Bioprocessing of Agricultural Waste (Banana Pseudostem) by Solid-State Fermentation (SSF) for Production of Cellulase

Cellulase was produced by Cellulomonas uda utilizing banana stem waste by solid state fermentation (SSF). The different parameters effects considered like particle size of substrate, pH(incubation and extraction), incubation period, temperature (incubation and extraction), media components (yeast extract and peptone) and moisture of substrate on the production of cellulase was investigated. The optimum activity of cellulase produced by Cellulomonas uda on banana waste for particle size (6.97 IU/min for 1mm), extraction pH (7.13 IU/min for pH 7), incubation pH(6.97 IU/min for pH 7), extraction temperature (7.10 IU/min for 500C), incubation temperature (7.20 IU/min for 450C), incubation period (7.20 IU/min on 3rd day), moisture content (7.12 IU/min for 100% ), peptone content ( 7.23 IU/min for 0.5 gm) and yeast extract content (7.18 IU/min for 0.30gm) was recorded. The cellulase produced by Cellulomonas uda by using banana stem waste indicates the socio-econic utilization of agricultural waste. After purification and characterization; cellulase enzyme can be used in industrial processes.


INTRODUCTION
Cellulose represents about 1.5 × 10 12 tons of biomass produced by photosynthesis and is most inexhaustible raw material source for different products [1]. Cellulose is the most dominating agricultural waste and is considered as abundant and renewable biopolymer present on planet earth [2]. Hydrolysis of lignocellulosic waste by microbial degradation and ultilization of resultant sugars (reducing) by fermentation process for production of bioproducts or biofuel will be most promising strategy for efficient utilization of this biomass [3]. Different class of Cellulases enzyme produced by a number of microorganisms helps in degradation of lignocellulosic waste through ferementation proce [4]. Media used in cellulose fermentation consists of cellulose in different degree of purity for submerged fermentation [5] and raw lignocellulosic substrate for solid state fermentation [6].
Cellulases hydrolyze cellulose (ß-1,4-D-glucan linkages) and produce as primary products glucose, cellobiose and cellooligosaccharides. There are three major types of cellulase enzymes [Cellobiohydrolase (CBH or 1,4-ß-D-glucan cellobiohydrolase, EC 3.2.1.91), Endo-ß-1,4-glucanase (EG or endo-1,4-ß-D-glucan 4-glucanohydrolase, EC 3.2.14) and ß-glucosidase (BG-EC 3.2. 1 [19], animal feeds [20] and food industry [21]. In breverage industry, cellulases enzyme is used in clarification and extraction of vegetable and fruit juices, fruit purees and nectars production, and in olive oil extraction [21], Glucanases enzyme are added in beer manufacturing to enhance the process of barley malting [22], exogenous hemicellulases and glucanases used in wine industry for color extraction and better maceration [21]. Cellulases and hemicellulases have been used for hand sheet strength properties and biomechanical pulp in the paper and pulp industry [23], deinking of recycled fibers [24]. Extracellular enzymes are also used in synthesis of silver nanoparticles [25,26] A most prospective use of cellulase is the conversion of cellulosic or lignocellulosic raw materials to other fermentable sugars and glucose, which acts as a microbial carbon source for different bioproduct production like single cell proteins and ethanol [31,32]. Commercial production of enzyme cellulases can be achieved by either solid [3] or submerged fermentation process [1] culture including batch, fed batch, and continuous fermentation processes [3].Various statistical tools or methods are used in order to optimize production of cellulase or biomolecules by using fermentation process [33,34,35]. The main agricultural waste of Jalgaon district is the banana fruit stalk and pseudo stem, also accumulated as waste, posing serious environmental problems. Banana fruit stalk contains total sugar (56.8%), starch (27.0%), reducing sugar (4.65%) and protein (4.3%) on a dry weight basis. The Banana pseudo stem consists of Hemicellulose 65%, Cellulose 64%, Lignin 19%, and Ash 15% on dry weight basis. The main objective of our research work was to effectively utilize the agricultural waste (banana pseudo-stem) for the production of cellulase from microbial strain by SSF. One-Factor at a Time (OFAT) study was conducted to select the optimize operating parameters and media components for the production of microbial cellulase.

Substrate Collection
The main agricultural waste of Jalgaon District (called land of Banana) is banana pseudostem. Pseudostem rich in cellulose accumulate as waste in the banana field. This pseudostem collected from the banana fields around Jalgaon city is utilized as substrate in SSF. Banana pseudostem collected from the fields near North Maharashtra University, Jalgaon.

