14th Workshop on the Developments in the Italian PhD Research on Food Science Technology and Biotechnology - University of Sassari, Oristano, September 16 – 18, 2009 Bioethanol production from agri-food lignocellulosic residues Guglielmo Santi (email@example.com) Dept. Agrobiology and Agrochemistry, University of Tuscia, via S.C. de Lellis, Viterbo, Italy Tutor: Dott.ssa. Silvia Crognale This PhD thesis research project is aimed at converting various agri-food lignocellulosic residues into bioethanol through fermentation. To this end, different ethanol-producing yeast strains and different hydrolysis methods will be assessed. The final purposes are the optimization and the scaling-up of the fermentative process with the selected strain and residue. Produzione di bioetanolo da scarti agroalimentari lignocellulosici Questo progetto di tesi di dottorato mira alla conversione di vari residui agroalimentari lignocellulosici in bioetanolo mediante processo fermentativo. A tale scopo verranno analizzati diversi ceppi di lievito produttori di etanolo e diversi metodi di idrolisi. Gli obiettivi finali sono l’ottimizzazione e lo scaling-up del processo fermentativo con il ceppo ed il residuo selezionati. 1. State-of-the-Art Energetic and environmental issues are driving the development of alternative fuels, such as bioethanol, for motor vehicles. Bioethanol production from agricultural products has been in practice for the past 80 years: current industrial processes mainly use sugarcane (Southern Hemisphere) or maize and other less common cereal grains (Northern Hemisphere). This kind of processes, however, has created a direct competition between food and energy sector for the utilization of such feedstocks. Since the price of the feedstock contributes more than 55% to the production cost, inexpensive materials such as agri-food lignocellulosic residues are being considered to make bioethanol competitive in the open market (del Campo et al., 2006). Extensive research over the past 20 years has been addressed towards making an efficient conversion of lignocelluloses into sugars for fermentation. Lignocellulosic agri-food wastes, such as cereal straw and hulls, are composed of carbohydrate polymers (mainly cellulose and hemicelluloses), lignin, and a remaining, smaller part of proteins, acids etc. The majority of the carbohydrates can be hydrolysed to single sugars and fermented to ethanol. However, in the case of lignocelluloses, a particular crosslinking (ester and ether linkages) between polysaccharides and lignin creates a barrier to the production and recovery of valuable materials. Thus, the necessary technology for ethanol production from this kind of residues differs from the one which is employed in the conventional starch-to-ethanol industry: an initial pretreatment stage (acid or alkaline hydrolysis, steam explosion, wet oxidation, etc.) is required to soften the material and break down the lignocellulosic structure in order to make it more susceptible to an enzymatic attack before fermentation (Mtui, 2009). All the pretreatment technologies are usually executed under severe reaction conditions with a large capital investment, high processing costs and great investment risks. Moreover, each technique has its own associated problems, particularly in relation to the ability to recycle expensive reagents. Other factors affecting the outcome of the pretreatment include lignin and hemicellulose content, cellulose fiber crystallinity and porosity of the waste materials. Thus, each kind of lignocellulosic residue requires a specific pretreatment in order to optimize the subsequent steps (Champagne, 2008; Wheals et al., 1999). During the pretreatment process, degradation compounds of pentoses and hexoses, primarily furfural and 5- hydroxymethyl furfural (5-HMF), are formed. These components are toxic and inhibit the succeeding enzymatic and fermentative processes. Therefore they should be removed or neutralized prior the fermentation. As an alternative, a fermenting organism with high inhibitor tolerance should be used (Palmqvist and Hahn-Hägerdal, 2000). A consortium of enzymes (endoglucanase, exoglucanase and β-glucosidase) is then needed to break down the carbohydrate polymers into their constituent monomers. Enzymatic hydrolysis can be improved through the optimization of substrate concentration, enzyme dosing and recycling, use of surfactants and enzyme