Scholarly article on topic 'Improvement of ethanol production from sweet sorghum juice under batch and fed-batch fermentations: Effects of sugar levels, nitrogen supplementation, and feeding regimes'

Improvement of ethanol production from sweet sorghum juice under batch and fed-batch fermentations: Effects of sugar levels, nitrogen supplementation, and feeding regimes Academic research paper on "Chemical engineering"

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{"Agricultural raw materials" / "Alternative energy source" / "Batch fermentation" / Bioethanol / "Ethanol-tolerant strain" / "Fed-batch fermentation" / "High gravity fermentation" / "Normal gravity fermentation" / "Nutrient supplementation" / " Saccharomyces cerevisiae " / "Sweet sorghum juice" / "Very high gravity"}

Abstract of research paper on Chemical engineering, author of scientific article — Niphaphat Phukoetphim, Apilak Salakkam, Pattana Laopaiboon, Lakkana Laopaiboon

Abstract Background Fermentation process development has been very important for efficient ethanol production. Improvement of ethanol production efficiency from sweet sorghum juice (SSJ) under normal gravity (NG, 160g/L of sugar), high gravity (HG, 200 and 240g/L of sugar) and very high gravity (VHG, 280 and 320g/L of sugar) conditions by nutrient supplementation and alternative feeding regimes (batch and fed-batch systems) was investigated using a highly ethanol-tolerant strain, Saccharomyces cerevisiae NP01. Results In the batch fermentations without yeast extract, HG fermentation at 200g/L of sugar showed the highest ethanol concentration (P E , 90.0g/L) and ethanol productivity (Q E , 1.25g/L·h). With yeast extract supplementation (9g/L), the ethanol production efficiency increased at all sugar concentrations. The highest P E (112.5g/L) and Q E (1.56g/L·h) were observed with the VHG fermentation at 280g/L of sugar. In the fed-batch fermentations, two feeding regimes, i.e., stepwise and continuous feedings, were studied at sugar concentrations of 280g/L. Continuous feeding gave better results with the highest P E and Q E of 112.9g/L and 2.35g/L·h, respectively, at a feeding time of 9h and feeding rate of 40gsugar/h. Conclusions In the batch fermentation, nitrogen supplementation resulted in 4 to 32g/L increases in ethanol production, depending on the initial sugar level in the SSJ. Under the VHG condition, with sufficient nitrogen, the fed-batch fermentation with continuous feeding resulted in a similar P E and increased Q P by 51% compared to those in the batch fermentation.

Academic research paper on topic "Improvement of ethanol production from sweet sorghum juice under batch and fed-batch fermentations: Effects of sugar levels, nitrogen supplementation, and feeding regimes"

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EJBT-00225; No of Pages 9

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Contents lists available at ScienceDirect

Electronic Journal of Biotechnology

Research article

Improvement of ethanol production from sweet sorghum juice under batch and fed-batch fermentations: Effects of sugar levels, nitrogen supplementation, and feeding regimes

Niphaphat Phukoetphim a, Apilak Salakkam b,c, Pattana Laopaiboon b,c, Lakkana Laopaiboon b,c *

a Graduate School, Khon Kaen University, Khon Kaen 40002, Thailand

b Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand c Fermentation Research Center for Value Added Agricultural Products, Khon Kaen University, Khon Kaen 40002, Thailand

ARTICLE INFO

ABSTRACT

Article history: Received 27 July 2016 Accepted 18 January 2017 Available online xxxx

Keywords:

Agricultural raw materials Alternative energy source Batch fermentation Bioethanol

Ethanol-tolerant strain Fed-batch fermentation High-gravity fermentation Normal gravity fermentation Nutrient supplementation Saccharomyces cerevisiae Sweet sorghum juice

Background: Fermentation process development has been very important for efficient ethanol production. Improvement of ethanol production efficiency from sweet sorghum juice (SSJ) under normal gravity (NG, 160 g/L of sugar), high gravity (HG, 200 and 240 g/L of sugar) and very high gravity (VHG, 280 and 320 g/L of sugar) conditions by nutrient supplementation and alternative feeding regimes (batch and fed-batch systems) was investigated using a highly ethanol-tolerant strain, Saccharomyces cerevisiae NP01.

Results: In the batch fermentations without yeast extract, HG fermentation at 200 g/L of sugar showed the highest ethanol concentration (PE, 90.0 g/L) and ethanol productivity (Qe, 1.25 g/L-h). With yeast extract supplementation (9 g/L), the ethanol production efficiency increased at all sugar concentrations. The highest PE (112.5 g/L) and QE (1.56 g/L-h) were observed with the VHG fermentation at 280 g/L of sugar. In the fed-batch fermentations, two feeding regimes, i.e., stepwise and continuous feedings, were studied at sugar concentrations of 280 g/L. Continuous feeding gave better results with the highest PE and Q of 112.9 g/L and 2.35 g/L-h, respectively, at a feeding time of 9 h and feeding rate of 40 g sugar/h.

Conclusions: In the batch fermentation, nitrogen supplementation resulted in 4 to 32 g/L increases in ethanol production, depending on the initial sugar level in the SSJ. Under the VHG condition, with sufficient nitrogen, the fed-batch fermentation with continuous feeding resulted in a similar PE and increased Qp by 51% compared to those in the batch fermentation.

