INTRODUCTION:

Plant cell wall is composed of cellulose, lignin, hemicellulose and pectin substances. Cellulose is the organic polymer from plant source consisting of β-1, 4-linked glucose units. They contain about 100 to more than 10,000 glucose units, linked by inter and intra chain hydrogen bonds that give rigid structural stability to cellulose¹.Because of its structural function, they are used as raw material for producing industrially important commodity products. The primary occurrence of cellulose is the existing lignocellulose material in forest, with wood as the most important source². Coir, pineapple, banana leaves, rice and wheat straw are used to produce cellulose-based materials. Modified cellulose such as rayon used as synthetic fiber³.

Cellulose have used to make food wrap to prevent food spoilage⁴.

Cellulose can also be produced by various bacterial species in nature. Plant cellulose and bacterial cellulose have same chemical structure, but differ in their physical and chemical properties. Bacterial cellulose (BC) is an excellent material used in many novel applications since its uses are expanding rapidly due to environmental benefits and specific strength properties. Increase in fuel and petroleum costs results in altered use of lignocellulosics to produce ethanol and other sugars by fermentation⁵. Cellulolysis, the process of breaking down cellulose into glucose units are used to produce glucose in large scale. Corn cob is a major component of agricultural, domestic waste and is composed mainly of cellulose. This cellulose is treated with Aspergillus niger and Penicillium decumbens in producing bioethanol⁶. Corn husk is an attractive substrate in producing cellulose as they contain high cellulose content⁷.

Enzymes are nature’s catalysis that speed up the reactions where most enzymes today are produced from the microorganisms⁸. The term cellulase is a group of three enzymes such as β- 1, 4-D-glucan cellobiohydrolase (EC 3.2.1.91), which cleaves cellobiosyl units from the ends of cellulose chains, endo-β-1,4-D-glucanase (EC 3.2.1.4), cleaves internal glucosidic bonds and β-1, 4-glucosidase (EC 3.2.1.21), cleaves glucose units from cell oligosaccharides⁹. The role of microorganisms has been highlighted in cellulase enzyme production. Microorganisms such as bacteria, yeast and fungi are capable of producing cellulase. However, bacteria gained more importance due to their high diversity. Cellulase-producing bacteria such as Pseudomonas fluorescens, Bacillus subtilIs, E. coli and Serratia marcescens can also be isolated from soil¹⁰. When fermenting cassava tubers, amylase-producing bacterial and fungal strains was isolated with increase in amylase activity¹¹. Cellulase was produced from Aspergillus niger by solid state fermentation using bio wastes such as sugarcane bagasse and wheat bran¹². The cellulolytic enzyme from cow dung isolated bacteria was obtained using Carboxymethyl cellulose clear zone (CMCZ) and Filter paper assay (FPA) techniques¹³. The purpose of this work was to explore the potential of cellulose degrading bacteria from soil for highest cellulase activity from corn waste and to examine bacterial growth at optimum working conditions.

MATERIALS AND METHODS:

Alkali extraction of sample:

Corn cob was collected from agriculture fields in padhma pazhamthirzolai, Trichy. Treatment of corn cob with alkali at suitable modest concentrations and temperatures can produce 50% of the original biomass such as extractives, hemicelluloses and lignin. During pre-treatment, adding the increased amount of alkali will solubilize the plant cell-wall and improve the enzymatic hydrolysis of cell-wall polysaccharide¹⁴. Cellulose waste was soaked in 100 ml of 6% NaOH separately and incubated at room temperature for 10 hrs with an agitation of 150 rpm and autoclaving at 121°C for 30 min. The residues were collected and washed extensively with tap water until neutral pH was reached, filtered and dried at 65°C for two days.