Microorganism
Microorganism was procured from National Center for Industrial Microorganism (NCIM), National Chemical Laboratory (NCL), Pune. Cellulomonas uda NCIM No. 2353 (Cellulase producer) is maintained on nutrient agar slant (Peptone 5 gm, Beef extract 3 gm, NaCl 5 gm, Distill water 1000 ml) of pH 7 at 4 0 C.The subculturing of microorganism was done regularly in the department laboratory.

Preparation of Substrate
Banana stem waste was considered as substrate for cellulase enzyme production was obtained from farm land located back side of North Maharashtra University, Jalgaon. The collected substrate was chopped in to smaller pieces and for 48hrs kept under sun light for sun drying. After sun drying, substrate was dried at 70 0 C for 24 hrs in hot air oven. The substrate was powdered by using electrical grinder.
The powdered substrates were passed through sieve shaker of 2mm mesh size. Substrate after sieve analysis were collected in conical flask of 250ml or petriplates. Salt solution was used to moisture the substrate with a composition of gm / 100 ml: peptone 0.5, yeast extract 0.3, Na 2 HPO 4 .2H 2 O 1.

Solid State Fermentation
Sterilized substrate was cooled to required temperature. Substrate of weighing 10gm in petridish and 15gm in 250ml was taken. Later inoculum of measuring 10 % (W/V) was added in the laminar air flow by using sterilized pipette.
Cellulomonas uda (NCIM No. 2353, Cellulase producer) was inoculated on banana stem. After inoculation, petriplates and conical flask was incubated at 37 0 C for 3days. Conical flasks were gently shaken for every 12 hrs for mixing of solid substrate with microorganism.

Extraction of Cellulase Enzyme
After fermentation process, 1:10 (W/V) of 0.1 M sodium phosphate buffer of pH 6.9 was mixed with fermented banana stem waste for extraction of crude cellulase enzyme. The mixture was kept at rotary flask shaker at 150 rpm for 60 minutes. The mixtue was filtered by using muslin cloth. Collected filtrate was centrifuged for 10 minutes at 10000rpm at 4 0 C. Collected supernatant was used as crude cellulase extract for estimating cellulase activity [13]. .

Cellulase Enzyme Assay
Activity of cellulase enzyme was estimated by filter paper method. Cellulase enzyme acts on cellulose present in filter paper to produce reducing sugar (glucose) and measured by DNSA method. Chemicals used for enzyme estimation were sodium phosphate buffer (pH 6.9) of 0.1 M, filter paper discs, DNSA, potassium sodium tartarate, glucose solution. 1 ml of crude enzyme extract was added to 30 mg of dry whatmann filter paper 1 and incubated the mixture for 1 hr at 50 0 C. Added 2.5ml of DNSA reagent and mixture was heated to 15min in a boiling water bath. Absorbance was measured at 530 nm. Standard graph of glucose was prepared. The enzyme activity was expressed as μmol of glucose released per minute.

Parameters Considered for SSF
The effect different parameters like particle size of substrate, pH (incubation and extraction), incubation period, temperature (incubation and extraction), media components (yeast extract and peptone) and moisture content on cellulase production was investigated.

Effect of Substrate Particle Size on
Cellulase Activity Effect of varying particle size on cellulase enzyme activity was considered by taking banana stem as solid substrate. Substrate from 0.100 to 2mm particle size was considered to its effect of different particle size on cellulase activity. Grinded substrate was sieved to different particle size by using sieve shaker. Sieves of different mesh size arranged in a decreasing order of mesh size as 2 mm , 1.4 mm ,1 mm , 0.850 mm, 0.425 mm, 212mm, 106 mm were arranged on a vibrator. Substrate collected on the sieve for example 1mm by passing through sieve of 1.4mm was considered as 1.4mm particle size.Different particle sized substrate was taken in 250 ml conical flask and SSF was performed for 36 hrs. at 37 0 C. Crude enzyme was extracted from each conical flask containing different particle sized substrate and cellulase activity was recorded. Fig. 1 shows thhe mean reading of cellulase activity against particle size.Substrate with larger particles have larger interparticle space there by providing better respiration/aeration efficiency. In contrast, substrate of smaller particle size results in accumulation of substrate, thereby interfere with microbial aeration/respiration by resulting in poor growth and cellulase enzyme production. For cellulase activity, increase in cellulase activity was resulted with decrease in particle size from 2mm to 1mm, but further decrease in particle size of substrate up to 0.106mm decreases cellulase activity. Optimal activity of 6.97IU/min was observed at 1mm particle size.
To investigate the effect different parameters like pH (incubation and extraction), incubation period, temperature(incubation and extraction), media components (yeast extract and peptone) and moisture content on the production of cellulase, 1mm substarte particle size was used for the SSF.