© 2017 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Bioethanol is an alternative energy source that is both renewable and environmentally friendly. It can be produced from agricultural raw materials such as corn grain, cassava, sugar cane, sugar cane molasses, and sweet sorghum, among others. Sweet sorghum, Sorghum bicolor (L.) Moench, is a potential alternative feedstock for bioethanol production because the juice from its stalks contains high levels of fermentable sugars, mainly sucrose, fructose, and glucose, and it has short life cycle of only 100-120 d. Moreover, it can be cultivated at almost all temperatures in tropical areas [1,2].

Saccharomyces cerevisiae is widely used in industrial ethanol production [3]. In addition to yeast strains, nutrients, and environmental conditions, the ability of yeast to produce ethanol also depends on the initial sugar concentration of the fermentation medium. In ethanol

* Corresponding author. E-mail address: lakcha@kku.ac.th (L Laopaiboon).

Peer review under responsibility of Pontificia Universidad Católica de Valparaíso.

fermentation, 1 mol of glucose can be converted to 2 mol of ethanol and 2 mol of carbon dioxide. Therefore, a medium containing a higher sugar concentration will give a higher ethanol concentration. Typically, sugar concentrations for ethanol fermentation are divided into normal gravity (NG) (< 180 g/L of sugar), high gravity (HG) (180-240 g/L of sugar), and very high gravity (VHG) conditions (>250 g/L of sugar) [4,5]. However, high sugar concentrations or VHG conditions cause an increased osmotic pressure, which has negative effects on yeast cells. Bafrncova et al. [6] reported that under appropriate environmental and nutritional conditions, S. cerevisiae could produce and tolerate high ethanol concentrations.

Fermentation process development has been very important for efficient ethanol production [7,8]. Ethanol fermentation can be performed in batch, fed-batch, and continuous modes. The batch fermentation is a closed culture system. Biomass and substrate are added into fermenter without removal of media during fermentation, and products are harvested at the end of the fermentation. The batch mode has disadvantages, particularly when microorganisms are either slow growing or strongly affected by substrate inhibition [9]. The

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fed-batch mode is started as a batch mode with a small amount of biomass and substrate in the fermenter. Then, a feeding medium is fed, stepwise or continuously, to the fermenter when most of the initially added substrate has been consumed. This process can increase the total substrate content in the fermenter while maintaining a low substrate concentration during fermentation to reduce the negative effects of osmotic pressure on yeast. The advantages of this process include reduction of substrate inhibition, higher productivity, shortened fermentation time, and reduction of toxic effects of the medium components, which are present at high concentrations [10]. Stepwise feeding of fed-batch fermentation was previously demonstrated to be effective in enhancing ethanol production and yield from sweet sorghum juice (SSJ) under HG conditions [8]. In the current study, stepwise and continuous feedings were examined under VHG conditions to determine if these regimes could enhance fermentation efficiency at very high initial sugar concentrations.

Ethanol produced by yeast is toxic to the yeast itself. To achieve high-level ethanol production, yeast strains that can produce and tolerate high ethanol concentration should be used. S. cerevisiae NP01 and S. cerevisiae ATCC 4132 are considered robust ethanol-producing strains because of their ability to produce high ethanol titers under HG and VHG conditions [2,11]. However, their ethanol tolerance has not been examined. In the current study, the ability of these yeast strains to tolerate ethanol at various concentrations was tested. Improvement of ethanol production efficiency from SSJ under NG, HG, and VHG conditions by nutrient supplementation and alternative feeding regimes (batch and fed-batch systems) was subsequently investigated.

2. Materials and methods

2.1. Microorganisms

2.2. Raw materials and ethanol production medium 128

Sweet sorghum cv. KKU40 was obtained from the Division of 129

Agronomy, Faculty of Agriculture, Khon Kaen University, Thailand. 130

To prevent bacterial contamination and improve storage stability 131

after extraction, the juice (17 °Bx) was heated to approximately 90°C 132

to concentrate to 65 °Bx, cooled, and stored at 4°C until use. It was 133

diluted with distilled water to 160, 200, 240, 280, and 320 g/L of sugar 134

and optionally supplemented with 9 g/L of yeast extract [13] before 135

use as an ethanol production (EP) medium. 136

2.3. Ethanol tolerance 137

S. cerevisiae NP01 or S. cerevisiae ATCC 4132 was inoculated into 138

50 mL of SSJ containing 100 g/L of sugar to attain an initial cell 139

concentration of ~5 x 107 cells/mL. Then ethanol was added to the 140

cultures at 0, 6, 9, 12, 15, and 18% (v/v). The setup was incubated 141

at 30°C and 100 rpm for 24 h. The yeast viability was measured at 142

regular time intervals. The yeast strain that showed higher ethanol Q4

tolerance was used in subsequent experiments. 144

2.4. Batch ethanol fermentation 145

EP media with and without 9 g/L of yeast extract were transferred 146

into 500-mL air-locked Erlenmeyer flasks with a working volume 147

of 400 mL and autoclaved at 110°C for 28 min [2]. The active cells 148

of the more ethanol-tolerant strain were inoculated into sterile EP 149

media to obtain an initial cell concentration of ~5 x 107 cells/mL. The 150

fermentation was performed at 30°C with an agitation rate of 100 rpm. 151

The samples were withdrawn at regular time intervals for analyses. 152

S. cerevisiae NP01 (accession number KP866701) was isolated from Loog-pang (Chinese yeast cake) for Sato (Thai rice wine) making and was identified by gene sequencing analysis using the D1/D2 domain of 26S rDNA [5], and S. cerevisiae ATCC 4132 was isolated from molasses distillery yeast. The yeasts were inoculated into 100 mL of yeast extract and malt extract (YM) medium (containing yeast extract, 3 g/L; malt extract, 3 g/L; peptone, 5 g/L; and glucose, 10 g/L) and incubated at 200 rpm and 30°C for 18 h. Then, the cultures (10% inoculum size) were transferred into 350 mL of SSJ containing 100 g/L of sugar [12] and incubated under the same conditions. After 15 h, the cells were harvested and used as inocula for ethanol fermentations.

