Front. Chem. Sci. Eng. https://doi.org/10.1007/s11705-018-1714-y

RESEARCH ARTICLE

Detoxification and concentration of corn stover hydrolysate and its fermentation for ethanol production Qing Li1, Yingjie Qin (✉)1,2, Yunfei Liu1, Jianjun Liu1, Qing Liu1, Pingli Li1, Liqiang Liu2 1 School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China 2 Chembrane Engineering & Technology, Inc., Tianjin 300308, China

© Higher Education Press and Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract Environmental and energy concerns have increased interest in renewable energy sources, particularly biofuels. Thus the fermentation of glucose from sulfuric acid-hydrolyzed corn stover for the production of bioethanol has been explored using a combined acid retardation and continuous-effect membrane distillation treatment process. This process resulted in the separation of the sugars and acids from the acid-catalyzed hydrolysate, the removal of most of the fermentation inhibitors from the hydrolysate and the concentration of the detoxified hydrolysate. The recovery rate of glucose from the sugar-acid mixture using acid retardation was greater than 99.12% and the sulfuric acid was completely recovered from the hydrolysate. When the treated corn stover hydrolysate, containing 100 g/L glucose, was used as a carbon source, 43.06 g/L of ethanol was produced with a productivity of 1.79 g/(L∙h) and a yield of 86.31%. In the control experiment, where glucose was used as the carbon source these values were 1.97 g/(L∙h) and 93.10% respectively. Thus the integration of acid retardation and a continuous-effect membrane distillation process are effective for the production of fuel ethanol from corn stover. Keywords corn stover, hydrolysate, acid retardation, continuous-effect membrane distillation, ethanol fermentation

1

Introduction

Ever-increasing environmental and energy issues have stimulated a large number of researchers to develop biofuels to help meet demands for clean energy sources Received November 17, 2017; accepted February 14, 2018 E-mail: [email protected]

[1]. A potential source for low-cost bioethanol production is lignocellulosic materials, which include wood biomass, agricultural and forestry residues, and biodegradable components from industrial and municipal wastes [2]. Corn stover, a globally abundant agricultural residue, has been used as a raw material in the production of bioethanol [3]. However lignocellulosic materials in the raw state are not easily fermentable and so they must be treated to release the monomeric sugars, which can then be converted into biofuels or other high value-added chemicals [4,5]. There are primarily two research directions for maximizing the utilization of lignocellulosic materials. One involves preparing a lignocellulose hydrolysate and then simultaneously converting all the sugars released from that hydrolysate to ethanol [6]. This requires a robust industrial fermentation microorganism that can simultaneously convert pentoses and hexoses to bioethanol. However, the microorganisms currently used for bioethanol production cannot metabolize xylose in the presence of glucose at a commercially meaningful level [7]. The other direction involves separating the three components of lignocellulose in a pretreatment process [1]; the sugars extracted from the hemicellulose can be used for dedicated pentose or furfural production, whereas the glucose extracted from the cellulose can be used for ethanol fermentation. Formic acid-based pretreatment has been used to effectively separate cellulose, lignin, and hemicellulose [8]. Cellulose and hemicellulose from corn stover can be hydrolyzed chemically or enzymatically [1,9]. Acid hydrolysis using either dilute or concentrated acid is a chemical process that can be used for either pretreatment before enzymatic hydrolysis or for the conversion of lignocellulose to monomeric sugars. Dilute acid hydrolysis has more advantages than concentrated acid hydrolysis such as higher sugar yields, milder operating conditions, shorter operating times, and fewer inhibitory compounds produced in the concentrated acid hydrolysate [10]. The removal of inhibitors prior to fermentation (called

