Chemical pretreatment and saccharification of … et al... · wood waste dump and recorded as high cellulase producer ... Fermentation of pretreated bagasse to ethanol ... Samples

Published by Basic Research Journal of Microbiology

Basic Research Journal of Microbiology ISSN 2354-4082 Vol. 4(1) pp. 01-11 February 2017 Available online http//www.basicresearchjournals.org Copyright ©2015 Basic Research Journal

Full Length Research Paper

Chemical pretreatment and saccharification of sugarcane bagasse for bioethanol fermentation by

Saccharomyces cerevisiae Y17 -KP096551

*Abdel-Hamied M. Rasmey1, Heba Hawary Hassan1, Akram A. Aboseidah1 and Omar A. Abdul-Wahid2

1Botany and Microbiology Department, faculty of Science, Suez University, Suez, Egypt.

2Botany Department, Faculty of Science, Suez Canal University, Ismailia, Egypt.

*Corresponding author Email: [email protected]

Accepted 23 February, 2017

ABSTRACT

Sugarcane bagasse is one of the most promising feedstock for ethanol production due to its abundance and low cost. The conversion of this agricultural waste into sugars and ethanol, considering its pretreatment strategies and biological transformation is the aim of this work. Five grams of milled bagasse was chemically pretreated using various concentrations of nitric and sulfuric acids, sodium hydroxide and hydrogen peroxide. The pretreated bagasse residue was saccharified by crude cellulase extracted from Trichoderma harzianum SC20. The maximum total reducing sugar yield was obtained by saccharification of bagasse hydrolysate pretreated with 0.8 M of sulfuric acid after one hour. The resulted pretreated bagasse was subjected to fermentation by the selected yeast isolate Saccharomyces cerevisiae Y17 KP096551. The highest ethanol concentration was obtained after a fermentation period 48 hours recording 1.34% (w/v) with a fermentation efficiency 51.81% and a volumetric productivity 0.275 gl

-1h

-1.

Keywords: Lignocellulose, fermentation, yeast, cellulose, Trichoderma, reducing sugar

INTRODUCTION Bioethanol is a promising type of biofuel that produced through fermentation of sugars and used as a partial gasoline replacement in many countries of the world (Sharma et al. 2004; Naik et al. 2010; Bhatia et al. 2014). Sugarcane bagasse is the most abundant by-product in Egypt (Ministry of Agriculture Egypt 2000). Recently, potential efforts have been directed towards the utilization of this cheap renewable agricultural resource as an alternative substrate for ethanol production (Bhatia and Paliwal 2011). Cellulose, hemicellulose and lignin are the key biomass polymers found in sugarcane bagasse

consisting about 50, 27.5 and 9.8% respectively (Kewalramani et al. 1988).

The lignocellulosic structure is more resistant to decay by the organisms and is nonperishable like soluble sugar and starch. So, it is required to alter the structure of cellulosic biomass to make cellulose more accessible to the enzymes that convert the carbohydrate polymers into fermentable sugars (Mosier et al. 2005). The key element in bioconversion process of lignocellulosic materials to useful products is the hydrolytic enzymes, mainly cellulases (Ojumu et al. 2003; Immanuel et al. 2006).


However, enzymatic hydrolysis alone is normally insufficient and requires physical pretreatment like steam treatment or chemical treatment (Yu 2002).

Various Mechanical, physical, chemical and biological processes have been used for pretreatment of lignocellulosic materials (Roche et al. 2009; Beszedes et al. 2011; Brodeur et al. 2011). Chemical pretreatments involves the use of different chemicals like dilute acids, alkalis, peroxides and organosolvents that affect lignocellulosic materials by increasing pore size and solubilizing lignin and hemicellulosic by increasing surface area (Rabelo et al. 2008; Martin et al. 2008).

There are two basic types of acid processes: dilute acid and concentrated acid. It has been known for many years that acids act as catalyst to hydrolyze cellulose and hemicellulose into simple sugars (Reddy et al. 2005). Some alkali can also be used for pretreatment of lignocellulosic materials and the effect of alkaline pretreatment depends on the lignin content of the materials (Fan et al. 1987). Hydrogen peroxide is the most studied that it degrades into hydrogen and oxygen and does not leave residues in the biomass (Uppal et al. 2011). The main goal of the present work was to ferment chemically pretreated sugarcane bagasse to ethanol using Saccharomyces cerevisiae Y17. MATERIALS AND METHODS Feedstock Sugarcane bagasse was collected in clean plastic bags from sugarcane juice shops and transferred immediately to Microbiological Research Lab., Botany and Microbiology Department, Faculty of Science, Suez University, Suez, Egypt. Sugarcane bagasse preparation: The collected sugarcane bagasse samples were immediately washed with tap water, air-dried in the lab for two hours and dried in a convection oven at 65ºC for 24 hours. The dried bagasse was milled to obtain particle size of approximately 1 mm to reduce cellulose crystallinity. Chemical pretreatment methods of bagasse Acidic pretreatment: The acid pretreatment of bagasse was conducted according to Manzoor et al. (2012). Diluted sulfuric and nitric acids have been used for sugar cane bagasse pretreatment. Five grams of bagasse were soaked in

