Combinational system for biodegradation of olive oil mill wastewater phenolics and high yield of bio-hydrogen production.

Olive oil mill wastewater (OMW) is a significant pollutant in the Mediterranean region. In the present contribution, we showed clearly that microorganisms (microalgae and OMW-microflora) activated the biodegradation of OMW-phenolics and produced a high yield of hydrogen (H2). In a closed incubation system, the appropriate adjustment of OMW-pH leads to the establishment of anoxic conditions through the oxygen consumption of microorganisms during the first incubation day. The biodegradation procedure of OMW-phenolics needs oxygen. Therefore, after the establishment of anoxic conditions, the biodegradation stopped and the activation of hydrogenases started, leading to a continuous high yield of bio-hydrogen production. If the cultivation system re-opened (oxygen enrichment), the OMW-phenolic biodegradation (oxygen dependent process) started again and therefore the detoxified OMW could be used for further biotechnological applications (production of biodiesel, bioalcohols, organic fertilizers, etc.). Apart from the environmental compatibility of the method and the sustainability of such a combinational application (OMW detoxification and high yield of hydrogen production) in the context of a green biotechnology approach, the cost/profit ratio appears to be particularly tempting and guarantees its widespread use in the near future. The present contribution proposes a solution to a major environmental problem by upgrading its solution to a high-value product.

Comparative biodegradation of all chlorinated phenols by the microalga Scenedesmus obliquus - The biodegradation strategy of microalgae.

This work presents the comparative biodegradation of all chlorinated phenolic compounds by the green alga Scenedesmus obliquus and determines the microalgal bioenergetic strategy. The microalga manages its energy reserves rationally by investing them, either on growth or on the biodegradation of the toxic compound. The microalga seems to follow two distinct detoxification strategies. In the first one, when microalgae are surrounded by relatively low toxic phenolic compounds (phenol, monochlorophenols, 2,4-dichlorophenol and 2,6-dichlorophenol), they use all, or at least more of their energy reserves to increase the biomass production and not the biodegradation. In the second one, when surrounded by higher toxic chlorophenols (meta-substituted dichlorophenols, trichlorophenols, tetrachlorophenols and pentachlorophenol) the microalgae invest more, or all of their energy reserves directly in the biodegradation of the toxic compounds, while less or no energy is invested in biomass increase. The microalga biodegraded in five days approximately 9% of the lower toxic phenol and 90% of the higher toxic pentachlorophenol. Considering our ability to interfere with microalgae energy reserves, which define their stress tolerance in the toxic environment, and knowing the microalgal bioenergetic strategy, we could easily use microalgae to biodegrade toxic wastes in the frame of a rational biotechnological approach in the near future.

Lichen as Micro-Ecosystem: Extremophilic Behavior with Astrobiotechnological Applications.

This work demonstrates the tolerance of lichen Pleurosticta acetabulum under extreme conditions similar to those encountered in extraterrestrial environments. Specifically, the impact of three extreme Mars-like conditions-complete dehydration, extremely low temperature (-196°C/77K), and oxygen depletion-on lichens was investigated. The symbiosis of mycobiont and photobiont partners creates a micro-ecosystem that ensures viability of both symbiotic partners under prolonged desiccation and extremely low temperatures without any cultivation care. Changes in the molecular structure and function of the photosynthetic apparatus, in the level of chlorophylls, polyamines, fatty acids, carbohydrates, ergosterol, efflux of K+, and DNA methylation ensure the ecological integrity of the system and offer resistance of lichens to above-mentioned extreme environmental conditions. For the first time, we also demonstrate that the unprecedented polyextremophilic characteristic of lichens could be linked to biotechnological applications, following exposure to these extreme conditions, such that their ability to produce a high yield of hydrogen was unchanged. All these support that lichens are (a) ideal model systems for a space mission to inhabit other planets, supporting also the aspect that the panspermia theory could be extended to incorporate in the traveling entities not only single organisms but micro-ecosystems like lichens, and (b) ideal model systems for astrobiotechnological applications (hydrogen production), such as in the development of bioregeneration systems for extraterrestrial environments.

Bioenergetic reprogramming plasticity under nitrogen depletion by the unicellular green alga Scenedesmus obliquus.

