A new local strain of
Yeast selection for fuel ethanol production over the past two decades, most bioethanol related researches in developing tropical countries have focused primarily on the isolation of local
Sugarcane molasses procured by Egyptian Sugar and Integrated Industries Company is used as carbon source for ethanol production in the Distillation Factories, El-Hawamdia, Giza, Egypt.
Sterilized 500 ml capacity conical flasks each contained 200 ml of medium containing (g/L) malt extract, 3, yeast extract, 3, peptone, 5 and sucrose, 30 was steam sterilized at 121°C for 15 minutes. Cooled to room temperature, then inoculated with a loop of yeast strain
Yeast cultures were prepared in separate seed fermenters of 300 L capacity. Molasses diluted to 4-5% (w/v) sugars content was supplemented with Urea (0.1%, and 0.2% DAP w/v, pH of the medium was adjusted to 4.6 (Pre optimized) using diluted NaOH and diluted H2SO4. The medium was steam sterilized. After cooling to 32°C ±2 two flat round bottom flasks from above inoculum strain of yeast were added and the seed fermenters were aerated to facilitate the growth of yeasts. At the end of first stage of 16 hours of continuous circulation, sample withdrawn from the sample valve was subjected to analyses to get
Batch culture system was employed for optimization of fermentation parameters for
During fermentation stage, all the parameters to be optimized were varied (Urea, diammonium phosphate (DAP), Orthophosphoric acid (OPA), magnesium sulfate and inoculum size). During optimization, temperature and pH were not adjusted.
Cell count optimization was performed by using yeast cell counts
Varying concentrations of urea, 10, 15, 20, 25, and 30 kg/65 m3 were added to the fermentation media in 65 m3 working volume fermenter inoculated with 10% v/v yeast inoculum cell counts
Varying concentrations of ADP (5, 10, 15, and 20 kg/65 m3) were added to the fermentation medium under above optimized urea level.
Varying concentrations of orthophosphoric acid (OPA) (5, 10, 15, and 20 kg/65 m3) were added to the fermentation media in 65 m3 working volume fermenter under optimized urea level.
Combinations of urea, ADP, and OPA, that is, 20, 10, and 5 kg/65 m3, respectively, were added to the fermentation medium.
Varying concentrations of magnesium sulfate (5, 10, 15, and 20 kg/65 m3) were added to the fermentation medium in 65 m3 working volume fermenter under the applied concentrations of urea, ADP, and OPA.
Varying sizes of inoculum (5, 10, 15, 20, and 25 v/v%) were used to inoculate the respective fermentation fermenters under optimized parameters of urea, DAP, OPA, and magnesium sulfate to investigate the effect of inoculum size on ethanol production.
Varying concentrations of molasses under optimized parameters of Urea, DAP, OPA and magnesium sulfate and inoculum size to investigate the effect of molasses concentrations on ethanol production on time.
The sugar concentration was determined by rapid method. The 5 mL of fermented sample was taken and dissolved in 100 mL of distilled water and mixed with 5 mL of conc. HCL acid and is heated at 70°C for a period of 10 min. The obtained sample was neutralized by adding NaOH and it was prepared to 1000 mL and taken into burette solution. The 5 mL of Fehling A and 5 mL of Fehling B were taken and mixed with 10 to 15 mL of distilled water in a conical flask and methylene blue indicator was added. The conical flask solution was titrated with burette solution in boiling conditions until disappearance of blue color. The sugar concentration was calculated by using the formula given below: Sugar Concentration (gm/L) = [(Dilution factor × Fehling factor)/Titrate value] × 100.
Ethanol content of the fermented samples was measured with ebulliometer approved in distillation factories.
Fermentation efficiency was calculated as the ethanol yield divided by the theoretical yield multiply by 100.
Cell count was determined using microscope with the help of haemocytometer. Cell viability was checked by using methylene blue indicator. The dead cells were stained with blue indicator while viable cells remained uncolored.
