Simultaneous production of cellulase and amylase by Aspergillus fumigatus IB-A1

Utilization of agricultural wastes for production of useful metabolites requires hydrolysis using both cellulase and amylase enzymes. We isolated Aspergillus fumigatus IB-A1 and evaluated its ability to simultaneously produce both cellulase and amylase. Although the isolate could produce both cellulase and amylase f rom either soluble starch or carboxymethyl cellulose, amylase activity was higher with soluble starch while cellulase activity was higher when carboxymethyl cellulose was used as the sole carbon source. With a mixture of carboxymethyl cellulose and soluble starch, both the amylase and cellulase activities increased with increase in the ratio of soluble starch. The optima ratio of carboxymethyl cellulose to soluble starch for cellulase and amylase activities were 0.7:0.3, and 0.4 to 0.6 respectively. For practical application, the optimum ratio of carboxymethyl cellulose to soluble starch in the production medium depends on the relative composition of cellulose and starch in the substrate to be hydrolyzed. The isolate was also able to ef f iciently produce both amylase and cellulase f rom cassava peel. With 10 g/L cassava peel, the cellulase and amylase activities were 6.122± 0.320 U/ml/min and 4.342± 0.210 U/ml/min respectively. When the cells were immobilized on loofa sponge and subjected to alternating air phaseliquid phase culture, cellulase and amylase production f rom cassava peel increased to 8.106± 0.620 U/ml/min and 5.206± 1.24 U/ml/min respectively. The optimum ratio of the air phase to the liquid phase was 3 hours of air phase to 21 hours of liquid phase.


Introduction
Majority of the agricultural wastes are lignocellulosic materials which are abundant and cheap biomass resources f or production of useful metabolites (Edama et al., 2014;Kongkiattikajorn, 2012;Pooja and Padmaja,2014). Thus, they have been used as carbon sources f or production of various products such as single cell protein, industrial enzyme, and f ine chemicals (Mohammed et al., 2013;Ndubuisi et al., 2018;Murata, et al., 2021). However, since cellulose, and starch are the major components of most of these agricultural wastes, their ef f icient utilization requires the use of both cellulase and amylase enzymes. The costs of these enzymes are usually very high, especially in most developing countries. Thus, sustainable utilization of these agricultural wastes requires that these enzymes must be cheaply produced. The cost of substrates used f or production of these enzymes represents a signif icant percentage of the f inal production costs. In the case of ethanol, for example, the cost of raw materials can represent 40-75% of the total production cost, depending on the type of f eedstock (Arijina et al., 2018). Thus, the cost of the upstream process can be signif icantly reduced by using cheap and easily available raw materials. It will be very interesting to develop a system where agricultural waste materials are used to produce enzymes that will again be used f or ef f icient conversion of the agricultural wastes to other usef ul materials. Furthermore, the production costs can be reduced by using microbial strains that can simultaneously produce both amylase and cellulase, and by improving their enzyme productivities.
Nigeria is the world largest producer of cassava and most of the produced cassava are utilized locally, leading to generation of huge amounts of cassava peels. Presently, the cassava peels pose a disposal problem and would even be more problematic in f uture with increased industrial production of cassava products such as garri, f lour, and starch. It is theref ore very important to convert these cassava peels into value-added products. The aim of this work was theref ore to use cassava peels to produce both cellulase and amylase enzymes that can be again used for ef f icient conversation of the cassava peels to other usef ul metabolites. The potentials of a thermophilic microorganism, Aspergillus fumigatus IB-A1 to simultaneously produce cellulase and amylase were f irst investigated using cellulose and starch as the carbon sources. Using this microorganism is expected to save costs since the two enzymes are produced simultaneously, the risk of contamination by mesophilic microorganisms, as well as the cost of reactor cooling will be reduced. The advantages of immobilizing microorganisms f or various processes have been documented. Thes e include: It allows f or re-use of bio-catalyst, it permits continuous cultures at high hydraulic retention time, high cell density can be achieved with consequent increase in productivity, and it makes downstream processing very easy. Cell immobilization techniques using biological materials is eco-f riendly and have many advantages over synthetic material (Hideno et al., 2007). It has been reported that loof a sponge (Luffa cylindrica) is an excellent carrier for immobilization of aerobic microorganisms (Roble et al., 2003a&b). It has also been demonstrated that alternating air phase-liquid phase system combines the advantages of solid-state cultures and submerged cultures (Roble et al., 2020;Ugwu et al., 2021). Thus, in order to f urther reduce the production costs, simultaneous production of cellulase and amylase by Aspergillus fumigatus in an alternating air phaseliquid phase system was investigated.

