Saccharification of Sawdust Masses from the Lagos Lagoon in Nigeria with Aspergillus niger Cellulase

JBM Sibiya1, NA Ndukwe2 and JPH van Wyk1*

1Department of Pharmacology and Therapeutics, Sefako Makgatho Health Sciences University, South Africa

2Department of Chemical Sciences, College of Basic and Applied Sciences, Mountain Top University, Magoki, Ogun State, Nigeria

Received: 20-Jul-2020 , Accepted: 30-Nov-2020

Keywords: A. niger, Waste cellulose, Cellulase, Sawdust, Saccharification

DOI: http://dx.doi.org/10.20510/ukjpb/8/i6/1607002257

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Abstract

The Lagos Lagoon in Nigeria is extensively polluted with sawdust produced by hundreds of sawmills located on the banks of the lagoon. In order to develop cellulose as a possible resource of bioenergy different masses of various sawdust materials was exposed to the saccharification action by a fungal cellulase source, Aspergillus niger. Five different masses of delignified and non-delignified sawdust materials have been bio-converted with A. niger cellulase into glucose, a fermentable sugar. The amount of sugar released has increased when increasing masses of waste cellulose was degraded although the percentage saccharification shown a decline when increasing masses was degraded. Delignification of sawdust was effective in terms of sugar production during A. niger cellulase catalyzed degradation as the bio-conversion of all delignified materials produced more sugar than the non-delignified materials. The highest amount of sugar was produced from 10 mg of Pterygota macrocarpa sawdust while the highest percentage of saccharification was calculated at 85% during bio-conversion of 2.0 mg of delignified sawdust from P. macrocarpa. Sawdust along the Lagos Lagoon is a major solid waste product that could be developed as a resource of bio-energy if the cellulose component of this material is effectively saccharified.

1 Introduction

The climate in Nigeria changes from tropical rain forest in the southern parts to savannah in the middle belt and dry or semi-dry in the northern parts which caused massive amounts of rainfall allowing trees to flourish all over the country.There are about 11 million hectares of forest and about 5.5 million hectares of other wooded land also available in Nigeria. The amount of wood waste produced in Nigeria is persistently extending, and if these wastes can be managed properly by no longer regarding it as trivial materials to be dumped but as useful resources of economic value from which energy, fuel and other by-products can be derived1. Sawmills form a large part of the industries that process these woods and are located in Lagos, Ekiti, Osun Cross River, Ondo, Oyo, Imo, Edo, Delta and Ogun States and jointly they represent more than 90% of sawmills found in the Nigeria according to Elijah and Elegbede2. The area of the Lagos Lagoon is approximately 6354,71 km2 and the circumference is about 285 km where about 104,000 m3 of sawdust is generated daily from various sawmills located along the lagoon forming one of the major sources of lignocellulose in the lagoon3.

Lignocellulosic materials included crop residues, grasses, sawdust, wood chips, organic portion of municipal solid wastes and these materials are abundantly available from nature, are renewable and of low cost4.  Depletion of fossil fuel sources and excess greenhouse gas production has become a concern and resulted in many scientists around the world to be interested in the development of non-conventional fuel derived from bio-renewable resources such as sugars, starches and lignocellulosic materials5.Incomplete combustion during burning of fossil fuels is creating a globally environmental crisis through the formation of harmful gases like carbon (IV) oxide (CO2), methane (CH4) and large amounts of nitrous oxides. To limit the production of these gases there is an alternative energy resource like bio-ethanol which is produced from lignocellulosic materials through fermentation, and it can be used as an alternative to the combustion of conventional fossil fuels6,7.Bio-ethanol contains an insignificant amount of sulfur when compared to gasoline and by mixing these two fuels the sulfur content in fuel will be reduced as well as the release of sulfur oxide, which is carcinogenic and which can cause acid rain8.

