INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 5, No 4, Copyright by the authors - Licensee IPA- Under Creative Commons license 3.

Transcription

1 INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 5, No 4, 2015 Copyright by the authors - Licensee IPA- Under Creative Commons license 3.0 Research article ISSN Utilization of cellulosic biomass as a substrate for the production of bioethanol PG and Research Department of Microbiology, PSG College of Arts and Science, Coimbatore, Tamilnadu, India raji.ajjii@gmail.com doi: /ijes ABSTRACT Energy is considered a prime agent in the generation of wealth and a significant factor in economic development. Recently there is renaissance in utilization of biomass for biofuel production employing cellulases and hence forth in obtaining better yields and novel activities. The present study deals with the bioconversion of cellulose from textile cotton waste into ethanol by using the methods of physical (Steam explosion method) and chemical pretreatment (Acid and Alkali), optimization of enzyme production and the ability to hydrolyze the cellulosic cotton biomass was also determined. The results of physical and chemical pretreatment revealed that the chemical pretreated substrate enriched the enzyme action, when compared to physical pretreatment method. The sugar analysis was achieved by DNSA method and the cellulose estimation was performed using Anthrone method. The enzyme production parameters such as temperature, ph, incubation time, inoculum concentration and agitation were optimized. The produced enzyme was partially purified by Dialysis followed by ammonium precipitation method and the ability to hydrolyze the cellulosic cotton biomass was also determined. The conditions for enzymatic hydrolysis were also optimized. Our energy systems should be renewable and sustainable, efficient and costeffective, convenient and safe. These problems make it urgent to develop an alternative energy resource that was both renewable and environmentally friendly. The purpose of the contemporary investigation was to recover the solid waste, improve the industrial application of cellulases and investigate the challenges in cellulase research exclusively in the direction of enlightening the process economics of energy production. Keywords: Cotton wastes, Pretreatment, Enzyme, Purification.. 1. Introduction Through the eternally increasing demand for energy and the fast diminishing petroleum resources, globally there is a better interest in substitute fuels, particularly liquid transportation energies (Wyman, 2007; Lynd., 2008). Energy crisis is one of the most serious threats towards the sustainability of human kind and civilization. Generally, bio-ethanol converted from edible source is called first-generation bio-ethanol (FGB). The most common disposal methods of the above waste include direct land application, composting and combustion. Waste textiles are mainly composed of cotton and viscose fibers, and holds, thanks to their cellulose content, a significant potential for production of different biofuels, such as biogas. The polymers of cellulose and hemicelluloses should be first released from the fibrils in pretreatment in order to have an effective hydrolysis (Zheng et al., 2009). Lignocellulose provides a cheap and abundant raw material, part of which can be converted to fermentable sugars through hydrolysis. Received on December 2014 Published on January

2 In developing countries, where adequate disposable technology is not much available, the cotton waste generated is mostly disposed off by scorching. Waste management is one of the biggest problems faced by textile industry. The wastes containing minute fibers which cause serious lung infection once it gets spread through air. They are often dumped as such or incinerated. A solution to this problem will be bioconversion of these wastes into ethanol using microorganisms present in the environment. Zheng et al., (2009) established a procedure for generating stimulated amorphous cellulose by acid pretreatment method. Silverstein et al., 2007 compared the effectiveness of sulphuric acid, sodium hydroxide and hydroden peroxide pretreatments for enzyme conversion of cotton stalks. When compared to the starch or sugar based resources, conversion of lignocelluloses is much more intricate due to their conflict to enzymatic attacks. Therefore, pretreatment is an important stage for cellulose bioconversion processes which helps to separate lignin and hemicellulose from cellulose, reduce the crystallinity of cellulose, and enhance the porosity of the materials. These process inturn supports the further enzymatic degradation (Vahid jafari 2011). The present study deals with the preliminary steps for the bioconversion of cotton wastes into bioethanol using pretreatment, production of enzyme, purification of produced enzyme and the determination of enzymatic hydrolysis. 2. Material and methods 2.1 Physical pretreatment Steam explosion method The steam explosion is typically initiated at a temperature of C (corresponding pressure, MPa) for several seconds to a few minutes before the material is exposed to atmospheric pressure. The biomass/steam mixture is held for a period of time to promote hemicellulose hydrolysis, and the process is terminated by an explosive decompression. The biomass/steam mixture is held for a period of time to promote hemicellulose hydrolysis, and the process is terminated by an explosive decompression. The process causes hemicellulose degradation and lignin transformation due to high temperature, thus increasing the potential of cellulose hydrolysis. Hemicellulose is thought to be hydrolysed by acetic and other acids released during steam-explosion pretreatment. The compositional analysis was done after physio chemical pretreatment using standard ASTM (1995) procedures. 2.2 Chemical pretreatment Acid and Alkali pretreatment About 50 ml of dilute sulphuric acid was prepared in concentration range of % at 0.5 % interval. The flasks added with 3 g of processed cotton wastes were separately autoclaved at 121 C for 30 min. The flasks containing the pretreated waste were then neutralized with distilled water. The samples were then estimated for the amount of glucose released according to DNSA method after pretreatment. The same procedure was carried out for alkali pretreatment. In order to dilute sulphuric acid, sodium hydroxide solution was used in alkali pretreatment. 2.3 Fungal and Enzymatic pretreatment 744

