Sample collection
Mucilage and pulp juice (one barrel, 200 L each) were collected from Bonga, Teppi, Goma II and Limu Kosa wet coffee processing factories (Ethiopia). The samples were kept in ice box and transported to the Addis Ababa Institute of Technology, School of Chemical and Bio-Engineering and kept in the Environmental Engineering and Bio-Innovative laboratories.
Chemicals
Ethyl alcohol (95%, Sigma-Aldrich, USA), sulfuric acid (98%, sd fine-chem. limited, Mumbai, India), sodium hydroxide (Abron Chemicals, India), hydrochloric acid (Abron Chemicals, India), methylene blue, Fehling solution (A and B), Pichia anomala (M4) (1% glucose, 0.5% peptone, 3% malt extract, and 2% agar–agar) and Whatman No. 1 filter papers were used in this research work.
Apparatus and equipments
Porcelain crucibles (50 mL), Erlenmeyer flasks (250 mL), round bottom flasks (250 mL), pH meter (3505-JENWAY, UK), balance (HCB1002-ADAM, UK), stove (seven star, Germany), oven (202-OA, Germany), centrifuge (Thermo Fisher Scientific, USA), refractometry (Bellingham and Stanley, UK), gas chromatography (DANI, model GC-1000, Italy) and furnace (SX-2.5-12, box type resistance furnace, China) were used in this research work.
Optimization of parameters
The procedure used for studying the optimization of all the parameters (including sterilization, hydrolysis time, temperature, acid concentration, fermentation time and temperature, distillation, and ethanol determination) is briefly described below. The effect of a particular parameter was studied by keeping all the parameters constant except the one under study.
To avoid microbial contamination, coffee waste was sterilized at 120 °C for 15 min, cooled to room temperature and kept in the fridge at 4 °C before use. The P. anomala (M4) was used for ethanol production optimization study. The yeast strain was maintained on agar slants (1% glucose, 0.5% peptone, 3% malt extract, and 2% agar–agar) was used to inoculate pre-fermenting media which contained 2% sugar and 3 g/L yeast extract and incubated for 12 h before use in the optimization studies (Viegas and Correia 1985). An aliquot of 100 mL of coffee waste were distributed into 250 mL conical flasks and pH was adjusted by using HCl and NaOH to 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 and 6.5. The flasks were sterilized at 120 °C for 15 min and upon cooling, 5 mL of the pre-fermentative yeast was inoculated to the sterilized media and there after incubated (without shaking) at an ambient temperature for 2 days. After every 12 h, 1.5 mL of the fermenting media was withdrawn to tubes. The tubes were centrifuged at 13,000 rpm for 4 min to get supernatant for ethanol analysis (Periyasamy and Venkatachalams 2009).
A series of experiments was performed for different hydrolysis times (30, 60, 90, and 120 min). In each experiment the hydrolysis time was kept constant as the fermentations times were varied from 12 to 48 h and the results were recorded. Suitable temperature for maximum production of ethanol was studied. Coffee waste was hydrolyzed with different concentrations of sulfuric acid (0.2, 0.4, 0.6, 0.8, 1 M and distilled water) in 500 mL Erlenmeyer flask and separately heated at 85, 100 and 115 °C for 1 h using oil thermostat. The hydrolysate obtained from hydrolysis of the coffee waste was collected and divided into four samples and used for the subsequent fermentation experiments. The fermentation was done at 12, 24, 36 and 48 h respectively withdrawing samples after 12 h, respectively (Dawson and Boopaty 2008). The ethanol produced was recovered and purified through distillation.
The ethanol produced from the fermentation process contains a significant quantity of water which must be removed. This was achieved by using the fractional distillation process by boiling the water and ethanol mixture. Since ethanol has a lower boiling point (78.3 °C) compared to that of water (100 °C), the ethanol turns into the vapor state before the water and it can be condensed and separated. The ethanol concentration in the distillate was determined gas chromatography (GC). Samples analyzed for ethanol production were taken at the end of fermentation after 12, 24, 36 and 48 h of incubation. Ethanol determination was done by using GC in which the sample was injected manually. Flame ionization detector was set at 280 °C. Separation was effected in a 30 m, 0.25 mm and 1 μm column (CP-SIL 8 CB) with the temperature maintained at 45–55 °C at a rate 2 °C/min for 5 min then at 10 °C/min to 200 °C. Column flow was employed at 1.5 mL/min with nitrogen as a carrier gas and hydrogen and compressed air as a combustion gases.
Optimized procedure for ethanol production
The coffee waste was sterilized at 120 °C for 15 min, cooled to room temperature and kept in the fridge at 4 °C before use. The P. anomala (M4) was used for ethanol production. The yeast strain was maintained on agar slants (1% glucose, 0.5% peptone, 3% malt extract, and 2% agar–agar) was used to inoculate pre-fermenting media which contained 2% sugar and 3 g/L yeast extract and incubated for 12 h before use. An aliquot of 100 mL of coffee waste was transferred into a 250 mL conical flask and pH was adjusted to 4.5 using HCl and NaOH. The flask was sterilized at 120 °C for 15 min and upon cooling, 5 mL of the pre-fermentative yeast was inoculated to the sterilized media and there after incubated (without shaking) at an ambient temperature for 2 days. Coffee waste was hydrolyzed with 0.4 M H2SO4 in 500 mL Erlenmeyer flask at 100 °C for 1 h using oil thermostat. The hydrolysate was fermented for 24 h. The ethanol produced was recovered and purified through distillation. The ethanol concentration in the distillate was determined GC.
Characterization of the sludge
The presence of nitrogen and trace elements in the fermentation media was determined according to the procedure described by Allen and Roche (1989). The minerals of the coffee waste were obtained by dry ashing method. About 5 g of coffee waste sludge was placed in a porcelain crucible and dried in an oven at 105 °C until a constant weight was obtained. The dried sludge was then placed in a muffle furnace and ignited at 550 °C for 2 h to get ash. The obtained ash was used for analysis of the minerals following the procedure described by Allen and Roche (1989).
Statistical analysis
All the data were analyzed using SPSS 20 software. The results are presented as mean values of triplicate analysis.