Cellulase Production by Wild-type Aspergillus niger, Penicillium chrysogenum and Trichoderma harzianum Using Waste Cellulosic Materials

Waste cellulosic materials (corncob, sawdust and sugarcane pulp) and crystalline cellulose induced cellulase production in wild strains of Aspergillus niger, Penicillium chrysogenum and Trichoderma harzianum isolated from a wood-waste dump in Lagos, Nigeria. Cellulose-supplemented media gave the maximum cellulase activity of 0.54, 0.67 and 0.39 units mg Protein-1 for A. niger, P. chrysogenum and T. harzianum respectively. The maximum enzyme activity for A. niger was obtained at 36 h of cultivation, while P. chrysogenum and T. harzianum gave their maximum enzyme activities at 12 and 60 h respectively. For the cellulosic wastes, highest enzyme activity was obtained with sawdust where A. niger, P. chrysogenum and T. harzianum gave the maximum enzyme activity of 0.30, 0.24 and 0.20 units mg Protein-1 respectively after 144 h of cultivation. A. niger recorded the highest enzyme activity with any of the three cellulosic materials followed by P. chrysogenum. It thus appears that the use of sawdust presents the best option for low-cost commercial production of cellulase using A. niger and P. chrysogenum as discussed herewith

GROWTH AND CELLULASE ACTIVITY OF WILD-TYPE ASPERGILLUS NIGER ANL301 IN DIFFERENT CARBON SOURCES

A wild-type Aspergillus niger (ANL301) isolated from wood-waste in Lagos, Nigeria, produces extracellular proteins with cellulase (EC 3. 2. 1. 4) activity. Three different carbon sources (Glucose, Cellulose and Sawdust) influenced the organism’s growth and the production of extracellular cellulase enzymes. Best growth was obtained with glucose at 72 hours of incubation. The peak mycelia weight of 1.56 mg/ mL obtained with glucose was about 3 times the maximum weight of 0.58 and 0.49 mg/ mL respectively obtained with cellulose and sawdust at 96 hours. The peak protein contents of the culture filtrates were 0.02, 0.15 and 0.69 mg/ mL respectively in the media containing glucose, cellulose and sawdust. There was no significant cellulase activity in the filtrates from glucose-containing media. The culture filtrates of the organism from cellulose- and sawdust-containing media yielded significant cellulase activities with maximum values of 105.6 Units /L (at 72 hours for cellulose) and 101.9 Units /L (at 144 hours for sawdust). There is a correlation between the protein content and cellulase activity of the culture filtrates. Sawdust can serve as a low-cost substrate for cellulase production by the organism

Properties of Endoglucanase of Penicillium chrysogemum PCL501

Crude extracellular enzyme from a 3-day culture of Penicillium chrysogenum (PCL 501), in basal medium containing cellulose as the sole carbon source, yielded 0.67 ± 0.03, 19.94 ± 1.30 and 8.50 ± 0.50 units mg protein-1 of 1, 4- â-endoglucanase, â-glucosidase and xylanase activity respectively. The crude enzyme was subjected to ammonium sulphate precipitation (80% saturation) and gel filtration. A purification-fold of 7.5 was achieved. Two active fractions of 1, 4 âendoglucanase (EC 3. 2. 1. 4), which exhibited about the same activity towards carboxymethylcellulose (CMC), were obtained and pooled for the subsequent analyses. The endoglucanase gave a Vmax of 10.0 ± 0.4 μmol min-1 mg protein-1 and Km of 11.8 ± 0.4 gL-1 with CMC. The enzyme was most active at pH of 4.5 – 5.0 and temperature range of 40 – 50 OC. The optimum pH was 4.9 while the Optimum temperature was 48 OC. Divalent metal ions and EDTA affected the enzyme activity at 2.0 mM concentrations. Mn2+ and Fe2+ had stimulatory effects on the enzyme whereas Mg2+, Cu2+, Zn2+, Hg2+ and EDTA inhibited the enzyme activity. The effect of Ca2+ was not significant. Over 3- fold increase in the enzyme activity was recorded with Mn2+. Percentage inhibition of 65.9 and 79.7 respectively was obtained with Hg2+ and EDTA. The organism appears to produce two types of endoglucanase which differed in their molecular weight but not significantly in their activity. The enzyme activity was highly stimulated by manganese ion and inhibited by the metal-chelating agent, EDTA

Isolation of Cellulolytic Microfungi Involved in Wood-Waste Decomposition: Prospects for Enzymatic Hydrolysis of Cellulosic Wastes

