The properties of extracellular cellulase obtained from Bacillus species (FIIRO Strain B223) on supplying cellulose from Brewers Spent Grain (BSG) were studied. Generally, a crude cellulase activity between 11,757 Units to 13,930 Units was observed for both sources of carbon (BSG and crystalline cellulose). The optimum pH and temperature of 5.0, and 30°C - 40°C for both sources of carbon was observed. Heavy metals such as copper, zinc, and iron inhibited the activities of B223 cellulase while the cellulase activities on alkaline earth metal (manganese) were moderate. The co-factor behaviour of manganese ion was also established. Local production of cellulase in Nigeria using local raw materials such as Brewers Spent Grain reduces enzyme cost; cost of finished products and increases gross domestic products.
There is an increasing interest in utilising cellulose wastes as feedstock for fermentation processes, thereby converting low cost starting materials into products of greater value. Most commercial cellulases are produced by submerged fermentation of the fungi and bacteria [
There are 2 main types of exo-cellulases (cellobiohydrolases, abbreviate CBH)―one type working processively from the reducing end, and one type working processively from the non-reducing end. Processive/Progressive cellulase: This cellulase continues to interact with a single polysaccharide strand. Non-progressive/Non-Processive: This cellulase will interact once then disengages and engages another polysaccharide strand. Cellobiose/beta- galactosidase: It hydrolyses the exo-cellulase product into individual monosaccharide. Oxidative cellulase: It depolymerises cellulose by radial reactions, as for instance cellobiose dehydrogenase (acceptor). Cellulose phosphorylases: It depolymerise cellulose using phosphate instead of water [
There is also report of effects of pH, temperature, and metallic ions on cellulase produced from Streptomyces sp., using fruit waste as substrate. The several waste utilisation programmes all over the World are geared towards converting wastes to wealth. This study reports the use of cellulose from Brewers Spent Grain (BSG) waste for submerged production of cellulase, and the temperature, pH, and metallic ion tolerance properties of the Bacillus cellulase.
The strain of choice was isolated from BSG undergoing bio-deterioration in the open environment. One (1) gram of the deteriorating BSG was weighed and aseptically transferred into the first test tube of 1 in 10 dilutions containing sterile distilled water as diluent. The pour plate technique as previously described by [
The bacterial strains with high cellulase producing ability were picked for identification and further investigation. Preliminary identification by morphological analysis was conducted by using light microscope after Gram staining, and spore staining. The Gram staining and spore staining method have been previously described [
A total of 21 strains of bacteria strains were obtained from deteriorating Brewers Spent Grain. Therefore, a preliminary qualitative analysis for cellulolytic activity was conducted by using Congo red dye. The bacteria were grown on Carboxymethyl Cellulose (CMC) agar which was prepared by adding 5% CMC into Nutrient Agar (Oxoid). The detailed screening procedure has been previously described [
Protein content of the enzyme extracts were determined by following the method of Lowry and colleagues [
The activity of crude cellulase in the centrifugal supernatant was determined according to the methods which had also been previously reported by [
This is the activity of an enzyme per milligram of total protein (expressed in units/mg/ml). Specific activity of the cellulase was determined using the formula below.
The Enzyme activity was measured by treating the enzyme-substrate mixture at various temperatures from 30˚C and 80˚C. Technically, the cellulase enzyme was diluted, and 0.5 ml of the cellulase diluted cellulase was gently pipetted into well-labelled test tubes previously loaded with o.5 gram of shredded filter paper. The enzyme- substrate reaction was incubated at 40˚C for 60 Minutes. In addition, other tubes prepared in this way were labelled, and each incubated at different temperatures (50˚C, 60˚C, 70˚C, and 80˚C) for 60 minutes in each case. Dinitro salicyclic acid (30 mls) was added into all the tubes to stop the reaction. The tubes were boiled further for 5 minutes at 100˚C to develop the red/orange colour. On cooling down to at least temperature of 10˚C, 0.2 ml of the boiled enzyme-substrate mixture was transferred into the curvette of a spectrophotometer (UN-ICAM) and absorbance read at 540 nm wavelength. This DNSA method has been previously used and reported [
The activity of the cellulase was measured at various pH using different pH values of citrate buffer. Technically, 0.5 ml of the citrate buffer at different pH (4 - 8) was mixed with 0.5 ml of the diluted enzyme in a well labelled test tube containing filter paper strip. The assay was done with DNSA method, and water bath incubation was carried out at 50˚C (optimum temperature).
