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Research Article - (2018) Volume 10, Issue 6

Millet Cobs: A Source of Microbial Enzymes

Arogunjo AO* and Arotupin DJ
Department of Microbiology, Federal University of Technology, Akure, Nigeria
*Corresponding Author: Arogunjo AO, Department of Microbiology, Federal University of Technology, Akure, Nigeria Email:

Abstract

Microbial enzymes have significant biotechnological application in industries. The purpose of this study was to isolate and identify microorganisms associated with millet cobs, determine the enzyme activity (lipase, protease, pectinase, cellulase and amylase) of the millet cob samples, screen the isolated microorganisms for enzyme production and determine the physicochemical parameters of the degrading medium. A total of seven bacteria and twelve fungi consisting of yeasts and moulds were isolated during the study. Day 20 of the degradation period has the highest enzyme activity for all the enzymes in the degraded millet cobs; lipase has the highest enzyme activity with a value of 0.496 mg/mL/min while protease has the lowest with a value of 0.003 mg/mL/min. All of the isolated microorganisms exhibited enzymatic activity except Zygosaccharomyces rouxii in which Bacillus spp were screened positive for all the enzymes assayed for. The temperature (ºC), pH and titratable acidity (%) ranged from 24.03- 28.47, 3.81-6.50 and 2.31-4.21 respectively. This study contributes to catalogue of microorganisms that has been identified as enzyme producers and provides additional information to support future research about the industrial potential of these microorganisms that may produce enzymes and other metabolites of industrial importance.

Keywords: Millet cobs; Enzymes; Bacteria; Fungi; pH, Temperature

Introduction

Many microorganisms such as bacteria, mould, and yeast produce a collection of multipurpose enzymes with extensive diversity of structures and industrial applications. Many microbial enzymes, such as amylases, cellulases, lipases, pectinases and proteases extracellularly produced. Amylases, starch-degrading enzymes, have numerous biotechnogical applications. These enzymes are used in textile and garments, paper industries, starch liquefaction, food, adhesive and sugar production and pharmaceuticals [1]. Cellulases, sugar degrading enzymes are used in textile industry for bio-polishing of fabrics and producing stonewashed look of denims, as well as in household laundry detergents for improving fabric softness and brightness [2]. Besides, they are used in animal feeds for improving the nutritional quality and digestibility, in processing of fruit juice and in baking, while de-inking of paper is yet another emerging application [3]. Lipases, lipid degrading enzyme are versatile tool for biotechnology. It is applicable in multiple industries such as agrochemical, pharmaceutical, cosmetic and perfume, taste and flavor industries, textile, food and dairy, detergent and surfactant industries, fat and oil, leather and paper production, chemical and waste water treatment [4]. Especially lipases are applied for biodiesel production [5]. Pectinases a group of enzymes that contribute to the degradation of pectin by various mechanisms. Acidic pectic enzymes are widely used in the production and clarification of fruit juices and wines [6]. They are also very important in maceration and solubilization of fruit pulps. Alkaline pectic enzymes have been used in several areas, including retting and degumming of fiber crops, textile processing, coffee and tea fermentations, paper and pulp industry, and oil extraction [7]. Proteases, enzyme which catabolizes protein by hydrolysis of peptide bonds are generally used in detergents, food industries meat processing, cheese making, silver recovery from photographic film, production of digestive and certain medical treatments of inflammation and virulent wounds [8-10]. They also have medical pharmaceutical applications [9]. The increase in world enzyme demand has led to sourcing for alternative substrate for the production of microbial enzyme; hence, agricultural wastes are readily accessible around the world as residual wastes for the production of these enzymes. In Nigeria, the most abundant renewable biomass resources include crop residues, such as corn straw, millet cobs, cassava peels, yam peels and rice husks.

Millets are a group of highly variable small seeded grasses, widely grown around the world as cereal crops or grains for fodder and human food. They do not form a taxonomic group, but rather a functional or agronomic one. Millets are important crops in the semi-arid tropics of Asia and Africa (especially in India and Nigeria), with 97% of millet production in developing countries and according to FAO statistics, the world production of millets was 26.7 million metric tonnes. The crop is favoured due to its productivity and short growing season under dry, high-temperature conditions [11,12]. Millet cobs are a great source of lignocellulosic biomass which is renewable, chiefly unexploited and inexpensive. Millets are a group of highly variable small seeded grasses, widely grown around the world as cereal crops or grains for fodder and human food. They do not form a taxonomic group, but rather a functional or agronomic one. Therefore, the present research work undertaken is to determine the suitability of millet cobs for microbial enzymes production.