Collection and preparation of the substrates
Cassava tubers were obtained f rom the Faculty of Agriculture, University of Nigeria, Nsukka, Enugu State, Nigeria. The tubers were harvested f rom variety 98/2101 at the age of 11 months, peeled and the f resh cassava peels were washed and sun dried. The dried cassava peels were ground into a f ine powder using a hand grinding machine. The powder was stored in a ref rigerator (4 o C) and aliquots were taken f or each experiment.

Microorganism and culture maintenance
A thermotolerant Aspergillus fumigatus IB-A1, previously isolated and maintained at the Department of Microbiology, University of Nigeria, Nsukka was used. The cultures were maintained on potato dextrose agar (PDA) slants at 4 o C and sub-cultured at two weeks intervals. The identif ication of the isolate has been described (Ezea et al., 2022).

Inoculum preparation
Inoculum was prepared according to Sivaramakrishnan et al., (2007). To a 7-day old culture slant, 10 ml of 0.1 % Tween 80 solution was added and the spores were dislodged using an inoculation needle under sterile condition. A 10 ml of spore suspension containing 2 x 10 7 spores per ml was used f or inoculation. The spore concentration was estimated using haemocytometer.

Evaluation of the ability of Aspergillus fumigatus IB-A1 for simultaneous production of cellulase and amylase from a mixture of carboxymethyl cellulose and soluble starch
The basal. medium was composed of 1 % carboxymethyl cellulose, 1 % soluble starch, 0.25 % NaNO3, 0.1% KH2PO4, 0.05 % MgSO4.7H2O, and 0.06 % CaCl2. One hundred (100) mL of the medium was prepared in 250 mL f lask, the pH was adjusted to 5.5 using 1 N NaOH and the medium sterilized at 121 o C f or 15 min. Af ter cooling, the f lask was inoculated with 10 mL of Aspergillus fumigatus spore suspension, containing 2 x 10 7 spores per mL and incubated at 45 o C f or 7 days. Thereaf ter, the culture broth was centrif uged and the supernatant used as the crude enzyme.
The optimum ratio of carboxymethyl cellulose to soluble starch for enzyme production was determined by varying the ratios of carboxymethyl cellulose to soluble starch in the medium, but the total concentration of the two was maintained at 1%.

Production of enzymes from cassava peel in submerged culture
Submerged culture was carried out using the basal medium without soluble starch and carboxymethyl cellulose but containing 1 % of cassava peel as the carbon source. One hundred milliliter of the medium was poured into 250 ml Erlenmeyer f lask and sterilized at 121 o C f or 15 minutes. The pH was adjusted to 5.0 or 6.0 using 1 N NaOH or 1 N HCl. Af ter cooling, 10 ml of the spore suspension containing 2 x 10 7 spores per ml was inoculated and incubated at 45 o C f or 5 days.

Alternating air phaseliquid phase culture
The f ungal isolates were immobilized on loofa sponge (Loofa cylindrica) as previously described (Ogbonna et al., 1994;Roble et al., 2003a&b;Roble et al., 2020;Ugwu et al., 2021). The dried outer cover of the loof a sponge was removed and the porous loof a sponge f iber was sliced and washed properly f ollowed by drying in air and in a hot oven at 105 o C f or 1 hour. A 10 ml of the spore suspension (2 x 10 7 spores per ml) was mixed properly with a sterilized viscous starch medium containing (per L) 50 g starch, 2.5 g NaNO3, 1.0 g KH2PO4, 0.5 g MgSO4.H2O, and 0.6 g CaCl2. The mixture was poured onto the loofa sponge and pre-incubated in air inside a bioreactor f or 3 days. Af ter germination of the f ungal spores, a medium containing (per L) 10 g sterilized cassava peel, 2.5 g NaNO3, 1.0 g KH2PO4, 0.5 g MgSO4.H2O, and 0.6 g CaCl2 was poured into the reactor and cultivated f or another 48 hours. The cultivation was then done under alternating air phase-liquid phase. Initially, the ratio of the air-phase to liquid phase was f ixed at 6h:18h ( Figure 2) and the ef f ects of the ratio of the air-phase to liquid phase was investigated by varying the ratios as described previously (Roble et al., 2020;Ugwu et al., 2021) (Figure 3).