Wood-wastes from dump-sites at Okobaba Saw-mills on the western part of the Lagos lagoon were examined for cellulolytic microorganisms. Cellulolytic microfungi were isolated from the wastes using minimal salt agar medium containing 0.2% (w/v) crystalline cellulose, sugarcane pulp, corn cob or saw-dust as sole carbon/energy source. The colonies of cellulolytic microfungi which appeared on the plates increased in size and number as the incubation period (days) increased. Among the fungal isolates were two pathogenic Aspergilli (A flavus and A fumigatus), three different black Aspergilli (herein designated as A.niger I, A.niger II and A.niger III), Botrytis cinerea, Fusarium species and Penicillium species. Cell-free filtrates of 7 – day cultures of A.flavus, A.niger I, A.niger II, B. cinerea and P.species grown on the minimal salt broth supplemented with crystalline cellulose as sole carbon/energy source showed very significant CM–cellulase activity. P. species gave a very high value that was over 4 times the value for the closest organism, A.niger II. There is a good propect for cellulase production using the virgin strain of P. species isolated from the wood-wastes

Kinetic Study and Characterization of 1,4-β-Endoglucanase of Aspergillus niger ANL301

Submerged fermentation of Aspergillus niger ANL 301 in basal medium containing cellulose as sole carbon source, yielded crude extracellular proteins with 0.54 ± 0.02 units mg protein-1 of 1,4-β-endoglucanase activity. Partial purification by ammonium sulphate precipitation (80% saturation) and gel filtration on Sephadex 25-300 gave two active fractions of 1,4-β-endoglucanase, which exhibited close activity towards carboxymethyl-cellulose (CMC). The pH profile of the pooled enzyme fractions showed three activity peaks at pH 3.5, 5.5 and 7.0. The enzyme was most active at pH 5.5 and showed optimal activity at 50°C. Vmax of 4.4 ± 0.4 µmol min-1 mg protein-1 and Km of 12.5 ± 0.4 gL-1 was obtained with CMC for the enzyme. Different divalent metal ions and EDTA affected the enzyme activity at 2.0 mM concentrations in different ways. Mn2+ and Fe2+ exhibited 253.4 and 24.0% stimulatory effects, respectively on the enzyme activity. Mg2+, Ca2+, Cu2+, and Zn2+ inhibited the enzyme by between 22.3 and 29.4%, whereas 75.0 and 71.3% inhibition were obtained with Hg2+ and EDTA, respectively. Manganese ion showed an exceptional activation of the 1,4-β-endoglucanase. The organism produced two types of 1,4-β-endoglucanase with different molecular weights

Agro-waste: a potential fermentation substrate for Penicillium chrysogenum

Common agro-wastes found in Lagos, Nigeria (cassava shavings, corncob, sawdust, and sugarcane pulp) were compared with glucose and lactose as fermentation substrates for Penicillium chrysogenum PCL501. Cassava shavings significantly (P<0.001) produced the highest amount of mycelia weight (0.43 ± 0.02 mg/ml) than all the other substrates. This was followed by corncob with peak mycelia weight of 0.33 ± 0.02 mg/ml. Peak mycelia weight of 0.27 ± 0.01 mg/ml was equally obtained with glucose and sugarcane pulp whereas lactose gave a slightly lower peak of 0.25 ± 0.01 mg/ml. Sawdust gave the least mycelia weight of 0.13 ± 0.01 mg/ml. Total sugar content of all the culture media steadily decreased as fungal growth progressed indicating that the organism utilized carbohydrates for growth and mycelia formation. Cultures containing cassava shavings and sawdust gave high protein peaks of 0.84 ± 0.05 and 0.65 ± 0.03 mg/ml respectively. Cultures containing corncob, glucose, lactose and sugarcane pulp yielded lower protein peaks of 0.37 ± 0.02, 0.30 ± 0.02, 0.24 ± 0.02 and 0.18 ± 0.01 mg/ml respectively. The results suggest that cassava shavings, corncob and sugarcane pulp could serve as cheap fermentation substrates for the growth of the fungus. Of all the substrates investigated, cassava shavings have the best potential to serve as substrate for fermentation by Penicillium chrysogenum PCL501. © 2009 International Formulae Group. All rights reserved

