Raganati, Francesca (2014) BUTANOL PRODUCTION BY FERMENTATION OF RENEWABLE FEEDSTOCKS. [Tesi di dottorato]

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Abstract

The study carried out during the present Ph.D. program aimed at investigating the Acetone-Butanol-Ethanol (ABE) production process by fermentation. The work was carried out at the Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriale of the University of Naples ‘Federico II’. The activities were articulated according to three paths: i) the characterization of the ABE fermentation process as regards kinetics and yields using different renewable resources (lignocellulosic biomass, high sugar content beverges and cheese whey); ii) the characterization of the ABE fermentation process according to the metabolic flux analaysis (MFA) and to the dynamic kinetic models; iii) the development of innovative continuous biofilm reactor for the ABE production. A commercial clostridia strain was investigated. Clostridium acetobutylicum DSM 792 was selected for its ability to produce ABE with satisfactory selectivity towards the butanol. The ABE fermentation process by adopting renewable resources. Characterization in terms of kinetics and yields. The study was aimed at the assessment of both the kinetics and the yields of cell growth and metabolites produced during the fermentation of: • sugars representative of hydrolized lignocellulosic biomass fermentation (glucose, mannose, arabinose and xylose); • sugars representative of high sugar content beverages (glucose, fructose and sucrose); • High sugar content beverages. Batch cultures of free C.acetobutyicum cells were inveastigated. Tests were focused on the preliminary characterization of the fermentation with the aim of highlighting the relevant features of the process. The fermentation was characterized in terms of kinetics and yields under a wide interval of operating conditions (substrate concentration, nutrient concentrations, ...). Characterization of the ABE fermentation process. MFA and dynamic kinetic models. The MFA and the kinetic dynamic methodology were adopted to characterize the ABE fermentation. Selected batch fermentation tests were carried out with the aim to charcaterize the time-evolution of the concentration of substrate, cells and metabolities. Tests were carried out at initial sugar concentration 60 g/L. The time- series of concentration were processed according the MFA and the kinetic model methodology. In particular: • the MFA was adopted to investigate the role of the main reaction steps of the Clostridium acetobutylicum metabolic pathway to convert reference sugars (glucose and xylose) of hydrolyzed lignocellulosic biomass into butanol. Results of batch fermentation tests carried out using glucose and xylose as carbon source were adopted for the flux assessment. The stoichiometric matrix of the model was characterized by a singularity that prevented the assessment of a unique set of fluxes of the primary metabolic activity. The non linear constrain proposed by Desai et al. (1999) relating the acetate and butyrate uptake fluxes was adopted to solve the model equation set. The MFA was proposed with reference to glucose and xylose as carbon source. The comparison of the assessed fluxes suggested the role of each reaction step as a function of the carbon source investigated. 5 • A kinetic dynamic model of acetone–butanol–ethanol (ABE) production by Clostridium acetobutylicum DSM 792 was proposed using the biochemical networks simulator COPASI. Effects of substrate were studied implementing the model with different sugars: glucose, mannose, fructose, sucrose, lactose, xylose and arabinose. If necessary, the metabolic pathway was modified according to the specific sugar. In particular, the Embden-Meyerhof-Parnas (EMP) pathway equations were used for hexose and disaccharide sugars while the pentose phosphate (PP) pathway equations were used for pentose sugars. Development of innovative continuous biofilm reactor for the ABE production. The study was aimed at the assessment of the butanol production in a C. acetobutylicum biofilm reactor. The activity aimed at the butanol production regarded the design, set-up and operation of a biofilm fixed bed reactor. Unsupplemented cheese whey was adopted as renewable feedstock. Operating conditions of the continuous tests were selected to maximize the butanol production and butanol selectivity. In particular, main activities were: § The selection of a pre-treatment process of the cheese-whey coupled with fermentation tests carried out under batch conditions to assess effects of the pre-treatment on fermentation performances; § Tests with the pre-treated cheese whey to characterize the fermentation process in terms of butanol production and butanol selectivity; § Design, set-up, and optimization of a fixed bed biofilm reactor for cheese whey conversion; § Tests with the biofilm reactor under continuous conditions. The success of operation of the Packed Bed Reactor (PBR) has fuelled the development of an innovative continuous biofilm reactor configuration: “carosello” of packed bed biofilm reactors connected in series. The reactor system has been equipped with a device to switch the feeding at the reactor according to a pre-set sequence. A mathematical model to support the bioreactor system design was developed.

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