3 For fungal hydrolysis, formerly known strains for cellulose breakdown (Trichoderma ressei- MTCC No 164) was obtained from Microbial Type Culture Collection Center, Chandigarh. The medium selection was carried out for cellulase enzyme production. Selection and standardization of medium for Cellulase Enzyme production The fungal organism was grown in three different medium (Czapek Dox broth medium, Reese and Mandels Mineral Salts Medium and Production medium) to determine the preeminent composition for cellulase enzyme production. The fungal isolate (T.ressei) was grown in all the above mentioned medium and incubated at 27 ºC for 5 days with constant shaking at 150rpm. After incubation, the culture flasks were retrieved and the culture broth was transferred to sterile centrifuge tubes and centrifuged at 8000 rpm for 15 mins. The centrifugation continues until the cells get settled. After that, the supernatant was filtered through a sterile nylon cloth and stored at 4 ºC as cellulase enzyme source. Extraction was done under sterile conditions to prevent any microbial contamination. The preeminent medium was selected based on the cellulase enzyme production and its activity. The cellulase enzyme activity was assayed by determining the glucose released according to Anthrone method Optimization of the Cellulase Enzyme production parameters Effect of incubation time on production of cellulase enzyme Cellulase enzyme production broth was prepared, inoculated with T.ressei culture and incubated for hrs. Culture flasks were retrieved after every 24 hrs and the cellulase production was assayed according to Anthrone method Effect of ph on production of cellulase enzyme The optimum ph for cellulase enzyme production was demonstrated by preparing the production medium with ph values ranging from 4.0 to 8.5 and incubated for 120 hrs. The flasks were inoculated with T.ressei and then incubated. After incubation, the culture filtrates were assayed for cellulase production according to Anthrone method Effect of temperature on cellulase enzyme production The temperature optimum was studied by incubating the production medium (ph 7) inoculated with T.ressei at varying incubation temperature such as C for 120 h. The culture filtrates were retrieved and assayed for cellulase production Effect of substrate concentration on cellulase production The effect of substrate concentration on enzyme production was determined by incubating the inoculated culture vessels with the production medium containing varied substrate (Cellulose) concentration from 0.5% to 5.0% at an interval of 0.5 % (separately) for 120 h and assaying the cell free supernatant at the end of fermentation. After incubation, the optimum substrate concentration for cellulase production was determined Effect of agitation condition on cellulase enzyme production 745

