Nowadays, renewable energy is one of the most discussed issues by the international scientific community. The unrestrained use of fossil fuels has raised relevant questions about sustainability and effects on the environment. Hydrogen (H2) is considered a promising fuel due to its thermodynamical properties and CO2-free combustion. Nevertheless, environmental problems arise when H2 is produced using energy deriving from fossil sources: only “green hydrogen” identifies a production 100% based on renewable energy. Currently, several microorganisms are known for their ability to produce H2 as a metabolic feature. Regarding microalgae, most of the information comes from Chlamydomonas reinhardtii. In this green microalgae, two different photosynthetic production pathways and one fermentative-like metabolism have been described concerning transitory H2 production. To extend H2 production one requirement is the creation of a hypoxic environment. This occurs when photosynthetic activity slows down or if there is an increase in mitochondrial respiration rates. Moreover, the electron flow should be directed preferentially towards the H2 evolution enzyme, hydrogenase. Concerning physiological conditioning, one of the most promising strategies for H2 production is sulphur deprivation from cultivation medium: within three days, anaerobiosis develops under saturated light. Chlamydomonas has also represented an excellent example for the development of different molecular strategies, that allow to overcome some limitations and extend the H2 production. One of the limitations is related to sunlight saturation and dissipation that also affect H2 production. Mutants with the truncated light-harvesting antenna (tla) in the chloroplast are subject to fewer phenomena of photoinhibition and light saturation. Another limitation is linked to the competitive pathways that remove electrons from hydrogenase. Pgrl1 (protein gradient regulation like 1) mutant showed improved H2 production reducing this phenomenon. Another relevant issue is the oxygen sensitivity of the hydrogenase enzyme. The use of an O2-tolerant clostridial [FeFe]-hydrogenase, expressed in C. reinhardtii, showed better enzymatic rate, as the bacterial hydrogenase had a lower inactivation rate in aerobiosis. The work carried out over the past three years was aimed primarily at the isolation and characterization of microalgal species in the Basilicata region for the identification of new biohydrogen producers. Particular attention has been given to the search for strains with good growth rates and able to use different carbon sources. Secondly, the physiological behaviour of single and double mutants of Chlamydomonas was analyzed concerning H2 production by modulating light condition without resorting to stress application, such as sulphur deprivation. Freshwater samples collected in different villages of the Basilicata region were used to isolate microalgae with different morphologies. Microscopical observations and molecular identification made it possible to identify the genus of the isolated pure colonies. The growth of the various strains was followed by different methods: absorbance and chlorophyll content proved to be effective and fast for monitoring cell growth over the days. This made it possible to evaluate the growth rates of the species under examination. Various tests were carried out to detect the production of H2. Bioreactors were kept in dark, limited light (12 PAR) or sulfur deprivation (with intense light, 100 PAR). All the experiments considered different carbon source too. The levels of H2 gas produced were daily assessed by gas chromatography by taking a sample of the airspace in contact with the liquid culture in the airtight bioreactors. Desmodesmus sp. and Haematococcus sp. strains demonstrated production of H2 similar to wild type Chlamydomonas (5-10 ml/litre of culture). Furthermore, the same production occurred similarly using acetate or glucose. For Chlamydomonas mutants, the experiments were conducted in collaboration with the University of Córdoba (Spain). Investigated mutants were tla3, pgrl1, and one engineered with Clostridium bacterial hydrogenase (clostr) and the relative combinations tla3 + pgrl1 and clostr + pgrl1 from genetic cross. In this case, the wild type, single and double mutant strains were subjected to different lighting conditions (12, 50, 100, 450 PAR). In particular, the combination tla3 + pgrl proved to be the best as it is capable of producing H2 even at light intensities that are generally less tolerated, opening up new application scenarios. The single mutant Clostr showed instead a fast hydrogenase activity in a replete media also proportionally with the increase of light. In conclusion, the algae isolated during the PhD project have shown interesting implications for the production of H2 such as the metabolic versatility regarding the use of the different carbon sources. This leads to the need to carry out a more in-depth investigation of the mechanisms underlying the metabolism of these microalgae both from a physiological and a molecular point of view. Regarding single and double Chlamydomonas mutants, knowledge about their behaviour in different light conditions and the feasibility of H2 production has been expanded

“Green microalgae biohydrogen production” / Limongi, Antonina Rita. - (2022 Mar 10).