The reaction of the enzyme and filter paper substrate was allowed to proceed at 50˚C with duplicate test tubes containing 10 mM MnSO4, CaSO4, MnSO4, ZnSO4, and FeSO4. Test tubes were labelled according to the source of carbon (BSG and Crystalline Cellulose), and above mentioned salts. The cellulase extract was diluted accordingly, and 0.5 ml of the diluted enzyme was pipetted into the labelled test tube already loaded with 0.5 gram of shredded filter paper. Thereafter, 0.5 ml of 10 mM solution of each of the above mentioned salts (one salt to one test tube) was pipetted to a test tube. The incubation of the reaction mixture was done at 50˚C, and the assay done using previously described DNSA Method [
The crude activity of crude cellulase from Bacillus species on utilization of cellulase from Brewers Spent Grain and from crystalline cellulose was studied at different hours of fermentation. The crude cellulase activities as cellulase from BSG was observed as 239 Units, 146OOU, 13930U, 13260U, 128320U and 13650U at 0, 24 h, 48 h, 72 h, 96 h and 120 hours respectively. The crude cellulase activities when crystalline cellulase was used as carbon source were 153U, 11757U, 12110U, 13015U, 13015U, 13850U, and 135000U at 0 h, 24 h, 48 h, 72 h, 96 h, and 120 h respectively (
There are extensive reports of cellulose with activity of 3.69 FPU/ml produced by solid state fermentation of defatted palm kernel cake by Bacillus cereus [
The protein content of the crude cellulase from BSG Cellulose Erlenmeyer flask was 3.3 Mg/ml on the zero hour but increased progressively to 10.4 mg/ml on the 48th hour. In addition, a slight decrease at 72 hour from 8.2 mg/ml to 5.97 Mg/ml on the 120 hour was also observed (
The specific enzyme activity when supplemented with BSG cellulase at Zero hour, 24 h, 48 h, 72 h, 96 h and 120 h was 182 U/Mg/ml, 1609 U, 13339, 1617.1 U/Mg/ml, 2746.6 U/Mg/ml and 2746.6 U/Mg/ml respectively. The specific cellulase activity on utilisation of crystalline cellulose as sole carbon source was 97.60 U/Mg/ml, 1567.1 Mg/ml, 1843.2 U/Mg/ml, 2135.3 U/mg/ml, 2308.3 U/mg/ml and 5133.1 U/Mg/ml respectively (
Hours of incubation | CRUDE CELLULASE ACTIVITIES (Units) BSG ± S.D | CRUDE CELLULASE ACTIVITIES (Units) CELL ± S.D |
---|---|---|
0 | 239 ± 0.34a | 153 ± 0.12b |
24 | 14600 ± 2.17a | 11757 ± 2.56b |
48 | 13930 ± 2.43a | 12110 ± 3.00b |
72 | 13260 ± 3.21a | 13015 ± 2.05a |
96 | 12832 ± 2.45a | 13850 ± 2.89b |
120 | 13650 ± 2.34a | 13500 ± 2.12a |
Carbon sources include; Brewers Spent Grains (BSG), and Crystalline Cellulose Powder; Values are average duplicate readings, S.D; Standard Deviation. Means followed by the same letter in a column are not significantly different (p < 0.05).
