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Studies on mould growth and biomass production using waste banana peel
Bioresource Technology 96 (2005) 1451–1456
Studies on mould growth and biomass production using waste banana peel J.P. Essien a
a,*
, E.J. Akpan b, E.P. Essien
a
Department of Botany and Microbiology, University of Uyo, P.M.B. 1017, Nigeria b Department of Biochemistry, University of Uyo, P.M.B. 1017, Nigeria Received in revised form 14 May 2004; accepted 6 December 2004 Available online 5 February 2005
Abstract Hyphomycetous (Aspergillus fumigatus) and Phycomycetous (Mucor hiemalis) moulds were cultivated in vitro at room temperature (28 + 20 C) to examined their growth and biomass production on waste banana peel agar (BPA) and broth (BPB) using commercial malt extract agar (MEA) and broth (MEB) as control. The moulds grew comparatively well on banana peel substrates. No significant difference (p > 0.05) in radial growth rates was observed between moulds cultivated on PBA and MEA, although growth rates on MEA were slightly better. Slight variations in sizes of asexual spores and reproductive hyphae were also observed between moulds grown on MEA and BPA. Smaller conidia and sporangiospores, and shorter aerial hyphae (conidiophores and sporangiophores) were noticed in moulds grown on BPA than on MEA. The biomass weight of the test moulds obtained after one month of incubation with BPB were only about 1.8 mg and 1.4 mg less than values recorded for A. fumigatus and M. hiemalis respectively, grown on MEB. The impressive performance of the moulds on banana peel substrate may be attributed to the rich nutrient (particularly the crude protein 7.8% and crude fat 11.6% contents) composition of banana peels. The value of this agricultural waste can therefore be increased by its use not only in the manufacture of mycological medium but also in the production of valuable microfungal biomass which is rich in protein and fatty acids. 2005 Elsevier Ltd. All rights reserved. Keywords: Mould growth; Biomass; Waste banana peel
1. Introduction A major problem experienced by agro-based industries in developing countries is the management of wastes. The disposal of agricultural wastes on land and into waterbodies are common, and has been of serious ecological hazards (Smith et al., 1987). Inefficient and improper methods of disposal of solid waste result in scenic blights, create serious hazards to public health, including pollution of air and water resources, accident hazards, and increase in rodent and insect vectors of disease, create public nuisances, otherwise interfere with *
Corresponding author. Tel.: +234 238 0233 28586. E-mail address: [email protected] (J.P. Essien).
0960-8524/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2004.12.004
community life and development (Tchobanoglous et al., 1993). The failure or inability to salvage and reuse such materials economically results in the unnecessary waste and depletion of natural resources (Selke, 1990; Tchobanoglous et al., 1993). To date emphasis is on biological conversion of plant wastes, especially agricultural wastes into added value products. Fungi are known organic waste decomposers and are generally capable of hydrolysing complex organic compounds as a major source of energy. This potential has been utilized for biomass production, organic waste disposal and its conversion into biofertilizers (Moreira et al., 1981; Obuekwe and Okungbowa, 1986). Studies on the economic importance of microorganisms have shown that many filamentous fungi
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J.P. Essien et al. / Bioresource Technology 96 (2005) 1451–1456
are major sources of industrial enzymes that could be used in the bioconversion of organic wastes to protein rich cell mass (biomass) which may be incorporated into rations for non-ruminants (Gonzales et al., 1989; Smith et al., 1989). Aspergillus fumigatus and Mucor hiemalis are among the most common stubborn food spoilage moulds encountered in tropical environment. Moulds are also known to produce high yields of nutritionally valued biomass when grown on organic waste as a source of carbon, energy and inorganic nitrogen (Gaden et al., 1976; Paterson, 1989). The importance of these moulds to us necessitates frequent researches on their activities in order to optimize their uses. However, such studies are seriously hindered by the high cost of analytical media. Banana (Musa sapientum) fruit peel is an organic waste that is highly rich in carbohydrate content and other basic nutrients that could support microbial growth. The potential economic benefits which may accrue from the use of this cheap nutrient material as a source of mycological research medium, and as substrates for production of valuable microfungal biomass have prompted the evaluation of the growth performance and biomass production of the two mould species on banana peel substrates.
2. Sources of banana peels The fruit peels used in this study were of the sweet banana variety. Musa sapientum fruit purchase from Uyo Main Market in Akwa Ibom State, Nigeria. The fresh, ripe fruit peels were used and when not used immediately, were stored at 5 C to arrest decay. Stored samples were utilized within 72 h of collection.
