ISSN: 1579-4377

PRODUCTION OF BIO-ETHANOL FROM CELLULOSIC COTTON WASTE THROUGH MICROBIAL EXTRACELLULAR ENZYMATIC HYDROLYSIS AND FERMENTATION
Arthe R, R. Rajesh*, E.M.Rajesh, R. Rajendran, S. Jeyachandran*,. PG & Research Department of Microbiology, PSG College of Arts & Science, Coimbatore, Tamil Nadu, India. *Department of Botany and Microbiology, A.V.V.M. Sri Pushpam College, Poondi, Thanjavur, Tamil Nadu, India. r_arthe@yahoo.co.in

ABSTRACT Global warming alerts and threats are on the rise due to the utilization of fossil fuels. Alternative fuel sources like bioethanol and biodiesel are being produced to combat against these threats. Bioethanol can be produced from a range of substrates. Cellulose rich substances like cotton waste which is generated in tons in agricultural countries like India can be used for the production of bioenergy in the form of bioethanol with the help of microbial catalytic enzyme cellulose. In this study, we studied the bioconversion of cellulosic cotton waste to ethanol with the help of cellulase enzyme synthesized from Trichoderma reesei (MTCC#164). Also the cotton based industries face difficulties in the safe disposal of the waste generated and hence this approach is aimed in order to produce wealth out of waste. Most cellulosic biomass is not fermentable without appropriate pretreatment methods and so dilute sulfuric acid pretreatment was applied to make the cellulose contained in the waste susceptible to cellulase enzyme. A range of acid pretreatment of cotton waste was made and the pretreated cotton waste samples were fermented with Saccharomyces cereviseae. The sample that was pretreated with 3% dilute sulfuric acid gave an ethanol yield of 8.9 g l-1 respectively. KEYWORDS Bio-ethanol, Cellulosic cotton, microbial, enzymatic hydrolysis, fermentation.

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INTRODUCTION Emission of green house gases is increasing everyday with the fast depleting oil resources. Utilization of fossil fuels in the form oil, natural gas, and coal, which modern society relies on for energy contributes significantly to global warming. Alternative fuels made from renewable resources, such as fuel ethanol, provide numerous benefits in terms of environmental protection, economic development, and national energy security (5). Much research is been poured in finding an alternative fuel through biological ways because of the positive environmental benefits of biofuels. Potential feedstocks for biofuel production include the cellulosic biomass as well as waste materials, occurring in abundance outside human food chain which can be obtained throughout the year and are relatively inexpensive. Cellulosic materials are renewable natural biological resources and generation of biobased products and bioenergy from such substances is important for the development of humans (20). Various industries across the world generate huge volumes of cellulosic waste which have an immense potential to be utilized for the production of several bio-products (6). These dedicated substances provide a low-cost and uniquely sustainable resource for production of many organic fuels and chemicals that can reduce greenhouse gas emissions, enhance energy security, improve the economy, dispose of problematic solid wastes, and improve air quality (28). There are mainly two processes involved in the conversion: hydrolysis of cellulose in the lignocellulosic biomass to produce reducing sugars, and fermentation of the sugars to ethanol (26). Cellulose can be effectively hydrolyzed and depolymerized in to fermentable sugars by the enzyme cellulase. Cellulase based strategies make the process of biorefinery more economical by means of utilizing cheaper substrates for enzyme synthesis (1, 20). A number of microorganisms are capable of producing extracellular cellulase enzyme and among which fungi are the widely used candidates for cellulase enzyme production. Currently most commercial cellulases are produced from Trichoderma sp. and Aspergillus sp (11). Cellulase is a term usually used to describe a mixture of cellulolytic enzymes whose synergistic action is required for effective breakdown of substrate to its monomeric units. The action of cellulases involves the concerted action of (i) endoglucanase(s), which randomly attacks the internal, 1,4-linkages, (ii) cellobiohydrolase, which cleaves off cellobiose units from the nonreducing ends of the glucan, and (iii) 3-glucosidase, which hydrolyzes cellobiose to glucose. Most of the -glucosidic bonds in naturally occurring lignocellulosic materials are inaccessible to cellulase enzymes and also cellulose in naturally occurring materials is closely associated with hemicellulose and other structural polysaccharides, and carbohydrate-rich microfibrils are surrounded by a lignin seal. The substrate usually requires a pretreatment process before being subjected to enzymatic breakdown which is aimed at increasing the susceptibility of cellulose to enzyme. Current leading pretreatment technologies include the use of dilute acid, hot water, flow through, ammonia fiber explosion (AFEX), ammonia recycle percolation, lime, microwave, steam explosion and organsolv which are intensively investigated (12, 14). The overall performance of cellulase mixture is highly dependant on the residual lignin present along with cellulose. Cellulosic residues such as wheat straw, corn stover, rice straw, corn cobs and other agricultural wastes are the most available renewable resources which store derived energy in their chemical bonds (22).

