Enhanced mannan-derived fermentable sugars of palm kernel cake by mannanase-catalyzed hydrolysis for production of biobutanol

Accepted Manuscript Enhanced mannan-derived fermentable sugars of palm kernel cake by mannanase-catalyzed hydrolysis for production of biobutanol Hafiza Shukor, Peyman Abdeshaihian, Najeeb Kaid Nasser Al-Shorgani, Aidil Abdul Hamid, Norliza A. Rahman, Mohd Sahaid Kalil PII: DOI: Reference:

S0960-8524(16)30901-4 http://dx.doi.org/10.1016/j.biortech.2016.06.084 BITE 16706

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

16 April 2016 21 June 2016 22 June 2016

Please cite this article as: Shukor, H., Abdeshaihian, P., Al-Shorgani, N.K.N., Hamid, A.A., Rahman, N.A., Kalil, M.S., Enhanced mannan-derived fermentable sugars of palm kernel cake by mannanase-catalyzed hydrolysis for production of biobutanol, Bioresource Technology (2016), doi: http://dx.doi.org/10.1016/j.biortech.2016.06.084

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1

Enhanced mannan-derived fermentable sugars of palm kernel cake by mannanase-catalyzed hydrolysis for production of biobutanol

Hafiza Shukor a,b, Peyman Abdeshaihian c, Najeeb Kaid Nasser Al-Shorganid, Aidil Abdul Hamide, Norliza A. Rahman a, Mohd Sahaid Kalil a,* a

Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia b

School of Bioprocess Engineering, Universiti Malaysia Perlis, Kompleks Pusat Pengajian Jejawi 3, 02600 Arau, Perlis, Malaysia c

Department of Microbiology, Masjed Soleyman Branch, Islamic Azad University, Masjed Soleyman, Iran d

Department of Applied Microbiology, Faculty of Applied Sciences, Taiz University, 6803 Taiz, Yemen e

School of Biosciences and Biotechnology, Faculty of Sciences and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia Email address of authors Hafiza Shukor: [email protected] Peyman Abdeshaihian: [email protected] Mohd Sahaid Kalil: [email protected] *Corresponding author Full name: Mohd Sahaid Kalil Tel: +60193345042 E-mail: [email protected] Address: Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia

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1

Abstract

2 3

Catalytic depolymerization of mannan composition of palm kernel cake (PKC) by mannanase

4

was optimized to enhance the release of mannan-derived monomeric sugars for further

5

application in acetone-butanol-ethanol (ABE) fermentation. Efficiency of enzymatic hydrolysis

6

of PKC was studied by evaluating effects of PKC concentration, mannanase loading, hydrolysis

7

pH value, reaction temperature and hydrolysis time on production of fermentable sugars using

8

one-way analysis of variance (ANOVA). The ANOVA results revealed that all factors studied

9

had highly significant effects on total sugar liberated (P<0.01). The optimum conditions for PKC

10

hydrolysis were 20% (w/v) PKC concentration, 5% (w/w) mannanase loading, hydrolysis pH

11

4.5, 45 oC temperature and 72 h hydrolysis time. Enzymatic experiments in optimum conditions

12

revealed total fermentable sugars of 71.54 ± 2.54 g/L were produced including 67.47 ± 2.51 g/L

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mannose and 2.94 ± 0.03 g/L glucose. ABE fermentation of sugar hydrolysate by Clostridium

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saccharoperbutylacetonicum N1-4 resulted in 3.27 ±1.003 g/L biobutanol.

15 16 17

Keywords

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Palm kernel cake; Enzymatic hydrolysis; Mannanase; Mannan-derived monomeric sugar;

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Biobutanol production

20 21 22

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1. Introduction

24

In recent years, increasing utilization of oil-derived fuels has brought about environmental

25

concerns including air pollution and global warming which are due to the high emission of

26

carbon dioxide (CO2), nitrous oxide (N2O) and other greenhouse gases (Rahimnejad et al., 2015;

27

Williams et al., 2012 ). Biofuel is known as a green and renewable alternative to the fossil fuel

28

to alleviate climate change effects and to diminish dependency on oil-based transportation fuels

29

(Kumar et al., 2016; Medipally et al., 2015). Butanol is a bio-based fuel and an adaptable

30

chemical which can be applied for the synthesis of a variety of industrial products.

31

Acetone-Butanol-Ethanol (ABE) process for biobutanol production is hampered by a high cost of

32

raw materials, which in turn affects the economics and feasibility of butanol production process

33

(Guan et al., 2016).

34

Lignocellulosic biomass serves the most appropriate feedstock for fermentation derived

35

biofuels such as butanol, owing to its environmental abundance, the high quantity of sugar

36

composition and a low price (Sindhu et al., 2016). Various agriculturally based lignocellulosic

37

substances have been studied as a potential carbon source for biobutanol production. The

38

lignocellulosic structure of the agro-industrial residues are mostly composed of cellulose and

39

hemicellulose which are connected with lignin by covalent bonds (Bansal et al., 2012).

40

Oil palm is a main economic crop in tropical and subtropical regions which is largely

41

utilized for the production of vegetable oil. Malaysia is one of the leading countries in the

42

production of palm oil in the world. It has been reported that in 2015, a quantity of 19,961,581

43

tonnes of crude palm oil was produced in Malaysia (MPOB, 2016).

44

Palm kernel cake (PKC) is one of the main waste products of palm oil industry which is

45

obtained after pressing and oil extraction process from palm kernels. According to the statistics

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presented by Malaysia Palm Oil Board (MPOB), 2,519,990 tones of PKC was produced from

47

palm oil industry of Malaysia in 2015 (MPOB, 2016) indicating that PKC is referred as a

48

renewable biomass feedstock for the production of bio-based chemicals. PKC contains a

49

relative high amount of carbohydrate which represents a potential raw material for biorefinery

50

system and industrial biochemical processes. Biochemical studies on PKC composition have

51

revealed that carbohydrate content of PKC is comprised of a high quantity of mannan. In this

52

regard, it has been estimated that mannan constitutes 57.8% of hemicellulose content of PKC

53

composition (Azman et al., 2016; Cerveró et al., 2010).