2.5. Fed-batch ethanol fermentation

Two feeding regimes for the fed-batch fermentation were used 154 under VHG conditions. The first regime was stepwise feeding. Here, 155 the fermentation was first performed in batch mode with sterile 156 EP medium using 50% of the total working volume [8,14]. After 12 or 157 24 h, an equal volume of fresh sterile EP medium was carefully added 158 into the flasks. The second regime was continuous feeding. Here, the 159 other half of fresh EP medium was fed continuously at flow rates of 1X 160 (10 g sugar/h), 2X (20 g sugar/h), and 4X (40 g sugar/h) to achieve 161 final total sugar concentrations in the range of a VHG condition. 162

,_, 9 ,_, 9

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I 7 ■ I 7

as 6 as 6

0 5 0 5

-Q 4 ■ -Q 4

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0 0

ro 2 ro 2

0 0

_1 1 ■ —'—1-1-— —,— -,-,— -,-,- _1 1

3 6 9 12 15 18 21 24 Time (h)

12 15 18 21 24

Time (h)

Control

Fig. 1. Time profiles of cell survival of S. cerevisiae NP01 (a) and S. cerevisiae ATCC 4132 (b) in the presence of ethanol at different concentrations.

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® 0.15 i

СП О 4= Ö a a M

0.10 -

0.05 -

Ethanol concentration (%)

ATCC 4132

Fig. 2. Comparison of the specific growth rates of S. cerevisiae NP01 and S. cerevisiae ATCC 4132 in the presence of ethanol at different concentrations.

fermentation broth was centrifuged at 13,000 rpm for 10 min to 168

remove solid particles. The supernatant was decanted, and its sugar 169

content was determined using a phenol sulfuric acid method [15]. 170

Ethanol concentration (PE, g/L) was analyzed by gas chromatography 171

[2]. The ethanol yield (YE/S) was calculated as the actual amount of 172

ethanol produced and expressed as g ethanol per g of sugars utilized 173

(g/g). The volumetric ethanol productivity (QE, g/L-h) was calculated 174

by dividing ethanol concentration produced (PE, g/L) by fermentation 175

time at which the highest ethanol concentration was attained. Nitrogen 176

in the fermentation broth was analyzed using a microwell ninhydrin 177

assay to determine free amino nitrogen (FAN) [16]. Glycerol, the main 178

by-product during ethanol fermentation, was quantified by HPLC 179

according to Sirisantimethakom et al. [17]. 180

The sugar consumption rate (g/L-h) in batch fermentations under 181

NG, HG, and VHG conditions was calculated for use in fed-batch 182

fermentations. It was determined from the sugars consumed during 183

the first 24 h of incubation. 184

3. Results and discussion 185

163 During the fed-batch fermentation, samples were obtained at regular

164 time intervals for analyses.

165 2.6. Analytical methods

166 The viable yeast cell numbers were determined by a direct counting

167 method using hemocytometer and methylene blue staining. The

3.1. Ethanol tolerance 186

When the NP01 and ATCC 4132 strains were subjected to ethanol 187 at the same concentrations, cell survival of both strains was similar 188 (Fig. 1). The yeast could grow in SSJ containing 100 g/L of sugar in the 189 presence of up to 6% ethanol. However, the growth at 6% ethanol 190 was lower than that in the absence of ethanol. The highest viable cell 191

CO ■>

36 48 60 Time (h)

320 280 240 200 160 120 80 40 0

160 g/L

- « - 200 g/L

■ 240 g/L

- -A - 280 g/L

■ -320 g/L

Fig.3. Batch culture profiles of viable cells (a), sugar (b: dashed lines), and ethanol (b: solid lines) during ethanol fermentation from SSJ containing 160-320 g/L of sugar without nutrient supplementation.

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tl.l Table 1

t Q1 Fermentation parameters of batch ethanol production from SSJ containing 160-320 g/L of sugar with and without 9 g/L of yeast extract supplementation.

