Enhanced bioethanol production by fed-batch simultaneous saccharification and co-fermentation at high solid loading of Fenton reaction and sodium hydroxide sequentially pretreated sugarcane bagasse

Accepted Manuscript Enhanced bioethanol production by fed-batch simultaneous saccharification and co-fermentation at high solid loading of Fenton reaction and sodium hydroxide sequentially pretreated sugarcane bagasse Teng Zhang, Ming-Jun Zhu PII: DOI: Reference:

S0960-8524(17)30048-2 http://dx.doi.org/10.1016/j.biortech.2017.01.028 BITE 17523

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

28 November 2016 11 January 2017 12 January 2017

Please cite this article as: Zhang, T., Zhu, M-J., Enhanced bioethanol production by fed-batch simultaneous saccharification and co-fermentation at high solid loading of Fenton reaction and sodium hydroxide sequentially pretreated sugarcane bagasse, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech. 2017.01.028

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Enhanced bioethanol production by fed-batch simultaneous saccharification and co-fermentation at high solid loading of Fenton reaction and sodium hydroxide sequentially pretreated sugarcane bagasse Teng Zhang, Ming-Jun Zhu * School of Bioscience and Bioengineering, South China University of Technology, Guangzhou Higher Education Mega Center, Panyu, Guangzhou 510006, People’s Republic of China ∗

Corresponding author, E-mail address: [email protected]; Tel: +8620 39380623;

Fax: +8620 39380601

Abstract: A study on the fed-batch simultaneous saccharification and co-fermentation (SSCF) of Fenton reaction combined with NaOH pretreated sugarcane bagasse (SCB) at a high solid loading of 10% ~ 30% (w/v) was investigated. Enzyme feeding mode, substrate feeding mode and combination of both were compared with the batch mode under respective solid loadings. Ethanol concentrations of above 80 g/L were obtained in batch and enzyme feeding modes at a solid loading of 30% (w/v). Enzyme feeding mode was found to increase ethanol productivity and reduce enzyme loading to a value of 1.23 g/L/h and 9 FPU/g substrate, respectively. The present study provides an economically feasible process for high concentration bioethanol production. Key words: Fed-batch SSCF; Sugarcane bagasse; High solid loading; Enzyme feeding

1. Introduction Bioethanol is a kind of clean energy which has a widespread use, especially, when it is added into the petroleum, the carbon dioxide emission can be greatly reduced, and therefore, the global warming will be alleviated to some extent (Agbor et al., 2011). In last decades, bioethanol production had been developed from the first generation to the second generation. Since the first generation bioethanol production consumed large amounts of starch-based crops which did not fit in with the idea of sustainability for mankind, it had been gradually replaced by the lignocellulose-based second generation bioethanol production (den Haan et al., 2013). There is a massive amount of lignocellulose on the planet. Lignocellulose is composed of three parts, i.e. cellulose, hemicellulose and lignin, among which cellulose and hemicellulose can be converted into fermentative sugars by microbes or enzymes (Liu et al., 2015). On the other hand, lignin becomes a block for enzymes to contact with the embedded cellulose. Sugarcane bagasse (SCB) is one of typical agricultural and industrial residues in large quantities (Zhou et al., 2016). It contains considerable holocellulose in the lignocellulose, which makes it an ideal kind of lignocellulosic material to be degraded to produce bioethanol. However, the raw material is hard to be discomposed without pretreatment, therefore, pretreatment is needed (Martín et al., 2007). Fenton reaction was found to be effective and economical in the pretreatment of some lignocellulosic material as well as eco-friendly for the environment since it could increase susceptibility of enzymes to the embedded cellulose (Jain and Vigneshwaran, 2012). Besides, the surrounding lignin