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detoxification) is essential for successful biofuel production from lignocellulosic hydrolysates [11]. Several methods, such as overliming [12], ion exchange resinbased adsorption [13], and peroxidase treatment [14], have been investigated for the detoxification of lignocellulosic hydrolysates. The economic feasibility of using acid hydrolysis to convert lignocellulose to ethanol depends on the cost of the sugar-acid separation and detoxification steps. Overliming is a common method for sugar-acid separation and for detoxification of the dilute acid-catalytic hydrolysate but the loss of sugars is large and the acid cannot be recycled [15]. To improve the economic efficiency of moderatestrength or concentrated acid hydrolysis-fermentation technologies, alternative techniques to separate the sugars and the acid are needed. Ion exclusion [16,17] has been investigated for this purpose and Nanguneri and Hester [16] performed a detailed economic analysis comparing the cost of lime precipitation and ion exclusion. They showed that ion exclusion is more economically feasible and environmentally friendly. However, ion exclusion processes still have some problems such as large consumption of eluents, complex equipment, and dilution of the sugar solution. Acid retardation [18] is a process for separating acids from their corresponding salts using an ion retardation resin. The acid retardation column, which is filled with a strongly basic anion exchange resin, adsorbs strong acids from an aqueous solution and excludes inorganic salts and other solutes. A previous study has shown that the acid retardation process can be used to separate a mixture of acid and sugar [19]. However, both the sugar and acid were dilute so it was necessary to concentrate the solutions before subsequent processing. This could be a capital- and energy-expensive prospect which would discourage the application of acid retardation to industrial processes. Membrane distillation is a separation process based on evaporation through a porous hydrophobic membrane. It can be used to concentrate nonvolatile sugars in hydrolysates and to remove some volatile inhibitors from hydrolysates. Chen et al. [20] found that vacuum membrane distillation could be used to remove 98% of furfural and 25% of acetic acid from a lignocellulosic hydrolysate and the ethanol yield was increased by 17.8%. By using a continuous-effect membrane distillation (CEMD), Yao et al. [21] showed that an aqueous solution of glucose was concentrated 12-fold to a final concentration of about 20 wt-% with a final gained output ratio (GOR) of 8.2. An aqueous solution of sulfuric acid was also concentrated by CEMD for recycling [19]. CEMD can be used for sugar concentration and sulfuric acid recovery, so it might improve the economic benefit of the bioethanol production process. In this work, acid retardation was investigated for the separation and purification of hydrolysate from corn stover; CEMD was studied for the concentration of the

hydrolysate and the recovery of sulfuric acid; then the concentrated hydrolysate was used as a carbon source for ethanol production using Saccharomyces cerevisiae CICC 1308. The aim was to evaluate the feasibility of using acid retardation and CEMD technology to produce renewable and green bioethanol from corn stover.

2

Materials and methods

2.1

Samples and chemicals

Corn stover, ground and screened to 0.2 – 0.4 mm, was obtained from Hebei Baoshuo Co., Ltd., Hebei Province, China. The raw material was kept in an oven at 40 °C until use. Its main constituents (% dry weight, w/w) were cellulose (45%), hemicellulose (31%), and lignin (17%), as reported in our previous work [19]. Chromatographic grade chemicals, such as anhydrous ethanol, glucose, formic acid, acetic acid, levulinic acid, furfural, and 5-hydroxymethylfurfural (5-HMF), were purchased from Heowns Biochemical Technology Co., Ltd., Tianjin, China. Peptone (≥98%), yeast extract (≥98%), MgSO4$7H2O (≥98%), K2HPO4 (≥98%), and KH2PO4 (≥98%) were purchased from Sangon Biotech Co., Ltd., Shanghai, China. Formic acid (≥88%), hydrochloric acid (≥38%), sulfuric acid (≥98%), glucose (≥99%), gallic acid (≥99%), sodium hydroxide (≥96%), and other analytical grade chemicals were purchased from Tianjin Jiangtian Chemical Technology Co., Ltd., Tianjin, China. All chemicals were used directly as purchased without further purification. Deionized water was prepared in the laboratory using a fourstage reverse osmosis system. 2.2

Schematic

Figure 1 shows the schematic for converting corn stover into bioethanol and other chemicals. First the corn stover was fractionated by pretreatment with a mixture of formic acid and hydrochloric acid which hydrolyzes the majority of the hemicellulose in the corn stover to xylose [22]. The xylose can then be dehydrated to produce furfural. The solid waste (lignin) obtained from the post-treatment of the hydrolysate can be directly used as fuel in subsequent procedures. After the pretreatment of the corn stover, the hydrolysate was filtered and the remaining solids which mainly containing cellulose were separated. These were then hydrolyzed using sulfuric acid as a catalyst. This hydrolysate was then filtered and the solid lignin waste was used as above. The liquid containing the sugars and acid was subjected to an acid retardation process to separate the two components. The resulting dilute sugar-containing eluent and dilute acid-containing eluent were then each concentrated by CEMD. Finally the concentrated sugar solution was used as the carbon source for ethanol

Qing Li et al. Detoxification and concentration of corn stover hydrolysate

fermentation. The sulfuric acid was reused in another treatment cycle. 2.3

3

Temperatures at the cold feed inlet, the cold feed outlet, the heated feed inlet, and the heated feed outlet were monitored using micro-thermometers.