4 To determine the effect of agitation on cellulase production, the production medium was kept under different shaking condition (0, 50, 100 and 150 rpm). After incubation the culture flasks were estimated for cellulase activity Purification of enzyme After optimizing the cellulase enzyme production system, the next step was to purify the enzyme to determine the characteristics of the enzyme. The crude enzyme synthesized under standardized conditions was extracted by centrifugation at 3000 rpm for 15 min followed by filtration through sterile nylon cloth Partial purification by ammonium sulphate precipitation The enzyme in the crude preparation was precipitated by the addition of ammonium sulphate to 80% saturation, followed by adding solid ammonium sulphate to 80% saturation (Iqbal et al., 2011). The mixture was left overnight at 4 ºC in a magnetic stirrer and centrifuged at 4000 rpm in a refrigerated centrifuge at 4 ºC for 30 min. The precipitate was redissolved in 10 ml of 0.02 M sodium phosphate buffer (ph 7.0). The partially purified enzyme was taken for dialysis. The dialysis bag was cut to the required length and allowed to boil for 10 min in 2% sodium bicarbonate and 1 mm EDTA (ph 8). The activated dialysis bag was used for the dialysis of the enzyme collected. About 10 ml of the partially purified enzyme obtained after the ammonium sulphate precipitation was dialyzed against 30 mm phosphate buffer (ph 7.4) at 4 ºC with three changes of buffer. The partially purified sample was assayed for enzyme activity. 2.4 Determination of Rate of Enzymatic Hydrolysis for cotton wastes The reaction was carried out in 0.05M Sodium Acetate buffer (ph 5). About 0.5g of pooled cotton waste sample was taken in 100ml flasks. About 20ml of sterile buffer and 0.2ml of the enzyme extract were added to the flask. An enzyme blank was also prepared without adding substrate and flasks were incubated at 50 C for 24 hours. The flasks were checked for the sugar content at 0 th hour and after 24 hours by means of DNSA method as mentioned in table 1. Table 1: Experimental plan for Enzymatic Hydrolysis of pooled cotton waste S.No Buffer (ml) Sample Enzyme Extract (ml) grams (A) grams pooled cotton waste sample (B) grams pooled cotton waste 0.2 (Commercially available sample (C) Cellulase enzyme) 3. Result and conclusion 3.1 Compositional analysis of untreated cotton waste The raw cotton wastes collected from textile mills were pooled together and their chemical composition were analyzed. Various components were analyzed in the cotton wastes such as moisture content, total sugars, acid insoluble residues, ash content and ethanol extractives. The compositions were mentioned in table

5 Table 2: Compositional analysis of cotton waste before pretreatment Compositional analysis Before pretreatment % Moisture 66 Ash 12.8 Acid insoluble residue 29.3 Total sugars 55.8 Ethanol Extractives 7.91 From the table 2, it was observed that the moisture content in the cotton wastes was predominant (66%), followed by total sugars in the cotton wastes was crucial (55.8%) and the acid insoluble residues (29.3%) respectively. The above results clearly indicate that the cotton waste with essential amount of sugars can be used as substrate for bioethanol production. The results obtained in this study were supported by Mahalakshmi et al., (2011) who similarly stated cotton wastes contained maximum total sugar concentration, followed by glucan and acid insoluble residues. The main composition of cotton gin waste is as follows: 22.40% of acid soluble lignin, % of cellulose, % of Hemicellulose, 10.2 % of ash content & 9.9 % of moisture content (Gupta 2009). 3.2 Compositional analysis of pretreated of Cotton waste The different cotton wastes used in the present study were processed mechanically and the chemical composition of the pooled cotton waste was determined according to the standard methods and the results were compared to the contents released before pretreatment and mentioned in table 3. The physiochemical pretreated samples exhibited and percentage of total sugars in compositional analysis. Steam explosion method i.e., destruction of a portion of the xylan fraction, incomplete disruption of the lignin carbohydrate matrix and generation of compounds inhibitory to microorganisms. Table 3: Compositional analysis of cotton waste after pretreatment Compositional analysis After pretreatment % Acid Pretreatment Alkali Pretreatment Physio chemical Pretreatment Moisture Ash Acid insoluble residue Total sugars Ethanol Extractives The result showed that acid pre-treated cotton waste was found to possess relatively higher percentage of total sugars (59.3%) compared to alkali pre-treated cotton wastes (46.80%).These observations agree with similar results obtained in previous works done by Mahalakshmi et al., (2011), Jiacheng Shen and Foster A. Agblevor (2009). Similarly, the percentage concentration of moisture and acid insoluble residues was also found to be relatively higher for acid pre-treated (77.80% and 30.31%) than for alkali pre-treated cotton wastes (72.2% and 24.86%). The correlation coefficient between the acid and alkali pre- 747

