Int.J.Curr.Microbiol.App.Sci (2015) 4(1): 484-494

ISSN: 2319-7706 Volume 4 Number 1 (2015) pp. 484-494 http://www.ijcmas.com

Original Research Article Assessment of microbial growth and survival in fresh rafia palmwine from Umuariaga community, Ikwuano L. G. A. Abia State, Nigeria

Obi C.N1*, Ogbulie, J.N2 and Nkwo, A.M1

1Department of Microbiology, College of Natural Sciences, Michael Okpara University of Agriculture, Umudike, P. M. B. 7267, Umuahia, Abia State, Nigeria. 2Department of Microbiology, Federal University of Technology Owerri, Nigeria, P. M. B. 1526 Owerri, Nigeria *Corresponding author

A B S T R A C T

The assessment of the presence, growth and survival of bacteria and yeasts in fresh raffia palm wine sample gave rise to the isolation of eight bacteria: Staphylococcus aureus, Escherichia coli, Lactobacillus spp, Micrococcus luteus, Serratia marcessens, Acetobacter spp, Bacillus and Streptococcus spp and four yeasts: Candida spp, Saccharomyces uvarum, Saccharomyces cerevisiae and Schizosaccharomyces pombe. The total bacterial count was in the range 6.08 x 103-3.48 x 103CFU/ml and the bacterial counts between 24-120 hrs of fermentation were not significantly different from the bacterial count in 0hr (fresh; P<0.05). Total coliform count was in the range 3 K e y w o r d s 4.60 x 10 0 CFU/ml. There was an initial rise in total yeasts counts after 24hrs (7.38 x 103- 8.22 x 103CFU/ml) and then a gradual decrease to 4.10 x 103 CFU/ml. The yeast count after 120 hrs of fermentation was significantly different (P<0.05) from counts Bacteria, between 0-72hrs. The growth and survival pattern of the bacteria isolates from 0- Growth, 120hrs showed that S. aureus was eliminated from the samples after 24hrs of growth Palm wine, while E. coli, M. luteus, Lactobacillus spp and Streptococcus spp were eliminated after Pathogens, 48hrs of growth. Serratia marcesens did not survive beyond 72hrs of fermentation Survival, while Bacillus and Acetobacter spp were present till the end of the fermentation. S. Yeasts cerevisiae, S. uvarum and S. pombe survived from 0-120hrs of fermentation while Candida spp was eliminated after 48hrs of growth. All the physicochemical parameters tested varied with respect to time. The pH values decreased from 6.8 -3.8. Fermentation temperature dropped from 25oC to 21.5oC after 24hrs and then fluctuated between 21.4oC-21.5oC till the end of the fermentation. The alcohol values of the palm wine samples increased steadily (1.6% v/v-15.10% v/v) from 0-120hrs of fermentation. There was a gradual increase in the moisture level (96.49% - 97.55%) as fermentation progressed. Bacterial and fungal pathogens which were present from the beginning of the fermentation as handling and processing contaminants were eliminated after some hours as fermentation progressed. Introduction

Palm-wine is the collective name for (Okafor, 1972). It is usually obtained from alcoholic beverages prepared from Raphia rinfera, R. hookeri and Elaeis fermented sap derived from various palm guineensis by methods described by Bassir