“Green microalgae biohydrogen production”

LIMONGI, Antonina Rita
2022-03-10

Abstract

Nowadays, renewable energy is one of the most discussed issues by the international scientific community. The unrestrained use of fossil fuels has raised relevant questions about sustainability and effects on the environment. Hydrogen (H2) is considered a promising fuel due to its thermodynamical properties and CO2-free combustion. Nevertheless, environmental problems arise when H2 is produced using energy deriving from fossil sources: only “green hydrogen” identifies a production 100% based on renewable energy. Currently, several microorganisms are known for their ability to produce H2 as a metabolic feature. Regarding microalgae, most of the information comes from Chlamydomonas reinhardtii. In this green microalgae, two different photosynthetic production pathways and one fermentative-like metabolism have been described concerning transitory H2 production. To extend H2 production one requirement is the creation of a hypoxic environment. This occurs when photosynthetic activity slows down or if there is an increase in mitochondrial respiration rates. Moreover, the electron flow should be directed preferentially towards the H2 evolution enzyme, hydrogenase. Concerning physiological conditioning, one of the most promising strategies for H2 production is sulphur deprivation from cultivation medium: within three days, anaerobiosis develops under saturated light. Chlamydomonas has also represented an excellent example for the development of different molecular strategies, that allow to overcome some limitations and extend the H2 production. One of the limitations is related to sunlight saturation and dissipation that also affect H2 production. Mutants with the truncated light-harvesting antenna (tla) in the chloroplast are subject to fewer phenomena of photoinhibition and light saturation. Another limitation is linked to the competitive pathways that remove electrons from hydrogenase. Pgrl1 (protein gradient regulation like 1) mutant showed improved H2 production reducing this phenomenon. Another relevant issue is the oxygen sensitivity of the hydrogenase enzyme. The use of an O2-tolerant clostridial [FeFe]-hydrogenase, expressed in C. reinhardtii, showed better enzymatic rate, as the bacterial hydrogenase had a lower inactivation rate in aerobiosis. The work carried out over the past three years was aimed primarily at the isolation and characterization of microalgal species in the Basilicata region for the identification of new biohydrogen producers. Particular attention has been given to the search for strains with good growth rates and able to use different carbon sources. Secondly, the physiological behaviour of single and double mutants of Chlamydomonas was analyzed concerning H2 production by modulating light condition without resorting to stress application, such as sulphur deprivation. Freshwater samples collected in different villages of the Basilicata region were used to isolate microalgae with different morphologies. Microscopical observations and molecular identification made it possible to identify the genus of the isolated pure colonies. The growth of the various strains was followed by different methods: absorbance and chlorophyll content proved to be effective and fast for monitoring cell growth over the days. This made it possible to evaluate the growth rates of the species under examination. Various tests were carried out to detect the production of H2. Bioreactors were kept in dark, limited light (12 PAR) or sulfur deprivation (with intense light, 100 PAR). All the experiments considered different carbon source too. The levels of H2 gas produced were daily assessed by gas chromatography by taking a sample of the airspace in contact with the liquid culture in the airtight bioreactors. Desmodesmus sp. and Haematococcus sp. strains demonstrated production of H2 similar to wild type Chlamydomonas (5-10 ml/litre of culture). Furthermore, the same production occurred similarly using acetate or glucose. For Chlamydomonas mutants, the experiments were conducted in collaboration with the University of Córdoba (Spain). Investigated mutants were tla3, pgrl1, and one engineered with Clostridium bacterial hydrogenase (clostr) and the relative combinations tla3 + pgrl1 and clostr + pgrl1 from genetic cross. In this case, the wild type, single and double mutant strains were subjected to different lighting conditions (12, 50, 100, 450 PAR). In particular, the combination tla3 + pgrl proved to be the best as it is capable of producing H2 even at light intensities that are generally less tolerated, opening up new application scenarios. The single mutant Clostr showed instead a fast hydrogenase activity in a replete media also proportionally with the increase of light. In conclusion, the algae isolated during the PhD project have shown interesting implications for the production of H2 such as the metabolic versatility regarding the use of the different carbon sources. This leads to the need to carry out a more in-depth investigation of the mechanisms underlying the metabolism of these microalgae both from a physiological and a molecular point of view. Regarding single and double Chlamydomonas mutants, knowledge about their behaviour in different light conditions and the feasibility of H2 production has been expanded
biohydrogen; microalgae; green algae; hydrogenase; biofuel.
“Green microalgae biohydrogen production” / Limongi, Antonina Rita. - (2022 Mar 10).
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11563/154466
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