Marine Microalgae Tetraselmis suecica as Flocculant Agent of Bio-flocculation Method

AbstractMicroalgae harvesting is an important part in microalgae cultivation system. Techniques for harvesting marine microalgae which are commonly used are centrifugation, filtration and flocculation. These techniques still have some disadvantages, such as not environment friendly, and high usage of energy and cost. Bio-flocculation harvesting technique using microalgae as a flocculant agent can be an alternative way to solve these problems. In this research, mixing of Tetraselmis suecica (flocculant) with Chlorella sp. and Nannochloropsis sp. (non-flocculant) in ratios of 1:4, 2:4, 3:4 and 4:4 (v/v) has been conducted to obtain percent recovery of marine microalgae harvest. The results showed that T. suecica as flocculant agent can fasten the harvesting of Chlorella sp. and Nannochloropsis sp. It was shown by the increase of percent recovery value of Chlorella sp. from 51.14 ± 1.07% to 67.34 ± 0.67% and Nannochloropsis sp. from 20.52 ± 1.17% to 42.43 ± 0.40% during the first hour of flocculating process. Our result showed that bio-flocculation is an environment friendly technique which can be applied to harvest marine microalgae

Harvesting of microalgae by bio-flocculation

The high-energy input for harvesting biomass makes current commercial microalgal biodiesel production economically unfeasible. A novel harvesting method is presented as a cost and energy efficient alternative: the bio-flocculation by using one flocculating microalga to concentrate the non-flocculating microalga of interest. Three flocculating microalgae, tested for harvesting of microalgae from different habitats, improved the sedimentation rate of the accompanying microalga and increased the recovery of biomass. The advantages of this method are that no addition of chemical flocculants is required and that similar cultivation conditions can be used for the flocculating microalgae as for the microalgae of interest that accumulate lipids. This method is as easy and effective as chemical flocculation which is applied at industrial scale, however in contrast it is sustainable and cost-effective as no costs are involved for pre-treatment of the biomass for oil extraction and for pre-treatment of the medium before it can be re-used

Biomass Production Potential of a Wastewater Alga Chlorella vulgaris ARC 1 under Elevated Levels of CO2 and Temperature

The growth response of Chlorella vulgaris was studied under varying concentrations of carbon dioxide (ranging from 0.036 to 20%) and temperature (30, 40 and 50°C). The highest chlorophyll concentration (11 μg mL–1) and biomass (210 μg mL–1), which were 60 and 20 times more than that of C. vulgaris at ambient CO2 (0.036%), were recorded at 6% CO2 level. At 16% CO2 level, the concentrations of chlorophyll and biomass values were comparable to those at ambient CO2 but further increases in the CO2 level decreased both of them. Results showed that the optimum temperature for biomass production was 30°C under elevated CO2 (6%). Although increases in temperature above 30°C resulted in concomitant decrease in growth response, their adverse effects were significantly subdued at elevated CO2. There were also differential responses of the alga, assessed in terms of NaH14CO3 uptake and carbonic anhydrase activity, to increases in temperature at elevated CO2. The results indicated that Chlorella vulgaris grew better at elevated CO2 level at 30°C, albeit with lesser efficiencies at higher temperatures

Long term outdoor operation of a tubular airlift pilot photobioreactor and a high rate algal pond as tertiary treatment of urban wastewater.

530 L high rate alga pond (HRAP) and 380 L airlift tubular photobioreactor (TPBR) were operated and compared in a urban wastewater treatment plant (WWTP), with the main purpose of removing nitrogen and phosphorous from the effluent of the WWTP while generating a valuable biomass. The photosynthetic activity in TPBR was during entire experiment higher than HRAP. The maximum areal productivity reached was 8.26 ± 1.43 and 21.76 ± 0.3 g SS m−2 d−1 for HRAP and TPBR respectively. Total nitrogen (TN) removal averaged 89.68 ± 3.12 and 65.12 ± 2.87% for TPBR and HRAP respectively, while for total phosphorus (TP) TPBR and HRAP averaged 86.71 ± 0.61 and 58.78 ± 1.17% respectively. The lipid content showed no significant differences (p < 0.05) between HRAP and TPBR averaging 20.80 ± 0.22 wt%. The main operating disadvantage of TPBR versus HRAP was the sever biofouling which forced to stop the experiment. Under the same conditions of operation TPBR was more limited at low temperatures than HRAP, and HRAP was more light limited than TPBR

Electromagnetic Biostimulation of Living Cultures for Biotechnology, Biofuel and Bioenergy Applications