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(1962). Raphia palms usually yield more fermentation process. The spp organisms sap than oil palms although Raphia palms have also been reported to originate from can only be tapped once in its life time several sources which include tapping because its terminal florescent is destroyed equipment, containers and the environment during tapping (Okafor, 1978). During etc (Faparunsi and Bassir, 1972a). fermentation, the sugars in the palm-sap are metabolized to alcohol and organic acids The fermentation of Raphia palm wine is with the result that sap loses the sweetness considered an inexpensive and effective (Okafor, 1975a). The type of bacteria means of food production in Nigeria, fresh present depends on the stage of fermentation palm sap is usually contaminate the juice as and the composition of the sap (Bassir 1962; is tapped and there are changes in Okafor, 1977). Although alcohol production biochemical composition of the palm wine is common among yeasts, it is rare among (Faparunsi and Bassir, 1972a). Palm wine bacteria (Ingraham and Ingraham 2004). loses its sweetness as the fermentation Yeasts are used to make most alcoholic continues and the original colourless juice beverages. Palm-wine is consumed in parts becomes milky. If not consumed or bottled of Africa, Asia and South America. In within 24 hours of production, it gets sour Nigeria, the two principal sources of sap for due to prolonged fermentation and sublime palm wine fermentation are the oil palm malo-lactic acid fermentation by the (Elaeis guineensis) and the Raphia palms bacterial microflora (Ezeronye, 2003). To (Raphia spp). produce palm wine, a succession of microorganisms occur mainly gram negative The quality of the wine is highly stable and bacteria. The wine contains about 3% depends among other factors on the genus of alcohol and since the bacteria and yeasts are palm from which the sap is obtained. When consumed live, it is a source of single cell Palmwine is examined under the protein and various vitamins. The shelf life microscope, a large number of yeast and of palm wine is short, so, it is best consumed bacteria are observed. One factor that with about 48hours, various methods have influenced the palm wine is the nature of the been devised to preserve palm wine such as yeast and other microorganisms it contains. pasteurization, use of chemicals like Sorbate The unfermented raphia Palm sap is clean, and Sulphite and other preservatives like sweet, colourless syrup containing about 10- Alstonia boonei (Egbu) and Saccoglottis 12% sugar which is mainly sucrose (Bassir gabonensis (Nche). 1962). Upon fermentation by the natural microbial flora, the sugar and other products The main objectives for this study include, (Obire, 2005), whereas the sap becomes isolating and characterizing the microbial milky-white due to the increase of microbial flora (and possible pathogens) in palm wine. suspension resulting from the prolific To ascertain the survival pattern of growth of the fermenting organisms microorganisms in palm-wine during the (Okafor, 1975ab). Palm wine is course of fermentation and to determine the characterized by an effervescence of gas physicochemical changes associated with resulting from the fermentation of the palmwine fermentation and storage. sucrose content (Bassir, 1962), by the fermenting organisms. Previous studies on Sample collection the microbiology of E. guineensis and R. hookeri have incriminated several bacterial Fresh palm wine samples (200ml) from and yeast flora to be involved in the Raphia palm (R. hookeri) was collected 485 Int.J.Curr.Microbiol.App.Sci (2015) 4(1): 484-494 from traditional palm wine trapper from morphology, methylene blue staining, sugar Umuariaga community, Ikwuano, Abia fermentation. State, Nigeria. The freshly tapped samples were collected using 6 pre-sterilized labeled Yeast viability staining 100 ml capacity sample bottles with perforated screw caps corresponding to 0, A smear of the yeast isolate was prepared, 24, 48, 72, 96 and 120 hours of heat fixed and covered with methylene blue fermentation. The perforated screw caps stain for 1 minute and then washed off with were plugged with sterile non-absorbent tap water. It was placed on a slide rack to cotton wool. The samples were transported drain and air dry and was later viewed with to the laboratory in a cooler supported with 40x objective lenses. packs of freezing mixture of salt and ice- block for analysis within 1 h of collection. Sugar fermentation This was to reduce considerably the rate of fermentation of the sample before the The test described by Lodder (1971) was analyses began (Bassir, 1962; Obire, 2005). carried out using 1.0g each of glucose, lactose, fructose, galactose and sucrose. Isolation of bacteria and yeast Both the inoculated and un-inoculated tubes (control) were then incubated for 48h at One ml (1ml) of the palm wine sample was 37°C (for bacteria) and at 22oC for 48-72 collected aseptically at 0, 24, 48, 72, 96 and hours (for yeasts). A colour change to 120 hours of fermentation and serially yellow showed acid production and was diluted in sterile peptone water and 0.1ml recorded as positive fermentation. aliquots of suitable dilution was inoculated in duplicates by spread plate method Spore staining (Cheesebrough, 1994) on MCA (for Total Coliform Count), Nutrient agar (NA) (for A smear of the organisms was made on a Total Bacterial Count) and Sabourand microscope slide and flooded with malachite Dextrose Agar (SDA) for Total Yeast Count green dye solution. It was steamed and (Cruickshank et al., 1982). The inoculated allowed to stand for 3 minutes before the plates were incubated aseptically at 30°C for dye was washed off and the smear counter 24hours for bacteria and 24-48 hours for the stained with 0.25% aqueous safranin and air yeast at 22oC. The recovered isolates were dried. The slide was viewed under the purified by sub-culturing and stored on agar microscope with ×40 objective lenses and slants at 4°C for characterization. then oil immersion objective lenses was Identification of isolates used for clearer view. Retention of the test dye colour indicates the presence of spores, Isolates were Identified using standard whereas vegetative cells will stain a red/ morphological characteristics and brown colour (Cheesebrough, 1994). identification keys (Barnett et al., 1990; Kregger, 1987) for yeasts and Bergey and Physiochemical tests Holt (1993) for bacteria. The tests used in the identification of bacteria include pH determination morphology, gram reaction, spore production, Biochemical test, and sugar The pH of the fermenting palm wine fermentation. The test used in the samples was measured using a pH meter identification of yeasts includes, (Hanna, Model Hi 9810). 486 Int.J.Curr.Microbiol.App.Sci (2015) 4(1): 484-494