3. Chemical analysis of banana peels The method for the determinations of dry matter, crude protein (Kjeldahl), crude fat, crude fibre, ash and total carbohydrate as well as the ascorbic acid content were those recommended by AOAC as described by Horwitz (1980) and Ranjhan and Krishna (1981). A known mass of the dry powdered samples was ashed at 600 C in a muffle furnace for 4 h. The ash was dissolved in 6 m HCl solution and the resulting solution was made to a definite volume and used for the determination of mineral elements. Phosphorus was estimated colorimetrically at 440 nm using the molybdovanadate reagent method of AOAC (1975). Sodium and potassium were determined with a flame photometer, while other mineral elements were determined using an Atomic Absorption Spectrophotometer (Horwitz, 1980). The determinations were carried out in triplicates.
4. Test organisms The moulds species Aspergillus fumigatus (Hyphomycete) and Mucor hiemalis (Phycomycete) used in this study were originally isolated from spoiled banana fruit using the spread plate technique (Heperkan and Alperden, 1998). The isolates were characterized and identified according to procedures described by Samson et al. (1981) and Pitt and Hocking (1985).
5. Preparation of banana peel media Fresh ripe banana fruit peels were obtained and dried under mild sunshine till dry. Dried samples were blended into powder and sieved with 0.5 mm2 wire mesh. The milled powder was stored in a sterile airtight glass container. 200 g of the banana powder was carefully weighed out into 800 ml of distilled water, boiled for 45 min and strained through two layers of cheese cloth. The turbid liquid obtained was then autoclaved separately at 10 psi/in. (115.2 C) for 10 min. The banana peel extract was used as banana peel broth (BPB) prepared for submerge cultures, used in the measurement of moulds biomass. On the other hand, the sterilized banana peel extract was aseptically mixed with sterilized agar (autoclaved at 15.16/in.2 at 121 C for 15 min) to produced banana peel agar (BPA). The BPA was used for radial growth rate analysis. The composition of the two media used in this investigation were as follows: (i) Banana peel agar (BPA) Banana peel extract = 200 g Agar powder = 15 g Distilled water = 1000 ml pH = 5.6 at 28 ± 2 C (ii) Banana peel broth (BPB) Banana peel extract = 200 g Distilled water = 1000 ml pH = 5.6 at 28 ± 2 C For comparative analysis, a conventional mycological media malt extract agar (MEA) and malt extract broth (MEB) were prepared according to the manufacturers (Oxoid) specification, and used as control.
6. Determination of mould growth The performance of the isolates on BPA and MEA was assessed by determining the radial growth rates of the culture at room temperature (28 ± 2 C). During this process, sterile 20 cm diameter-petridishes containing 20 ml of the basal nutrient agar (BPA) per plate were inoculated centrally with 5 mm diameter agar plugs (inocula)
J.P. Essien et al. / Bioresource Technology 96 (2005) 1451–1456
cut with the aid of a sterile cork borer from the margin of 2 day old actively growing colonies of Aspergillus fumigatus and Mucor hiemalis maintained on the appropriate medium (BPA or MEA). The length and width of the reproductive hyphae (conidiophore in A. fumigatus and sporangiophore in M. hiemalis) as well as the diameter of the asexual spores (conidia and sporangiospores) of the isolates cultured on BPA and MEA were determined microscopically using calibrated ocular micrometer.
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Table 1 Proximate properties (% dry matter, DM) of banana peels Parameters
Banana peelsa
Cocoyam peelsb
Cassava peelsb
Sweet potatob
Dry matter Crude protein Crude fat Crude fibre Total ash Carbohydrate Moisture
14.08 7.87 11.60 7.68 13.44 59.51 78.4
17.62 9.56 2.05 32.02 12.17 – –
27.94 5.27 1.18 20.97 5.93 –
11.73 6.33 1.34 0.34 4.55 – –
a b
Values are mean of three determinations. Adapted from Oyenuga (1968).
7. Estimation of biomass weight The submerge culture technique was used. Spores suspensions were prepared from slope cultures of the test fungi as described by Sterne and McCarver (1978) and Agina (1991). The cultures were flooded with sterile physiological saline. The asexual spores were dislodged into the solution using a sterile inoculating needle bent slightly at the tip. The spore suspension were then filtered to remove mycelial fragments and clumps of spores using four layers of sterile cheese cloth, and then diluted to obtained spore concentrations of about 1 to 1.2 · 106 spores per ml. The spore concentrations were determined by direct counting in a haemocytometer (Agina, 1991; Essien, 2002). Two sets of twelve 250 ml Erlenmeyer flasks (one set containing 50 ml of banana peel broth (BPB) and the other set containing the same volume of malt extract broth (MEB)) were inoculated with 1ml aliquot of the mould spores. The flasks were incubated at 28 ± 2 C in an orbital incubator with agitation at 180 rpm. Biomass production on the BPB and MEB substrates was measured weekly by determining the mycelial dry weight. In this case the mycelium of the cultures were harvested by filtration, washed with several changes of cold distilled water, dried to a constant weight in a draft oven at 60 C and then weighed in a chemical balance.