HOURS | PROTIEN CONTENT BSG + S.D | (mg/ml) CELL + S.D | SPECIFIC ACTIVITIES (u/mg/ml) BSG + S.D CELL + S.D | |
---|---|---|---|---|
0 | 3.33 ± 0.45a | 1.57 ± 0.91b | 182.0 ± 0.56a | 97.60 ± 0.45b |
24 | 9.07 ± 1.98a | 5.10 ± 1.46b | 1609.7 ± 2.89a | 1567.1 ± 4.6b |
48 | 10.4 ± 1.03a | 6.57 ± 1.87b | 13339.4 ± 6.04a | 1843.2 ± 4.7b |
72 | 8.2 ± 1.11a | 7.11 ± 1.56a | 1617.1 ± 4.23a | 2135.3 ± 4.8ab |
92 | 6.67 ± 1.34a | 6.0 ± 1.56a | 2746.6 ± 4.19a | 2308.3 ± 5.2ab |
120 | 5.97 ± 1.15a | 2.63 ± 1.06b | 2286.4 ± 6.00a | 5133.1 ± 6.7b |
S. D; Standard deviation. Values are average of duplicate readings, BSG; Brewers Spent Grain. Means followed by the same letter in a column are not significantly different (p < 0.05).
Carbon source/hour of incubation | Temperatures (˚C) | |||||
---|---|---|---|---|---|---|
30 ± S.D | 40 ± S.D | 60 ± S.D | 70 ± S.D | 80 ± S.D | ||
BSG/0 HR | NS | NS | NS | NS | NS | |
CELL/0 HR | NS | NS | NS | NS | NS | |
BSG/24 HR | 2024.28 ± 3.61a | 1518.21 ± 2.10a | 519.16 ± 0.19a | 1134.30 ± 0.26a | 397.00 ± 0.01a | |
CELL/24 HRS | 1986.75 ± 3.06b | 1489.56 ± 2.10a | 487.99 ± 0.26a | 1134.30 ± 0.26a | 349.00 ± 0.11a | |
BSG/48 HRS | 1605.61 ± 2.79ab | 1316.6 ± 2.96b | 441.40 ± 0.47a | 1301.23 ± 2.4b | 570.71 ± 0.03b | |
CELL/48 HRS | 2656.03 ±2.79c | 1728.53 ± 1.14ab | 878.56 ± 0.86b | 1156.37 ± 0.97a | 963.01 ± 0.26ab | |
BSG/72 HRS | 2046.03 ±3.19a | 1351.15 ± 1.09b | 410.17 ± 0.26a | 1299.93 ± 0.96a | 738.31 ± 0.14abab | |
CELL/72 HRS | 2301.32 ± 3.69c | 2533.11 ± 3.61c | 634.66 ± 0.03ab | 1456.95 ± 0.96b | 1473.31 ± 0.14c | |
BSG/96 HRS | 2764.52 ± 2.87cd | 2645.88 ± 3.14c | 842.41 ± 0.03b | 1898.39 ± 1.12ab | 771.22 ± 0.39ab | |
CELL/96 HRS | 3007.28 ± 3.87d | 2578.61 ± 2.86c | 1206.87 ± 0.12c | 1569.59 ± 0.06ab | 903.50 ± 0.33ab | |
BSG/120 HRS | 2850.06 ± 1.06cd | 2545.17 ± 2.87c | 742.34 ± 0.14b | 2021.56 ± 0.18c | 881.53 ± 0.46ab | |
CELL/12O HRS | 7567.85 ± 1.79e | 6168.63 ± 3.07d | 212.40 ± 0.06a | 2482.50 ± 0.28cd | 1579.77 ± 0.11c | |
NS; Not studied. CELL; crystalline cellulose powder, BSG; cellulose from brewers spent grain. Values are units of enzyme/cellulase. Means followed by the same letter in the column are not significantly different (p < 0.05) (Duncan multiple range test).
Generally, the specific activity of cellulase produced from crystalline cellulase was highest at 5133.1 U/Mg/ ml on the 120 hour of fermentation as against a specific activity of 2286.4 U/Mg/ml on the 120 hour of fermentation. This significantly higher specific activity of the crystalline cellulose could be because the crystalline cellulose is more refined than the crude BSG cellulose.