Temperature Acetobacter spp, Bacillus and Streptococcus spp and four yeasts genera: Candida spp, The temperature of the fermenting samples Saccharomyces uvarum, Saccharomyces was measured at room temperature using cerevisiae and Schizosaccharomyces pombe mercury-in-glass thermometer. (Tables 1 and 2). Agu et al., (1993) stated that palm wine is rich in non-pathogenic Moisture content microorganisms, so, the presence of E. coli (a pathogen) in fresh palm wine suggest This was based on removal of water from faecal contamination through the untreated the palm wine samples. A crucible was water that is traditionally used to dilute the washed and dried in hot air oven at 100°C palm wine and thereby increase the quantity and later cooled in a desiccator. The crucible for market sales. Bacillus spp and S. aureus was weighed and 10 ml of the samples have been reported to be mostly found as added to it and then transferred to hot air food contaminants and their presence in oven at 70-80°C for 2hrs and 100°C for the large amount could result to food poisoning next 4 hours. After weighing, the moisture (Whong et al., 2006). The fruit fly loss was measured at any given time (Drosophila melanogaster) is an important interval. factor in the contamination of the product ab initio. The insect habours yeasts on its body Alcohol content and transfers the same from ripped fruits to the palm wine which attracts its attention via An alcohol meter was used in the the natural aroma of the fresh product. The determination of the alcohol content of the microbial counts showed and initial bacterial palm wine before and after fermentation by count of 6.08 x 103 CFU/ml (0hr), but the finding the differences between the two count progressively decreased as numbers (original gravity (OG) and terminal fermentation moved on to 120hrs to a count gravity (TG). Using the specific gravity, the of 3.48 x 103CFU/ml. However, the density of the Palm wine was then bacterial counts at 120 hrs, 96 hrs, 72hrs, determined. A hygrometer was used to 48hrs, and 24hrs of fermentation were not measure the specific gravity of the solution significantly different when compared to the after a temperating to the room temperature. bacterial count in 0hr (fresh; P<0.05). The The original gravity (OG) and the terminal same trend in microbial count decrease was gravity were then determined. The noted for the coliform count beginning from difference between the two infers how much 4.60 x 103 CFU/ml (0 hr). But there was alcohol is in the palm wine thus: zero count for the coliforms at the end of the 120hrs of fermentation. This showed that the % Alcohol = (1.05 x {OG-TG) /TG) x 100 E. coli was totally eliminated from the palm 0.79 wine due to a mixture of parameters Result and Discussion produced in the palm wine by the bacteria (acids; Fig. 1) and yeasts (alcohol, CO2; Fig. The assessment of microbial presence, 3). The coliform counts at various stages of growth and survival in fresh palm wine the fermentation were significant (P<0.05) samples gave rise to the isolation of eight indicating that the various factors produced bacterial genera: Staphylococcus aureus, the palm wine which contributed to the Escherichia coli, Lactobacillus spp, killing of the coliforms had profound effect Micrococcus luteus, Serratia marcessens, on the organisms. The complete elimination