8. Results and discussion Table 1 shows the proximate composition (% dry matter) of banana peels compared with those of cocoyam, cassava and sweet potato. The protein content of banana peels (7.87% DM) was higher than those of sweet potato (6.33% DM), and cassava (5.29% DM) but lower than cocoyam peels (9.56% DM) (Oyenuga, 1968). This high protein content of banana peels is an indication that the waste could serve as a possible alternative substrate for cultivation of fungi. Since protein is necessary for microbial growth, therefore supplementation with vegetable protein will support large scale cultivation of mould for the production of valuable microfungal biomass and probably for enzyme and anti-
biotic production. Aspergillus oryzae is used in the manufacture of soya sauce. A. fumigatus in the production of antibiotic fumigillin, A. flaviceps convert antitussive alkaloid glaucine to 5-(+)-glaucine which is preferred, while Mucor species are used in the manufacture of industrial amylase (Trease and Evans, 1989). Fat content was higher in banana peels (11.60% DM) than others. Fats are vital to the structure and biological functions of cells and are used as alternative energy source. Aspergillus species are particularly liable to grow in oil seed meals and in cereals (Trease and Evans, 1989). The high fat content would support its growth on banana peel medium. The crude fibre content (7.6% DM) of banana peels was lower than 32.02% DM and 20.97% DM reported in cocoyam and cassava peels respectively. Fibre contain appreciable amount of nutrients which are released slowly in further enzymic action. High fibre content reduce the rate of glucose and fat absorption in biological cells (Mottram, 1979). It is implied here that the low fibre content may likely favour the growth of fastidious organisms such as Mucor species. The ash content of banana peels (13.44% DM) compare very favourably with that of cocoyam peels (12.17% DM). Ash is a reflection of the amount of mineral elements contained in the sample. The carbohydrate content of the banana peels is significant and could serve as the main carbon source for microbial growth. The elemental composition of banana peels is shown in Table 2. The amount of potassium, phosphorus, magnesium and sodium are high, while iron and calcium contents are low. In view of the importance of calcium and iron to some microorganisms, salts of the elements could be incorporated into the media during formulations. Some of these elements serve as prosthetic groups
Table 2 Mineral and ascorbic acid content (mg/100 g) of banana peels Ca
Na
P
K
Fe
Mg
S
Ascorbic
7
34
40
44
0.93
26
12
18
Values are mean of triplicate determinations.
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of some enzymes. The presence of ascorbic acid indicates the inhibitory properties of the substrate against bacteria. The mean radial growth rates of the moulds shown in Figs. 1 and 2 were derived from the linear relationship of the colony diameter with time. In all the treatments the radial growth rate of the moulds increased exponentially with increase in incubation time. No significant difference in radial growth rates of moulds was observed between BPA and MEA although growth on MEA was slightly better than on BPA. The result may be ascribed to the formulated nature of MEA. It is expected that BPA would also support vigorous radial growth of fungi when properly formulated. Slight variation in sizes of asexual spores were also observed between moulds cultured on BPA and MEA with the later supporting the production of larger spores. The conidia of A. niger and sporangiospores of M. hiemalis cultured on BPA were smaller in diamter than the values recorded for A. fumigatus and M. hiemalis grown on MEA (Table 3). However, the disparity in the influence of BPA and MEA on the growth of the moulds was markedly evident with the length of the asexual reproductive hyphae (conidiophores and sporangiophores). On BPA the length of conidiophore in A. fumigatus and sporangiopohore in M. hiemalis were shorter than the values observed respectively for same moulds, grown on MEA. The biomass weight of the test moulds obtained with BPA were only 1.8 mg and 1.4 mg less than the values recorded respectively for A. fumigatus and M. hiemalis
Fig. 2. Radial growth of Mucor hiemalis on banana peel agar (BPA) and malt extract agar (MEA) at 28 ± 2 C: (d—d) BPA, (—) MEA.
grown on MEB for 4 weeks (Fig. 3). However in both cases the biomass produced by the moulds increased linearly with growth. Similar observations have earlier been reported by Smith and Berry (1978) and Aseigbu et al. (1996). Fungi are known to have different enzymatic responses to plant based substrates. Under favourable conditions the extracellular enzymatic activities are affected by both the components of the medium and temperature of incubation (Aseigbu et al., 1996). The low biomass production rates of the moulds on BPA may be ascribed to the presence of certain catabolic compounds or nutrients in banana peels which may suppress or are not enough to support mycelial biomass production while inducing high enzymatic activities leading to degradation of the substrate. Such catabolic products which were not determined in this study, may not be readily utilized by aerobic fungi grown under submerge condition.