MAIN CONCLUSION: Simultaneous nitrogen depletion and 3,4-dichlorophenol addition induce a bioenergetic microalgal reprogramming, through strong Cyt b 6 f synthesis, that quench excess electrons from dichlorophenol's biodegradation to an overactivated photosynthetic electron flow and H 2 -productivity. Cellular energy management includes "rational" planning and operation of energy production and energy consumption units. Microalgae seem to have the ability to calculate their energy reserves and select the most profitable bioenergetic pathways. Under oxygenic mixotrophic conditions, microalgae invest the exogenously supplied carbon source (glucose) to biomass increase. If 3,4-dichlorophenol is added in the culture medium, then glucose is invested more to biodegradation rather than to growth. The biodegradation yield is enhanced in nitrogen-depleted conditions, because of an increase in the starch accumulation and a delay in the establishment of oxygen-depleted conditions in a closed system. In nitrogen-depleted conditions, starch cannot be invested in PSII-dependent and PSII-independent pathways for H2-production, mainly because of a strong decrease of the cytochrome b 6 f complex of the photosynthetic electron flow. For this reason, it seems more profitable for the microalga under these conditions to direct the metabolism to the synthesis of lipids as cellular energy reserves. Nitrogen-depleted conditions with exogenously supplied 3,4-dichlorophenol induce reprogramming of the microalgal bioenergetic strategy. Cytochrome b 6 f is strongly synthesized (mainly through catabolism of polyamines) to manage the electron bypass from the dichlorophenol biodegradation procedure to the photosynthetic electron flow (at the level of PQ pool) and consequently through cytochrome b 6 f and PSI to hydrogenase and H2-production. All the above showed that the selection of the appropriate cultivation conditions is the key for the manipulation of microalgal bioenergetic strategy that leads to different metabolic products and paves the way for a future microalgal "smart" biotechnology.

Bioenergetic strategy of microalgae for the biodegradation of tyrosol and hydroxytyrosol.

Olive mill wastewater has significant polluting properties due to its high phenolic content [mainly tyrosol (trs) and hydroxytyrosol (htrs)]. Growth kinetics and a series of fluorescence induction measurements for Scenedesmus obliquus cultures showed that microalgae can be tolerant of these phenolic compounds. Changes in the cellular energy reserves and concentration of the phenolic compounds adjust the "toxicity" of these compounds to the microalgae and are, therefore, the main parameters that affect biodegradation. Autotrophic growth conditions of microalgae and high concentrations of trs or htrs induce higher biodegradation compared with mixotrophic conditions and lower phenolic concentrations. When microalgae face trs and htrs simultaneously, biodegradation begins from htrs, the more energetically demanding compound. All these lead to the conviction that microalgae have a "rational" management of cellular energy balance. Low toxicity levels lead to higher growth and lower biodegradation, whereas higher toxicity levels lead to lower growth and higher biodegradation. The selection of appropriate conditions (compatible to the bioenergetic strategies of microalgae) seems to be the key for a successful biodegradation of a series of toxic compounds, thus paving the way for future biotechnological applications for solving complicated pollution problems, like the detoxification of olive mill wastewater.

Lichen symbiosis: nature's high yielding machines for induced hydrogen production.

Hydrogen is a promising future energy source. Although the ability of green algae to produce hydrogen has long been recognized (since 1939) and several biotechnological applications have been attempted, the greatest obstacle, being the O2-sensitivity of the hydrogenase enzyme, has not yet been overcome. In the present contribution, 75 years after the first report on algal hydrogen production, taking advantage of a natural mechanism of oxygen balance, we demonstrate high hydrogen yields by lichens. Lichens have been selected as the ideal organisms in nature for hydrogen production, since they consist of a mycobiont and a photobiont in symbiosis. It has been hypothesized that the mycobiont's and photobiont's consumption of oxygen (increase of COX and AOX proteins of mitochondrial respiratory pathways and PTOX protein of chrolorespiration) establishes the required anoxic conditions for the activation of the phycobiont's hydrogenase in a closed system. Our results clearly supported the above hypothesis, showing that lichens have the ability to activate appropriate bioenergetic pathways depending on the specific incubation conditions. Under light conditions, they successfully use the PSII-dependent and the PSII-independent pathways (decrease of D1 protein and parallel increase of PSaA protein) to transfer electrons to hydrogenase, while under dark conditions, lichens use the PFOR enzyme and the dark fermentative pathway to supply electrons to hydrogenase. These advantages of lichen symbiosis in combination with their ability to survive in extreme environments (while in a dry state) constitute them as unique and valuable hydrogen producing natural factories and pave the way for future biotechnological applications.