Materials

Collection of sample

Pearl millet (Pennisetum glaucum, Linn) was obtained from a farmland in Gusau, Zamfara State, Nigeria. The millet cobs were kept in a sterile air tight polythene bags and transported to the Microbiology Postgraduate Laboratory, Federal University of Technology, Akure for further analysis.

Preparation of millet cobs

The millet cobs were sun-dried for 3 weeks and grinded into powder using an electric blender (Binatone Blender). The grinded millet cobs were divided into two portions; A and B. Portion A was left undegraded, while Portion B was degraded for 20 days.

Liquid substrate degradation

In the submerged substrate degradation, 10.0 g of the portion B of the substrate was naturally degraded by soaking in 100.0 mL of sterile distilled water. Liquid substrate degradation was carried out during which analyses such as: microbial analysis; pH, temperature and titratable acidity determination and enzyme activity were carried out at 4 days interval during millet cobs degradation.

Isolation and identification of microorganisms

Nutrient agar (NA), nutrient broth (NB), De Man Rogosa and Sharpe Agar (MRSA), De Man Rogosa and Sharpe broth (MRSB), Potato dextrose agar (PDA), Potato dextrose broth (PDB) were prepared according to manufacturer’s specification for the isolation of bacteria and fungi. Serial dilution was carried out according to the standard method of Fawole and Oso for the isolation of bacteria (×106 cfu/mL) and fungi (×104 sfu/mL) [13]. Conventional methods were used in identification of the isolated bacteria and fungi using the standard methods of Fawole and Oso [13]. Molecular identification of isolated bacteria and yeasts were carried out using the standard methods of Tamura, (2011) [14].

Determination of pH

The electrode of the meter was aseptically inserted into the media after standardization with the appropriate buffer (4 and 7 solutions). The electrode was then left in the media for 3 minutes to stabilize after which the pH was read at the same time on the meter scale.

Determination of temperature

Determination of temperature was done using a thermometer. The thermometer was inserted into the substrate 4 day interval to monitor the temperature.

Determination of total titrable acidity (TTA)

The total titratable acidity of the fermenting extrudates was determined every four day interval as described by AOAC (2012) [15]. Two grams (2 g) of the sample was weighed into 20 ml of sterile distilled water and filtered. 10 mL of the filtrate was measured and few drops of phenolphthalein indicator added. This was titrated with 0.1 M sodium hydroxide (NaOH) solution and the titre values in milliliter were added from the burette.

Enzyme activity

The enzyme activity of both the undegraded and degraded millet cobs sample was determined using the techniques for cellulase, protease, lipase, pectinase and amylase respectively [16-20].

Microbial screening for enzyme production

Culture media specific to each enzyme were used for primary screening of enzymes production by following the methods for cellulase, protease, lipase, pectinase and amylase respectively [21-25].

Statistical analyses

The experimental design was done in triplicate using complete randomization. The data obtained were subjected to analysis of variance (ANOVA) and the means were separated using Duncan’s New Multiple Range Test.

Results

Microbial isolation and identification

The total bacterial counts (106 cfu/ml), fungal mean counts (104 sfu/ml) is shown in Figure 1 and 2. The total bacterial count in the undegraded sample was 7.33, while that of fungi was 8.67. The total bacterial counts for the degraded sample ranged from 28.00 to 34.33, while the range for fungi was 7.00-36.00. The conventional identification of microorganisms isolated from the millet cobs samples is shown in Tables 1-3. The frequency of occurrence of isolated microorganisms is shown in Tables 4 and 5. Molecular identification for acillus cereus, acillus subtilis, acillus licheniformis, Corynebacterium fasciens, Lactoacillus lactis, Lactococcus lactis and Flavobacterium columnare respectively which were later identified using 16S rDNA analysis to be acillus licheniformis strain ZULMMI012, acillus subtilis strain b17a, Macrococcus carouselicus strain H8B16, acillus cereus strain pBCO_5, Lactoacillus brevis strain 14.8.28, Lactococcus lactis subsp. lactis strain Mast_19 and Flavobacterium ferrugineum while that of yeasts: Saccharomyces cerevisae, Candida albicans, Candida krusei, Geotrichum albidium and Zygosaccharomyces rouxii respectively which were also identified using 16S rDNA analysis to be Candida albicans strain h70b, Saccharomyces cerevisiae strain K289-3A, Rhodotorula mucilaginosa strain G20, Saccharomyces cerevisiae strain BY4742 and Kluyveromyces marxianus strain GX-15 respectively (Figure 3a and 3b).

microbial-biochemical-technology-millet

Figure 1: Bacterial counts isolated from millet cobs during degradation.