9. Conclusion
Fig. 1. Radial growth of Aspergillus fumigatus on banana peel agar (BPA) and malt extract agar (MEA) at 28 ± 2 C: (d—d) BPA, (—) MEA.
Obviously, the two mould species differed in their physiological response to the banana peel substrates. The variation in individual species response to growth substrate is a genetically determined property which indicate that the substrate can be physiologically optimized for a particular species of mould. Similar observa-
J.P. Essien et al. / Bioresource Technology 96 (2005) 1451–1456
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Table 3 Size (lm ± SD) of the reproductive structures of test moulds Aspergillus fumigatus
Diameter of conidium/sporangiophore Length of conidiophore/sporangiophore Width of conidiophore/sporangiophore
Mucor hiemalis
BPA
MEA
BPA
MEA
1.6 ± 0.5 240 ± 49 5.1 ± 1.01
2.1 ± 0.4 321 ± 41 7.4 ± 1.51
3.3 ± 1.8 314 ± 78 6.2 ± 2.50
5.7 ± 1.3 433 ± 48 7.4 ± 1.51
tions have earlier been reported by Smith and Berry (1978). The technique adopted for measuring the growth performance of moulds on banana peel substrates were found to be adequate when compared to their growth on malt extract based media. Consequently, it was possible to delimit the mean radial growth and biomass weights, which were, characteristic of each organism. It was also possible to estimate the sizes of the reproductive hyphae and spores of moulds grown on banana peel substrates. These findings is in concert with the observations of Boddy et al. (1985) and Agina (1991). In view of the persisting economic recession and escalating cost of substrate for microbial cultivation especially in third
world countries. There is an urgent need to explore organic materials as alternative sources of microbial growth medium. Banana peel offers a good option if researches on the possibilities of augmenting its nutritional status are carried out, otherwise the potential of these moulds to utilize the substrate could be harnessed for effective waste management. Acknowledgements The working facilities at the Microbiology Laboratory of University of Uyo are gratefully acknowledged. The chemical analyses were performed at the Faculty Central Laboratory, Mr. N.A. Udofia is thanked for practical assistance, and Dr. R.M. Ubom for statistical advice. References
Fig. 3. Average biomass production by Aspergillus fumigatus and Mucor hiemalis on banana peel broth (BPB) and malt extract broth (MEB): ( ) BPB, (h) MEB, (I) ±standard deviation.
Agina, S.E., 1991. Effect of reduced water activities of growth media on growth rate characteristic of some xerophilic fungi. Nigerian Journal of Botany 4, 13–21. AOAC, 1975. Method of Analysis, 12th ed. The Association of Official Analytical Chemists, Washington, DC. Aseigbu, F.O., Paterson, A., Smith, J.E., 1996. Inhibition of cellulose saccharification and glyco-linin-attacking enzymes of five Lignocellulose—degrading fungi by ferric acid. World Journal of Microbiology 12, 16–21. Boddy, L., Gibbson, O.M., Grumdy, M.A., 1985. Ecology of Daldinia concentrica: effect of abiotic variables on mycelial extension and inter specific interaction. Transaction of the British Mycological Society 85, 201–211. Essien, J.P., 2002. Mycotoxigenic moulds in maize stores in Nigeria Mud rhumbus. Tropical Science 49, 154–158. Gaden, E.L., Mande, M.H., Reese, E.T., Spano, L.A., 1976. Enzymatic conversion of cellulosic materials: technology and application. Biotechnology and Bioengineering 6, 275–313. Gonzales, A.E., Martinez, A.T., Almendoros, G., Gribergs, F., 1989. A study of yeast during the delignification and fungal transformation of wood into cattle feed in Chilean rainforest. Antonio Van Leuwenhoek 55, 221–236. Heperkan, D., Alperden, I., 1998. Mycological survey of chicken feed and some feed ingredients in Turkey. Journal of Food Protection 51 (10), 807–810. Horwitz, W., 1980. Official Methods of Analysis, 13th ed. The Association of Official Analytical Chemists, Washington, DC. Moreira, A.R., Phillips, J.A., Humphrey, A.E., 1981. Production of cellulases in Thermospora sp. Biotechnology and Bioengineering 23, 1339–1348. Mottram, R.F., 1979. Human Nutrition, third ed. Edward Arnold Publishers, Great Britain, pp. 58–59, 159.
