Accepted Manuscript

Simultaneous saccharification and fermentation of delignified lignocellulosicbiomass at high solid loadings by a newly isolated thermotolerant Kluyveromy-ces sp. for ethanol production

Madhuri Narra, Jisha P. James, Velmurugan Balasubramanian

PII: S0960-8524(14)01738-6DOI: http://dx.doi.org/10.1016/j.biortech.2014.11.116Reference: BITE 14323

To appear in: Bioresource Technology

Received Date: 29 October 2014Revised Date: 27 November 2014Accepted Date: 28 November 2014

Please cite this article as: Narra, M., James, J.P., Balasubramanian, V., Simultaneous saccharification andfermentation of delignified lignocellulosic biomass at high solid loadings by a newly isolated thermotolerantKluyveromyces sp. for ethanol production, Bioresource Technology (2014), doi: http://dx.doi.org/10.1016/j.biortech.2014.11.116

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Simultaneous saccharification and fermentation of delignified lignocellulosic biomass at 1

high solid loadings by a newly isolated thermotolerant Kluyveromyces sp. for ethanol 2

production 3

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Madhuri Narraa*

, Jisha P Jamesa, Velmurugan Balasubramanian

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a Sardar Patel Renewable Energy Research Institute, P. Box No.2, Vallabh Vidyanagar, 388 9

120, Gujarat, India 10

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* Corresponding author. Tel.: +91 2692 231332, 235011; fax: +91 2692 237982 18

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E-mail addresses: madhuri68@gmail.com (M. Narra) 20

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Abstract 1

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Simultaneous saccharification and fermentation studies were carried out using thermotolerant 3

newly isolated Kluyveromyces sp. with three different delignified lignocelllulosic biomass viz. 4

rice straw, wheat straw and sugarcane bagasse at 5-15% solid loading and 6-12 FPU g-1

substrate 5

enzyme loading for different time intervals 0-72 h at 42οC. Maximum ethanol achieved from rice 6

straw, wheat straw and sugarcane bagasse with in-house crude cellulases from Aspergillus 7

terreus was 23.23, 18.29 and 17.91 mg mL-1

at 60 h with 10% solid load and 9 FPU g-1

substrate 8

enzyme loading. Tween 80 1% (v/v) enhanced the ethanol yield by 8.39, 9.26 and 8.14% in rice 9

straw, wheat straw and sugarcane bagasse, respectively. External supplementation of β-10

glucosidase to the crude as well commercial cellulases produced maximum theoretical ethanol 11

yield of 71.76, 63.77, 57.15 and 84.56, 72.47, 70.55% from rice straw, wheat straw and 12

sugarcane bagasse, respectively. 13

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Key words: Lignocellulosic biomass; Delignification; Thermotolerant yeast strain; Simultaneous 16

saccharification and fermentation; Ethanol 17

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1. Introduction 1

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Lignocellulosic biomass such as rice straw, wheat straw and sugarcane bagasse are abundant 3

and are cheap renewable resources of carbohydrates for microbial conversion to fuels and 4

chemicals (Brijwani et al., 2010; Borbala et al., 2013). They primarily consist of lignin and the 5

carbohydrate polymers cellulose and hemicelluloses; however the composition of each 6

component varies with the feed stock used. Since lignocellulosic materials are very complex, not 7

one pretreatment method can apply for all the materials. Different pretreatment methods 8

including chemical, physical, physico-chemical and biological have been developed for 9

lignocellulosic waste pre-treatment. Among all pretreatment methods steam, dilute acid, alkaline 10

and oxidative pretreatment methods have been widely employed. Dilute acid pretreatment has 11

become an up to date technology for pre-treating any lignocellulosic biomass. Acid hydrolysis 12

removes the hemicellulosic portion and some portion of lignin, the leftover of the lignin remains 13

intact to the cellulosic substrate (Kaya, Heitmannand Thomas, 2000). During enzymatic 14

hydrolysis of lignocellulosic biomass, cellulase components, β-glucosidase and endoglucanase 15

have more binding affinity towards lignin than to the carbohydrates, resulting in a lower 16

efficiency of saccharification. Hence, to achieve maximum hydrolysis of cellulosics an 17

appropriate delignification treatment of biomass is required. Alkali treatment processes are 18

generally very effective in the pre-treatment of agricultural residues such as rice straw and 19

herbaceous crops (Chen et al., 2007). It generally utilizes lower temperatures, pressures and time 20

compared to other pre-treatment technologies. The most commonly used alkali sodium 21

hydroxide has been extensively studied for many years, and it has been shown to disrupt the 22

lignin structure of the lignocellulosic biomass, increasing accessibility of enzymes to cellulose 23

and hemicelluloses. 24

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The cellulose fraction of lignocellulosic biomass can be converted to ethanol by either 1

separate enzymatic hydrolysis and fermentation (SHF) processes or simultaneous 2

saccharification and fermentation (SSF). SSF is more favoured because it offers benefits such as 3

improved ethanol yields by reducing the product inhibition excerted by saccharification products 4

and also eliminates the need for separate reactors for saccharification and fermentation, which 5

results in cost reduction (Wang et al., 2013; Molaverdi et al., 2013; Scordia et al., 2013). On the 6

other hand, SSF suffers from a hitch, which is a different optimum temperature of the 7

hydrolysing enzymes and fermenting microorganisms. Many researchers indicated in their 8

publications that the optimum temperature for enzymatic hydrolysis is 40-50οC, while the 9

microorganisms with good ethanol productivity and yield do no usually tolerate this high 10

temperature. This problem has usually been tackled by using thermotolerant microorganisms 11

