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Fuel 116 (2014) 699–702 Contents lists available at ScienceDirect Fuel journal homepage: www.else vier.com/l

Contents lists available at ScienceDirect

Fuel

journal homepage: www.else vier.com/l ocate/fuel

Fuel journal homepage: www.else vier.com/l ocate/fuel Enzymatic hydrolysis of microwave alkali pretreated rice

Enzymatic hydrolysis of microwave alkali pretreated rice husk for ethanol production by Saccharomyces cerevisiae , Scheffersomyces stipitis and their co-culture

Anita Singh a , b , , Somvir Bajar a , Narsi R. Bishnoi a

a Department of Environmental Science and Engineering, Guru Jambheshwar University of Science and Technology, Hisar 125001, Haryana, India b Department of Environmental Science, Central University of Jammu, Jammu and Kashmir, India

Central University of Jammu, Jammu and Kashmir, India highlights Rice husk used as feedstock for ethanol

highlights

Rice husk used as feedstock for ethanol production. Crude unprocessed enzymes were used for saccharification. Concentrated enzymatic hydrolyzate used for ethanol production by yeast. Fermentation was accomplished by Saccharomyces cerevisiae, Scheffersomyces stipitis and their co-culture.

article info

Article history:

Received 6 January 2012 Received in revised form 15 August 2013 Accepted 27 August 2013 Available online 5 September 2013

Keywords:

Rice husk

Saccharomyces cerevisiae

Scheffersomyces stipitis

Hydrolyzate

Fermentation

abstract

Rice husk is one of the abundant lignocellulosic feed stocks in the world. It contains mixtures of pentose and hexose sugars, so selected for ethanol production. The microwave alkali pretreated rice husk enzy- matically hydrolyzed with crude unprocessed hydrolytic enzymes. Concentrated enzymatic hydrolyzate having initial reducing sugar concentrations of 10 g/L, 25 g/L, 50 g/L and 70 g/L were used for ethanol pro- duction. Fermentation was accomplished by the action of yeast Saccharomyces cerevisiae, Scheffersomyces stipitis and their co-culture. Ethanol yield obtained with S. cerevisiae were 0.3 g/g, 0.35 g/g, 0.40 g/g and 0.39 g/g; with S. stipitis were 0.24 g/g, 0.27 g/g, 0.36 g/g and 0.35 g/g; and with co-culture of both were 0.33 g/g, 0.36 g/g, 0.42 g/g and 0.40 g/g with different sugar concentrations. Maximum ethanol yield was found to be 0.42 g/g with co-culture, 0.40 g/g with S. cerevisiae and 0.36 with S. stipitis at initial reducing sugar concentration of 50 g/L.

2013 Elsevier Ltd. All rights reserved.

1. Introduction

Lignocelluloses from agricultural, industrial and forest residuals account for the majority of the total biomass present in the world and are regarded as the largest known renewable carbohydrate for ethanol production [1] . The key requirements for an economical lignocellulosic ethanol process include: efficient pretreatment methods of lignocelluloses, availability of low-cost hydrolytic en- zymes, and use of optimal microbial strains capable of converting hexose and pentose sugars to ethanol at high rates, yields, and final concentrations [2] . Rice husk is agricultural waste, accounting for about one fifth of the annual gross rice, 545 million metric tons, of the world [3] . Rice husk predominantly contains hemicellulose

Corresponding author at: Department of Environmental Sciences, Central University of Jammu, Jammu-180011, Jammu and Kashmir, India. Tel.: +91 9354312123/+91 9419294912; fax: +91 1662 276240. E-mail addresses: anitasaharan@gmail.com (A. Singh), nrbishnoi@gmail.com (N.R. Bishnoi).

