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Open Access Journal of Microbiology & Biotechnology Research Article 12 min read

Screening of Fungi from Disposed Maize Cobs for Amylase Production

Oguche SO*, Iliyasu MY, Ahmad A, Abba M, Karamba KI, Haruna US, Na’Allah AJ and Agbo E B
* Corresponding author
ISSN: 2576-7771  10.23880/oajmb-16000207  Received: November 29, 2021  Published: December 15, 2021
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Keywords
Amylase Fungi Maize Cobs Penicillium Chrysogenum Rhizopus Oryzae
Abstract

Background of Study: Microorganisms in particular have been regarded as treasure of useful enzymes. There is a great variation between various genera as to their ability to produce a specific enzyme for the production of particular enzymes varies with the particular medium and pH. Place and Duration of Study: The study was conducted at the Department of Microbiology Laboratory, Abubakar Tafawa Balewa University, ATBU, Bauchi Nigeria, from November, 2020 to October, 2021. Aim: This study was aimed to isolates fungi from disposed maize cobs and evaluates its potentials to produce amylase. Methods: Twelve samples each was collected from three different areas; market place, farmland and residential areas in Bauchi metropolis, (a total of 36 samples in all) using precise aseptic techniques. Each sample was collected using clean polythene bag, transported to the lab and aseptically blended. One gram of each sample was aseptically weighed and placed in a test tube containing sterile water; it was then allowed to stand for 30 minutes. One ml of the stock solution was serially diluted and 10ml dilution of each sample was plated on Potato Dextrose Agar (PDA) media. The plate was incubated at 25°C within a period of three, five and seven days during which they were monitored and examined, to isolate the required fungi species. The isolates were tested for amylolytic activity using 1% iodine and screen for amylase production by pre-treatment and solid state fermentation, then α-amylase activity finally determined. Results: Amylase-producing fungi were isolated from maize cobs collected from residence, market and farm areas in Bauchi metropolis. The ability of ten (10) fungal isolates recovered, (Mucor racemosus, Aspergillus niger Penicillium chrysogenum, Rhizopus oryzae, Microsporum sp, Trichoderma sp, Nocardia sp, Monilla sp, Fusarium sp and Chaetomum sp) to degrade starch was determined. Three (3) of the fungal isolates Aspergillus niger Penicillium chrysogenum, Rhizopus oryzae, had the highest frequency of (20%) each. Four (4) of the fungal isolates (Mucor racemosus, Aspergillus niger, Penicillium chrysogenum and Rhizopus oryzae) showed zone of clearance on starch agar medium, the fungi isolates were selected and subjected to various temperatures, incubation time and pH ranges for amylase production. The results showed that Penicillium chrysogenum and Rhizopus oryzae have maximum amylase activity at temperature 35°C, incubation time 96hrs (4days), pH 5.5 and temperature 30°C,incubation time 96hrs(4days) and pH 5.0 respectively. Penicillium chrysogenum produced 46.3μ/ml, and Rhizopus oryzae, produced 30.8μ/ml of amylase. Conclusion: The results of this work proved Penicillium chrysogenum to be the best producer of amylase compared to Rhizopus oryzae. Isolation of amylase producing fungi from maize cobs from residence, market and farm areas will help in the bioremediation of environment, which could have caused environmental pollution. It is recommended that Penicillium chrysogenum and Rhizopus oryzae, are suitable fungi for amylase production. While Maize cobs can be used as substrate for commercial enzymes production.

Introduction

The main cereal crop grown in many parts of Nigeria is Maize (Zea mays). It is used primarily as staple food for human consumption, animal feeds and a raw material for industrial purposes [1]. Maize cob is an agricultural waste which is currently used as substrate for combustion. Due to its high contents of chlorine, the combustion has to take place in continuously monitored industrial power plants [2]. In recent years cellulolytic and amylolytic enzymes are used in Nigeria industries for the production and processing of chemicals, foods, and manufactured goods such as papers, rayon and cellophane among others [3]. Amylase is among the most important enzymes that hydrolyze starch releasing several products including dextrin, and progressively smaller polymers of glucose units [4]. Alpha-amylase (EC3.2.1.1, α-1, 4-glucan-4-glucanohydrolysis), catalyzes the endo-cleavage of the α-1,4-glucoside linkages and the release of short oligosaccharides and a- limit dextrin, this enzyme is used commercially for the production of sugar syrups from starch which consist of glucose, maltose and higher oligosaccharides [5, 6, 7]. Amylase can be derived from several source such as plants, animals, fungi and bacteria , fungal sources have gained much attention in industrial sector and large number of them are available commercially [8].