mixtures (Champagne, 2008). Several reports and reviews have been published about the production of ethanol through fermentation: the most frequently used microorganism for fermenting ethanol in industrial processes is Saccharomyces cerevisiae, which has proved to be very robust and well suited to the fermentation of glucose from lignocellulosic hydrolysates. Due to the large proportion of xylose in this kind of hydrolysates, and the inability of S. cerevisiae to ferment xylose to ethanol, the use of yeasts that are capable of cofermenting pentoses and hexoses in hydrolyzates offers an opportunity for a more efficient utilization of the hemicellulose component of agri-food 14th Workshop on the Developments in the Italian PhD Research on Food Science Technology and Biotechnology - University of Sassari, Oristano, September 16 – 18, 2009 lignocellulosic residues. Among the xylose fermenting yeasts, some species of Pichia such as P. stipitis show high ethanol yields under low oxygen level (Telli-Okur and Eken-Saraçoğlu, 2008). Generally, economic restrictions force industrial processes to work in a very small range of operating conditions. Thus, it is very important to define the optimum conditions to achieve sufficient profitability and, in the case of bioethanol, to find a strain that is tolerant to high temperatures (in order to reduce cooling costs) and to high concentrations of ethanol, for an easier recovery of the final product (Yan and Shuzo, 2006). 2. PhD Thesis Objectives and Milestones Within the overall objective mentioned above this PhD thesis project can be subdivided into the following activities according to the Gantt diagram given in Table 1: A1) Screening of Saccharomyces cerevisiae and Pichia anomala strains: in shaken flasks, using a model and defined medium: evaluation of growth and ethanol production with glucose as sole carbon source (A1.1); evaluation of any possible inhibitory effect due to the presence of pentoses in the medium (A1.2); evaluation of the ability of P. anomala strains to grow on pentoses as sole carbon source (A1.3); A2) Study and selection of agri-food wastes hydrolysates: hydrolysis of the residues with different methods (chemical or enzymatic) (A2.1) and analysis of their chemical composition by gas chromatography (A2.2). Optimization of the hydrolysis conditions, evaluation of different temperatures, reaction times and acid concentrations (A2.3). Comparison between the selected waste and a non agri-food waste, in order to evaluate the suitability of the agri-food residue (A2.4); A3) Set up of the fermentative process with the selected strain and residue: optimization (kind of fermentor, stir condition, aeration, medium composition, pH) (A3.1) and scaling-up from laboratory scale to a higher scale, with a preliminary evaluation of yield, productivity and feasibility (A3.2); A4) Results processing. Writing and editing of the PhD thesis: scientific papers and/or oral communications. Table 1 Gantt diagram for this PhD thesis project. Months 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Activity A1) Screening of yeast strains 1) hexose 2) hexose + pentose 3) pentose A2) Selection of hydrolysates 1) Hydrolysis 2) Chemical composition analysis 3) Optimization of the method 4) Comparison with non-food waste A3) Set up of the fermentative process 1) optimization 2) scale up A4) Thesis and paper preparation 3. Selected References Champagne P (2008) Bioethanol form agricultural waste residues, Environ Prog 27: 51-57. Del Campo I, Alegrìa I, Zazpe M, Echeverrìa M, Echeverrìa I (2006) Dilute acid hydrolysis pretreatment of agri-food wastes for ethanol production, Ind Crop Prod 24: 214-221. Mtui GYS (2009) Recent advances in pretreatment of lignocellulosic wastes and production of value added products, Afr J Biotechnol 8: 1398-1415. Palmqvist E, Hahn-Hägerdal B, (2000) Fermentation of lignocellulosic hydrolyzates. I: inhibition and detoxification. Bioresource Technol 74: 17-24. Telli-Okur M, Eken-Saraçoğlu N, (2008) Fermentation of sunflower seed hull hydrolysate to ethanol by Pichia stipitis, Bioresource Technol 99: 2162–2169. Wheals AE, Basso LC, Alves DMG, Amorim HV (1999) Fuel ethanol after 25 years, Trends Biotechnol 17: 482-487. Yan L, Shuzo T (2006) Ethanol fermentation from biomass resources: current state and prospects, Appl Microbiol Biot 69: 627-642.
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