Influence of Cellulose-Cellulase Enzyme Digestibility in the Production of Glucose from Lignocellulosic Biomass Waste

A comprehensive understanding of the factors and ability of cellulase enzyme to completely hydrolyze the structural lignocellulosic biomass has been a major research focus over the years. A comparative analysis of cellulose-cellulase digestibility of Kraft-pretreated sawdust from twenty different Nigeria wood wastes was carried out with increasing enzyme (Trichorderma viride) loading of 0.4 mg cm−3, 1.0 mg cm−3, 2.0 mg cm−3, and 4.0 mg cm−3 at constant substrate application of 10 mg cm−3 biomass concentration, temperature of 40°C and 2 h incubation period. This was carried out in order to establish the optimum cellulase-cellulose ratio for maximum biodegradation of the lignocellulosic biomass to produce glucose, a fermentable sugar. The influence of the cellulose-cellulase interaction from 0.4 mg cm−3 to 1.0 mg cm−3 enzyme treatment resulted in 144% increase in glucose yield from E. suaveolen and 121% from S. pustlatas. while 2.0 mg cm−3 cellulase concentration gave 674%, 641% and 617% increase from E.suaveolen, H. ciliate and A. germinans respectively. A general trend of increasing sugar formation was observed with an increasing enzyme loading due to enhanced cellulose accessibility by the cellulase enzyme leading to effective saccharification of the lignocellulosic materials for more sugar production. However, further increase of 4.0 mg cm−3 enzyme concentration failed to produce a commiserate amount of fermentable sugar.


INTRODUCTION
The effective use of microbial cellulase in the biodegradation of lignocellulosic biowaste materials has contributed immensely in bioenergy and bioproducts development.Furthermore, the utilization of abundant and renewable lignocellulosic wastes provides viable feedstocks for large scale and sustainable biofuel economy [1].In cellulose-cellulase interactions, enzymic hydrolysis remains a major factor in the biodegradation of lignocellulosic biomass and its efficiency in bioenergy development [2].The proper management of cellulosic wastes has become important tool for reduction of environmental pollution caused by incessant dumping and burning of these waste materials.Maximizing their potentials as biofeedstocks for industrial and economic advancement has become very desirable [3,4].Forestry lignocellulosic materials (wood/pulp fibers), agro-industrial residues and organic wastes (waste paper) have been widely identified as potential renewable feedstocks for large-scale glucose formation in bioenergy production and other industrial applications [5,6].Research on bioconversion of lignocellulosics is currently focused on enzymatic hydrolysis of the carbohydrate portion of the cellulosic biomaterials into glucose, followed by fermentation process for bioenergy production [7,8].
Bioconversion processes that enhance the digestibility of the enzyme system for efficient and maximum enzyme-substrate interaction with optimal product yield at low operation cost are essential for downstream biomass-bioethanol applications [9,10].A major contributing factor for successful enzymatic conversion of lignocellulosic biomass into fermentable sugars is the accessibility of the β (1-4) glycosidic bonds in cellulose to cellulase enzymes [9,11,12].The degree of crystallinity of lignocellulosic biomass is an important factor in the substrate enzyme interaction for a successful hydrolytic process [13].The enzymatic hydrolysis of any cellulosic biomass is a biochemical reaction initiated by highly specific cellulase enzymes [14].Cellulase enzymes consist of mixture of several enzymes, among which at least three major groups are actively involved in the enzymatic hydrolysis of cellulosic biomaterials [15,16].These include; β -1-4endoglucanase (EC 3.2.1.4.) which specializes in the attacks of regions of low crystallinity in the cellulose fibers thereby creating free chain ends, β -1-4-exoglucanase or cellobiohydrolase (EC 3.2.1.91.) which consolidates the further degradation of cellulose molecule by removing cellobiose units from the free chain ends and β-glucosidase or cellobiase (EC 3.2.1.21.) which hydrolyzes cellobiose to produce fermentable sugar (glucose) [17].Enzymatic hydrolysis has proved to be a heterogeneous reaction that requires the synergistic action of all the enzyme components, biocatalytic influence of any reaction process or medium that will facilitate the enzyme digestibility of the crystalline cellulose up to the systematic degradation of the cellobiose into fermentable glucose [18].Fundamental bioconversion processes that could be affected by both the physicochemical properties of the lignocellulosic substrate such as cellulose crystallinity, degree of polymerization, surface area that determine its accessibility by the enzyme and digestibility of the enzyme components to completely hydrolyze the cellulose fraction for glucose formation have been reported [19,20].The rate of enzymatic hydrolysis of cellulose has been reported to decreases rapidly with conversion process, leading to low yields of glucose, long processing times, and high enzyme consumption [21].The rate of enzymatic hydrolysis as dependent factor on enzyme digestibility and adsorption of the substrates with the effectiveness of the adsorbed enzymes instead of the diffusive mass transfer of enzyme has also been reported [22].Lignin removal also increases enzyme effectiveness by eliminating non-productive adsorption site and by increasing access to holocellulose [23,24].Several reasons have been presented to support this observation.These include thermal instability of cellulase enzymes [25], cellulose-cellulase hydrolysis product inhibition, cellulase inactivation, substrate transformation into a less digestible form, for example recrystallization of the originally amorphous cellulose component into a crystalline fraction and the heterogeneous structure of the polysaccharide substrate [26].It has also been shown that lignin and acetyl groups in hemicellulose are significant barriers for cellulase enzymes digestibility in accessing the lignocellulosic fiber matrix, as well as crystallinity effects on the efficiency of enzyme interaction with cellulose fraction of the lignocellulosic biomass material during enzymatic hydrolysis [13].The steric hindrance on hydrolytic enzymes during enzyme-substrate hydrolysis could lead to inefficient and poor carbohydrate digestibility with resultant effect on the amount of glucose produced [27,28].

