Resumen de Innovative approaches for enhancing the cost-efficiency of biological methane abatement
Methane (CH4), which is considered the second most important greenhouse gas (GHG), represents nowadays 18 % of the total EU-28 GHG emissions and possesses a global warming potential 25 times higher than that of CO2 (in a 100 yr horizon). The methane produced during the anaerobic digestion of organic waste (wastewater and organic solid waste) can be collected and combusted for the generation of electricity and/or heat, thus replacing fossil fuels. However, methane can be only used for energy generation when its concentration in the emission is higher than 20 %. Unfortunately, more than 56 % of the anthropogenic CH4 emissions contain CH4 concentrations below 5 %, which significantly limits the implementation of energy recovery-based treatment technologies. Therefore, cost-efficient abatement methods to mitigate the pernicious environmental effects of residual CH4 emissions are required.
In this regard, despite the environmental relevance of CH4 and forthcoming stricter regulations, the development of cost-efficient and environmentally friendly GHG treatment technologies is nowadays very limited. Hence, even state-of-the-art physical/chemical technologies are either costly or inefficient when treating the emissions of the largest sources of CH4 (waste treatment, livestock farming, mining, fuel combustion). In this context, despite biological technologies have been consistently proven as a low-cost, efficient, and environmentally friendly method for the treatment of gas emissions containing malodours or industrial volatile organic compounds, the application of conventional biotechnologies for the abatement of this GHG is nowadays severely limited by: 1) its poor mass transport from the gas to the microbial community as a result of its poor aqueous solubility 2) the lack of cost-competitive abatement methods for the treatment of diluted CH4 emissions 3) the limited knowledge of the microbial communities and biodegradation kinetics of methane at the trace level concentrations typically encountered during the treatment of diluted CH4 emissions.
The most important limitation of biological methane abatement is caused by the low aqueous solubility of this GHG (Henry’s law constant (H) = 30 at 25ºC). This limited mass transport entails process operation at high empty bed gas residence times, which significantly increases both the investment and operating costs of methane treatment biotechnologies. In this regard, the implementation of a new concept of biological gas treatment based on the direct gas-cell transport of CH4 could enhance the mass transport of this hydrophobic GHG. Multiple innovative and high-performance technologies for biological gas treatment have emerged in the last 10 years: two-phase partitioning bioreactors (TPPB), membrane bioreactors, fungal-based biofilters and Taylor flow bioreactors. Unfortunately, the number of studies assessing the potential of these bioreactors for the treatment of CH4 is still scarce.
In this regard, a part of the research carried out on the present thesis focused on the comparative performance evaluation of conventional and two-phase partitioning stirred tank reactors for methane abatement. This research demonstrated for the first time the capability of methanotrophs to grow inside the non-aqueous phase (NAP) and identified the optimum cultivation conditions for enrichment of hydrophobic methanotrophs (high dilution rates and low agitation rates). The potential of the hydrophobic methanotrophs enriched was assessed in a single-phase stirred tank reactor and in a two-phase stirred tank reactor containing 60 % of silicone oil. Contrary to what it was expected, the results showed that the single-phase stirred tank reactor achieved higher elimination capacities (EC up to ≈3 times) than the TPPB. This might be due to the low affinity of silicone oil for methane, which is only 10 times higher than that of CH4 for water, as well as to the limitation in nutrients or water activity inside the NAP, which can cause limitations in biological activity.
Based on the above results, alternative biotechnologies were developed, implemented and evaluated along this thesis, with a focus on biological methods that could make this process more environmentally friendly and cost-competitive. More specifically, the present thesis focused on the valorization of dilute methane emissions via CH4 bio-conversion into ectoine, which is a cyclic imino acid used in the pharmaceutical industry due to its high effectiveness as stabilizer of enzymes, DNA-protein complexes and nucleic acids, with a retail value of approximately US$1000 kg-1. Despite its potential, ectoine is only currently produced biotechnologically by Halomonas elongate through a long fed-batch fermentation process called bio-milking, which consists of sequential hypo and hyper osmotic shocks. However, this process is still inefficient due to the high cost of the sugar-based substrate used. In 1999, the team of Khemelenina and co-workers described a new species of haloalkalophilic bacterium, Methylomicrobium alcaliphilum 20Z, capable of synthesizing ectoine from methane. However, little was known about the influence of environmental conditions on the bioproduction of this secondary metabolite when combined with the abatement of diluted CH4 emissions. In addition, no study addressing the continuous production of ectoine by M. alcaliphilum 20Z has been carried out to date using methane as the sole carbon and energy source.