Experimental apparatus

The acid retardation resin was a strongly basic anion exchange resin in the Cl– form, TULSION® A-32-Fine mesh, purchased from THERMAX, Inc., Pune, India. The resin was washed three times with deionized water before use. The jacketed glass column that held the resin was purchased from Shanghai Huxi Analysis Instrument Factory Co., Ltd., Shanghai, China. The inner diameter of the column was 35 mm, the height was 1.05 m, and the total volume was 1.001 L. The CEMD membrane module was made from polytetrafluoroethylene porous hollow fibers and polypropylene dense-wall hollow fibers. It was provided by PureSea Spring Membrane Technology Co., Ltd., Tianjin, China. The internal and external diameters of the porous and dense-wall fibers were 0.33 and 0.63 mm and 0.40 and 0.50 mm, respectively. The effective length of the module was 1.05 m, the effective membrane area (based on the internal diameter of the porous fiber) was 0.63 m2, the number of porous fibers was 580, the number of densewall fibers was 1160, and the packing density was 45%. The module was pressure-tested by water to ensure no leakage [23]. The CEMD experimental apparatus is depicted in Fig. 2. The feed solution was circulated and delivered by a magnetic pump; flow rates were measured using a rotameter and regulated by adjusting the backflow rate.

Fig. 1

2.4 2.4.1

Experimental procedure Pretreatment

Corn stover was pretreated with a mixture of formic acid and hydrochloric acid (100:1, v/v) at a liquid-solid ratio of 10:1 (v/w) in a 60 °C water bath with oscillation for 3 h, according to the procedure described by Huang et al. [8]. After cooling to ambient temperature, the mixture was filtered by vacuum filtration. The formic acid and hydrochloric acid were recovered from the filtrate by vacuum distillation. The solid fraction (pretreated corn stover) was washed with deionized water, and dried at 105 °C for 4 h. 2.4.2

Corn stover hydrolysis

Sulfuric acid hydrolysis of the pretreated corn stover (cellulose) was divided into two stages and performed in stirred-tank reactors. First, 72 wt-% sulfuric acid was mixed with the treated stover at a liquid-solid ratio of 4:1 (v/w), at 25 °C, at a stirring rate of 150 r/min and with a reaction time of 2 h. Second, deionized water was added to dilute the liquid to a sulfuric acid concentration of 20 wt-%, and the temperature was quickly increased to 100 °C and maintained for 1 h. After cooling to ambient

The schematic for converting corn stover to bioethanol and other chemicals

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24 h and then inoculated (10% v/v) and incubated in a 250mL shake flask at 30 °C and 150 r/min for 12 h. A range of fermentation conditions were investigated to produce ethanol efficiently with Saccharomyces cerevisiae CICC 1308. The optimum conditions were obtained using an orthogonal design with the objective of maximizing the ethanol yield, which is equal to the ratio of the ethanol content to the theoretical maximum ethanol content. The detoxified and concentrated CSH was sterilized in an autoclave sterilizer at 115 °C for 30 min before it was used for fermentation. In the control experiments, glucose was used as the carbon source. All experiments were performed independently in triplicate and all results are expressed as mean values. 2.5 2.5.1 Fig. 2

Apparatus for continuous-effect membrane distillation

temperature, the liquid fraction (corn stover hydrolysate, CSH) was filtered, and stored at 4 °C. 2.4.3

Acid retardation

The acid and sugar in the CSH were separated using acid retardation. The resin in the column was equilibrated with deionized water before loading. The eluent from the column was collected every minute using a fraction collector. When the CSH, a sugar-acid mixture, was passed through the resin bed, the sugar-rich stream (the sugar eluent) eluted first, followed by an acid-rich stream (the acid eluent). 2.4.4

Concentration of eluents by CEMD

The sugar and acid eluents from the CSH were then concentrated by CEMD. For the first hour, the feed-out stream and the distillate were returned to the feed reservoir to attain a steady state. Then the distillate was collected in another reservoir, and the feed solution cycled between the module and the feed reservoir was concentrated continuously. 2.4.5