Bleaching:

Corn waste was bleached with the hypochlorite-hydrogen peroxide process similar to the method described by Ibrahim et al.¹⁵

Estimation of cellulose from treated sample:

Cellulose content was estimated by the method of Uppdegraff et al. To about 100 mg of pretreated cellulose wastes, about 5ml of Nitric reagent was added to boil and cooled. It was centrifuged at 5000 rpm for 5min. The pellet was washed with distilled water and 67% sulphuric acid was added. To 1ml of the each diluted solution, 10ml of freshly prepared ice cold Anthrone reagent was added and boiled in a boiling water bath for 10 min at 100˚C. Absorbance was recorded at 600nm¹⁶.

Isolation of bacteria:

Soil sample were collected for the isolation of cellulose degrading bacteria from Aalavanthanaillur village, Trichy, Tamilnadu, India. Thermo intolerant bacteriawas removed from the soil by serial suspension in sterile water and heated for 15 minutes at 80°C. After suitable dilutions the samples were incubated in nutrient agar plates for 48 hours at 30°C as methods described by Sethi et al.¹⁰

Screening of cellulolytic bacteria:

Based on morphological and biochemical characteristics, cellulolytic bacteria were identified and were inoculated in Carboxy methyl cellulose (CMC) medium. Identified cellulolytic species were screened for cellulase enzyme production by submerged fermentation process¹⁷.

Crude enzyme production and its Estimation:

Submerged fermentation procedure was followed to produce crude enzyme and the method used was described by Sharma et al ¹⁸. Finally, the broth was centrifuged at 1600rpm for 20 min at 4°C. The supernatant obtained was used as a crude enzyme source. Total cellulase activity in the culture filtrate was determined according to the standard method described by Hankin et al¹⁹.

Optimization of Culture Conditions for Enzyme Production:

Effect of pH, temperature, incubation period, substrate concentration and inoculum were carried out as methods described by mandels et al²⁰.

RESULTS AND DISCUSSION:

Estimation of cellulose from treated sample:

The collected samples after pre-treated with alkali and bleached, yellow substrate changed its colour into pale yellow as shown in Figure 1. Treated sample was estimated for cellulose production using nitric acid reagent where the pre-treated corn waste contains 97mg of cellulose.

a b

Figure 1: Corn waste sample before treatment (a) and after treatment (b) with alkali and bleaching.

The most important industrial material is cellulose and other wood products. The waste from industries can cause environmental pollution. Proper utilization of these wastes by convert them into useful by-products may eliminate the adverse effects caused by them²¹. Bioethanol was produced from rice straw using cellulase enzyme hydrolysis obtained from bacterial source²². Rice chaff is a polished substance extracted from Oryza sativa L.and by solid state fermentation of rice chaff by Penicillium chrysogenum, fibrinolytic enzyme was produced for use in thrombolytic therapy²³. In the present study, corn waste was selected as it is a rich source of cellulose, so that diverse group of cellulolytic microorganisms can utilize them. Further, it is easily available with ease of processing. Even cost effectiveness plays an important role for its selection.

Isolation and screening of cellulolytic bacteria:

To get the cellulolytic bacteria, 50 bacterial strains were isolated from soil through culturing them in appropriate medium and screened them for cellulase activity. On screening fifty bacterial isolates using CMC agar medium, twelve isolates showed cellulolytic activity by forming zone formation. Out of twelve cellulolytic bacteria, bacterial strain (9) showed highest zone formation (0.8cm) and its cellulase activity was found as 0.480(u/ml). To identify the resulted bacterial isolate, various biochemical tests were performed and the bacterial strain showed various positive characteristics such as motility, indole utilization,Voges proskauer, Citrate utilization, glucose fermentation, catalase, oxidase and nitrate reduction tests.The identified organism is Pseudomonas species as shown in Figure 2.