54

Mannan is known as an important hemicellulose in PKC which is mainly found as a

55

linear (1

56

content of PKC is linked to galactose by (1

57

galactomannan (Cerveró et al., 2010). Mannanase , a hemicellulolytic enzyme, is categorized

58

under the glycosyl hydrolase family 5, 26 and 113 that hydrolyzes the mannan backbone by

59

random cleavage of β-mannosidic linkages to release mannobiose units with different quantities

60

of other manno-oligosaccharides (Eom et al., 2016; Wang et al., 2016).

61

4) linked β-D-mannopyranose backbone. Moreover, a low quantity of mannan 6) linked α-D-galactopyranose to form

Enzymatic saccharification of lignocellulosic substances is a key approach for the

62

production of fermentable sugars for further utilization in biofuel production. Enzymatic

63

hydrolysis entails the conversion of carbohydrate polymers into monomeric sugars. The

64

efficiency of enzymatic degradation of polysaccharides can be affected by different factors

65

including substrate concentration, enzyme loading, pH of hydrolysis reaction and incubation

66

temperature of enzymatic hydrolysis (Geng et al., 2015; Sun and Cheng, 2002). In order to attain

67

the highest effect of enzymatic hydrolysis on carbohydrate substances, the optimization of

68

reactor conditions is necessary. In this view, when enzyme hydrolysis is not performed under

5 69

optimum conditions, elevated enzyme preparations may be consumed compared to that in

70

optimized conditions, which in turn influences the overall cost of the hydrolysis process for the

71

release of fermentable sugars. Hence, the optimization of enzymatic hydrolysis of cellulosic

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biomass could be of economic interest for researchers to produce biofuel from biomass

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feedstocks in an economically viable process.

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However, little work has been carried out to hydrolyze mannan content of PKC to release

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mannan-derived

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depolymerization of mannan content of PKC by mannanase enzyme for biobutanol production in

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ABE process is very limited.

78

monomeric sugars.

Furthermore,

the current

knowledge

about

the

Considering PKC as a potential mannan rich source and the high abundance of PKC with

79

a low price in Malaysia, the current study was performed to utilize PKC for the extraction of

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mannan-derived reducing sugars by mannanase-catalyzed hydrolysis process. The operating

81

conditions of enzyme reaction were optimized to achieve the highest amount of fermentable

82

sugars which subsequently were utilized for the production of butanol by Clostridium

83

saccharoperbutylacetonicum N1-4 in ABE fermentation process.

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2. Materials and methods

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2.1. PKC preparation and enzymatic hydrolysis

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PKC was utilized as mannan-based substrate for enzymatic hydrolysis. PKC was obtained from

87

FELDA Pasir Gudang, Johor, Malaysia. PKC was ground and passed through a 600 µm mesh

88

sieve to obtain fine and uniform PKC particles. Enzymatic hydrolysis of PKC was carried out in

89

a 250 mL Schoot Duran bottle using mannanase enzyme (Habio Bioengineering Co, Ltd).

90

Enzyme solution was prepared by dissolving defined amount of solid form of mannanse in

6 91

0.2mM sodium acetate buffer (pH 4). Different quantities of PKC were dissolved in enzyme

92

solution to make a working volume of 200 ml. The effect of different reaction factors on PKC

93

hydrolysis such as PKC concentration, enzyme loading, reaction temperature, pH value of

94

enzymatic reaction and hydrolysis reaction time were studied. Table 1 shows the range of levels

95

used for factors considered in enzymatic hydrolysis of PKC by mannanase. The effect of factors

96

tested on mannanase hydrolysis of PKC was studied by measuring the amount of fermentable

97

sugars released. All experiments of enzymatic hydrolysis of PKC were conducted at an agitation

98

rate of 170 rpm.

99

2.2. Microorganism preparation

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Microbial strain Clostridium saccharoperbutylacetonicum N1-4 was obtained from

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Biotechnology Lab in the Chemical and Process Engineering Department, Universiti

102

Kebangsaan Malaysia (UKM). C. saccharoperbutylacetonicum N1-4 is one of the most

103

important butanol producing bacteria with high efficiency for the synthesis of butanol using

104

sugar-based substrates. In this regard, this strain has successfully been employed for the efficient

105

production of butanol in ABE fermentation using various sugar sources and agro-industrial waste

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biomass (Al-Shorgani et al., 2015; Chen et al., 2013; Noguchi et al., 2013) . For inoculums

107

preparation, a volume of 1 mL stock culture was transferred into potato glucose (PG) medium

108

with subsequent heat shock and cool shock in which culture was heated at boiling water (100 oC)

109

for 1 min and immediately placed at cold water for 30 second to activate spore form of clostridial

110

cells. Seed culture was incubated anaerobically for 1-2 days at 30°C. Inoculum preparation was

111

carried out by transferring 10% (v/v) of the seed culture into tryptone–yeast extract–acetate

112

(TYA) medium and incubating culture at 30°C for 18 h under anaerobic conditions.

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In this regard, 10% (v/v) microbial inoculum has been found as a suitable inoculation of C.

114

saccharoperbutylacetonicum N1-4 for butanol production in ABE process (Al-Shorgani et al.,

115

2015; Noguchi et al., 2013 ) . PG medium consisted of following components (g/L): potato, 150;

116

glucose, 10; CaCO3, 3 and (NH4)2SO4, 0.5. Moreover, TYA medium was composed of chemicals

117

are as follows (g/L): tryptone, 6; yeast extract, 2; ammonium acetate, 3; KH2PO4, 0.5;

118

MgSO4.7H2O, 0.3, and FeSO4.7H2O, 0.01 (Al-Shorgani et al., 2015) .

