PRODUCTION OF LACCASE BY FUNGI ISOLATED FROM SOIL VIA SUBMERGED FERMENTATION USING CORN COB AS SUBSTRATE

One of the limitations of large scale application of laccase (EC 1.10.3.2) is the inability to produce them in large quantity at an affordable cost. This study was carried out to screen indigenous fungi for their ability to produce laccase using the locally available substrate. Five soil samples were collected and diluted serially, 0.1 mL of the 10 and 10 dilutions were inoculated onto Potato dextrose agar (PDA) plates. The fungal isolates were identified based on their macroscopic and microscopic characteristics. The isolates were then screened for laccase production by growing them on PDA containing tannic acid as an indicator compound. The laccase producing isolates were further screened for their ability to utilize corn cob as a substrate for laccase production. Ten fungal species were isolated and identified as Trichoderma viridae (3), Trichoderma harzianum (3), Aspergillus niger (2), Fusarium sp. (1) and Penicillium sp. (1). Only two of the isolates namely T. viridae and T. harzianum were found to be laccase producers. Both laccase producing fungal species were able to utilize corn cob as substrate for laccase production. T. viridae had higher enzyme activity (2.228 U/mL) than T. harzianum (2.1583 U/mL) after 9 days of incubation. Laccase producing fungi were isolated in this study and they were able to use corn cob as substrate for laccase production.


INTRODUCTION
Lignocellulosic materials are the major component in plants and consist of cellulose, hemicellulose and lignin. Lignin is an important barrier that protects hemicellulose and cellulose from enzymatic attack (Wang et al., 2019). Fungi are capable of breaking these barriers by the production of a different extra lignocellulosic enzyme such as laccases, lignin peroxidase and manganese (Emre and Ozfer, 2013).
Industries such as food agriculture, forestry, paper pulp and timber release large lignocellulosic waste. These waste cause serious environment pollution but rather than been burned they can be reused due to their rich sugar contents. The chemical composition of this lignocellulosic material /waste make them a crucial and cost-effective fermentation medium for biotechnological applications (Sahadevan et al., 2013). Numerous microorganism especially fungi make use of cellulose and hemicellulose components of lignocellulosic waste while only limited number of the organism such as white rot fungi are capable of degrading lignin due to its high resistance to microbial attack (Kantharaj et al., 2017).
In other to exploit the use of lignocellulosic wastes, several methods are employed for the separation of these materials, these processes are the physical, chemical and biological process. The physical process involves the use of microwave pretreatment, the chemical process involves the use of acid and alkali while biological process usually involves the use of an enzyme (biocatalyst) which improve the superiority of pretreatment processes (Brustarr M, Bakenhus, 2008). The use of biocatalyst is more preferred simply because they overcome the drawback of decrease in the quality of polymer and release of by-products that inhibit the fermentation of resulting sugars (Kantharaj et al., 2017).
Laccase (EC 1.10.3.2) is one of the oldest known enzymes and was first discovered in 1883 in the sap of Japanese lacquer tree Rhus vernicifera, leading to it polymerization and the product used commonly as Japanese lacquer varnish (Mendoza, 2011). They are copper-containing polyphenol oxidases which are capable of degrading both phenol and non-phenol substrate. They degrade lignin by oxidation of phenolic units of the lignin to phenolic radicals which cleave aryl-C while non-phenolic substrates are oxidized in the presence of an auxiliary substrate (Brijwani andVadlani, 2011, Kantharaj et al., 2017). Laccase is one of the three main ligninases but has peculiar characteristics of catalyzing the oxidation of lignin component using molecular oxygen as electron acceptor which reduced to water. Laccase uses electron mediator to degrade complex structures (Call and Muncke, 1997).
In industries like paper and pulp industry, certain chemical like chlorine and oxygen-based chemical oxidant are required for separation of lignin from lignocellulosic waste. Preparation of paper at an industrial level face certain problems such as cost recycling and toxicity which remain unresolved (Solomon, 1996). Laccase has great potential application in several areas of food and other industries, however, one of the limitation of large scale application of laccase is the lack of capacity to produce enzyme in large volume at an affordable cost (Shraddha et al., 2011). The use of agricultural waste such as corn cob, yam peels, rice straw, rice husk and agro-waste as a substrate for fermentation can increase the yield of enzyme production at low cost and will reduce the use of chemicals for degradation of lignincontaining wastes, thereby reducing the risk of hazardous chemicals been release into the environment (Sadh et al., 2018). Thus, the aim of this study was to isolate and characterize laccase producing fungi from the soil and screen for their ability to utilize corn cob as a substrate for the production of laccase enzymes that can be used for the chemical degradation of lignin-containing waste.