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Figure 2: Fungal counts isolated from millet cobs during degradation.

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Figure 3a: Phylogenic tree of bacteria isolated from millet cobs during degradation.

microbial-biochemical-technology-yeasts

Figure 3b: Phylogenic tree of yeasts isolated from millet cobs during degradation.

Laboratory Ref. No B1 B2 B3 B4 B5 B6 B7
Colony/Morphology              
Colour Creamy Creamy Creamy Yellow Creamy Creamy Yellow
Surface Opaque Opaque Opaque Opaque Opaque Opaque Translucent
Cell Characteristics
(microscopy)
Straight Rods Opaque Straight Rods Single, straight and short rods Straight single rod on short chain Cocci in pairs Singly cocci
Biochemical Test              
Gram’s reaction +ve +ve +ve +ve +ve +ve -ve
Catalase Test +ve +ve +ve +ve -ve -ve -ve
Motility +ve +ve +ve -ve -ve -ve -ve
Spore +ve central +ve central +ve central -ve -ve -ve -ve
Indole Production +ve -ve -ve -ve -ve +ve -ve
Starch hydrolysis +ve +ve +ve -ve -ve -ve +ve
Citrate utilization +ve +ve +ve -ve -ve -ve -ve
Methyl red -ve -ve -ve -ve -ve -ve -ve
Sugar Fermentation              
Glucose A- A- A- AG A- A- AG
Fructose A- A- A- -- AG A- A-
Maltose A- A- A- AG AG A- AG
Lactose -- -- -- AG AG A- A-
Sucrose A- A- -- AG AG A- AG
Mannitol A- A- -- -- -- -- A-
Probable Bacterium acillus licheniformis acillus subtilis acillus cereus Corynebacterium fascians Lactoacillus lactis Lactococcus lactis Flavobacterium columnare

Key: +ve = Positive, -ve = Negative, AG = Acid and Gas, A- = Gas, -- = Acid and gas absent

Table 1: Morphological and Biochemical Characteristics of Bacterial Isolates from millet cobs.

Isolates Cultural characteristics Spores/conidia arrangement under the microscope Identity of isolates
F1 Colonies  are blue-green with a suede-like surface consisting of a dense felt of conidiophores Uniserate and columnar conidial heads with the phalides limited to the upper two third of the vessicle  and curving to the roughly  parallel to each other Aspergillus fumigatus
F2 Spores are granular, flat, often with radial grooves, yellow at first but quickly becomes bright to yellow-green with age Conidia are globose to subglobose, pale green and conspicuously echinulate Aspergillus flavus
F3 Spores are white at initial stage and turn grey with maturity Hyphae is typically branching and form septate Aspergillus candidus
F4 Conidia vary in colour from white to grey Chains of single-celled conidia produced in basipetal succession from a specialized conidiogenous cell Scorpulariopsis brevicaulis
F5 Conidia  grows rapidly, resemble cotton candy and darken with age Mycelia are marked by numerous stolons connecting groups of long sporangiophores Rhizopus stolonifer
F6 Conidia appears orange Microconidia are absent, chlamydospores are intercalary, exceptionally terminal, spherical to ovoidal Fusarium poae

Table 2: Morphological Characteristics of Mould isolates from biodegraded millet cobs.

Laboratory Ref. No Cell shape Morphology Biochemical Properties
Fermentation/Assimilation
Yeast Identity
Ascospore
Present/Absent Shape Spore Pseudomycelium Mycelium Glucose Fructose Sucrose Lactose Maltose Nitrate
Y1 Oval + Oval + + - FA FA FA -A FA - Sacharomyces cerevisae
Y2 Cylindrical + Oval + + - FA FA FA - FA + Candida albicans
Y3 Cylindrical + Ovoid + + - FA FA FA FA FA - Candida krusei
Y4 Cylindrical + Short Cylindrical + - + FA FA -A -A FA + Geotrichum albidium
Y5 Spherical + Spherical + + - FA FA FA -A FA - Zygosaccharomyces rouxii

Key: + = Present, - = Absent, FA= Fermentation and Assimilation, -A= Assimilation only

Table 3: Morphological and Biochemical Characteristics of Yeasts isolated from millet cobs samples.