The surge of interest in bioenergy has been marked with increasing efforts in research and development to identify new sources of biomass and to incorporate cutting-edge biotechnology to improve efficiency and increase yields. It is evident that various microorganisms will play an integral role in the development of this newly emerging industry, such as yeast for ethanol and Escherichia coli for fine chemical fermentation. However, it appears that microalgae have become the most promising prospect for biomass production due to their ability to grow fast, produce large quantities of lipids, carbohydrates and proteins, thrive in poor quality waters, sequester and recycle carbon dioxide from industrial flue gases and remove pollutants from industrial, agricultural and municipal wastewaters. In an attempt to better understand and manipulate microorganisms for optimum production capacity, many researchers have investigated alternative methods for stimulating their growth and metabolic behavior. One such novel approach is the use of electromagnetic fields for the stimulation of growth and metabolic cascades and controlling biochemical pathways. An effort has been made in this review to consolidate the information on the current status of biostimulation research to enhance microbial growth and metabolism using electromagnetic fields. It summarizes information on the biostimulatory effects on growth and other biological processes to obtain insight regarding factors and dosages that lead to the stimulation and also what kind of processes have been reportedly affected. Diverse mechanistic theories and explanations for biological effects of electromagnetic fields on intra and extracellular environment have been discussed. The foundations of biophysical interactions such as bioelectromagnetic and biophotonic communication and organization within living systems are expounded with special consideration for spatiotemporal aspects of electromagnetic topology, leading to the potential of multipolar electromagnetic systems. The future direction for the use of biostimulation using bioelectromagnetic, biophotonic and electrochemical methods have been proposed for biotechnology industries in general with emphasis on an holistic biofuel system encompassing production of algal biomass, its processing and conversion to biofuel

"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 of biodegradation of the phenolic compounds by the green alga Scenedesmus obliquus: biotechnology applications