(Keikhosro et al., 2006). Use of such microbes for high temperature fermentation will minimize 12

operational costs with respect to maintaining growth temperature in reactors, reducing chances of 13

contamination and facilitate recovery of products (Singh et al., 1998; Araque et al., 2008; 1998; 14

Yanase et al., 2010). Significant research has been done to identify microbial strains such as 15

Saccharomyces cerevisiae F111, Kluyveromyces marxianus WR12 and Saccharomyces 16

cerevisiae D5A that can grow at high temperatures (Abdel Fattah et al., 2000; Spindler et al., 17

1988). Due to their potential applications, the screening of yeast strains able to ferment sugars 18

obtained from lignocellulosic material at temperatures above 35οC with high ethanol yields has 19

become a necessity. 20

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Although rice straw, wheat straw and sugarcane bagasse are considered as an attractive 22

feedstock for bio-ethanol production, a very few publications on ethanol production from these 23

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lignocellulosic biomass using SSF by thermotolerant yeast strain are available. In the present 1

study, results of SSF of the pretreated lignocellulosic biomass to ethanol by a newly isolated 2

thermotolerant hexose fermenting yeast strain have been reported. The effect of solid load, 3

enzyme load and time required for ethanol yields were studied using in-house cellulases 4

produced from Aspergillus terreus and the role of supplementation of β-glucosidase on ethanol 5

yields were also evaluated. 6

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2. Methods 1

2.1. Biomass and chemicals 2

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Lignocellulosic biomass i.e. rice straw and wheat straw were procured from local farms of 4

Anand. Sugarcane bagasse was procured from a local sugar factory. The biomass were milled 5

and screened to achieve the size of less than 5-mm mesh prior to pretreatment (Finex, India). 6

The raw materials were washed thoroughly with tap water till clean and colorless, air dried and 7

stored at room temperature in air tight containers. All the reagents, media and chemicals used 8

were of analytical grade (Qualigens, Himedia, Merck and Sigma etc.). 9

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2.2. Chemical pretreatment of rice straw 11

One fifty grams of pre-sized rice straw, wheat straw and sugarcane bagasse were mixed with 12

750 mL, 1500 mL and 3000 mL of 2% and 4% H2SO4to obtain the substrate to acid ratio (w/v) 13

of 1:5, 1:10 and 1:20, respectively. Pretreatment experiments were carried out at 121οC for 30 14

and 60min. The acid hydrolysates after dilute acid pretreatment were recovered by filtering the 15

contents through double layered muslin cloth. The acid pretreated solid residues were washed 16

with tap water till neutral pH was achieved and sun dried prior to delignification. Delignification 17

was done with 0.5% NaOH at 121οC for 30 min. Substrate to alkali ratio maintained was 1:20. 18

The delignified biomass was then filtered through double layered muslin cloth and cellulosic 19

residue was neutralized with tap water till neutral pH. The solid residues were sun dried and 20

used immediately either for SSF studies or stored at 4οCin air tight bags. 21

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2.3. Yeast strain isolation and identification 1

Screening for hexose fermenting yeast strains were carried out from fruit waste and soil 2

samples from sugar factories. The samples were serially diluted and plated on hexose 3

fermenting medium containing (g L-1

): glucose, 30.0; yeast extract, 3.0; peptone, 5.0; agar, 20.0 4

at pH 6.0±0.2. The plates were incubated at 30, 42 and 45οC for 2-4 days. Colonies that grew 5

and produced ethanol at 42οC were isolated, purified and maintained on Yeast extract peptone 6

dextrose agar slants. The slants were stored at 4ºC until further use. Partial sequencing of 7

purified strain was carried out at National Collection for Industrial Microorganisms (NCIM), 8

Pune, India. The genetic analysis was performed using Molecular Evolutionary Genetic analysis 9

(MEGA) phylogenetic software (Tamura et al., 2013). The dendrogram was constructed using 10

Neighbor Joining algorithm (Saitou et al., 1987). The distances were calculated using Kimura 2-11

parameter algorithm (Kimura, 1980). Basic Local Alignment Search Tool (BLAST) was used to 12

infer functional and evolutionary relationships between sequences as well as identify members of 13

gene families. (i) Initial search was carried out to find potentially closely related sequences 14

using the BLASTN program. (ii) Pairwise alignment to calculate the sequence similarity values 15

between the query sequence and the sequence identified in step (i). 16

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2.4. Inoculum preparation 18