Ethanol fermentation

Saccharomyces cerevisiae CICC 1308 was obtained from the China Center of Industrial Culture Collection. The strain was stored on a slant culture medium at 4 °C. The growth medium was composed of 50 g/L glucose, 5 g/L peptone, 5 g/L yeast extract, 1 g/L MgSO4∙7H2O and 1 g/L KH2PO4. The fermentation medium was same as the growth medium except for that is also contained a carbon source. The strain was cultured in a growth medium for

Analytical methods Chemical analysis

The concentrations of glucose and inhibitors (formic acid, acetic acid, levulinic acid, furfural, and 5-hydroxymethylfurfural) were determined by liquid chromatography (Agilent 1200 series, USA) using a refractive index detector. Separations were carried out using an Aminex HPX-87H column (Bio-Rad, USA) at 65 °C. The mobile phase was 0.5 mmol/L sulfuric acid at a flow rate of 0.5 mL/min. The concentration of total phenolics was measured spectrophotometrically using the Folin-Ciocalteu method [24]. The ethanol concentration was determined using gas chromatography (Shimadzu GC-2014, Japan) with a WondaCap Wax column (30 m
320 µm
0.25 µm, Shimadzu) and a hydrogen flame ionization detector. 2.5.2

Performance parameters

Resolution (R) and recovery of sugar or acid (Rs, Ra) were used to characterize the operational performance of the acid retardation process. Resolution was used to evaluate the separation of the sugar-acid mixtures in the acid retardation process and was calculated as R¼

2ðta – ts Þ w a þ ws

(1)

where ta and ts are the retention times of the acid and sugar, respectively, and wa and ws are the peak widths of the acid and sugar peaks, respectively [19]. The recovery rates of sugar and acid by the acid retardation process were calculated as Rs ¼

Me,s V C
100% ¼ e,s e,s
100%, Mf ,s Vf Cf ,s

(2)

Ra ¼

Me,a V C
100% ¼ e,a e,a
100%, Mf ,a Vf Cf ,a

(3)

Qing Li et al. Detoxification and concentration of corn stover hydrolysate

where Rs and Ra are the recovery percentages of the sugar and acid, respectively; Mf,s and Me,s are the masses (g) of the sugar in the feed and the sugar in the eluent, respectively; Mf,a and Me,a are the masses (g) of the acid in the feed and the acid in the eluent, respectively; Cf,s and Ce,s are the concentrations (g/L) of the sugar in the feed and the sugar in the eluent, respectively; Cf,a and Ce,a are the concentrations (g/L) of the acid in the feed and the acid in the eluent, respectively; and Vf, Ve,s, and Ve,a are the volumes (L) of the feed and the corresponding sugar and acid eluents respectively [19]. The permeation flux (J), GOR, and rejection rate of glucose (Rg) were used to characterize the operational performance of the CEMD process. The value of J indicates the productivity of the CEMD process and can be calculated from J¼

Fd , S

(4)

where J is the permeation flux (L/(h∙m2)), Fd is the volumetric flow rate (L/h) of the distillate, and S is the effective membrane area (m2) based on the inner diameter of the porous fibers [19]. The GOR was used to evaluate the thermal efficiency of the CEMD process and can be calculated from GOR ¼

ΔHJS
d , ðTh,i – Tc,o ÞCp Ff
f

(5)

where DH is the evaporation heat (kJ/kg) of the feed stream, Ff is the feed volumetric flow rate (L/h), ρf and ρd are the densities (kg/m3) of the feed and the distillate, respectively, Cp is the specific heat capacity (kJ/(kg$°C)) of the feed, and Tc,o and Th,i are the feed temperatures (°C) at the exit of the dense-wall fibers and the entrance of the porous fibers, respectively [19]. The rejection rate of glucose (Rg) indicates the degree of separation in the CEMD and can be calculated from   Cd,g Rg ¼ 1 –
100%, Cf ,g

(6)

where Rg is the rejection rate of glucose, and Cf,g and Cd,g are the concentrations (g/L) of glucose in the feed and the distillate, respectively [24].