Studies on cellulase produced by wild strains of Aspergillus niger, Penicillium chryseogenum, and Trichoderma harzianum using a submerged fermentation method recorded a maximum activities of 0.30, 0.24 and 0.20 Units/Mg protein for cellulase from A. niger, P. chryseogenum, and T. harzianum respectively at 144 hours of incubation using Saw dust as substrate [
In another related study, a novel thermo-tolerant endoglucanase from Bacillus strains with the highest specific activity of 1.1 U/Mg proteins on utilisation of 8% carboxymethyl cellulose was reported [
The temperature tolerance properties of the Bacillus cellulase were also studied at different time intervals with respect to the source of cellulose. The temperature studies were not carried on cellulase harvested on the zero hour because activity was grossly low. Cellulase activities in both sources of cellulose (BSG and crystalline cellulose) were high on incubation of enzyme and substrate at 30˚C The BSG and crystalline cellulose options had activities of 2850.06 and 6168.63 Units respectively (
There are several studies on tolerance of cellulase to different temperature. The study reported the production of cellulase by a cellulolytic bacterium identified as Cellulomonas sp ASN2, with an optimum temperature of 60˚C [
There are studies on cellulase from microorganisms associated with the gut of giant African snail (Archachatina marginata) [
The effect of various pH on the activities of cellulase was also studied and reported. pH was varied between 3.0 and 8.0 for both cellulase produced using BSG cellulose and refined cellulose powder, and the optimum pH was observed at 5.0. This implies that the catalysis mediated by cellulase of B223 strain is best achieved at pH 5.0 (
The optimum pH of 5.0, and 5.5 for cellulase produced from Penicillium artrovenetum in a submerged fermentation condition was documented by other workers [
Carbon source/hour of incubation | Different pH values | ||||||
---|---|---|---|---|---|---|---|
3.0 ± S.D | 4.0 ±S.D | 4.8 ± S.D | 5.0 ± S.D | 6.0 ± S.D | 7.0 ± S.D | 8.0 ± S.D | |
BSG/0 HRS | NS | NS | NS | NS | NS | NS | NS |
CELL/0 HRS | NS | NS | NS | NS | NS | NS | NS |
BSG/24 HRS | 135.24 ± 0.11a | 287.94 ± 0.19b | 1605.47 ± 0.26b | 1112.48 ± 0.32a | 889.99 ± 2.40a | 1269.54 ± 0.91a | 397.00 ± 0.26a |
CELL/24 HRS | 129.47 ± 0.46a | 119.60 ± 0.26a | 1780.00 ± 0.27b | 1162.47 ± 0.46a | 804.00 ± 2.61a | 1280.71 ± 0.94a | 392.00 ± 0.11a |
BSG/48 HRS | 163.60 ± 0.26a | 95.12 ± 0.81a | 1340.24 ± 0.81a | 1008.26 ± 0.27a | 939.78 ± 0.23a | 528.86 ± 0.60b | 570.71 ± 0.19b |
CELL/48 HRS | 66.25 ± 0.27b | 96.36 ± 0.03a | 1842.96 ± 0.03c | 1915.24 ± 0.21b | 1403.30 ± 0.26b | 1247.24 ± 0.67a | 963.64 ± 0.29ab |
BSG/72 HRS | 231.63 ± 0.47ab | 453.60 ± 0.37c | 1616.56 ± 0.47b | 1307.72 ± 0.04ab | 926.50 ± 0.28a | 1143.65 ± 0.21a | 738.31 ± 0.84ab |
CELL/72 HRS | 93.82 ± 0.29b | 419.43 ± 0.29c | 2135.76 ± 2.61d | 1782.56 ± 0.32b | 1716.33 ± 0.80c | 1335.54 ± 0.26ab | 1473.51 ± 0.30c |
BSG/96 HRS | 563.58 ± 0.28c | 337.14 ± 0.07ab | 2746.23 ± 2.09de | 2295.03 ± 0.33c | 2046.70 ± 0.80bc | 1874.66 ± 0.03c | 771.22 ± 0.30ab |
CELL/96 HRS | 1490.4 ± 0.14d | 316.56 ± 0.06ab | 2308.22 ± 0.26d | 2499.47 ± 0.26c | 1503.64 ± 2.49b | 1147.52 ± 0.34a | 903.50 ± 0.36ab |
BSG/120 HRS | 391.06 ± 0.11ab | 490.48 ± 0.27c | 2286.68 ± 0.41d | 1855.86 ± 0.85b | 1252.70 ± 2.69b | 1663.64 ± 0.26c | 881.53 ± 0.14ab |
CELL/120 HRS | 2437.36 ± 0.03e | 646.95 ± 0.84d | 5115.45 ± 0.47f | 5025.17 ± 0.91d | 2828.54 ± 2.80d | 5055.26 ± 3.15d | 1579.77 ± 2.97c |
Means followed by the same letter in the column are not significantly different (p < 0.05) (Duncan multiple range test). Values are units of cellulase.