Rasmey et al. 02 various concentrations of acids solution (0.4, 0.8, and 1 M) at a solid to liquid ratio of 1:20 (w/v) in 250 ml Erlenmeyer flask. In addition, a distilled water control was used in the experiment. The flasks were placed on a rotary shaker at 150 rpm for 30 minutes to ensure that all of the bagasse came into contact with the treatment solution. The flasks were then autoclaved at 121

oC for

different time intervals (15, 30, and 60 minutes). The residue was removed from the solutions by filtration through cheesecloth and then triple-rinsed for 30 minutes in distilled water to obtain neutral pH. The residue was then dried in an oven at 65

oC for one day. All

experiments were made in triplicates. Alkaline pretreatment The alkaline pretreatment was done as the previous treatment process except that the five grams of the bagasse were soaked in various concentrations of sodium hydroxide (NaOH) solution (0.5, 1.0, and 2.0 M) along with control (distilled water) according to Malik et al. (2010). Oxidative pretreatment Different concentrations (0.0, 1.0, 2.0, and 5%) of hydrogen peroxide (H2O2) solution were prepared and adjusted for different three pH values 8, 11.5 and 13 using NaOH (Dawson and Boopathy, 2008). Saccharification of bagasse Fungal isolate Trichoderma harzianum SC20 previously isolated from wood waste dump and recorded as high cellulase producer (Abdul Wahid et al. 2015) was used in this study. Cellulase production in submerged culture Trichoderma harzianum SC20 was grown on synthetic medium described by Chinedu and Okochi (2011); this medium composed of (g/l): carboxymethyl cellulose, 10.0; NaNO3 3.0; KH2PO4, 1.0; KCI, 0.5; MgSO4.7H2O, 0.5; FeSO4. 7 H2O, 0.01 and supplemented with 1.0 ml of a trace element solution containing 0.1% and 0.05% (w/v) of ZnSO4 and CuSO4, respectively. The pH of the medium was adjusted to 5.5. Conical flasks (250ml) containing 100 ml of the respective medium were autoclaved at 121

oC for 15 minutes, cooled and

inoculated with one disc of 5.0 millimeter diameter of the


03. Basic Res. J. Microbiol. 4 days old culture of the tested isolate grown on PDA plates using a sterile cork borer. The cultures were incubated for 10 days at 30

oC on a rotary shaker (150

rpm for 10 days. The culture broth was filtered through Whatman No.1 filter paper and the clear filtrate was centrifuged at 4000 rpm for 15 min. The clear supernatant was collected and used as crude cellulase enzyme in the further experiments. Saccharification process Saccharification experiments were performed in 250 ml Erlenmeyer flasks separately containing the pretreated bagasse and crude cellulase enzyme at ratio (1g substrate: 25ml crude enzyme). The flasks were sealed with aluminum foil and incubated at 50

oC under

continuous agitation at 150 rpm for 48 hours. Hydrolysates were then transferred in screw-capped tubes, autoclaved at 121

oC for 15 minutes and

centrifuged to remove solid particles. Estimation of total reducing sugars The total reducing sugar liberated during the enzymatic assays was quantified by the dinitrosalicylic acid (DNS) method (Miller, 1959; Narkprasom et al., 2013) using glucose as a standard. Fermentation of pretreated bagasse to ethanol Yeast inoculum preparation Saccharomyces cerevisiae Y17-KP096551 previously isolated and identified by the same authors for its ability to produce bioethanol from different substrates was used in this study. Active cultures for fermentation experiments were prepared by growing the yeast isolate in YPD (Wickerham, 1951) broth medium for 48 h at 30∘C in shaking incubator at 150 rpm. The inoculum was transferred at the rate of 15 % to the ethanol production medium. Fermentation process The cellulase enzyme treated bagasse sample that showed maximum release of reducing sugars as compared to other pretreatments was further subjected to ethanol fermentation. Fifty ml of the hydrolysate were added to the following fermentation medium: 10g/l of yeast extract; 3.0g/l of potassium dihydrogen phosphate; 2.91g/l of disodium hydrogen phosphate; 2.5g/l of ammonium chloride; 0.25g/l of magnesium sulfate;