could be degraded to some extent by the radicals like hydroxyl radicals produced in the reduction-oxidation reaction (Arantes et al., 2011; Soudham et al., 2014). In our previous studies (Zhang and Zhu, 2016), SCB after Fenton reaction followed by NaOH extraction (FT-AE) was found to be easily degraded in the simultaneous saccharification and co-fermentation (SSCF) test. Therefore, the SCB was still pretreated by the same method in the research. SSCF is an excellent processing technology to produce bioethanol from lignocellulose since it could improve bioethanol production by alleviating the end-product inhibition (Saha et al., 2011) and shortening the processing time (Jin et al., 2012) as well as ameliorating the consumption of pentose (Gubicza et al., 2016) compared with traditional separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). However, when the solid loading became very high, the ethanol concentration did not increase significantly or even decrease at a higher solid loading as a result of a high viscosity and poor mass transfer (Erdei et al., 2013). In order to overcome the problems, the fed-batch mode had been tried in the SSCF (Zhang et al., 2009; Jin et al., 2013), in which substrate or enzyme was fed more than one time to decrease the initial viscosity of the broth. However, most papers usually concerned about the effects of substrate feeding on the fermentation and there was only a few research involved enzyme feeding (Hoyer et al., 2010; Koppram and Olsson, 2014). Here, in the paper, a comprehensive study of fed-batch SSCF including enzyme and substrate feeding had been carried out. Besides, a much higher solid

loading of 30% (w/v) had also been investigated to better apply to the industrial level. 2. Material and methods 2.1. Material The raw SCB was provided by the Guangzhou Sugarcane Industry Research Institute (Guangzhou, China). The composition of raw SCB was of 32.2%, 20.6% and 22.4% for cellulose, hemicellulose and lignin, respectively. Fenton’s reagents including FeCl2·4H2O and H2O2 (30%, w/w) were purchased from Guangzhou Chemical Reagent Factory (Guangzhou, China). The commercial cellulase Cellic® CTec2 was kindly provided by Novozymes (China) Investment Co.Ltd. Filter paper activity of cellulase was 110 FPU/mL and pNPG activity of β-glucosidase was 2743 U/mL. 2.2. Pretreatment FT-AE pretreatment was carried out according to the paper (Zhang and Zhu, 2016). Briefly, 5 g of SCB (< 0.1 mm in diameter) was added to the flask, followed by supplementation of 50 ml of 20 mM Fe2+, and stirred to make the SCB well-distributed. Then 50 ml of 10% (w/v) H2O2 was poured into the flask to initialize the Fenton reaction, the reaction duration was 6 h. After the Fenton pretreatment, the suspension was filtrated to obtain the solid residue. The residue was washed to the neutral pH and dried. Finally, the Fenton pretreated SCB was ground to less than 0.1 mm in diameter. The harvested SCB was soaked in 1% (w/w) NaOH solution with a solid-liquid ratio of 1:20 for 1 h under 80℃. After the pretreatment, the suspension was filtrated, and the obtained residue was washed to the neutral pH, then dried and ground to the size of less

than 0.1 mm. The composition of the pretreated SCB was of 58.1%, 14.1% and 9.0% for cellulose, hemicellulose and lignin, respectively. 2.3. Simultaneous saccharification and co-fermentation 2.3.1. Microorganism strains The yeast SHY07-1 was used as an inoculum which could turn glucose and xylose into ethanol (Zhu et al., 2012). 2.3.2. Culture media YPD medium (10 g/L yeast extract; 20 g/L peptone; 20 g/L glucose) was used as seed medium. The growth medium (fermentation medium) contained (NH4)2SO4 2 g/L, yeast extraction 5 g/L, KH2PO4 5 g/L, CaCO3 2 g/L and MgSO4·7H2O 0.5 g/L. The pH was adjusted to the neutral pH. 2.3.3. Fermentation The SSCF test was performed in 25 ml serum bottles with a working volume of 15 ml. Desired amount of FT-AE pretreated SCB was added into the bottle followed by pouring into about 13 ml of fermentation medium. The bottle was sealed by a rubber stopper with an aluminum lid crimped on the top. The medium was autoclaved at 115 ℃ for 20 min. Later, 1.5 ml of inoculum (OD600≈2.0) at a dosage of 10% (v/v) was inoculated to the medium along with enzyme addition. The enzyme was added at a loading of 15 FPU/g SCB except that there was a special instruction. Bottles were incubated in a rotary shaker at 30℃, 180 rpm for 120 h during which 0.1 ml of samples

were withdrawn periodically. During fermentation, the system was outgassed to the atmospheric pressure periodically by a syringe. Ethanol fermentation efficiency (η) and ethanol yield (Y) was calculated according to the following equation (1) and equation (2), respectively.