Bacterial isolates Degradation Period (day) Occurrence (%)
Undegraded sample 4 8 12 16 20  
acillus cereus + + + - - - 17.79
acillus licheniformis + + - - - - 14.65
acillus subtilis + + + - - - 16.76
Corynebacterium fasciens - - - + + + 12.77
Flavobacterium ferrugineum - + + + + - 9.79
Lactoacillus lactis - + + + + + 16.10
Lactococcus lactis - + + + + + 12.14
Mould isolates Degradation Period (day) Occurrence (%)
Undegraded sample 4 8 12 16 20  
Aspergillus flavus + + + - - - 16.41
Aspergillus fumigatus + + - - - - 16.37
Aspergillus candidus - + + + + - 20.39
Fusarium poae - - - + + + 17.03
Rhizopus stolonifer + + + + - - 17.07
Scopulariopsis brevicaulis - - - + + + 12.73
Yeast isolates Degradation Period (day) Occurrence (%)
Undegraded sample 4 8 12 16 20  
Candida albicans + + - - + + 25.76
Candida krusei + + - - - - 19.18
Geotrichum albidum - -   + + + 20.83
Saccharomyces cerevisiae - - + + + + 23.90
Zygosaccharomyces rouxii - + - - - - 10.33

Table 4: Frequency of occurrence of microbial isolates from millet cobs during degradation.

Conventional identification of isolates Sequence with best
match Accession no
Sequence based with
Similar strain
Base Pair Similarity (%)
acillus cereus MH411110.1 acillus licheniformisstrain ZULMMI012 1260 100
acillus subtilis SPP54602 acillus subtilisstrain b17a 1453 95
acillus licheniformis NR_044927.1 Macrococcus carouselicus strain H8B16 1549 85
Corynebacterium fasciens CP009965.1 acillus cereusstrain pBCO_5 4983 100
Lactoacillus lactis KX301062.1 Lactoacillus brevis strain 14.8.28 826 94
Lactococcus lactis JQ953678.1 Lactococcus lactis subsp. lactis strain Mast_19 678 99
Flavobacteriun columnare AF335328.1 Flavobacteriun ferrugineum 589 100
Saccharomyces cerevisiae KP674770.1 Candida albicans strain h70b 510 80
Candida albicans AF058447.1 Saccharomyces cerevisiae strain K289-3A 1227 97
Candida krusei KY296083.1 Rhodotorula mucilaginosa strain G20  613 97
Geotrichum albidium NM_001181492.1 Saccharomyces cerevisiae  strain S288C 1227 99
Zygosaccharomyces rouxii FJ896138.1 Kluyveromyces marxianus strain GX-15 556 99

Table 5: Conventional and Molecular Identification of microorganism isolated from Millet Cobs during degradation.

Temperature and pH variation of the millet cob samples

Figures 4 and 5 shows the temperature (oC) and pH variation of both the undegraded and degraded millet cob samples respectively. The highest temperature value was recorded on day 16 with value 28.47 ± 0.03 while the lowest temperature was recorded on day 4 with a value of 24.03. The control sample has the highest pH with a pH of 6.50 while the lowest pH was recorded on day 20 of the biodegradation period with a pH of 3.81.

microbial-biochemical-technology-degradation

Figure 4: Temperature of the millet cobs during degradation.

microbial-biochemical-technology-cobs

Figure 5: pH of the Millet cobs during degradation.

Titratable acidity of the millet cob samples

Figure 6 show that the undegraded sample (2.31) has the lowest titratable acidity (%), while the highest was on day 8 (4.21). There were no significant difference (P˃0.05) between the values of days; 12 (4.00), 16 (4.05) and 20 (4.07), however, there was significant difference (P<0.05) between these days of degradation and day 8.

microbial-biochemical-technology-acidity

Figure 6: Titratable Acidity of the millet cobs during degradation.

Enzyme activity and microbial screening for enzyme production

The enzyme activities of the millet cob a sample is shown in Figure 7. Lipase has the highest enzyme activity which was recorded on day 20 of the degradation period. The enzyme activities for all the enzymes assayed for were low in the control sample. All the isolated microorganisms exhibited enzymatic activity except Z. rouxii (Table 6).

microbial-biochemical-technology-millet-cobs

Figure 7: Enzyme activity of millet cobs during degradation.

Bacteria Pectinase Lipase Cellulase Amylase Protease
B. cereus + + + + +
B. licheniformis + + + + +
B. subtilis + + + + +
Corynebacterium fascians - + + + +
Flavobacterium ferrugunieum - - - - +
Lactoacillus lactis - + + + +
Lactococcus lactis - + + + +
Fungi Pectinase Lipase Cellulase Amylase Protease
Saccharomyces cerevisae + + + + +
Geotrichum albidum + - - - -
Aspergillus fumigatus + - + + +
Aspergillus flavus + - + + +
Candida krusei + - + - +
Aspergillus candidus + - + + -
Scorpulariopsis brevicaulis + - + - -
Rhizopus stonifer + - + + +
Candida albicans + + - + +
Fusarium poae + - - - +
Zygosaccharomyces rouxii - - - - -

Table 6: Microbial screening for enzyme production.