6 treated cotton waste was found to be with high degree of positive correlation (r=0.9967) thereby confirming that there was not much difference in the acid and alkali pretreatment. 3.3 Effect of ph on cellulase production by Trichoderma.reesei Thousands of microorganisms have the ability to grow on cellulose. Many of them grow quite rapidly, but only few produce extracellular cellulase that is capable of converting the native crystalline cellulose to sugars in vitro (Mandels, M.; Weber, J, 1969). Trichoderma reesei (MTCC 164) are the excellent sources of cellulase suitable for practical applications. Cellulase is an inducible enzyme in Trichoderma, with highest yields obtained when the fungus is grown on cellulose rich medium. The shake flasks, with nutrients and inoculum, were adjusted and controlled at different ph (3.5, 4.0, 4.5,5.0, 5.5,6.0 and 6.5) and incubated for 6 days. During incubation, samples were withdrawn for every 24 h and analyzed for the enzyme levels. Figure 1: Effect of initial ph on cellulase production by Trichoderma reesei Maximum activity was reached at ph 4.5 (Figure 1). The Filter paper (FP) activity increased with increasing incubation time. Enzyme activities were found to be higher with the mycelial inoculum compared to the spore inoculum and the absorbance were observed in UV-Vis Spectrophotometer. Inoculum age was also found to be important. The yield of cellulase in a cellulose culture is reduced unless a second more readily metabolized substrate is added. 3.4 Effect of incubation time on cellulase production by Trichoderma. reesei The effect of time on cellulase production was studied by incubating the production media for 7 days in orbital shaking incubator and determined for optimum time for maximum cellulase production. The extracellular enzyme produced in the media was determined by estimating the cellulase activity at different time duration (1, 2, 3, 4, 5, 6, and 7 days) and cellulase activities of 0.2, 1.02, 4.3, 6.4, 7.1, 7.8 and 6.6 IU/ml were obtained, respectively (Figure 2). The time of fermentation had a great effect on enzyme production as the maximum cellulase activity was found as 7.8 U/ml on 6th day. The highest cellulase level of 1.88, 1.53 and 2.40 IU/mL of cellulase activity was achieved on the 4th day of the fermentation period by Trichoderma harzianam and Phanerochaete chrysosporium respectively (Khan et al., 2007). Ojumu et al. (2003) found that the highest level of cellulase activity occurred at the 12th hr of 748

7 fermentation by Aspergillus flavus. It was perceived that a high concentration of reducing sugar was released on the 4th day of the fermentation (Khan et al., 2007). Similar drift was also reported in cellulase production using Trichoderma sp. Appropriate cultivation time was significant for growth and production (Liu and Yang, 2007). The time of fermentation had a great effect on enzyme production, as the cellulase activity of 7.8 IU/ml was obtained after 6 days of fermentation. However, further increase in the incubation time, reduced the enzymes production. It might be due to the depletion of macro- and micronutrients in the fermentation medium with the lapse in time, which stressed the fungal physiology resulting in the inactivation of secretory machinery of the enzymes. Figure 2: Effect of incubation time on cellulase production by Trichoderma reesei In addition, the substances were initially more susceptible, making a rapid rise in enzymes biosynthesis. But with the prolongation of cultural time, the susceptible portions were completely hydrolyzed by microorganisms, which inhibited the enzyme secretion pathways (Nochure et al., 1993). 3.5 Effect of temperature on cellulase production by Trichoderma reesei Incubation temperature plays an important role in the metabolic activities of microorganism. Temperature optimization was carried out by incubating the fermentation flask at 20 C, 25 C, 30 C, 35 C, 40 C, 45 C, 50 C, 55 C, 60 C and 65 C and the cellulase activities were found to be 6.2, 7.3, 6.9, 5.2, 2.9, 2.2, 1.6, 0.7, 0.1 and 0.0 IU/ml, respectively. (Figure 3) Thus, as shown in figure 12, maximum cellulase production was observed at 25 C, as the temperature increased the cellulase production gradually reduced, finally there was no enzyme production seen after 60 C. Any change, either increase or decrease in temperature resulted in the gradual decrease in protein production (Ikram-ul-Haq et al., 2006). A higher temperature alters the cell membrane composition and stimulates protein catabolism, thus causing cell death. The incubation temperature is a factor regulating the enzyme synthesis (Liu and Yang, 2007). Similarly Liu and Yang (2007) reported that maximum cellulase production of Trichoderma koningii was observed in the temperature range of C. 749

