1. Biofuel from pectin-rich biomass 1.Author’s profile 2.Executive Summary 3.Background of papers 4.Summary2.Executive Summary 3.Background of papers 4.1 Article 1 4.2 Article 24.1 Article 14.2 Article 2 5. Comparative Analysis 6. References5. Comparative Analysis 6. References

2. Author’s profile Hello everyone! My name is Hu Shi and I am currently doing a major in Biotechnology and Biomedical Science. My topic is fermentation of pectin-rich biomass with normal type and recombinant ethanologens to produce fuel ethanol as a biofuel. I chose this topic as I found the idea that pectin-rich biomass, an under- utilized waste product, can augment many countries’ ethanol supplies by capitalizing on this already established feedstock. It somehow can be an potential renewable resource for the liquid fuel in the future. Author’s profile Executive summary Backgrou nd of papers Summary -Article 1Article 1 Summary contd -Article 2Article 2 Compara tive Analysis Referenc es Hu Shi 32082998 Summary contd -Article 1Article 1 Summary -Article 2Article 2 Table5 Summary contd2 -Article 1Article 1

3. Executive summary Currently, pectin-rich biomass is sold as animal feed at a low value, this work will compare two scientific articles related to the production of ethanol by using these types of pectin-rich biomass: sugar beet pulp, citrus waste and galacturonic acid. Fermentation of these materials have been conducted with a variety of ethanologens, including yeasts and some bacteria as well as some of recombinant bacteria under various conditions. The aim is to understand how effective of the different species of ethanologens ferment the pectin-rich biomass as compared to the recombinant ones and their degradation products as well in order to optimise ethanol production. Author’s profile Executive summary Backgrou nd of papers Summary -Article 1Article 1 Summary contd -Article 2Article 2 Compara tive Analysis Referenc es Summary contd -Article 1Article 1 Summary -Article 2Article 2 Table5 Summary contd2 -Article 1Article 1

4. Background of papers With a worldwide increase in the consumption and gradual diminishing of fossil fuels, the search for renewable energy sources has also become increasingly important. Bio-fuels such as ethanol have attractive prospects in the industry. Pectin-rich biomass, an under-utilized waste product of industry, can augment the world’s ethanol supplies by capitalizing on this already established feedstock(Meredith C. Edwards & Joy Doran-Peterson,2012). Currently, the biofuel ethanol can be produced by a series of pathways, the figure can be seen below. Author’s profile Executive summary Backgrou nd of papers Summary -Article 1Article 1 Summary contd -Article 2Article 2 Compara tive Analysis Referenc es Summary contd -Article 1Article 1 Summary -Article 2Article 2 Table5 For the purpose of this study the main focus will be on the sugar platform which utilize hydrolysis of pectin-rich residues followed by ethanolic fermentation by various bacteria. Summary contd2 -Article 1Article 1

5. Summary – Article1 Overview The industrial processing of fruits resulting in pectin-rich waste produces a favorable biomass for ethanol production. Most sugar beet pulp fermentations were conducted using autoclaving at 121 °C for 20 minutes to minimize contamination, followed by enzymatic digestion and fermentation of the resulting carbohydrates. Therefore several kinds of ethanologens were introduced to see the efficiency of the fermentation process. Material Three most studied types of pectin-rich biomass: sugar beet pulp, citrus waste and apple pomace. Fermentations of these materials have been conducted with a variety of ethanologens including Saccharomyces cerevisiae, Escherichia coli, and other ethanologens, such as Kluyveromyces marxianus, E. chrysanthemi and K. oxytoca. Author’s profile Executive summary Backgrou nd of papers Summary -Article 1Article 1 Summary contd -Article 2Article 2 Compara tive Analysis Referenc es Summary contd -Article 1Article 1 Summary -Article 2Article 2 Table5 Pectin-rich biomass as feedstock for fuel ethanol production Meredith C. Edwards & Joy Doran-Peterson Summary contd2 -Article 1Article 1