Discussion

Variation observed in names given using conventional and molecular methods of identification of microorganisms agrees with the findings of Frickmann [26]. They reported differences in names allotted using conventional and molecular methods of identification. However, the results of this study demonstrated clearly the interest and feasibility to introduce the 16SrRNA gene sequencing method in identification of bacteria and yeasts. Combination of conventional techniques and molecular approach will improve microbiological study and verification, allowing for exclusive and effective identification of microorganisms as against using only conventional method of identification.

The lignocellulosic structure of millet cobs maybe a factor responsible for the low microbial population present particularly in the undegraded sample [27-29]. The presence of bacteria such as B. cereus, B. licheniformis and B. subtilis in the undegraded sample agrees with the findings of Kunchala when sorghum and pearl millet from semiarid tropics were characterized for potential probiotic bacteria [30]. The presence moulds such as A. fumigatus, A. flavus and R. stolonifer, in the undegraded sample could be a result of them being millet cobs microflora which agrees with the finding of Badau when A. nidulans, A. niger, R. arrhizus were isolated from unmalted millet grains. Bamigboye also isolated A. flavus, A. niger, Rhizopus oryzae from corn cobs [31,32]. Slightly acidic environment of the millet cobs may be responsible for their adaptation [33,34]. The dominant population of A. fumigatus, A. flavus particularly in the undegraded sample maybe due to the presence of nutrients available within the millet cobs for utilization. Nasrin attested to this when combination of molasses and jackfruit were used as a substrate for mutant strain of A. niger for citric acid production [34]. The high counts of bacteria, mould and yeast in the millet cobs during degradation may be due to the high concentration of nutrients which agrees with the findings of Arotupin that arrays of microorganisms could increase in population due to the nutrient rich nature of the substrate, thus, supporting the growth and proliferation of microorganisms [35]. Low pH could be responsible for the increased fungal population [36].

Lactoacillus lactis and Lactococcus lactis are vital organisms for fermentation process which may be responsible for their presence from during degradation. Kalui documented in a review that these bacteria are essential in spontaneously fermenting cereal based food and that these microorganisms produce lactic acid as an important product from the energy yielding fermentation of sugars [37]. Ogbonnaya and Chukwu also attested to this; that majority of lactic acid bacteria isolated from ‘Akamu’ which is made from maize, a cereal belonged to the Genera: Lactoacillus [38]. Hence, these microorganisms may be implicated as partly responsible for initiating acidification during degradation of millet cobs. Saccharomyces cerevisae and Aspergillus spp may as well be responsible for lactic acid production which concurs with the documentation of Kalui that some yeasts (Saccharomyces) and moulds (Penicillium, Aspergillus and Botrytis) too produce lactic acid [37].

Lactococcus spp also produce ammonia from arginine [37]. All these potential bye-product of L. lactis may be responsible for the reduction of other bacterial population which was obtained in the undegraded sample. These conditions created by Lactoacillus lactis and Lactococcus lactis could favour the growth of fungi particularly on day 4 and beyond, thus, Fungi, Lactoacillus and Lactococcus metabolise sugars within the millet cobs which is converted to organic acids.

The growth and population of other microorganisms maybe due to the creation of enabling environment which favour their survival, growth and development of these set of microorganisms. Lactobacilli lactis and Lactococcus latis and mould had the highest population counts towards the end of the degradation period. These conform to the report of Ogbonnaya and Chukwu [38]. Saccharomyces spp, a cabtree positive yeast could utilize the sugars present within the millet cobs for the facilitation of the fermentation process in order to accumulate ethanol in the presence of oxygen particularly during early days of degradation period [39-41]. Candida albicans disappearing on day 8 may be due to the decrease in the available sugars within the millet cobs which might have facilitated their survival, growth, development and proliferation, however, S. cerevisiae becoming dominant on day 12 may be due to the environmental conditions that facilitate their survival, growth, development and proliferation. Candida spp, a cabtree negative yeast catabolizes sugars into carbon dioxide in the presence of oxygen may create an enabling environmental condition which may have facilitated the occurrence of S. cerevisae during degradation [42]. However, the reduction in the numbers of S. cerevisiae on days 16 and 20 maybe due to their preference for sugars [42]. Increase in the number of Candida spp over Saccharomyces spp on day 16 and 20 maybe due to their utilization of sugars. The reduction in yeast population of days 16 and 20 as compared to day 4 may be due to reduced oxygen [39-41].