Agricultural and industrial waste such as cereals, straw, leaves, and maize cobs contribute to environmental pollution, the local production of enzymes using these locally available agricultural wastes as substrates reduce the cost of importation and encourage self-reliance [9]. These waste are highly underutilized in Africa including Nigeria and most part of the country, they are mostly used for animal feeds. Proper biotechnology utilization of these agricultural wastes in the environment will help to eliminates pollution and transforming them into useful by- product and ameliorate the problem they cause [9]. Fungal sources have gained much attention in industrial sector and a large number of them are available commercially [10]. Filamentous fungi, such as Aspergillus oryzae and Aspergillus Niger, produce considerable quantities of enzymes that are used extensively in the industry. An oryzae has received increased attention as a favourable host for the production of heterologous proteins because of its ability to secrete a vast amount of high value proteins and industrial enzymes, e.g.-amylase [11].

Materials and Methods

Isolation of Amylase-Producing Fungi

Maize cobs were collected from different areas in Bauchi LGA of Bauchi state using precise aseptic techniques. The pour plate method as described by Cheesbrough M [12] was adopted. One (1) gram of each sample was aseptically weighed and placed in a test tube containing sterile water, it was allowed to stand for 30 minutes. One (1) ml of the stock solution was serially diluted and 10ml dilution of each sample was plated on Potato Dextrose Agar (PDA) media. The plate was incubated at 25oC for a period of 3-7 days during which they will be actually monitored and examined.

Screening for Amylase Producing Fungi by Starch-reaction test

The fungi isolates were inoculated into a starch agar plate using the streaking plate method, and incubated at room temperature for 72hrs and further tested for amylolytic activity, after which the plates were completely flooded with approximately 5ml of 1% 1grams iodine(2g Potassium iodide,1g Iodine dissolved in 300ml distilled water) using Pasteur pipette to screen for amylase production, zone of clearance around the growth was observed and measured as coefficient of the diameter of the fungal colony formed and that of the hydrolysis zone around the colony as reported by Maki M and Soares, et al. [13, 14].

( ) diameter of thecolony The hydrolytic coefficient Hc diameter of theentire zoneof hydrolysis including the fungal colony = The maximum value of Hc is 1, when there is no activity. The lower the value, the higher the activity. And the range is taking as; 0-0.33cm (+++), 0.34 – 0.66 cm (++), and 0.67- 0.99cm (+). When there is no activity the ratio between the colonies to the hydrolysis zone diameter is 1. Therefore it is assigned negative (-) Positive results of fungi isolates indicates yellow colour or clearing, while blue-black indicates negative, where starch has not been degraded [15].

Production of Amylase Using the Positive Isolates

The production medium used for the positive isolates were soluble starch (50)g, yeast extract(0.5)g, KH2PO4 (10) g,(NH4)2SO4 (10.5)g,MgSO4 (0.3)g,CaCl2 (0.5)g, FeSO4 (0.013)g, MnSO4 (0.004)g, ZnSO4 (0.004)g, CuCl2 (0.0067)g. In addition to this, agricultural waste product, maize cobs was used as solid state medium. The pH was adjusted to 6.5, and the media was sterilized in an autoclave for 15min at 121oC.The media was inoculated with a loop-full of fungal spore suspension (0.5ml) and incubated at 30oC in an orbital shaker set at 100rpm for 72hrs. The media was centrifuged at 5,000g for

15 min. Crude enzyme served as the enzyme source and the enzyme activity was assayed using the Dinitrosalicylic acid method (DNS) as described by Kwatia S, et al. [16].

Optimization of the Cultural Conditions

The factors such as pH, temperature, under different incubation time, affecting production of enzymes were optimized by varying the parameters using one factor at a time (OFAT). The experiment was conducted in 250ml Erlenmeyer flask, containing 150ml of production medium for each parameter. After sterilization by autoclaving, the flask was cooled to a temperature below 40°C and inoculated with culture and maintained under various culture conditions as described by Bedan D, et al. [17].