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2.3. ABE fermentation

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ABE fermentation for biobutanol production was conducted in a 100 mL serum bottle with a

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working volume of 50 mL. PKC hydrolysate liberated from enzymatic hydrolysis reaction was

123

used as a substrate for biobutanol production by C. saccharoperbutylacetonicum N1-4. ABE

124

fermentation medium was prepared by an addition of P2 medium components into 1 litter of

125

PKC hydrolysate, followed by sterilization of medium at 121°C for 15 min using an autoclave.

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The chemical composition of P2 medium was as follows (g/L): yeast extract, 1; MnSO4.4H2O,

127

0.01; K2HPO4,0.75 ; MgSO4.7H2O, 0.4; KH2PO4,0.75 ; FeSO4.7H2O, 0.01; NH4NO3, 2;

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Cysteine, 0.5; and 1ml solution containing 1mg/L 4-aminobenzoic acid and 80µg/L biotin.

129

Subsequently, anaerobic conditions inside the bottles were provided by sparging oxygen-free

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nitrogen to the void space of the bottles for 10 min using a membrane air filter (0.22 µm pore

131

size). All air filter and air tube set were sterilized by the autoclave prior to utilization.ABE

132

fermentation medium was inoculated by transferring 10% (v/v) of fresh inoculum of

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C.saccharoperbutylacetonicum N1-4. Inoculated culture medium was incubated at the

134

temperature of 30oC for 240 h. Initial pH of ABE fermentation medium was adjusted to 6.5 using

135

a solution of 6 M NaOH.

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2.4. Analytical methods

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Sugars concentrations were measured by a high performance liquid chromatography (HPLC

138

12000 Series, Agilent technologies, Palo Alto, CA, USA) using Rezex RPM-Monosaccharide

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Pb+2 (8%) column (300 × 7.8 mm ID) with a refractive index detector (RID) at 60oC and a flow

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rate 0.5mL/min using a water as a mobile phase. Concentrations of solvents (acetone, butanol

141

and ethanol) were measured using a gas chromatograph (7890A GC-System; Agilent

142

Technologies, Palo Alto, CA, USA) equipped with a flame ionization detector and a 30-

143

mcapillary column (Equity 1; 30 m×0.32 mm×1.0µm film thickness; Supelco, Bellefonate, PA,

144

USA). The oven temperature was programmed to increase from 40°C to 130°C at a rate of

145

8°C/min. The injector and detector temperatures were set at 250°C and 280°C, respectively.

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Helium, as the carrier gas, was set at a flow rate of 1.5mL/min. Biochemical composition of PKC

147

such as protein, fat, carbohydrate, and nutrient was measured by UNIPEQ Sdn. Bhd. (870956-

148

D), Blok A, M-MTDC Pusat Teknologi, UKM.

149 150

2.5. Statistical analysis

151 152

The experimental data were analyzed using Statistical Package for Social Sciences (SPSS)

153

analytical tool based on one-way analysis of variance (ANOVA) to determine the significant

154

effects of the factors tested on the total fermentable sugar production. Turkey’s Post Hoc tests

155

were carried out to know the multiple comparisons and interaction between the different levels of

156

factors under consideration.

157 158 159

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3. Results and discussion

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3.1. Analysis of PKC composition

162 163

Compositional analysis for PKC shows that this substrate contains a high amount of

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carbohydrate compared to crude protein and fat. Obviously, the carbohydrate content formed

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60.3% (w/w) of PKC particles, followed by crude protein and fat representing 22% and 4.6%

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(w/w), respectively. Similar to this study, Cerveró et al. (2010) found that carbohydrate

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constitutes 50% of PKC. High carbohydrates content is a favorable property for PKC to be used

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as a promising carbohydrate rich feedstock in the production of fermentable sugars with an

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application in biobutanol synthesis.

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On other hand, the protein composition of PKC was relatively high (22g/100g PKC)

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which make it a potential substrate for animal feed utilization. PKC also contained certain types

172

of nutrient and minerals such as manganese (161.56 mg/kg PKC), phosphorus (0.84 mg/kg

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PKC), calcium (533.52 mg/kg PKC), iron (310.35 mg/kg PKC), potassium (6190.86 mg/kg

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PKC) and sodium (382.52 mg/kg PKC). The presence of heavy metals such as arsenic

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(0.018mg/kg PKC) and cadmium (0.024 mg/kg PKC) at low concentrations seems not to give

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deleterious effects on the production of biobutanol.

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3.2. Effect of reaction conditions on hydrolysis of PKC using mannanase enzyme

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3.2.1. Effect of PKC concentration

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Substrate concentration plays a crucial role in enzymatic hydrolysis so that it affects the

181

efficiency of enzymatic depolymerization of many lignocellulosic materials. In order to study the

182

effect of PKC concentration on enzymatic saccharification of PKC by mannanase, hydrolysis

183

experiments were carried out using different PKC concentrations ranging from 5 to 20% (w/v)

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with the fixed mannanase enzyme loading of 5% ( w/wpkc) at 45oC and pH 4.5 for 72h. Because

185

of the insolubility nature of lignocellulose, this heterogeneous hydrolysis reaction was carried

186

out directly by physical contact between the enzyme and the substrate. As depicted in Fig. 1, it is

187

evident that PKC concentration proportionally affected the total fermentable sugar released. As

188

can be found, the increment in PKC concentration resulted in a direct increase in the production

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of simple sugars, which indicated that no substrate inhibition effect on enzyme hydrolysis

190

reaction occurred. In this regard, it has been noted that the extent of substrate inhibition depends

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on the ratio of the total amount of substrate to enzyme so that increasing substrate concentration

192

to a certain value may cause substrate inhibition, which in turn leads to the lower hydrolysis

193

efficiency (Sun and Cheng, 2002).