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Obuekwe, C.C., Okungbowa, J.O., 1986. Assessment of biomass production potential of some fungal isolates. Nigerian Journal of Microbiology 66 (1–2), 120–130. Oyenuga, V.A., 1968. Nigeria Food and Feeding Stuffs: Their Chemistry and Nutritive Value, third ed. Ibadan University Press, Nigeria, p. 34. Paterson, A., 1989. Biodegradation of lignin and cellulolytic materials. In: Olds, R.J. (Ed.), Biotechnology for Livestock Production. Plenum Press, New York, pp. 245–261. Ranjhan, S.K., Krishna, G., 1981. Laboratory Manual for Nutrition Research. Vikas Publishing House, New Delhi, pp. 23–64. Samson, R.A., Hoekstra, E.S., Vanooerschot, C.A.N., 1981. Introduction to Foodborne Fungi. Centre Bureau Voor Schimelcultures, Netherlands. Selke, S.E., 1990. Package and the Environment: Alternative, Trends and Solutions. Technomic Publishing Company, Lanchester, PA.
Smith, J.E., Anderson, J.G., Senior, E., Aiddo, K., 1987. Bioprocessing of lignocellulose. Philosophical Transactions of the Royal Society of London, Series A 321, 507–521. Smith, J.F., Claydo, N., Love, M.N., Alban, M., Wood, D.A., 1989. Effect of substrate depth on extra cellulase cacase production of Agaricus bisporus. Mycological Research 93, 292–296. Smith, J.J.E., Berry, D.R., 1978. Developmental Mycology: In Filamentous Fungi. Halsted Press Book, London, pp. 74–83. Sterne, R.E., McCarver, T.H., 1978. Osmotic effects on radial growth rate and specific growth rate of three soil fungi. Canadian Journal of Microbiology 24, 1432–1437. Tchobanoglous, G., Theisen, H., Vigil, S., 1993. Integrated Solid Waste Management: Engineering Principles and Management Issues. McGraw-Hill, New York, pp. 3–22. Trease, G.E., Evans, W.C., 1989. A Textbook of Pharmacognosy, 13th ed. Bailliere, Tindall, London, pp. 180–181.
Studies on mould growth and biomass production using waste banana peel J.P. Essien a
a,*
, E.J. Akpan b, E.P. Essien
a
Department of Botany and Microbiology, University of Uyo, P.M.B. 1017, Nigeria b Department of Biochemistry, University of Uyo, P.M.B. 1017, Nigeria Received in revised form 14 May 2004; accepted 6 December 2004 Available online 5 February 2005
Abstract Hyphomycetous (Aspergillus fumigatus) and Phycomycetous (Mucor hiemalis) moulds were cultivated in vitro at room temperature (28 + 20 C) to examined their growth and biomass production on waste banana peel agar (BPA) and broth (BPB) using commercial malt extract agar (MEA) and broth (MEB) as control. The moulds grew comparatively well on banana peel substrates. No significant difference (p > 0.05) in radial growth rates was observed between moulds cultivated on PBA and MEA, although growth rates on MEA were slightly better. Slight variations in sizes of asexual spores and reproductive hyphae were also observed between moulds grown on MEA and BPA. Smaller conidia and sporangiospores, and shorter aerial hyphae (conidiophores and sporangiophores) were noticed in moulds grown on BPA than on MEA. The biomass weight of the test moulds obtained after one month of incubation with BPB were only about 1.8 mg and 1.4 mg less than values recorded for A. fumigatus and M. hiemalis respectively, grown on MEB. The impressive performance of the moulds on banana peel substrate may be attributed to the rich nutrient (particularly the crude protein 7.8% and crude fat 11.6% contents) composition of banana peels. The value of this agricultural waste can therefore be increased by its use not only in the manufacture of mycological medium but also in the production of valuable microfungal biomass which is rich in protein and fatty acids. 2005 Elsevier Ltd. All rights reserved. Keywords: Mould growth; Biomass; Waste banana peel
1. Introduction A major problem experienced by agro-based industries in developing countries is the management of wastes. The disposal of agricultural wastes on land and into waterbodies are common, and has been of serious ecological hazards (Smith et al., 1987). Inefficient and improper methods of disposal of solid waste result in scenic blights, create serious hazards to public health, including pollution of air and water resources, accident hazards, and increase in rodent and insect vectors of disease, create public nuisances, otherwise interfere with *
Corresponding author. Tel.: +234 238 0233 28586. E-mail address: [email protected] (J.P. Essien).