η=

Ethanol concentrat ion, g/L × 100 % Solid loadings × glucan % × 1.11× 0.51 + Solid loadings × xylan % × 1.14 × 0.46, g/L

Y=

Ethanol concent ration , g/L solid loading initial , g/L

(1)

(2)

where 1.11 is the coefficient of glucose obtained from glucan; 1.14 is the coefficient of xylose obtained from xylan; 0.51 is the coefficient of ethanol obtained from glucose; and 0.46 is the coefficient of ethanol obtained from xylose. 2.4. Analytical methods 2.4.1. SCB component analysis The SCB component analysis was conducted according to the methods recommended by the National Renewable Energy Laboratory (NREL, 2008). 2.4.2. Analysis of broth products The broth products were analyzed by Waters 2414 HPLC (Milford, LA, the US) equipped with refractive index detector and Aminex HPX-87H column ((Bio-Rad, Hercules, CA). The mobile phase was 5 mM H2SO4 and the flow rate was 0.6 ml/min. The running time was kept for 30 min. 2.4.3. Data analysis The data were analyzed statistically by SPSS software for Duncan’s multiple range

tests. The software SPSS 17.0 (SPSS Inc. Chicago) was used for all statistical analysis. [* statistically significant (p < 0.05)]. 3. Results and discussion 3.1. Fed-batch SSCF of SCB at different final solid loadings under different feeding strategies 3.1.1. Final solid loading of 10% (w/v) As shown in Table 1, three feeding strategies had been used along with the batch mode (the control), i.e, enzyme feeding, substrate (SCB) feeding and combination of both at a final SCB loading of 10% (w/v). In enzyme and SCB feeding mode, enzyme and SCB were fed proportionally each time and the percentage of SCB added was the same with that of the enzyme. As shown in Fig.1A, the batch mode had the advantages over the other fed-batch modes regardless of the initial fermentation rate or the final ethanol production. Similar result was also found by Sugiharto (Sugiharto et al., 2016) and Liu (Liu and Chen, 2016). The ethanol fermentation efficiency reached 96.8% in 72 h, indicating that the SSCF of the pretreated SCB is highly efficient. However, it seemed that a solid loading of 10% (w/v) was not high enough to get an adverse impact on the fermentation performance in such a system. Besides, the initial activity of enzymes in the system was not affected at a solid loading of 10% (w/v). The specific results were calculated and shown in the Table 2. Particularly, in the feeding modes, enzyme feeding got a significant superiority over the other two on the fermentation performance. Moreover, as long as the substrate feeding participated, the fermentation

performance seemed to weaken a lot either on the final ethanol concentration or productivity. This might be attributed to the gradual loss of enzyme activity (Hegedus and Nagy, 2015). When the SCB was fed later, the actual enzyme activity might be much lower than that of the initial. Besides, as the ethanol consistently produced in the broth, the yeast activity might also be much lower than the initial. Therefore, its ethanol production rate was lower than the batch mode. Since the mass transfer for the solid loading of 10% (w/v) was verified to be excellent in the system, therefore, a higher solid loading was tested in the following experiment. 3.1.2. Final solid loading of 14% (w/v) In order to raise the ethanol concentration to meet the needs of industrial requirements as well as investigating the potential of fed-batch mode, a higher solid loading of 14% (w/v) was tested. The fed-batch strategies were shown in the Table 1. The feeding strategies had also been improved according to the previous test and more SCB had been added in the initial period of fermentation. Additionally, the enzyme and SCB feeding were accomplished within 48 h. As shown in Fig.1B, the batch mode still seemed to get a better fermentation performance over the feeding modes. The ethanol concentration and fermentation efficiency reached 53.75 g/L and 95.32%, respectively, indicating that the mass transfer was still very good when the final solid loading of SCB ran up to 14% (w/v). However, the discrepancy of fermentation performance between the batch and fed-batch modes became smaller, especially for the SCB feeding mode, the fermentation efficiency improved a lot, achieving a value of 93.61%. The detailed