3

Results and discussion

3.1 Acid retardation for sugar-acid separation and detoxification of the CSH

Table 1 and Fig. 3 show a comparison of the sugar/acid separation performances for CSH and for a glucose and sulfuric acid solution (GSS) by acid retardation at a bed volume (V0) of 1 L, a feed volume (Vf) of 200 mL, a flow

5

velocity of 10 mL/min, and a temperature of 50 °C. The two feeds (CSH and GSS) contained the same amount of glucose and sulfuric acid. As shown in Fig. 3, the sugar and acid in both feeds were effectively separated with resolutions of 1.56 and 1.58 which can be considered a complete separation [25]. A resolution of 1.5 corresponds to a baseline separation for a touching-band situation, and R≥1.50 can be considered complete acid and sugar separation [25]. However, the sugar-acid separation for CSH needed more deionized water, had a longer elution time and had a slightly lower resolution. This may be because the inhibitors in the CSH interacted with the acid retardation resin and became tightly adsorbed onto the resin, which delayed the flow of sulfuric acid out of the column. As seen from Table 1, the glucose recovery rate the CSH was 99.12%, which was comparable to that from the GSS (99.16%). The sugar loss (0.88%) is much less than that obtained with an overliming method, where the sugar loss was 8.7%4.5% [16]. The sulfuric acid recovery rate from CSH was 98.92%, which is higher than that obtained using ion exchange chromatography, where the acid recovery rate was 92% – 95% [11]. In fact, all the sulfuric acid contained in the hydrolysate was either recovered or utilized during the ethanol production process. The small amount of sulfuric acid remaining in the sugar solution served as nutrients during the ethanol fermentation after it was neutralized by Mg(OH)2 and K2HPO4. Table 1 Sugar-acid separation performance of CSH and GSS by acid retardation Feed

Glucose /(g∙L–1)

Sulfuric acid /wt-%

R

Rs /%

Ra /%

4.98

1.56

99.12

98.92

5.16

1.58

99.16

99.01

Feed

Eluent

Feed

Eluent

CSH

38.48

20.96

20.00

GSS

38.48

21.93

20.00

During the acid retardation process, the pH of the sugar eluent was gradually decreased during the elution and the sugar eluent was collected until its final pH was ca. 2.3. As the acid retardation progressed, the pH of the eluent further decreased to a minimum and then gradually increased. In the later-eluting acid process, the acid eluent was collected until its final pH reached ca. 3.2. The sugar eluent was then directly concentrated using CEMD, and then the desired amounts of K2HPO4 and Mg(OH)2 were added to meet the requirements for KH2PO4 and MgSO4 in the fermentation medium. The regenerated resin column was then for reuse. The content of sugar and inhibitors in the original CSH, the sugar eluent, and the concentrated sugar solution are listed in Table 2. The main inhibitors in the CSH were organic acids with a total concentration of 4.678 g/L. Phenols were a minor component with a concentration of ca. 0.1 g/L. However, phenols can inhibit ethanol fermentation, especially phenolic compounds with low molecular weights. Clark and Mackie studied the effect of

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which represents an increase of about five-fold. During this time both J and GOR slightly decreased. At the end of process the GOR value was 11.86 which corresponds to the energy efficiency of a conventional 14-effect evaporator [19]. The highest values of J and GOR were 4.84 and 15.27 L/(m2∙h) respectively. During the concentration of the dilute sugar eluent, no sugar was detected in the distillate indicating that Rg remained at 100%. In addition, the concentrated sugar solution only contained a trace amount of phenolic compounds (Table 2) so it may be directly useable as a carbon source for ethanol fermentation. 3.2.2

The acid eluent was subjected to CEMD for 5.5 h and the results are shown in Fig. 5. The sulfuric acid was concentrated about nine times, with a final concentration of 43.43 wt-% and during this CEMD process J sharply decreased with increasing sulfuric acid concentration. The maximum values for GOR and J were 14.51 and 4.50 L/(m2∙h) respectively. There was almost no sulfuric acid in the final distillate. The concentrated acid solution was then further concentrated to ca. 72 wt-% by high-temperature evaporation. The concentration of inhibitors in the acid eluent, the concentrated acid solution (43 wt-%), and highly concentrated acid solution (72 wt-%) are listed in Table 3. The CEMD process did not result in large changes in the concentrations of formic acid, acetic acid, and 5-HMF. However the concentrations of levulinic acid and phenols increased several times. This is because most volatile inhibitors were removed during the CEMD process, however the semi-volitale and unvolitale compounds can only removed partially from the feed [24]. The concentrations of inhibitors in the highly concentrated acid solution were much lower than those in the concentrated acid solution. There are two main reasons for these reductions. First, when the acid was heated to 170 °C during the evaporation process, the hydrogen bonds formed between

