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1 1 Question 1 The company SweetEthanol produces ethanol and high-fructose corn syrup (HFCS) from corn grain. Instead of using the traditional wet milling process, where the wet distilled grains (WDG) are obtained after simultaneous saccharification and fermentation and distillation, the process is modified for the production of a liquid glucose hydrolysate, which is separated from the solids before fermentation into ethanol or enzymatic conversion into HFCS. The solids (and liquid retained) are in this case used for enzyme production on-site both as single substrate or mixed with a portion of fresh cooked grains. Enzymes are all produced on-site. Yeast and enzymes are recycled from liquid fractions. The company SweetEthanol processes a total of 120.000 t (dry matter) of corn grain per year, estimating a total of 5% used for on-site enzyme production, 60% for ethanol and 35% for HFCS. a) Draw a flowchart with the main operation units of the biotechnological process used by the company SweetEthanol. b) Which enzymes are produced by SweetEthanol. Justify your answer by connecting the enzymes to the respective operations units described in a). c) Assuming a 90% starch content of the corn grain, an enzymatic starch conversion of approx. 95% in the production of the glucose hydrolysate, the carbon loss in solid/liquid separation accounted in the 5% used for on-site enzyme production, and a fermentation yield of 95%, calculate the amount of ethanol produced per year. d) Briefly describe the main operation units potentially used for the enzyme production and enzyme recycling processes, including those for enzyme recovery and concentration. e) Provide a possible explanation for the company’s decision of modifying the traditional wet milling process by the one described. Question 2 To increase business competitiveness, the Board of Directors of SweetEthanol decided to implement a process able to convert 150.000 t of corn stover/cobs/husk into ethanol (second-generation ethanol). You are hired to design and implement a process where on-site enzyme production is again considered. The corn stover/cobs/husk has an average of 15% water content. The dry matter content is composed of 45% cellulose and 30% hemicellulose. a) Draw a flowchart with the main operation units proposed and explain the main constrains of the process to be economical feasible. b) Assuming 90% overall yield for the conversion of cellulose into ethanol, how much ethanol would be generated from this fraction. 2 2 c) Which kind of organism could be used to convert both the cellulose and hemicellulose into ethanol? Estimate how much more ethanol can be produced from the hemicellulose fraction (assume reasonable enzymatic hydrolysis and fermentation yields). Question 3 To further increase business competitiveness, the Board of Directors of SweetEthanol is evaluating alternative bio-based products for their portfolio. They are approached by two other companies willing to provide new yeast strains able to efficiently produce succinic acid or isobutanol from glucose. The Board of Directors seeks your advice regarding these offers. The current production cost for bio-based succinic acid is estimated to be 1.8-2.3$ per kg, which is even lower than the production cost from chemical processes (around 2.5$ per kg), but the Board of Directors still consider it too high. a) Calculate the amount of succinic acid that could be produced if 25% of the corn grain (30,000 t/y) is used as substrate (again, consider 90% starch content and an enzymatic starch conversion into glucose of approx. 95%). Justify your answer in relation to the metabolic pathway(s) used. b) How would you reply to the concerns of the Board of Directors in relation to the production costs of succinic acid? What could be the advantages of integrating succinic acid in their product portfolio? What are the main advantage(s) of employing a yeast strain in relation to the fermentation, recovery and purification of succinic acid compared with bacterial producers (e.g. Actinobacillus succinogenes or Escherichia coli)? c) The company providing the yeast strain for isobutanol production claims that the overall isobutanol yield is similar to the ethanol yield of the current corn grain-toethanol process. Point out the advantage of implementing the isobutanol process and explain which modification(s) to the ethanol process need(s) to be implemented? Question 4 Lactic Acid Bacteria (LAB) are used in multiple food and industrial biotechnology processes. a) Briefly describe the generic process for the production of LAB to be used in the dairy industry. b) Explain the process of yogurt production. c) One big dairy industry is considering the biological production of polylactic acid from their wastewaters. Which operation units need to be implemented and what are the relevant characteristics of the microorganism for this process. d) What is the traditional fate of dairy wastewaters? Briefly explain the process. 3 3 Question 5 The biodiesel and glycerol market has shown an exponential increase in the last decade. a) Describe the process of the transesterification and explain the possible issues related to the use of animal fat or algae biomass as a feedstock. How could these issues be overcome? b) Describe the main differences between HVO and biodiesel, in terms of industrial production, feedstock used and fuel characteristics. c) According to the stoichiometric reaction, transesterification requires that 3 moles of alcohol catalyst to react for each mole of triglyceride. In praxis however, the industries will use an excess of alcohol (5-6 moles per mole of triglyceride). Which could be the reasons for this and what effect would you expect it to have on the production costs? d) What do you think is the main challenges of 3rd generation biodiesel at the moment? e) Make an analysis of the biodiesel market today, considering the last 10 years trends, the types of feedstock used, and the different competing technologies in place. What factors can affect the high cost and continuous oscillations of biodiesel on the market?

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