0960-8524/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2004.12.004
community life and development (Tchobanoglous et al., 1993). The failure or inability to salvage and reuse such materials economically results in the unnecessary waste and depletion of natural resources (Selke, 1990; Tchobanoglous et al., 1993). To date emphasis is on biological conversion of plant wastes, especially agricultural wastes into added value products. Fungi are known organic waste decomposers and are generally capable of hydrolysing complex organic compounds as a major source of energy. This potential has been utilized for biomass production, organic waste disposal and its conversion into biofertilizers (Moreira et al., 1981; Obuekwe and Okungbowa, 1986). Studies on the economic importance of microorganisms have shown that many filamentous fungi
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J.P. Essien et al. / Bioresource Technology 96 (2005) 1451–1456
are major sources of industrial enzymes that could be used in the bioconversion of organic wastes to protein rich cell mass (biomass) which may be incorporated into rations for non-ruminants (Gonzales et al., 1989; Smith et al., 1989). Aspergillus fumigatus and Mucor hiemalis are among the most common stubborn food spoilage moulds encountered in tropical environment. Moulds are also known to produce high yields of nutritionally valued biomass when grown on organic waste as a source of carbon, energy and inorganic nitrogen (Gaden et al., 1976; Paterson, 1989). The importance of these moulds to us necessitates frequent researches on their activities in order to optimize their uses. However, such studies are seriously hindered by the high cost of analytical media. Banana (Musa sapientum) fruit peel is an organic waste that is highly rich in carbohydrate content and other basic nutrients that could support microbial growth. The potential economic benefits which may accrue from the use of this cheap nutrient material as a source of mycological research medium, and as substrates for production of valuable microfungal biomass have prompted the evaluation of the growth performance and biomass production of the two mould species on banana peel substrates.
2. Sources of banana peels The fruit peels used in this study were of the sweet banana variety. Musa sapientum fruit purchase from Uyo Main Market in Akwa Ibom State, Nigeria. The fresh, ripe fruit peels were used and when not used immediately, were stored at 5 C to arrest decay. Stored samples were utilized within 72 h of collection.
3. Chemical analysis of banana peels The method for the determinations of dry matter, crude protein (Kjeldahl), crude fat, crude fibre, ash and total carbohydrate as well as the ascorbic acid content were those recommended by AOAC as described by Horwitz (1980) and Ranjhan and Krishna (1981). A known mass of the dry powdered samples was ashed at 600 C in a muffle furnace for 4 h. The ash was dissolved in 6 m HCl solution and the resulting solution was made to a definite volume and used for the determination of mineral elements. Phosphorus was estimated colorimetrically at 440 nm using the molybdovanadate reagent method of AOAC (1975). Sodium and potassium were determined with a flame photometer, while other mineral elements were determined using an Atomic Absorption Spectrophotometer (Horwitz, 1980). The determinations were carried out in triplicates.
4. Test organisms The moulds species Aspergillus fumigatus (Hyphomycete) and Mucor hiemalis (Phycomycete) used in this study were originally isolated from spoiled banana fruit using the spread plate technique (Heperkan and Alperden, 1998). The isolates were characterized and identified according to procedures described by Samson et al. (1981) and Pitt and Hocking (1985).
5. Preparation of banana peel media Fresh ripe banana fruit peels were obtained and dried under mild sunshine till dry. Dried samples were blended into powder and sieved with 0.5 mm2 wire mesh. The milled powder was stored in a sterile airtight glass container. 200 g of the banana powder was carefully weighed out into 800 ml of distilled water, boiled for 45 min and strained through two layers of cheese cloth. The turbid liquid obtained was then autoclaved separately at 10 psi/in. (115.2 C) for 10 min. The banana peel extract was used as banana peel broth (BPB) prepared for submerge cultures, used in the measurement of moulds biomass. On the other hand, the sterilized banana peel extract was aseptically mixed with sterilized agar (autoclaved at 15.16/in.2 at 121 C for 15 min) to produced banana peel agar (BPA). The BPA was used for radial growth rate analysis. The composition of the two media used in this investigation were as follows: (i) Banana peel agar (BPA) Banana peel extract = 200 g Agar powder = 15 g Distilled water = 1000 ml pH = 5.6 at 28 ± 2 C (ii) Banana peel broth (BPB) Banana peel extract = 200 g Distilled water = 1000 ml pH = 5.6 at 28 ± 2 C For comparative analysis, a conventional mycological media malt extract agar (MEA) and malt extract broth (MEB) were prepared according to the manufacturers (Oxoid) specification, and used as control.