6. Summary contd – Article1 Results Early fermentations were conducted with S. cerevisiae. Some advantages of using S. cerevisiae include its ability to tolerate high substrate concentrations and high ethanol concentrations, as well as relatively low pH and oxygen levels, making it a robust organism for the fermentation process. Detailed results can be obtained from Table3 E. coli does not tolerate ethanol as well as S. cerevisiae. Therefore, E. coli and commercial enzymes required for degradation of the plant cell wall (which have an acidic optimum pH) cannot reach their maximum activities simultaneously during biomass fermentation. Other different strains of E. coli were also compared based on their yields and requirement of pH as well as parameters.. Detailed results can be obtained from Table4 Author’s profile Executive summary Backgrou nd of papers Summary -Article 1Article 1 Summary -Article 2Article 2 Compara tive Analysis Referenc es Pectin-rich biomass as feedstock for fuel ethanol production Meredith C. Edwards & Joy Doran-Peterson Summary contd -Article 1Article 1 Table5 Summary contd -Article 2Article 2 Summary contd2 -Article 1Article 1

7. Summary contd – Article1 Results contd Other organisms have been used for ethanol production from pectin-rich materials as well. Detailed results can be obtained onTable5(Shown on next slide). Research has focused on strains that are thermotolerant, which can produce their own cell wall degrading enzymes or organisms that are able to metabolize a wide variety of sugars. Both bacterial ethanologens use the mixed acid fermentation pathway to metabolize sugars and will produce organic acid co-products similarly to E. coli. Discussion All of the ethanologens used in the experiment produce some acetate during pectin-rich biomass fermentations. The production of side products like acetate decreases the amount of ethanol. Fermenting pectin-rich residues is citrus waste specific, the presence of the inhibitor Dlimonene which produce from citrus would also decrease ethanol yields. It has been suggested that gram-negative organisms, like E. coli, tend to be more resistant to some terpenes, including limonene, which indicate the increase of ethanol yields. Author’s profile Executive summary Backgrou nd of papers Summary -Article 1Article 1 Summary contd -Article 2Article 2 Compara tive Analysis Referenc es Pectin-rich biomass as feedstock for fuel ethanol production Meredith C. Edwards & Joy Doran-Peterson Summary contd -Article 1Article 1 Table5 Summary -Article 2Article 2 Summary contd2 -Article 1Article 1

8. Table5 Author’s profile Executive summary Backgrou nd of papers Summary -Article 1Article 1 Summary contd -Article 2Article 2 Compara tive Analysis Referenc es Summary contd -Article 1Article 1 Summary -Article 2Article 2 Table5 Summary contd2 -Article 1Article 1

9. Summary – Article 2 Overview Pectin-rich residues from sugar beet processing contain significant carbohydrates and insignificant amounts of lignin. Beet pulp was evaluated for conversion to ethanol using recombinant bacteria as biocatalysts. The three recombinant bacteria evaluated in this study, Escherichia coli strain KO11, Klebsiella oxytoca strain P2, and Erwinia chrysanthemi EC 16 pLOI 555, ferment carbohydrates in beet pulp with varying efficiencies. Materials and Methods Two genes from Z. mobilis encoding alcohol dehydrogenase and pyruvate decarboxylase were introduced into three bacteria: E. coli, Klebsiella oxytoca, and Erwinia chrysanthemi EC 16, with various conditions provided, fermentation experiments were conducted in a six-station magnetic stirrer was placed beneath a water bath and maintained at 100 rpm. Galacturonic acid fermentations were conducted at a pH of 6.0 and temperature at 30°C. Ethanol concentration was determined by gasliquid chromatography as previously described.Fermentation broths from galacturonic acid fermentations were examined for organic acid production by high-performance liquid chromatography with ultraviolet detection. Author’s profile Executive summary Backgrou nd of papers Summary -Article 1Article 1 Summary contd -Article 2Article 2 Compara tive Analysis Referenc es Summary contd -Article 1Article 1 Summary -Article 2Article 2 Table5 Fermentations of Pectin-Rich Biomass with Recombinant Bacteria to Produce Fuel Ethanol JOY BETHUNE DORAN,* JENNIFER CRIPE,MISTY SUTTON, AND BRIAN FOSTER Summary contd2 -Article 1Article 1