parameters involved with the fermentation performance could be seen in the Table 2. It seemed that all the feeding improvements just worked. Therefore, the following tests under a higher solid loading will comply with this direction. 3.1.3. Final solid loading of 20% (w/v) The fed-batch strategies at a final SCB loading of 20% (w/v) was listed in the Table 1, and the feeding time intervals and the overall feeding period were continuously shortened to 12 h and 24 h, respectively. As illustrated in the Fig.1C, the enzyme feeding mode began to gain superiority over the batch mode in both ethanol concentration and productivity, with a value of 59.08 g/L and 1.231 g/L/h, respectively, while it was of 53.55 g/L and 0.446 g/L/h for the batch mode (Table 2). Besides, the enzyme feeding mode showed significant advantage over SCB feeding when the solid loading increased to 20% (w/v), suggesting that enzyme activity and stability started to play a more vital role than the mass transfer in the system. However, the result was not in accordance with the traditional view that mass transfer was the most important factor during the fermentation at high solid loading (de Cassia Pereira et al., 2016; Unrean et al., 2016). In order to further validate the phenomenon, fed-batch SSCF at a final solid loading of 30% (w/v) was conducted in the following experiment. 3.1.4. Final solid loading of 30% (w/v) A higher solid loading was key for the decrease of processing cost, since higher solid loading enabled a higher potential ethanol concentration, which would apparently reduce the energy consumption in the distillation operation (Romaní et al., 2012). To

the best of our knowledge, there were very few papers concerned about a solid loading of 30% (w/v) in batch or fed-batch SSF (SSCF). Here, fed-batch SSCF at a final solid loading of 30% (w/v) was conducted. The feeding strategies were shown in the Table 1. The time courses of ethanol concentration were illustrated in Fig.1D. Since there was no free liquid before the first 36 h, it was nearly impossible to sample for the batch mode. Therefore, the sampling began at 48 h when the slurry was liquefied distinctly. As shown in Fig.1D, the enzyme feeding mode still achieved a minor superiority over the batch mode in ethanol concentration and productivity with an ethanol concentration of 84.04 g/L and a fermentation efficiency of 69.55% (Table 2). Compared with the other two feedings, the enzyme feeding still had an obvious advantage, which suggested that enzyme activity and yeast viability played a dominant role, instead of mass transfer in such a system. However, the enzyme feeding mode seemed not to further significantly enhance ethanol concentration under such a high solid loading condition compared with the batch mode (p>0.05). It might also be attributed to the poor yeast viability in the high ethanol concentration environment (Rudolf et al., 2005). Moreover, the fermentation performance of the batch mode did not further get worse compared with the solid loading of 20% (w/v). On the one hand, it suggested that the Fenton reaction combined with NaOH extraction pretreated SCB was very easy to be degraded since a high ethanol concentration of 83.25 g/L was still obtained under such a high solid loading of SCB. On the other hand, it could be due to the special reaction system. A lab-scale airtight system, in which carbon dioxide was consistently produced and further