MATERIALS AND METHODS Collection of Samples
The soil samples used in this study were collected from 5 different locations of Botanical Garden, Department of Biological Sciences, Ahmadu Bello University, Zaria. The samples were collected in sterile polythene bags and brought to the laboratory for further study. Corn cobs were purchased from Samaru market, Zaria. The corn cobs were crushed and ground. The sample was then stored at room temperature in polythene bags.

Isolation and Identification of Fungi
One gram of the soil was weighed and dispensed into 10 mL of sterile distilled water. The suspension was mixed and serially diluted from 10 -1 to 10 -6 dilution. Then 0.1 mL of 10 -5 to 10 -6 dilutions were spread on the surface Potato Dextrose Agar (PDA) separately and incubated at 25 o C for 7 days. Distinct fungal colonies were sub-cultured until the pure culture was obtained. The cultures were maintained on PDA slant at room temperature (Adiveppa and Basappa, 2015). The Fungi isolates were identified based on their macroscopic and microscopic characteristic using a colour Atlas of Mycology by Walsh et al. (2018).

Qualitative screening for laccase production
Mycelium from each fungal strain was inoculated on PDA plate containing 0.5 % tannic acid as an indicator compound for laccase production. The plates were incubated at 25 o C and observe daily for 12 days. Formation of reddish-brown halo around the fungal isolate on the plate indicated laccase production (Adiveppa and Basappa, 2015).

Laccase production by submerged fermentation using corn cob as a substrate Inoculum preparation
The slants of the laccase producing fungal isolates were used to prepare spore suspension by the addition of 2 mL of sterile distilled water to 7 days old slant culture of the fungal isolates. With aid of a sterilized wire loop the spores were dislodged and counted with a haemocytometer to obtain a concentration of 1.0 x 10 6 spore/mL (Buddolla et al., 2008).

Medium preparation and fermentation
Determination of the ability of the laccase producing fungal isolates to use corn cob as a substrate for laccase production was carried out by growing the isolates using 250 mL Erlenmeyer flask containing 100 mL of mineral salt medium with the following composition (g/L): KCl, 0.5; MgSO4.7H2O, 0.5; (NH4)2HPO4, 0.5; NaH2PO4, 0.5; CaCl.2H2O, 0.01; FeSO4.7H2O, 0.001; ZnSO4.7H2O, 0.002 and 10 g corn cob as was designed by Suprabha et al. (2008). The pH was adjusted to 5.0 and autoclaved at 121 o C for 15 mins.
The 2 mL spore suspension was transferred into the sterilized mineral salt medium and incubated at 25 o C in a rotatory shaker at 150 rpm for 9 days (Sanjeeviravar et al., 2015). 2 mL of the fermentation medium were taken from each conical flask at an interval of 3 days. The laccase activity was determined using guaiacol assay method.

Determination of laccase activity using Guaiacol assay method
This assay is based on the oxidation of guaiacol by laccase enzyme which results in the development of reddish-brown colour which is used to measure laccase activity using spectrophotometer at 450 nm. The reaction mixture was prepared as follows: (a) 1 mL of 2 mM Guaiacol. (b) 3 mL of 10 mM Sodium acetate buffer. (c) 1 mL of fungal supernatant which serves as enzyme source. A blank which contains 1 mL of distilled water instead of the enzyme was also prepared. The mixture was incubated at 30 o C for 15 mins and the absorbance was read at 450 nm using UV spectrophotometer (Abd El Monssef et al., 2016).