194

As can be seen from Fig. 1, the concentration of mannose was enhanced gradually almost

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two times from 4.53 ± 0.45 g/L to 9.56 ± 0.09 g/L when PKC concentration increased from 5%

196

to 10%. Obviously, a rise in PKC concentration from 10% to 15% and 20% improved the

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quantity of mannose liberated to a lower extent. As can be found, the elevated PKC

198

concentration had no notable effects on glucose concentration. It can be related to the fact that

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mannanase used in the enzymatic hydrolysis of PKC was aimed at depolymerizing mannan

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structure of PKC to release mannose to a large extent. In this context, glucose could be liberated

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from galactomannan component in PKC polysaccharide which includes a linear mannan

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backbone with (1-6) linked α-D-galactopyranose side groups (Cerveró et al., 2010). A quantity

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of 16.07 ± 3.54 g/L of mannose and 18.85 ± 4.07 g/L of total sugars were liberated when a

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maximum of 20% (w/v) PKC concentration was used in enzyme hydrolysis by mannanase.

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It is noteworthy that too high increase in substrate concentration will lead to limited

206

mixing and the reduction in homogeneity of hydrolysis reactor. This causes insufficient mass and

11 207

heat transfer, which substantially lowers the efficiency of hydrolysis ( Geng et al., 2015). The

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one-way ANOVA analysis of results for the effect of PKC concentrations on mannanase

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hydrolysis showed that PKC concentration had a significant effect (P<0.05) on total fermentable

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sugar production (Table 2). In this view, Sun and Cheng (2002) have indicated that a rise in

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substrate concentration normally results in the enhancement of the release of monomeric sugars

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during hydrolysis time because of increment in the higher hydrolysing substrate.

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3.2.2. Effect of mannanase loading

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The cost of enzyme is a critical factor in enzymatic hydrolysis of lignocellulosic biomass and it

215

contributes to the total cost of biofuel production so that the optimization of enzyme loading

216

leads to reduce biobutanol product cost (Newman et al., 2013). In order to evaluate the effect of

217

mannanase loading, different enzymatic hydrolysis experiments were performed using a fixed

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concentration of PKC (5% w/v) dissolved in enzyme solution (pH 4.5), at the reaction

219

temperature of 45oC for 72 h of hydrolysis reaction time. The effect of mannanase loading was

220

studied in the range of 0.5% to 10% (w/w of PKC). Fig. 2 illustrates the results obtained for

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mannanas loading experiment. As is evident, a similar trend in the enhancement of mannose,

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glucose and total sugar concentration occurred when mannanase loading for PKC hydrolysis

223

increased from 0.5 to 10% so that sugar concentrations increased up to 9.39 ± 0.33 g/L of

224

mannose and 11.62 ± 0.42 g/L of total sugar when 10% (w/w) mannanase loading was utilized.

225

This result may be attributed to the fact that the elevated mannanase loading could

226

increase the enzyme binding capacity to mannan with the higher diffusion of mannanase in the

227

solution, which in turn enhanced the conversion of mannan to mannose. In an attempt for

228

enzymatic hydrolysis of heat treated Eucalyptus globulus wood, the utilization of cellulolytic

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enzymes with enzyme loading of 20 g/g substrate at 48.5 oC for 96 h resulted in the production

230

of 35.1 g/L glucose as the main monomeric sugar hydrolysate (Romani et al., 2010).

231

Similar to the effect of PKC concentration on enzymatic hydrolysis, the results of

232

statistical analysis revealed that generally, mannanase loading had a significant effect on the

233

improvement of the total sugar concentration (P<0.01) (Table 2). On the other hand, multiple

234

comparison result by SPSS showed that the production of the fermentable sugars at enzyme

235

loading of 5% (w/w) had no a significant difference (P=0.86) to that when 10% (w/w)

236

mannanase loading was used in which a total sugar of 11.31 g/L and 11.62 g/L were produced,

237

respectively (Fig. 2). Obviously, higher enzyme loading resulted in the enhancement of the

238

fermentable sugars, however, it should be taken into consideration that the cost of enzyme

239

preparation is a bottleneck in the hydrolysis process and it is essential to minimize enzyme

240

loading to attain economically viable hydrolysis process. Thus, the mannanase loading of 5%

241

was selected as the optimum enzyme loading for PKC hydrolysis.

242

3.2.3. Effect of pH value

243

Effect of pH of reaction medium on the enzymatic hydrolysis of PKC by mannanase was studied

244

using sodium acetate buffer at four different pH values, ranging from pH 4 to pH 5.5. For this

245

experiment, the enzymatic hydrolysis was carried out using 5% (w/v) PKC concentration with an

246

enzyme load of 0.5% (w/w PKC) at a temperature of 45oC for 72 h of enzymatic saccharification

247

time. Fig. 3 depicts the experimental results obtained for enzymatic hydrolysis of PKC at pH

248

values tested. It is obvious that increasing pH value of hydrolysis medium from 4 to 4.5

249

decisively enhanced the quantity of fermentable sugars to the maximum concentrations. As

250

shown in Fig. 3, the concentration of mannose was improved from 0.81 ± 0.00 g/L at pH 4 to the

13 251

highest level of 4.53 ± 0.45 g/L at pH 4.5 with the highest total sugar concentration of 6.47 ±

252

0.44 g/L.

253

Further increase in pH from 4.5 to 5.0 decreased the monomeric sugar production (Fig.

