CONTACT : BARTHOLOMEW SAANU ADELEKE microbade@yahoo.com 190 Abstract Cassava is a tuber crop mainly cultivated in Africa countries. The presence of unwanted microorganisms can complicate the control of the fermentation process, which can lead to the production of objectionable odors in steeping water. Microbial and physical analyses during fufu production were performed on the steeping water. Bacteria count decreased with an increase in the fermentation time. The highest bacterial count of 6.6 × 103 CFU/mL, while the least count of 2.2 × 103 CFU/ml was recorded, respectively. The bacteria isolated include Bacillus subtilis, Pseudomonas species, Lactobacillus fermentum, Proteus mirabilis, and Klebsiella sp. The pH value of 3.32 was recorded on day four, while the temperature was constant during the fermentation process. A high value of 45.2 mg/L total solids was recorded on day four, while the least value of 15.0 mg/L was obtained on day one. The effect of fermenting microorganisms under a controlled environment reduces the foul odor usually perceived during fufu production. Hence, the survival of these microorganisms at low pH can inhibit the growth of unwanted microorganisms, thus contributing to the acceptability of the cassava product, i.e. fufu. ISSN : 2580-2410 eISSN : 2580-2119 Characterization of Bacteria Isolates from Fermented Cassava Steeping Water Olalekan Blessing Balogun 1, Bartholomew Saanu Adeleke 2*, Ibukun I. Owoseni 1 1 Department of Biological Sciences, Joseph Ayo Babalola University, Ikeji-Arakeji, Nigeria 2 Department of Biological Sciences, Olusegun Agagu University of Science and Technology, Okitipupa, Nigeria Introduction Cassava (Manihot esculenta) is a tuber crop serving as a cheap source of carbohydrates and other nutrients in the diet of the teeming population in Africa and Asia, which provides energy for about 500 million people (Achi & Akomas, 2006). Bamidele et al. (2015) posited cassava as a supplementary staple food to more than 200 million Africans aside from its uses as livestock feeds. Nigeria is ranked first and largest producer of cassava but with less export compared to Thailand (Otekunrin & Sawicka, 2019). Cassava production and processing are usually concentrated in the hands of numerous smallholder farmers located primarily in the South and Central regions of Nigeria. Cassava tuber consists of 64 - 87% starch depending on the growth stage or at maturity, but with low protein, fats, vitamins, and minerals (Aloys & Hui Ming, 2006). Cassava tubers are composed of starch OPEN ACCESS International Journal of Applied Biology Keyword Cassava; fermentation process; food contamination; steeping water; starter culture Article History Received 05 October 2021 Accepted 30 December 2021 International Journal of Applied Biology is licensed under a Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. International Journal of Applied Biology, 5(2), 2021 191 depending on the growth stage or at maturity and the starch content compared to other starchy carriers (Lindeboom et al., 2004). Adequate processing of cassava tubers by traditional means has yielded to a variety of edible products, such as fufu, ‘Akpu’, Lafun, Garri, Abacha, and Tapioca. Fermentation is one of the oldest and most important traditional food processing and preservation techniques. Employing this technique enhances the nutrient content and reduces the anti- nutrient contents in cassava (Oyetayo, 2006). In Nigeria, cassava has been processed into many fermented and unfermented products in many ways. Some of the fermented products include cassava flour (lafun), which is produced by drying and milling fermented cassava tubers, cassava flakes (garri), which is produced by grating, soaking, fermenting, and roasting cassava mash. Other products include fermented cassava slurry used to produce “fufu”. The product of interest of this research is fufu, a fermented cassava mash, which comes as a wet mash or a dry powder (Adegbehingbe et al., 2019) and is most commonly consumed in the Eastern and Southern regions of Nigeria. Fermentation of cassava involves the steeping of cassava roots in water for 3 to 4 days, which softened the root to disintegrate the tissue structures in contact with linamarin which is located in the cell walls by the action of linamarase (Adeleke & Olaniyi, 2018). This enzyme hydrolyses linamarin to glucose and cyanohydrins and subsequently breaks down to ketone and HCN (Aloys & Hui-Ming, 2006). Traditionally, African fermented foods and products, for instance, garri and fufu can be obtained from a series of operational procedures, which include grating, dewatering, fermenting, and roasting and these processes generate waste among which is steeping water, wastewater, and solid waste. However, the focus of this study is on steeping water. About one-third of the cassava tubers harvested in Nigeria are utilized domestically for fufu production (Essers et al., 1995). It is bulky, easily contaminated by microorganisms, and cannot be stored for long due to its high water content, thus susceptible to easy spoilage. The inefficient method of preservation of wet cassava mash can be linked to the microbial growth that causes undesirable odor, and sometimes total spoilage. Nigeria has a large, sustainable, and expanding market for cassava fufu, with a population of over 180 million people (Ezedinma et al., 2006). In Nigeria, the consumption pattern varies according to ecological zones as Garri, a roasted granule is widely accepted in