0.08g/l of calcium chloride; 4.3g/l of citric acid and 3.0g/l of sodium citrate (Bawa et al., 2008). The production medium pH was adjusted to 4.5 using the citrate buffer dissolved in the medium. The bottles were autoclaved and inoculated with S. cerevisiae Y17 then incubated under anaerobic conditions. Samples were taken at different fermentation periods (24, 48, 72, 96, 120 and 144 hours). Ethanol determination Ethanol produced in the fermentation medium was estimated by potassium dichromate (K2Cr2O7) oxidation method according to Balasubramanian et al. (2011). The volumetric ethanol productivity (Qp) was calculated according to Onsoy et al. (2007).

Statistical analysis Analysis of variance (ANOVA) was performed using CoStat V. 6.311 (CoHort software, Berkeley, CA94701). Ethanol production mean values were compared at 5% significance level using Tukey’s test. Least significant difference (LSD) test was used to test the significant differences between the whole means of different groups and compared with the critical difference at the 5% level. RESULTS Chemical pretreatments of sugarcane bagasse Different pretreatment methods were investigated including: diluted acids, alkalis and hydrogen peroxide (oxidative) pretreatments. The obtained results indicated that the strongest degradation indicated by weight loss was obtained when sugarcane bagasse was subjected to the highest chemical concentrations and times. Figures (1 and 2) showed that there is a significant difference between the tested pretreatments where the greatest weight loss (76 and 66 %) were achieved at one molar nitric acid and sulfuric acid, respectively, after autoclaving for 60 minutes. Also, data represented in figure (3) showed that both of the reaction time and the sodium hydroxide concentration were significant factors on the bagasse weight loss. It was also verified that the greatest weight loss (68%) was obtained at 2 M sodium hydroxide after autoclaving for 60 minutes.

The results presented in figures (4, 5 and 6) showed that the bagasse yield decreased as the hydrogen peroxide concentration increased. Statistical analysis of the data, using three way ANOVA, verified that the significant difference between all the pretreatments and the bagasse weight loss was a function of chemical


Rasmey et al. 04

Figure 1. Bagasse weight loss % at different sulfuric acid concentrations for various autoclaving time intervals.

Figure 2. Bagasse weight loss % at different nitric acid concentrations for various autoclaving time intervals.

Figure 3. Bagasse weight loss % at different sodium hydroxide concentrations for various autoclaving time intervals.


05. Basic Res. J. Microbiol.

Figure 4. Bagasse weight loss % at different hydrogen peroxide concentrations and different pH values at 24 hours.




Rasmey et al. 06

Figure 7. Total reducing sugars (mg/ml) yield in the bagasse hydrolysates at different sulfuric acid concentrations for various reaction times.

Figure 8. Total reducing sugars (mg/ml) yield in the bagasse hydrolysates at different nitric acid concentrations for various reaction times.

concentration, pH value and reaction time. The combination effect of high pH value, high concentration and reaction time obtained the strongest degradation of bagasse indicated by weight loss. The maximum weight loss was recorded when bagasse was treated with 5% hydrogen peroxide at pH 13 for 72 hours resulted in 78 % loss of weight and 22% bagasse yield. This weight loss percentage was the greatest among all the pretreatments had been used in this investigation. Saccharification of the chemically pretreated bagasse The total sugar yield is actually the most important yield since it deals with all process steps from the raw bagasse to fermentable sugars. Determination of reducing sugars gave a total picture of the combined effect of both pretreatment and enzymatic hydrolysis. Different

chemical pretreatments exhibited various effects on the total reducing sugar yield. Figures (7 and 8) showed that the saccharification of sulfuric acid pretreated bagasse gave the maximum reducing sugar yield in this study. It was observed that during sulfuric acid hydrolysis, an increase of time reaction improves reducing sugars production. Statistical analysis of the data, using three way ANOVA, showed that a significant difference was between the various acids pretreatments; and the maximum sugar yield was occurred at concentration 0.8 M of sulfuric after reaction time of 60 minutes with a total reducing sugar of approximately 1.66 mg/ml. On the other hand, the saccharification of nitric acid pretreated bagasse showed a decrease of total reducing sugars with the increase of nitric acid concentrations; and the maximum sugar yield was occurred at concentration 0.4 M of nitric acid after reaction time of 30 minutes with a total reducing sugar of approximately 1.64 mg/ml.

The amount of reducing sugar released after


07. Basic Res. J. Microbiol.

Figure 9. Total reducing sugars (mg/ml) yield in the bagasse hydrolysates at different sodium hydroxide concentrations for various reaction times.