254

3). Further rise in pH value from 5 to 5.5, resulted in a low increase in the total sugar level from

255

5.95 ± 0.55 g/L to 6.19 ± 0.29 g/L which was still low compared to that at pH 4.5. The different

256

effect of pH values on the efficiency of enzyme reaction could be attributed to the fact that the

257

variations of pH value result in the changes of ionic form of active site, which leads to

258

denaturation of enzyme’s active site. This phenomenon affects the hydrolysis of substrate in

259

catalyzed reaction (Dhabhai et al., 2012). As can be observed from Table 2, the consideration of

260

one–way ANOVA result showed that the pH value of reaction medium had a significant effect

261

on the production of fermentable sugars using mannanase enzyme. The pH value of 4.5 appeared

262

to be the most suitable pH value for the mannanase-catalyzed hydrolysis of PKC. In this regard,

263

the studies on the enzymatic saccharification of lignocellulosic raw materials revealed that the

264

pH values of 4.8 was the optimum pH for enzymatic hydrolysis of cellulosic content of olive tree

265

waste (Cara et al., 2008).

266

3.2.4. Effect of reaction temperature

267

Temperature is an important factor which drastically affects enzyme hydrolysis process. In order

268

to study the effect of reaction temperature on enzyme hydrolysis of PKC, enzymatic experiments

269

were carried out at a varied temperature range of 40- 55°C. The trend of production of

270

monomeric sugars with the different temperature studied is presented in Fig. 4. As can be seen,

271

the total reducing sugar production was improved from 4.85 ± 0.75 g/L to a maximum

272

concentration of 6.47 ± 0.44 g/L when hydrolysis temperature increased from 40 to 45°C. The

273

increasing temperature from 40 to 45°C favored the hydrolysis of mannan content of PKC to

14 274

release maximum mannose concentration of 4.53 g/L, which consequently enhanced glucose

275

production up to 1.9 g/L. However, further increment in temperature from 45°C to 55oC reduced

276

the total sugar concentration to 2.03 ± 0.12 g/L.

277

The adverse effect of the elevated hydrolysis temperature on monomeric sugar release

278

could be attributed to temperature inactivation or thermal denaturation phenomenon in which a

279

rise in temperature higher than a critical temperature (in present case 45°C) could decrease the

280

efficiency of enzyme owing to enzyme denaturation, since high temperature tends to change the

281

protein folding (Dhabhai et al., 2012).

282

As shown in Fig. 4, in this study temperature of 45°C seemed to be the most appropriate

283

temperature for the mannanase enzyme hydrolysis of PKC. Statistical analysis using one-way

284

ANOVA analysis of the results showed that temperature of hydrolysis reaction had a significant

285

effect (P<0.01) on total sugar production from PKC (Table 2). In this context, it was noted that

286

the mannanase enzyme of Man5A which was obtained from Gloeophyllum trabeum CBS900.73

287

could retain its saccharolytic effect at temperature of 45°C, while it lost 50% of its initial activity

288

at 50 oC so that it could not tolerate temperature of 60 oC and lost its activity completely at this

289

temperature (Wang et al., 2016). The study on the enzyme hydrolysis by a wild-type mannanase,

290

namely Man-PrtAC3 showed that this enzyme was able to tolerated well the incubation

291

temperature range of 35 to 45 oC with a relatively high activity, however, the enzyme efficiency

292

decreased drastically as the incubation temperature was elevated from 45 to 70 oC (Eom et al.,

293

2016). The mild temperature determined for enzymatic hydrolysis of PKC (45°C) was consistent

294

with the optimum temperature reported by Öhgren et al. (2007) who performed enzymatic

295

saccharification of polysaccharide content of corn stover at the optimum hydrolysis temperature

296

of 45 oC. The studies conducted by Dhabhai et al. (2012) for the liberation of fermentable sugars

15 297

from pure cellulose showed that the utilization of cellulose-degrading enzymes in the hydrolysis

298

of substrates tested released the highest glucose sugar at an optimum temperature of 50 °C.

299 300

3.2.5. Effect of hydrolysis time

301

Hydrolysis time is known as one of the main factors that have a pivotal effect on enzyme

302

hydrolysis. In order to investigate the effect of the hydrolysis time on fermentable sugar

303

concentration during mannanase hydrolysis of PKC, the hydrolysis reaction was conducted for

304

96 h and samples were taken out every 24 h where enzymatic reaction was performed at 5%

305

(w/v) PKC, 0.5% (w/w) mannanase loading, 45oC temperature and pH medium of 4.5. The

306

fermentable sugar concentrations produced through the hydrolysis of PKC at different hydrolysis

307

time within 4 days (96 h) are illustrated in Fig. 5. Basically, the production of fermentable sugars

308

was enhanced when hydrolysis process proceeded. Mannose concentration increased almost two

309

folds from 24 h to 48 h with the mannose concentration of 1.09± 0.092 g/L and 2.87 g ± 0.272

310

g/L, respectively. Similar observation was found when reaction time continued from 48 h to 72 h

311

in which mannose was enhanced up to 4.53 ± 0.45 g/L.

312

As can be found, the maximum production of reducing sugars occurred at 72 h enzymatic

313

hydrolysis time with a total sugar production of 6.47 ± 0.44 g/L, mannose concentration of 4.53

314

± 0.45 g/L and glucose concentration of 1.90 ± 0.012 g/L. A further increment in hydrolysis time

315

up to 96 h had a deleterious effect on the release of glucose (1.19 g ± 0.056 g/L) and mannose

316

(3.75± 0.431 g/L) with a total sugar production of 4.98 ± 0.49 g/L. The analysis of one-way

317

ANOVA (Table 2) showed that the hydrolysis time had a significant effect on total sugar

318

production from PKC during mannanase enzyme hydrolysis (P<0.01). It is obvious that 72h of

319

hydrolysis is an optimum hydrolysis time for hydrolysis of PKC by mannanase. Similar

320

optimum hydrolysis reaction time (72 h) was reported in enzymatic hydrolysis process of a

16 321

number of cellulosic feedstocks such as olive tree waste, corn stover (Cara et al., 2008; Öhgren

322

et al., 2007). With regard to the effect of hydrolysis time on enzymatic saccharification of

323

mannan composition of PKC, the low hydrolysis time reveals an economical aspect for

324

fermentable sugar production since the lower reaction time makes hydrolysis reaction less energy

325

intensive process and reduces total energy costs.