t1.3 t1.4 Initial sugar (g/L) Fermentation parameter*

Sc (%) Pe (g/L) Qe (g/L-h) Ye/s (g/g) t (h) FAN'n't'ai (mg/L) FANconsumed (%)

t1.5 160 89.8 ± 0.3g 71.3 ± 0.3a 1.49 ± 0.13d 0.50 ± 0.04f 48 183.0 ± 0.3a 81.0 ± 0.4h

t1.6 200 88.8 ± 0.8f 90.0 ± 0.1d 1.25 ± 0.01c 0.50 ± 0.00f 72 190.1 ± 0.2b 73.0 ± 0.8g

t1.7 240 78.5 ± 0.1c 88.0 ± 0.2c 1.05 ± 0.00b 0.47 ± 0.01d 84 199.4 ± 2.5c 64.7 ± 1.1f

t1.8 280 72.0 ± 0.6b 83.2 ± 1.3b 0.99 ± 0.02a 0.41 ± 0.01a 84 208.0 ± 0.6d 63.6 ± 0.7e

t1.9 320 64.2 ± 1.0a 83.0 ± 0.0b 0.99 ± 0.00a 0.42 ± 0.00a 84 220.8 ± 0.6e 65.3 ± 0.4f

t1.10 160 + YE 91.1 ± 0.2h 70.9 ± 0.8a 1.97 ± 0.10f 0.48 ± 0.03e 36 516.6 ± 0.9f 59.0 ± 0.6d

t1.11 200 + YE 93.2 ± 0.8' 93.8 ± 1.2e 1.95 ± 0.02f 0.45 ± 0.03c 48 529.0 ± 0.3g 55.5 ± 1.1c

t1.12 240 + YE 93.0 ± 0.4' 102.2 ± 0.9f 2.13 ± 0.04g 0.45 ± 0.00c 48 538.3 ± 2.2h 54.4 ± 0.7b

t1.13 280 + YE 86.9 ± 0.2e 112.5 ± 0.7g 1.56 ± 0.01e 0.46 ± 0.00c d 72 544.2 ± 2.5' 54.0 ± 1.3a

t1.14 320 + YE 81.6 ± 0.4d 112.0 ± 0.1g 1.56 ± 0.00e 0.44 ± 0.00b 72 560.3 ± 1.6? 53.4 ± 1.6a-b

15 The experiments were performed in triplicate and the results were expressed as mean ± SD.

16 a b c d e f g h> 1 andj: values with same letter within the same column are not significantly different using Duncan's multiple range test at 0.05 level of significance.

17 * SC = sugar consumption, PE = ethanol concentration, QE = ethanol productivity, YE/s = ethanol yield, FANinitial = initial FAN concentration, FANconsumed = FAN consumption,

18 t = fermentation time and YE = 9 g/L of yeast extract.

192 concentration with no ethanol addition was 2.5 to 2.9 x 108 cells/mL,

193 whereas it was 1.7 to 1.8 x 108 cells/mL in the presence of 6% ethanol Q5 at 24 h. No growth was observed at 9% and 12% ethanol for NP01 and

195 ATCC 4132,respectively, after 24 h. The viable cell counts of NP01

196 and ATCC 4132 under these two conditions were relatively constant

197 during the first 24 h. It seemed that NP01 showed better ethanol

198 tolerance at 15% ethanol. It could survive for 6 h with ~36% survival

rate, whereas ATCC 4132 could survive for only 4 h at this ethanol 199 concentration, with only ~8% survival rate. However, neither strain 200 could survive after 30 min of exposure to 18% ethanol. 201

The effects of ethanol concentration on the specific growth rates (1) 202 of S. cerevisiae NP01 and ATCC 4132 are shown in Fig. 2. With no ethanol, 203 the 1 of NP01 and ATCC 4132 were similar (0.166-0.168/h). At 6% 204 of ethanol concentration, the 1 of NP01 and ATCC 4132 were lower 205

Time (h)

(0 TO 3 CO

320 280 240 200 160 120 80 40 -0

, . . 160 g/L

120 I- 100

80 60 40 20 0

- -Â.- 280 g/L

, ■ ■ 320 g/L

Fig. 4. Batch culture profiles of viable cells (a), sugar (b: dashed lines), and ethanol (b: solid lines) during ethanol fermentation from SSJ containing 160-320 g/L of sugar and 9 g/L of yeast extract.

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160 200 240 280 320

Sugar concentration (g/L)

^m no YE ^m YE

Fig. 5. Glycerol production in batch ethanol fermentation from SSJ containing 160-320 g/L of sugar with and without yeast extract (YE) supplementation.

206 ( 0.153 and 0.116/h, respectively). When the ethanol concentrations

207 were further increased, ^ decreased sharply. The inhibition of yeast

208 growth at 9-12% of ethanol was almost complete. Similar results

209 were observed by Zhang et al. [18], who found that the end product

210 (ethanol) was shown to be the primary factor inhibiting yeast growth

211 and fermentation activity because the yeast completely stopped

212 growing and fermenting when the exogenous ethanol concentration

213 exceeded 70 g/L (~ 9%, v/v).

214 Ethanol tolerance of yeast depends on not only the yeast strain used

215 but also the composition of the growth medium. In the current study,

216 higher ethanol tolerance of the two yeast strains may be obtained if

217 they were cultured in an enriched medium. This was supported by

218 Kumar et al. [19], who reported that S. cerevisiae could tolerate up to

219 15% ethanol for 48 h in a yeast extract-peptone-glucose medium. In

220 this experiment, SSJ containing 100 g/L of sugar was used to mimic

221 real conditions during ethanol fermentation from SSJ. According to the

222 current experiment, NP01 could grow and tolerate ethanol better than

223 ATCC 4132. Therefore, NP01 was selected for use in the subsequent

224 experiments.