"Rational" management of dichlorophenols biodegradation by the microalga Scenedesmus obliquus.

The microalga Scenedesmus obliquus exhibited the ability to biodegrade dichlorophenols (dcps) under specific autotrophic and mixotrophic conditions. According to their biodegradability, the dichlorophenols used can be separated into three distinct groups. Group I (2,4-dcp and 2,6 dcp - no meta-substitution) consisted of quite easily degraded dichlorophenols, since both chloride substituents are in less energetically demanding positions. Group II (2,3-dcp, 2,5-dcp and 3,4-dcp - one meta-chloride) was less susceptible to biodegradation, since one of the two substituents, the meta one, required higher energy for C-Cl-bond cleavage. Group III (3,5-dcp - two meta-chlorides) could not be biodegraded, since both chlorides possessed the most energy demanding positions. In general, when the dcp-toxicity exceeded a certain threshold, the microalga increased the energy offered for biodegradation and decreased the energy invested for biomass production. As a result, the biodegradation per cell volume of group II (higher toxicity) was higher, than group I (lower toxicity) and the biodegradation of dichlorophenols (higher toxicity) was higher than the corresponding monochlorophenols (lower toxicity). The participation of the photosynthetic apparatus and the respiratory mechanism of microalga to biodegrade the group I and the group II, highlighted different bioenergetic strategies for optimal management of the balance between dcp-toxicity, dcp-biodegradability and culture growth. Additionally, we took into consideration the possibility that the intermediates of each dcp-biodegradation pathway could influence differently the whole biodegradation procedures. For this reason, we tested all possible combinations of phenolic intermediates to check cometabolic interactions. The present contribution bring out the possibility of microalgae to operate as "smart" bioenergetic "machines", that have the ability to continuously "calculate" the energy reserves and "use" the most energetically advantageous dcp-biodegradation strategy. We tried to manipulate the above fact, changing the energy reserves and as a result the chosen strategy, in order to take advantage of their abilities in detoxifying the environment.

Bioenergetic strategy for the biodegradation of p-cresol by the unicellular green alga Scenedesmus obliquus.

Cultures from the unicellular green alga Scenedesmus obliquus biodegrade the toxic p-cresol (4-methylphenol) and use it as alternative carbon/energy source. The biodegradation procedure of p-cresol seems to be a two-step process. HPLC analyses indicate that the split of the methyl group (first step) that is possibly converted to methanol (increased methanol concentration in the growth medium), leading, according to our previous work, to changes in the molecular structure and function of the photosynthetic apparatus and therefore to microalgal biomass increase. The second step is the fission of the intermediately produced phenol. A higher p-cresol concentration results in a higher p-cresol biodegradation rate and a lower total p-cresol biodegradability. The first biodegradation step seems to be the most decisive for the effectiveness of the process, because methanol offers energy for the further biodegradation reactions. The absence of LHCII from the Scenedesmus mutant wt-lhc stopped the methanol effect and significantly reduced the p-cresol biodegradation (only 9%). The present contribution deals with an energy distribution between microalgal growth and p-cresol biodegradation, activated by p-cresol concentration. The simultaneous biomass increase with the detoxification of a toxic phenolic compound (p-cresol) could be a significant biotechnological aspect for further applications.

High yields of hydrogen production induced by meta-substituted dichlorophenols biodegradation from the green alga Scenedesmus obliquus.