0016-2361/$ - see front matter 2013 Elsevier Ltd. All rights reserved.

and cellulose and these can be hydrolyzed into sugars at approxi- mately 0.426 g of sugars per 1 g of dry rice husk [4] . With further process the sugars can be fermented into ethanol at about 0.21 g of ethanol per 1 g of dry rice husk [5] . However, due to the close association of cellulose and hemicellulose with lignin in the plant cell wall, pre-treatment is necessary to make these carbohydrates available for enzymatic hydrolysis and fermentation [6] . A pre- requisite to the biological conversion of lignocellulosic biomass to ethanol is to release of the cellulose portion (and subsequently glucose) from the tightly woven lignocellulosic structure [7] . Pre- treatment includes physical, chemical, biological and thermal methods and their combinations. High temperature acid treatment can hydrolyze cellulose to yield sugars, this process will result in formation of degradation products like 5-hydroxymethyl furfural which will interfere with microbial fermentation [6] . Pretreatment enhance the accessibility of enzymes and digestibility of holocellu- lose components because of the salvation and the saphonication during alkaline pretreatment [8] . Alkali pretreatment is a typical chemical pretreatment method and a combination of microwave

700

A. Singh et al. / Fuel 116 (2014) 699–702

irradiation with alkali may be an alternative pretreatment ap- proach for lignocellulosic materials at lower temperature to solve the pretreatment problem [8] . The cellulose component resulted from the pre-treatment process can be converted to ethanol in a two-step process where the cellulose is first converted to glucose sugars by hydrolysis; the resulting sugars can in turn be converted to ethanol by fermentation [9] . The hydrolysis of polysaccharides is usually catalyzed by hydro- lytic enzymes, because enzymatic hydrolysis produces better yields than acid-catalyzed hydrolysis [7] . Cellulases are mixture of at least three different enzymes: endoglucanase (EG, endo-1,4- d-glucanohydrolase, or EC 3.2.1.4.) which attacks regions of low crystallinity in the cellulose fiber, creating free chain-ends; exoglu- canase or cellobiohydrolase (CBH, 1,4-b-d-glucan cellobiohydro- lase, or EC 3.2.1.91.) which degrade the molecule further by removing cellobiose units from the free chain-ends; and b -glucosi- dase (EC 3.2.1.21.) which hydrolyzes cellobiose to produce glucose, in much smaller amounts [10]. However, the most widely used cellulase from Trichoderma reesei is poor in cellobiase, and thus restricts the conversion of cellobiose to glucose [9] . The accumula- tion of cellobiose will cause severe feedback inhibition to the cellulase reaction [11] . Therefore, cellulase complex system is crucial to raise enzymatic hydrolysis yield. Glucose and xylose are the two dominant sugars in lignocellulosic hydrolyzates after saccharification. Both need to be fermented efficiently, but current approaches are inefficient, since no native microorganisms can convert all sugars into ethanol at high yield [1] . The lack of indus- trially robust microbes for co-fermentation of glucose and xylose has been a major technical barrier. Researchers have taken two different approaches to solve this problem either by using geneti- cally engineered microbes or co-culture of microbes [2] . The present study conducted with enzymatic hydrolysis of microwave alkali pretreated rice husk biomass by crude enzymes produced in-house by Aspergillus heteromophus. Enzymatic hydro- lyzate was fermented by Saccharomyces cerevisiae, Scheffersomyces stipitis and their co-culture for ethanol production.

2. Methods

2.1. Material and microorganism

Microwave alkali pretreated rice husk were used for present study [8] . Yeast S. cerevisiae MTCC 174 was purchased from Insti- tute of Microbial Technology, Chandigarh (IMTEC), India and S. sti- pitis NCIM No. 3497 from National Collection of Industrial Microorganism’’ (NCIM) National Chemical laboratory, Pune, Maharashtra, India. The yeast cultures was maintained on YEPD agar slants and sub-cultured weekly. Yeast for inoculation was grown in 1 L Erlenmeyer flasks filled with 400 mL of medium con- taining 50 g/L glucose, 20 g/L peptone and 10 g/L yeast extract. After incubation at 30 C and 150 rpm for 48 h, the cell suspension was aseptically collected by centrifugation (7200 g; 10 min) at 4 C and suspended in 0.9% NaCl for further use.