225 3.2. Batch ethanol fermentation

226 The changes of viable yeast cell count and sugar and ethanol

227 concentrations during batch fermentation from the EP media without

228 nutrient supplementations under NG, HG, and VHG conditions are

229 shown in Fig. 3. The viable cell concentration increased during the first

230 12 h and remained constant in the experiments with initial sugar

231 concentrations of 160-240 g/L. At higher initial sugar concentrations

232 (280-320 g/L), the viable cell counts decreased after 72 h, which

233 might have been due to osmotic and ethanol stress [4]. The residual Q6 sugar increased with increasing initial sugar concentration. The sugar

235 consumption (SC) was about 90% when the initial sugar concentrations

were 160 and 200 g/L (Table 1). The sugar consumption and ethanol 236 productivity (Qe) decreased with increasing initial sugar concentration, 237 indicating that high substrate concentration might lower the yeast 238 fermentation capacity. The highest ethanol concentration was obtained 239 with an initial sugar of 200 g/L. However, the sugar was not completely 240 consumed at all concentrations, implying that essential nutrients 241 might be insufficient (Table 1). Therefore, yeast extract was used to 242 supplement the EP media and thereby improve sugar consumption 243 and ethanol production. 244

When SSJ was supplemented with 9 g/L of yeast extract (Fig. 4), 245 the viable cell counts at all conditions increased during the first 246 24 h, except with 160 g/L of initial sugar. These values dramatically 247 decreased after 48 h. It was found that fermentation of SSJ with 248 nutrient supplementation gave higher viable cell count and ethanol 249 concentration. This suggested that yeast extract could promote cell 250 growth, which in turn resulted in enhanced ethanol production. 251 However, the viable cell counts under nutrient supplementation 252 decreased more severely during the later stage of the fermentation 253 compared to those with no supplementation, which might have been 254 due to ethanol toxicity to the yeast cells (Fig. 3a and Fig. 4a). 255

FAN was used in this study to monitor the utilization of nitrogen 256 during the fermentation process. FAN is a collective term that refers to 257 individual amino acids and small peptides of up to 3 units, which have 258 been found essential for yeast growth [20]. Adequate provision of FAN 259 resulted in higher rates of sugar uptake and consequently higher 260 ethanol concentrations [21,22]. The availability and consumption of 261 FAN in this study are given in Table 1. The initial FAN concentrations 262 in the media were slightly different because of the varying amounts 263 of concentrated SSJ juice used to prepare the EP media (data not 264 shown). In the media without yeast extract supplementation, the 265 initial values ranged from 183.0 to 220.8 g/L. The ability of the yeast to 266 consume FAN was found to decrease with increasing initial sugar 267 concentration from 81.0 to 64.7%, when the initial sugar concentration 268 was increased from 160 to 240 g/L. Comparing with the sugar 269 consumption (SC, %), a correlation between SC and FAN consumption 270 was observed. However, this correlation was not observed under the 271 HG and VHG conditions with 240-320 g/L of initial sugar. Even so, the 272 percentage of SC decreased with increasing initial sugar concentration. 273 FAN utilization was similar, ranging from 63.6 to 65.3%. When the 274 juices were supplemented with 9 g/L of yeast extract, the initial FAN 275 concentrations were in the range of 516.6-560.3 mg/L (9 g/L yeast 276 extract contained 334-339 mg/L FAN). The utilization of FAN in the 277 supplemented media was approximately double that in the media 278 without yeast extract. It was found to slightly decrease from 59.0 to 279 53.4% when the concentration of the initial sugar was increased from 280 160 to 320 g/L. The presence of yeast extract, i.e. FAN, in the media 281 resulted in higher sugar consumption by up to 17.4% with the same 282 initial sugar concentration (Table 1). This was considered the main 283 reason for the enhanced yeast growth and ethanol production during 284 a shorter fermentation time. 285

Table 1 summarizes the important fermentation parameters 286 in ethanol production from SSJ with and without yeast extract 287 supplementation. With yeast extract supplementation, the SC values 288 were higher, particularly at higher initial sugar concentrations, than 289

t2.1 Table 2

t2.2 Four regimes used in fed-batch fermentations by stepwise feeding with an initial working volume of 50%.

t2.3 Regime* Initial sugar Feeding Sugar concentration in Sugar concentration in the Summation of sugar

concentration (g/L) time (h) feeding medium (g/L) broth after feeding (g/L) concentration (g/L)

t2.4 1 (FB1:200,24,280) 200 24 356 200 280

t2.5 2 (FB2:200,24,320) 200 24 434 240 320

t2.6 3 (FB3:240,24,320) 240 24 413 240 320

t2.7 4 (FB4:200,12,280) 200 12 356 240 280

t2.8 * FBI:200,24,280 = fed-batch fermentation: initial sugar, 200 g/L; feeding time, 24 h; all sugar, 280 g/L, FB2:200,24,320 = fed-batch fermentation: initial sugar, 200 g/L; feeding time,

t2.9 24 h; all sugar, 320 g/L, FB3:240,24,320 = fed-batch fermentation: initial sugar, 240 g/L; feeding time, 24 h; all sugar, 320 g/L, and FB4:200,12,280 = fed-batch fermentation: initial sugar,

t2.10 200 g/L; feeding time, 12 h; all sugar, 280 g/L.