326

3.3. Enzymatic hydrolysis of PKC by mannanase in optimum conditions

327

The experimental results obtained were used to determine optimum conditions for enzymatic

328

hydrolysis of PKC by mannanase. The optimum conditions determined were a PKC

329

concentration of 20% (w/v), mannanase loading of 5% (w/w), hydrolysis pH value of 4.5,

330

reaction temperature of 45 oC and hydrolysis time of 72 h.

331

In order to determine the amount of fermentable sugars released in optimum conditions

332

of mananase-catalyzed hydrolysis of PKC, a set of enzymatic hydrolysis experiments were

333

carried out in triplicate under optimum conditions determined. Experimental results revealed that

334

a total fermentable sugar of 71.54 ± 2.54 g/L was produced which included mostly mannose with

335

the concentration as high as 67.47 ± 2.51 g/L and glucose to a lower extent (2.94 ± 0.03 g/L).

336

The results obtained in this study showed that increasing temperature from 40 to 45 oC had a

337

favorable effect on the hydrolysis process of PKC and the liberation of sugar hydrolysate (Fig.

338

4). This could be related to the better solubility of substrate at higher incubation temperature,

339

resulting in higher hydrolysis efficiency (Ahirwar et al., 2016). Furthermore, the variations in the

340

hydrolysis of mannan fraction of PKC under different incubation temperature could be

341

associated to the effect of reaction temperature on the activity of mannanase during hydrolysis

342

process. It has been indicated that the optimum temperature of the majority of mannanases lies in

343

the range of 40-75 oC and only a small number of mannanases exhibit their hydrolytic effects at

17 344

temperature optima of ≥80 oC (Yang et al., 2015). It is postulated that mannanase loses its

345

activity at the temperatures higher than optimum point which could be attributed to the

346

irretrievable inactivation and conformational change of mannanase at higher temperatures

347

(Esmaeilipour et al., 2012; Wang et al., 2016). On the other hand, the pH value of enzyme

348

reaction had a notable effect on the mannanase efficiency in the degradation of mannan content

349

of PKC (Fig. 3). As different types of mannanase have an optimum pH value for their activities,

350

mannanases may exhibit different stability or susceptibility at varied pH values. It has been

351

found that the most reported mannanases have a optimum pH value in the range of 3-7.5 so that

352

they are inactive and unstable at highly acidic pH values (lower than 3) and alkaline conditions

353

(pH values higher than 8) (Chai et al., 2016; Yang et al., 2015). This phenomenon can be due to

354

the disruption of hydrogen and ionic bonds which are essential for maintaining the active

355

conformation of the hydrolytic enzyme (Esmaeilipour et al., 2012). It is evident from Fig. 5 that

356

there was a reciprocal correlation between hydrolysis time and the release of sugars in

357

mannanase-catalyzed saccharification which corroborated the fact that longer time of hydrolysis

358

of PKC led to rise mannanase efficiency to a large extent at the optimum hydrolysis reaction

359

time determined (72 h). However, too high reaction time reduced the liberation of fermentable

360

sugars. It could be attributed to the point that the activity and stability of the enzymes are the

361

function of hydrolysis time, as such, the hydrolytic enzymes lose their activity and stability as

362

hydrolysis proceeds (Ahirwar et al., 2016; Eom et al., 2016).

363

An attempt was made by Cerveró et al. (2010) to hydrolyze palm kernel cake to liberate

364

mannan-derived sugars for further utilization in ethanol production. It was found that a mixture

365

of two mannanase preparations (Gammanase–Mannaway mixture) could release a maximum

18 366

total monomeric sugar when the mannanase mixture was reacted with 2.3 mg/g PKC at 50 oC

367

and pH value of 5.4 for 96 h.

368

In the study fulfilled by Kumar et al. (2013) for the production of monomeric sugars from

369

cellulosic feedstocks, it was revealed that enzymatic saccharification of de-oiled jatropha by

370

cellulolytic enzymes using enzyme loading of 10% (v/v) led to liberate a total sugar quantity of

371

12.9 g/L. The optimization of a multi-enzyme mixture of seven cellulose degrading enzyme

372

preparations for the hydrolysis of steam-exploded corn stover led to release 15.5 g/L of glucose

373

(Zhou et al., 2009).

374

3.4. Biobutanol production from mannan–derived monomeric sugars of PKC

375

In this study monomeric sugar hydrolysate obtained from the enzymatic hydrolysis of PKC in

376

optimum conditions determined was utilized to produce biobutanol in ABE process. ABE

377

fermentation was conducted by C. saccharoperbutylacetonicum N1-4. It has been found that C.

378

saccharoperbutylacetonicum N1-4 is capable of fermenting various types of pentose and hexose

379

sugars originated from lignocellulosic materials (Al-Shorgani et al., 2015; Chen et al., 2013).

380

Thus, the batch ABE fermentation was carried at 30oC for 240 h. Fig. 6 depicts the sugar

381

consumption by clostridial cells and production of butanol from fermentable sugars obtained. It

382

is obvious that mannose and glucose were concurrently utilized by the strain C.

383

saccharoperbutylacetonicum N1-4. Although the concentration of mannose was higher than

384

glucose concentration, glucose was depleted fully at a shorter time. This can be attributed to the

385

fact that glucose has been found as the superior fermentable sugar for solvent producing

386

clostridial cells compared to other pentose and hexose sugars.

387

In an attempt to produce butanol from cellulosic substances, pretreated corn stover was

388

subjected to hydrolytic enzymes including cellulase, β-glucosidase and xylanase that resulted in

19 389

liberating a total sugar of 39 g/L. Subsequently, ABE fermentation of the released sugar

390

hydrolysate by Clostridium beijerinckii P260 revealed that at the end of ABE process all glucose

391

was consumed by clostridial cells, while 4.13 g/L xylose and 0.21 g/L galactose remained

392

unused in the culture medium (Qureshi et al., 2014).