Kraft Pulping of the Wood Sawdust to Generate Cellulose Fibers.
2 kg each of the sawdust (2.8-5.0 mm particle size) from the twenty Nigerian woods investigated was subjected to Kraft pulping process with 350 g of NaOH and 140 g NaS2.The Kraft pulping chemicals was dissolved in 8 L water and the delignification of the sawdust lignocellulosic materials was carried out in a rotary steel digester at 170 0 C and a pressure of 200 kPa for 1.45 h at a cooking liquor to wood ratio of 4:1.After the Kraft pretreatment, the extracted cellulose fibres were washed in turn with deionized water until they are free of the Kraft reagents [29,30].

Cellulase Enzyme Catalysis in the Production of Glucose
The cellulose enzyme interaction was evaluated by treating in triplicate a constant biomass concentration of 10 mg cm -3 with increasing dose of T. viride cellulase enzyme (10 mg cm -3 stock solution) of 0.4 mg cm -3 (20 μL), 1.0 mg cm -3 (50 μL), 2.0 mg cm -3 (100 μL) and 4.0 mg cm -3 (200 μL) respectively.The enzymatic process was activated with 0.40 cm 3 of 5 mM Tris (hydroxymethyl aminomethane buffer solution, pH 4.5 and catalysis was carried at constant temperature of 40 0 C in 2h [5].The amount of glucose released during cellulose hydrolysis was determined by the dinitrosalicyclic acid (DNS) method at 546 nm [6,31].The amount of fermentable sugars obtained from bio-conversion of Kraft cellulose obtained from twenty sawdust wood species at different enzyme concentrations 0.4 mg cm -3 , 1.0 mg cm -3 2.0 mg cm -3 and 4.0 mg cm -3 (20 μL, 50 μL, 100 μL and 200 μL) was investigated (Table 1).It was observed that after the enzymatic hydrolysis of twenty Kraft cellulose with 0.4 mg cm -3 T. viride cellulase the highest concentration of sugar released was obtained during the saccharification of cellulose from L. alata at a glucose concentration of 2.31 mg cm -3 .This was followed by 1.02 mg cm -3 to 1.61 mg cm -3 glucose concentration released from the Kraft cellulose of the following eight wood sawdust species I. asarifolia, S. gabonensis, P. angolensis, T. superba, T. scleroxylon, U. guineensis, N. diderrichii and C. pentadra.The lowest sugar concentration of 0.47 mg.mL -1 was obtained by the enzymatic hydrolysis of cellulose from A. germinans wood waste.The remaining eleven Kraft cellulose on treatment with 0.4 mg cm -3 T. viride cellulase enzyme produced average glucose concentration of 0.7 mg cm -3   The highest sugar concentration released after the enzymatic conversion of the twenty Kraft cellulose with 1.0 mg cm -3 Trichoderma viride cellulase was again released from L. alata cellulose to give 86% increase in sugar formation of the L. alata wood cellulose from 0.4 mg cm -3 bioconversion to 1.0 mg cm -3 enzyme treatment.The second highest sugar concentration of 3.25 mg cm -3 was produced from C. pentadra cellulose.The influence of the cellulose-cellulase interaction resulted in 144% increase in glucose from E.