Lignocellulose is a complex carbohydrate polymer that consists of three principal components namely cellulose, hemicellulose and lignin. Cellulose, a linear and crystalline homopolymer is made up of repeating sugar units of glucose linked by β-1,4-glucosidic bonds. Hemicellulose is a heteropolymer composed of D-xylose, D-arabinose, D-glucose, D-galactose, and D-mannose that is short and highly branched. Lignin, a hydrophobic substance is firmly bound to these two carbohydrate polymers and protects them from microbial attack9. Lignin also makes it difficult for the enzymatic hydrolysis of cellulose because of the covalent bonds between lignin and cellulose thus makes woody plants (woody biomass) harder to treat as compared to herbaceous crops such as grass biomass10.  For lignocellulosic biomass to be converted into value added products like bio-fuels it requires a multi-step process that includes pre-treatment (mechanical, chemical, or biological), enzymatic hydrolysis and fermentation. Pre-treatment is an essential process in the breakdown of the lignin barrier for the recovery of cellulose, which could be converted into fermentable sugars by enzymatic hydrolysis11.  The best possible pre-treatment procedure of lignocellulose should be inexpensive, be able to remove most of the lignin, potent for various lignocellulosic substrates and glucan loss should be minimal12. Cellulase catalyzed hydrolysis is becoming a suitable way for the breakdown of lignocelluloses into simple sugars because of its low energy demands, gentle environmental conditions and production of fewer fermentation inhibitor products, which are generated13. When compared with other micro-organisms, it is known that Aspergillus niger and Trichorderma viride are more effective for the production of the three components of the cellulase complex enzyme which are endoglucanase, exoglucanase and b-glucosidase14. These components exhibit a synergistic action during the hydrolysis of biomass into fermentable sugars.  Glucose, fructose and sucrose are mainly the fermentable sugars present in sugar based raw materials and yeasts or other fermenting microorganisms can be used to convert these soluble sugars into ethanol by fermentation without the need of further treatment15,16.

The current research investigated the relative saccharification of non-delignified and delignified sawdust from five different trees along the Lagos Lagoon. The saccharification was performed with A. niger cellulase with the relative amount of sugar released from each sawdust sample and the percentage saccharification of each wood material, determined.

2 Materials and methods

2.1 Delignification of Sawdust

To ensure a maximum cellulose exposure to the cellulase enzyme the various sawdust materials were delignified by subjecting 2 kg of each of the different sawdust materials (2.8-5.0 mm particle size) to 350 g of NaOH and 140 g Na2S during the Kraft pulping process. The Kraft pulping chemicals was dissolved in 8 L water and the delignification of the lignocellulosic materials (sawdust) was carried out in a rotary steel digester at 1700C and a pressure of 200 kPa for 1.45 h at cooking liquor to wood ratio of 4:1. After the Kraft pretreatment, the extracted cellulose fibers were washed in turns with deionized water until they were free of the Kraft reagents17.  To remove residual lignin from these Kraft-treated cellulose all these sawdust materials (10 g) were treated with 30 % hydrogen peroxide (60 ml) at 40°C for 25-30 min.  

2.2 Sawdust substrates and cellulase enzyme

Non-delignified and delignified sawdust samples from five different trees (Erythropleum suaveolens, Symphona globulifera, Ricindendron heudelotii, Pterygota macrocarpa, Milicia excelsa) obtained along the Lagos Lagoon were used in this study. Different masses of each waste cellulose material (2, 4, 6, 8 and 10 mg) were transferred in triplicate into test tubes.  Commercially obtained A. niger cellulase enzyme (0.1g) was dissolved in 0.005 mol.dm-3 pH 5.0 tris buffer resulting in an enzyme solution concentration of 2.0 mg.ml-1.

2.3 Cellulase incubation, DNS analyses and Statistical Analyses

The weighed sawdust materials were transferred in triplicate into tubes and  incubated in a water bath with the A. niger cellulase enzyme solution (200 ul) and Tris buffer solution (800 ul) for 2h at a temperature of 500C. To determine the concentration of sugars released during saccharification of the sawdust materials a standard glucose calibration curve was constructed using the DNS methodand glucose standard solutions at concentration of 0.50 mg.ml-1, 2.00 mg.ml-1, 4.00 mg.ml-1, 6.00 mg.ml-1 and 8.00 mg.ml-1 18. The DNS reagent (1.5 ml) was transferred to each test tube containing the cellulase-sawdust mixture (1.0 ml) and was incubated in a boiling waterbath for 10 min. After cooling the reaction mixture down to room temperature the resulting colour intensity was read at 520 nm and used to construct a calibration curve. The sugar concentration of each incubation was derived from the calibration curve constructed with the glucose standards. Excel was used to determine the average sugar concentration as well as the standard deviation for each set of incubations when the various paper materials were treated with the A. niger cellulase with the average sugar concentration values and standard deviation indicated in each graph.

3 Results and Discussions

The accumulation of sawdust along the Lagos Lagoon in Nigeria is a major threat to environmental quality with water resources and air mostly polluted by this waste product produced by numerous sawmills located on banks of the lagoon. Although research has been conducted on developing this major environmental pollutant into a feedstock for chemical procedures, the problem is not yet solved but is rather intensifying19,20.