393

Fig. 6 also illustrates biobutanol production from PKC hydrolysate. The production of

394

biobutanol was low during early ABE fermentation. As ABE process proceeded, butanol

395

concentration was drastically enhanced up to the maximum quantity of 3.27±1.003 g/L at 240h

396

ABE fermentation time. In this view, it has been observed that ABE fermentation is a biphasic

397

process in which two distinct phases, namely acidogenesis and solventogenesis occur

398

consecutively in microbial cells of biobutanol producing strains. In acideogenic phase, organic

399

acids such as acetic acid and butyric acid are synthesized where clostridial strains initiate cell

400

growth. In the next step, acideogenic phase switches to solventogenic phase so that solvents

401

(acetone, butanol and ethanol) are produced by reassimilation of organic acids produced (Amiri

402

et al., 2014). The highest yield and productivity of butanol was obtained at 72 h with values as

403

high as 0.071 ± 0.004 g/g sugar consumed and 0.026 ± 0.004 g/L/h, respectively, while

404

maximum butanol quantity (3.27±1.003 g/L) was produced at 240 h of ABE process. This

405

indicated that higher yield and productivity of biobutanol is favorable for economically viable

406

production of biobutanol with reducing the process time. It was observed that biobutanol

407

production by Clostridium beijerinckii BA101 from enzyme treated corn fiber using cellulose

408

degrading enzymes resulted in 6.5 g/L butanol with a yield and productivity of 0.26 g/g sugar

409

consumed and 0.09 g/L/h, respectively (Qureshi et al., 2008).

410

20 411

Table 3 enumerates the amount of total fermentable sugar liberated from various

412

cellulosic agricultural residues and similar substrate of palm kernel cake such as oil palm

413

decanter cake, palm kernel shell, palm pressed fibre and oil palm empty fruit branch with the

414

hydrolysis type applied. It is evident that the hydrolysis of PKC by mannanase resulted in the

415

production of a relatively high quantity of the total fermentable sugar compared to the total

416

reducing sugar released from the similar cellulosic feedstocks to PKC. This indicates that PKC

417

could serve as a potential lignocellulosic substance to provide a sustainable sugar rich source for

418

the production of butanol and other biofuels.

419

The variations in the butanol production obtained could be attributed to the various

420

agricultural residues utilized, different microorganisms inoculated, and varied culture conditions

421

applied in ABE process. It is worthy of note that the higher production of total fermentable sugar

422

from lignocellulosic biomass is of prime interest for biofuel synthesis. Hence, the utilization of

423

low enzyme preparations with liberating higher quantity of total sugar exhibits economically

424

feasible production of biofuels.

425 426

3.5 Economic evaluation of butanol production from PKC

427

The economic production of butanol could be evaluated by considering its financial viabilities,

428

and the up-stream and downstream processing costs. It has been reported that ABE-derived

429

butanol price could be varied from 0.59 $/L to 1.05 $/L (Okoli and Adams, 2014). Among the

430

parameters affecting butanol fermentation economy, the feedstock cost constitutes a substantial

431

percentage of butanol prices. According to Azman et al. (2016), the potential energy content of

432

PKC has been estimated as 1.36 MJ per kg of PKC with the average estimated price of 0.07 USD

433

for each MJ energy generated from one kg PKC, which indicates the potential generation of

434

butanol from PKC costs 2.40 USD / kg butanol, considering a lower heat value of 34.32 MJ per

21 435

kg of butanol. In this view, the reduction of the production costs concerning operational

436

variables, enzymatic saccharification and product recovery make production of butanol from

437

PKC more economically attractive.

438

4. Conclusion

439

PKC is a promising mannan rich bioresource that can be exploited for extraction of fermentable

440

sugars. Enzymatic hydrolysis of mannan content of PKC by mannanse was enhanced under

441

optimum conditions of hydrolysis in which PKC concentration, mannanase loading , hydrolysis

442

pH value, temperature and hydrolysis time of 20% (w/v), 5% (w/w), 4.5, 45 oC and 72 h,

443

respectively were utilized. Total fermentable sugar of 71.54 ± 2.54 g/L was produced in

444

optimum conditions. ABE fermentation of monomeric sugars by C.saccharoperbutylacetonicum

445

N1-4 led to 3.27±1.003 g/L biobutanol with 0.071 ± 0.004 g/g biobutanol yield and 0.026 ±

446

0.004 g/L/h biobutanol productivity.

447

Acknowledgements

448

This work was supported by the grant UKM-GUP-2013-037.

449

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450

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451

optimization and functional characterization of thermostable β-mannanase from Malbranchea

452

cinnamomea NFCCI 3724 and its applicability in mannotetraose (M 4 ) generation. J. Taiwan

453

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456

Yusoff, W.W.M., 2015. Process optimization of butanol production by Clostridium

457

saccharoperbutylacetonicum N1-4 (ATCC 13564) using palm oil mill effluent in acetone-

458

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459

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acetone, butanol, and ethanol production. Bioresour. Technol. 152, 450-456.

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4. Azman, N.F., Abdeshahian, P, Kadier, A., Shukor, H., Al-Shorgani, N.K.N, Hamid, A.A.,

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Kalil, M.S., 2016. Utilization of palm kernel cake as a renewable feedstock for fermentative

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Bioresour. Technol. 99, 5915–5922.

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hydrothermally pretreated Eucalyptus globulus wood. Bioresour. Technol. 101, 7806-8712.

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Pandey, A., 2016. Development of a combined pretreatment and hydrolysis strategy of rice straw

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33. Wang, C., Zhang , J., Wang, Y., Niu, C., Ma, R., Wang, Y., Bai, Y., Luo, H., Yao, B., 2016.

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Biochemical characterization of an acidophilic b-mannanase from Gloeophyllum trabeum

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CBS900.73 with significant transglycosylation activity and feed digesting ability. Food. Chem.