10. Summary contd – Article2 Results Galacturonic Acid Fermentation Fermentation with galacturonic acid indicate that E. coli KO11 gave the highest ethanol yield(Table 1). All strains produced some ethanol and varying amounts of acetic acid. Recombinant Erwinia produced the greatest amounts of acetic acid and succinic acid. Yield for ethanol production was lowest for K. oxytoca P2, which also produced both acetic and succinic acid. Fermentation with Sugar Beet Pulp: Pressed or Pelletized Fermentations with sugar beet pulp either pressed or dried and pelleted were performed with and without fungal enzymes. The result is shown inTable2, which suggest that E. coli KO11 had the highest yield for ethanol production, but did not possess any hydrolytic enzymes to contribute to a bioconversion process. Discussion Enzymatic processes are currently expensive, but they can operate with high yields and generate few inhibitory side products. Enzymatic hydrolysis of pectin-rich processing residue such as sugar beet pulp appears to be much more amenable to enzymatic degradation than lignified cellulosic substrates. E. coli KO11 appears to show the most promise for a biocatalyst, even though it does not produce any enzymes on its own for the degradation of plant material. E. chrysanthemi and K. oxytoca both possess enzymes in the native state that could help degrade plant material; however, these enzymes do not appear to confer any advantage to these organisms in our process. Author’s profile Executive summary Backgrou nd of papers Summary -Article 1Article 1 Summary contd -Article 2Article 2 Compara tive Analysis Referenc es Summary contd -Article 1Article 1 Summary -Article 2Article 2 Table5 Fermentations of Pectin-Rich Biomass with Recombinant Bacteria to Produce Fuel Ethanol JOY BETHUNE DORAN,* JENNIFER CRIPE,MISTY SUTTON, AND BRIAN FOSTER Summary contd2 -Article 1Article 1

11. Comparative Analysis The scientific research from both Meredith C. Edwards et al. and JOY BETHUNE DORAN et al focus on the fermentation of pectin-rich biomass by using the different bacteria. Their working aim to find the effectiveness of different types of bacteria, how they convert the pectin-rich waste to ethanol and their by-products. The study conducted by Meredith C. Edwards et al were relatively simple, as it was straight away compared different yeast and bacteria strains which were used to ferment the biomass. Some of by-products also mentioned, such as acetate acid and Dlimonene which would decrease the amount of ethanol. E.coli was proved to be the currently best option for pectin-rich biomass fermentation. It can metabolize all of the sugars present in the biomass and has been engineered to produce high ethanol yields with limited unwanted co- products. The research performed by Joy Bethune Doran et al aimed at providing a more specific choices bacteria, as they used three types of recombinant bacteria and made comparison between them in order to see a better efficiency. However, as this experiment required a certain load of enzyme, the whole process could be relatively expensive. On the other hand, they could operate with high yields and generate few inhibitory side products, such as acetic acid and succinic acid. They also believed that with a furthermore modification such as replacement of biocatalyst and optimization of conditions, this may become a more- efficient process. Author’s profile Executive summary Backgrou nd of papers Summary -Article 1Article 1 Summary contd -Article 2Article 2 Compara tive Analysis Referenc es Summary contd -Article 1Article 1 Summary -Article 2Article 2 Table5 Summary contd2 -Article 1Article 1

12. References Image reference: http://web.extension.illinois.edu/ethanol/cellulosic.cfm http://jcwinnie.biz/wordpress/?p=845 Context reference: Meredith C. Edwards & Joy Doran-Peterson, Pectin-rich biomass as feedstock for fuel ethanol production,2012. JOY BETHUNE DORAN,* JENNIFER CRIPE,MISTY SUTTON, AND BRIAN FOSTER, Fermentations of Pectin-Rich Biomass with Recombinant Bacteria to Produce Fuel Ethanol,2000. Doran JB, Cripe J, Sutton M, Foster B (2000) Fermentations of pectinrich biomass with recombinant bacteria to produce fuel ethanol. Wilkins MR, Suryawati L, Maness NO, Chrz D (2007a) Ethanol production by Saccharomyces cerevisiae and Kluyveromyces marxianus in the presence of orange-peel oil. Author’s profile Executive summary Backgrou nd of papers Summary -Article 1Article 1 Summary contd -Article 2Article 2 Compara tive Analysis Referenc es Summary contd -Article 1Article 1 Summary -Article 2Article 2 Table5 Summary contd2 -Article 1Article 1