enhance the mixture of the suspension and abate the viscosity of the broth, was beneficial for mass transfer. The controls without yeast inoculation at different SCB loadings were set to compare sugar release with those of co-fermentation. It was observed (data not shown) that sugars such as cellobiose, glucose and xylose significantly accumulated at all SCB loadings. At a SCB loading of 30% (w/v), the final (120 h) concentrations of sugars reached 19.93 g/L, 123.29 g/L and 27.04 g/L for cellobiose, glucose and xylose, respectively. However, no sugar accumulation (<1 g/L) was found in the co-fermentation at a final SCB loading of 10% (w/v), 14% (w/v) and 20% (w/v). When the SCB solid loading increased to 30% (w/v), there was only a slight sugar accumulation with a concentration of 5.71 and 4.96 g/L for glucose and xylose, respectively, suggesting that SSCF at a very high solid loading of the sequentially pretreated SCB could efficiently ferment the released sugars, thus leading to a high ethanol concentration. The mass balance for the whole process of lignocellulosic ethanol production with a solid loading of 30% (w/v) was shown in Fig.2. As it was calculated, 1 Kg of raw SCB could finally be converted to 0.122 Kg ethanol, which was equal to a volume of 0.156 L anhydrous ethanol. The conversion ratio was a little lower than the reported literatures (Su et al., 2013; Kang et al., 2015) because of the significant mass loss (about 50%) of sequential pretreatment. However, as it is known that the cost for lignocellulosic ethanol production mainly depends on enzyme cost and ethanol recovery cost, therefore, a

higher ethanol concentration with lower enzyme loading is of the most importance. An ethanol concentration of above 80 g/L with a lower enzyme loading was obtained in this study; therefore, it was still much competitive against the previously reported paper. 3.2. Effects of different enzyme feeding time intervals and feeding times As revealed in the above research, enzyme feeding showed an obvious advantage over the other feeding modes. Besides, at a solid loading of 30% (w/v), enzyme feeding still gained higher ethanol productivity than that of batch mode though the maximum ethanol concentration did not increase significantly. However, conventional research usually concentrated on the substrate feeding (Zhu et al., 2014; Carrasco et al., 2011). Therefore, a more scientific enzyme feeding strategy turned to be very vital for a higher ethanol concentration. As it was known that enzyme activity and stability might decrease as the time elongated during the fermentation, different enzyme feeding time intervals and feeding times might have a close relation with the total enzyme activity. Therefore, effects of different enzyme feeding time intervals and times were investigated at a solid loading of 30% (w/v). The enzyme feeding strategies at different enzyme feeding time intervals were shown in Table 3, ∆t values ranged from 0 to 48 h were set up. As depicted in Fig.3A, the final ethanol concentrations reached about 80 g/L for all groups, and there was no significant difference (p>0.05) among them, which suggested that the enzyme feeding time interval had no significant impact on the final ethanol concentration at a solid loading of 30% (w/v). The enzyme feeding strategies at different enzyme feeding times

were shown in Table 4. As illustrated in Fig.3B, there was still no significant difference (p>0.05) among all the groups, which suggested that feeding times had no significant impact on the final ethanol concentration. Therefore, on the whole, it could be concluded that enzyme feeding had no significant impact on the final ethanol concentration. 3.3. Effects of reduced enzyme loadings Despite that enzyme feeding mode could not enhance the final ethanol concentration; however, the ethanol productivity could be elevated. Hence, enzyme feeding at a reduced loading without sacrificing the ethanol production was enlightened. As exhibited in Table 5, different percentages of enzyme loading reduction were investigated. Enzyme was averagely fed for 6 times. The enzyme loading descended gradiently in each experimental group compared with the control. When the enzyme loading decreased by 40%, i.e, 9 FPU/g SCB (Fig.4), the ethanol concentration showed no significant decline (p> 0.05), suggesting that a 40% reduction of enzyme loading had no significant impact on the final ethanol concentration. Nevertheless, when the enzyme loading decreased by 50%, the ethanol concentration began to decline significantly (p<0.05), indicating that enzyme feeding mode did get a threshold with an enzyme loading of approximately 9 FPU/g SCB, which was of a relatively economical loading for the lignocellulosic ethanol production. 4. Conclusion Fenton reaction combined with NaOH extraction pretreated SCB achieved a good

fermentation performance under a high solid loading of 30% (w/v). Besides, fed-batch SSCF showed no significant effect on the enhancement of final ethanol concentration at a high solid loading of 10% ~ 30% (w/v) in the reaction system of 25 ml airtight bottle. However, enzyme feeding mode could significantly enhance the ethanol productivity and reduce the enzyme loading, which was a promising strength for the industrial-scale lignocellulosic ethanol production. Acknowledgment The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China [grant nos. 51478190 and 51278200], Guangdong Provincial Natural Science Foundation Key Project [grant no. 2014A030311014], Guangdong Provincial Science and Technology Program [grant no. 2013B010102002] and Guangzhou Science and Technology Program [grant no.201510010288].