2.2. Enzymatic hydrolysis

Enzymatic hydrolysis was done by crude unprocessed enzyme [9]. Enzymatic hydrolysis of microwave alkali pretreated rice husk (50 g) was carried out in 3 L bioreactor (BioAge, Mohali, India) (equipped with agitator for stirring and temperature controller) containing 1.5 L citrate buffer (50 mM, pH 5.0 ± 0.2, 50 ± 0.5 C) at 100 rpm. The cellulosic substrate was soaked in citrate buffer for 2 h before adding enzymes. Sodium azide was also added at a concentration of 0.005% to restrict any microbial growth during the course of enzymatic hydrolysis. The substrate soaked in citrate

buffer was supplemented with crude enzyme (activities of exoglu- canase 8.2 FPU/g, endoglucanase 198.2 IU/g, xylanase 134.3 IU/g and b-glucasidase 147.8 IU/g [8]). Samples were withdrawn at various intervals, centrifuged and supernatant analyzed for total reducing sugar released. The hydrolyzate was concentrated by evaporation in rotatory evaporator to reducing sugar content of 10 g/L, 25 g/L, 50 g/L and 70 g/L.

2.3. Fermentation

Concentrated enzymatic hydrolyzate of different sugar concen- tration was used for ethanol production. The hydrolyzate was inoc- ulated with 2% v/v of a 48 h old seed culture of S. cerevisiae, S. stipitis and co-culture (1:1) of both yeast. Incubation was carried out in stoppered flasks at room temperature (28 ± 2 C) without agitation. 5 ml samples were withdrawn after 36 h (optimized fer- mentation time) [9] , centrifuged for 10 min at 4 C at 10,000 rpm and supernatant was analyzed for ethanol and residual reducing sugar contents.

2.4. Analytical methods

Total reducing sugar in enzymatic hydrolyzate of biomass was estimated by DNS method [12] . The enzymatic hydrolyzate was concentrated by the methods of Dehkhoda et al. [13] . The estima- tion of ethanol was done by Spectrophotometer [14] .

2.5. Fermentation parameters

The ethanol volumetric productivity (Q p ) (g p /l/h) was calculated as the ratio of ethanol concentration (g/L) at the end of the run to the fermentation time (t, h). The yield of ethanol (Y p/s ) to con- sumed sugar (g p /g s ) was defined as ratio of ethanol concentration to the sugar consumption (S o –S f , S 0 initial sugar concentration and S f final sugar concentration). Sugar conversion (%) calculated as a ratio of sugar consumption to the initial sugar concentration. The efficiency of sugar conversion to ethanol (g, %) has been esti- mated by the ratio of ethanol yield to theoretical value of ethanol yield (0.51 g/g).

3. Results and discussion

3.1. Enzymatic hydrolysis and its concentration

Pretreated rice husk used in present study contains 46% cellu- lose and 20% hemicelluloses [8] . The sugar content production depends on the enzyme used for hydrolysis. The data of the reduc- ing sugar concentration resulted from the enzyme hydrolysis of the alkali pre-treated rice husk using hydrolytic enzymes from A. heteromorphus was shown in Fig. 1 Enzymatic hydrolysis using

16 14 12 10 8 6 4 2 0 12 24 48 72 96 120
16
14
12
10
8
6
4
2
0
12
24
48
72
96
120
Reducing sugar relaesed (g/L)

Incubation time (h)

Fig. 1. Reducing sugar production during enzymatic hydrolysis over time.

A. Singh et al. / Fuel 116 (2014) 699–702

701

cellulases does not generate inhibitors like in acid hydrolysis and the enzymes are very specific for cellulose [7] . Maximum reducing sugar 13.5 g/L was obtained at 72 h after that decrease observed. The microwave pretreated biomass with alkali has a lower degree of crystallinity which enhances the enzymatic hydrolysis and hence facilitates the conversion of the cellulose into reducing sug- ars [8] . This higher increase of reducing sugars in alkali pretreated substrates may also be attributed to the release of lignin moieties during alkali treatment which in turn enhanced accessibility of cel- lulose to the enzymes [15] . Saha et al. [16] studied enzymatic sac- charification of alkaline peroxide pretreated rice hulls and found sugar yield 428 ± 12 mg/g (90% yield). Saha et al. [17] studied enzy- matic saccharification of lime pretreated rice hulls and found the maximum yield of monomeric sugars 154 ± 71mg g 1 (32% yield). Concentration of rice husk hydrolyzate was carried out to increase the sugar concentration for ethanol fermentation. Concentration of rice husk hydrolyzate was carried out by rotatory evaporator (vac- uum distillation unit) from 13.5 g/L to 70 g/L. The hydrolyzate was evaporated to with the main purpose of increasing the sugar con- tent [18] . The concentrated hydrolyzate was used for ethanol pro- duction with initial reducing sugar concentrations of 10 g/L, 25 g/L,

50 g/L and 70 g/L.