8 Figure 3: Effect of temperature on cellulase production by Trichoderma reesei 3.6 Effect of static and agitated condition on cellulase production by Trichoderma. reesei To determine the effect of agitation on cellulase production, the production media were kept under different shaking condition (0, 50, 100 and 150 rpm). After incubation the culture extract from all flasks were estimated for cellulase activity and the results were shown in figure 4. There was a strong influence on agitation on cellulase production; the cellulase production was high in shaking condition then in static condition. Figure 4: Effect of static and agitated conditions on cellulase production by T. reesei The maximum cellulase yield of about 8.33IU/ml was obtained in the flask kept at 150 rpm. The enzyme production in the flask kept at shaking condition of 0, 50 and 100 was 7.47 IU/ml, 7.70 IU/ml and 8.00 IU/ml respectively. When the shaking speed was increased beyond 150 rpm, a slight drop in enzyme production was observed, which could be due to the fact that the increase in the rpm level has resulted in the coagulation of the organisms to form lumps and decrease in rate of mass transfer. Similar result was reported by Lejeune and Baron, (1995) that the enzyme production of T. reesei was strongly affected by the agitation 750

9 and at the higher agitation rates the enzyme production was dropped. On the other hand, lower agitation speed of less than 130 rpm resulted in low growth, which thus resulted in low enzyme production. This could be due to low amount of dissolved oxygen in the cultivation medium. 3.7 Purification of cellulase enzyme The harvested enzyme was then purified to study its characteristics of the enzyme. Crude enzyme produced under standardized conditions was centrifuged to extract the enzyme. 3.8 Partial purification by ammonium sulphate precipitation Ammonium sulphate precipitation was carried out at 80% saturation. Precipitate obtained was redissolved in sodium phosphate buffer. The precipitated enzyme was dialyzed against 30 mm phosphate buffer with three changes of buffer. Cellulase enzyme activity was calculated according to DNSA method and enzyme activity was found to be 1.18 IU/ml/min. (Iqbal et al., 2011). 3.9 Determining the rate of Enzyme Hydrolysis The Rate of enzymatic hydrolysis of cotton waste samples were computed from concentration of glucose released per hydrolysis time: Where, v = enzyme hydrolysis rate (mg/ml glucose per hour) Glut = Concentration of glucose at time, t (mg/ml), Glu0 = Initial glucose concentration at time = 0 h (mg/ml), t = hydrolysis time (h), and to = time = 0 hour (h). The rate and fidelity of the indigenous enzyme on the pooled cotton waste samples were calculated based on the sugar released with the formula mentioned and the results are mentioned in Table 4. The results revealed that the sample B and C exhibited similar glucose reaction rate. Table 4: Determination of rate of enzymatic hydrolysis S No Sample Reaction Rate (moles glucose released per hour) 1 Sample A Sample B Sample C