6. Determination of mould growth The performance of the isolates on BPA and MEA was assessed by determining the radial growth rates of the culture at room temperature (28 ± 2 C). During this process, sterile 20 cm diameter-petridishes containing 20 ml of the basal nutrient agar (BPA) per plate were inoculated centrally with 5 mm diameter agar plugs (inocula)
J.P. Essien et al. / Bioresource Technology 96 (2005) 1451–1456
cut with the aid of a sterile cork borer from the margin of 2 day old actively growing colonies of Aspergillus fumigatus and Mucor hiemalis maintained on the appropriate medium (BPA or MEA). The length and width of the reproductive hyphae (conidiophore in A. fumigatus and sporangiophore in M. hiemalis) as well as the diameter of the asexual spores (conidia and sporangiospores) of the isolates cultured on BPA and MEA were determined microscopically using calibrated ocular micrometer.
1453
Table 1 Proximate properties (% dry matter, DM) of banana peels Parameters
Banana peelsa
Cocoyam peelsb
Cassava peelsb
Sweet potatob
Dry matter Crude protein Crude fat Crude fibre Total ash Carbohydrate Moisture
14.08 7.87 11.60 7.68 13.44 59.51 78.4
17.62 9.56 2.05 32.02 12.17 – –
27.94 5.27 1.18 20.97 5.93 –
11.73 6.33 1.34 0.34 4.55 – –
a b
Values are mean of three determinations. Adapted from Oyenuga (1968).
7. Estimation of biomass weight The submerge culture technique was used. Spores suspensions were prepared from slope cultures of the test fungi as described by Sterne and McCarver (1978) and Agina (1991). The cultures were flooded with sterile physiological saline. The asexual spores were dislodged into the solution using a sterile inoculating needle bent slightly at the tip. The spore suspension were then filtered to remove mycelial fragments and clumps of spores using four layers of sterile cheese cloth, and then diluted to obtained spore concentrations of about 1 to 1.2 · 106 spores per ml. The spore concentrations were determined by direct counting in a haemocytometer (Agina, 1991; Essien, 2002). Two sets of twelve 250 ml Erlenmeyer flasks (one set containing 50 ml of banana peel broth (BPB) and the other set containing the same volume of malt extract broth (MEB)) were inoculated with 1ml aliquot of the mould spores. The flasks were incubated at 28 ± 2 C in an orbital incubator with agitation at 180 rpm. Biomass production on the BPB and MEB substrates was measured weekly by determining the mycelial dry weight. In this case the mycelium of the cultures were harvested by filtration, washed with several changes of cold distilled water, dried to a constant weight in a draft oven at 60 C and then weighed in a chemical balance.
8. Results and discussion Table 1 shows the proximate composition (% dry matter) of banana peels compared with those of cocoyam, cassava and sweet potato. The protein content of banana peels (7.87% DM) was higher than those of sweet potato (6.33% DM), and cassava (5.29% DM) but lower than cocoyam peels (9.56% DM) (Oyenuga, 1968). This high protein content of banana peels is an indication that the waste could serve as a possible alternative substrate for cultivation of fungi. Since protein is necessary for microbial growth, therefore supplementation with vegetable protein will support large scale cultivation of mould for the production of valuable microfungal biomass and probably for enzyme and anti-
biotic production. Aspergillus oryzae is used in the manufacture of soya sauce. A. fumigatus in the production of antibiotic fumigillin, A. flaviceps convert antitussive alkaloid glaucine to 5-(+)-glaucine which is preferred, while Mucor species are used in the manufacture of industrial amylase (Trease and Evans, 1989). Fat content was higher in banana peels (11.60% DM) than others. Fats are vital to the structure and biological functions of cells and are used as alternative energy source. Aspergillus species are particularly liable to grow in oil seed meals and in cereals (Trease and Evans, 1989). The high fat content would support its growth on banana peel medium. The crude fibre content (7.6% DM) of banana peels was lower than 32.02% DM and 20.97% DM reported in cocoyam and cassava peels respectively. Fibre contain appreciable amount of nutrients which are released slowly in further enzymic action. High fibre content reduce the rate of glucose and fat absorption in biological cells (Mottram, 1979). It is implied here that the low fibre content may likely favour the growth of fastidious organisms such as Mucor species. The ash content of banana peels (13.44% DM) compare very favourably with that of cocoyam peels (12.17% DM). Ash is a reflection of the amount of mineral elements contained in the sample. The carbohydrate content of the banana peels is significant and could serve as the main carbon source for microbial growth. The elemental composition of banana peels is shown in Table 2. The amount of potassium, phosphorus, magnesium and sodium are high, while iron and calcium contents are low. In view of the importance of calcium and iron to some microorganisms, salts of the elements could be incorporated into the media during formulations. Some of these elements serve as prosthetic groups
Table 2 Mineral and ascorbic acid content (mg/100 g) of banana peels Ca
Na
P
K
Fe
Mg
S
Ascorbic
7
34
40
44
0.93
26
12
18
Values are mean of triplicate determinations.