The Kluyveromyces sp. inoculum was grown for 12 h at 42±2ºC in a culture medium 19

containing (g-1

); glucose, 30.0; yeast extract, 3.0; peptone, 5.0; (NH4)2HPO4, 0.25 at pH 6.0±0.2 20

(Chen et al., 2007). At the end of incubation the contents of the flask was collected aseptically 21

centrifuged and used for SSF. Cells were cultured to an optical density of 0.6-0.8 at 620nm. The 22

flasks were inoculated with 1% (v/v) of a 12 h seed culture of Kluyveromyces sp. 23

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2.5. Cellulase preparation 1

SSF was performed with three forms of enzyme preparations i.e. crude cellulases from 2

Aspergillus terreus, commercial cellulases from Trichoderma reseei ATCC 26921 and 3

commercial cellobiase from Aspergillus niger. Crude cellulase used for SSF was indigenously 4

produced by Aspergillus terreus under solid state fermentation as described earlier (Narra et al., 5

2012 and 2014) and contained FP (0.98 ± 0.13 U mL-1

), β-glucosidase (5.2 ± 0.30 U mL-1

), 6

respectively. The commercial cellulase preparation, which was from Trichoderma reseei 7

hadcellulase activity of 25 FPU mL-1

and Commercial cellobiase had β-glucosidase activity of 8

266 UmL-1

. 9

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2.6. Simultaneous saccharification and fermentation of cellulosic solid residues 11

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Simultaneous saccharification and fermentation of delignified rice straw, wheat straw and 13

sugarcane bagasse were carried out at 42οC using cellulases produced by in-house isolated fungal 14

strain Aspergillus terreus where the fungal strain produces glucose from pretreated biomass 15

(cellulose) and the newly isolated yeast strain Kluyveromyces sp. simultaneously converted the 16

glucose to ethanol. For better conversion, prehydrolysis was carried out by liquefying 17

lignocellulosic biomass at 42οC for 6 h, and then newly isolated yeast strain Kluyveromyces sp. 18

was added under sterile conditions. 19

SSF was performed with cellulosic solid residues of rice straw, wheat straw and sugarcane 20

bagasse at 42°C in 50 mL capacity oakridge wide mouth bottles with a total system of 20 mL 21

(0.05M citrate buffer, pH 4.8). Four different sets of experiments were carried out with all the 22

three cellulosic residues. In set 1: Cellulosic solid residues were hydrolysed by the crude 23

cellulases produced by Aspergillus terreus at different substrate loads (5-15% w/v), different 24

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time intervals (0-72 h) and different enzyme loads (6, 9 and 12 FPUg-1

substrate). In set II: 1

Cellulosic substrates with 10% solid loading were hydrolysed with commercial cellulases with 9 2

FPUg-1

substrate enzyme load. In set III: Cellulosic substrates with 10% solid loading were 3

hydrolysed with addition of 50% commercial β-glucosidase to the crude cellulases. In set IV: 4

Cellulosic substrates with 10% solid loading were hydrolysed with addition of 50% commercial 5

β-glucosidase to the commercial cellulases. The experimental flasks with 5% (w/v) substrate 6

load were agitated at 120 rpm in shaker-water bath and the flasks with 10% and 15% were 7

loaded on the rotating assembly for 72 h. 8

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2.7. Analytical methods 10

FP activity was measured according to IUPAC recommendations employing filter paper 11

(Whatman No.1) as substrate (Ghose, 1994). The release of reducing sugars in 60 min at 50oC at 12

pH 4.8 (0.05 M sodium acetate buffer) was measured as glucose equivalent using dinitrosalysilic 13

acid method. One unit of FP activity is measured as the amount of enzyme liberating 1 µmol of 14

glucose per min. β-glucosidase assay was carried out by using p-nitrophenyl - β-D-15

glucopyranoside (PNPG, Sigma Chemical Co.) as substrate at 50oC for 30 min. The reaction 16

was terminated by addition of 4 mL NaOH -glycine buffer (0.2 M, pH 10.6). The total reducing 17

sugars were determined by the dinitrosalysilic acid method (Miller, 1959). Lignin, cellulose and 18

hemicellulose contents of the untreated and pre-treated rice straw, wheat straw and sugarcane 19

bagasse were analysed according to Goering and Vansoest (1975). 20

The samples from SSF were withdrawn every 12 h, centrifuged at 10,000 x g for 15 min and 21

the supernatant was analyzed for residual sugars and ethanol using high performance liquid 22

chromatography (Shimadzu, Japan) equipped with a refractive index detector (RID) and packed 23

with an Aminex-HPX-87 column (Biorad, Hercules, USA, CA) with dimension of 300 mm x 7.8 24

10

mm. Samples were eluted using 5 mM H2SO4 with the flow rate of 0.6 mL min-1

. Column 1

temperature was maintained at 65ºC. The theoretical yield of ethanol was calculated as 2

described by Naveen et al. (2011). 3

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% theoretical yield= (EtOHt) - (EtOHo) 6