Crude protein was measured by micro-Kejldahel method [
Varying concentrations of urea were added as nitrogen supplement for yeast growth (Table
Effect of varying concentrations of urea (as nitrogen source) on ethanol production by
Urea kg/65 m3 | Initial pH | EOH% v/v | RS% | FE% | Final pH | Viable yeast cells ×108 | Yeast yield (g/L) |
---|---|---|---|---|---|---|---|
10 | 4.6 | 7.8 | 3.11 | 70.1 | 4.9 | 2.55 | 10.4 |
15 | 4.6 | 8.1 | 2.85 | 72.8 | 5.0 | 2.75 | 10.8 |
20 | 4.7 | 8.3 | 2.60 | 74.6 | 5.1 | 2.90 | 11.4 |
25 | 4.7 | 8.4 | 2.46 | 75.5 | 5.2 | 2.90 | 11.4 |
30 | 4.8 | 8.2 | 2.66 | 73.7 | 5.4 | 2.85 | 11.2 |
Varying concentrations of DAP were used as phosphorus and supplementary nitrogen source to promote yeast growth and increase ethanol production (Table
Effect of varying concentrations of DAP on ethanol production by
DAP kg/65 m3 | Initial pH | EOH% v/v | RS% | FE% | Final pH | Viable yeast cells ×108 | Yeast yield (g/L) |
---|---|---|---|---|---|---|---|
5 | 4.7 | 8.5 | 2.16 | 76.4 | 5.2 | 2.95 | 11.1 |
10 | 4.7 | 8.7 | 1.96 | 78.2 | 5.2 | 3.00 | 11.4 |
15 | 4.8 | 8.9 | 1.66 | 80.1 | 5.3 | 3.10 | 11.8 |
20 | 4.8 | 8.9 | 1.64 | 80.1 | 5.4 | 3.10 | 11.6 |
Phosphate limitation has been shown to affect cell growth and biomass formation as well as directly affecting fermentation rate [
Effect of varying concentrations of OPA on ethanol production by
OPA kg/65 m3 | Initial pH | EOH% v/v | RS% | FE% | Final pH | Viable yeast cells ×108 | Yeast yield (g/L) |
---|---|---|---|---|---|---|---|
5 | 4.7 | 8.6 | 1.72 | 77.3 | 4.9 | 3.00 | 11.2 |
10 | 4.7 | 8.8 | 1.56 | 79.1 | 4.8 | 3.20 | 11.4 |
15 | 4.8 | 9.1 | 1.32 | 81.8 | 4.8 | 3.40 | 11.7 |
20 | 4.8 | 9.0 | 1.46 | 81.7 | 4.6 | 3.20 | 11.6 |
Date presented in Table
Effect of combination of urea, of DAP, and OPA on ethanol production by
kg/65 m3 | Initial pH | EOH% v/v | RS% | FE% | Final pH | Viable yeast cells ×108 | Yeast yields (g/L) |
---|---|---|---|---|---|---|---|
Urea 20* | 4.7 | 8.3 | 2.6 | 74.6 | 51 | 2.90 | 11.4 |
*+DAP10** | 4.7 | 8.7 | 1.74 | 78.2 | 5.2 | 3.15 | 11.8 |
**+OPA 5 | 4.8 | 9.3 | 1.32 | 83.6 | 5.1 | 3.45 | 12.1 |
Varying concentrations of magnesium sulfate were supplement under the above optimized levels of urea, DAP and OPA for yeast growth (Table
Effect of varying concentrations of magnesium sulfate on ethanol production by
Magnesium sulfate kg/65 m3 | Initial pH | EOH% v/v |
RS% | FE% | Final pH | Viable yeast cell ×108 | Yeast yield (g/L) |
---|---|---|---|---|---|---|---|
5 | 4.7 | 9.5 | 1.40 | 85.4 | 4.8 | 3.60 | 12.4 |
10 | 4.7 | 9.6 | 1.32 | 86.3 | 4.8 | 3.60 | 12.4 |
15 | 4.8 | 9.6 | 1.36 | 87.1 | 5.0 | 3.60 | 12.2 |
20 | 4.8 | 9.5 | 1.40 | 85.4 | 5.0 | 3.55 | 12.1 |