During the cellulase catalyzed bio-conversion of the various sawdust materials the amount of sugar released from the delignified material was higher than the sugar concentration obtained from the non-delignified samples. This observation was evident with sawdust from all five different trees and during treatment of the incubated masses with the cellulase enzyme. Another uniform tendency observed with all incubations was that the relative percentage saccharification of the various sawdust materials decreased when increasing masses were saccharified. The percentage saccharification of the delignified material of all sawdust samples was higher than the corresponding sugar concentration released from the non-delignified material. When sawdust from non-delignified E. suaveolens (Fig 1) was treated with the cellulase enzyme the sugar concentration varied between 0.78 mg.ml-1 obtained from the lowest mass (2 mg) and 1.21 mg.ml-1 released from the highest mass (10 mg). The amount of sugar released from the highest mass was 53% higher than the amount of sugar released from the lowest non-delignified mass. When the delignified sawdust was exposed to the cellulase enzyme the concentration of the released sugar increased from 1.16 mg.ml-1 obtained from the lowest sawdust mass to 2.26 mg.ml-1 released from sawdust with the highest mass of 10 mg. The percentage increase in sugar production from lowest to highest concentration was 94% when degrading the delignified material. A decreasing tendency of percentage saccharification when increasing masses of E. suaveolens was degraded indicated that the non-delignified material was 39% saccharified when 2 mg of sawdust was degraded and 12% when 10 mg of the substance was exposed to the enzyme action. A similar trend was observed when the delignified material was exposed to the cellulase enzyme and the percentage saccharification decreased from 58 % when the lowest mass was exposed to 22% when the highest mass was hydrolysed by this enzyme action.

Figure 2 reflects the cellulase catalyzed degradation of sawdust from P. macrocarpa when the amount of sugar released by degradation of the non-delignified material increases from 1.3 mg.ml-1 to 2.7 mg.ml-1 when the lowest mass (2 mg) and highest mass (10 mg) were bio-converted, respectively. The respective sugar concentration released from the lowest mass and highest mass of the delignified materials was 1.6 mg.ml-1 and 2.8 mg.ml-1. The percentage increase in sugar formation from the lowest mass to the highest mass of the non-pretreated material was 107% whilst the corresponding value for the pretreated material was 75%. The decrease in percentage saccharification with increasing mass exposed to cellulase treatment for the non-pretreated material change from 65% obtained with the 2 mg sample to 12% calculated during degradation of the 10 mg sample. When degrading the delignified material the percentage saccharification decreased from 85% obtained from the 2 mg to 26% released from the highest mass of 10 mg.

When sawdust from M. excelsa (Fig 3) was degraded by A. niger cellulase the sugar concentration released from the 2 mg sample was 1.34 mg.ml-1 whilst a sugar concentration of 2.17 mg.ml-1 was released by the highest mass of 10 mg. These values represent an increase of 61% degradation from the lowest sugar concentration to the highest concentration. When the delignified material was exposed to the cellulase action, the sugar concentration increased from 2.0 mg.ml-1 to 3.0 mg.ml-1 during the bio-conversion of  2 mg and 8 mg sawdust samples, respectively. The amount of sugar produced increased by 50% from the lowest mass (2 mg) to the highest mass (8 mg). The decreasing percentage of saccharification when increasing masses of the material was degraded changed from 65% obtained from the lowest mass of 2 mg to 27% when the highest mass of 10 mg was degraded. This decreasing percentage saccharification was also experienced with the delignified material and it decreased from 80% obtained with the lowest mass of 2 mg to 21% calculated when the highest mass of 10 mg was bio-converted into sugar by the A. niger cellulase enzyme.

During the degradation of S. globulifera (Fig 4) sawdust with A. niger cellulase the difference in sugar production between delignified and lignified materials was small in amounts compared to the values obtained from the other sawdust materials. Increasing amounts of sugar were produced from both delignified and non-delignfied materials when increasing masses of these materials was bio-converted. The lowest amount of sugar released from 2 mg of non-delignified sawdust was 1.21 mg.ml-1 and a sugar concentration of 1.95 mg.ml-1 was obtained from 10 mg of this material. The lowest mass of delignified S. globulifera sawdust produced sugar at a concentration of 1.16 mg.ml-1 whilst the concentration released from the 10 mg substrate was higher at a concentration of 2.81 mg.ml-1. Similar to the degradation of the other sawdust materials both the non delignified and delignified materials showed a decrease in the percentage saccharification when increasing masses of this sawdust material was degraded by A. niger cellulase. The percentage degradation of the non-pretreated material when 2 mg was saccharified changed from 58% to 19% when 10 mg of the substrate was degraded. A similar pattern was observed during the saccharification of the delignified material when 60% bio-conversion were obtained from 2 mg sawdust to 21% when 10 mg of the same material was degraded.