-------------------------------------- 7

0.51 x (f (biomass) x 1.11) x 100 8

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where (EtOHt) is the concentration of ethanol at time t, (EtOHo) is the initial ethanol 10

concentration, f is the glucan fraction of dry biomass, (biomass) is dry mass concentration and 11

1.11 is the conversion factor for glucan to glucose. 12

3. Results and discussion 13

3.1. Isolation and identification of an efficient thermotolerant hexose fermenting yeast strain 14

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The selection was carried out on a total of 13 yeast strains. All the yeast strains grew in the 16

maintenance medium at 30οC. Only two strains grew at 42

οC and no strain grew at 45

οC. The 17

strains that grew at 42οC were incubated in the production medium and evaluated for biomass 18

and ethanol production. The isolated strains were found capable of growing and fermenting 19

glucose at 42οC with an ethanol yield of 85-89%in 36 h (data not shown). The potential isolate 20

which showed higher ethanol yield of 89%was isolated from fruit waste and was identified at 21

NCIM, Pune, India. The isolate showed closest homology to Kluyveromyces sp. (nearest species 22

marxianus) using ITS rDNA gene sequencing. Phylogenetic tree based on ITS rDNA gene 23

sequences (669 bp) showing relationship of strain with other type species or members of genus 24

(Table 1and Fig. 1a). The numbers at nodes represent bootstrap values (based on a resampling of 25

1000) (Felsenstein, 1985). Bootstrap values above 50% are shown. The genbank accession 26

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numbers of sequences of each reference species are listed in parenthesis. Bar at the end of tree 1

indicates nucleotide substitutions per site. Gaps in sequencing alignment and missing data were 2

omitted. After trimming there were 443 positions in final sequence set used to infer evolutionary 3

relationships. Sequence data for the newly isolated yeast strain was shown in Fig. 1b. 4

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3.2. Compositional analysis of lignocellulosic biomass 6

The compositional analysis of untreated and delignified lignocellulosic biomass is shown in 7

Table 2.Rice straw, wheat straw and sugarcane bagasse contained (41.02 ± 1.45%; 38.50 ± 8

1.07%; 39.00 ± 1.83%) cellulose, (28.47 ± 1.91%; 27.00 ± 1.36%; 25.00 ± 1.44%) 9

hemicellulose, (9.20 ± 1.12%; 12.82 ± 1.27%; 14.21 ± 1.62%) lignin and (7.04 ± 1.21%; 6.93 ± 10

1.17%; 8.14 ± 1.15%) moisture, respectively. After pretreatment, the solid biomass of rice straw, 11

wheat straw and sugarcane bagasse contained (80.00 ± 1.58%; 67.00 ± 1.60%;70.00 ± 12

1.32%)cellulose, (3.00 ± 1.91%; 6.54 ± 1.42%; 4.36 ± 1.44%) hemicellulose and (2.01 ± 1.16%; 13

4.98 ± 1.97%; 5.13 ± 1.48%) lignin, respectively. 14

3.3. Pretreatment 15

Rice straw, wheat straw and sugarcane bagasse yielded maximum reducing sugars(110, 90 16

and 95mg mL-1

) when treated with 4% H2SO4at 121°C for 30 min and the substrate to acid ratio 17

was 1:5 (w/v). As the time of treatment increased from 30 min to 60 min, no significant 18

difference in the reducing sugar yield was observed (102, 85 and 91 mg mL-1

).The acid 19

hydrolysate was used for ethanol fermentation studies using pentose fermenting yeast strain (data 20

not shown).In order to achieve higher reducing sugar yield, the acid treated biomass were further 21

delignified with 0.5% NaOH at 121°C for 30 min. The alkaline pretreatment step led to 22

remarkable reduction in lignin content and substantial increase in cellulose content in the 23

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pretreated biomass feedstock (Table2). This finding was consistent with earlier reported results 1

on effects of delignification by alkaline pretreatment for lignocellulosic biomass rice straw, 2

wheat straw and rape seed straw (Narra et al., 2012; Nopparat et al., 2013). Mild alkali 3

pretreatment at 121°C removed 78.16, 61.15 and 63.90% lignin from the rice straw, wheat straw 4

and sugarcane bagasse. Lignin removal increases enzyme effectiveness by eliminating 5

nonproductive adsorption sites and increasing access to cellulose and hemicelluloses (Lu et al., 6