The increased population of A. flavus, A. fumigatus and R. stolonifer in day 4 may be as a result of the reduced lignocellulosic component of the millet cobs [27-29]. The increased moisture content of the degraded millet cob as the degradation progressed could be responsible for the reduction in the fungal population [43]. The growth of A. candidusand F. poae on days 8 and 12 respectively succeeding A. fumigatus maybe a result of factors that may not favour its growth such as low relative humidity [44]. The availability of light may be responsible for the growth of A. fumigatus and A. flavus particularly in the undegraded sample, degraded sample of day 4 which also agrees with the findings of Shehu and Bello while studying the effect of environmental factors on the growth of Aspergillus spp associated with stored millet grains [44]. The growth of A. flavus, though in small population on day 8 shows that A. flavus has a higher tolerance level than A. fumigatus in darkness. The presence of A. candidus from day 8 to day 20 with a relative progressive rise in population could be as a result of increase in the dark condition as the degradation process progresses [44]. The occurrence of R. stolonifer on day 8 to day 12 could be as a result of these factors (ability to grow under low relative humidity and in the dark). Scopulariopsis brevicaulis occurred on day 12 and day 20 in which it was the only mould that grew on day 20 of the degraded millet cobs. Hence, S. brevicaulis have the highest survival rate of growing in the dark and in low relative humidity. Also, as the biodegradation period increases, relative humidity and light intensity decreases. Thus, these factors could be applicable to bacteria and yeast that grew on the degraded millet cobs.

Millet cobs could serve as a fermentation media for the production of enzymes. Adeleke documented that the production of enzymes by microorganisms in the fermentation media depend on the availability of suitable and utilizable substrate [45]. Rashid concurred that major and minor elements contained within a substrate can be degraded or synthesized by microorganisms using various enzymes [43]. Also, the capacity of microorganisms to produce extracellular enzymes is influenced by environmental conditions such as temperature, pH, aeration, inoculums age and the presence of inducer or repressor substrates [46,47].

The low amount of enzyme activity of all the enzymes assayed for particularly in the undegraded sample may be due to complex structure such as cellulose, hemicellulose and lignin which is important for its utilization and digestibility [33,48-50]. Solid state fermentation for the production of enzymes offers advantages over the conventional method of submerged fermentation [51]. Submerged method of fermentation used may also be attributed to the low enzyme activities [52]. The progressive increase in enzyme activities may be due to the hydrolytic (water) effect on the millet cobs which may increase the surface area and remove hemicellulose. This is in agreement with the findings of Rodolfo that water treatments at elevated temperatures (200-230ºC) and pressures can increase the biomass surface area and remove hemicellulose [53]. The enzyme activity of the millet cobs may have increased if it temperature and pressure were raised.

The presence of cellulase in millet cobs agrees with the separate reports of Sethi and Philip which states that enormous amounts of agricultural, industrial and municipal cellulose wastes contains cellulose [54,55]. The increased cellulase activity on day 4 may be due to the low amount of sugars recorded particularly glucose which may inhibit the production of cellulase while the low cellulase activity on day 12 and the undegraded sample may be due to the increased glucose composition inherent within the millet cobs. It could also be due to the reduced amount of disaccharide cellobiose which could be present within fermented millet cobs which seems to be a more potent inhibitor of cellulase [56]. Payne et al. documented that a large number of compounds such as glucose, mannose, galactose, xylose, ethanol and various ions can act as possible inhibitor for cellulose [57]. The undegraded sample offered reduced accessibility to cellulose and hemicellulose and degradability for enzymatic or chemical action which agrees with the findings of Barakat and Gao [56,58].

The presence of acillus spp may contribute to the production of cellulase in the degrading millet cobs which were shown by the zone of hydrolysis in their screening for cellulase production. Separate findings of Akhtar and Saowapar agrees with this finding when different species of acillus produced cellulose [21,29]. Shilpa and Pethe also isolated different cellulolytic bacteria from the soil in which B. thurigenesis showed the highest zone of hydrolysis when these bacteria were optimized at different condition [59]. The pH may also be a limiting factor to the low production of cellulase within the millet cobs during degradation. Shilpa and Pethe documented that acillus subsp subtilis A-54 has optimum pH of 6.5 and stable in pH range of 6.5-8.0 [59]. Sadhu and Maiti stated that the optimum temperature for acillus sp to produce cellulase in high quantity is between 37ºC to 55ºC [60]. Aspergillus fumigatus, A. flavus and A. candidus that were positively screened for cellulase production agrees with the separate findings of Jabasingh, Liu, Amorea and Faracoa that A. acculeatus, A. fumigatus, A. niger were confirmed to be producers of cellulose [61-63]. Yeasts such as S. cerevisae and Candida spp have shown capacity to produce cellulase [64].