both rural and urban areas. Interestingly, it can be consumed without any additives or with a variety of additives, such as sugar, groundnut, fish, meat, and stew (Graffham et al., 2019). The presence of unwanted microorganisms complicates the control of the fermentation process, thus leading to the production of objectionable odors in steeping water (Omar et al., 2000). Therefore, to provide basic information on the microorganism present in the fermenting substrates, there is a need to understand the type of the fermenting microbes in the steeping process to ensure improvement in the quality of cassava and cassava products. Therefore, this study was designed to isolate, enumerate, and identify microorganisms present in cassava steeping water and also determine the physical properties of the steeping water. Materials and Methods Sample collection and processing This study was carried out in February 2017. Cassava tubers were obtained from a cassava processing site in Ikeji-Arakeji inside sterile plastic bags and then transported to the International Journal of Applied Biology, 5(2), 2021 192 Department of Microbiology, Joseph Ayo Babalola University, Osun State, Nigeria for further processing, such as washing, peeling, cutting, and re-washed with sterile distilled water. Fermentation and microbial analysis Cassava tubers were fermented for four days inside fermenting vessels containing sterile distilled water at room temperature. Microbial analyses were performed daily by pipetting 1 mL of the sample stock solution and then serially diluted up to the appropriate dilutions. One milliliter from the dilutions, 10-2 and 10-4 was pipetted, gently dispensed into Petri dishes, and then pour plated using Nutrient Agar (NA), Salmonella Shigella agar (SSA), Mannitol salt agar (MSA), MacConkey agar (MA) and Eosin Methylene Blue agar (EMB). The Petri plates were incubated at 37oC for 24 hours to determine bacterial growth and morphological details. Pure cultures were obtained by repeated streaking of the bacterial inoculum on the fresh bacteriological media and stored on slants inside the refrigerator for further use. Furthermore, the pure culture was subjected to various morphological and biochemical characterization tests, such as catalase, oxidase, and indole, hydrogen sulfide, coagulase, and sugar fermentation, to determine the identity of the bacteria isolates (Adeleke et al., 2017). Physical properties The physical properties of distilled water were used in the soaking of the cassava were carried out in the laboratory. The physical parameters determined were pH, temperature, total dissolved solids, total suspended solids, and total solid according to the modified method of and Afuye & Mogaji (2015) and Adegbehingbe et al. (2019). Statistical analysis Data were reported as average of triplicate determinations and analysed using Analysis of Variance (ANOVA) on SPSS. Duncan's multiple tests at 5% level significance were used to determine the significant differences among the samples. Results and Discussion Total bacteria count The population of bacteria isolated from the cassava steeping water indicated a reduction from day one to the last day of the fermentation process. The bacterial count ranged from 2.2 × 103 CFU/mL to 6.6 x 103 CFU/mL. Day one had the highest bacterial count of 6.6 × 103 CFU/mL while the least bacterial count of 2.2 × 103 CFU/mL was recorded on day four (Table 1). Cassava is usually processed into various products through the fermentation process to increase the shelf life, easy packaging, and transportation for economic purposes (Aro, 2008). Processing of cassava by submerged state fermentation techniques by traditional methods and beyond specified fermentation time usually produce mash, which contains a foul odor resulting from the uncontrolled fermentation and storage techniques (Oyewole & Odunfa, 1988). During the process of retting of cassava, a decrease in the total viable count of aerobic mesophiles was observed and this can be linked to the increase in acidity of the fermentation medium (Adegbehingbe et al., 2019). Also, an increase in acidity of the medium can cause a decrease in the growth of pathogenic-like microorganisms (Ojo et al., 2019). International Journal of Applied Biology, 5(2), 2021 193 Nine bacterial isolates were identified based on colonial and cellular morphological characterization. The identifiable bacterial isolates include, Klebsiella pneumonia, Lactobacillus species, Bacillus subtilis, Proteus mirabilis, Pseudomonas spp, Pseudomonas aeruginosa and Staphylococcus aureus (Table 2). Table 1: Mean population of bacteria from the steeping water Days Mean CFU/mL x 103 1 66 6.6 2 50 5.0 3 30 3.0 4 22 2.2 Key: CFU - Colony-forming unit International Journal of Applied Biology, 5(2), 2021 194 Table 2. Characterization and identification of isolates Is o la te c o d e Colonial morphology Staining reactions Biochemical tests Sugar fermentation C it r a te u ti li z a ti o n P r o b a b le i so la te s S h a p e E le v a ti o n C e ll s h a p e C e ll a rr a n g e m e n t C o lo u r G ra m s ta in in g S p o re s ta in in g C a ta la se t e st In d o le p ro d u c ti o n C o a g u la se t e st H 2 s p ro d u c ti o n S u c ro se L a c to se G lu c o se IA CR RD R S CR +ve -ve -ve - ve -ve -ve -veG -veG +veG +ve Lactobacillus fermentum IB CR RD SR C WH - ve -ve + ve - ve - ve - ve +ve +ve + ve -ve Klebsiella pneumoniae IC CR RD R S CR - ve -ve + ve - ve - ve - ve - veG - veG -veG +ve Pseudomonas