2002). On the other hand, solubilization of other components in the aqueous alkali solution 7

increased the cellulose content (Kumar et al., 2009). Nopparat et al. (2013) reported that alkaline 8

pretreatment increased the proportion of cellulose by 79.6% in rice straw. The present data also 9

shows that the cellulose content was found to be increased by 95.02, 74.02 and 79.48% in rice 10

straw, wheat straw and sugarcane bagasse, respectively and most of the pentosan were also 11

solubilized during pretreatment (Table 2). 12

3.4. Simultaneous saccharification and fermentation 13

3.4.1. Influence of substrate concentration 14

Maximum ethanol yield from rice straw, wheat straw and sugarcane bagasse (23.23, 18.29 15

and 17.91 mg mL-1

) was achieved at 60 h with 10% solid load (Fig.2 a, b & c). As the 16

concentration of solid load increased from 10 to 15%, the ethanol yield was decreased to 19.24, 17

14.84and 13.84 mg mL-1

, respectively. The decline in ethanol yield beyond the optimal substrate 18

levels could be due to substrate inhibition, which substantially lowers the rate of fermentation 19

(Xin et al., 2010). Many of the previously published reports have shown that high substrate 20

concentration usually caused decrease in hydrolysis yield due to the product inhibition, and the 21

extent of substrate inhibition depends on the ratio of the total substrate to enzyme loaded (Wang 22

et al., 2011; Xin et al., 2010). Higher ethanol yields at lower solid loads may also be due to 23

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lower glucose accumulation by the end of fermentation compared to higher solid loads. Similar 1

observations were also reported by Naveen et al. (2011) that at 8% solid load with Accellerase 2

1500, higher ethanol yields were observed compared to 12% solid load in SSF of pretreated 3

switchgrass by Kluyveromyces marxianus IMB3 after 168 h. 4

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3.4.2. Influence of reaction time and enzyme loading 6

In order to determine the optimum enzyme loading and time required in SSF, three enzyme 7

loadings (6, 9 and 12 FPU g-1

substrate) were tested at 42°C for different time intervals 0-72 h 8

with 10% solid loading. The time courses of SSF processes of rice straw, wheat straw and 9

sugarcane bagasse to ethanol are presented in (Fig.3 a, b & c). It can be seen that the ethanol 10

production increased gradually and declined thereafter. The highest ethanol concentration 11

(23.23, 18.29 and 17.91 mg mL-1

) was obtained from rice straw, wheat straw and sugarcane 12

bagasse, respectively, which was equivalent to 51.29, 48.22 and 45.19% of maximum theoretical 13

yield (Fig.3a, b & c). Kluyveromyces sp. produced higher concentrations of ethanol as the 14

enzyme loadings in SSF were increased from 6 to 9 FPU g-1

substrate as shown in Fig.3. The 15

increase in the enzyme loading above 9 FPU g-1

substrate did not increase the ethanol yields may 16

due to saturation occurred above an optimum enzyme loading. In agreement to our studies, 17

Spinder et al. (1988) studied the effect of different enzyme loadings in SSF ranging from 7-21 18

FPU g-1

substrate and found that saturation occurred above an enzyme loading of 20 FPU g-1

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substrate. Naveen et al. (2011) also reported that higher ethanol yields were obtained in SSF of 20

Kanlow switchgrass by thermotolerant Kluyveromyces marxians IMB3 with an enzyme loading 21

above 0.7 mL g-1

glucan after 120 h. 22

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3.4.3. Effect of surfactants 1

Simultaneous saccharification and fermentation of rice straw, wheat straw and sugarcane 2

bagasse without surfactant yielded 23.23, 18.29 and 17.91 mg mL-1

of ethanol and achieved 3

51.29, 48.22 and 45.19%maximum theoretical conversion. Addition of 1% Tween 40 and 4

Tween 80 to the reaction mixture increased the ethanol yield by 3.83, 5.52, 2.55%, and 8.39, 5

9.26 and 8.14%, respectively in comparison to the control at 60 h and at 10% solid loading 6

(Fig.4). Maximum ethanol yield from rice straw, sugarcane bagasse and wheat straw were found 7

to be 25.18, 19.57 and 18.19 mg mL-1

, respectively with 1% Tween 80. Different mechanisms 8

have been proposed to explain the positive effect of surfactant addition on the enzymatic 9

hydrolysis of cellulose (Kaar and Holtzapple. 1998). Surfactants could change the nature of the 10

substrate, e.g. by increasing the available cellulose surface or by the removing inhibitory lignin. 11

The studies conducted at our laboratory showed that 1%Tween 80 enhanced the saccharification 12

yield by 13.25%, when mild alkali pretreated rice straw was enzymatically hydrolysed by 13

cellulases produced from Aspergillus terreus (Narra et al., 2012). Kaar and Holtzapple (1998) 14

and Zhu et al. (2014) similarly have found that Tween 80 increased rate and extent of hydrolysis. 15

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3.5. Simultaneous saccharification and fermentation studies with in-house and commercial 17

cellullases 18

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In order to produce higher ethanol yields, four sets of experiments were carried out at 10% 20

solid load as described in methods section i.e. using crude cellulases, commercial cellulases and 21

supplementation of commercial β-glucosidase to crude cellulases as well commercial cellulases. 22