3.2. Ethanol production by mono-culture of yeast

Glucose and xylose are two main dominant sugars in lignocellu- losic hydrolyzate. 10–70 g/L reducing sugar concentration was used for fermentation by S. cerevisiae, S. stipitis and co-culture of both for ethanol production. The sugar consumptions seemed to depend on the yeasts’ abilities to utilize the different sugars in the enzymatic hydrolyzates [2] . Ethanol production by S. cerevisiae with variation of reducing sugar concentration was shown in Ta- ble 1 . Hexose and pentose sugars are the primary reactant in yeast metabolism. When reducing sugar concentration increased from

10 to 25 g/L, ethanol conc. enhanced by 57%, and when sugar conc.

increased from 25 to 50 g/L, ethanol enhanced by 56% and when sugar concentration increased from 50 to 70 g/L then ethanol conc. increased by 42 %. Under fermentative condition, the rate of etha- nol production is related to the available sugar concentration [19,20] . At very low substrate concentration, the yeast starved and productivity decreases [21] . An important secondary effect of higher sugar content is catabolite repression of the oxidative path-

ways [22] . Ethanol yield to consumed sugar was shown in Table 1 and highest yield was obtained with reducing sugar level 50 g/L. The result of ethanol yield of this study was in agreement with pre- vious report on fermentation of lignocellulosic hydrolyzates using yeast. Meghawati et al. [4] studied maximum ethanol concentra- tion 2.63% (v/v) indicating 92.18% ethanol yield from rice husk using Baker’s yeast. Latif and Rajoka [23] conducted simultaneous and saccharification and fermentation of corn cobs to produce eth- anol with yield of 0.42 g/g (2.1% v/v and 82.20 % ethanol yield)

using S. cerevisiae. Fermentation of glucose–xylose mixtures of lignocellulosic biomass using S. cerevisiae by Govindaswamy and Vane [24] produced ethanol yield of 0.49 g/g (2.45% v/v and 95.89% ethanol yield). Ethanol yield obtained from Lantana camara hydrolyzate (34.75 ± 1.54 g/L sugars) using yeast was 17.7 ± 0.96 g/L (±1.87% v/v and ± 93.93% ethanol yield) [25] . Saha et al. [16] studied enzymatic saccharification of rice hulls and found ethanol concentration was 8.0 ± 0.2 g/L with a yield of 0.20 g/g hulls in the case of simultaneous saccharification and fermentation by the E. coli strain. Xylose fermentation has been identified as an important factor for economically viable bioethanol production due to the large pro- portion of xylose in lignocellulosic material [26] . The yeasts used for industrial fermentations ( Saccharomyces sp.) are not capable of fermenting xylose. The successful fermentation of xylose would increase the ethanol production [1] . So, S. stipitis used for the production of ethanol from lignocellulosic hydrolyzate. Table 2 showed that ethanol production from rice husk hydrolyzate by S. stipitis . Table 2 shows that when reducing sugar concentration increased from 10 to 25 g/L, ethanol conc. enhanced by 60%, and when sugar conc. increased from 25 to 50 g/L, ethanol enhanced by 57% and when sugar concentration increased from 50 to 70 g/L then ethanol conc. increased by 41 %. Ethanol yield to consumed sugar was shown in Table 2 and highest yield was obtained with reducing sugar level 50 g/L.

3.3. Ethanol production by co-culture of yeast

Co-culture fermentation for ethanol production from lignocel- lulosic biomass can increase ethanol yield and production rate, shorten fermentation time, and reduce process costs, and it is a promising technology although immature [1,2] . According to a US Department of Energy (DOE) study, co-culture bioconversion is a very plausible and potentially high-payoff opportunity for eth- anol production [27] . Table 3 shows that ethanol production from rice husk hydrolyzate by co-culture of S. cerevisiae and S. stipitis. Table 3 showed that when reducing sugar concentration increased from 10 to 25 g/L, ethanol conc. enhanced by 54.5%, and when

Table 2 Fermentation parameters obtained in fermentation of microwave alkali pretreated rice husk hydrolyzate with S. stipitis.

