Increase of alcohol production by optimizing aerobic

bioreactor culture medium using produced vinasse from

alcohol distillation unit waste

Zahra Mahdavi1, Valilollah Babaeipour2*, Hadi Moshiri Aydenlou3

1 Master Student of Chemical Engineering-Biotechnology, Faculty of Chemistry and

Chemical Engineering, Malek Ashtar University of Technology, Tehran, Iran2* Associate Professor of Chemical Engineering-Biotechnology, Faculty of

Chemistry and Chemical Engineering, Malek Ashtar University of Technology,

Tehran, Iran, vbabaeipour@mut.ac.ir3PhD in Chemical Engineering from Amirkabir University of Technology, Research

and Development Director of Bidestan Alcohol and Food Industries Company ،

Qazvin

biotech12-07250463

Introduction

• Ethanol is a clear, colorless and volatile liquid, with wide applications in various fields of home, industrial and medical.

• Fermented ethanol is made by Saccharomyces cerevisiae using sugar raw materials. This process consists of two parts, both aerobic

and anaerobic.

• Aerobic process The goal of yeast reproduction is the anaerobic process of alcohol production. The performance of anaerobic

bioreactors is strongly influenced by aerobic reactors.

• In order to achieve a very high and competitive yield of bioethanol production by Saccharomyces cerevisiae, several bottlenecks in

the process of alcoholic fermentation must be overcome. One of these bottlenecks is the modification and optimization of yeast

proliferation conditions to achieve high cell count.

• Variables such as temperature, sugar concentration, pH, stirring rate and inoculation rate, aeration intensity and nutrient source affect

the production time of biomass and consequently on the alcoholic degree of fermentation.

• Yeast for growth and reproduction needs to compounds such as carbohydrates, protein, fat, phosphorus, sulfur, sodium, calcium,

magnesium and other minerals and vitamin sources.

• Most alcohol factories use molasses and glucose syrup or fructose syrup to supply carbon sources, to supply nitrogen sources from

the corn steep liquor (CSL) and ferric ammonium phosphate, and to supply phosphate from diamonium phosphate. The quality of the

variable CSL affects the efficiency of alcohol production.

• The effluent of the distillation unit of ethanol production plants is a rich source of nitrogen that can be considered as an alternative to

CSL, fertilizers used in culture medium.1

Materials and Methods

2

• Materials used: Dry bakery yeast, purchased from Iran Maye Company with the brand name of Gol Maye and raw molasses

used from beet molasses from Hamedan Sugar Factory (with 70 brix and 50% sugar and pH equal to 7), glucose syrup and

fructose ( With 50 brix and 50% sugar and pH equal to 3.5) used from glucose and pure fructose, respectively, urea fertilizer

with 46% w / w nitrogen, diamonium phosphate fertilizer (with 41% usable phosphorus and 18% total nitrogen ), Silicone

based antifoam, 98% sulfuric acid (Merck), 98% caustic soda (Avin Company)

• Measurements include: yeast growth, measuring sugar and alcohol levels

• method: The design consisting of two different levels was examined in 31 experiments based on the response surface statistical

method with a central composite designs (CCD) in Table 1 on the amount of live yeast. All experiments in a system consisting

of 10 parallel bioreactors with a working volume of 1 liter with aeration performed in this study are as shown in Figure 1.

parameters Level 1 Level 2

Molasses to glucose ratio 0/8 2

Vinasse (g/l) 40 80

di ammonium phosphate(g/l) 0/1 0.4

urea(g/l) 0/1 0.4

Table 1: Selection of four factors in two levels to design the experiment using the central square response surface method

Figure 1: Image of the culture medium fermenter

Results and Discussion

3

• The design of the RSM test is as shown in the table 2.

• In this design, after adding the inoculum and aerating the

culture medium, the amount of cells is counted at the end.

RunOrder G/M Vinasse DAP Urea LIVE cell× 𝟏𝟎𝟔

1 0.875 0.2 0.1 0.3 7.0

2 0.875 0.2 0.3 0.1 5.5

3 1.250 0.4 0.2 0.2 11.0

4 1.250 0.4 0.2 0.2 11.4

5 1.250 0.4 0.2 0.2 11.0

6 1.625 0.2 0.1 0.3 8.0

7 1.250 0.4 0.2 0.2 11.0

8 1.250 0.4 0.4 0.2 11.0

9 1.625 0.6 0.1 0.3 34.0

10 0.875 0.6 0.3 0.1 18.0

11 1.625 0.2 0.3 0.1 7.0

12 1.250 0.0 0.2 0.2 0.0

13 0.875 0.2 0.1 0.1 9.0

14 0.875 0.6 0.1 0.3 26.0

15 0.875 0.6 0.1 0.1 28.0

16 1.625 0.6 0.3 0.3 30.0

17 0.875 0.6 0.3 0.3 30

18 1.250 0.4 0.0 0.2 22.5

19 1.625 0.2 0.3 0.3 8.2

20 1.625 0.2 0.1 0.1 6.7

21 1.625 0.6 0.1 0.1 27.0

22 1.250 0.4 0.2 0.2 11.0

23 1.250 0.8 0.2 0.2 27.0

24 1.250 0.4 0.2 0.4 17.0

25 1.250 0.4 0.2 0.2 11.0

26 0.875 0.2 0.3 0.3 8.0

27 0.500 0.4 0.2 0.2 20.5

28 1.625 0.6 0.3 0.1 13.0

29 1.250 0.4 0.2 0.2 11.0

30 1.250 0.4 0.2 0.0 15.0

31 2.000 0.4 0.2 0.2 13.0

Table 2: Conditions and results of experiments designed based on the statistical method of

response surface with central composition algorithm

Results and Discussion

4

• R-sq This equation is 95.01, so we can say that the equation is valid.