When crude and commercial cellulases were used alone rice straw, wheat straw and sugarcane 23

bagasse produced 23.23, 18.29 and 17.91 mg mL-1

and about 24.63, 19.83 and 19.05mg mL-

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1ethanol corresponding to 51.29, 48.22 and 45.19% and 54.38, 52.44 and 48.07% of maximum 1

theoretical yield (Fig. 5a, b & c). It was observed that the difference in ethanol yield was not 2

significant when these two forms of enzymes were used. Addition of commercial β-glucosidase 3

(50%) to crude cellulases and commercial cellulases increased the ethanol yield by 39.90, 32.24, 4

25.63%, and 55.49, 38.19, 46.76%, in rice straw, wheat straw and sugarcane bagasse, 5

respectively, in 60h (equivalent to maximum theoretical yield 71.76, 63.37, 57.15 and 84.56, 6

72.47, 70.55%). However, the ethanol production was slightly declined after 60 h of incubation 7

(Fig. 5a, b & c). The higher ethanol yields at 60 h may be due to external supplementation of β-8

glucosidase ensured better cellobiose conversion to glucose monomers and simultaneously 9

metabolized by the yeast strain, thereby alleviating problems caused by production inhibition. 10

This result is in good agreement with that reported by Hari Krishna et al. (2001). The substrates 11

were of the following order in terms of rate of conversion: rice straw > wheat straw > sugarcane 12

bagasse. 13

The utilization of both cellulose and hemicellulosic sugars present in typical lignocellulosic 14

biomass hydrolysate is essential for the economical production of ethanol. Based on the results 15

obtained in the present study, 1 kg raw rice straw, wheat straw and sugarcane bagasse contained 16

410, 385 and 390 g cellulose. This amount can theoretically enough for production of 232, 217 17

and 220 g ethanol. Considering the best achieved yield in the current work as 84.56%, 72.47 and 18

70.55% from rice straw, wheat straw and sugarcane bagasse can yield 196 g or 248 mL, 157g or 19

198 mL and 155 g or 196 mL of ethanol, respectively. Besides cellulose fermented to ethanol 20

through SSF processes, as hemicelluloses accounts for one third of the total carbohydrates in 21

native lignocellulosic biomass, fermentation of xylose in the liquid fraction to produce ethanol 22

are also being conducted in the laboratory using Pichia stipitis 3498 (NCIM, Pune, India) and 23

16

found that 55.07%, 52.06% and 58.95% conversion efficiency with rice straw, wheat straw and 1

sugarcane bagasse, respectively. 2

The best ethanol yield obtained from Mucor indicus was 73.58%, which was from rice straw 3

in anaerobic SSF at 5% solid load (Keikhosro et al., 2006). Punnapayak and Emert. (1986) 4

studied SSF of alkali-pretreated rice straw with Pachsolen tannophilus and Candida brassicae. 5

They achieved less than 30% theoretical ethanol yields from rice straw. Naveen et al. (2011) 6

reported that at 8% solid load with Accellerase 1500, higher ethanol yield of 86% were achieved 7

in SSF of pretreated switchgrass by Kluyveromyces marxianus IMB3 after 168 h. The total 8

ethanol concentration in SSF of wheat straw pretreated with steam explosion and alkaline 9

peroxide was 51.5 g/L and an overall yield of 81.1% (Chen et al., 2008). Comparison of 10

simultaneous saccharification and fermentation of pretreated substrates by various enzyme 11

sources (Table 3). 12

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4. Conclusion 1

The present study was undertaken to assess the ethanol production from pre-treated 2

lignocellulosic biomass by simultaneous saccharification and fermentation process using a newly 3

isolated thermotolerant yeast strain. The selected yeast strain is able to grow at higher 4

temperatures than conventional yeast strain Saccharomyces cerevisiae and produced higher 5

ethanol yields when supplemented with β-glucosidase. Maximum theoretical yield was achieved 6

with rice straw followed by wheat straw and sugarcane bagasse. Furthermore, any improvement 7

in pretreatment as well as the SSF will enhance the yield of ethanol from lignocellulosic 8

biomass. 9

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Acknowledgements 16

The authors are thankful to the Director, Sardar Patel Renewable Energy Research Institute 17

(SPRERI), VallabhVidyanagar, Gujarat for allowing us to carry out research at SPRERI. The 18

financial support from Department of Biotechnology (DBT), Government of India is highly 19

acknowledged. 20

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15

16

17

18

19

20

21

22

23

23

Legends 1

Fig. 1(a)shows phylogenetic relationship of isolated yeast strain; (b) Sequence data 2

(ITS4_SC_NC141014) 3

4

Fig.2.Simultaneous saccharification and fermentation of (a) rice straw, (b) wheat straw and (c) 5

sugarcane bagasse at different substrate loads. 6

7

Fig. 3.Simultaneous saccharification and fermentation of (a) rice straw, (b) wheat straw and (c) 8

sugarcane bagasseat different enzyme loads and at different time intervals. 9

10

Fig. 4.Effect of non-ionic surfactants on simultaneous saccharification and fermentation of rice 11

straw, wheat straw and sugarcane bagasse. 12

13

Fig. 5.Comparison of simultaneous saccharification and fermentation of (a) rice straw, (b) wheat 14

straw and (c) sugarcane bagasse with in-house and commercial enzymes. 15

16

17

18

19

20

21

22

23

24

25

26

24

Table 1 Relationship of isolated strain with other type species or members of genus 1