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Cotton is one of the important cash crops of India and the cotton based textile mills situated especially in southern region generate huge volumes of fibrous cotton waste rich in cellulose everyday. These cotton wastes containing minute fibers when been suspended in air may cause serious manifestations affecting mainly lungs. They are often dumped as such or incinerated. Currently, sugar industry wastes are utilized as raw material for biofuelbioethanol production in India. Waste management is a major problem faced by cotton industries and hence utilization of cotton waste for the production of value added product bioethanol is a sound method for the safe disposal of wastes. The fibrous cotton waste is usually comprised of 97-98% cellulose and has less or no lignin apart from containing impurities. Optimal mild pretreatment method is required to make the cellulosic cotton fibers present in the waste susceptible to attack by the cellulolytic enzymes. This study was therefore initiated to 1) recycle the cotton waste and produce fermentable sugars, 2) evaluate the pretreatment efficacy with sulfuric acid that can yield more sugar release from cotton waste upon enzymatic hydrolysis and 3) check whether sugars released are of fermentable category with Saccharomyces cervisiae. MATERIALS AND METHODS Substrate Cotton waste of D1 phase was obtained from Sri Kannapiran Mills Pvt. Ltd., Coimbatore, India. Prior to pretreatment the waste containing lengthy cotton fibers was processed mechanically to reduce the length of fibers. The minor impurities were removed by means of washing and were oven dried at 40C overnight. Substrate Pretreatment About 200ml of dilute sulfuric acid was prepared with a concentration range of 0%, 0.5%, 1%, 1.5%, 2%, 2.5% and 3% in separate 500ml Erlenmeyer flasks. The flasks were added with 5g of processed cotton separately and autoclaved for 30 minutes. The flasks were then neutralized by washing with distilled water and the samples were dried separately for further use. Cellulase Enzyme Production (7, 17, 25) Trichoderma reesei (MTCC # 164) was cultured on Potato Dextrose Agar Plates, incubated at 28 C for seven days for the development of spores. The basal medium used for growth of Trichoderma reesei and cellulase production was Reese and Mandels Mineral salts solution containing a pH of 4. About 200 ml of the basal medium was prepared in two sets of 500 ml Erlenmeyer flasks, autoclaved for 30 minutes and seeded with a spore suspension of Trichoderma reesei. The flasks were incubated at 28 C for 5 days. Extraction of Enzyme After 5 days of incubation the flasks were retrieved from shaker. Culture broth was transferred to sterile centrifuge tubes, centrifuged until mycelia settled and supernatant was filtered in a sterile nylon cloth. Extraction was done under sterile conditions to prevent any microbial contamination. The crude filtrate containing enzyme is assayed for its activity in terms of filter paper units through DNS method.

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Enzymatic Hydrolysis of Pretreated Cotton Waste The reaction was carried out in 50mM Sodium Acetate buffer (pH 5) and 2.5g of each of samples were taken in separate 500ml flasks containing 250 ml of buffer. About 2.5g of the untreated sample was also taken in a separate flask. The flasks were added 0.5ml of the enzyme extract. An enzyme blank was also prepared without adding substrate and flasks were incubated at 50C for 24 hours. The flasks were estimated for the sugar content at 0th hour and after 24 hours by means of DNSA method. Determination of Cellulose Conversion Percentage (10) C.C. = [ (Glut - Glu0) / Cellulose ]* 100% Where, C.C. = Cellulose Conversion: Concentration of glucose released in time, t per amount of concentration of available cellulose (mg/mL glucose / mg/mL cellulose), Glut = Concentration of glucose at time, t (mg/mL), Glu0 = Initial glucose concentration at time = 0 h (mg/mL), and Cellulose = Concentration of available cellulose (mg/mL). Determination of Rate of enzyme Hydrolysis Enzyme hydrolysis rates were computed as concentration of glucose released per hydrolysis time: dS = Glut - Glu0 dt t - to enzyme hydrolysis rate (mg/mL glucose per hour) Concentration of glucose at time, t (mg/mL), Initial glucose concentration at time = 0 h (mg/mL), hydrolysis time (h), and time = 0 hour (h). v= Where, v Glut Glu0 t to = = = = =

Fermentation Saccharomyces cervisiae was selected for the fermentation of released sugars. Heavy inoculums of 1ml of the yeast enriched in Saborauds Dextrose Broth was transferred to all the flasks containing the released sugars and were kept for fermentation at 28C for 72 hours on a shaker at 120rpm. The alcohol content of all the flasks upon incubation were estimated at 24 hours time intervals as outlined by Caputi et al., (2). RESULTS AND DISCUSSION Based on the literature and due to the efficient enzyme production at laboratory studies, Trichoderma reesei was selected as the best cellulase producing strain. Trichoderma reesei was cultured in Reese and Mandels Mineral Salts Medium and incubated at 28C under shaking conditions. The Cellulolytic activities of the flasks were monitored at 24 hours interval for 7 days and estimated in terms of FPU. The results are presented in Figure. 1. It is clearly illustrated from the figure that there was a steady increase in the enzyme activity as the incubation time increase up to 120 hours. The activity was estimated to be 0.52U during 120 hours of incubation. After which the enzyme extract was prepared and stored.