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Lists of figures Fig.1. Time courses of ethanol concentrations in fed-batch SSCF at a final SCB loading of 10% (w/v) (A), 14% (w/v) (B), 20% (w/v) (C) and 30% (w/v) (D) under different feeding strategies. Fig.2. Mass balance for sequential pretreatment and SSCF. Fig.3. Final ethanol concentrations of enzyme fed-batch SSCF at different feeding time intervals (A) and feeding times (B). Fig.4. Final ethanol concentrations of enzyme fed-batch SSCF at reduced loadings of enzymes.

Lists of tables Table 1 Enzyme and SCB feeding strategies in fed-batch SSCF Table 2 Ethanol concentration, fermentation efficiency, ethanol yield and ethanol productivity of different feeding samples at a final SCB loading of 10% (w/v), 14% (w/v), 20% (w/v) and 30% (w/v) under different feeding strategies Table 3 Enzyme feeding strategies at different enzyme feeding time intervals Table 4 Enzyme feeding strategies at different enzyme feeding times Table 5 Enzyme feeding strategies at reduced enzyme loadings

Figures

A

B

C

D

Fig.1. Time courses of ethanol concentrations in fed-batch SSCF at a final SCB loading of 10% (w/v) (A), 14% (w/v) (B), 20% (w/v) (C) and 30% (w/v) (D) under different feeding strategies.

Sugarcane bagasse (SCB)

Sequential pretreatment

SSCF (30% w/v of SCB)

1 Kg dry matter

0.437 Kg dry matter

0.122 Kg ethanol

Cellulose Hemicellulose Lignin

0.322 Kg 0.206 Kg 0.224 Kg

Cellulose

0.254 Kg

Anhydrous ethanol 0.156 L

Hemicellulose Lignin

0.062 Kg 0.039 Kg

ρethanol= 0.781 g/mL (30℃)

Fig.2. Mass balance for sequential pretreatment and SSCF.

A

B

Fig.3. Final ethanol concentrations of enzyme fed-batch SSCF at different feeding time intervals (A) and feeding times (B). Data are shown with error bars indicating ± standard deviation. [*: statistically significant (p<0.05)].

Fig.4. Final ethanol concentrations of enzyme fed-batch SSCF at reduced loadings of enzymes. Data are shown with error bars indicating ± standard deviation. [*: statistically significant (p<0.05)].

Tables Table 1 Enzyme and SCB feeding strategies in fed-batch SSCF Samples

Final substrate loading (w/v)

Batch Enzyme feeding SCB feeding

10%

Enzyme and SCB feeding Batch Enzyme feeding SCB feeding

14%

Enzyme and SCB feeding Batch Enzyme feeding SCB feeding Enzyme and SCB feeding

20%

Batch Enzyme feeding SCB feeding Enzyme and SCB feeding

30%

SCB loading (w/v), enzyme loading (FPU/g SCB) 0h

48 h

96 h

10%, 15.0

-

-

10%, 4.5

6.0

4.5

3%, 15.0

4%

3%

3%, 4.5

4%, 6.0

3%, 4.5

0h

24 h

48 h

14.0%, 15 14.0%, 6

6

3

5.6%, 15

5.6%

2.8%

5.6%, 6

5.6%, 6

2.8%, 3

0h

12 h

24 h

20% ,15 20%, 6 8%, 15 8%, 6

6 8% 8%, 6

3 4% 4%, 3

0h

12 h

24 h

30% ,15

-

-

30%, 6

6

3

12%, 15

12%

6%

12%, 6

12%, 6

6%, 3

Table 2 Ethanol concentration, fermentation efficiency, ethanol yield and ethanol productivity of different feeding samples at a final SCB loading of 10% (w/v), 14% (w/v), 20% (w/v) and 30% (w/v) under different feeding strategies Samples