Lactococcus lactis and Lactoacillus lactis being protease producers agrees with the finding of Hnin that proteolytic activity is an important characteristic of lactic acid bacteria [65]. acillus spp production of protease agrees with the findings of Hamza and Woldesenbet when acillus sp. Cab44 was observed to hydrolyse casein [65]. Alemu reported that B. licheniformis, B. firmus, B. alcalo B. subtilis and B. thuringiensis were producers of protease [66]. All the Aspergillus spp being producer of protease agrees with the report of Alemu that A. flavus, A. miller, A. niger and P. griseofulvinare were protease producers [66]. Oyeleke documented that A. flavus and A. fumigatus were able to produce extracellular protease [67]. Oyeleke observed that when A. flavus and A. fumigatus were subjected to the same temperature of 30ºC, A. flavus was able to produce the highest amount of protease. Rhizopus stolonifer, a fungus also screened positive for protease production agrees with the findings of Devi that moulds of genera Aspergillus, Penicillium and Rhizopus are useful for producing proteases, as several species of these genera are generally regarded as safe [68]. Sharp decrease of protease activity from day 12 to day 16 could be due to the ability of microorganisms to utilize sugar faster than protein. Dash et al. reported that acillus sp isolated from soil has maximum protease activity at optimum pH of 9.0. acillus amyloliquefaciens, B. subtilis, B. licheniformis and B. stearothermophilus were reported to produce protease at 37-60°C [68]. Optimum temperature for protease production in acillus spp has also been reported at 35-80°C [69]. Oyeleke documented that the optimum temperature for proteases production for both A. flavus and A. fumigatus was 30°C [67]. Oyeleke documented that optimum pH for A. flavus is 8.0 while A. fumigatus is 5.0. Devi reported optimum pH of 8.5 for protease production by A. niger, although, this is at variance with Siala et al. that reported optimum temperature of 60 and 90°C and optimum pH of 3.0 and 9.0 for proteases produced by A. niger [70,71]. These factors could be responsible for protease showing low activity.

The various acillus spp isolated from the millet cob samples are the best producer of lipase based on the zone of hydrolysis. acillus licheniformis strains, B. licheniformis -Ht7 have been identified to be good producer of lipase [72]. Lactic acid bacteria isolated from the millet cobs during degradation exhibiting lipase activity concurred with the documentation that Lactoacillus sp is a producer of lipase which is used for meat degradation [73]. Candida albicans and A. flavus, screened positive for lipase production agrees with the documentation of Singh that C. Antarctica, C. lipolytica and A. flavus are producers of lipase [74]. Singh et al. also observed that different lipase producing bacteria and fungi produced maximum lipase between pH 5.0 and 10.0 [74]. Padmapriya et al. observed lipase production increased with increase in temperature from 30 to 40°C and maximum production of lipase was obtained at 40°C and production declined at 50°C with different acillus spp and Lactoacillus spp [73]. Microorganisms were not subjected to these conditions, yet, lipase activity was highest among the enzymes evaluated.

Fungi are better producers of pectinase when compared to bacteria based on the findings of this research. Only acillus spp isolated where indicated to be a producer of pectinase. This agrees with different documentations of Bayoumi et al., Geetha et al., Mukesh et al., Darah et al., Raju and Divakar, Roosdiana et al., Kavuthodi et al. and Reddy et al. that most of the Bacterial isolates (mostly acillus spp and Pseudomonas spp) such as; Pseudomonas fluorescence and B. subtilis, acillus sp. MFW7, B. cereus, B. licheniformis, B. cereus, acillus sp. MBRL576, B. firmus I-4071, Enterobacter aerogenes NBO2, Pseudoalteromonas haloplanktis strain ANT/505, Paeniacillus xylanolyticus, B. firmus, B. firmus (P1), B. coagulans (P13), B. endophyticus (P57) and B. vietnamensis (P58), B. subtilis BKDS1, Enterobacter sp. PSTB-1 and Staphylococcus aureus were reported as good pectinase producers [75-82]. acillus subtilis being a producer of pectinase agrees with the report of Mariam and Aruna that B. subtilis strain arium 1115 produced the highest quantity of extracellular pectinase out of the arrays of acillus spp assayed for [83]. Many strains of acillus have been previously reported to produce extracellular pectinases, such as B. subtilis Strain NVFO 19 (El-sayed, 2015), marine B. subtilis, B. subtilis BKDS1, B. sphaericus MTCC 7542, B. sp. MBRL576, B. licheniformis KIBGEIB21, B. cereus, B. subtilis, B. stearothermophilus, B. firmus and B. cereus [80-89]. pH could also be a factor for low pectinase production in which acidic pH values were observed during the degradation of the millet cobs. According to the finding of Mariam and Aruna, B. subtilis strain arium 1115 produced the highest amount of pectinase at pH 9 (21.44 U/mL) as against pH 3 (0.61 U/mL) [83]. This could be a limiting factor for pectinase production especially for acillus spp isolated from the millet cobs sample but some species like acillus sp. MBRL576 and B. circulans have optimum pH at 4.0 and 7.0 respectively [81,88]. Temperature is another factor to consider for pectinase production. acillus species have demonstrated slightly higher temperature for pectinase production such as marine B. subtilis and B. circulans at 40°C [86,81]. However, findings show B. cereus, B. firmus I-10104, B. cereus and B. endophyticus and B. coagulans exhibited maximum pectinase production at 37°C [90-92]. Simran and Vijay attested that acillus subtilis isolated from different agro-industrial wastes were able to produce pectinase at a temperature of 35ºC and at pH 7.0 [93]. The temperature range of millet cobs during degradation fall short of these values.