Metal Ions (10 mM) | CELLULASE ACTIVITIES BEFORE EXPOSURE TO METALS (U/Mg/Ml) | CELLULASE ACTIVITIES BEFORE AFTER TO METALS (U/Mg/Ml) | ||
---|---|---|---|---|
BSG ± S.D | CELL ± S.D | BSG ± S.D | CELL ± S.D | |
CU | 2746.60 ± 0.31a | 2308.03 ± 0.06a | 1382.26 ± 0.94b | 1378.34 ± 0.81ab |
Fe | 2746.60 ± 0.26a | 2308.03 ± 0.04a | 1542.44 ± 0.97ab | 3719.53 ± 0.92c |
Mn | 2746.60 ± 0.27a | 2308.03 ± 0.21a | 4763.76 ± 0.62c | 5599.08 ± 0.03d |
Zn | 2746.60 ± 0.41a | 2308.03 ± 0.09a | 1038.18 ± 0.06a | 1193.68 ± 0.26a |
Mg | 2746.60 ± 0.03a | 2308.03 ± 0.37a | 1506.84 ± 0.08ab | 1503.64 ± 0.01ab |
Means followed by the same letter in the column are not significantly different (p < 0.05) (Duncan multiple range test).
not tally with the optimum pH of the B223 Bacillus cellulase produced by submerged fermentation of cellulose abundant Brewers Spent Grain (BSG).
In addition, the effect of metallic ions on the B223 cellulase was investigated. Heavy metals such as copper reduced the specific activity of cellulase from 2746.60 to 1382.62 U/Mg/ml. Iron, Zinc and magnesium reduced the specific activity of the cellulase (
On the contrary, manganese at 10 mM concentration was able to increase the B223 cellulase activity from 2746.60 to 4763.76 and 2308.03 to 5599.08 U/Mg/ml for BSG cellulose and crystalline cellulose options respectively (
Only two sources of carbon were tried in this experiment. Results obtained showed that there is no statistical significance between the experimental group that contained refined crystalline cellulose and Cellulose from Brewers’ Spent Grain. This by implication shows that the crude Brewers’ Spent Grain is a good raw material, and if refined further can as well provide more cellulose to organisms carrying out fermentation than the refined crystalline cellulose. This is the right time for Nigerian Government to pay adequate research attention to research and development in agro-allied processing.
The production of cellulase using cellulose from BSG is established, and the possibility of packaging of B223 cellulase by incorporating manganese ion as co-factor is hereby established in this study. This local production of cellulase from local sources is a noteworthy contribution as it reduces enzyme cost, cost of final product, increases Gross Domestic Products (GDP), and saves foreign exchange.
Frank AnayoOrji,Ekaette NdukaDike,Adekunle KolawoleLawal,Abdulateef OmoniyiSadiq,YewandeSuberu,Ariyo CalebFamotemi,Agatha IsiomaUgbana,FolakeFashola,BlossomIta,Samuel OlakiitanOlatope,Emoleila EjiyaItoandoan,Abimbola OlajumokeAdefiranye,Gloria NwakaeghoElemo, (2016) Properties of Bacillus species Cellulase Produced Using Cellulose from Brewers Spent Grain (BSG) as Substrate. Advances in Bioscience and Biotechnology,07,142-148. doi: 10.4236/abb.2016.73013