Jamonline / 2(2); 2012 / 214222 Research Article

Srivastava AK and Pushpa Agrawal

Journal of Atoms and Molecules

An International Online Journal
ISSN 2277 1247

CELLULOSE HYDROLYSIS BY CELLULOMONAS FIMI AND ETHANOL PRODUCTION BY ZYMOMONAS MOBILIS Ajeet Kumar Srivastava*, Pushpa Agrawal Department of Biotechnology, R V College of Engineering, R.V. Vidyaniketan Post, Bangalore, India, Received on: 21-04-2012 Abstract: lignocellulosic material is one of the most abundant and renewable resource on earth for conversion to readily utilizable hydrolyzate conversion .Degradation of lignin is a prerequisite for rendering cellulose for action by cellulose hydrolyzing enzymes. While various pretreatments have been proposed for lignin degradation use of oxidizing agent such as inorganic oxidants ozone, per acetic acid and hydrogen peroxide has been reported as effective for lignin degradation and cellulose hydrolysis.It is proposed to employ mechanical disintegration of paddy straw followed by treatment with MnSO4 and H2O2 as they enhance the susceptibility of rice straw to enzymatic saccharification. To initiate the production of industrially important products from lignocellulosic biomass, bioconversion of the lignocellulosic components into fermentable sugars is necessary that could be used for ethanol production. Cellulomonas is known to produce both cellulases and hemicellulases and it is used for the saccharification . This present study reports on the microbial pretreatment and saccharification of the agricultural residues like rice straw (raw material) using a bacterial strain Cellulomonas fimi (MTCC-24) and ethanol production by Zymomonas mobilis. Key Words: Cellulose hydrolysis, ethanol production, lignocellulosic material, saccharification, fermentable sugars, microbial pretreatment. Introduction: * Corresponding author Ajeet Kumar Srivastava, Email: ajeeth@rvce.edu.in Tel: +91 9916830878 The natural energy resources such as fossil fuel, petroleum and coal are being utilized at a rapid rate and these resources have been estimated exhaust over a few years. Revised on: 26-04-2012 Accepted on: 29042012

Therefore, alternative energy resources such www.jamonline.in 214

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Jamonline / 2(2); 2012 / 214222 as ethanol, methane, and hydrogen are being considered. A worldwide interest in the utilization of bioethanol as an energy source has stimulated studies on the cost and efficiency of industrial processes for ethanol production. Lignocellulose is an abundant material created from solar energy and renewable resources on earth, which makes them attractive for production of sugar through saccharification process.

Srivastava AK and Pushpa Agrawal cellulose at the ends of glucan chains by C. fimi cellobiohydrolases CbhA and CbhB is strongly suggested by hydrolysis experiments using cellooligosaccharides [6,7,8,9,10].

Previous studies have indicated that CenA attacks susceptible linkages in soluble CMC in a relatively nonprocessive manner[11] i.e., the enzyme dissociates from the substrate after each hydrolytic event. While CenB and CenD attack CMC in a similar way,C. fimi CenC seems to act in a more processive fashion [12,13]. Therefore, CenC activity was analyzed in order to determine if the enzyme behaves in a similarly processive manner on cellulose. Previous determinations of

Lignocellulose is composed of three main fractions like cellulose (~ 45% of dry weight), hemicellulose (~30% of dry weight) and lignin (~25% of dry weight). In these waste products (rice straw, wheat straw and rice husk) cellulose and hemicellulose are closely associated with lignin in the plant cell wall [1]. Cellulose, the most abundant polymer on earth is composed of thousands of molecules of anhydroglucose linked by (1, 4)glycosidic bonds. Cellulose can be effectively hydrolyzed and depolymerized into

molecular size distribution during hydrolysis have shown that the choice of substrate is an important consideration [14]. In this study used surface/volume ratios and substrate heterogeneity, which are associated with the use of substrates like rice straw [15, 16]. Objective: 2.1.To used Potential availability of some agricultural residues in India like rice straw. 2.2.To achieve high yields of fermentable sugar 2.3.From cellulosic components using

fermentable sugars by the enzyme cellulase [2, 3, 4, 5]. A number of microorganisms are capable of producing extracellular cellulase enzyme and among which Cellulomonas fimi widely used candidates for cellulase enzyme production. Cellulomonas fimi produces at least six -1, 4-glucanases, of which four (CenA, CenB, CenC, and CenD) are

microorganisms 2.4.To determine the effect of sugar yield using a Cellulomonas fimi bacteria. 2.5.To achieve high yields of ethanol at economic level using Zymomonas mobilis bacteria

endoglucanases and two (CbhA and CbhB) appear to be cellobiohydrolases that are the functional equivalents of Trichoderma reesei CBHI and CBHII. The preferential attack of