Effect of Extraction pH on Cellulase Activity
Extraction pH and its effects on cellulase activity was performed by SSF. Cellulase was extracted by buffers of pH from 4 to 9. For pH 4 and 5, 0.2M Acetate buffer was used. For pH 6, 7, and 8 Phosphate buffer of 0.1M was used. For pH 9, 0.2M Glycine buffer was used. To study the effect of extracting pH and also to optimize the condition for pH, the cellulase enzyme activity was recorded. Change in the concentration of hydrogen ion will have considerable influences on enzyme activity.
Each enzyme will have show its maximum activity at its optimum pH. Amino acids ionic charges particularly at the substrate binding site and active site, etc is influenced by presence of hydrogen ions which inturn influence the enzyme activity. For cellulase activity, increase in enzyme activity was observed from pH 4 to 7 increases, further increase in pH up to 9, decreases enzyme activity. Optimal cellulase activity of 7.13IU/min was observed at pH 7 as shown in

Effect of Incubation pH on Cellulase Activity
Effect of ph (incubation) on cellulase enzyme activity was performed by SSF. Among different physicochemical parameters, growth medium ph have a major role in inducing enzyme secretion and changes in morphological structure in the organism. Medium pH varies due to consumption of substrate (eg: protein hydrolysis) and production of metabolites like organic acids. Cellulase activity increases with, increase in ph from 4 to 7, further increase in ph up to 9 decreases cellulase activity. At pH 7 optimal cellulase activity of 6.97 IU/min was observed.

Effect of Extraction Temperature on Cellulase Activity
SSF was performed to study the effect of extraction temperature on cellulase activity.

Effect of Incubation Period on Cellulase Activity
SSF was performed by altering incubation time/period from 1 day to 10 days at 45 0 C. For cellulase activity, incubation time/period from day 1 to day 3, increases cellulase activity. Further incubation time/period from 3 rd day to 10 th day, cellulaser activity was decreased. At 3 rd day of incubation period, optimum cellulase activity of 7.20IU/min was observed. This may be due to availability of required moisture content in the solid substrate, whereas drastic decrease in cellulase activity was observed after 3 rd day of incubation due to decrease in moisture content of the substrate.

Effect of Substrate Moisture Content on Cellulase Activity
SSF was performed to study the effect of substrate moisture content on cellulase activity.Different moisture content of substrate was achieved by adding salt solution of w/v from 50 to 120%. In SSF, growth of microorganism and enzyme secretion is influenced by initial moisture contents of the substrate. Presence of moisture in the substrate helps microorganism to access nutrients present in substrate thereby increases microorganism growth. Presence of moisture in substrate influences the physicochemical properties of the substrate, thereby effect the production of enzyme. Higher moisture content of the substrate results in substrate particles to stick together and thereby affecting the diffusion of oxygen between the substrate pores. This in turn reduces the growth of microorganism due to steric hindrance caused due to reduced porosity of the substrate and thus decrease in enzyme production.
Lower substrate moisture content decreases the solubility of nutrients of the substrate, low degree of swelling and high water tension. For cellulase activity, moisture content from 50% to 100% increases cellulase activity, further change in

Effect of Yeast Extract and Peptone Content on Cellulase Activity
Addition of nitrogen source like yeast extract and peptone enhances the secretion of hydrolytic enzymes. In this present study, effect of varying concentrations of yeast extract and peptone was checked on cellulase activity. Increase in concentration of yeast extract from 0.06gm to 0.30gm and concentration of peptone from 0.1gm to 0.5gm, showed increase in cellulase activity. Further increase in concentration of yeast extract to 0.48 gm and peptone concentration from 0.5 to 1.0 gm, decreased cellulase activity. Optimum activity of 7.23IU/min was observed at 0.5 gm of peptone concentration and 7.18IU/min at 0.30 gm of yeast extract concentration as shown in Fig. 8 and 9 respectively.

CONCLUSION
Banana stem waste provides a low cost feed stock for biological production of cellulase. In present study, cellulase produced by Cellulomonas uda (NCIM No.2353) was obtained from NCIM, NCL, Pune. The maximum activity of cellulase produced by Cellulomonas uda on banana waste was recorded for different parameters like particle size, pH, temperature, incubation period, moisture content, peptone and yeast extract content. Experimental setup showed that cellulase production can be done by using banana pseudo-stem; instead of burning them in the agriculture field, which otherwise causes serious environmental concerns by increasing the air pollution.Thus, our research highlighted the use of banana stem waste as potential economic source for the production of cellulase by solid state fermentation.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.