Six species of marine microalgae (Dunaliella salina, Phaeodactylum tricornutum, Synechococcus sp., Nannochloropsis salina, Isochrysis galbana and Porphyridium purpureum) have been cultivated in the presence of phenolic compounds (tyrosol, p-coumaric acid, caffeic acid) typical of olive mill waters (OMWs) in order to select the optimal algae for phytodepuration and enhancement of the aforementioned wastewater. D. salina was the most suitable and was therefore selected because it was able to grow in the presence of all three compounds and to tolerate oxidative stresses caused by these molecules (measured through Comet assay on non-acclimatized cells). Subsequently, D. salina was acclimatized to a growth medium containing a mixture of the three phenolic compounds (each at the concentration used for selection). The microalga has proven to be able to grow. A significant increase in carbohydrates was observed exponentially compared to the control grown in the absence of phenolic compounds. The ratio between chlorophyll a and b (index of the stability of photosynthetic antennae) and the algal biomass reduction index (ratio between the absorbances detected at FTIR of saturated and unsaturated functional groups in organic matter), other than values in control cells, suggested, however, the presence of oxidative stress. From the concentration data of the phenolic compounds measured in the aqueous medium during algal growth it was noted that D. salina does not degrade any of the three compounds used and does not modulate antioxidant activity over time (measured through the DPPH assay). The alga was subsequently acclimatized and grown in OMW diluted to 10 % (v/v) with standard growth medium and exposed to two different irradiances, 100 (LL) and 300 (HL) μmol of m-2 s-1 photons. In the LL condition, algae had a 70 % reduction in growth rate of cells grown in standard medium alone, while HL did not significantly affect algal growth compared to control. Again, a significant increase in carbohydrates was observed in the algal biomass grown at HL, while the amounts of pigments remain unchanged. Through the Comet assay (on cells not acclimatized to OMWs) it was observed that after 8 days of exposure to AV, oxidative damage to the genetic material of D. salina is greatly reduced compared to what was observed after 1 day of exposure, suggesting an effective defense against oxidative stress caused by OMWs. Despite the observed response, D. salina was unable to inhibit or significantly modify the oxidizing activity of phenolic compounds contained in OMWs during acclimatization (as measured by the DPPH assay). Given the promising cellular response shown by D. salina in this thesis, studies aimed at favoring the removal of phenolic compounds by this microalgal species (through adaptive evolution in the laboratory / ALE, for example) could allow the exploitation of this alga to enhance the OMW as a growth medium and at the same time degrade the phenolic compounds present, finally, obtain valuable biomass in a sustainable way.
Sei specie di microalghe marine (Dunaliella salina, Phaeodactylum tricornutum, Synechococcus sp., Nannochloropsis salina, Isochrysis galbana e Porphyridium purpureum) sono state coltivate in presenza di composti fenolici (tirosolo, acido p-cumarico, acido caffeico) tipici di acque di vegetazione (AV) allo scopo di selezionare l’alga ottimale per la fitodepurazione e valorizzazione delle citate acque reflue. D. salina è risultata la più idonea ed è stata quindi selezionata perché capace di crescere in presenza di tutti e tre i composti e di tollerare gli stress ossidativi causati da tali molecole (misurati attraverso Comet assay su cellule non acclimatate). Successivamente, D. salina è stata acclimatata ad un mezzo di crescita contenente una miscela dei tre composti fenolici (ciascuno alla concentrazione utilizzata per la selezione). L’alga si è dimostrata in grado di crescere. Un aumento significativo dei carboidrati è stato osservato in fase esponenziale rispetto al controllo cresciuto in assenza di composti fenolici. Il rapporto tra la clorofilla a e b (indice della stabilità delle antenne fotosintetiche) e l’indice di riduzione della biomassa algale (rapporto tra le assorbanze rilevate all’FTIR dei gruppi funzionali saturi e insaturi nella materia organica), diversi dai valori in cellule di controllo, hanno suggerito tuttavia la presenza di stress ossidativo. Dai dati di concentrazione dei composti fenolici misurati nel mezzo acquoso durante la crescita algale si è notato che D. salina non degrada nessuno dei tre composti utilizzati e non modula l’attività antiossidante nel tempo (misurata attraverso il saggio del DPPH). L’alga è stata successivamente acclimatata e cresciuta in AV diluite al 10 % (v/v) con mezzo di