Xylanase production by Aspergillus niger ANL 301 using agro - wastes

Xylanase production by wild-type Aspergillus niger ANL301, newly isolated from wood-waste, was monitored at 24 h intervals for a period 168 h in media containing different carbon sources. The carbon sources were oat-spelt xylan (Fluka) and three agro-wastes (sawdust, sugarcane pulp and wheat bran). Highest xylanase activity of 6.47 units/mL was obtained at 96 h in media containing wheat bran as sole carbon source. Maximum activity value for the media containing sugarcane pulp was 0.95 units/mL obtained also at 96 h. Sawdust and oat spelt xylan gave the peak enzyme activities of 0.65 and 0.80 units/mL respectively at 120 h. High protein yield was obtained in media containing the agro-wastes, with wheat bran giving the highest value of 1.14 mg/mL at 96 h. The maximum specific xylanase activities were 3.86, 3.37, 5.69, and 9.36 units/ mg protein for sawdust, sugarcane pulp, wheat bran and oat spelt xylan, respectively. Out of the three agro-wastes used in this study, wheat bran holds greatest promise for low cost production of the xylanase enzyme

CELLULASE PRODUCTION BY WILD STRAINS OF ASPERGILLUS NIGER, PENICILLIUM CHRYSOGENUM AND TRICHODERMA HARZIANUM GROWN ON WASTE CELLULOSIC MATERIALS.

Waste cellulosic materials (corncob, sawdust and sugarcane pulp) and crystalline cellulose induced cellulase production in wild strains of Aspergillus niger, Penicillium chrysogenum and Trichoderma harzianum isolated from a wood-waste dump in Lagos, Nigeria. Cellulose-supplemented media gave the maximum cellulase activity of -1 0.54, 0.67 and 0.39 units mg Protein for A. niger, P. chrysogenum and T. harzianum respectively. The maximum enzyme activity for A. niger was obtained at 36 hours of cultivation, while P. chrysogenum and T. harzianum gave their optimal enzyme activities at 12 and 60 hours respectively. Of the three cellulosic wastes, best enzyme -1 activity was obtained with sawdust. Maximum enzyme activity of 0.30, 0.24 and 0.20 units mg Protein respectively was obtained with A. niger, P. chrysogenum and T. harzianum at 144 hours of cultivation using the substrate. A. niger gave the highest enzyme activity with any of the three cellulosic materials followed by P. chrysogenum. It thus appears that the use of sawdust presents the best option for low-cost commercial production of cellulase using A. niger and P. chrysogenum as discussed herewit

Xylanase production by Penicillium chrysogenum (PCL501) fermented on cellulosic wastes

Xylanase production by Penicillium chrysogenum PCL501, newly isolated from wood-wastes, was monitored at 24 h intervals for a period 168 h in media containing four different carbon sources (oatspelt xylan, wheat bran, sawdust, and sugarcane pulp). The highest xylanase activity of 6.47 Units mL-1 was obtained at 96 h in media containing wheat bran whereas media containing sugarcane pulp gave a peak value of 1.39 Units mL-1 at 144 h. Sawdust and xylan gave a peak xylanase activity of 1.35 and 0.79 Units mL-1 respectively at 120 h. Maximum protein released in xylan-containing media was 0.38 mg mL-1. Higher protein yield was obtained in media containing the agro-wastes, with wheat bran giving the highest value of 1.14 mg mL-1. The maximum specific xylanase activities were 2.59, 8.52, 16.06, and 9.36 Units mg Protein -1 for sawdust, sugarcane pulp, wheat bran and xylan respectively. Out of the three agro-wastes used in this study, wheat bran holds the greatest promise for cost-effective production of the xylanase enzyme. The carbon source is the highest inducer of the enzyme in the fungus

Plant Waste Hydrolysis by Extracellular Enzymes of Aspergillus niger and Penicillium chrysogenum: Effect of Ammonia Pretreatment

Aspergillus niger (ANL301) and Penicillium chrysogenum (PCL 501) cultured in basal media with cellulose as sole carbon source yielded extracellular enzymes which partially hydrolyzed sawdust and sugarcane pulp into simple sugars. Pre-treatment of sawdust by ammonium hydroxide steeping increased the yield of simple sugars. The reducing sugars released from the pretreated sawdust by the crude enzymes of A. niger (ANL301) and P. chrysogenum (PCL 501) were 3.58% and 7.02% of the total hydrolysable sugars respectively. This is in contrast to the 0.92% and 1.02% of the total hydrolysable sugars released respectively by the enzymes of A. niger (ANL301) and P. chrysogenum (PCL 501) from the non-pretreated sawdust. Enzymatic hydrolysis of sugarcane pulp by the crude enzymes was not significantly affected by ammonia pre-treatment. Reducing sugars released from non-pretreated sugarcane pulp by the crude enzymes of A. niger (ANL301) and P. chrysogenum (PCL 501) were respectively 4.17% and 5.08% of the total hydrolysable sugars