Crude enzyme preparation
Culture broth samples were taken at time intervals and f iltered using Whatman number 1 f ilter paper. The f iltrate was used as the crude enzyme f or determination of the cellulase and amylase activities.

Determination of cellulase activity
Cellulase activity (CMCase) was assayed using a method of Mendel and Weber (1969) and Ghose (1987). The activity was estimated using 1 % solution of carboxymethyl cellulose (CMC) in 0.05 M citrate buf f er (pH 4.5). The reaction mixture contained 1 ml citrate buf f er, 0.5 ml of the 1% CMC and 0.5 ml of the crude enzyme. The reaction was carried out at 50 o C f or 30 min. The reaction was stopped by addition of 3 ml of 3, 5 dinitrosalicylic acid reagents (DNSA) and boiled f or 10 minutes f ollowed by addition of 3 ml of distilled water.
Thereaf ter, the absorbance was taken at 540 nm (Miller, 1959). One unit of endoglucanase activity was def ined as the amount of enzyme releasing 1 µmol of reducing sugar f rom carboxymethyl cellulose per ml per min under the assay conditions. A calibration curve was established with glucose f rom which reducing sugars were calculated.

Determination of amylase activity
Amylase activity was determined according to Demoraes et al. (1999) using 0.5 ml of 0.5 % gelatinized soluble starch, buf f ered with 0.2 ml of 0.05 M citrate buf f er (pH 4.8). The reaction mixture contained 0.2 ml citrate buf f er, 0.5 ml of substrate solution (gelatinized soluble starch) and 0.3 ml of the crude enzyme. The reaction was carried out at 40 o C f or 30 min. The reaction was stopped by addition of 1 ml of 3, 5 dinitrosalicylic acid reagents (DNSA) and boiled f or 10 minutes f ollowed by addition of 3 ml of distilled water. Thereaf ter, the absorbance was taken at 540 nm (Miller, 1959). One unit of amylase activity was def ined as the amount of enzyme releasing 1 µmol of reducing sugar (glucose equivalent) f rom soluble starch per ml per min under the assay conditions. Table 1 shows that Aspergillus fumigatus IB-A1 was able to simultaneously produce cellulase and amylase f rom a mixture of carboxymethyl cellulose and soluble starch. In a medium containing 1 % soluble starch, the maximum cellulase and amylase activities were 1.143± 0.081 U/ml/min and 2.144± 0.240 U/ml/min respectively. When cultivated with 1 % carboxymethyl cellulose, the maximum cellulase and amylase activities were 2.913± 0.030 U/ml/min and 1.558± 0.172 U/ml/min respectively. In other words, using starch as the carbon source led to higher amylase activity while production of cellulase was f avoured by using CMC as the carbon source. However, when cultivated in a medium containing 0.5 % soluble starch and 0.5 % carboxymethyl cellulose, the maximum cellulase and amylase activities were 2.746± 0.170 U/ml/min and 2.177± 0.321 U/ml/min respectively. As shown in Table 1, it is noted that the total enzyme activity was higher when a mixture of soluble starch and CMC was used as the carbon source. When Aspergillus fumigatus IB-A1 was cultivated in the medium in which a mixture of soluble starch and carboxymethyl cellulose was replaced with either soluble starch only or carboxymethyl cellulose only, the cells grew well on the loof a sponge as revealed by physical observation. However, the growth was better in a medium containing cellulose than in the medium containing soluble starch. The results are in agreement with those reported by Khokhar et al. (2011) on the amylase and cellulase production by Aspergillus and Penicillium species. Several species of f ungi have been reported to be able to produce both amylase and cellulases in both submerged and solid-state cultures ) and the relative proportions of the enzymes depend on the species and the substrate used.