Fig. 3 Sugar-acid separation by acid retardation: (a) CSH; (b) GSS

vanillin on the yeast growth and found growth to be completely inhibited at a concentration of 5 g/L, with approximately 50% inhibition at 1.3 g/L [26]. After the acid retardation treatment, the organic acids and furfural inhibitors in the CSH were almost completely removed, and the removal rate of phenolic compounds was > 90%. The final concentrated sugar solution only contained 0.019 g/L of total phenols, which is not enough to significantly inhibit ethanol fermentation [26]. 3.2

Concentration of the eluents

3.2.1

Concentration of the acid eluent

Concentration of the sugar eluent by CEMD

The variations of J, GOR, Rg, and glucose concentration as a function of CEMD operating time are shown in Fig. 4. Over the course of 4 h of operation, the dilute sugar eluent was gradually concentrated from 20.96 g/L to 103.22 g/L

Table 2 Compositions of non-detoxified CSH and sugar eluent after acid retardation and concentrated sugar solution after CEMD Substance Glucose

Non-detoxified CSH /(g∙L–1) 38.479

Total organic acids

4.678

Formic acid

3.483

Acetic acid

0.310

Sugar eluent /(g∙L–1) 20.956 b)

Removal rate /% NA

a)

Concentrated sugar solution /(g∙L–1) 103.215

100

ND

ND

100

ND

ND

100

ND

ND

Levulinic acid

0.885

ND

100

ND

Furfural

0.003

ND

100

ND

5-HMF

0.164

ND

100

ND

Phenols

0.097

0.004

92.495

0.019

NA

99.124

NA

100

Sugar yield (%) a) not applicable; b) not detected

Qing Li et al. Detoxification and concentration of corn stover hydrolysate

7

very small amounts of inhibitors, it could be reused for further acid hydrolysis of corn stover. The lignin obtained from the corn stover could be used directly as a fuel [28] to provide heat for a hightemperature evaporator to further concentrate the sulfuric acid to 72 wt-%. The high-temperature steam released from the evaporator could also be further used as a heat source for CEMD which would greatly reduce the overall energy consumption. Finally it should be noted that a large amount of distillate containing inhibitors was produced during the production of the highly concentrated acid solution. These inhibitors could probably be removed from the distillate using conventional biological methods [29], and then the distillate could be used in the acid retardation process after filtration.

Fig. 4 Variation of J, GOR, Rg, and glucose concentration during CEMD for concentrating sugar eluent. Experiment conditions: Tc,i = 35 °C, Th,i = 98 °C, Ff = 25 L/h

3.3

Ethanol fermentation

3.3.1 3.3.1.1

Fig. 5 Variation of J, GOR, and sulfuric acid concentration during CEMD for concentrating acid eluent. Experiment conditions: Tc,i = 35 °C, Th,i = 98 °C, Ff = 25 L/h

the water and formic, acetic, and levulinic acids became weaker which increased the volatility of these organic acids resulting in their loss through evaporation. Second, as the concentration of sulfuric acid gradually increased, sulfuric acid can be combined with more water molecules, so the activity coefficient of the furfurals, phenols, and other nonpolar compounds in the solution significantly increased, leading to an increase in their volatilities [27]. Since the highly concentrated acid solution contained only

Optimization of ethanol fermentation conditions Single-factor experiments

Figure 6 shows the fermentation performance with time for various initial glucose concentrations. As the initial glucose concentration was increased from 50 to 150 g/L, the ethanol concentration drastically increased. When the initial glucose concentration was greater than 150 g/L, the ethanol concentration only increased a little within 24 h, and it was even less than that in the initial glucose concentration of 150 g/L for 48 h. If the initial concentration of glucose is too high then the growth and metabolism of the yeast cells is hindered because of substrate repression and production inhibition [30,31]. Previously it has been reported that ethanol can directly inhibit the ethanol production pathway; when the ethanol concentration was higher than 70 g/L, the cell mass was reduced by 80% [31]. At a fermentation time of 24 h, the maximum ethanol yield (93.20%) was obtained with an initial glucose concentration of 100 g/L. Therefore, the optimal initial glucose concentration was determined to be 100 g/L. Figure 7 shows the effect of fermentation temperature, agitation rate, initial pH, and inoculum size on ethanol

Table 3 Concentration of inhibitors in acid eluent, concentrated acid solution and highly concentrated acid solution Acid eluent /(g∙L–1)