When R. heudelotii sawdust(Fig 5) was treated with A. niger cellulase the non-delignified material did not produce an increasing amount of sugar when increasing masses were bio-converted. The amount of sugar produced varied between the concentration of 1.2 mg.ml-1 and 1.4 mg.ml-1. Saccharification of the delignified material resulted in the production of an increase in the amount of sugar produced when an increased amount of sawdust was converted. During this bio-conversion process the sugar concentration increased from 1.7 mg.ml-1 obtained from the 2 mg sample to 2.6 mg.ml-1 sugar obtained from the 10 mg sawdust material. The percentage saccharification of both non-delignified and delignified sawdust materials showed a decline in sugar production when increasing masses of the material was degraded. Degradation of the delignified material decreased from 80% when 2 mg of the material was degraded to 24% when 10 mg of this substance was bioconverted. A decrease from 62% (2 mg) to 21% (10 mg) was obtained during degradation of the non-delignified material.

When comparing the amount of sugar produced from the different types of waste cellulose materials it was observed that the highest concentration of sugar obtained from the non-delignified material was calculated at 2.7 mg.ml-1 obtained from 10 mg of P. macrocarpa (Fig 2). The second highest was obtained from 10 mg of M. excelsa (Fig. 3) at a concentration of 2.17 mg.ml-1 and the lowest sugar concentration obtained from non-delignified sawdust was calculated at the concentration of 0.78 mg.ml-1 released from 2 mg of E. suaveolens (Fig. 1). The highest sugar concentration released from the delignified material was calculated at 3.0 mg.ml-1 obtained during the degradation of 8 mg of M. excelsa (Fig. 3) followed by 2.8 mg.ml-1 released during the degradation of P. macrocarpa (Fig. 2). A concentration of 1.16 mg.ml-1 was calculated as the lowest sugar concentration released from both delignified sawdust from E. suaveolens (Fig 1) and S. globulifera (Fig 4).

The percentage increase in sugar production from the delignified sawdust relative to the non-delignified material for each mass of sawdust incubated with A. niger cellulase is indicated in table 1.

All sawdust materials showed an increase in percentage saccharification obtained from the delignified material relative to the non-delignified material. During saccharification of 2 mg of S. globulifera the non-delignified material resulted in a higher sugar production as the delignified substance. The highest increase in sugar formation was obtained during the bio-conversion of 8 mg of R. heudelotii sawdust which resulted in a 145% increase of sugar produced from the delignified material relative to the non-delignified substance. The second and third highest increase in sugar formation was also obtained from R. heudelotii sawdust when 10 mg and 8 mg resulted in an increase of 116% and 118%, respectively. The lowest percentage increase in sugar formation was experienced with 10 mg of P. macrocarpa resulting in a 4% increase followed by 4 mg of S. globulifera producing a 7% increase and 10 mg of M. excelsa in a 11% increase of sugar production from the delignified material relative to the non-delignified substance.

The development of alternative and renewable energy resources such as sawdust would become more topical as the negative effect of environmental pollution and fossil fuel combustion on the global population becomes more evident. In order to manage these negative environmental issues, it is important that the amount of solid waste produced is limited and that a substitute for fossil fuel is identified and developed. The cellulose component of organic solid waste could be developed as a suitable alternative in energy resource by degrading this bio-polymer into glucose that could act as a feedstock for many renewable formation products such as bio-ethanol21, bio-pharmaceuticals22 and bio-chemicals23. The negative effect of sawdust along the Lagos Lagoon is well documented and this major organic solid waste material can be effectively utilized if the cellulose component is successfully bio-degraded into glucose a fermentable sugar24. Such a solid waste development would not only have a positive effect on the environment but will also limit the negative effect on the local population who are already exposed to polluted air as a result of the burning of sawdust.

4 Conclusions

Sawdust a potential bio-energy resource and major organic solid waste product are produced in great amounts along the Lagos Lagoon in Nigeria. In order to utilize the cellulose component of this waste material various masses of sawdust from different trees have been saccharified with cellulase from A. niger. A trend of increasing sugar production with increasing sawdust mass degraded was observed although the percentage saccharification decreased when increasing masses was bio-degraded by the cellulase enzyme system. From this investigation, it can be concluded that the cellulose component of sawdust from the Lagos Lagoon in Nigeria can be utilized as a potential resource for bio-energy production and bio-product development. The cellulase concentration to mass of sawdust ratio incubated is, however, an important variable that should be optimized in order to ensure maximum sugar production from sawdust of different trees.

5 Conflict of interests

No conflict of interest among all authors of this manuscript.

6 Author’s contribution

Collection and delignification of samples was done by NA, laboratory incubations and data analyses were performed by JBM while the research was planned and manuscript written by JPH. All authors approved the publication.

7 References

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