Lanzillo, Fabiana (2023) Syngas fermentation for chemicals production. [Tesi di dottorato]

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Abstract

The PhD project had the objective to analyse syngas fermentation by Clostridium carboxidivorans. In particular, the focus was on the optimization of the process in terms of cell and product yield and cost of the fermentation. C. carboxidivorans is a gram positive bacterium that was isolated for the first time from an agricultural setting lagoon in Oklahoma. It is one of few bacteria able to ferment syngas and to use CO or CO2 + H2 as carbon and energy source to produce high added value products. The bacterium is able to produce acids like acetic acid, butyric acid, and hexanoic acid and solvents like ethanol, butanol, and hexanol. Solvents are very important products because it is possible to use them like biofuels. C. carboxidivorans is one of the few microorganisms that is able to produce butanol using syngas. Butanol is a highly promising biofuel and has several advantages. It is less hygroscopic and less corrosive than ethanol, it has a higher caloric content than ethanol and, moreover, it is possible to use it without modification of the infrastructures and engines. Batch cultures The first part of the project was performed in serum bottles (270 mL). The aim of this part was to assess the effects of the CO partial pressure in the headspace of the fermenter on the microorganism growth kinetics and on the fermentation performances. Tests to characterize cell growth and acids/solvents production under different CO pressure were carried out using batch fermenters. The CO pressure in the headspace was set between 0.5 and 2.5 atm, setting the liquid/gas volumetric ratio at 0.28 and 0.92, respectively, discovering that the best performance in terms of ethanol/butanol production was obtained with VL/VG = 0.28 at initial PCO = 1.7 atm. The effects on growth kinetics and on fermentation performances of several medium components was analyzed in batch cultures. Different concentration of yeast extract, nitrogen source, several reducing agents – at different concentration - and the different concentration and composition of the trace metal solution have been investigated. The concentration of yeast extract (YE) was increased up to 3 g/L. The reducing agents investigated were: Cysteine-HCl 0.6 g/L, pure Cysteine 0.6 g/L, Sodium Sulphide (Na2S) 0.6 g/L, Cysteine-Sodium Sulphide 0.6 g/L and Cysteine-Sodium Sulphide 0.72 g/L. The metal solution concentration was decreased down the 25% of the standard value. The results pointed out that: the best concentration of YE for the growth and production is 1 g/L; the supplementation of Na2S produced higher solvent concentrations; the beneficial effects of Na2S were mitigated when it was mixed with cystein; the supplementation of metals to the medium was confirmed to be fundamental for the bacterial growth and production of acids and solvents. Concentration of metals set at less than 75% with respect to the standard value (stock solution) was not compatible with an appreciable solvent production. Gas-fed stirred tank reactor Tests were carried out by continuously feeding pure CO (10 mL/min) to the culture of C. carboxidivorans in a mechanically stirred tank reactor (non-pressurized) The reactor was kept at constant temperature (35°C) and under constant agitation by rotating impellers (250 rpm) to favour the mass transfer between gas and liquid phase. The fermentation test was performed setting the initial pH at 5.75. It was proved that the production of ethanol and butanol was much more effective than in batch reactor. However, the microorganism used both the direct and the indirect route to produce alcohols, so the final acid concentration acids was limited. After 80 h, the concentration of all metabolites approached a constant value, suggesting some possible inhibitory effect impeding progression of fermentation process. It is possible that absence of any specific compound could have hindered the progression of fermentation process. Continuous stirred tank reactor An experimental activity regarded the development of a continuous stirred tank reactor, to investigate the effect of different liquid dilution rate in a range between 0.035-0.25 h-1, and different pH (5.0; 5.4, 5.6, 5.8, 6.0) using a constant flux (10 mL/min) of pure CO or a synthetic syngas (65% CO,10% CO2, 25% H2). The experiments were carried out to find the best conditions to maximize the growth and the yield of acids and solvents. Models Syngas fermentation is characterized by several potential advantages and these advantages make the process more and more attractive for industrial applications. However, strain enhancement, efficient product separation and integrated optimization of process parameters are only few aspects that can be improved from the point of view of process systems engineering. The number of models to support the syngas fermentation available in the literature is still limited and only some authors have attempted to propose kinetic expressions to fit experimental data. For this reason, a model was developed and parameters were assessed by processing experimental data from batch experiments and from literature (Fernandez at al., 2016a). A vector of 31 kinetic/yield parameters to describe the fermentation system was assessed. The soundness of the model was assessed by measuring the effect of a perturbation on the parameters, in terms of final concentration of cells and metabolites. The effect of some operating conditions (agitation speed, gas feeding flow-rate and filling ratio of the reactor) of industrial interest was investigated. A product inhibition model based on the Ierusalimsky model was developed to evaluate the results obtained in the CSTR. The results obtained from tests carried out at pH set between 5.0 to 6.0, dilution rates between 0.034 and 0.167 h-1 were used. The model correctly represents the growth kinetics of Clostridium carboxidivorans grown in CSTR fed at a constant flow of CO. Transformation of C. ljungdahlii A six-months period abroad was spent at the microbiology and biotechnology laboratory lead by professor Dürre at the University of Ulm, Germany. The project carried out during these months involved the engineering of a wild-type strain of Clostridium ljungdahlii. The aim was to provide the micro-organism of interest with the necessary genes to enable it to produce butanol, a metabolite that the WT strain is unable to produce. The strategy employed was to use two plasmids containing one 6 genes and the other 4, which were designed and constructed for use in the transformation of C. ljungdahlii.

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