2

3

4

Description Max score Total score Query cover E value Ident Accession

Kluyveromycesmarxianus strain PAZ

18S ribosoma RNA gene, partial

sequencing; internal transcribed spacer

1, 5.8S ribosomal RNA genes, and

internal transcribed spacer 2, complete

sequence; and 28S ribosomal RNA gene,

partial sequence

1205 1205 99% 0.0 99% KF964549.1

Kluyveromycesmarxianus 26S ribosomal RNA, 18S ribosoma RNA

genes, internal transcribed spacer 1, 5.8S

ribosomal RNA gene, and internal

transcribed spacer 2, complete sequence;

and 26S ribosomal RNA gene, partial

sequence

1199 1199 99 0.0 99% AF543841.1

Kluyveromycesmarxianus isolate VA

116042-03 18S ribosoma RNA gene,

partial sequence; internal transcribed

spacer 1, 5.8S ribosomal RNA gene, and

internal transcribed spacer 2, complete sequence; and 28S ribosomal RNA gene,

partial sequence

1194 1194 99% 0.0 99% AY939806.1

Kluyveromycesmarxianus strain DAOM

21636, internal transcribed spacer 1,

partial sequence; 5.8S ribosomal RNA

gene, complete sequence; and internal

transcribed spacer 2, partial sequence

1166 1166 96% 0.0 99% JN942845.1

Kluyveromycesmarxianus genomic

DNA containing ITS1, 5.8S rRNA gene

and ITS2, strain AMB5130

1158 1158 95% 0.0 99% HG532087.1

Kluyveromycesmarxianus strain Y2 18S ribosomal RNA gene, partial sequence;

internal transcribed spacer 1, 5.8S

ribosomal RNA gene, and internal

transcribed spacer 2, complete

sequence; and 26S ribosomal RNA

gene, partial sequencing

1158 1158 99% 0.0 98% JF715191.1

25

Table 2 Compositional change of rice straw, wheat straw and sugarcane bagasse before and after

delignification

1

2

3

4

5

6

7

8

9

10

11

12

Constituents Rice straw Wheat straw Sugarcane bagasse

Untreated Delignified Untreated Delignified Untreated Delignified

Cellulose, %

41.02±1.45

80.00±1.58

38.50±1.07

67.00±1.60

39.00±1.83

70.00±1.32

Hemicellulose, % 28.47 ±1.91 3.00±1.91 27.00±1.36 6.54±1.42 25.00±1.44 4.36±1.44

Lignin, % 9.20±1.12 2.01±1.16 12.82±1.27 4.98±1.97 14.21±1.62 5.13±1.48

Increase in cellulose

content , %

- 95.02±1.91 - 74.02±2.13 - 79.48±1.84

Reduction in lignin

content, %

- 78.16±1.54 - 61.15±1.98 - 63.90±1.69

Solubilized content

of hemicellulose, %

- 89.46±2.01 - 75.77±1.62

- 82.56±1.89

26

Table 3 1

Comparison of simultaneous saccharification and fermentation of pretreated substrates by 2

various enzyme sources 3

Source Substrate Strain Temperature

Maximum

theoretical

ethanol yield

References

Celluclast,

Novozyme 188

Antigonum

Leptopus (linn)

leaves

Saccharomyces

cerevisiaeNRRl-

Y-132

38.5οC 69.21% Hari Krishna

et al., 2000

Commercial

cellulase from (BTXL, ASA

Spezialenzyme

GmbH (Germany)

Rice straw

Rice straw

Avicel

Rhizopus oryzae

Mucor indicus

Saccharomyces

cerevisiae

38οC

38οC

38

οC

73.58%

67.62%

75.91%

Keikhosro

et al., 2006

Celluclast,

Novozyme 188

Wheat straw Recombinant

E.coliFBR5

37οC 45.00% Saha and

Cotta, 2006

Trichoderma viridae

Cellulase

Wheat straw Saccharomyces

cerevisiae

40οC 81.20% Chen et al.,

2008

Accellerase 1500 Switch grass Kluyveromyces

marxianus IMB3

45οC 86.00%

Naveen

et al., 2011

Celluclast,

Novozyme 188

Sweet sorghum

stalk

Mucor indicus 37οC

85.60% Molaverdi

et al., 2013

Alpha amylase and

beta glucanase

Cassava flour Saccharomyces

cerevisiae

30οC

86.10%

(lab scale)

83.60%

(pilot scale)

Nguyen

et al., 2014

Cellulase from

Trichoderma reesei

26291 (Sigma) + β-glucosidase from

Aspergillus niger

(Sigma)

Cellulase from

Aspergillus terreus+ β-glucosidase from

Aspergillus niger

(Sigma)

Rice straw

Wheat straw

Sugarcane

bagasse

Rice straw

Wheat straw

Sugarcane bagasse

Kluyveromyces

sp.