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.Με την παρούσα εργασία για πρώτη φορά αποδεικνύεται ότι δεν έχει τόσο σημασία η επιλογή του οργανισμού, όσο η επιλογή των κατάλληλων εκείνων συνθηκών που θα αλλάξουν το βιοενεργητικό ισοζύγιο και θα επιτρέψουν την ενεργοποίηση των απαραίτητων μεταβολικών μηχανισμών για τη βιοαποικοδόμηση τοξικών ενώσεων.Η βιοενεργητική στρατηγική βιοαποικοδόμησης φαινολικών ενώσεων από το μονοκύτταρο χλωροφύκος Scenedesmus obliquus, που πραγματεύεται η παρούσα διατριβή, έδειξε ότι πρόκειται για μια ξεκάθαρα θερμοδυναμικά φωτοελεγχόμενη διαδικασία. Η επιλογή του καταλληλότερου μονοπατιού βιοαποικοδόμησης γίνεται με βάση το ενεργειακό ισοζύγιο του χλωροφύκους. Αυτό το ενεργειακό ισοζύγιο καθορίζεται από πληθώρα βιοτικών και αβιοτικών παραμέτρων. Η θέση (ortho, meta ή para) και ο αριθμός των υποκαταστατών στο φαινολικό δακτύλιο, τα φαινόμενα του συντονισμού και της επαγωγής, που ελέγχουν τη συμπεριφορά του υποκαταστάτη ως δότη ή δέκτη ηλεκτρονίων, καθορίζουν τις ενεργειακές απαιτήσεις για τη βιοδιάσπαση των φαινολικών ενώσεων, ενώ η εξωγενής πηγή του άνθρακα (ανόργανου ή/και οργανικού) και η ένταση της φωτονιακής ακτινοβολίας είναι οι σημαντικότεροι από τους παράγοντες που ελέγχουν [μέσω μη κυκλικής και κυκλικής φωτοφωσφορυλίωσης στο χλωροπλάστη, αναπνευστικής αλυσίδας δια του κυτοχρωμικού (COX) ή/και του εναλλακτικού (AOX) μονοπατιού στο μιτοχόνδριο και της χλωροαναπνοής (PTOX) στο χλωροπλάστη] τα ενεργειακά αποθέματα του χλωροφύκους που θα επενδυθούν για τη βιοαποικοδόμηση φαινολικών ενώσεων διαφορετικής τοξικότητας και δυσκολίας βιοδιάσπασης.Η απλούστερη φαινολική ένωση, η φαινόλη, χρησιμοποιείται από το χλωροφύκος Scenedesmus obliquus ως εναλλακτική πηγή άνθρακα, ενώ σε συνδυασμό με την κατάλληλη ένταση φωτονιακής ακτινοβολίας, επιτυγχάνεται ολοκληρωτικός μεταβολισμός της (100% βιοαποικοδόμηση). Αυξάνοντας το βαθμό δυσκολίας της βιοδιάσπασης, προσθέτοντας έναν υποκαταστάτη αλογόνο (Cl, Br, I) στο φαινολικό δακτύλιο, η βιοαποικοδόμηση γίνεται μέσω συμμεταβολισμού και πραγματοποιείται σε δύο διακριτές φάσεις. Κατά την πρώτη φάση αποσπάται ο υποκαταστάτης (αλογόνο) και κατά τη δεύτερη φάση λαμβάνει χώρα η σχάση του φαινολικού δακτυλίου. Μία επιπλέον απόδειξη ενός τέτοιου μηχανισμού βιοαποικοδόμησης φαινολικών ενώσεων με έναν υποκαταστάτη αποτελεί η βιοαποικοδόμηση των μεθυλφαινολών, που αποδείχτηκε ότι μετά από την απόσπαση του υποκαταστάτη (δηλαδή της μεθυλομάδας) από το φαινολικό δακτύλιο δημιουργείται μεθανόλη, ενισχύοντας τη φωτοσυνθετική δραστηριότητα του χλωροφύκους, αφού είναι αποδεδειγμένο (από προηγούμενα δημοσιεύματα του εργαστηρίου μας) ότι η μεθανόλη μεταβολίζεται σε CO2. Η βιοαποικοδόμηση είναι μια οξειδοαναγωγική αντίδραση, οπότε και το είδος του υποκαταστάτη καθορίζει σε μεγάλο βαθμό τη βιοαποικοδομησιμότητά του. Όσο πιο κοντά είναι ένας υποκαταστάτης δέκτης ηλεκτρονίων (π.χ. μία νιτροομάδα) σε έναν υποκαταστάτη δότη ηλεκτρονίων (υδροξύλιο φαινόλης), τόσο πιο έντονη είναι η βιοαποικοδομησιμότητα. Αντίθετα όσο πιο κοντά είναι μια ομάδα δότης ηλεκτρονίων (π.χ. μία μεθυλομάδα) σε έναν υποκαταστάτη δότη ηλεκτρονίων (υδροξύλιο φαινόλης), τόσο πιο δύσκολη είναι η βιοαποικοδομησιμότητα. Όταν πάλι ο υποκαταστάτης δεν καθορίζεται αυστηρά ως δότης ή δέκτης ηλεκτρονίων (π.χ. αλογόνο) από τα φαινόμενα του συντονισμού ή της επαγωγής, αλλά ανάλογα με τις συνθήκες επικρατεί άλλοτε η ιδιότητα του δότη και άλλοτε η ιδιότητα του δέκτη, τότε η βιοαποικοδομησιμότητα της φαινολικής ένωσης είναι παντού η ίδια, αφού φαίνεται να επιλέγεται από το χλωροφύκος η βέλτιστη διαχείριση των καταστάσεων.Η επιπλέον αύξηση του βαθμού δυσκολίας της βιοαποικοδόμησης φαινολικών ενώσεων από το χλωροφύκος, που επιτυγχάνεται με την προσθήκη ενός δεύτερου υποκαταστάτη στο φαινολικό δακτύλιο (διχλωροφαινόλες), διαφοροποιεί το επίπεδο βιοαποικοδόμησης ανάλογα με το πόσα meta-υποκατεστημένα χλώρια υπάρχουν στο φαινολικό δακτύλιο. Αποδείχθηκε ότι σε συνθήκες αυξημένης τοξικότητας (διχλωροφαινόλες) το χλωροφύκος επενδύει περισσότερη ενέργεια στη βιοαποικοδόμηση και λιγότερη στην ανάπτυξη, ενώ το αντίθετο συμβαίνει όσο μικρότερη είναι η τοξικότητα της φαινολικής ένωσης (μονοχλωροφαινόλες).Το πρώτο βήμα της βιοαποικοδόμησης των διχλωροφαινολών είναι η αναγωγή τους. Η ανηγμένη μορφή των 2,3-, 2,5- και 3,4-διχλωροφαινολών, βάσει του οξειδοαναγωγικού τους δυναμικού, μπορεί να ενσωματωθεί στη φωτοσυνθετική αλυσίδα μεταφοράς ηλεκτρονίων πριν από τη δεξαμενή της πλαστοκινόνης (PQ), τροφοδοτώντας με ηλεκτρόνια -ως δότης ηλεκτρονίων- το PSI, παρεμποδίζοντας ολοκληρωτικά την ενεργότητα του PSII και κατ’ επέκταση την παραγωγή Ο2. Η μοναδική συγκυρία της συνδυαστικής δράσης ανοξίας και αποκλειστικής ενεργοποίησης του PSI, επάγει την υδρογενάση και την παραγωγή H2.Συνδυαστικά οι εν λόγω διχλωροφαινόλες μπορούν να μεταφέρουν ηλεκτρόνια και απευθείας σε πρωτόνια, σχηματίζοντας επιπλέον μοριακό υδρογόνο (Η2) σύμφωνα με την αντίδραση: 2H+ + Dred → H2 + Dox, δικαιολογώντας τις ιδιαίτερα υψηλές συγκεντρώσεις του παραγόμενου υδρογόνου σε σύγκριση με τον ανοξικό μάρτυρα (έως και 125 φορές υψηλότερες). Πρόκειται για μία βιοενεργητική στρατηγική του χλωροφύκους για τη βιοαποικοδόμηση των εν λόγω διχλωροφαινολών που δημιουργεί τις μοναδικές εκείνες συνθήκες που επιτρέπουν μία «πράσινη» βιοαποικοδόμηση τοξικών ενώσεων με την ταυτόχρονη παραγωγή υψηλών συγκεντρώσεων βιο-υδρογόνου (Η2) για περαιτέρω βιοτεχνολογική εκμετάλλευση