The leitmotif of this Ph.D. thesis is represented by carbon dioxide (CO2) recycling via biological production of methane (CH4). This work particularly focuses on the physiology of three hydrogenotrophic methanogens, Methanothermobacter marburgensis (M. marburgensis), Methanothermococcus okinawensis (M. okinawensis) and Methanococcus maripaludis S2 (M. maripaludis), that can be used as catalysts for biological methane production (BMP) process. This CO2 recycling method is challenging due to an inefficient transfer rate of molecular hydrogen (H2) from the gas phase to the liquid phase. Thus, the biocatalyst performance is limited by H2 availability in the liquid medium. However, several factors, as strain type and media requirements, operating conditions, and reactor design, can contribute to the success of CO2 conversion to CH4. Understanding the physiology of methanogens is a powerful tool for developing a scalable BMP process. Therefore, a novel study on the role of trace metals in pure cultures of M. okinawensis and M. marburgensis respectively is herein proposed. Experimental method of this study included an in silico analysis, closed batch, and fed-batch cultivations. In silico analysis revealed genomic differences among the transport systems and enzymes related to the methanogenesis pathway of these two methanogens. The importance of Fe as metal cofactor in methanogenesis emerged from the in silico analysis and it has been confirmed by the closed batch and fed-batch experiments. M. okinawensis responded to rising concentrations of trace element (TE) by increasing specific growth rate (µ, h-1) and volumetric productivity of methane (MER, mmolL-1h-1) during closed batch cultivation. Furthermore, M. okinawensis shown growth and CH4 in fed-batch cultivation. On the base of fed-batch cultures results, M. marburgensis was prioritized and applied for CO2-based BMP process optimization. It has been proposed a new feeding strategy based on exponential fed-batch cultivation where different medium-, TE- and sulphide dilution rates combinations, and different CO2/H2 inflow rates corresponded to a defined run. The specific setting of each run produced different responses from M. marburgensis. In this context, a MER of 476 mmol L-1 h-1 and µ of 0.69 h-1 were eventually achieved at highest H2/CO2 gassing rate and ratio. However, if these factors mitigate the limitation due to the H2 mass transfer on one side, they also reduce CH4 purity in the offgas on the other side. The combined effect of increasing TE dilution and H2/CO2 gassing rates positively affected the biomass and biomass concentration. Among trace elements, there are heavy metals whose toxicity is higher than others. Heavy metals can seriously affect the functionality of microorganisms, and therefore compromise their performances as biocatalysts of a bio-based process. Not only metals, but also organic compounds, such as carboxylic acids, can damage cells survival. Thus, the second experimental part of this thesis deals with inhibition studies on pure culture of M. maripaludis in closed batch cultivation. Despite the potential applications of M. maripaludis, the knowledge surrounding this strain runs out of lab-scale studies concerning the physiology and toxicology of heavy metals and VFAs. Therefore, M. maripaludis growth and productivity were tested by using copper (Cu), zinc (Zn), acetate (Ac) and propionic acid (Pr) as potential inhibitors of microbial activity. The culture was totally inhibited at concentration of 30, 70 and 100 mgL-1 of Cu and 0.7 and 1 gL-1 of Zn. However, M. maripaludis shows tolerance to 3, 7 and 10 mgL-1 of Cu with different extent. The addition of 0.3 gL-1 of Zn to the medium, rather promoted the biomass build-up of M. maripaludis and cancelled the effect of Cu when used together in the medium. In this study, it has been supported that the inhibition by Cu is due to a reduced or suppressed activity of the CODH/ACS complex producing acetyl-CoA intermediate. Acetyl-CoA is the precursor of many metabolic subsystems (e.g. lipid, amino acids, nucleotides pathways) and its alteration would interfere with them. While CODH/ACS activity is supported by CO2 and methanogenesis intermediate, the other way to produce acetyl-CoA is based on the acetate:CoA ligase. The relevance and the tolerance to rising concentrations of Ac and Pr was also investigated and quantified via HPLC analysis. Concentration of 5 and 10 mgL-1 of acetate did not inhibit nor growth neither productivity. Interestingly, the deprivation of acetate not only impacted on the growth rate but also on methanogenesis in M. maripaludis. In absence of Ac, the same concentrations of Pr caused a slow-down of the growth, while productivity was not touched. This study sheds light on the individual and combined impact of Cu, Zn, acetate and propionic acid on the metabolism of M. maripaludis. Furthermore, an attempt to define a possible mechanism which regulates specific acetate capture is provided in this study and the relevance of acetate:CoA ligase respect to CODH/ACS complex for acetyl-CoA synthesis is herein discussed. The information collected in this study are essential to improve the process efficiency of CO2 conversion to CH4 and extend the knowledge on the physiology of certain compounds. The tendency of these methanogens to adapt to adverse conditions, most of the time, offers the possibility to improve the engineering aspects of a limited process toward an unlimited one. Moreover, as a future activity, this thesis proposed the use of a 10-bar pressure bioreactor which has been projected in the frame of the Ph.D. research with a view to improving the success of biological CH4 production.
|Titolo:||From CO2 to CH4 via biological methanation|
|Data di pubblicazione:||11-mag-2018|
|Appare nelle tipologie:||8.1 Doctoral thesis Polito|