• According to the statistical size of p-value, a significant probability for the obtained vinasse index (0.000), vinasse has

the greatest effect on yeast proliferation. Urea and diamonium phosphate are also somewhat effective. On the other hand,

parameters that are greater than (0.05) their significant effect is not very low or significant and can be ignored.

According to the effect of the parameter of glucose to molasses ratio which is 0.388, it can be interpreted that in both

cases, whether using molasses or using glucose syrup, the same result is obtained.

• Figure (a) shows that the ratio of glucose to molasses is not very effective on cell yeast cell count. It can be concluded

that these results can be achieved separately with each of these two substrates.

• Figure (b) shows the effect of vinasse factor on live yeast cell count. As can be seen, the Venus factor is very effective

and its effect is positive

• Figure c The effect of diamonium phosphate is decreasing and increasing its amount reduces the amount of live yeast.

• (Figure d) shows a positive effect of low urea on reproduction.

• In the optimal state of the factors, the ratio of glucose to molasses 2, vinasse and urea reached 80 and 0.4 (g / l),

respectively, the cell content reached 70-80 106 / ml. Using this medium in anaerobic and aerobic conditions with Brix

22 10% alcohol was produced in 72 hours.

Figure or Table

5

0

5

10

15

20

25

30

0/5 1/5Mea

n o

f L

IVE

cel

l*10

6

G/M Ratio

(a)

0

5

10

15

20

25

30

0 2 4 6 8Mea

n o

f L

IVE

cel

l*1

06

Vinasse*10 (gr/lit)

(b)

0

5

10

15

20

25

30

0 0/1 0/2 0/3 0/4

Mea

n o

f L

IVE

cel

l*1

06

DAP (gr/lit)

(c)

0

5

10

15

20

25

30

0 0/2 0/4Mea

n o

f L

IVE

cel

l*1

06

Urea (gr/lit)

(d)

Figure 2: (a) effect of glucose to molasses ratio (G / M), (b) effect of vinasse, (c) effect of diamonium phosphate, (d) effect of urea fertilizer on the

number of live yeast cells

The amount of living

cells

(Number in ml)

Degree of

alcohol

(V / V)

UREA

(gr/lit)

DAP

(gr/lit)

VINASSE

(gr/lit)G/M Ratio

75× 𝟏𝟎𝟔1000802

Table 5: Amount of yeast in aerobic state and alcohol production in anaerobic bioreactor

under optimal conditions in 10 liter bioreactor

Conclusion

6

• Yeast in the process of alcohol production and reproduction needs a source of carbon and nitrogen Carbon sources are

supplied from molasses and glucose and nitrogen sources are supplied from urea fertilizer, diamonium phosphate and

CSL liquid and required phosphorus is also supplied from the source of diamonium phosphate fertilizer.

• The variable quality of these components or their lack of timely supply causes many problems in the production process

and reduces its efficiency. Urea fertilizer and phosphate fertilizer are two materials required by the agricultural industry

and their supply is not easily possible.

• The quality of CSL supplied by corn processing plants is highly variable and non-uniform.

• Low efficiency, large amounts of effluent are produced with high BOD and COD.

• Use a mixture of vinasse and molasses and glucose syrup (as rich sources of nitrogen and carbon) in the culture

medium:

1) eliminate the need to use urea, phosphate and CSL fertilizers to a large extent.

2) Vinasse is an by-product of the alcohol production process, its use as a source of nitrogen.

3) Eliminated to a large extent the environmental problems of the alcohol factory.

4) Solve CSL problems and the need to use salts that are costly to produce.

References

• Deodhar, A., & Shinagare, K. (1990). Biostil-a continuous fermentation-distillation system. Paper presented at the Technical Papers of the Fortieth Annual Convention of the Deccan Sugar Technologists' Association, Part 1.

• Fadel, M., Abdel- Naser, A. Z., M, M., M, S. H., & A, M. A.-A. (2014). Recycling of vinasse in ethanol fermentation and application in Egyptian distillery factories. African Journal of Biotechnology,13(47), 4390-4398. doi:10.5897/ajb2014.14083

• Jacques, K. A., Lyons, T. P., & Kelsall, D. R. (2003). The alcohol textbook. a reference for the beverage, fuel and industrial alcohol industries. pp. 88-89.

• Mahr un, N., Khan, M. A., Sarwar, M., Lee, W. S., Lee, H. J., Ki, K. S., Kim, H. S. (2006). Influence of Corn Steep Liquor on Feeding Value of Urea Treated Wheat Straw in Buffaloes Fed at Restricted Diets. Asian-Australasian Journal of Animal Sciences, 19(11), 1610-1616. doi:10.5713/ajas.2006.1610

• Mohd Azhar, S. H., Abdulla, R., Jambo, S. A., Marbawi, H., Gansau, J. A., Mohd Faik, A. A., & Rodrigues, K. F. (2017).Yeasts in sustainable bioethanol production: A review. Biochemistry and Biophysics Reports, 10, 52-61. doi:10.1016/j.bbrep.2017.03.003

• Taiwo, A., Madzimbamuto, T., & Ojumu, T. (2018). Optimization of Corn Steep Liquor Dosage and Other Fermentation Parameters for Ethanol Production by Saccharomyces cerevisiae Type 1 and Anchor Instant Yeast. Energies, 11(7), 1740. doi:10.3390/en11071740

7