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fc ■a 9.0

£ n 8.0

■0 7.5

5» O 7.0

ta 160

til 120

1À 100

LLJ 20

12 24 36 48 60 72 84 Time (h)

12 24 36 48 60 72 84 Time (h)

B 280 g/L

Time (h)

FB1:200, 24, 280

FB2:200, 24, 320

FB3:240, 24, 320

FB4:200, 12, 280

those with no nutrient supplementation (Table 1). At initial sugar 290 concentrations of 200-240 g/L with yeast extract supplementation, 291 the SC increased to 93%, indicating that yeast extract may help 292 alleviate osmotic stress due to a high sugar concentration resulting in 293 higher Q E. However, substrate inhibition still markedly occurred at 294 initial sugar concentrations of 280-320 g/L resulting in only 82-87% SC. 295 With yeast extract supplementation, the SC, PE and Q E values markedly 296 increased at all initial sugar concentrations. The highest ethanol 297 production efficiency was obtained at an initial sugar concentration 298 of 280 g/L. The PE, QE, and YE/S values were 112.5 g/L, 1.56 g/L-h, and 299 0.46 g/g, respectively, at 72 h. At an initial sugar concentration of 300 240 g/L or lower, yeast extract markedly promoted both PE and QE, 301 whereas at higher initial sugar concentrations (280-320 g/L), nutrient 302 supplement promoted PE but the rate of ethanol production or QE was 303 reduced. This might have been due to substrate inhibition under VHG 304 conditions. 305

In the process of ethanol fermentation by S. cerevisiae, the main 306 by-product is glycerol. It is a metabolite that regulates osmotic 307 pressure produced by high concentration of sugar and ethanol in 308 the fermentation process [23,24]. Fig. 5 shows glycerol production 309 from the EP media with and without yeast extract. The glycerol 310 concentration increased with increasing sugar concentration. At 160 311 and 200 g/L of sugar, glycerol production levels were similar regardless 312 of the presence of yeast extract, indicating that the stresses under both 313 conditions were similar. At higher initial sugar concentrations, glycerol 314 concentrations under yeast extract supplementation were significantly 315 higher than those without nutrient supplementation. This might have 316 been due to high osmotic stress coupled with ethanol stress on yeast 317 cells at high sugar concentrations. The highest glycerol concentration 318 (PG, 17.1 g/L) was detected in the broth containing the highest initial 319 sugar and ethanol concentrations (SSJ containing 320 g/L of sugar and 320 9 g/L of yeast extract). 321

From the batch ethanol fermentation, SSJ containing 280 g/L of 322 sugar and 9 g/L yeast extract gave relatively high PE (112.5 g/L). 323 However, the residual sugar was ~37 g/L (~86.9% SC) with a QE 324 of only 1.56 g/L-h. Therefore, to improve sugar consumption and 325 ethanol production efficiency, the fed-batch fermentation was further 326 investigated. 327

3.3. Sugar consumption rate under NG and HG conditions

Fig. 6. Profiles of viable cell counts (a), sugar (b), and ethanol (c) under fed-batch fermentation by stepwise feeding of SSJ (280 and 320 g/L of all sugar) at feeding times of 24 and 12 h; B = batch system and FB = fed-batch system.

In fed-batch fermentations, the initial sugar concentration used in Q7

batch fermentation was used to prevent substrate inhibition. Feeding 330

of the substrate was initiated when most of the substrates had been 331

consumed and the yeast growth was still in the exponential phase 332

[25]. Before studying fed-batch fermentation, the sugar consumption 333

rates under NG and HG conditions were calculated. The sugar 334 concentration in SSJ containing 160-240 g/L of initial sugar and 9 g/L 335

yeast extract (NG and HG conditions) decreased sharply during the 336

first 24 h (Fig. 4b). The sugar consumption rate during 24 h of batch 337

fermentations with an initial sugar concentration of 160 g/L was the 338

t3.1 Table 3

t3.2 Fermentation parameters of fed-batch ethanol fermentation using a stepwise feeding from SSJ under VHG conditions (280 and 320 g/L of all sugar) at feeding times of 24 and 12 h.

t3:3 t3:4 Regime Fermentation parameter***

Sc (%) Pe (g/L) Qe (g/L-h) Ye/s (g/g) Pg (g/L) t (h)

t3:5 B280* 86.9 ± 0.2d 112.5 ± 0.7e 1.56 ± 0.01c 0.46 ± 0.00c 13.9 ± 0.0e 72

t3.6 FB1:200, 24, 280** 77.0 ± 0.7b 101.5 ± 0.0c 1.41 ± 0.00c 0.47 ± 0.00c 8.9 ± 0.2b 72

t3.7 FB2:200, 24,320** 62.8 ± 0.5a 88.7 ± 0.0b 1.23 ± 0.00b 0.44 ± 0.00b 8.8 ± 0.1a 72

t3:8 FB3:240, 24, 320** 62.7 ± 1.8a 85.6 ± 1.9a 1.19 ± 0.03a 0.42 ± 0.00a 9.6 ± 0.0c 72

t3:9 FB4:200,12, 280** 80.7 ± 0.9c 107.1 ± 0.0d 1.49 ± 0.03d 0.46 ± 0.02c 11.4 ± 0.0d 72

t3.12 t3.13 t3.14

The experiments were performed in triplicate and the results were expressed as mean ± SD.

a b c d ande: means followed by the same letter within the same column are not significantly different using Duncan's multiple range test at the level of 0.05. * B280 = batch fermentation at 280 g/L of sugar with 9 g/L of yeast extract supplementation. ** See Table 2.