Figure 10. Total reducing sugars (mg/ml) yield in the bagasse hydrolysates at different hydrogen peroxide concentrations and pH values after pretreated for 24 hours.

saccharification of the sodium hydroxide pretreated bagasse decreased with the increase of NaOH concentration (figure 9). A significant difference was between the various alkaline pretreatments; and the maximum sugar yield was occurred at concentration of 0.5 M NaOH following reaction time of 30 minutes, with a total reducing sugar of approximately 1.40 mg/ml.

Statistical analysis of the data reported in figures (10, 11 and 12) showed that a significant difference was between the various pretreatments; and the maximum total reducing sugar yield was occurred when bagasse pretreated with 2 % hydrogen peroxide at pH 11.5 following reaction time of 48 hours, with a total reducing sugar of approximately 0.794 mg/ml. On the other hand, it was noticed that the hydrogen peroxide bagasse pretreatment (5% H2O2, pH 13 for 72 hours) had the greatest weight loss but with minimum total reducing sugars concentration (0.508 mg/ml). From the previous results, we can estimate that the maximum total reducing

sugar yield occurred following saccharification of bagasse hydrolysate pretreated with 0.8 M of sulfuric acid after reaction time of 60 minutes. This pretreatment was selected for the bagasse subjected to fermentation process. Bioethanol production from pretreated bagasse hydrolysate Evaluation of ethanol production from the bagasse hydrolysate is the final performance in this study. The resulted hydrolysate of 150g bagasse pretreated with sulfuric acid was saccharified by by Trichoderma harzianum SC20 and used for fermentation by Saccharomyces cerevisiae Y17 to produce ethanol (figure 13). The highest ethanol concentration was obtained after a fermentation period 48 hours recording 1.32% (w/v) with a fermentation efficiency 51.81% and a


Rasmey et al. 08

Figure 11. Total reducing sugars (mg/ml) yield in the bagasse hydrolysates at different hydrogen peroxide concentrations and pH values after pretreated for 48 hours.

Figure 12. Total reducing sugars (mg/ml) yield in the bagasse hydrolysate at different hydrogen peroxide concentrations and pH values after pretreated for 72 hours.

Figure 13. Ethanol production level (EC) % (w/v) and the volumetric ethanol productivity (QP) by the isolate Saccharomyces cervisieae Y17 following saccharification of sulfuric acid pretreated bagasse hydrolysate.


09. Basic Res. J. Microbiol. volumetric productivity equal to 0.275 gl

-1h

-1. It was

noticed that further increase in the fermentation period resulted a high decrease in the ethanol produced by Saccharomyces cerevisiae (Y17) with a reduction in the fermentation efficiency and volumetric ethanol production rate. The fermentation efficiency calculation based on bagasse hydrolysate initial sugar concentration (approximately 50g/l), this initial sugar concentration resulted from the pretreatment and the saccarification of 150g milled bagasse. DISCUSSION Different studies were conducted to develop a suitable pretreatment process for sugarcane bagasse to separate the lignocellosic cell wall arrangement and mild enough to avoid a significant chemical degradation of biomass components (Kuhad and Singh, 1993; Sun and Cheng, 2002; Canetieri et al., 2007; Cardona et al., 2010). In the present investigation, several chemical pretreatments had been applied on milled sugarcane bagasse. The recorded results showed that there was a decline in yields, and hence, an increased loss of weight in function of both of chemical concentration and reaction times. The amount of weight lost following chemical pretreatment of residue was due to lignin removal (Candido et al., 2012; Bhatia et al., 2014; Irfan et al., 2011).

Saccharification of the pretreated bagasse had been the second step in the ethanol production process. Saccharification process is an important step because it determines the amount of glucose produced as a substrate to be fermented into ethanol (Muthuvelayudham and Viruthagiri, 2007; Patel et al., 2007). The enzymatic saccharification step is cost-prohibitive because of the high cost of the commercial enzymes (Abo-State et al., 2014). A large number of microorganisms are capable of degrading cellulose; one of the most promising species is Trichoderma harzianum which has an efficient cellulase production system (Ahmed et al., 2009; Castro et al., 2010). Therefore, in this study; a low cost crude cellulase enzyme was prepared from Tticoderma harzianum SC20 for the saccharification process.