Figure 2: Zone of cellulose utilization by cellulase producing bacterial strain (Pseudomonas sp.) grown on Luria agar supplemented with 0.1 %CMC

Degradation of cellulosic materials is a complex process requiring participation by a number of microbial enzymes. Habitats that contain these substrates are the best sources to find these microorganisms. Cellulose is actually degraded by an enzyme cellulase that is naturally produced by several microorganisms, commonly by bacteria and fungi. Screening for the isolates with cellulolytic activity revealed that the bacteria were more prolific producers of the enzyme²⁴. Similarly, L-asparaginase was produced from groundnut oil cake by employing Aspergillus sp. under solid-state fermentation²⁵. Pseudomonas aeruginosa may involve efficiently in dispersant utilization (solvent-based and water-based) and biodegradation²⁶. In screening of new antibiotics, several actinomycetes were isolated from soil samples²⁷. Antibacterial compoundswere produced by some actinomycetes that was isolated from sedimented water²⁸.

Enzyme production and its optimization:

Pseudomonas sp. showing cellulolytic activity was used for enzyme production assays on liquid medium. After 72 hrs of culture, the supernatant was separated by centrifugation and used to evaluate total cellulase activity. The cellulase enzyme produced by selected bacterial strain is 0.480(U/ml). In order to determine the optimum pH value for the bacterial enzyme, the activity of the enzyme was assayed between the pH 2 to 10. The optimum pH was 6 as shown in Table 1.

Table 1: Effect of pH on enzyme production

S.No	pH	Enzyme U/ml
1	2	0.165±0.013
2	4	0.183±0.009
3	6	0.221±0.009
4	8	0.116±0.014
5	10	0.098±0.009

Cellulase production was expressed and gradually increased as the pH values increased from 4 to 6 and reached its maximum at pH of 6 with production of about 0.221 U/ml of cellulase respectively. Our results are in accordance with Bakare et al. who have optimum pH values around 6 to 7. pH values were adjusted using NaOH/HCl, to assess the effect of pH on cellulase enzyme production²⁹.

The production media was prepared with all the nutritional components with the alternative in the temperature alone varying from (20 °C, 40 °C, 60 °C, and 80 °C) was inoculated with the culture and incubated. After incubation the cellulase activity was estimated. The optimum temperature was found to be 40˚C for cellulase production as shown in Table 2.

Table 2: Effect of temperature on enzyme production

S.No	Temperature (˚C)	Enzyme U/ml
1	20	0.027±0.003
2	40	0.240±0.002
3	60	0.168±0.008
4	80	0.086±0.014

The enzyme activity of selected strain was gradually increased with the increase in temperature and the highest cellulase activity was found to be 0.240U/ml at 40°C. This temperature influences the enzyme production at increased rate by altering the cell membrane properties. The result is in agreement with the other reports on bacterial enzyme production³⁰.

To find the suitable concentration of substrate (corn waste), production was carried out using different concentration such as 0.5, 1.0, 1.5 and 2g/100ml of the production medium. Cellulase is an inducible enzyme and it is affected by the nature of the substrates used for production. Corn waste of 1g showed higher enzyme production of about 0.097 U/ml as shown in Table 3.

Table 3: Effect of substrate concentrationon enzyme production

S.No	Substrate Concentration (g)	Enzyme U/ml
1	0.5	0.098±0.002
2	1	0.097±0.007
3	1.5	0.120±0.001
4	2	0.068±0.004

These substrates of cellulase enzyme production under mechanical shaking increased the medium aeration and permitted better contact between the substrate and the microorganisms causing significant differences in favour of the quantity of enzyme produced in agitated system. The submerged fermentation technique has been widely used in the production of cellulases and the other enzymes. The production of cellulase from corn waste in submerged fermentation under optimum conditions were performed where the enzyme production was higher.

The production medium was inoculated with culture containing various inoculum concentrations (0.5-2ml). The cellulase production was higher at 1ml of inoculum as shown in Table 4.