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of some enzymes. The presence of ascorbic acid indicates the inhibitory properties of the substrate against bacteria. The mean radial growth rates of the moulds shown in Figs. 1 and 2 were derived from the linear relationship of the colony diameter with time. In all the treatments the radial growth rate of the moulds increased exponentially with increase in incubation time. No significant difference in radial growth rates of moulds was observed between BPA and MEA although growth on MEA was slightly better than on BPA. The result may be ascribed to the formulated nature of MEA. It is expected that BPA would also support vigorous radial growth of fungi when properly formulated. Slight variation in sizes of asexual spores were also observed between moulds cultured on BPA and MEA with the later supporting the production of larger spores. The conidia of A. niger and sporangiospores of M. hiemalis cultured on BPA were smaller in diamter than the values recorded for A. fumigatus and M. hiemalis grown on MEA (Table 3). However, the disparity in the influence of BPA and MEA on the growth of the moulds was markedly evident with the length of the asexual reproductive hyphae (conidiophores and sporangiophores). On BPA the length of conidiophore in A. fumigatus and sporangiopohore in M. hiemalis were shorter than the values observed respectively for same moulds, grown on MEA. The biomass weight of the test moulds obtained with BPA were only 1.8 mg and 1.4 mg less than the values recorded respectively for A. fumigatus and M. hiemalis
Fig. 2. Radial growth of Mucor hiemalis on banana peel agar (BPA) and malt extract agar (MEA) at 28 ± 2 C: (d—d) BPA, (—) MEA.
grown on MEB for 4 weeks (Fig. 3). However in both cases the biomass produced by the moulds increased linearly with growth. Similar observations have earlier been reported by Smith and Berry (1978) and Aseigbu et al. (1996). Fungi are known to have different enzymatic responses to plant based substrates. Under favourable conditions the extracellular enzymatic activities are affected by both the components of the medium and temperature of incubation (Aseigbu et al., 1996). The low biomass production rates of the moulds on BPA may be ascribed to the presence of certain catabolic compounds or nutrients in banana peels which may suppress or are not enough to support mycelial biomass production while inducing high enzymatic activities leading to degradation of the substrate. Such catabolic products which were not determined in this study, may not be readily utilized by aerobic fungi grown under submerge condition.
9. Conclusion
Fig. 1. Radial growth of Aspergillus fumigatus on banana peel agar (BPA) and malt extract agar (MEA) at 28 ± 2 C: (d—d) BPA, (—) MEA.
Obviously, the two mould species differed in their physiological response to the banana peel substrates. The variation in individual species response to growth substrate is a genetically determined property which indicate that the substrate can be physiologically optimized for a particular species of mould. Similar observa-
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Table 3 Size (lm ± SD) of the reproductive structures of test moulds Aspergillus fumigatus
Diameter of conidium/sporangiophore Length of conidiophore/sporangiophore Width of conidiophore/sporangiophore
Mucor hiemalis
BPA
MEA
BPA
MEA
1.6 ± 0.5 240 ± 49 5.1 ± 1.01
2.1 ± 0.4 321 ± 41 7.4 ± 1.51
3.3 ± 1.8 314 ± 78 6.2 ± 2.50
5.7 ± 1.3 433 ± 48 7.4 ± 1.51
tions have earlier been reported by Smith and Berry (1978). The technique adopted for measuring the growth performance of moulds on banana peel substrates were found to be adequate when compared to their growth on malt extract based media. Consequently, it was possible to delimit the mean radial growth and biomass weights, which were, characteristic of each organism. It was also possible to estimate the sizes of the reproductive hyphae and spores of moulds grown on banana peel substrates. These findings is in concert with the observations of Boddy et al. (1985) and Agina (1991). In view of the persisting economic recession and escalating cost of substrate for microbial cultivation especially in third
world countries. There is an urgent need to explore organic materials as alternative sources of microbial growth medium. Banana peel offers a good option if researches on the possibilities of augmenting its nutritional status are carried out, otherwise the potential of these moulds to utilize the substrate could be harnessed for effective waste management. Acknowledgements The working facilities at the Microbiology Laboratory of University of Uyo are gratefully acknowledged. The chemical analyses were performed at the Faculty Central Laboratory, Mr. N.A. Udofia is thanked for practical assistance, and Dr. R.M. Ubom for statistical advice. References
Fig. 3. Average biomass production by Aspergillus fumigatus and Mucor hiemalis on banana peel broth (BPB) and malt extract broth (MEB): ( ) BPB, (h) MEB, (I) ±standard deviation.
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