10.2. 2022. ethanol. P7roposal. PPT.pptx

ethanol preparation

1. Production of native fermented beverages from Agricultural solid waste by using saccharomyces cerevisiae Laboratory of Food Microbiology Dept of Food Engineering Dhaka University of Engineering & Technology, Gazipur 17-Jan-22 1

2. Objective of the study Production of native alcoholic beverage production Bioprocessing of agricultural solid waste to alcoholic fermented products Production bioethanol for solve energy crisis Solve environmental pollution by using waste products 17-Jan-22 2

3. Introduction This study investigates the potential of ethanol production from agro wastes. Agro waste from sugarcane Saccharum officinarum (sugarcane baggasse , sugarcane bark) and maize plant Zea mays (corncob, corn stalk, corn husk) Ethanol production by Saccharomyces cerevisiae is affected not only by fermentation conditions (temperature, pH and sugar concentration). The basic formula for making ethanol from sugar glucose is as follows: 3

4. Introduction The improvement of living standard urges the hunt for sustainable energy in order to meet energy consumption across the world. The use of fossil fuels as the main energy resources caused the arising of worldwide problems such as environmental pollution and global warming. These led to the finding of environmentally friendly, renewable and sustainable energy by government, industrial and energy sector. Among renewable energies, priority was given to liquid biofuels as it represents about 40% of the total energy consumption in the world. The use of liquid biofuels contributes to the reduction of greenhouse gas emissions, creation of job opportunities, regional development and supply security. 17-Jan-22 4

5. Introduction The process begins with the yeast breaking down the different forms of sugar in the wort. The types of sugars typically found in wort are the monosaccharides glucose and fructose. These sugars contain a single hexose, which is composed of 6 carbon atoms in the molecular formula C6H12O6. Typical disaccharides in the wort are galactose, sucrose, and maltose. The third type of fermentable sugar in the wort is a trisaccharide . This trisaccharide is formed when three monosccharides join together. 17-Jan-22 5

6. Hypothesis Bioethanol is one of the most commonly used biofuels in transportation sector to reduce greenhouse gases. S. cerevisiae is the most employed yeast for ethanol production at industrial level though ethanol is produced by an array of other yeasts, bacteria, and fungi. Ethanol is an attractive transportation fuel due to its high octane content, compatibility with fuel employed in internal combustion engines, and ease of integration with existing alcohol production and fuel distribution infrastructure. The international atomic energy agency estimated that as much as 13.3% of current global energy consumption is renewable energy of which 10% is bioenergy , 2.3% is hydro-energy and about 1% is from sources such as solar, wind, heat and geothermal. 6

7. Materials and Method: Machine Conical flask Shaker incubator Air laminar flow cabinet UV- Spectrophotmeter Fermenter ( Solida Biotechnology, Munich, Germany • Model: Biobench control- UCG20-G4-MM-ORBB09-17) 17-Jan-22 7

8. Materials and Method: Reagents and strain YPD media( Bacteriological peptone,=20g/L, Glucose= 20g/L, Yeast extract= 10g/L) Glucose Glycerol Strain: Saccharomyces cerevisiae 17-Jan-22 8

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10. Ethanol Production Yield Optimization for Fermentation condition in Vitro (Flask) System Yield Vs Time: 24 h, 48 h, 72 h Yield Vs pH: 4, 5, 6, 6.5 Yield Vs Temperature: 20, 25, 30, 35 °C Yield Vs RPM: 50, 100, 150, 200 Yield VS Glucose: 10g/L, 15g/L, 20g/L 17-Jan-22 10

11. ESTIMATION OF GLUCOSE BY GOD -POD METHOD Principal: Glucose oxidase (GOD) oxidizes the specific substrate β- D- glucose to gluconic acid and hydrogen peroxide (H2O2) is liberated. Peroxidase (POD) enzyme acts on hydrogen peroxide to liberate nascent oxygen (O2), then nascent oxygen couples with 4- amino antipyrine and phenol to form red quinoneimine dye.

12. Continue… The intensity of the colour is directly proportional to the concentration of glucose present in plasma. The intensity of colour is measured by colorimeter at 530 nm or green filter and compared with that of a standard treated similarly. Final colour is stable for at least 2 hours if not exposed to direct sunlight.

13. Reagents 1. Glucose colour reagent; it contains GOD, POD, 4- amino antipyrine , phenol & phosphate buffer (pH 7.5) 2. Glucose standard solution, Concentration = 100 mg/dl. 100 mg of anhydrous glucose is dissolved in 100 ml of distilled water Or D-Glucose Determination Kit/ GOD-POD Kit

14. Procedure Pipette into clean, dry test tube labelled as Blank (B), Standard (S) and Test (T). Then add the solution in each of test tubes separately as shown in table below.

15. Data and Calculation Plasma Glucose standard concentration is 100 gm/dl

16. Reagents used for reducing measurement Substrate solution : standard solution of 1000µg/ mL concentration was prepared by dissolving 100mg of glucose in 100ml distilled water. 3, 5 dinitrosalicylic acid (DNS) solution: reagent was prepared by dissolving 10.0 g of 3, 5-DNS, 2.0g of phenol and 0.5 g of sodium sulphite in 500 ml of 2% NaOH solution and then diluting it to 1 liter with distilled water. The reagent was filled and stored in dark coloured bottle. Potassium sodium tartarate (Rochelle salt): 40 g of potassium sodium tartarate was dissolved in distilled water and the volume was made to 100 ml.