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197, 474-481.

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34. Yang, H., Shi, P., Lu, H., Wang, H., Luo, H., Huang, H., Yang, P., Yao, B., 2015. A

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thermophilic b-mannanase from Neosartorya fischeri P1 with broad pH stability and significant

566

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35. Zhou, J., Wang, Y-H., Chu, J., Luo, L-Z., Zhuang, Y-P., Zhang, S-L., 2009. Optimization of

569

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570

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571

Figure caption

572 573 574

Fig. 1 Effect of PKC concentration on fermentable sugar production in hydrolysis process by

575

mannanase

576

Fig. 2 Effect of mannanase loading on fermentable sugar production from PKC in hydrolysis

577

process

578

Fig. 3 Effect of pH value on fermentable sugar production from PKC in hydrolysis process by

579

mannanase

26 580

Fig. 4 Effect of reaction temperature on fermentable sugar production from PKC in hydrolysis

581

process by mannanase

582

Fig. 5 Effect of hydrolysis time on fermentable sugar production from PKC in hydrolysis process

583

by mannanase

584

Fig. 6 The profile of sugar consumption and biobutanol production by Clostridium

585

saccharoperbutylacetonicum N1-4 using mannan-dervide fermentable sugars obtained from PKC

586

hydrolysis by mannanase

587

588

589

27 590 20 18 16 Sugar (g/L)

14 12 10

Glucose

8

Mannose

6

Total sugar

4 2 0 5

10 15 PKC concentration (% w/v)

591 592 593 594 595

596

597

Fig. 1

20

28 598 599 600 14 12

Sugar (g/L)

10 8 Glucose 6

Mannose Total sugar

4 2 0 0.5

1 5 Enzyme loading (% w/w)

601 602 603 604 605

606

607

Fig. 2

10

29 608 609 7 6

Sugar (g/L)

5 4 Glucose 3

Mannose

2

Total sugar

1 0 4

4.5

5 pH value

610 611 612 613 614

615

616

Fig. 3

5.5

30 617 7 6 Sugar (g/L)

5 4 Glucose 3

Mannose

2

Total sugars

1 0 40

45 50 Temperature (Co)

618 619 620 621

622

623

Fig. 4

55

31 624 7 6

Sugar (g/L)

5 4 Glucose 3

Mannose

2

Total sugar

1 0 24

48 72 Hydrolysis time (h)

625 626 627 628

629

630

Fig. 5

96

32 631 632 633 634 Total Sugars Glucose Butanol Mannose

70 Sugars (g/L) Butanol (g/L)

60 50

635 636 637

40

638

30 20

639

10

640

0 0

24

48

72

96

120 144 168 192 216 240641 Time (h)

642 643 644 645

646

647

Fig. 6

33 648 649

Table 1 Experimental factor and their respective levels used for the hydrolysis of PKC by

650

mannanase Factors

651 652

653

654

Levels

PKC concentration, % (w/v)

5, 10, 15, 20

Mannanase loading, % (w/wpkc)

0.5, 1, 5, 10

pH

4, 4.5, 5, 5.5

Temperature (oC)

40, 45, 50, 55

Hydrolysis time (h)

24, 48, 72, 96

34 655 656 657 658

Table 2 ANOVA results for showing the significance of the effect of different factors tested on total fermentable sugar produced from hydrolysis of PKC by mannanase

659 660

Factor

661

PKC concentration

662

Degree of freedom

F-value

176.11

7

11.42

0.020

Mannanase loading

36.91

7

114.45

0.001

663

pH value

29.01

7

65.30

0.001

664

Reaction temperature

21.63

7

19.35

0.008

665

Hydrolysis time

18.17

7

37.18

0.002

666

667

668

Sum of squares

P-value (Prob>F)

35 669 670 671

Table 3 Production of fermentable sugars and biobutanol from different lignocellulosic feedstocks

672 673 674

Microbial strains

Substrate

Hydrolysis type

Amiri et al., 2014

6.04

Qureshi et al., 2014

6.50

Qureshi et al., 2008

Alakali pretreatment + 99.93 cellulase

6.04

Razak et al. 2013

Alkali pretreatment + 29.37 cellulase

1.94

Ibrahim et al., 2015

C. acetobutylicum

Rice straw

C. beijerinckii

Corn stover

679 680

C. beijerinckii

Corn fiber

681 682

C. acetobutylicum

Oil palm decanter cake

683 684

C. acetobutylicum

Oil palm empty fruit branch

685 686

C. acetobutylicum

Palm pressed fibre

687 688

C. acetobutylicum

Palm kernel shell

689

C.acetobutylicum

Felled oil palm trunk

690

C. saccharoperbutylacetonicum Palm kernel cake Mannanase

692 693 694 695

696

697

References

80.3

675 676 677 678

691

Total sugar Biobutanol (g/L) (g/L)

Organosolve pretreatment + 31 cellulose degrading enzymes Acid pretreatment + cellulase with xylanase

39.0

Hot water pretreatment + 25 cellulose degrading enzymes

Steam explosion pretreatment + 38.75 cellulase

3.1

Steam explosion pretreatment + 30 cellulase

1.5

Sangkharak et al., 2016

14.4

Komonkiat and Cheirsilp, 2013

Acid hydrolysis

30 71.54

3.27

Sangkharak et al., 2016

This study

36 698 699 700

701 702

703

704

705

706

Graphical abstract

37

707 708

Highlights

709 710

● Palm kernel cake (PKC) was hydrolyzed by mannanase.

711

●The effect of operating factors on the enzymatic hydrolysis of PKC was studied.

712

● Mannose was the main fermentable sugar released with glucose to a lower extent.

713

●The total sugar of 71.54 ± 2.54 g/L was produced in optimum conditions.

714

●Maximum biobutanol production of 3.27±1.003 g/L was obtained.

715 716