Biofuels
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Comparison of different pretreatment methods for efficient conversion of bagasse into ethanol Asma Siddique, Ambreen Gul, Muhammad Irfan, Muhammad Nadeem & Quratulain Syed To cite this article: Asma Siddique, Ambreen Gul, Muhammad Irfan, Muhammad Nadeem & Quratulain Syed (2017) Comparison of different pretreatment methods for efficient conversion of bagasse into ethanol, Biofuels, 8:1, 135-141, DOI: 10.1080/17597269.2016.1215066 To link to this article: http://dx.doi.org/10.1080/17597269.2016.1215066
Published online: 05 Aug 2016.
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Date: 01 February 2017, At: 07:13
BIOFUELS, 2017 VOL. 8, NO. 1, 135141 http://dx.doi.org/10.1080/17597269.2016.1215066
Comparison of different pretreatment methods for efficient conversion of bagasse into ethanol Asma Siddiquea, Ambreen Gula, Muhammad Irfan
a,b
, Muhammad Nadeema and Quratulain Syeda
a Food & Biotechnology Research Center (FBRC), Pakistan Council of Scientific and Industrial Research (PCSIR) Laboratories Complex Ferozpur Road Lahore Pakistan 54600, Lahore, Pakistan; bDepartment of Biotechnology, University of Sargodha, Sargodha, Pakistan
ABSTRACT
ARTICLE HISTORY
Biofuel production is popular with biotechnologists due to the continuously increasing need for energy. One major issue is to get maximum fuel production for minimum cost. In our studies we focused on both factors and developed a method for bioethanol production by investing in low cost chemicals. We compared three chemical pretreatments (NaOH, Na2SO3 and H2O2) at 121 C temperature and 15 psi pressure for delignification to evaluate the efficacy of chemicals in pretreatment. NaOH and Na2SO3 showed comparable percentage delignification (81.3 and 80.17% respectively) while H2O2 showed 67.4% delignification. Endogenously produced cellulolytic enzyme with 30 IU/mL CMCase and 22 IU/mL FPase units was used for saccharification of exposed cellulose. An indigenously developed strain of Saccharomyces cerevisiae SC36 (developed in a peptone free high gravity media) was used to ferment saccharified sugars into ethanol at 37 C fermentation temperature. We were able to convert 27% of the total bagasse (as per dry mass basis) into ethanol with NaOH pretreatment followed by 26% bagasse (as per dry mass basis) into ethanol with Na2SO3 pretreatment and 1% ethanol was obtained from H2O2 treated bagasse.
Received 5 March 2016 Accepted 4 July 2016
Introduction In Pakistan, 50 million tons of annual sugarcane production gives rise to almost 10 million tons of bagasse. [1] Since bagasse is a by-product of the cane sugar industry, the quantity of production in each country is in line with the quantity of sugarcane produced.[2] Pakistan is the 5th largest country which produces millions of tons of bagasse annually. Sugarcane bagasse production in Pakistan on the basis of cane crushed in 20082009 was 17,835,000/m ton per annum or 7431 m ton/h.[3] To reduce the dependency on oil and conventional fuels, research into lignocellulosic biofuel has received a great deal of attention. One of the major lignocellulosic materials to be considered in tropical countries is sugarcane bagasse, the fibrous residue obtained after extracting the juice from sugarcane in the sugar production process. The cellulose of the lignocellulosic biomass can easily be converted into fermentable sugars by the action of cellulolytic enzymes. However, the low bioconversion efficiency of the lignocellulosic materials into fermentable sugars is an enormous challenge for biotechnology. Untreated lignocellulose is hard to hydrolyze due to the crystalline structure of the cellulose and due to the presence of lignin.[4] Lignin hampers the approach of cellulolytic enzyme towards cellulose. Pretreatment is required to alter the structural and CONTACT Muhammad Irfan
Bagasse; pretreatment; saccharification; fermentation; Saccharomyces cerevisiae; ethanol
chemical composition of the lignocellulosic biomass to facilitate rapid and efficient hydrolysis of carbohydrates to sugars.[5] A wide range of physical (comminution, hydrothermolysis), chemical (acid, alkali, solvents, ozone), physico-chemical (steam explosion, ammonia fibre explosion), and biological pretreatment techniques have been used to improve the accessibility of enzymes to cellulosic fibres (Figure 1).[6] Alkali pretreatment has been considered a promising approach for delignification of biomass.[7] During alkaline pretreatment the first reactions taking place are solvation and saponication, making it more accessible for enzymes. Alkali also changes the cellulose structure to a denser and thermodynamically more stable form than the native cellulose. It is also used in small amounts.[8,9] Oxidative pretreatment and the use of other chemicals (sodium sulphite, sodium sulphide) is also a common practice.[10,11] Hendriks and Zeeman [8] concluded that there is little differentiation between the economic performances of all pretreatment options, but the optimization of enzyme blends with pretreated material and the conditions of hydrolysis may result in great differentiation in process economy. Solid state fermentation is an attractive process to produce cellulase economically due to its lower capital investment and lower operating expenses.[12,13]
[email protected];
[email protected]
© 2016 Informa UK Limited, trading as Taylor & Francis Group
KEYWORDS
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Figure 1. The process of ethanol production from lignocellulosic substrate.