Hydrogen is a highly promising energy source with important social and economic implications. The ability of green algae to produce photosynthetic hydrogen under anaerobic conditions has been known for years. However, until today the yield of production has been very low, limiting an industrial scale use. In the present paper, 73 years after the first report on H(2)-production from green algae, we present a combinational biological system where the biodegradation procedure of one meta-substituted dichlorophenol (m-dcp) is the key element for maintaining continuous and high rate H(2)-production (>100 times higher than previously reported) in chloroplasts and mitochondria of the green alga Scenedesmus obliquus. In particular, we report that reduced m-dcps (biodegradation intermediates) mimic endogenous electron and proton carriers in chloroplasts and mitochondria, inhibit Photosystem II (PSII) activity (and therefore O(2) production) and enhance Photosystem I (PSI) and hydrogenase activity. In addition, we show that there are some indications for hydrogen production from sources other than chloroplasts in Scenedesmus obliquus. The regulation of these multistage and highly evolved redox pathways leads to high yields of hydrogen production and paves the way for an efficient application to industrial scale use, utilizing simple energy sources and one meta-substituted dichlorophenol as regulating elements.

Inductive and resonance effects of substituents adjust the microalgal biodegradation of toxical phenolic compounds.

The type and the position of the substituent in the phenolic ring, the bond dissociation energy and the exogenously supplied carbon source as well as the inductive and resonance effect phenomena of the substituents adjust the biodegradability of the phenolic compounds. The comparative biodegradation study of mono-nitrophenols (electron acceptors) and mono-methylphenols (electron donors) revealed that it is a completely photoregulated process. The closer the donor group (OH(-)) of the phenolic ring is to the acceptor group (NO(2)(-)), the higher the biodegradation values are (2-nitrophenol>3-nitrophenol>4-nitrophenol); the further the donor group (OH(-)) of the phenolic compound is from the second donor group (CH(3)(+)), the higher the biodegradation values are (2-methylphenol<3-methylphenol<4-methylphenol). However, there are compounds without a specific role of acceptor or donor such as mono-iodophenols, where a type of counteraction between the inductive and resonance effect determines the behavior of the substituent. This fact combined with the presence of the hydroxyl group in the phenolic ring gave the observed stabilization in the biodegradation results of mono-iodophenols (2-iodophenol approximately 3-iodophenol approximately 4-iodophenol).

Bioenergetic changes in the microalgal photosynthetic apparatus by extremely high CO2 concentrations induce an intense biomass production.

Unicellular green alga Chlorella minutissima, grown under extreme carbon dioxide concentrations (0.036-100%), natural temperature and light intensities (Mediterranean conditions), strongly increase the microalgal biomass through photochemical and non-photochemical changes in the photosynthetic apparatus. Especially, CO(2) concentrations up to 10% enhance the density of active reaction centers (RC/CS(o)), decrease the antenna size per active reaction center (ABS/RC), decrease the dissipation energy (DI(o)/RC) and enhance the quantum yield of primary photochemistry (F(v)/F(m)). Higher CO(2) concentrations (20-25%) combine the above-mentioned photochemical changes with enhanced non-photochemical quenching of surplus energy, which leads to an enhanced steady-state fraction of 'open' (oxidized) PSII reaction centers (q(p)), and minimize the excitation pressure of PSII (1 - q(p)) under very high light intensities (approximately 1700 micromol m(-2) s(-1) maximal value), avoiding the photoinhibition and leading to an enormous biomass production (approximately 2500%). In conclusion, these extreme CO(2) concentrations - about 1000 times higher than the ambient one - can be easily metabolized from the unicellular green alga to biomass and can be used, on a local scale at least, for the future development of microalgal photobioreactors for the mitigation of the factory-produced carbon dioxide.

Bioenergetic strategy of microalgae for the biodegradation of phenolic compounds: exogenously supplied energy and carbon sources adjust the level of biodegradation.

The biodegradation of phenolic compounds by microalgae seems to be not a simple feature of a particular organism, but mostly a bioenergetic process depending on the growth conditions, especially on the exogenously supplied energy (carbon and light) sources. By using chlorophyll fluorescence induction measurements to estimate the molecular structure and function of the photosynthetic apparatus and therefore the tolerance/sensitivity of microalgae incubated with phenols, it can be assumed that, at least in low concentrations, phenol have no toxic effects on the cultures and can be used as alternative carbon source in them. Halophenols (chlorophenols, bromophenols and iodophenols) are quite toxic for the microalgal cultures. In halophenols the first step of the biodegradation is the split of the halogen substituent (dehalogenation). This is strongly determined by the bond dissociation energy of the corresponding substituent and therefore the energetic requirement for the biodegradation of halophenols increases following the sequence: iodophenol