sp. ID CR F R C WH - ve -ve + ve - ve - ve - ve - ve - ve + ve +ve Proteus mirabilis IE IR F R C CR + ve +ve + ve - ve - ve + ve +veG +veG +veG +ve Bacillus subtilis IF CR RD SR C WH - ve - ve + ve - ve - ve -ve + ve + ve +ve -ve Klebsiella pneumoniae IG CR CV CC C LY + ve -ve + ve - ve + ve - ve + ve + ve + ve -ve Staphylococcus aureus IH CR RD SR S CR + ve -ve - ve - ve - ve - ve - veG - veG +veG -ve Lactobacillus sp. II CR RD R S GR -ve -ve + ve -ve - ve - ve - ve - ve - ve - ve Pseudomonas aeruginosa International Journal of Applied Biology, 5(2), 2021 195 Ezedinma et al. (2006) reported the amylolytic activity of Bacillus subtilis, which produces an enzyme necessary for the breakdown of starch to sugar needed for the growth of other fermenting microorganisms, including lactic acid bacteria. In the report of Freire et al. (2015), the authors reported some members of Enterobacteriaceae during the fermentation of cassava, which is similar to the results obtained in this study. The presence of Proteus spp and Klebsiella spp could underline microbial activities in the rotting of cassava roots which was consistent with the findings of Achi & Akomas (2006). Bacillus spp and Lactobacillus spp were detected at the later stage of the retting process due to their persistence and ability to grow in an acidic medium. Obilie et al. (2003) and Essers et al. (1995) reported the involvement of Bacillus species in the textural modification of cassava roots, which cause a softening of the cassava tissues, which is evident in this study. The presence of Staphylococcus aureus, a normal skin microflora in the cassava steeping water may originate from human contact, contamination, poor hygienic conditions, and post-contamination (Olopade et al., 2014). Identification of S. aureus from diverse fermenting substrates has been reported in many studies (Nout, 1994; Fowoyo & Ogunbanwo, 2017; Anyogu et al., 2021). However, the results from this study agree with the findings of Oyetayo (2006), who reported some pathogenic bacteria from fermenting cassava steeping water. Pseudomonas spp and Proteus spp have also been reported in the fermentation of cassava, due to their enzyme activities in the reduction of cyanide (Izah et al., 2018). The Lactobacillus fermentum isolated from this study agreed to the findings of Adeleke et al. (2017) who reported L. fermentum from the fermented cassava peels. The presence of these bacteria can contribute significantly to the fermentation process of cassava wastewater for desirable output. Table 3 shows the physicochemical properties of steeping water. A pH value of 3.67 was recorded at day one while at day four a pH value of 3.32 was recorded, respectively. Table 3. Physicochemical properties of the steeping water Parameters Days One Four pH 3.67 3.32 Temperature (oC) 26.2 26.2 International Journal of Applied Biology, 5(2), 2021 196 The temperature was constant. Figure 1 shows the chemical properties of the steeping water. The high total solids value of 45.2 mg/L was obtained at day four while the lowest total dissolved solids of 15.0 mg/L were obtained at day one of the fermentation processes. In this study, the pH of the retting liquor (steep water) was acidic after 24 hours of the fermentation process. This explains the high count of lactic acid bacteria in the later stage of the fermentation, probably due to increased acidity of the medium, which favored the growth of the microorganisms (Adegbehingbe et al., 2017b). The decrease in pH recorded throughout the fermentation period may be associated with the fermentation by Lactobacillus fermentum. An increase in the acidity and decrease in pH of tuber fermenting medium has been documented in several studies (Oboh, 2006; Olufemi & Murtala, 2015; Adegbehingbe et al., 2017a). The results of the effect of temperature on cassava retting showed that the optimum temperature was 26˚C. This is an indication that temperature had a very strong effect on the retting time. Moreover, the container should be covered immediately after soaking the cassava to prevent contamination and allow the chemical reaction to take place. Furthermore, this study showed the chemical parameters of fermented cassava steeping water. The values of total solids were higher in the fermented samples during the fermentation process. A total suspended solids value of 9.28 mg/L in cassava effluents has been reported by (Lawal et al., 2018). Proper measures should be taken to treat cassava wastewater for irrigation purposes instead to be indiscriminatingly discharged into the environment. Figure 1. Chemical properties of steeping water. Key: TSS - total suspended solids, TDS - total dissolved solids, TS - Total solids 0 5 10 15 20 25 30 35 40 45 50 TSS TDS TS C o m p o si ti o n ( m g /L ) Chemical parameters da… da… International Journal of Applied Biology, 5(2), 2021 197 Conclusions In conclusion, this study has revealed microorganisms involved in the fermentation of cassava steeping water. Ensuring quality control during fufu production by soaking cassava tubers is essential to reduce contaminants from the fermenting substrates. The presence of Lactobacillus species in the fermenting medium can be beneficial in softening the cassava tubers and inhibiting the growth of other pathogens which might cause foul odor during fufu production. The physical properties of the steeping water reveal the survival rate of the isolated microorganism fermentation medium. 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