Statistical analysis
ANOVA was performed to analyze the statistical significance of the differences observed in the laccase activities produced by T. harzianum and T. viridae using IBM SPSS Statistics. P ≤ 0.05 was considered statistically significant. Table 1 shows the macroscopic and microscopic appearances of the fungal isolates. Isolates DBS-1, DBS-6 and DBS-10 were identified as T. harzianum while DBS-2, DBS-5 and DBS-7 were identified as T. viridae. Two isolates (DBS-4 and DBS-8) were A. niger while DBS-3 and DBS-9 were identified as Penicillium sp. and Fusarium sp. respectively. Table 2 shows the distribution of the fungal species isolated from a soil sample. The percentage occurrences of T. harzianum and T. viridae were 30 % each while that of A.

Characterization of fungal isolate
niger was 20 %. The least occurring species were Penicillium sp. (10 %) and Fusarium sp. (10 %). Table 3 shows the result of the screening for laccase producing ability of the fungal isolates. Isolate DSB-1 (T. harzianum) and DSB-2 (T. viridae) were positive for laccase production while the remaining isolates were negative for laccase production.

Qualitative screening for laccase production ability of the isolates
Laccase production by submerged fermentation using corn cob as a substrate Figure 1 shows the laccase activities produced by the two positive isolates at 3 days interval for 9 days. At day 3, 6 and 9 days of incubation, T. viridae had higher laccase activity (1.1821, 1.3860 and 2.2280 U/mL respectively) compared to T. harzianum (0.7209, 1.0353 and 2.1583 U/mL) . However, this difference was not statistically significant (p = 0.6159).

DISCUSSION
Fungi have successfully inhabited the soil due to their ability to adopt to adverse conditions by adopting various forms (Sun et al., 2005). They are able to degrade lignocellulosic wastes by producing a wide variety of extracellular enzymes (Žifčáková et al., 2016). Fungi exist in the soil as spore and germinate upon inoculation on a mycological medium and incubation. T. viridae, T. harzianum, A. niger, Fusarium sp. and Penicillium sp. were the fungal species isolated from the soil samples collected in this study. These fungal genera were among the most abundant in soil. The presence of these fungal isolates in the soil suggested they can produce a wide variety of extracellular enzymes, hence they can break down all kinds of organic matter and regulate the balance of carbon and nutrients (Frac et al., 2018).
The laccase producers in this study were T. viridae and T. harzianum. These findings are in line with the result of Abd El Monssef et al. (2016) who reported that of all the isolates screened from biodeteriorated parchment, only Trichoderma harzianum was found to be laccase producer but disagree with the findings of Kumar et al. (2011) where T. viridae and T. harzianum isolates screened were not laccase producer. Only one out of the three isolates of T. harzianum was found to be laccase producer, this is likely due to strain variation among members of the same species.
Increase in laccase activity was observed with an increase in the incubation period for both T. viridae and T. harzianum. This implies that the isolates were able to use corn cob as a substrate for laccase production. Adiveppa and Basappa (2015) also reported an increase in laccase production with an increase in the incubation period. However, the highest enzyme activity observed in this study (2.228 U/mL) was higher than 0.718 U/mL produced by Marasmius sp. as observed by Adiveppa and Basappa (2015). This difference might be due to difference in enzyme production ability of different fungal species. Contrary to this finding, higher laccase activity (28.2 U/mL) was reported by Amanpreet et al. (2017). This wide difference was because Amanpreet et al. (2017) used optimized conditions and added enzyme inducers (2 mM CuSO4 and 1 % MgSO4) to the Czapek Dox medium used.
The higher activities exhibited by T. viridae as compared to T. harzianum in this study may be due to differences in enzyme production ability of different species of the same genus. This difference was however not statistically significant.

CONCLUSION
In conclusion, ten different fungal species were isolated from a soil sample and belong to the genus Trichoderma, Penicillium, Fusarium and Aspergillus. The fungal species with the highest frequency of occurrence were T. viridae (30 %) and T. harziaum (30 %) while the least occurring species were Penicillium sp. (10 %) and Fusarium sp. (10 %). T. viridae (DSB-2) and T. harziaum (DSB-1) were found to be laccase producing fungi while the remaining eight isolates were not. T. viridae (DSB-2) was a better laccase producer compared to T. harziaum (DSB-1). Both laccases producing isolate were able to utilize corn cob substrate for laccase production.