Trichoderma reesei is widely used as a cellulase producer in solid state fermentation using different biomasses (sugarcane bagasse, rice straw, wheat straw) as substrate. Our country’s growing fuel demand stimulated us to exploit the potential of a native resource for alternate fuel (biofuel). Indigenous production will definitely reduce the cost of the product. The purpose of pretreatment optimization is to screen the best solvent for maximum delignification and cellulose exposure by simultaneously less degradation of released glucose. Different types of acidic and alkaline chemicals have been the focus of research studies, which, despite their advantages for the fermentation
industry, are not safe for the environment. Therefore, the current study included a human and environmental friendly ‘green’ chemical for the optimization of the complete process of lignocellulosic ethanol fermentation. Three different pretreatment chemicals including sodium sulphite were used to evaluate their efficacy towards delignification of bagasse and ethanol production. The conversion of cellulose to ethanol was calculated after enzymatic saccharification and fermentation by yeast (Saccharomyces cerevisiae). This study aimed to develop a proper pretreatment technique for maximum saccharification to produce ethanol from sugarcane bagasse and make it a costeffective process.
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Experimental
(CMC) in 0.05 M citrate buffer and pH 4.8. After incubation at 50 C for 30 min, the reducing sugar content was assayed by 3,5-dinitrosalicylic acid (DNS).[19,20]
Lignocellulosic biomass Sugarcane bagasse was used as a source of lignocellulosic biomass, which was kindly provided by Shakarganj Sugar Mills (Pvt.) Limited, Jhang Road, Faisalabad. The biomass was washed and dried to remove the unwanted particles and then milled into powder form (2 mm) with a hammer beater mill.
Pretreatment of biomass The pretreatment method described by Irfan et al. [14] was used after some modifications. The substrate was washed and dried to remove the unwanted particles and then milled into powder form (2 mm) with hammer beater mill. Ten gram substrate was soaked in different concentrations of Na2SO3 (0.510%, w/v), NaOH (0.53%, w/v) and H2O2 (15%, v/v) solutions at the ratio of 1:10 (solid:liquid) for 2 h at room temperature. The samples were then heated at 121 C for 60 min at 15 psi. Samples were filtered and solid residues were washed to neutrality.
Cellulose and lignin estimation The cellulose content was estimated by the method of Gopal and Ranjhan.[15] Lignin content was estimated by the method described by Milagres.[16] Delignification was calculated using the following formula:[17] Delignification ð%Þ D
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Lu £100 Lu ¡ Lt
where Lu D Lignin (untreated sample), and Lt D Lignin (treated sample).
Cellulolytic enzyme production and extraction For enzyme production, 10 g of untreated bagasse was taken in a 250 mL flask with 20 ml of Vogel’s salt media.[18] This was autoclaved at 121 C and 15 psi for 20 min. The experiment was run in triplicate. Into the cooled medium was added 5 ml of freshly prepared inoculum of Trichoderma reesei and the flask was incubated at 30 C. Maximum enzyme production was achieved by conducting time-scale experiments for enzyme production. The enzyme was harvested after 96 h of inoculation with 100 ml (for each flask) of 0.5 M sodium citrate buffer (pH 4.8).