17. Reducing sugar determination Procedure One ml of appropriately diluted solution (500-1000 µg ml -1 ) sample was taken in a test tube to which 3 ml of DNS reagent was added. The tubes were boiled in a boiling water bath for 15 minutes. One ml of Rochelle was added to these test tubes and tubes were cooled to room temperature and used for measuring optical density at 540 nm.

18. Agricultural waste sampling procedure and Location

19. For preservation strain: glycerol 19

20. Ethanol Purification by Distillation After fermentation, the fermented liquor or ‘beer’ contains alcohol as well as low boiling point volatile compounds such as acetaldehydes, esters, and the higher boiling, fusel oils. The alcohol is obtained after several steps. First, steam is passed through the beer which is said to be steam-stripped. Secondly, the dilute alcohol solution is passed into the center of a multi-plate aldehyde column in which the following fractions are separated: esters and aldehydes, fusel oil, water, and an ethanol solution containing about 25% ethanol. Thirdly, the dilute alcohol solution is passed into a rectifying column where a constant boiling mixture, an azeotrope, distills off at 95.6% alcohol concentration. 17-Jan-22 20

21. Reactions Kinetics Determination Fig: Representative invertase production by mutant MLD10 of S. cerevisiae in industrial-scale fermenters from 180 g sugars l−1 in fermenter (filled circle), 150 g sugars l−1 (filled triangle), and 120 g sugars l−1 (inverted filled triangle) 17-Jan-22 21

22. Condition of Ethanol production in Bioreactors (Fermenter) About 30% of inoculum (cell density 3 × 106 ml-1) will be generally employed. It will be added to the fermenter by pumping or gravity. This addition of inoculum to the fermenter is called as pitching. The following environmental factors like incubation time and temperature will to be suitably maintained and controlled in order to achieve optimum yield. The time required for the maximum yield of ethanol to be 30 to 72 hours , which largely depends upon the specific gravity of the fermentation liquid. Fermentation generally starts within few hours of yeasts inoculation. The process becomes rapid after 24 hours. Maintain temperature 25-35°C. 17-Jan-22 22

23. Ethanol Production from Agricultural Solid Waste (Sugar cane) Ethanol as most important alcohol can be produced by converting the sugar content of any starchy material into alcohol with the evolution of carbon dioxide (CO2) under controlled environmental conditions. The process is an anaerobic fermentation in accordance with embden-meyerhoff pathway (EMP) catalysed by enzymes produced by bacteria and fungi. The fermentation process is essentially the same process used to make alcoholic beverages. In this process yeast and heat are used to break down complex sugars into more simple sugars, producing ethanol. Starchy materials are first hydrolysed to fermentable sugars, and subsequently fermented with the required yeast species to produce ethanol. During the fermentation process, part of the sugar is assimilated by the yeast cells and part is transformed into glycerol, acetaldehydes and lactic acid. 17-Jan-22 23

24. Continue…. Production of ethanol from ligno -cellulosic materials such as corncob, cornstalk, cornhusk, sugarcane bagasse and sugarcane bark though faces challenges, but can substitute bio-ethanol production from edible food substances. The energy produced is both renewable and available in large quantities throughout the world. It would also allow agricultural land to be used more efficiently and at the same time prevent competition with food supplies. Until recently the problem was that the complex mixture of sugars that make up these left over materials could not be efficiently converted into ethanol by Saccharomyces cerevisiae because they have a very strong crystalline structure surrounded by lignin which makes it difficult for enzyme accessibility. 24

25. Results Figure 1 shows the flowchart of ethanol production that was adopted in the processing of the 5 different raw materials. Each point in the production line are critical control points that must be carefully monitored for quality control of products and reproducibility of the process…………… 17-Jan-22 25

26. Discussion 17-Jan-22 26

27. Conclusion Saccharomyces cerevisiae has grown with humans for many thousands of years. Today it has become significant in different ways specifically in the production of ethanol as a biofuel. Perhaps Saccharomyces cerevisiae is the future answer to our fossil fuel problem. 17-Jan-22 27

28. Future Direction 1. There should be the development of an environmentally friendly pretreatment procedure. 2. Highly effective enzyme systems for conversion of pretreated waste to fermentable sugars. 3. Effective microorganism to convert multiple sugars to ethanol. 17-Jan-22 28

29. Reference [1] Sun, Y.and Cheng, J. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour . Technol., 2002, 83, 1–11. [2] Martín, C., Galbe , M., Wahlbom , C.F., Hahn- Hägerdal , B., Jönsson , L.J. Ethanol production from enzymatic hydrolysates of sugarcane bagasse using recombinant xylose-utilizing Saccharomyces cerevisiae . Enzyme and Microbial Technology., 2002, 31, 274–282. [3] Zhang, M.J., Wang, F., Su, R.X., Qi, W. and He, Z.M. Ethanol production from high dry matter corncob using fed-batch simultaneous saccharification and fermentation after combined pretreatment. Bioresour . Technol., 2010, 101, 4959–4964. [4] Gould, J.M. and Freer, S.N. High-efficiency ethanol production from lignocellulosic residues pretreated with alkaline H2O2. Biotechnology and Bioengineering., 1984, 26, 628–631. [5]https://www.researchgate.net/publication/303316970 Production of bioethanol from agricultural waste. 29

30. Acknowledgement Industrial Microbiology Laboratory, Institute of Food Science and Technology (IFST), BCSIR, Bangladesh. For Donating Saccharomyces cerevisiae strain. Institute of Energy, DUET, Gazipur. Dept of Chemical and Food Engineering. DUET, Gazipur basic research fund for research. 30