Microorganism and culture maintenance
Aspergillus niger strain was obtained from Department of Microbiology University of Nigeria, Nsukka. The cultures were maintained on potato dextrose agar (PDA) slants at 40 C and sub-cultured at intervals.
Inoculum preparation
The inoculum was prepared according the method of Ezea et al. (2015), the spores of Aspergillus niger was harvested from potato dextrose Agar slant using a sterile solution of 0.01% Tween 80. The inoculation wire loop was used to dislodge the spores and to ensure proper mixing of the culture with the Tween 80. A 5 ml of 5 × 107 spores/ml was counted using haemocytometer.
Pretreatments of wild cocoyam flour
Wild cocoyam (Caladium bicolor) was obtained from a bush and farm land at Nsukka in Enugu State of Nigeria. The wild cocoyam was peeled, sundried, ground and sieved into fine powder (flour) using Muslim cloth. The flour was thermally pretreated to gelatinize the starch by suspending in 100 ml of the basal nutrient medium. The sample was pretreated with an autoclave at 121 °C for 15 min.
Submerged fermentation of wild cocoyam flour
Submerged fermentation was carried out in a modified method Ezea et al. (2015), in 250 ml foam-plugged Erlenmeyer flask. A 10 g wild cocoyam tuber flour was weighed using DENVER digital weighing balance, Model: MXX-123 USA and suspended in 100 ml nutrient medium containing NH4NO3, 2 g/l; KH2PO4, 0.2 g/l; ZnSO4·7H2O, 0.01 g/l; Fe(SO4)2·7H2O, 0.01 g/l and MgSo4·7H2O, 0.5 g/l before pretreatment. The sample was inoculated with 5 ml of Aspergillus niger spores and incubation at 30 °C for 144 h under rotary incubator shaker (model: VWR International by B. Bran Scientific & Instrument Company England) at 225 rotations per minutes.
Effect of wild cocoyam flour concentration on citric acid production
Effect of wild cocoyam flour concentration on citric acid production was carried by suspending different percentage of wild cocoyam flour ranging from 5 to 25% in 100 ml nutrient medium into 250 ml foam-plugged Erlenmeyer flask and incubated under rotary incubator shaker (model: VWR International by B. Bran Scientific & Instrument Company England) at 225 rotations per minutes (rpm) for 144 h.
Effect of wild cocoyam pretreatment time on citric acid production
The wild cocoyam flour was thermally pretreated to gelatinize the starch by suspending 10 g in 100 ml of the basal nutrient medium. The suspension was pretreated in an autoclave at 1210C for 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min. Thereafter the medium was allowed to cool before inoculation and incubated for 144 h in a rotary shaker at 225 rpm (model: VWR International by B. Bran Scientific & Instrument Company England).
Effect of inoculums size on citric acid production from wild cocoyam
The effect of inoculums size on citric acid production from wild cocoyam was carried out by inoculating different inoculums concentrations from 5 to 25% into 250 ml foam-plugged Erlenmeyer flask and incubated under rotary incubator shaker (model: VWR International by B. Bran Scientific & Instrument Company England) at 225 rotations per minutes (rpm) for 144 h.
Effect of initial pH on citric acid production from wild cocoyam
The effect of initial pH on citric acid production from wild cocoyam was carried out by adjusting the pH to 3.5, 4.5, 5.5, 6.5, 7.5 and 8.5 using 0.1 M HCl and 0.1 M NaOH before Pretreatment.
Effect of incubation temperature on citric acid production from wild cocoyam
The effect of incubation temperature on citric acid production form wild cocoyam by Aspergillus niger was carried under rotary incubator shaker (model: VWR International by B. Bran Scientific & Instrument Company England) at 225 rotations per minutes, under the following temperature 200 C, 250 C, 300 C, 350 C and 400 C for 144 h.
Analytical techniques
Citric acid was estimated using pyridine acetic anhydride method as reported by Marrier and Boulet (1958). A 1 ml of diluted culture filtrate along with 1.30 ml of pyridine was added in the test tube and swirled briskly. Then 5.70 ml of acetic anhydride was added in the test tube. The test tube was placed in a water bath at 32 °C for 30 min. The absorbance was measured on a Spectrophotometer 722S B. Bran Scientific and Instrument Company, England at 420 nm against the blank and the citric acids of the samples were estimated with reference standard. The pH of the sample was determined using digital pH meter (DENVER Instrument, Model: UB-10058245 ultraBASIC USA).
Statistical analysis
Data obtained were subjected to one-way analysis of variance (ANOVA) and the means were separated using the least significant difference.