487 Int.J.Curr.Microbiol.App.Sci (2015) 4(1): 484-494 of E. coli from the palm wine is very Saccharomyces chevalieri and Pichia significant as it correlates with the fact that membrabefaciens. Van Pee and Swing cases of ill healths associated with E. coli (1971) reported S. cerevisiae and are very rare among the population that Saccharomyces pastorianus as the dominant consumes palm wine. For the yeasts, there yeasts in palm wine samples in Congo. It is was an initial increase in the population possible that differences in the chemical count between 0-24hrs of fermentation (7.38 composition of palm wine tapped from the x 103- 8.22 x 103CFU/ml: Tab. 3) suggesting felled and upright trees favour the complete that the yeasts were best adapted to the domination of the yeast biota by S. nutritional status of the fresh palm wine. cerevisiae in palm wine. According to This supports the findings of Okafor (1990) Ayernor and Matthews (1971), the methods which revealed that fresh palm wine of palm wine tapping and collection of palm contains several sugars (sucrose, fructose, sap influence the microbial content of the raffinose) and various growth factors like sap. Saccharomyces cerevisiae has been vitamin C and B12. After the first 24hrs, there confirmed in the present work as the was a gradual decrease in the yeast count dominant yeast species responsible for the obviously due to the competition for fermentation of palm wine tapped from the nutrient, oxygen and space that must have felled palm trees (Table 2). In matured palm ensued among the microorganisms as wine samples, only S. cerevisiae was fermentation progressed. The yeast count isolated and this species appear to after 120 hrs of fermentation was completely dominate the fermentation of significantly different (P<0.05) from counts palm wine in the felled palm trees. It was at the 0hrs, 24hrs, 48hrs and 72hrs. The high also reported that Lactobacillus spp were bacteria, yeast and coliform counts indicated responsible for the sour taste of palm wine inadequate hygienic conditions during in this study. Earlier, Bassir (1968) had collecting, dilution, processing and storing. reported L. plantarum and L. Microbial contamination during and or post mesenteriodesas being responsible for the processing can also result in spoilage or souring of palm wine tapped from the live persistence of some bacteria in palm wine. upright palm tree. The presence of The presence of micro-organisms in palm Lactobacilli in palm wine samples in wine can sometimes influence the stability Nigeria have also been reported by Faparusi of the product and its hygienic quality. and Bassir (1971) and Okafor (1975).

The genus Saccharomyces was the organism Four (4) yeasts were isolated from this of importance in palm wine as revealed in study, they include; Candida spp, this study, by its numerical predominance. Saccharomyces cerevisae and Due to its superior fermentative ability, Saccharomyces uvarum, Saccharomyces may have adapted to growth Schizosaccharomyces pombe all were in the special condition of the palm-sap positive to sucrose, maltose, glucose and (Faparusi and Bassir, 1991). Earlier, fructose and negative to lactose respectively. Faparusi (1973) and Okafor (1972) had reported S. cerevisae and The growth and survival pattern of the Schizosacchoromyces pombe and bacteria isolates from 0-120hrs showed that Saccharomyces uvarum as the dominant S. aureus was eliminated from the samples yeasts in palm wine, whilst Fahwehinmi after 24hrs of growth while E. coli, M. (1981) also reported the presence of luteus, Lactobacillus spp and Streptococcus

488 Int.J.Curr.Microbiol.App.Sci (2015) 4(1): 484-494 spp were eliminated after 48hrs of growth. The alcohol values of the palm wine Serratia marcesens was not isolated again samples increased steadily from 0-120hrs of after 72hrs of fermentation while Bacillus fermentation due to the activities of and Acetobacter spp were present till the Lactobacillus spp and Acetobacter spp. The end of the fermentation. lowest alcohol value (0hr) was 1.6%v/v while the highest level of alcohol recorded Saccharomyces cerevisiae, S. uvarum and was 15.10% v/v and that at 120 hrs of Schizosaccharomyces pombe were present in fermentation. Normal palm-wine contains the palm wine from 0-120hrs of approximately 0.5% to 7. 1% ethanol fermentation and can be said to be of major depending on its stage of fermentation importance in the fermentation of palm (Bassir, 1968; Okafor, 1978). Further wine. Candida spp (a pathogen) was increase in alcohol concentration beyond eliminated 48hrs of growth and activity 10% resulted in inhibition and possible possibly due to the increased production of elimination of the bacterial and yeast alcohol by the S. cerevisiae, S. uvarum and isolates from the palm wine. Schizosaccharomyces pombe and organic acids by the bacteria. This could be seen Moisture content expresses the amount of from the fact that the pH and alcohol values water present in the palm wine. Moisture of the palm wine increased after 48 hrs content determines the shelf life of the sap. (Figures 1 and 3). Earlier, Faparunsi (1973) The lower the moisture content, the longer and Okafor (1972) reported S. cerevisiae the expected shelf life, while the higher the and Schizosaccharomyces pombe as the moisture content, the shorter the expected dominant yeast in palm wine while Sanni shelf life. The gradual increase in the and Lonner (1993) reported to have isolated moisture level recorded in this work Saccharomyces cerevisiae, Saccharommyces (96.49% - 97.55%: Fig. 4), must have been uvarum and Candida spp from palm wine from the respiratory activities of aerobic which is in consonance with the findings of bacteria found in the palm wine which led to the research work. the formation of water as oxygen is finally reduced to water in the electron transport All the physicochemical parameters tested system. Given the high moisture content of varied with respect to time. The pH values the palm wine analyzed here the palm wine decreased (increase in acidity, Fig. 1) with will deteriorate rapidly. time from 6.8 -3.8 till 120hrs of fermentation and this is attributed to the From the result obtained, palm wine is a activities of Acetobacter spp which is known natural habitat for the growth of to produce acetic acid (since Lactobacillus microorganism, it could be deduced from spp was eliminated after 48hrs of this study that the microorganisms fermentation). The further production of associated with the fermentation of palm acid by Acetobacter spp usually makes the wine were mainly lactic acid bacteria, acetic palm wine very sour and unfit for human acid bacteria, some enterobactericea and consumption as affirmed by yeast. The increased rate of their isolation Theivendirarajah and Chrystopher (1987). further proved them to be the most important in the fermentation of palm wine. During fermentation, the palm wine Lactic acid bacteria were considered to be temperature dropped from 25oC to 21.5oC responsible for the rapid acidification of the and then fluctuated between 21.4oC-21.5oC. product as the acetic acid bacteria were not