Initial

Residual

Sugar

Ethanol

Qp

Yp/

Efficiency of

sugar

sugar

consumption

(g

p /l)

(g

p /

s

sugar

(g

s /l)

(g

s /l)

(%)

l/h)

(g

p /

conversion

 

g

s )

to ethanol

 

(%)

10

5

50

1.2 ± 0.1

0.03

0.24

47.6

25

14

44

3.0 ± 0.5

0.08

0.27

52.9

50

30

40

7.1 ± 0.9

0.20

0.36

70.5

70

36

48.5

12.2 ± 1.2

0.36

0.35

68.6

Time 36 h.

Table 1 Fermentation parameters obtained in fermentation of microwave alkali pretreated rice husk hydrolyzate with S. cerevisiae.

Initial

Residual

Sugar

Ethanol

Qp

Yp/

Efficiency of

sugar

sugar

consumption

(g

p /l)

(g

p /

s

sugar

(g

s /l)

(g

s /l)

(%)

l/h)

(g

p /

conversion

 

g

s )

to ethanol

 

(%)

10

5

50

1.5 ± 0.1

0.04

0.3

58.8

25

15

40

3.5 ± 0.5

0.10

0.35

68.6

50

30

40

8.1 ± 1.1

0.22

0.40

78.4

70

34

51.4

14.0 ± 1.3

0.36

0.39

76.5

Time 36 h.

Table 3 Fermentation parameters obtained in fermentation of microwave alkali pretreated rice husk hydrolyzate with co-culture of S. cerevisiae and S. stipitis.

Initial

Residual

Sugar

Ethanol

Qp

Yp/

Efficiency of

sugar

sugar

consumption

(g

p /l)

(g

p /

s

sugar

(g

s /l)

(g

s /l)

(%)

l/h)

(g

p /

conversion

 

g

s )

to ethanol

 

(%)

10

2.5

75

2.5 ± 0.1

0.06

0.33

64.7

25

10

60

5.5 ± 0.5

0.15

0.36

70.5

50

20

60

12.6 ± 1.1

0.31

0.42

82.3

70

18

74.3

20.8 ± 1.5

0.54

0.40

78.4

Time 36 h.

702

A. Singh et al. / Fuel 116 (2014) 699–702

sugar conc. increased from 25 to 50 g/L, ethanol enhanced by 56.3% and when sugar concentration increased from 50 to 70 g/L then ethanol conc. increased by 39%. Ethanol yield to consumed sugar was shown in Table 3 and highest yield was obtained with reduc- ing sugar level 50 g/L. Kordowaska-Wiater et al. [28] studied fer- mentation of glucose/xylose mixture by co-culture of P. stipitis CCY39502 and S. cerevisiae V 30 under batch fermentation ethanol yield 0.39 g/g and ethanol productivity 0.39 g/L/h. Rouhollah et al. [29] studied fermentation of glucose/ xylose mixture by co-culture of P. stipitis CCUG18492 and S. cerevisiae under batch fermentation and obtained ethanol productivity 0.41 g/L/h. Tanig- uchi et al. [30] found ethanol productivity 0.39 g/L/h under batch fermentation by co-culture of P. stipitis CBS5773 and S. cerevisiae No. 7 using glucose/ xylose mixture. Taniguchi et al. [31] reported ethanol production by co-culture of P. stipitis and a respiratory- deficient mutant of S. cerevisiae of variable concentrations in synthetic medium containing glucose and xylose under batch fermentation and found ethanol productivity of 0.50 g/L/h. Co-culture fermentation provides the opportunity to achieve simultaneous conversion of glucose and xylose, maximize sub- strate utilization rate, increase ethanol yield and production rate, and reduce process costs [2] .

4. Conclusions

Crude unprocessed on-site produced enzymes were used for hydrolysis of microwave alkali pretreated rice husk. The reducing sugar produced during hydrolysis were concentrated and used for ethanol production by S. cerevisiae and S. stipitis and their co- culture. Highest ethanol production with co-culture was 20.8 g/L and co-culture of S. cerevisiae and S. stipitis produced 32% more eth- anol than S. cerevisiae alone and 41% more ethanol than S. stipitis alone. Rice husk is an abundant agricultural residue with low commercial value, can be used for ethanol production.

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