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Jamonline / 2(2); 2012 / 214222 Material and Methods: Raw materials Rice straw was procured from a rice mill in Bangalore, It was cleaned dried and powdered to 100 mesh size in a ball mill. Powder of raw material was used as carbon source. Microorganisms Cellulomonas fimi and Zymomonas mobilis were procured from MTCC, Chandigarh. Culture medium Media for Cellulomonas fimi: Beef Extract 1.0g, Yeast Extract 2.0g, Peptone 5.0g, NaCl - 5.0g, Agar 15g, Distilled water 1 litre. Media for Zymomonas mobilis: Glucose 80g, Yeast Extract 10g, KH2PO41.0g, MgSO4.7H2O0.5g, Agar-15g, Distilled Water-1 litre. Standardisation of pre-treatment process Three samples of Cellulosic material (Rice straw Powder) was suspended in three different sterilized conical flask of hydrogen peroxide solution containing MnSO4 at concentration of the molar ratio 1: 100 against H2O2 and were allowed to stand for 2 hrs at three temperatures viz. 25C, 50C, 72C and continuously stirred using glass stirrer. The pretreated material were washed several times with deionised water and then dried with hot air oven for 24 hrs at 70C then dry fibrous cake was weighed which further processed for microbial saccharification All rights reserved 2011

Srivastava AK and Pushpa Agrawal Microbial saccharification Saccharification was carried out using 10ml of active bacterial culture (incubated for 24 hrs, in orbital shaker), 1.8g of raw material and 90ml media so that volume of broth becomes 100ml. The experimental setup was kept in the orbital shaker for saccharification to occur. After each 24hrs levels of reducing sugar in the above setup were checked quantitatively by DNStest [17]. Determination of total carbohydrate The carbohydrate content of pretreated raw materials in the culture broth was measured by Anthrone method [18]. Fermentation Culture filtrate was further inoculated with Zymomonas mobilis strain and allowed for fermentation for seven days [19]. After fermentation it was filtered and ethanol content was determined. Ethanol estimation Determination of ethanol content was done by spectrophotometric method [20] Results and Discussions: 1) Rice straw pretreatment method was standardised for easy access of the substrate surface for microbial action. 2) The alkali pretreatment has yielded optimum reducing sugar at 72C using Cellulomonas fimi.

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Jamonline / 2(2); 2012 / 214222 3) Increase in the microbial concentration shows increase in reducing sugar level. 4) Delignificationand depolymerisation of Rice straw was needed in order to utilise it as a potential substrate in

Srivastava AK and Pushpa Agrawal analysis of CMC hydrolysis. CenC appeared to show a more processive action, relative In to the the other present

endoglucanases.

investigation we have compared the activities of CenA, CenC, CbhA, and CbhB by determining the molecular size

saccharification. 5) The chemical composition of Rice straw is lignin (24.8 %), Cellulose (34 %), and hemicellulose (28.2%). The total sugar estimated before pre treatment (3.74 mg/g). 6) The percentage loss of weight of Rice straw due to pretreatment can be

distribution

products

obtained

by

hydrolysis of rice straw. 10) For all enzymes, it is evident that substantial solubilization of BMCC

occurred without major changes in the overall shapes of distribution profiles. Loss of high-DP cellulose was slightly more pronounced for CenA than for CbhA and CbhB, as expected for a randomly acting endoglucanase, but the the activity of CenA is not easily

attributed to the dissolved lignin content to larger extent and hemicellulose and cellulose molecule to the some extent. 7) Lignin forms the barrier for

microorganisms to utilise cellulose as carbohydrate for it growth. The

distinguished from the activities of the two cellobiohydrolases when rice straw is used as the substrate 11) The data support our earlier suggestion that C. fimi CbhA and CbhB correspond to similar pairs of cellobiohydrolases seen in fungal cellulase systems and, more generally, that aerobic fungi and bacteria have similar types of cellulase systems. 12) It has been reported that the bacteria Zymomonas mobilis which gives a high ethanol yield, tolerates high ethanol concentrations and can ferment arabinose and xylose (18% of ethanol).