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Int.J.Curr.Microbiol.App.Sci (2015) 4(1): 484-494 isolated in the palm wine sample on the first responsible for the fermentation of palm 3 days. Saccharomyces species has been wine. The physicochemical parameters confirmed as the dominant yeast species tested varied with respect to time.

Table.1 Identification of bacterial isolates from palm wine samples

S/ Colonial Gram Cell

N morphology rxn shape e s e e a e e e e e l s s s s

s Isolate s e s a u e o t a o o e l l o t o g t a r t r c d a c o l r i a t c o c u t a u d l x a o i a p u r n C C C I S L M S F G O

1 Smooth, with entire Cocci + + - - - AG AG A A A - Staphylococcus margin +round and + in G aureus milky colonies on cluster nutrient agar 2 Round purple _ Short + - - + - AG AG - - A - Escherichia coli colonies with rod in G methallic green singles sheen on EMB

3 Small and smooth + Rod - - - - - A A A A A - Lactobacillus with white colonies spp

4 Round and yellow + Cocci + - - - - - A A A - - Micrococcus colonies on in luteus Nutrient agar clusters 5 Round and mucoid _ Short + - + + - - A A A A - Serratia red colonies on rod in marcescens nutrient agar clusters

6 Mucoid brown _ Short + ------A A - Acetobacter spp colonies rod

7 Raised, irregular + Short + - + - + - - A - A - Bacillus spp white colonies on rod in nutrient agar chains

8 Small colourless + Cocci - - - - - A AG - A A - Streptococcus colonies in G spp chains

Keys: A= acid; AG= acid & gas; + = positive; - = negative

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Table.2 Identification of yeast isolates from palmwine samples e e e e s e s s s s o o o t o o c t t c r

S/N Morphology Microscopic features l Isolate u c c u l a a r u G L S M F

1 Round and creamy Single oval cells were seen, + - + + + Saccharomyces colonies on SDA some in pairs and elongate, cerevisiae Also budding was pronounced, spores seen 2 Whitish colonies, Single round cells seen, spores + - + + + Candida spp not were absent well developed pseudomycellium 3 Creamy colonies Single oval cells were seen, + - + + + Schizsaccharomyces with whitish edges. some cells were elongated pombe Pseudomycelium was while others were in pairs absent.