Table 4: Effect of Inoculum on enzyme production

S.no	Inoculum (ml)	Enzyme U/ml
1	0.5	0.080±0.002
2	1	0.117±0.007
3	1.5	0.141±0.001
4	2	0.054±0.006

The culture used for inoculation in the fermentation medium must be in healthy, active state and of optimum size, possibly in the log phase, thus it will be in its high rate for substrate conversion.Enzyme production was tested with different pH, different temperature, different substrate concentration and incoculum concentration. Based on the results, the fermentation media has been designed and the production of cellulase was carried out.

CONCLUSION:

Cellulolytic bacteria isolated from soil sample were screened for cellulase enzyme production with 12 strains showed cellulolytic activity and Pseudomonassp. showed higher zone formation which was identified by performing biochemical test. Corn waste when used as a substrate results in increased enzyme production. With optimized condition of pH, temperature, substrate concentration and incoculum concentration, industrial production of cellulase is reliable with Pseudomonas sp. However, further studies are required for the enzyme purification.

REFERENCES:

1. Gupta P, Samant K, Sahu A. Isolation of Cellulose-Degrading Bacteria and Determination of Their Cellulolytic Potential. International Journal of Microbiology. 2012; 2012: 578925.

2. Milala MA, Shugaba A, Gidado A, A.C E, Wafar JA. Studies on the use of agricultural wastes for cellulase enzyme production by Aspegillus niger. Research Journal of Agriculture and Biological Sciences. 1(4); 2005: 325-328.

3. Lim S, Won Son T, Lee D-W, Kuk Park B, Min Cho K. Novel regenerated cellulose fibers from rice straw. Journal of Applied Polymer Science. 82(7); 2001: 1705 - 1708.

4. Ryu DDY, Mandels M. Cellulases: Biosynthesis and applications. Enzyme and Microbial Technology. 2(2); 1980: 91-102.

5. Zhuo L, Ho S-H, Sasaki K, Den Haan R, Inokuma K, Ogino C, et al. Engineering of a novel cellulose-adherent cellulolytic Saccharomyces cerevisiae for cellulosic biofuel production. Scientific Reports. 2016: 24550.

6. Saliu BK, Sani A. Bioethanol potentials of corn cob hydrolysed using cellulases of Aspergillus niger and Penicillium decumbens. EXCLI Journal.11; 2012: 468-479.

7. Reddy N, Yang Y. Natural Cellulose Fibers from Corn Stover. Springer. 2015: 5-8.

8. Murugaiah K, Kesavan R, Dhanaraj TS. Optimization of amylase production from Bacillus subtilis and Pseudomonas aeruginosa using sugarcane bagasses by submerged fermentation. Research J. Science and Tech. 4(2); 2012: 86-89.

9. Wang H, Squina F, Segato F, Mort A, Lee D, Pappan K, et al. High-Temperature Enzymatic Breakdown of Cellulose. Applied and Environmental Microbiology. 77(15); 2011: 5199-5206.

10. Sethi S, Datta A, Gupta BL, Gupta S. Optimization of cellulase production from bacteria isolated from soil. ISRN Biotechnol. 2013;2013:985685.

11. Liu T, Williams DL, Pattathil S, Li M, Hahn MG, Hodge DB. Coupling alkaline pre-extraction with alkaline-oxidative post-treatment of corn stover to enhance enzymatic hydrolysis and fermentability. Biotechnology for Biofuels. 7; 2014: 48-55.

12. Swarnakaran Hemalatha, Roja Madhuri Machineni. Specific determination of amylase activity in crude extracts from fermented cassava (Manihot esculenta) tubers. Research J. Science and Tech. 3(4); 2011: 192-196.

13. Tara Chand, Fanish K. Pandey, Shruti Dhingra, Manoj K. Sharma. Production of Industrially Significant Enzymes from Bio-Wastes Using Aspergillus niger by Solid State Fermentation. Res. J. Pharm. Dosage Form. and Tech. 6(1);2014: 33-36.

14. Ranjani M., Rajan S, Murugesan AG. Isolation and optimization of cellulase enzyme by Ocrobacterium sp from Cow dung. Research J. Science and Tech. 5(2); 2013: 255-258.

