Uklanjanje fenola iz otpadnih voda sortama zelene salate (Lactuca sativa L.)

Abstract

U ovoj doktorskoj disertaciji ispitivana je mogućnost uklanjanja fenola iz rastvora, korišćenjem različitih sorti zelene salate, sa ciljem njihove upotrebe u procesu fitoremedijacije. Ispitivano je 11 različitih sorti zelene salate, čija semena su klijala na podlogama sa različitim koncentracijama fenola (0-400 mgL-1) tokom 20 dana. Pokazano je da dve sorte zelene salate mogu klijati na visokim koncentracijama fenola. Sorta Ljubljanska ledenka (LJL) klija na koncentraciji fenola do 350 mgL-1 i sorta Nansen (N) podnosi koncentraciju fenola do 300 mgL-1. Dalja istraživanja aktivnosti antioksidativnih enzima peroksidaza (POX), katalaza (CAT), kao polifenol oksidaza (PPO) rađena su na klijancima koji su gajeni na inhibitronim koncentracijama fenola (IK50), odnosno onim koncentracijama fenola, specifičnim za svaku sortu, na kojima klija 50% semena. Rezultati su prikazani za četiri sorte zelene salate i pokazano je da je aktivnost POX relativno niska, dok je aktivnost CAT i PPO viša u odnosu na klijance koji su gajeni bez prisustva fenola. Uporedo je praćena i morfologija klijanaca koji su gajeni na IK50 fenola. Praćen je tempo uklanjanja fenola iz otpadne vode i analiziran je tokom gajenja odrasle zelene salate u hidroponičnim uslovima. Odabrane su dve sorte zelene salate (LJL i N) koje su pokazale najveću otpornost na prisustvo fenola, i gajene su na koncentraciji fenola u rastvoru od 200 mgL-1. Ova koncentracija fenola je najviša koncentracija ispod IK50 na kojoj klija 100% semena. Pomenuta koncentracija fenola dodavana je u hidroponični rastvor na početku eksperimenta (početna koncentracija fenola) ili svaka dva dana tokom trajanja eksperimenta (konstantna koncentracija fenola). Praćena je promena koncentracije fenola u rastvoru posle 2, 4, 6, 8 i 10 dana gajenja, kao i aktivnost enzima (POX, CAT, SOD i PPO) u korenu i listovima zelene salate. Obe sorte zelene salate uklanjaju fenol iz rastvora za osam dana, a brzina uklanjanja fenola je veća kod sorte LJL u odnosu na sortu N. Fenol konstantne koncentracije biljke obe sorte nisu u mogućnosti da u potpunosti uklone, a kapacitet za uklanjanje fenola kontantne koncentracije gubi se posle osam dana gajenja. Kod svih ispitivanih enzima, osim POX, primećen je porast aktivnosti tokom gajenja biljaka na fenolu. Pored promena u enzimskoj aktivnosti, primećena je promena u koncentraciji prolina, hlorofila, ukupnih pigmenata, kao i anatomiji korena zelene salate koja je gajena na fenolu. Uspešno je dobijena kultura hairy roots zelene salate genetičkom transformacijom pomoću Agrobacterium rhizogenes A4M70GUS, a najveći prinos biomase imala je linija Ljubljanska ledenka 18 (LJL18), koja je dalje gajena na različitim koncentracijama fenola u rastvoru, da bi se ispitala mogućnost fitoremedijacije pomoću kontinualne kulture korenova. Pokazano je da linija LJL18 uklanja fenol početne koncentracije od 100 mgL-1 iz rastvora za deset dana, dok kultura netransformisanih korenova iste sorte može ukoliniti najviše 50 mgL-1 fenola. Pored praćenja morfologije transformisanih korenova gajenih na fenolu, analizirana je i aktivnost enzima (POX, CAT, SOD i PPO) posle 2, 4, 6, 8 i 10 dana gajenja. Primećena je značajna promena u aktivnosti enzima tokom gajenja hairy roots na početnoj ili konstantnoj koncentraciji, kao i razlika u odnosu na netransformisane korenove podvrgunte istom tretmanu. Rezultati ove doktorske disetracije dokazuju da se zelena salata može uspešno koristiti za uklanjanje fenola iz otpadnih voda korišćenjem celih biljaka ili hairy roots kulture.

In this doctoral dissertation, the possibilities of use of various cultivars of lettuce have been investigated for removal of dissolved phenol in the process of phytoremediation. Seeds of 11 different cultivars of lettuce germinated on media with different phenol concentrations (0-400 mgL-1) over 20 days. It has been shown that seeds of two cultivars of lettuce can germinate even on high phenol concentrations: Seeds of Ljubljanska ledenka (LJL) can germinate on 350 mgL-1, while Nansen (N) tolerates as high as 300 mgL-1. Further investigation of antioxidant enzyme proxidases (POX), cataleses (CAT) and polyphenol oxidase (PPO) activity was carried out on seedlings grown on inhibitory concentrations of phenol (IC50), that is, the cultivar-specific concentration of phenol reducing the germination efficiency to 50% for the seeds of a given cultivar. Results are shown for four lettuce cultivars, revealing that POX activity is relatively low, while CAT and PPO activity show higher values compared to seedlings grown without phenol. Morphological features of seedlings grown on IC50 of phenol were also recorded. Dynamics of phenol removal from waste water were recorded in adult lettuce plants grown hydroponically. Two lettuce cultivars that showed the highest tolerance to phenol, were chosen for hydroponic growth at 200 mgL-1 phenol, which is the highest concentration on which 100% germination has been recorded for these cultivars. This concentration of phenol was either established in the hydroponic solution once, at the beginning of the experiment (this was called the "starting phenol concentration") or maintained by supplying additional phenol to the solution every two days (which was called the "constant phenol concentration"). Changes in phenol concentration in the solution, as well as enzyme activity (POX, CAT, SOD and PPO) in lettuce roots and leaves were recorded after 2, 4, 6, 8 and 10 days of growth. Both lettuce cultivars are able to completely remove the starting concentration of phenol from the solution within eight days, the cultivar LJL being more efficient than N. Plants of both cultivars were unable to completely remove the phenol at constant concentration. Furthermore, they lost the capacity for removing constant concentrations of phenol after eight days. All the investigated enzymes except POX show a trend of activity increase over the course of growth on media containing phenol. Beside changes in enzyme activity, changes in endogenous concentrations of proline, chlorophyll, total pigments, as well as changes in root anatomy of lettuce grown on phenol, were also recorded. A culture of lettuce hairy roots was successfully established with the use of Agrobacterium rhizogenes A4M70GUS, with the highest value for biomass yield being recorded for the clone Ljubljanska ledenka 18 (LJL 18), which was further cultivated on different concentrations of phenol in the solution, in order to investigate the potential of use of continuous root cultures for phytoremediation purposes. It has been shown that LJL18 can remove the 100 mgL-1 starting concentration of phenol from the solution within 10 days, while the culture of non-transformed roots of the same cultivar can remove only 50 mgL-1 phenol. Significant changes in POX, CAT, SOD and PPO activity were recorded over the course of hairy roots growth on starting or constant concentrations of phenol, as well as a difference in comparison to non-transformed roots growing in the same conditions. The results of this doctoral dissertation show that lettuce, either in the form of intact plants or hairy roots cultures, can be successfully used for removing phenol from waste waters.