Estimation of filter paper (FPase) activity The FPase activity of the indigenously produced enzyme was determined by incubating 1 ml of diluted enzyme with 1£6 cm strip of filter paper (Whatman no.1) in 0.05 M citrate buffer (pH 4.8). After incubation at 50 C for 30 min, the reducing sugar content was assayed by 3,5-dinitrosalicylic acid. Concentration of sugar released was determined by standard curve of glucose.[19,20]
Enzymatic saccharification From all the pretreated materials the best delignified materials were chosen for enzymatic saccharification. For that, 1 g untreated (control), 3.5% Na2SO3, 3% H2O2 and 2.5% NaOH treated bagasse and 50 ml crude enzyme were added in each flask and incubated at 50 C for 6 h in a water bath shaker at 50 rpm. Released reducing sugars were estimated through a DNS method as described by Miller.[19] Hydrolysis rate was determined by the formula illustrated by Niu et al.:[21] Hydrolysis rate ð%Þ D
Reducing sugars ðgÞ£0:9 £100 Substrate ½Cellulose ðgÞ C Hemicellulose ðgÞ
Ethanol fermentation A previously developed thermotolerant and ethanol tolerant Saccharomyces cerevisiae strain SC36 was employed for ethanol fermentation of saccharified biomass under micro-aerobic conditions. The strain SC36 was previously optimized for thermotolerance and high gravity fermentation.[22] Saccharified bagasse was filtered under aseptic conditions to remove the spent biomass residue and the filtrate was supplemented with yeast extract (0.5%) and MgSO4.7H2O (0.3%). To the fermentation broth a 2% inoculum of freshly grown yeast cells were added and incubated at 37 § 1 C for 72 h. Ethanol production was monitored every 24 h by the dichromate method as described by Caputi et al. [23] and substrate consumption was analyzed by the DNS method as described by Miller.[19]
Statistical analysis Estimation of carboxymethylcellulase (CMCase) activity For estimation of CMCase units, 1 ml crude enzyme (filter separated) was incubated with 1 ml of 1% substrate
All experiments were conducted in triplicate and the mean of three values was used for graph preparation. The data analysis and graph preparation was performed using MS Excel software.
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Figure 2. The effect of different pretreatments on delignification, cellulose and lignin content of sugarcane bagasse.
Results and discussion Pretreatment Bagasse was exposed to NaOH, Na2SO3 and H2O2 for the pretreatment process. The efficiency of the pretreatment process was evaluated in terms of percentage delignification of the raw biomass upon exposure to aforementioned chemicals (see Figure 2). According to the results there is no significant difference between the NaOH and Na2SO3 resulting in 72 and 71% delignification of bagasse respectively whereas H2O2 depicted only 57% delignification of bagasse. The three chemicals have three different modes of actions with lignin solubilization. The alkali NaOH increases the porosity of biomass by de-esterification of cross-linked bonds between lignin and xylan. Oxidative delignification as a result of H2O2 treatment results in loosening of the lignocellulosic matrix.[24] The mode of action of Na2SO3 is not well
documented. It might contribute to pretreatment by formation of thio-radicals, which ultimately end in residual phenolic compounds of lignin. Idrees et al. reported that Na2SO3 increases the hydrophilicity of the lignin by its sulphonation and decreasing the xylan and cellulose crystallinity.[10] Cellulose exposure is also described in Figure 2 where NaOH, Na2SO3 and H2O2 caused 71, 69 and 56.2% cellulose exposure respectively.
Saccharification Glucose released after enzymatic saccharification of delignified material is shown in Figure 3. In comparison to untreated bagasse, the NaOH treated bagasse resulted in maximum free glucose after enzymatic saccharification in a solid state fermentation process by Trichoderma reesei. It can be concluded that NaOH treatment resulted in much less inhibitory compounds which resulted in maximum saccharification as
Figure 3. Total glucose content released after different pretreatments on sugarcane bagasse.