15. Ibrahim M, Agblevor F, K. El-Zawawy W. Isolation and characterization of cellulose and lignin from steam-exploded lignocellulosic biomass. Bioresources. 5(1); 2010: 397-418.

16. Updegraff DM. Semimicro determination of cellulose inbiological materials. Analytical Biochemistry. 32(3); 1969: 420-424.

17. Shaikh NM, Patel AA, Mehta SA, Patel ND. Isolation and screening of cellulolytic bacteria inhabiting different environment and optimization of cellulase production. Universal Journal of Environmental Research and Technology. 3(1); 2013: 39-49.

18. Sharma P, Bajaj B. Production and partial characterization of alkali-tolerant xylanase from an alkalophilic Streptomyces sp. CD3. Journal of Scientific and Industrial Research. 64(9); 2005: 688-697.

19. Hankin L, Anagnostakis SL. Solid media containing carboxymethylcellulose to detect CX cellulose activity of micro-organisms. J Gen Microbiol. 98(1); 1977: 109-115.

20. Mandels M, Weber J. The Production of Cellulases. Cellulases and Their Applications. Advances in Chemistry. American Chemical Society.95; 1969: 391-414.

21. Menetrez MY. The Potential Environmental Impact of Waste from Cellulosic Ethanol Production. Journal of the Air & Waste Management Association. 60(2); 2010:245-250.

22. Aditiya HB, Sing KP, Hanif M, Mahlia TMI. Effect of Acid Pretreatment on Enzymatic Hydrolysis in Bioethanol Production from Rice Straw. International Journal of Technology. 6(1); 2015: 1-5

23. Gopinath SM, Suneetha TB, Ashwini Patil GM. Rice Chaff a Novel Substrate for Fibrinolytic Enzyme Production by Solid State Fermentation Using Penicillium chrysogenum SGAD12. Research J. Science and Tech. 3(5); 2011: 261-263.

24. Nigam PS. Microbial Enzymes with Special Characteristics for Biotechnological Applications. Biomolecules. 3(3); 2013:597-611.

25. Sreenivasulu V, Jayaveera KN, Mallikarjuna Rao P. Solid-State Fermentation for the Production of L-Asparaginase by Aspergillus Sp. Research J. Pharmacognosy and Phytochemistry. 1(1); 2009: 21-25.

26. Moersidik SS, Pratiwi ZR, Zulkifliani. Influence of Pseudomonas aeruginosa Presence in the Biodegradability study of Solvent‐based and Water‐based Dispersant in Oil Spill Handling. International Journal of Technology. 4(3); 2013: 1-4.

27. Chandrashekhara S, Nanjwade BK, Goudanavar PS, Manvi FV, Shamrez Ali M. Isolation and Characterization of Antibiotic Production from Soil Isolates by Fermentation. Research J. Pharma. Dosage Forms and Tech. 2(1); 2010: 32-36.

28. Sumathi R, Saravana kumar A, Rajeswari R, Pavani S. Isolation, Purification, Partial Characterization And Antibacterial Activities of Compound Produced by some Actinomycetes from Sedimented Waters. Research J. Pharm. and Tech.2 (3);2009: 521-526.

29. K. Bakare M, Adewale I, Ajayi A, O. Shonukan O. Purification and characterization of cellulase from the wild-type and two improved mutants of Pseudomonas fluorescens. African Journal of Biotechnology. 4(9); 2005: 898-904.

30. Nkohla A, Okaiyeto K, Olaniran A, Nwodo U, Mabinya L, Okoh A. Optimization of growth parameters for cellulase and xylanase production by Bacillus species isolated from decaying biomass. Journal of Biotech Research. 8; 2017: 33-47.

Received on 30.05.2018 Modified on 09.06.2018

Research J. Pharm. and Tech 2018; 11(9): 4024-4028.

DOI: 10.5958/0974-360X.2018.00740.0