Effect of Temperature on Enzyme Produced: Effect of temperature on enzyme production was investigated by inoculating fermentation medium which was later incubated at 25°C, 30°C, 35°C, 40°C and 50°C Effect of pH on Enzyme Produced: The effect of pH was studied by maintaining other culture conditions and the medium at constant while varying the pH of the medium at 4.0, 4.5, 5.0, 5.5 and 6.0 Effect of Incubation Time: The effect of incubation time was studied by varying the medium for 24,48,76,96 and 120 hours.

Results and Discussion

Fungal Isolates Obtained

Micro-organisms in particular have been regarded as treasure of useful enzymes. There is a great variation between various genera as to their ability to produce a specific enzyme, the production of particular enzymes varies with the particular medium, temperature, pH and incubation time. In recent years, the uses of microorganisms have become a huge importance to food, textile, baking and detergent industries and sparked a large interest into the exploration of enzyme activity in microorganisms [18]. This study was focused on assessing the ability of several fungi in producing amylase, at different temperature, time and pH. The finding of the study are presented and interpreted as follows: Table 1 shows sample locations and amount of samples collected from three (3) different areas namely residential, farm land and market areas, fifteen (15) samples were collected from each location, where a total of thirty six (36) samples were collected. Ten (10) suspected fungi species namely, Mucor racemosus, Aspergillus Niger Penicillium chrysogenum, Rhizopus oryzae, Microsporum sp, Trichoderma sp, Nocardia sp, Chaetomum sp, Fusarium sp, and Monilla sp were isolated from maize cobs.

SamplelocationNumber of samples collected (n=36)Fungal specieisolated
A(residential)12Rhizopus oryzae, Penicillium chrysogenum, Nocardia sp,
Aspergillus niger
B (Farm land)12Mucorracemosus, Aspergillusniger, Microsporum sp
Chaetomum sp, Trichoderma sp, Monilla sp
C (Market place)12Chaetomumsp, Fusariumsp Rhizopusoryzae

Table 1: Fungi isolated from disposed maize cobs from different locations. KEY: A, B, C = Locations.

Amylolytic Potential of the Fungal Isolates

The amylolytic activity tested based on starch – iodine reaction of fungi on starch agar medium (Table 2). Out of 10 fungi isolated (39.5%) showed amylolytic activity, these organisms include Aspergillus Niger Mucor racemosus, Penicillium chrysogenum, Rhizopus oryzae. Aspergillus Niger having the highest amylase activity as shown by the zone of clearance (0.21cm) followed by Penicillium chrysogenum (0.33cm) while Mucor racemosus showed the lowest amylase activity (0.69cm).

FungiZone of clearanceStarch-iodine reaction test interference
Rhizopus oryzae++(0.48cm)+ve
Fusarium sp--ve
Mucor racemosus+(0.69cm)+ve
Monilla sp--ve
Penicillium chrysogenum+++(0.33cm)+ve
Aspergillus niger++++(0.21cm)+ve
Microsporum sp--ve
Trichoderma sp--ve
Nocardia sp--ve
Chaetomum sp--ve

Table 2: Amylolytic potential of fungal isolates based on starch-iodine reaction test. KEYS: The sign (+) indicates the positive

Table 2: Amylolytic potential of fungal isolates based on starch-iodine reaction test. KEYS: The sign (+) indicates the positive screening test and (-) indicates a negative screening test. The number of (+) mark ranging from 1+ to 3+ indicates the level of hydrolysis by the zone of clearance around the colony. However, 6(60.1%) of the fungi grown on the starch agar medium and tested for their amylase activity based on starch agar reaction test were negative and did not show any zone of clearance these fungi include Microsporum sp, Trichoderma sp, Nocardia sp, Chaetomum sp, Fusarium sp, Monilla sp, these correspond with the work of [19] who identified amylolytic activity from several fungal species isolated from soil and Aspergillus sp possess the highest amylase activity producing extracellular amylase using (wheat, rice, and black gram bran).