10 4. Conclusion India is a fast growing economy with an inherent increase in demand for energy. The country with a positive outlook towards renewable energy technologies and committed to the use of renewable sources to supplement its energy requirements. The country is one among the few nations to have a separate ministry for renewable energy which address the development of biofuels along with other renewable energy sources. The solution to renewable transportation fuels may not exclusively be bio-ethanol, but it will definitely play a significant part. The country lacks mature technologies for ethanol production from lignocellulosic biomass and though biomass itself is cheap, the costs of its processing are relatively higher. Various magnum stanches in such technologies comprises the pretreatment of biomass, enzymatic saccharification of the pretreated biomass, and fermentation of the hexose and pentose sugars released by the hydrolysis and saccharification. One of the major difficulties that would be faced by bio-ethanol technology developers as well as future entrepreneurs will be the choice of feedstock. Though India generates a huge amount of biomass residues as agro, solid waste and forest residues, the only feasible feedstock among these would be the solid waste predominantly cotton wastes residues due to problems in pooling and logistics. The main objective of this research was to break the sugars, which is present in the solid waste i.e., cotton waste using pretreatment, Enzymatic saccharification and Microbial Fermentation. In this present study, the major strides of bioethanol production were carried out. The possibility of converting cotton wastes to bioethanol via pretreatment and enzymatic hydrolysis. However, they need a pretreatment for the better yield of sugar release which would help to provide more yield of bioethanol on further fermentation. It must also be accentuated that the use of textile cotton waste as a secondary fuels are essential to fulfil the energy requirements of the biorefinery with hydrolysis processes. Acknowledgement The Authors would like to thank Department of Biotechnology (DBT). This work was financially supported by the Department of Biotechnology (DBT), Government of India. 6. References 1. Wyman, C.E., (2007), What is (and is not) vital to advancing cellulosic ethanol. Trends biotechnology, 25, pp Lynd, L.R., Laser, M.S., Bransby, D., Dale, B.E., Davison, B., Hamilton, R., Himmel, M.,Keller, M., McMillan, J.D., Sheehan, J., Wyman, C.E., (2008), How biotech can transform biofuels. Nature biotechnology, 26, pp Zheng, Y., Pan, Z. & Zhang, R., (2009), Overview of biomass pretreatment for cellulosic ethanol production. International journal of agricultural and biological engineering, 2(3), pp Silverstein, R.A. Chen, Y., Sharma- Shivappa, R.R., Boyette, M.D., and Osborne J., (2007), comparison of chemical pretreatment methods for improving saccharification of cotton stalks. Bioresource technology, 98, pp Vahid Jafari, Sara R. Labafzadeh, Alistair King, Ilkka Kilpeläinen, Herbert Sixta and Adriaan van Heiningen., (2014), Oxygen delignification of conventional and high 752

11 alkali cooked softwood Kraft pulps, and study of the residual lignin structure RSC Advances, 4, pp Iqbal, H. M. N., Ahmed, I., Zia, M. A. & Irfan, M., (2011), Purification and characterization of the kinetic parameters of cellulase produced from wheat straw by Trichoderma viride under SSF and its detergent compatibility. Advances in Bioscience and Biotechnology, 2, pp Mahalakshmi M, Ankayarkanni J, Rajendran R, Rajesh R., (2011), Bioconversion of cotton waste from textile mills to bioethanol by microbial saccharification and fermentation. Annals of biological research 2(3), pp GK Gupta., (2009)., Master of Technology in Biotechnology and Medical Engineering, Thesis, Department of Biotechnology and Medical Engineering, National Institute of Technology (Rourkela). 9. Jiacheng Shen and Foster A. Agblevor., (2009), Ethanol production from mixture of cotton gin waste and recycled paper sludge. ASABE annual meeting, Reno, Nevada, June Mandels M., Weber J., (1969), Production of cellulases. Advances in chemistry Series, 95, pp Zuber Khan and Anjani K. Dwivedi., (2013), Fermentation of biomass for production of Ethanol: A review, Universal journal of environmental research and technology, 3(1), pp Liu, Q., Peng, S. L., Bi, H., Zang, Y., Li, Z. A., Ma, W. H. & Li, N. Y., ( 2005), Decomposition of leaf litter in tropical and subtropical forests of Southern China. Journal of tropical forest science, 17 (4), pp Nochure, S.V., Roberts, M.F. and Demain, A.I., (1993), True cellulases production by Clostridium thermocellum grown on different carbon sources, Biotech. Letters, 15, pp Ikram-Ul-Haq, Kiran Shahzadi, Uzma Hameed, Muhammad Mohsin Javed and Qadeer,M.A., (2006), Solid state fermentation of cellulases by locally isolated Trichoderma harzianum for the exploitation of agricultural byproducts. Pakistan Journal of biological Sciences, 9(9), pp Lejeune R J., Nielsen and G.V. Baron., (1995), Morphology of Trichoderma ressei QM 9414 in Submerged cultures, Biotechnology and Bioengineering, 47, pp