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0.6

Filter Paper Units (IU/ml)

0.5 0.4 0.3 0.2 0.1 0 1 2 3 Days of Incubation 4 5 Series1

Figure 1. Influence of Time on Cellulase Enzyme Synthesis in Reese and Mandels Mineral Salts Medium using Trichoderma reesei. -- represents the Enzyme activity.

Acid pretreatment had a greater influence on the sugar release through enzymatic hydrolysis of the cotton waste. An increase in acid severity in terms of concentration resulted in higher sugar releases and the results are presented in the Figure. 2. The figure shows that there was a sudden increase in the release of sugars when the concentration of acid used for pretreatment was increased from 0 to 0.5%. The amount of sugar released with the 0% acid treated cotton waste was about 55mg/ml and 295mg/ml when 0.5% acid was used which increased to a maximum of 400mg/ml gradually when the acid concentration was increased up to 3%.
450

Sugar Released upon Enzymatic Hydrolysis (mg/ml)

400

350

300

250
Series1

200

150

100

50

0 0 0.5 1 1.5 2 2.5 3

Severity of Acid Pretreatment on Cotton Waste (%)

Figure 2. Efficiency of Acid Pretreatment on Enzymatic Hydrolysis of Cotton Waste. -- represents the amount of sugar released upon enzymatic hydrolysis.

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The percentage of cellulose converted and the available form of substrate after enzymatic hydrolysis of various concentrations of acid pretreated cotton waste samples were calculated based on the amount of sugar released. The results are presented in the Table 1. The results show that the percentage of cellulose conversion increased with the increased concentrations of acid used for pretreatment and it was also noted that the available form of substrate reduced with increased concentrations of acid. The cellulose conversion percentage increased from 2.2% to 16% when the acid concentration increased from 0 to 3% during pretreatment. The available form of substrate decreased from 97.8 % to 84% during the enzymatic saccharification of pretreated cotton waste.
Table. 1. Effect of Acid Pretreatment on Cellulose Conversion and Available Substrate after Enzymatic Hydrolysis

SNo

Sample-Acid used for Pretreatment (%) 0.0 0.5 1.0 1.5 2.0 2.5 3.0

Cellulose Conversion (%)

Available Substrate (%)

S1 S2 S3 S4 S5 S6 S7

2.2 11.8 12.8 13.6 14.2 15.2 16.0

97.8 88.2 87.2 86.4 85.8 84.8 84

S1 to S7 Pretreated cotton waste samples in different acid concentrations

The rate and fidelity of the indigenous enzyme on the acid treated samples were calculated based on the sugar released and the results are mentioned in Table 2. The rate of reaction increased with increase in acid concentration used for pretreatment purpose. The fidelity of the enzyme was found to be 0.0229 for 0% acid pretreated cotton waste which increased to a maximum of up to 0.1666 with 3% acid used for pretreatment.

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Table. 2. Rate of Enzymatic Hydrolysis for Acid Pretreated Samples

SNo

Sample-Acid used for Pretreatment (%) 0.0 0.5 1.0 1.5 2.0 2.5 3.0

Reaction Rate (moles glucose released per hour) 0.0229 0.1229 0.1333 0.1416 0.1479 0.1583 0.1666

S1 S2 S3 S4 S5 S6 S7

S1 to S7 Pretreated cotton waste samples in different acid concentrations

The feasibility of bio-alcohol production of cellulosic wastes especially cotton waste was made in this study. From the previous studies it was noted that increased acid concentration used for the pretreatment purpose enhanced the sugar release during enzymatic hydrolysis. Fermentation studies also proved that increased acid concentration yielded more amount of alcohol. Alcohol content of about 8.9 gl-1 (Table 3) was detected in the 72 hours old fermentation broth containing the hydrolysed sample pretreated with 3% sulfuric acid. This study proved that sugars released by means of enzymatic hydrolysis of pretreated cotton waste are of fermentable category and alcohol can be produced efficiently using a suitable fermenting strain Saccharomyces cervisiae.
Table. 3. Alcohol Production on Acid Pretreated Cotton Waste through Fermentation with Saccharomyces cervisiae.

SNo

Sample-Acid used for Pretreatment (%)

Ethanol gl-1

S1 S2 S3 S4 S5 S6 S7

0.0 0.5 1.0 1.5 2.0 2.5 3.0

1.4 3.8 4.9 5.6 6.8 7.7 8.9

S1 to S7 Pretreated cotton waste samples in different acid concentrations.

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