| Abstract |
The current contribution provides for the first time proof that selection of appropriate conditions of microorganisms for the biodegradation of toxic compounds is of greater importance than selection of individual organisms. We showed that the bioenergetic balance of microalgae adjusts the selection of the optimal pathway and the activation of the necessary metabolic mechanisms for the biodegradation process.
The bioenergetic strategy for biodegradation of phenolic compounds by the unicellular green alga Scenedesmus obliquus, described herein, has been proven beyond doubt to be a thermodynamically, photoregulated process. Selection of the appropriate biodegradation pathway is determined by the energy balance of the alga, which in turn is determined by a range of biotic and abiotic parameters. The position (ortho, meta or para) and the number of substituents in the phenolic ring, as well as the resonance and induction effects, which control whether the substituent will act towards donating or receiving electrons, determine the energy demand for the biodegradation of phenolic compounds. In addition, the exogenous carbon source (organic or/and inorganic) and photon intensity are the primary factors that regulate [via linear and cyclic electron flows in photosynthesis, respiratory chain via cytochrome or/and alternative oxidase in the mitochondria, as well as chlororespiration (PTOX) in the chloroplast] the energy reserves of the green alga, which will be contributed towards biodegrading phenolic compounds with varying degrees of toxicity and dissociation strength.
The simplest phenolic compound, phenol, is used by the unicellular green alga Scenedesmus obliquus, as an alternative carbon source, whereas in combination with the appropriate photon intensity total metabolism (100% biodegradation) is achieved.
Increase of the biodegradation difficulty, by adding a halogen substituent (Cl, Br, I) in the phenolic ring, forces the microalga to degrade the phenolic compounds by means of cometabolism, which is carried out in two distinct phases. During the first phase the substituent (halogen) is dissociated, while during the second phase the phenolic ring is broken down. The biodegradation mechanism of methylphenols provides further evidence for the existence of a process for the biodegradation of phenolic compounds with one substituent, such as the one described above. After the dissociation of the substituent (the methyl group) from the phenolic ring, methanol is produced supporting the photosynthetic activity of the microalga, since it has been proven (from previous publications of our laboratory) that methanol is metabolized to CO2.
Since biodegradation is a RedOx process, the type of the substituent determines how easy it will be to degrade the compound. In cases where substituents acting as electron acceptors (e.g. nitro-group) are located close to a substituent acting as an electron donor (phenol hydroxide), biodegradation is achieved. In fact the distance between two such substituents determines how easy it will be to biodegrade the compound, closer distance leading to more intense biodegradation. On the contrary when an electron donor group (e.g. a methyl-group) is located nearby a substituent acting as an electron donor (phenol hydroxide), it renders the biodegradation process harder. On the other hand, when the substituent cannot be classified as an electron donor or acceptor, (e.g. halogen) from the resonance and induction effects, but according to the conditions, acts as a donor or an acceptor, then the phenolic compound is degraded with similar ease, since the green alga selects the optimal strategy.
Further increase in the biodegradation difficulty of phenolic compounds by the green alga Scenedesmus obliquus, achieved by the addition of a second substituent in the phenolic ring (dichlorophenols), alters the extent of the biodegradation process in correlation to the number of meta-substituted chlorides present in the phenolic ring. It has been proven that in conditions of increased toxicity (dichlorophenols) the green alga invests more energy for the biodegradation process and less energy for growth, whereas for less toxic phenolic compounds (monochlorophenols) ratio between biodegradation and growth is reversed.
The first step in the biodegradation of dichlorophenols is their reduction. The reduced form of 2,3-, 2,5-, and 3,4-dichlorophenols can be inserted in the photosynthetic electron transport chain, right before the plastoquinone (PQ) pool, based on its RedOx potential. There it acts as an electron donor, feeding PSI with electrons, and in doing such, inhibiting the activity of PSII and therefore, O2 production. This unique event of combined anoxia and exclusive activation of PSI, induces hydrogenase activity and hydrogen (H2) production.
The dichlorophenols in question can also transfer electrons directly to protons, and in doing such, form molecular hydrogen (H2) according to the reaction 2H+ + Dred→ H2 + Dox. This justifies the particularly high concentrations of produced hydrogen compared to the anoxic control (up to 125 times higher)
The bioenergetic strategy of the microalga for the biodegradation of the dichlorophenols in question creates the unique conditions, which allow a “green” biodegradation procedure of toxic compounds and in the same time production of large quantities of bio-hydrogen (H2) for further biotechnological applications.
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Βιοενεργητική στρατηγική βιοποικοδόμησης φαινολικών ενώσεων από το Χλωροφύκος Scenedesmus obliquus - Βιοτεχνολογικές προεκτάσεις