crescita standard ed esposta a due irradianze differenti, 100 (LL) e 300 (HL) µmol di fotoni m-2 s-1. Nella condizione LL le alghe hanno presentato una riduzione del tasso di crescita pari al 70 % del tasso di cellule cresciute in solo mezzo standard, mentre la condizione HL non ha influenzato significativamente la crescita algale rispetto al controllo. Nuovamente, un aumento significativo di carboidrati è stato osservato nella biomassa algale cresciuta ad HL, mentre le quantità di pigmenti rimangono invariate. Tramite il Comet assay (su cellule non acclimatate ad AV) si è osservato che dopo 8 giorni di esposizione alle AV, i danni ossidativi al materiale genetico di D. salina sono notevolmente ridotti rispetto a quanto osservato dopo 1 giorno di esposizione, suggerendo una efficace difesa dagli stress ossidativi causati dalle AV. Nonostante la risposta osservata, D. salina non è stata in grado di inibire o modificare significativamente l’attività ossidante dei composti fenolici contenuti nelle AV durante l’acclimatazione (misurata tramite il saggio del DPPH). Vista la promettente risposta cellulare mostrata da D. salina in questa tesi, studi mirati a favorire la rimozione dei composti fenolici da parte di questa specie microalgale (tramite evoluzione adattativa in laboratorio/ALE, per esempio) potrebbero permettere lo sfruttamento di quest’alga per valorizzare le AV come mezzo di crescita e contemporaneamente degradare i composti fenolici presenti, infine ottenere biomassa pregiata in maniera sostenibile.
ESPOSIZIONE DI MICROALGHE MARINE A COMPOSTI FENOLICI: EFFETTO SULLA CRESCITA E POSSIBILE APPLICAZIONE NELLA FITO-DEPURAZIONE
DRIGO, FILIPPO
2021/2022
Abstract
Six species of marine microalgae (Dunaliella salina, Phaeodactylum tricornutum, Synechococcus sp., Nannochloropsis salina, Isochrysis galbana and Porphyridium purpureum) have been cultivated in the presence of phenolic compounds (tyrosol, p-coumaric acid, caffeic acid) typical of olive mill waters (OMWs) in order to select the optimal algae for phytodepuration and enhancement of the aforementioned wastewater. D. salina was the most suitable and was therefore selected because it was able to grow in the presence of all three compounds and to tolerate oxidative stresses caused by these molecules (measured through Comet assay on non-acclimatized cells). Subsequently, D. salina was acclimatized to a growth medium containing a mixture of the three phenolic compounds (each at the concentration used for selection). The microalga has proven to be able to grow. A significant increase in carbohydrates was observed exponentially compared to the control grown in the absence of phenolic compounds. The ratio between chlorophyll a and b (index of the stability of photosynthetic antennae) and the algal biomass reduction index (ratio between the absorbances detected at FTIR of saturated and unsaturated functional groups in organic matter), other than values in control cells, suggested, however, the presence of oxidative stress. From the concentration data of the phenolic compounds measured in the aqueous medium during algal growth it was noted that D. salina does not degrade any of the three compounds used and does not modulate antioxidant activity over time (measured through the DPPH assay). The alga was subsequently acclimatized and grown in OMW diluted to 10 % (v/v) with standard growth medium and exposed to two different irradiances, 100 (LL) and 300 (HL) μmol of m-2 s-1 photons. In the LL condition, algae had a 70 % reduction in growth rate of cells grown in standard medium alone, while HL did not significantly affect algal growth compared to control. Again, a significant increase in carbohydrates was observed in the algal biomass grown at HL, while the amounts of pigments remain unchanged. Through the Comet assay (on cells not acclimatized to OMWs) it was observed that after 8 days of exposure to AV, oxidative damage to the genetic material of D. salina is greatly reduced compared to what was observed after 1 day of exposure, suggesting an effective defense against oxidative stress caused by OMWs. Despite the observed response, D. salina was unable to inhibit or significantly modify the oxidizing activity of phenolic compounds contained in OMWs during acclimatization (as measured by the DPPH assay). Given the promising cellular response shown by D. salina in this thesis, studies aimed at favoring the removal of phenolic compounds by this microalgal species (through adaptive evolution in the laboratory / ALE, for example) could allow the exploitation of this alga to enhance the OMW as a growth medium and at the same time degrade the phenolic compounds present, finally, obtain valuable biomass in a sustainable way.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12075/10546