevisiae to high concentrations of galactose is important to increase the production of ethanol from K. alvarezii.

Fig. 4C shows the results of fermentation with non-adapted K. marxianus. The sugar concentration at the start of fermentation was 36.8 g/l. Glucose was consumed during the first 48 h, and then galactose was consumed, with 17.4 g/l remaining after 144 h. The final ethanol concentration was 6.7 g/l, with YEtOH = 0.18 and Y%T = 35.3%. This is similar to the result for non-adapted S. cerevisiae owing to the repression of galactose uptake by glucose.

Fig. 4D shows the results of fermentation with adapted K. marxianus. The sugar concentration at the start of fermentation was 37.2 g/l. Glucose and galactose were consumed simultaneously. Glucose was consumed during the first 48 h, and 7.1 g/l of galactose remained after 144 h. The final concentration of ethanol was 11.6 g/l, with YEtOH = 0.31 and Y%T = 60.8%.

Wu et al. [26] reported fermentation of the red seaweed Pterocladiella capillacea hydrolyzate (12% (w/v)) using K. marxianus, with a maximum ethanol yield coefficient of 0.17. This result is consistent with our yield coefficient from the fermentation with non-adapted K. marxianus. Therefore, the adaptation of K. marxianus to high concentrations of galactose is a promising method for increasing the ethanol yield coefficient.

Fig. 4E shows the results of fermentation with non-adapted C. lusitaniae. The initial galactose concentration was 25.0 g/l, and the initial glucose concentration was 11.6 g/l. The glucose consumption rate was lower than those of non-adapted S. cerevisiae and K. marxianus, requiring 72 h. Little galactose was consumed because of the repression of galactose uptake by glucose. Thus, an ethanol concentration of 6.0 g/l was obtained, with YEtOH = 0.16 and Y%T = 31.4%.

When C. lusitaniae was adapted to high concentrations of galactose, both glucose and galactose were consumed simultaneously, as shown in Fig. 4F. An ethanol concentration of 13.4 g/l was obtained, with YEtOH = 0.37 and Y%T = 72.5%; only 5.3 g/l of galactose remained. Ra et al. [22] reported fermentation of the red seaweed Gracilaria verrucosa using adapted and non-adapted C. lusitaniae. Their results also showed that the adapted yeast increased the sugar uptake compared with non-adapted yeast and the ethanol yield coefficient increased from 0.29 to 0.43. This is consistent with our work, showing that a higher ethanol yield coefficient was obtained with the adapted C. lusitaniae.

Our results show that K. alvarezii is a promising biomass resource for bioethanol production. The optimal pretreatment conditions were 364 mM H2SO4, with a 12% (w/v) slurry at 121℃ for 60 min, and the optimal conditions for saccharification were 16 U/ml of Celluclast 1.5L at 45℃ for 48 h. We adapted S. cerevisiae, K. marxianus, and C. lusitaniae to high concentrations of galactose and found that these yeasts resulted in significantly higher concentrations of ethanol production compared with non-adapted strains. The maximum ethanol concentration was 15.8 g/l, with YEtOH = 0.43 and YT% = 84.3%, obtained using SHF with adapted S. cerevisiae.