*** SC = sugar consumption, PE = ethanol concentration, Q = ethanol productivity, YE/s = ethanol yield, PG = glycerol concentration and t = fermentation time.

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lowest (6.16 g/L-h), whereas these values with 200 and 240 g/L of initial sugar were similar at 7.22 and 7.32 g/L-h, respectively. Therefore, initial sugar concentrations of 200 and 240 g/L were used in the fed-batch fermentations.

3.4. Fed-batch ethanol fermentation

In this research, two feeding regimes were studied:

3.4.1. Stepwise feeding

SSJ media containing 200 and 240 g/L of sugar and 9 g/L of yeast extract were used as EP media in fed-batch fermentations, employing 50% of the initial working volume [8]. According to Fig. 4b, the remaining 50% of the medium was fed at 12 and 24 h during which time the yeast cells were still active. Four regimes were conducted, and the overall sugar concentrations in the EP media were in VHG conditions at 280 and 320 g/L as shown in Table 2.

The viable cell counts continued to increase until fresh medium was fed to the flask at either 12 or 24 h (Fig. 6a). The cell concentration decreased after feeding fresh medium and then slightly increased. However, the maximum cell number after the feeding did not reach the maximum values that were obtained before feeding. The viable cell counts were relatively constant, except in Regime 4 (FB4). At 48 h, the viable cell count at feeding time at 12 h was higher than that at 24 h. In comparison to the control (batch system), the viable cell count of the fed-batch system at feeding time of 12 h and the control were similar until 72 h.

Changes in sugar and ethanol concentrations in the EP media under various fed-batch fermentations were different (Fig. 6b and c). The sugar and feeding time affected the PE, QE, and YE/S (Table 3). At a feeding time of 24 h, the SC in FB1 was higher than that in FB2 and FB3, resulting in a higher Pe. At feeding time of 12 h (FB4), the Sc and PE were higher than those at feeding time of 24 h. In FB1 and FB4 (overall sugar concentration of 280 g/L), the feeding time at 12 h (FB4) gave higher values of ethanol production, with the PE and QE of 107.1 g/L and 1.49 g/L-h, respectively (Table 3).

However, the SC and PE of FB4 were lower than those of the control (batch system) (Table 3). Glycerol concentrations at a feeding time of 24 h (8.8 to 9.6 g/L) were lower than that at a feeding time of 12 h (11.4 g/L). This might have been due to lower ethanol concentrations at feeding time of 24 h. Glycerol concentrations under all fed-batch conditions were lower than those under batch fermentation (13.9 g/L) (Table 3). This, again, might have been due to the lower stresses of high sugar and ethanol concentrations [26].

The results showed that the fed-batch fermentation with 1:1 stepwise feeding at feeding times of 12 and 24 h could not improve ethanol production efficiency from SSJ compared to that in the batch fermentation. To improve fed-batch ethanol production, continuous feeding was studied at a feeding time of 12 h.

3.4.2. Continuous feeding

According to the stepwise feeding fed-batch fermentation, FB4 (initial sugar, 200 g/L; feeding time, 12 h; overall sugar concentration, 280 g/L) gave the highest SC, PE, and QE values (Table 3). Therefore, the conditions used in FB4 were applied in continuous feeding.

The fed-batch fermentation by continuous feeding was performed in a 2-L fermenter. It was started by filling 50% of working volume of the fermenter with SSJ containing 200 g/L of initial sugar and 9 g/L of yeast extract. As discussed in Section 3.3, the sugar consumption rate at the initial sugar of 200 g/L was 7.22 g/L-h. Therefore in the fed-batch fermentation after 12 h, fresh medium (360 g/L of sugar) was fed continuously at 1X (27 mL/h, 10 g sugar/h) and 2X (54 mL/h, 20 g sugar/h). The results showed that the viable cell counts under these regimes were higher than those of the control during the first 12 h, which might have been due to lower osmotic stress. However, after 24, the viable cell counts under all conditions were similar

(Fig. 7a). After 24 h, the SC and PE of the fermentation at feeding time 401 of 12 h and the feeding rate 2X [FB2X(12)] were higher than those of Q9 1X [FB1X(12)] (Fig. 7b and c). However, these values at feeding time of 403 12 h were similar to the batch control. Therefore, the feeding was 404 started earlier, at 9 h, and the feeding rates of 2X and 4X (108 mL/h, 405 40 g sugar/h) were further investigated to improve ethanol production 406 (Fig. 8). The results showed that at a feeding time of 9 h, the feeding 407 rate of 4X gave better sugar consumption and ethanol production rate 408 than 2X (Table 4). 409

In the fed-batch fermentation with continuous feeding, feeding 410 time and feeding rate affected PE and QE (Table 4). The best conditions 411

- 40 -

ffl 20-

36 48 Time (h)

B 280 g/L

24 36 48 Time (h)

—A— FB1X(12)

FB2X(12)

Fig. 7. Profiles of viable cell counts (a), sugar (b), and ethanol (c) under fed-batch fermentation by continuous feeding of SSJ (280 g/L of all sugar) at a feeding time of 12 h and feeding rate of 1X (10 g sugar/h) and 2X (20 g sugar/h); B = batch system and FB = fed-batch system.