In this context, a first attempt assessing the influence of operational conditions on the bio-conversion of CH4 into ectoine by the methanotrophic ectoine-producing strain M. alcaliphilum 20 Z was carried out in this thesis. The results obtained demonstrated that a proper selection of the environmental parameters (temperature, CH4, Cu2+ and NaCl concentration) during M. alcaliphilum 20Z cultivation was crucial to simultaneously maximize both, the intra-cellular production and excretion of ectoine and CH4 abatement. Hence, concentrations of 20 % CH4, 6 % NaCl, 25 µM Cu2 and a temperature of 25 ºC supported a maximum intra-cellular ectoine production yield of 67 ± 4 mg g biomass-1. On the other hand, extra-cellular ectoine concentrations were detected at high Cu2+ concentrations despite this methanotroph has not been previously classified as an ectoine-excreting strain. These promising results supported further research in order to implement the bio-conversion of CH4 into ectoine in a continuous system capable of creating value out of GHG mitigation. In this regard, a study of ectoine production during the continuous abatement of diluted emissions of CH4 by M. alcaliphilum 20Z in stirred tank reactors under non-sterile conditions was carried out during the development of this thesis. In this research, NaCl concentration was identified as the main factor influencing the accumulation of intra-cellular ectoine in continuous mode, with high salt concentrations of 6% NaCl inducing average intra-cellular ectoine yields of 37 ± 4 mg g biomass-1. Moreover, it was observed that process operation at high agitation rates (600 rpm) damaged cell integrity, with a subsequent decrease in both CH4 removals and ectoine yields. Thus, this study demonstrated the feasibility of producing ectoine in continuous bioreactors during methane treatment and identified the best operational conditions of the process. These results encouraged further research in order to implement CH4 biorefineries for the production of ectoine in a fed-batch fermentation using a bio-milking approach. The results demonstrated that M. alcaliphilum 20Z exhibited a rapid response to osmotic shocks in batch and continuous mode, which resulted in the release of the accumulated ectoine under hyposmotic shocks and the immediate uptake of the previously excreted ectoine during hyperosmotic shocks. The intra-cellular ectoine yield obtained in this study under optimal operational conditions accounted for 70 ± 14 mg g biomass-1 under steady state, and constituted the first proof of concept of ectoine bio-milking coupled to CH4 abatement from diluted emissions.
Although at this point, the perspective of a future methane-based biorefinery was promising, the implementation of these CH4 bio-conversion processes was still limited by physical and biological limitations. Thus, the low growth rate of M. alcaliphilum 20Z and its fragility against mechanical stress hampered the process productivities obtained. These findings lead to explore the enhancement of the cost-effectiveness of the bio-conversion of methane into ectoine by i) co-producing multiple metabolites using methane as the sole carbon and energy source, ii) isolating new haloalkaliphilic methanotrophs able to produce ectoine and iii) determining the best culture conditions to obtain high bio-conversion rates of methane.
In this context, a bubble column bioreactor configuration was used to assess the co-production of multiple metabolites of high-added market value during CH4 mitigation using an enriched methanotrophic consortium (consisted mainly of the genera Halomonas, Marinobacter, Methylophaga and Methylomicrobium). This consortium was capable of accumulating the same concentration of ectoine than M. alcaliphilum (80 mg ectoine g biomass-1) with the concomitant synthesis of hydroxyectoine (up to 13 mg hydroxyectoine g biomass-1) and exopolysaccharides (up to 2.6 g EPS g biomass-1). Unfortunately, bioplastics were not accumulated inside the methanotrophic consortium based on the absence of a fasting period that allowed the bacterial synthesis of PHAs. This research represented the first proof of concept of a methane biorefinery based on the multi-production of high profit margin substances using methane as the sole carbon and energy source. Additionally, this thesis also focused on the enrichment and isolation of novel haloalcaliphilic methanotrophic strains able to support high productivities of ectoine during CH4 abatement, which resulted in the discovery of two novel methane oxidizing bacterial species that were not previously identified as methanotrophic bacteria (Alishewanella and Halomonas). Based on a dissimilarity of at least 98 % in the 16SrDNA sequences with their closest relatives, these bacteria were described as two novel strains (representing novel species): Alishewanella sp. strain RM1 and Halomonas sp. strain PGE1. Halomonas sp. strain PGE1 presented higher ectoine yields (70-92 mg ectoine g biomass-1) than those generally obtained in M. alcaliphilum 20Z (37.4 to 70.0 mg ectoine g biomass-1), although the growth of Halomonas sp. strain PGE1 was likely inhibited by the production of a toxic metabolite.
Finally, the influence of Cu2+ and CH4 concentrations and CH4 mass transfer rate during culture enrichment on the community structure and the CH4 biodegradation kinetics was studied in this thesis in order to standardize the enrichment conditions for highly efficient methanotrophs. The results obtained demonstrated that an increase in Cu2+ concentration from 0.05 to 25 μM increased the qmax and Ks of the communities enriched by a factor of ≈ 3. In addition, high Cu2+ concentrations supported the growth of more adapted methanotrophs and less diverse bacterial communities. However, no clear effect of CH4 concentration on the population structure or on the biodegradation kinetics of the communities enriched was recorded at the two CH4 concentrations studied (4 and 8%). This study showed the key role of enrichment conditions to develop microbial communities capable of maintaining a high efficiency and robustness, overcoming some of the current limitations of biotechnologies and improving methane abatement performance.
The results obtained in the present thesis open up a new door of possibilities towards a more cost-effective methane treatment based on the bio-conversion of this GHG into products with a high profit margin, which could completely switch the current conception of GHG abatement technologies.