Kluyveromyces

sp.

42οC

42οC

84.56%

79.64%

78.45%

71.76%

63.37%

57.15%

This study

This study

27

Fig.1

(a) 1

2

CTTCCGTAGGTGAACCTGCGGAAGGATCATTAAAGATTATGAATGAATAGATTACTGGGGGAATCGTCTGA 3

ACAAGGCCTGCGCTTAATTGCGCGGCCAGTTCTTGATTCTCTGCTATCAGTTTTCTATTTCTCATCCTAAA 4

CACAATGGAGTTTTTTCTCTATGAACTACTTCCCTGGAGAGCTCGTCTCTCCAGTGGACATAAACACAAAC 5

AATATTTTGTATTATGAAAAACTATTATACTATAAAATTTAATATTCAAAACTTTCAACAACGGATCTCTT 6

GGTTCTCGCATCGATGAAGAACGCAGCGAATTGCGATATGTATTGTGAATTGCAGATTTTCGTGAATCATC 7

AAATCTTTGAACGCACATTGCGCCCTCTGGTATTCCAGGGGGCATGCCTGTTTGAGCGTCATTTCTCTCTC 8

AAACCTTTGGGTTTGGTAGTGAGTGATACTCGTCTCGGGTTAACTTGAAAGTGGCTAGCCGTTGCCATCTG 9

CGTGAGCAGGGCTGCGTGTCAAGTCTATGGACTCGACTCTTGCACATCTACGTCTTAGGTTTGCGCCAATT 10

ACGTGGTAAGCTTGGGTCATAGAGACTCATAGGTGTTATAAAGTCTACGCTGGTGTTTGTCTCCTTGAGGC 11

ATACGGCTTAACCAAAACTCTCAAAAGTTTG 12

(b) 13

14

15

16

28

0

5

10

15

20

25

30

0 12 24 48 60 72

Eth

an

ol

(mg m

L-1

)

Time (h)

5% (w/v) 10% (w/v) 15% (w/v)

0

2

4

6

8

10

12

14

16

18

20

0 12 24 48 60 72

Eth

an

ol

(mg

mL

-1)

Time (h)

5% (w/v) 10% (w/v) 15% (w/v)

0

2

4

6

8

10

12

14

16

18

20

0 12 24 48 60 72

Eth

an

ol

(mg m

L-1

)

Time (h)

5% (w/v) 10% (w/v) 15% (w/v)

Fig.2

(a)

1

2

3

4

5

6

7

8

9

10

11

12

(b)

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

(c) 29

30

31

32

33

34

35

36

37

38

39

29

02468

101214161820

0 12 24 48 60 72

Eth

an

ol

(mg

mL

-1)

Time (h)

6 FPU g-1 substrate 9 FPU g-1 substrate 12 FPU g-1 substrate

02

468

1012

14161820

0 12 24 48 60 72

Eth

an

ol

(mg m

L-1

)

Time (h)

6 FPU g-1 substrate 9 FPU g-1 substrate 12 FPU g-1 substrate

0

5

10

15

20

25

30

0 12 24 48 60 72

Eth

an

ol

(mg m

L-1

)

Time (h)

6 FPU g-1 substrate 9 FPU g-1 substrate 12 FPU g-1 substrate

Fig.3

(a)

1

2

3

4

5

(b) 6

7

8

9

10

11

(c)

12

13

14

30

0

5

10

15

20

25

30

Rice straw Wheat straw Sugarcane bagasse

Eth

an

ol

(mg m

L-1

)

Surfectants (% v/v)

Control Tween 40 Tween 80

Fig.4

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

31

0

5

10

15

20

25

30

12 24 48 60 72

Eth

an

ol

(mg

mL

-1)

Time (h)

In-house cellulases In-house cellulases + commercial β

Commercial cellulases Commercial cellulases + commercial β

0

5

10

15

20

25

30

12 24 48 60 72

Eth

an

ol

(mg m

L-1

)

Time (h)

In-house cellulases In-house cellulases + commercial β

Commercial cellulases Commercial cellulases + commercial β

0

10

20

30

40

50

12 24 48 60 72

Eth

an

ol

(mg m

L-1

)

Time (h)

In-house cellulases In-house cellulases + commercial β

Commercial cellulases Commercial cellulases + commercial β

Fig.5

(a)

1

2

3

4

5

6

(b) 7

8

9

10

11

12

13

14

15

(c) 16

17

18

19

20

21

22

23

33

Research Highlights 1

� Assessment of ethanol production from delignified lignocellulosic biomass by SSF 2

� SSF at high solid loadings by a newly isolated thermotolerant yeast strain 3

� Supplementation of β-glucosidase ensured better cellobiose conversion to glucose 4

5

6