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t is 9.0

о 8.5

о с 8 8.0

■Q 7.5

SI о 7 0

та 160

<0 120

Si 20 -

24 36 48 Time (h)

24 36 48 Time (h)

24 36 48 Time (h)

B 280 g/L -A- FB2X(9)

for ethanol production were to start feeding at 9 h at a rate of 412 40 g sugar/h. Under these conditions, the PE, QE, and YE/S values were 413 112.9 g/L, 2.35 g/L-h, and 0.47 g/g, respectively, at 48 h. Comparison 414 of ethanol production between batch and fed-batch fermentations 415 revealed that the PE and YE/S values in the fed-batch fermentation at 416 9 h and feeding rate of 40 g sugar/h were not different from those 417 of the batch system, but the QE of the latter was higher because the 418 fermentation time was shortened from 72 to 48 h. Moreover, the 419 glycerol concentration decreased from 13.9 to 12.3 g/L compared to 420 that in the batch control (Table 4), indicating that stresses under the 421 fed-batch fermentation were less. 422

In the fed-batch process, the fermentation was initiated with a 423 sugar concentration in the range of HG conditions (initial sugar 424 concentration of 200 and 240 g/L). Then, the feed medium containing 425 high sugar concentration was fed to attain overall sugar concentrations 426 in the range of VHG conditions. Therefore, this process can avoid 427 substrate inhibition of cell growth. In the current study, the fed-batch 428 fermentation with continuous feeding improved ethanol productivity 429 by ~51%. To further improve sugar consumption and ethanol production 430 efficiency, aeration may be supplied [27] and/or some essential trace 431 elements or osmoprotectant could be added to the EP medium [3,12]. 432 Moreover, increasing the initial cell concentration may also improve 433 ethanol productivity [28]. 434

4. Conclusions 435

S. cerevisiae NP01 and ATCC 4132 could tolerate up to 12% (v/v) 436 ethanol without loss of cell viability. At 15% ethanol, NP01 showed 437 higher ethanol tolerance than ATCC 4132. In batch ethanol 438 fermentations from SSJ, yeast extract supplementation promoted yeast 439 growth, leading to an increase in ethanol production and reduced 440 fermentation time, especially under HG and VHG fermentations. 441 In fed-batch fermentations with continuous feeding, apart from 442

nitrogen supplementation, feeding time and feeding rate were the key 443 parameters to improve ethanol production efficiency under VHG conditions. In this study, continuous feeding starting at 9 h with a feeding rate of 40 g sugar/h gave the highest ethanol production efficiency.

FB4X(9)

Financial support

This study was supported by the Higher Education Research Q10 Q11 Promotion and National Research University Project of Thailand 450 through the Biofuels Research Cluster of Khon Kaen University (KKU), 451 Office of the Higher Commission Education; and Center for Alternative 452

Energy Research and Development, KKU, Thailand. 453

Fig. 8. Profiles of viable cell counts (a), sugar (b), and ethanol (c) under fed-batch fermentation by continuous feeding of SSJ (280 g/L of all sugar) at a feeding time of 9 h and feeding rate of 2X (20 g sugar/h) and 4X (40 g sugar/h); B = batch system and FB = fed-batch system.

Conflict of interest

The authors declare no conflict of interest.

t4.l Table 4

t4.2 Fermentation parameters of fed-batch ethanol fermentation under a VHG condition (280 g/L of all sugar) with continuous feeding (starting at 9 and 12 h at different feeding rates).

Condition

Fermentation parameter '

t4.4 Se (%) Pe (g/L) Qe (g/L-h) Ye/s (g/g) Pg (g/L) t (h)

t4.5 B280 86.9 ± 0.2e 112.5 ± 0.7d 1.56 ± 0.01a 0.46 ± 0.00b 13.9 ± 0.0e 72

t4.6 FB1X(12) 77.8 ± 0.8a 98.2 ± 1.1a 1.64 ± 0.01b 0.45 ± 0.00a 10.9 ± 0.3b 60

t4.7 FB2X(12) 81.0 ± 1.9b 111.1 ± 1.3b 1.85 ± 0.00c 0.47 ± 0.00c 8.9 ± 0.1a 60

t4.8 FB2X(9) 84.7 ± 1.4c 112.1 ± 0.7c 1.87 ± 0.01d 0.47 ± 0.00c 11.8 ± 0.2c 60

t4.9 FB4X(9) 85.6 ± 1.2d 112.9 ± 0.1e 2.35 ± 0.00e 0.47 ± 0.01c 12.3 ± 0.4d 48

t4.10 t4.11 t4.12 t4.13 t4.14

a b c d ande: values with the same letter within the same column are not significantly different using Duncan's multiple range test at 0.05 level of significance.

* B280 = batch fermentation at 280 g/L of sugar with 9 g/L of yeast extract supplementation, FB1X(12) = fed-batch fermentation at feeding time of 12 h and feeding rate of10gsugar/h, FB2X(12) = fed-batch fermentation at feeding time of 12 h and feeding rate of 20 g sugar/h, FB2X(9) = fed-batch fermentation at feeding time of 9 h and feeding rate of 20 g sugar/h, and FB4X(9) = fed-batch fermentation at feeding time of 9 h and feeding rate of 40 g sugar/h. ** See Table 3.

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Acknowledgments

The authors would like to thank Assistant Prof. Dr. Paiboon Danviruthai, Faculty of Technology, KKU, for providing the NP01 strain and Associate Prof. Dr. Prasit Jaisil, Faculty of Agriculture, KKU, for providing sweet sorghum juice.

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