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Figure 4. The effect of different pretreatments on saccharification and conversion of cellulose fibres to sugars from sugarcane bagasse.
compared to Na2SO3 and H2O2 treatment. The enzymes derived from Trichoderma reesei are best characterized and often used for enzymatic saccharification. Additionally, the use of agro-industrial waste as cheap fermentation substrate makes the process very economical.[12,24] Enzyme produced by solid state fermentation of bagasse yielded 30 IU/mL of CMCase and 22 IU/mL of FPase activity. This enzyme was used for the hydrolysis of exposed cellulose in pretreated samples to sugars. During the hydrolysis process, 0.5 g glucose/g of substrate was released from NaOH treated samples followed by Na2SO3 treated samples (0.44 g glucose/g of substrate) as illustrated in Figure 4. Conversion efficiency of substrate into glucose from H2O2 treated samples was less than the other two types of treatment. These results are in agreement with the study by Silversein et al.[25] Conversion of cellulose to glucose and saccharification of all treated samples were found to be parallel to the delignification of all treated samples. NaOH treated samples showed less lignin content after treatment (see Figure 2). It was concluded that maximum cellulose was exposed in NaOH treated bagasse, which directly influenced the contact of enzyme with cellulose, which resulted in maximum percentage saccharification (see Figure 4). Despite a tremendous amount of research in the utilization of lignocellulosic substrate for biofuel production, there is still a lack of efficient technology which can result in 100% conversion of cellulosic substrate into monomeric sugars.[26]
Ethanol fermentation As the industrial processes need high product yield and minimum investment both in terms of media components and energy consumption, ethanol fermentation was developed for high product formation in low cost medium.[27] This is only possible if the yeast strain can be modified to work in the presence of high gravity media and at high temperature.[28] For this reason, Saccharomyces cerevisiae SC36 was developed to tolerate the stressed environment and used for ethanol fermentation of the enzymatic hydrolysates. This SC36 strain was able to ferment 300 g/L glucose at 37 C at pH 5.0.[22] After the fermentation process, 60% of sugars from Na2SO3 treated and 54% of sugars from NaOH treated samples were converted to ethanol. Figure 5 explains the complete process results. Comparing the final production of ethanol from all four samples, 27% of Na2SO3 treated, 26% of NaOH treated and 2% of H2O2 treated sample was finally converted into ethanol (see Figure 5). Although the substrate to product conversion after H2O2 treatment was not very high as compared to Na2SO3 and NaOH, it was significant when compared to the untreated sample. In the untreated control, 1 g bagasse produced 0.029 g ethanol, which was 2.9% conversion of substrate into product. The saccharification of the treated bagasse, however, showed maximum yield with the NaOH treated sample but the result of ethanol fermentation was high with the Na2SO3 treated bagasse. This clearly implies that in
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Figure 5. Summary of ethanol production from sugarcane bagasse after different pretreatments.
the case of Na2SO3 treatment the inhibitory compounds are either much minimized or they are not affecting the ethanol fermentation potential of the yeast strain. The high salt content after NaOH treatment may have resulted in decreased ethanol yield during the fermentation process.[28] Khuong et al. [29] pretreated sugarcane bagasse with 0.8% NaOH for 60 min at 121 C and obtained ethanol production of 4.5 g/l after 24 h of fermentation with Phlebia sp. MG60. A recent study suggested that alkaline sulphite pretreatment of corn stover is a suitable process for production of fermentable sugars.[30,31]
Acknowledgement The authors would like to thank the Ministry of Science and Technology (MoST), Islamabad, Pakistan, for providing the financial support to carry out this research work under PSDP project entitled Production of Bioenergy from Plant Biomass.
Disclosure statement No potential conflict of interest was reported by the authors.
ORCID Muhammad Irfan
http://orcid.org/0000-0003-2955-4237
Conclusion The chemical treatment of bagasse was tested at different levels from saccharification to ethanolic fermentation which highlighted the best chemical for lignocellulosic ethanol fermentation. Researchers are continuously seeking the most effective, safe and ecofriendly chemical for this purpose. The efficiency of the chemical was measured on the basis of substrate to sugar conversion and sugar to ethanol conversion. In our study, sodium sulphite was found to be the most promising chemical for the pretreatment of bagasse and subsequent ethanol production. Apart from being an effective pretreatment substance, this chemical has already been used for meat and fruits preservation, chlorine detoxification in pools and many other industrial processes hence rendering it a safe or ‘green’ chemical.
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