Concentrated acid solution /(g∙L–1)

Highly concentrated acid solution /(g∙L–1)

Formic acid

0.867

0.966

0.210

Acetic acid

0.077

0.064

0.016

Levulinic acid

Substance

0.220

0.874

0.105

Furfural

ND

0.003

ND

5-HMF

0.041

0.059

0.012

Phenols

0.022

0.187

0.015

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Fig. 6 (a) Glucose concentration; (b) ethanol yield; (c) ethanol concentration; and (d) ethanol productivity as a function of time for various initial glucose concentrations. Fermentation conditions: temperature, 30 °C; agitation rate, 150 r/min; initial pH, 5.0; inoculum size, 10%

Fig. 7 Ethanol concentration, productivity, yield, and glucose utilization rate for a 24-h fermentation with different: (a) temperature, (b) agitation rate, (c) initial pH, and (d) inoculum size. Fermentation conditions: initial glucose concentration, 100 g/L; temperature, 30 °C; agitation rate, 150 r/min; initial pH, 5.0; inoculum size, 10%

fermentation using Saccharomyces cerevisiae CICC 1308 with a carbon source of 100 g/L glucose for a 24-h fermentation. Among these factors, temperature had the

greatest influence on the ethanol fermentation. The highest ethanol concentration and yield were 47.43 g/L and 93.37% respectively at a fermentation temperature of

Qing Li et al. Detoxification and concentration of corn stover hydrolysate

30 °C. The optimum temperature for free Saccharomyces cerevisiae fermentation has been reported to be about 30 °C [32,33], which is consistent with these results. An agitation rate of 175 r/min resulted in a maximum ethanol yield of 93.36% and a pH of 5.0 was optimal for the glucose utilization rate, ethanol concentration, productivity, and yield. This is in accordance with the fact that the optimum pH of free yeast ethanol fermentation is 4.8 – 5.0 [34]. The highest ethanol yield of 93.36% was obtained for an inoculum size of 10%. 3.3.1.2

Orthogonal test

To further optimize the fermentation conditions, including temperature, inoculum size, agitation rate and initial pH, an orthogonal test for ethanol fermentation with the carbon source of 100 g/L glucose was carried out, and the ethanol yield was considered as the optimization objective. Table 4 gives the L9 (34) orthogonal experiment design and results. The fermentation temperature has the greatest effect on the ethanol yield, and the order of influence for all the factors was temperature > inoculum size > pH > agitation rate. The optimal condition for the maximum ethanol yield was A2B2C3D2 which corresponds to a temperature of 30 °C, an inoculum size of 10%, an agitation rate of 175 r/min, and a pH of 5.0. These results are consistent with those of the single-factor experiments. 3.3.2 Ethanol production from the detoxified and concentrated CSH

Ethanol fermentation of the detoxified and concentrated

9

CSH was conducted in a 5-L stirred tank bioreactor using Saccharomyces cerevisiae CICC 1308 and the results are shown in Table 5 and Fig. 8. The strain was grown in glucose-based medium (control) and detoxified and concentrated CSH-based medium. In the control experiment, an ethanol concentration of 47.29 g/L was obtained after 24 h and 43.06 g/L was obtained for the detoxified and concentrated CSH for the same time period. The CSH treated by acid retardation and CEMD resulted in an ethanol yield of 86.31%, less than that (93.10%) of the control experiment. These differences are probably due to the traces of phenolic inhibitors or other inhibitory compounds that remained in the detoxified and concentrated CSH. In addition, the CSH that had not undergone detoxification and concentration was also subjected to fermentation but it exhibited no growth or ethanol production. This is mainly because the concentration of sulfuric acid was so high in the untreated CSH that a large amount of sodium sulfate was produced when the CSH was neutralized by sodium hydroxide [12]. Such a high salt content completely inhibits the growth of yeast and the production of ethanol. Ethanol fermentation results obtained using various microorganisms and different types of substrates have been reported in the literature [9,35,36] and a few are summarized in Table 6. Among them, the highest ethanol yield of 83.5% (corresponding to 42.60.8 g ethanol per 100 g glucose) was obtained from pretreated palm fronds, which were hydrolyzed with Cellicthe H-Tech2 [36]. Heinonen et al. [9] obtained an ethanol yield of 74.3% from spruce cellulose with concentrated acid hydrolysis

Table 4 Orthogonal design of ethanol fermentation conditions No.