RESULTS AND DISCUSSION
suaveolen and 121% from S. pustlatas, while the lowest glucose concentration of 0.69 mg cm -3 was released from M .excelacellulose.Seven of the Kraft cellulose of the following wood sawdust namely S. gabonensis, P. angolensis, T. superba, T. scleroxylon, U. guineensis, N. diderrichii, I. asarifolia gave average sugar concentration of 2.48 mg cm -3 .The average glucose concentration produced from the remaining ten Kraft cellulose was in the range of 1.05 mg cm -3 to 1.61 mg cm -3 .
The highest sugar concentration released from the bioconversion of the Kraft cellulose at 2.0 mg cm -3 enzyme catalysis was again released from the same L.alata cellulose with glucose concentration of 9.49 mg cm -3 to give 312% increase in sugar formation pattern of the wood waste biomass from 0.4 mg cm -3 to 2.0 mg cm -3 enzyme application.The second highest sugar producing wood cellulose was obtained from C. pentadra with sugar concentration of 8.16 mg cm -3 .This was followed by sugar concentrations of 7.71 mg cm -3 and 7.43 mg cm -3 produced from T. superba and U. guineensis cellulose respectively.The highest percentage increase in glucose as a result of enzyme solubilization of the cellulose after treatment with 2.0 mg cm -3 cellulase concentration was 674%, 641% and 617% from E.suaveolen, H. ciliate and A. germinans respectively.This was followed by another increase in percentage yield of glucose at 515%, 513% and 503% from, R. heudelotii, K. ivorensis and S. globulifera respectively.with The lowest sugar concentration of 2.08 mg cm -3 produced from M. excelsa wood cellulose while the remaining Kraft cellulose released average sugar concentration of 5.5 mg cm -3 After the 4.0 mg cm -3 enzymatic hydrolysis of the Kraft cellulose from the twenty different sawdust wood species with Trichoderma viride cellulase, it was also observed that the highest sugar concentration was again released from M. alata cellulose at a concentration of 9.49 mg cm -3 .It was however observed that almost the percentage increase in sugar formation was produced from the biodegradation of this M. alata cellulose between 0.4 mg cm -3 to 2.0 mg cm -3 (310.82%)Trichoderma viride enzyme treatment and 0.4 mg cm -3 to 4.0 mg cm -3 (313.41%)enzyme concentration.Increase in the concentration of the Trichoderma viride cellulase enzyme up to 4.0 mg cm -3 at a constant M. alata cellulose interaction did not lead to further increase in the amount of sugar produced by the biomass.This same trend was also observed in the sugar releasing pattern of the remaining nineteen Kraft wood cellulose where further increase in the enzyme concentration failed to produce a commiserate increase in fermentable sugar.This suggests that the biomass-cellulase saturation point for the wood cellulose-Trichoderma viride cellulase enzyme interaction was actualized at 2.0 mg cm -3 enzyme treatment.
A cellulase system could act differently on cellulose from non-identical cellulose materials as described during the bioconversion of wastepaper into fermentable sugars [17].The optimization of the cellulosecellulase ratio during the bioconversion of cellulose is an important variable to monitor during the process of developing waste cellulose as a resource of renewable energy.
A general trend of increasing sugar production with increasing enzyme loading was concluded with a strong increase in saccharification from 20 uL enzyme to 100 uL enzyme.A little change in sugar formation was obtained with all substrate when treated with 200 uL enzyme compared to the degree of saccharification when exposed to 100 uL of enzyme.It can also be concluded that optimum enzyme-cellulase ratio for maximum degradation is in the range of 200 uL enzyme incubated with 10 mg cellulose substrates.

CONCLUSION
Lignocellulosic biomass waste as a recalcitrant structural cellulose component can be converted into glucose with cellulase enzymes.However, the understanding of cellulose-cellulase digestibility ration is very important in the effective solubilization of cellulose molecule for the production of high yield of fermentable sugars at relatively low enzyme concentration.

Table 1 :
Concentrations of glucose released (mg/mL) from different twenty sawdust cellulose on incubation with Trichorderma viride cellulase at different enzyme loading with constant biomass interaction

Table 2 :
Percentage increase in glucose concentration (mg cm -3 ) from the hydrolysis of different twenty sawdust cellulose with T. viride cellulase after the lowest cellulase concentration of 0.4 mg cm -3 and 1.0 mg cm -3 at constant biomass

Table 3 :
Percentage increase in glucose concentration (mg cm -3 ) from the hydrolysis of different twenty sawdust cellulose with T. viride cellulase after the lowest cellulase concentration of 0.4 mg cm -3 and 2.0 mg cm -3 at constant biomass