All the fungal isolates that were screened positive for the production of pectinase may be responsible for the progressive increase in pectinase activity. Arotupin et al. revealed that A. repens is capable of hydrolyzing pectin. The hydrolysis of pectin by A. flavus, A. versicolor and A. niger have been reported [94,95]. Arotupin et al. revealed that temperature at 30ºC is optimum for the production of pectinase by A. repens [94].

Amylase activity in the millet cobs during degradation may be as a result of the presence of high concentration of hemicellulose carbohydrate particular starch within the millet cobs. This agrees with the findings of Singh et al. when amylase and xylanase content of rice bran, corn cob, wheat bran, wheat straw, and sugarcane bagasse were described in relation with the composition of starch and hemicellulose [95]. The progressive increase in amylase activity in the fermented millet cobs from day 4 to day 20 could be due to the availability of microorganisms such as B. cereus, B. licheniformis, B. subtilis, Lactoacillus lactis, Lactococcus lactis, A. flavus, A. fumigatus, A. candidus, Fusarium poae, Scorpulariopsis brevicaulis, C. albicans, and S. cerevisae in the degrading substrate. This consortium of microorganisms may contribute to the high amylase production. Akpomie et al. reported that acillus spp and Lactoacillus sp have ability to produce amylase [25]. Prakash and Jaiswal reported that B. subtilis, B. stearothermophilus, B. lecheniformis and B. amyloliquefaciens are known to be the good producers of thermostable α- amylase [96]. Sundarapandiyan and Jayalakshmi isolated, characterized and screened acillus subtilis SJ33 from marine environment for amylase production [97]. It is also to be known Prakash and Jaiswal stated in one of their findings that thermophilic bacterium B. stearothermophilus offers an alternative for commercial production of thermostable α-amylases [96]. Hence, acillus spp are known to be commercial producer of amylase. Filamentous fungi, such as A. oryzae and A. niger produce considerable quantities of enzymes that are used extensively in the industry [98]. Progressive increase in temperature could also be another factor that led to the progressive increase in amylase production which may be a satisfactory condition that made the microorganisms to produce more amylase. Akpomie et al. attested to this fact that gradual increase in amylase activity was observed from 26 to 45ºC and beyond this range it declined [25]. Thus, temperature contributes to the factor responsible for the secretion of amylase. Cavalheiro et al. documented that high enzymatic activity for amylase production by Gongronella butleri was between pH ranges of 4.0-5.5 [99]. Nwagu and Okolo reported that amylase produced by A. fumigatus maintained 94% of its activity at pH 4.5-6.5 for 24 hours [99]. de-Souza and Magalhães reported that A. niger has important hydrolytic capacities in amylase production and its tolerance of acidity (pH<3) could be a factor in the production of amylase by different Aspergillus spp isolated [100]. Sujeeta et al. reported that amylase activity of yeast isolates may increase at a temperature of 30ºC. The temperatures obtained during the degradation of the millet cobs were below this [101].

Conclusion

A Millet cob shows amylase, cellulytic, pectinolytic, lipolytic, proteolytic and amylytic activities when degraded by inherent microorganisms. Further studies on the millet cobs particularly on improving the microbial enzymatic activities in order to explore their potent value in commercial application should be carried out.

References

Citation: Arogunjo AO, Arotupin DJ (2018) Millet Cobs: A Source of Microbial Enzymes. J Microb Biochem Technol 10: 124-133.

Copyright: © 2018 Arogunjo AO, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.