EVALUATION OF MICROORGANISMS FROM AGRICULTURAL WASTES FOR MANNANASE PRODUCTION AND EFFECTS ON THE GROWTH PERFORMANCE OF CLARIAS GARIEPINUS

Abstract

Mannanase is an enzyme complex which breaks down mannan to mannooligosaccharide, mannose, glucose and galactose. It has been used widely in commercial food processing, textile industry, animal feed formulation, prebiotic preparation and laundry detergents. Microorganisms associated with agricultural by-products obtained from local markets, farmfield and domestic sources were investigated for their ability to produce mannanase of industrial application. The associated microorganisms were isolated and counted by standard microbiological methods. Quantitatively, mannanase production was performed in mineral salt solution into which mannan containing substrate had been incorporated as the sole carbon source and enzyme activity was determined by dinitrosalicylic acid method. Optimization of process parameters were performed using one factor at a time technique. Purification of crude mannanase was done by standard biochemical methods. The chemical composition of fungal and mannanase treated agricultural wastes was determined according to standard chemical methods. The mineral composition of fungal and mannanase treated wastes was determined using atomic absorption spectrophotometer method. The four bacteria and six fungi isolated exhibited mannanase activity on locust bean gum agar medium. In fermentation broth, all the bacterial isolates exhibited mannanase activity ranging from 0.278 to 13.889 U/ml for static, 0.556 to 13.426 U/ml for shaken and 3.060 to 103.200 U/ml for solid state fermentation respectively. Similarly, all the fungal isolates showed mannanase activity which were between 0.370 and 21.667 U/ml for static, 0.278 and 3.3.982 U/ml for shaken and 5.000 and 18.056 U/ml for solid state fermentation. The enzyme from Penicillium italicum was purified by ammonium sulphate precipitation, ion exchange chromatography (DEAE-Sephadex-A-50) and gel filtration (Sephadex G-150). Fractionation of ammonium sulphate precipitated β-mannanase from P. italicum on sephadex A-50 produced one major activity peak. Further fractionation of the enzyme on ion exchange with Sephadex G-150 yielded one activity peak. A pH of 5.0 was optimum for purified enzyme activity and relatively stable between 40 to 100 min of incubation at this pH. The optimum temperature was 70oC and 100% thermostable for 40 min after which a slight decline in activity was observed. The apparent Km for the hydrolysis of LBG from Lineweaver-Bulk plot was approximately 0.29 mg/mL, while the Vmax was 0.13 μmol/min/mL. The addition of salts and organic compounds at 10 mM and 40 mM respectively caused inhibition of enzyme activity. At 20 mM, enzyme activity was enhanced by FeSO4.7H2O, SDS and ZnSO4. 7H2O, while others caused inhibition of enzyme activity. The incubation of enzyme with CaCl2 and FeSO4.7H2O at 60 mM enhanced enzyme activity, while others caused inhibition. In mono and co-culture treatment with P. italicum and T. oedocephalis, crude protein and fat contents increased significantly (P<0.05) with increase in fermentation time, while reduction occurred in moisture and crude fibre contents in all the fermented wastes when compared with the unfermented control treatments. However, co-fungal treatments resulted in higher crude protein and fat content, while lower values were obtained for crude fibre and moisture content when compared with that of mono-culture for all the experiments. The cellulose, hemicelluloses and lignin contents decreased significantly in monoculture of P. italicum, T. oedocephalis and co-culture of P. italicum and T. oedocephalis when compared to those of unfermented wastes (control treatments). The mineral contents of the fermented wastes varied with the types of treatment. The mannanase treated wastes showed significant increase (P<0.05) in crude protein, fat and ash contents, while remarkable reduction occurred in crude fibre and moisture content in all the treated wastes when compared with untreated wastes. The amount of anti-nutrient contents between mannanase treated wastes varied significantly with the time of treatment. Mannanase treatment showed significant reduction in fibre fractions (lignin, cellulose and hemicelluloses) in almost all the mannanase treated wastes. Co-fungal solid state fermentation was employed for the formulation of fish feed. Co-fungal solid state fermentation of rice bran was carried out using P. italicum and T. oedocephalis. Proximate composition of the experimental diets revealed that the highest percentage crude protein, moisture content and lowest crude fiber and ash content in T1 (control/ reference diet). A feeding trial was conducted for 58 days to evaluate the effect of partial replacement of fish meal with co-fungal treated and untreated rice bran on the growth performance, nutrient utilization and body composition of African Catfish Clarias garipienus (6.04±0.5g initial mean weight). After 58 days of rearing, the survival rate (%) ranged from 53.33% in T4 (D50T) to 80% in T1 (D0) and T2 (D20T). Maximum weight gain (84.312±3.83g), percentage weight gain (2.40±0.06%), specific growth rate (1.51±9.13), protein intake (2.89±0.31) and protein efficiency ratio (4.67±0.00) were observed in T1 (D0). The percentage crude protein, fat and ash contents of the carcass obtained from all the treatments were higher than values of these parameters before feeding trial. However, of all the diets evaluated in this study, T4 (D50T) had the highest percentage crude protein and lowest fat content of 49.05±0.89% and 7.28±0.51% respectively. Hemoglobin, packed cell volume, red blood cell, lymphocytes, mean cell hemaglobin and mean cell volume were highest in fish fed diets T4 (D50T) and T2 (D20T) in comparison with others treatments. Clarias garipienus fed T2 (D20T) had 1%, while others had 0%. Fish fed T3 (D20U) had the highest erthrocyte sedimentation rate while the lowest was observed in T5 (D50U). Histopathological examination revealed varied degrees of pathological changes in the organs (gill, kidney, liver and skin) of fish fed different inclusion diets. Analysis of the nucleotide sequences obtained in this study using the Basic Alignment Search Tool (BLAST) program revealed 88-89% and 99% nucleotide sequence identity with corresponding sequences of Penicillum italicum and Moniliella oedocephalis synonym Trichosporonoides oedocephalis respectively, available on the Genbank database. In this investigation, microbial isolates evaluated for mannanase production from agro-wastes elaborated considerable mannanase activity and this could be exploited for economic uses. The purified β-mannanase was active over a wide pH and temperature, and its stability implies that the enzyme will be useful in processes that are subjected to extreme conditions. Therefore, this enzyme can withstand some harsh conditions encountered during industrial operations. The low Km value obtained suggests that the purified β-mannanase has high affinity toward substrates and can possibly be used for prebiotic and animal feed formulation. Animal nutrition point of view, agricultural wastes are not suitable feed ingredients as they are deficient in their digestible protein, high in anti-nutrient content and rich in high molecular mannan. However, this study has shown an improvement in the nutritive values of agricultural wastes when subjected to mannolytic fungal and mannanase treatment.