Optimum carboxymethyl cellulose to soluble starch ratio for enzyme production
The optimum ratio of carboxymethyl cellulose to soluble starch f or enzyme production was determined by varying their ratio in the medium. Cultivation of Aspergillus fumigatus IB-A1 in a medium containing 0.7% carboxymethyl cellulose and 0.3 % soluble starch gave the highest cellulase activity of 2.907± 0.32 U/ml/min while the medium containing 0.4% cellulose and 0.6 % starch gave the highest amylase activity of 2.442± 0.21 U/ml/min (Fig 1). It is noted that the cellulase activities increased with increase in CMC ratio up to 70% but the activity remained high even when the medium contained 0.9% CMC and 0.1% starch. On the other hand, amylase activity increased with increase in the concentration of soluble starch up to 0.6% and remained high even in a medium containing 0.9% starch and 0.1% CMC. Thus, the optimum ratio of soluble starch and CMC would depend on the relative concentrations of cellulose and starch in the substrate to be hydrolyzed. When about the same cellulase and amylase activities are desired, the ratio of CMC to soluble starch in the medium should be maintained between 4:6 and 5:5.

Comparison of enzymes production from cassava peel in submerged and in alternating air phaseliquid phase cultures
In a submerged culture, cellulase and amylase production by Aspergillus fumigatus IB-A1 were 6.122± 0.320 U/ml/min and 4.342± 0.210 U/ml/min respectively. However, in the alternating air phase -liquid phase culture, the cellulase and amylase activities increased to 8.106± 0.620 U/ml/min and 5.206± 1.24 U/ml/min respectively (Figure 2). This shows that alternating air phase-liquid phase cultivation led to a signif icant increase in the cellulase and amylase activities over the values obtained in submerged culture. Morphological observation showed that higher cell growth was achieved in alternating air phase-liquid phase culture than submerged culture. This may have contributed to the higher enzymes production in the alternating air phase-liquid phase than that of submerged. It is noteworthy that regardless of the culture system used, the activities of the two enzymes were higher when cassava peel was used as the carbon source when compared with the use of either soluble starch, CMC or a mixture of soluble starch and CMC.  Cellulase activity The ef f ects of the ratio of the air phase to liquid phase on enzyme production are shown Figure 3. Exposing the immobilized cells to 3 hours in air phase and 21 hours in submerged culture broth resulted in the maximum cellulase and amylase activities of 8.55± 0.32 U/ml/min and 5.285± 0.34 U/ml/min respectively. Regardless of the ratio of the air phase to the liquid phase investigated, the cellulase activity remained higher than that of amylase activity. However, cellulase appeared to be more sensitive to the ratio of the air phase to the submerged phase ( Figure 3). In this system, the f unction of the air phase is mainly f or aeration of the immobilized cells (exposure to air) while the liquid phase is to supply the soluble nutrients and to extract the produced enzymes into the culture broth. A major advantage of the alternating air phaseliquid phase culture is that it reduces the problem of oxygen limitation, which is a major problem in submerged cultures. It combines the advantages of submerged cultures and those of the solid-state cultures (Roble et al., 2020;Ugwu et al., 2021).
Figure2. Comparison of submerged and alternating air phase-liquid culture in cassava peel medium. The ratio of air phase to liquid phase was 6h:18h.

Cellulase activity
Many previous workers have reported higher enzyme production by immobilizing cells in loofa sponge. Samia et al (2013) reported higher specif ic βglucosidase activity by cell immobilized on loof a sponge (Luffa cylindrical) than f ree cell. Also, Velichkova et al (2014) reported that immobilization of Aspergillus awamori K-1 and Trichoderma viride SL-45 on loof a sponge led to higher cellulase activity when compared with that of the suspended cells. However, the present study is the f irst report on simultaneous production of amylase and cellulase in an alternating air phase-liquid phase culture. It is noted that both the cellulase and amylase activities were higher in this culture system when compared with the conventional suspended submerged cultures.

CONCLUSION
Aspergillus fumigatus IB-A1is capable of simultaneous production of cellulase and amylase in media containing either soluble starch, CMC, a mixture of the two or cassava peel as the carbon sources. The activities of the two enzymes were higher when cassava peel was used as the carbon source. Production of the two enzymes were f urther enhanced by alternatingly exposing the cells to air phase and liquid phase.

Conflict of interest
There is no conf lict of interest