Figure 1: Effect of pH in the production of amylase using Penicillium chrysogenum.
Click to enlarge
Figure 1: Effect of pH in the production of amylase using Penicillium chrysogenum.
Figure 2: Effect of pH in the production of amylase using Rhizopus oryzae.
Click to enlarge
Figure 2: Effect of pH in the production of amylase using Rhizopus oryzae.

In this study, Figure 1 shows Penicillium chrysogenum having an optimum pH of 5.5 the amylase activity produced was (43.6±11.1μ/ml). Aspergillus sp, such as A. oryzae, A. ficuum and A. Niger exhibit optimal production at pH 5.0–6.0 in Solid state fermentation [20]. The effect of pH (Figure

2) shows that Rhizopus oryzae with the pH of 5.0 and the amylase activity produced was (25.5±9.5μ/ml). According to Kathiresan K, et al. [21] during production of α-Amylase from P. fellutanum optimum pH was found to be within the range of 6.0 to 7.0.

Figure 3: Effect of temperature in the production of amylase using Penicillium chrysogenum.
Click to enlarge
Figure 3: Effect of temperature in the production of amylase using Penicillium chrysogenum.

The present research recorded 35°C as optimum temperature for Penicillium chrysogenum as shown in Figure 3 with amylase activity of (46.3±15.1μ/ml).An optimal temperature of 50⁰C was observed when temperature was varied within a range of 30°C-90°C for production of α-Amylase by Aspergillus oryzae [22] When solid state fermentation was carried out for production of the enzyme by Aspergillus niger a range of 30-65°C, the optimum temperature was found to be 60°C [23] this did not agree with the recent findings for Penicillium chrysogenum. Figure

4 shows optimum temperature of Rhizopus oryzae at 30°C, amylase production was (30.8±14.9μ/ml). According to Adeniran AH, et al. [24] optimum temperature for amylase production was found ranging between 25°C and 37°C for the mesophilic fungi, which agrees with earlier findings, that influence of temperature on amylase production is related to the growth of microbes. Sethi S, et al. [25] also reported the influence of temperature on extra cellular enzymes secretion. This could probably be as a result of varying of the functional properties of the cell membrane.

Figure 4: Effect of temperature in the production of amylase using Rhizopus oryzae.
Click to enlarge
Figure 4: Effect of temperature in the production of amylase using Rhizopus oryzae.

The temperature has been observed to significantly influence enzymes production by the fungi [26]. Hence, the resistance to extreme temperatures, PH and pressure by the microorganisms and synthesis of particular enzymes are necessary for their adaptive response [27]. The microbial presences in such condition are of enormous importance for the exploration of biodiversity in biotechnology [14].

Conclusion

The use of solid state fermentation for the production of α-amylase using Penicillium chrysogenum and Rhizopus oryzae, is an economical process and is very simple to apply. The substrate maize cobs support the production of amylase production using Penicillium chrysogenum and Rhizopus oryzae under solid state fermentation. However, this substrate made enzyme production high therefore maize cobs is a good substrate for the production or synthesis for α- amylase using Penicillium chrysogenum and Rhizopus oryzae by solid state fermentation.

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@article{oguche2021,
  title   = {Screening of Fungi from Disposed Maize Cobs for Amylase Production},
  author  = {Oguche SO, Iliyasu MY, Ahmad A, Abba M, Karamba KI, Haruna US, Na’Allah AJ and Agbo E B},
  journal = {Open Access Journal of Microbiology & Biotechnology},
  year    = {2021},
  volume  = {6},
  number  = {4},
  doi     = {10.23880/oajmb-16000207}
}
Oguche SO, Iliyasu MY, Ahmad A, Abba M, Karamba KI, Haruna US, Na’Allah AJ and Agbo E B (2021). Screening of Fungi from Disposed Maize Cobs for Amylase Production. Open Access Journal of Microbiology & Biotechnology, 6(4). https://doi.org/10.23880/oajmb-16000207
TY  - JOUR
TI  - Screening of Fungi from Disposed Maize Cobs for Amylase Production
AU  - Oguche SO, Iliyasu MY, Ahmad A, Abba M, Karamba KI, Haruna US, Na’Allah AJ and Agbo E B
JO  - Open Access Journal of Microbiology & Biotechnology
PY  - 2021
VL  - 6
IS  - 4
DO  - 10.23880/oajmb-16000207
ER  -