Saccharification of diluted acid pretreated bagasse hydrolysate yielded the highest total reducing sugar among various pretreatments (alkaline and oxidative) that had been used. Different chemical pretreatments exhibited different effects on the total reducing sugar yield (Abo-State et al., 2013). It was noticed that the maximum reducing sugar yield was following the saccharification of bagasse pretreated with 0.8 M of sulfuric acid after reaction time of 60 minutes with a total reducing sugar of approximately 1.66 mg/ml. The results showed that dilute acid pretreatment improved the enzymatic hydrolysis process which is in agreement with

Kumar et al. (2009). The dilute sulfuric acid pretreatment can achieve high reaction rates and significantly improve cellulose hydrolysis. Esteghlalian et al. (1997) estimated that dilute acid effectively removes and recovers most of the hemicellulose as dissolved sugars, and glucose yields from cellulose increase with hemicellulose removal to almost 100% for complete hemicelluloses hydrolysis. Further increase in acid concentration and reaction time decreased the sugar yield; this might be due to the degradation of monomeric sugars (xylose, glucose) at high concentration and reaction times according to Bhatia et al. (2014). The present results are also in consistent with Lavarack and Griffin (2002); Kumar et al. (2009) who reported that pretreatment with acid hydrolysis can result in improvement of enzymatic hydrolysis of lignocellulosic biomasses to release fermentable sugars and sulfuric acid at concentrations usually below 4wt%, has been of the most interest in such studies as it is inexpensive and effective.

It was noticed that the maximum sugar yield occurred at concentration of 0.5 M NaOH with reaction time of 30 minutes with a total reducing sugar of approximately 1.40 mg/ml. Further increase in reaction time showed a decrease in the sugar yield. Fan et al. (1987) reported that dilute NaOH treatment of lignocellulosic materials has been found to cause swelling, leading to an increase in internal surface area, a decrease in the degree of polymerization, a decrease in crystallinity, separation of structural linkages between lignin and carbohydrates, and disruption of the lignin structure. Hydrogen peroxide greatly enhanced its susceptibility to enzymatic hydrolysis (Kumar et al., 2009), furthermore, It was shown that the alkaline-oxidative pretreatment, in which NaOH and H2O2 were used, was a very effective method for improving the enzymatic hydrolysis (Saha and Cotta, 2006). Our data reported that the maximum total reducing sugar yield occurred when bagasse pretreated with 2% hydrogen peroxide at pH 11.5 following reaction time of 48 hours, with a total reducing sugar of approximately 0.794mg/ml. These results was similar to Dawson and Boopathy (2008) who reported that the alkaline pretreatment with 2% hydrogen peroxide and soaking for 48 hours removed most of lignin compared to several other combination. It was also observed that the minimum total reducing sugars concentration was obtained when the bagasse pretreated with 5% H2O2 and pH 13 for 72 hours. It is worth mentioning that this pretreatment had the greatest weight loss. This might due to the degradation of simple sugars (xylose, glucose) at the high hydrogen peroxide concentration, pH and reaction times (Bhatia et al., 2014). Uppal et al. (2011) reported that the application of oxidizing agents produces soluble lignin compounds that inhibit the conversion of hemicelluloses and cellulose to ethanol. There is also loss of sugar due to the occurrence of non-selective oxidation. By comparing the oxidative and acids


pretreatments, the results indicated that diluted acid pretreatments were more effective than alkaline hydrogen peroxide pretreatment and these results are in agreement with Dawson and Boopathy (2008).

Ability of the fermenting microorganisms to utilize the whole range of sugars available from the hydrolysate is vital to increasing the economic competitiveness of cellulosic ethanol (Kumar et al., 2009). Bioethanol production from the bagasse hydrolysate was the final performance in this study. The sulfuric acid pretreated bagasse hydrolysate resulted from the saccharification by Trichoderma harzianum SC20 was used for fermentation by yeast isolate S. cerevisiae (Y17) to produce ethanol. The highest ethanol concentration from the bagasse hydrolysate was obtained after a fermentation period 48 hours recording 1.32% (w/v) with fermentation efficiency 51.81% and a volumetric productivity equal to 0.275g l

-1h

-1. These results were in

consistent with Ingale et al. (2014). Low yields of ethanol resulted from the fermentation of lignocellulosic materials might be due to presence of inhibitory compounds, whose composition and concentration depend on the type of lignocellulosic material, chemistry and nature of the pretreatment and mode of hydrolysis process (Taherzadeh and karimi, 2007). The variation in the pretreatment method, saccharification conditions and the yeast strain used in fermentation process might also effect on the ethanol yield. Conflict of interest The authors declare no conflicts of interest. REFERENCES Abdul Wahid OA, Abo-Seidah AA, Hawary HH (2015). Optimization of

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Chemical pretreatment and saccharification of … et al... · wood waste dump and recorded as high cellulase producer ... Fermentation of pretreated bagasse to ethanol ... Samples