Ethanol yield and production of coproducts have a major relationship during ethanol fermentation. Extensive studies have been carried out to investigate the effect of yeast inoculation rate to help the yeast cells overcome the bacterial cells on the basis of size and number. Effect of varying inoculum sizes on ethanol yield was studied under optimized parameters, urea 20 kg, DAP 10 kg, OPA 5 kg, and magnesium sulfate 10 kg/65 m3 working volume fermenter. Maximum ethanol content was found at an inoculation rate of 20% v/v. Results have shown that at 20% inoculation rate, ethanol content was 9.8% (v/v) (Table
Effect of varying inoculum size on ethanol production by
Inoculum size% (v/v) | Initial pH |
EOH% v/v |
RS% | FE% | Final pH | Viable yeast cell ×108 | Yeast yield (g/L) |
---|---|---|---|---|---|---|---|
5 | 4.7 | 9.4 | 1.71 | 84.5 | 4.8 | 3.15 | 12.2 |
10 | 4.7 | 9.6 | 1.36 | 86.3 | 4.8 | 3.60 | 12.4 |
15 | 4.8 | 9.7 | 1.28 | 87.2 | 5.0 | 3.75 | 13.1 |
20 | 4.8 | 9.8 | 1.22 | 88.1 | 5.2 | 3.80 | 13.2 |
25 | 4.8 | 9.6 | 1.64 | 86.3 | 5.2 | 395 | 13.6 |
Varying doses of sugar cane molasses contained varying sugars concentration were applied to study the effect of sugar level in fermentation medium on the ethanol yield on time under the above optimized levels of urea, DAP. OPA, magnesium sulfate and inoulum size (Table
Effect of varying sugarcane molasses concentrations on time on ethanol production by
Sugar% (w/v) | Fermentation time (hrs) | FE% | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
16 | 18 | 20 | 22 | 24 | |||||||
EOH% (v/v) | RS% | EOH% (v/v) | RS% | EOH% (v/v) | RS% | EOH% (v/v) | RS% | EOH% (v/v) | RS% |
||
16 | 8.2 | 1.34 | 8.6 | 1.32 | 8.6 | 1.22 | 8.5 | 1.22 | 8.5 | 1.22 | 86.9 |
17 | 8.7 | 1.46 | 8.9 | 1.28 | 9.1 | 1.28 | 9.3 | 1.28 | 8.8 | 1.28 | 86.5 |
18 | 9.4 | 1.68 | 9.9 | 1.22 | 9.8 | 1.22 | 9.8 | 1.22 | 9.8 | 1.22 | 88.1 |
19 | 9.2 | 1.86 | 9.4 | 1.56 | 9.7 | 1.96 | 9.8 | 1.74 | 9.8 | 1.74 | 83.4 |
20 | 8.9 | 3.45 | 9.4 | 2.84 | 9.7 | 2.96 | 9.8 | 2.86 | 9.8 | 2.86 | 79.2 |
21 | 8.2 | 4.65 | 8.4 | 3.55 | 9.4 | 3.20 | 9.6 | 2.20 | 9.6 | 2.20 | 73.9 |
22 | 7.8 | 6.24 | 8.2 | 5.42 | 8.6 | 3.22 | 9.2 | 3.24 | 9.4 | 3.24 | 69.1 |
The chemical composition of the yeast on basis of dry weight was protein 34.6% and ash 8.2%. Our study was applied in distillery factory distilled about 1600 m3 daily producing about 20 tons of fodder yeast. Yeasts are a rich source of protein and B-complex vitamins. They have been used successfully as a complementary protein source in fish diet [
The results of our study showed that under optimum conditions. However, optimization of process parameters improved ethanol production by the local yeast strains of
The authors deeply thanks the engineering H. K. Hassan Chairman & Managing Director of Egyptian Sugar & Integrated Industry Company. Also all thanks to Chairman and members of Hawamdia Distillation Factories.