Temperature A

Inoculum size B

Agitation rate C

pH D

Ethanol yield /%

1

1 (25 °C)

1 (5%)

1 (125 r/min)

1 (4.5)

75.232

2

1 (25 °C)

2 (10%)

2 (150 r/min)

2 (5.0)

86.201

3

1 (25 °C)

3 (15%)

3 (175 r/min)

3 (5.5)

83.434

4

2 (30 °C)

1 (5%)

3 (175 r/min)

2 (5.0)

91.943

5

2 (30 °C)

2 (10%)

1 (125 r/min)

3 (5.5)

90.566

6

2 (30 °C)

3 (15%)

2 (150 r/min)

1 (4.5)

92.611

7

3 (35 °C)

1 (5%)

2 (150 r/min)

3 (5.5)

81.931

8

3 (35 °C)

2 (10%)

3 (175 r/min)

1 (4.5)

86.810

9

3 (35 °C)

3 (15%)

1 (125 r/min)

2 (5.0)

86.723

K1

244.867

249.107

252.521

254.654

NA

K2

275.121

263.577

260.743

264.868

NA

K3

255.465

262.768

262.188

255.931

NA

k1

81.622

83.036

84.174

84.885

NA

k2

91.707

87.859

86.914

88.289

NA

k3

85.155

87.590

87.399

85.310

NA

R

10.085

4.824

3.222

3.405

NA

Q

A2

B2

C3

D2

NA

10

Front. Chem. Sci. Eng.

Table 5 Fermentation of detoxified and concentrated CSH using Saccharomyces cerevisiae CICC 1308 Initial glucose /(g∙L–1)

Residual glucose / (g∙L–1)

Ethanol concentration /(g∙L–1)

Ethanol productivity /g∙(L$h) –1

Ethanol yield /%

Control (glucose)

100.0

0.406

47.289

1.970

93.102

Detoxified and concentrated CSH

100.0

2.175

43.061

1.794

86.311

Experiment

Fig. 8 Ethanol concentration, productivity, yield and glucose concentration for ethanol fermentation of: (a) glucose, (b) hydrolysate. Fermentation conditions: initial glucose concentration, 100 g/L; temperature, 30 °C; agitation rate, 175 r/min; initial pH, 5.0; inoculum size, 10%

even though the hydrolysate also contained a small amount of xylose. Obviously, the ethanol yield of 86.3% obtained in this study with Saccharomyces cerevisiae CICC 1308 is higher than those reported in Table 6. Therefore, these results demonstrate that combining acid retardation and CEMD can effectively be utilized for bioethanol production from corn stover.

4

Conclusions

Acid retardation is an effective method for separating sugar

and acid and removing most of the inhibitors in a sulfuric acid-catalytic hydrolysate obtained from corn stover. Almost all the inhibitors were removed, 99.12% of the sugar was recovered, and the sulfuric acid contained in the hydrolysate was completely recovered or used. The dilute sugar and acid solutions were concentrated using CEMD. Although J and GOR decreased to some extent as the sugar or acid concentration in the concentrate increased, the final values of J and GOR were still moderately high. The integration of CEMD and high-temperature evaporation enabled the recycling of the sulfuric acid. After treatment by acid retardation and CEMD, the CSH containing

Table 6 Ethanol produced from different types of substrates by various microorganisms Strain S. cerevisiae VTTB-08014

Substrate

Mode

Sugar /(g∙L –1)

Ethanol yield /%

Ref.

Spruce

Batch

43.3

74.3

9

41.8

64.7

Birch a)

S. cerevisiae

Corn stover

SHF

S. cerevisiae

Palm fronds

SHF

b)

NM

NM

Eucalyptus chips S. cerevisiae CICC 1308

Corn stover

a) separate hydrolysis and fermentation; b) not mentioned

67.4

35

83.5

36

71.4 Batch

100

86.3

This study

Qing Li et al. Detoxification and concentration of corn stover hydrolysate

100 g/L glucose produced 43.06 g/L of ethanol with a yield of 86.31% in batch fermentation by Saccharomyces cerevisiae CICC 1308. The ethanol concentration and yield were 91.06% and 92.71% respectively of the corresponding values obtained in the control experiment. CSH treated by acid retardation and CEMD can effectively be utilized as a renewable feedstock for ethanol fermentation. Acknowledgements The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China (Grant No. 21376175).

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