Amorphous nature of substrate after pretreatment was due the removal of lignin. 8) Powdering the substrate increases the surface area and the pore size of the particle necessary for the absorption of moisture and penetration of microbes. 9) The cellulose-degrading system of C. fimi contains at least six cellulases. Four of these enzymes (CenA, CenB, CenC, and CenD) were designated as

endoglucanases and two (CbhA and CbhB) were designated as

cellobiohydrolases, based largely on All rights reserved 2011 www.jamonline.in 217

Jamonline / 2(2); 2012 / 214222

Srivastava AK and Pushpa Agrawal

Table 1: Initial composition of the raw materials in mg/gm SI. No 1 Raw materials Rice straw Total sugar 1.89 Reducing sugar 1.81 Non reducing sugar .08

Table 2 After 24 hrs Control (O.D) 0.0 0.0 0.0 Test (O.D) .005 .002 .001 % of reducing sugars .054 .033 0.00 % of ethanol 5 3 0

Sl. No

Sample 250C 500C 720C

Conc. g/ml

1 2 3

5 3 0

Table 3 After 48hrs Control (O.D) 0.0 0.0 0.0 Test Conc. g/ml (O.D) .075 .045 .015 80 50 20 sugars .88 .56 .22 % of reducing % of ethanol 7 4 2

Sl. No

Sample

1 2 3

250C 500C 720C

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Jamonline / 2(2); 2012 / 214222

Srivastava AK and Pushpa Agrawal

Table 4 After 72hrs Control (O.D) 0.0 0.0 0.0 Test (O.D) .44 .37 .29 490 380 290 Conc. g/ml % of reducing sugars 5.4 4.2 3.2 % of ethanol 15 13 10

Sl. No

Sample

1 2 3

250C 500C 720C

Table 5 After 120hrs Control (O.D) 0.0 0.0 0.0 Test Conc. g/ml (O.D) .545 .625 .74 550 620 730 sugars 6.1 6.9 8.2 % of reducing % of ethanol 16 10 12

Sl. No

Sample

1 2 3

250C 500C 720C

Table 6 After 168hrs Control (O.D) 0.0 0.0 0.0 Test Conc. g/ml (O.D) .56 .70 .90 555 620 854 sugars 6.16 7.66 9.32 % of reducing % of ethanol 9 14 18

Sl. No

Sample

1 2 3

250C 500C 720C

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Jamonline / 2(2); 2012 / 214222

Srivastava AK and Pushpa Agrawal

Variation In Concentration of Reducing Sugar with Time and Temperature

900 800 700 600 500 400 300 200 100 0 24 48 Sample A 25 'C Sample C 70'C 72 120 Time (hrs) 168

Concentration (ug/ml)

Sample B 50 'C

Conclusion It is seen that pretreatment with H2O2 and MnSO4 effectively expels Lignin and Hemicellulose and allow the microorganisms to utilize the cellulose without any hindrance to produce reducing sugar .Sugar conversion from cellulosic materials holds great potential due to the widespread availability, abundance and relatively low cost. In this article Cellulomonas fimi used for saccharification and Zymomonas mobilis used for ethanol production. Many laboratories around the world are involved in research on the different aspects of natural biodegradation of cellulosic All rights reserved 2011

materials. Consequently, processes that use microorganisms are being developed to explore the potential for their

biotechnological application because high cost of cellulase is one of the major hindrance to make the process commercialized. This can be reduced by adopting cellulase producing microorganisms. Acknowledgement The authors thank Prof. B.S. Satyanarayana, Principal R.V. College of Engineering for his constant encouragement and support

throughout the research work.

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