Batch

Enzyme feeding

SCB feeding

Enzyme and SCB feeding

Solid loading % (w/v)

Ethanol concentration (g/L)

Fermentation efficiency (%)

Ethanol yield (g/g)

Ethanol productivity (g/L/h)

10

39.00

96.83

0.39

0.54

14

53.75

95.32

0.38

0.56

20

53.55

66.48

0.27

0.47

30

83.25

68.90

0.28

0.69

10

38.60

95.83

0.39

0.27

14

51.66

91.61

0.37

0.54

20

59.08

73.34

0.30

1.23

30

84.04

69.55

0.28

0.88

10

29.66

73.63

0.30

0.21

14

52.78

93.61

0.38

0.44

20

47.96

59.54

0.24

0.57

30

70.45

58.31

0.24

0.59

10

39.05

96.94

0.39

0.27

14

47.99

85.10

0.34

0.40

20

49.32

61.23

0.25

0.59

30

68.68

56.84

0.23

0.57

Table 3 Enzyme feeding strategies at different enzyme feeding time intervals Time intervals

Enzyme feeding loading 0h *

12 h

24 h

36 h

48 h

60 h

72 h

84 h

96 h

120 h

－

－

－

－

－

－

－

－

－

∆t=0 h

E0

∆t=12 h

2/5 E0

2/5 E0

1/5 E0

－

－

－

－

－

－

－

∆t=24 h

2/5 E0

－

2/5 E0

－

1/5 E0

－

－

－

－

－

∆t=36 h

2/5 E0

－

－

2/5 E0

－

－

1/5 E0

－

－

－

∆t=48 h

2/5 E0

－

－

－

2/5 E0

－

－

－

1/5 E0

－

E0 represented the total enzyme loading of 15 FPU/g substrate.

Table 4 Enzyme feeding strategies at different enzyme feeding times Feeding times

Enzyme feeding loading 0h

12 h

24 h

36 h

48 h

60 h

72 h

96 h

120 h

1

E 0*

－

－

－

－

－

－

－

－

2

1/2 E0

－

－

－

－

1/2 E0

－

－

－

3

1/3 E0

－

－

1/3 E0

－

－

1/3 E0

－

－

4

1/4 E0

－

1/4 E0

－

1/4 E0

－

1/4 E0

－

－

5

1/5 E0

－

1/5 E0

－

1/5 E0

－

1/5 E0

1/5 E0

－

6

1/6 E0

1/6 E0

1/6 E0

1/6 E0

1/6 E0

1/6 E0

－

－

－

E0 represented the total enzyme loading of 15 FPU/g substrate

Table 5 Enzyme feeding strategies at reduced enzyme loadings Reduced enzyme loading (%)

Enzyme feeding loading 0h

12 h

24 h

36 h

48 h

60 h

0

1/6 E0

1/6 E0

1/6 E0

1/6 E0

1/6 E0

1/6 E0

14.3

1/7 E0

1/7 E0

1/7 E0

1/7 E0

1/7 E0

1/7 E0

25.0

1/8 E0

1/8 E0

1/8 E0

1/8 E0

1/8 E0

1/8 E0

33.3

1/9 E0

1/9 E0

1/9 E0

1/9 E0

1/9 E0

1/9 E0

40.0

1/10 E0

1/10 E0

1/10 E0

1/10 E0

1/10 E0

1/10 E0

50.0

1/12 E0

1/12 E0

1/12 E0

1/12 E0

1/12 E0

1/12 E0

E0 represented the total enzyme loading of 15 FPU/g substrate. The fermentation time was 120 h.

Highlights > A high ethanol concentration above 80 g/L was obtained. > Enzyme feeding could significantly enhance the ethanol productivity. > Enzyme feeding could significantly reduce the enzyme loading.