4 White colonies were Cells were ovals with a pear + + + + + Saccharomyces seen shape uvarum Pseudomycellium Keys: + = Positive; - = Negative

Table.3 Total microbial counts of palm wine samples (log10cfu ml-1)

Age of palmwine (hrs) Bacterial count Coliform count Yeast count 0 (fresh) 6.08 33.8a 4.60 24.2d 7.38 14.9ab

24 5.30 24.8a 3.52 7.73ab 8.22 12.3d

48 5.12 17.9a 2.60 b 7.76 20.7cd

72 4.94 23.4a 1.92 19.6bc 6.02 14.7bc

96 3.92 17.5a 3.0 6.71cd 5.20 8.57ab

120 3.48 a 0.00 0.00d 4.10 4.0a

a, b, c, d superscript have level of significance. Similar subscript means no significance difference while different subscript means significant differences

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Table.4 Growth and survival pattern of bacterial isolates in palm wine (hrs)

Organism 24 48 72 96 120

Staphylococcus aureus + + - - - -

Bacillus spp + + + + + + Escherichia coli + + + - - - Serratia marcessans + + + + - - Micrococcus luteus + + + - - - Acetobacter spp + + + + + + Streptococcus spp + + + + - - Lactobacillus spp + + + - - - Key: + = Presence; - = Absence

Table.5 Growth and survival pattern of yeast isolates in palm wine (hrs) Organism 0 24 48 72 96 120

Saccharomyces cerevisiae + + + + + +

Candida spp + + + - - -

Schizosaccharomyces pombe + + + + + +

Saccharomyces uvarum + + + + + +

Key: + = Presence; - = Absence

Figure.1 pH of fermenting palm wine samples

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Figure.2 Temperature of palm wine samples (oC)

Figure.3 Alcohol content of palm wine samples (%)

Figure.4 Moisture content of palm wine samples (%)

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References In: Julet, M (Edition) Introduction to Microbiology; A Case History Ayernor, G.K.A and Mathews, J. S. (1971) Approach. Third Edition, Books/ The sap of the Palm ELaeisguineensis cole-thomson, calif. USA. Pp 270- Jacq as raw material for alcohol 276. fermentation in Ghana. Trop Sci Kreger-Van Rig, N.J.N (1987).The yeasts; rd 13,71-83. A Taxonomic study 3 Editon. Barnette, J., Payne, R and Yarrow, D Elsevier Science Netherlands; Pp 89- (1990), yeast characteristics and 90. identification, 2nd Edition. Cambridge Lodder, J (1970). The yeasts a taxonomic nd Press; Pp 605-610. study, 2 Edition, North Holland Bassir, O (1962). Observation on the Amsterdam. fermentation of palm wine.West Obire, O (2005). Activity of Zymomonas African Journal of Biological spp in palm sap obtained from three Chemistry; 6:5-21. areas in Edo state, Nigeria. Journal of Bassir, O (1968). Some Nigerian Wines. Applied Science and Environmental West African Journal of Biological Management; 9:25-30. Applied Chemistry: 10: 41 45. Okafor, N (1972). Palm wine Yeasts from Bergey, D.H and Holt, D.H (1993). parts of Nigeria. Journal of Food Bergeys Manual of Determinative Science and Agriculture; 23:1399- Bacteriology, the Williams and 1407. Wilkins Company Baltimore; Pp 812- Okafor, N (1975a). Microbiology of 821. Nigeria palm wine with particular Cheesbrough M (1994). Medical reference to bacteria. Journal of Laboratory Manual for Tropical Applied Bacteriology; 38:81-88. Country Volume 11: Okafor, N (1975b) Preliminary Microbiology. Butterworth, Heinemann microbiological studies on the Ltd Cambridge. pp 56-58 preservation of palm wine. Journal of Cruikshank, R., Duiguid, P.J., Mamon, Applied Bacteriology; 43:159-161. P.C., Swarm, A.H.R (1982). Medical Okafor, N (1977). Microorganisms Microbiology, The practice of associated with cassava fermentation medical microbiology.Churchill for Garri production. . Journal of living stored Edinburgh; Pp 587. Applied Bacteriology; 42:279-284. Ezeronye, O.U (2003) Hybridization of Okafor, N (1978). Microbiology and palm wine yeasts (Saccharomyces Biochemistry of oil palm wine. cerevisae) with brewers yeast by Advanced Applied Microbiology: 24: protoplast fusion; Journal of 237 254. Biological Resource and Okafor, N (1978). Microbiology and Biotechnology; 1:53-62. biochemistry of oil palm wine. Faparunsi, S.I and Bassir, O (1972a). Journal of Applied Bacteriology; Factors affecting palm wine. Period 42:279-284. of Tapping. West African Journal of Okafor, N (1987) Industrial Microbiology st Biotechnology and Applied 1 Edition, University of Ife Press Ile Chemistry; 15:17-23. Ife, Nigeria; Pp 67-71. Ingram, J.L and Ingraham, C.A (2004). Benefits and Uses of microorganisms.

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