Applications of microalgae/cyanobacteria for wastewater treatment and product recovery

Environmental pollution and fresh water crisis are the main causes constituting wastewater treatment necessary. Wastewaters are emerging from various production processes that intend to meet the human needs, such as nutrition, and they contain high concentrations of organic load, nutrients and water. During their treatment physicochemical and/or biological processes with bacteria even though they are considered very effective for the removal of organic matter, they can not achieve the complete removal of nitrogen and phosphorus while energy consumption (aeration) is also required for their treatment, secondarily producing sludge. Microalgae offer an alternative and efficient solution in wastewater treatment, by assimilating simultaneously different organic and inorganic components, including heavy metals, which are utilized for the growth and production of valuable biomass. Therefore biological treatment of wastewaters using microalgae can achieve significant pollutant removal and biomass production, which is suitable feedstock for bioenergy and/or other commercial products production (i.e. fertilizers, animal feed) in an environmentally friendly way. Due to their great phylogenetic diversity, microalgae can intracellularly accumulate a multitude of chemical components and thus being an important source of biomolecules, which are exploited in a range of commercial applications. In particular, microalgal biomass contains high amounts of proteins, lipids, carbohydrates and pigments. Their lipids can be used in the field of biofuels for biodiesel production. Their carbohydrates can be used as a source of carbon (C) for the fermentation industries, by replacing simple sugars and lignocellulosic biomass, while they have also been used for bioethanol production. Moreover, their long-chain polyunsaturated fatty acids play an important role in nutrition, improving the beneficial properties of food supplements intended for humans (i.e. vitamins) or animals (i.e. aquafeeds, animal feeds). Research studies report that proteins and pigments derived from microalgae are desirable for applications in the pharmaceutical and cosmetic industries, as they contribute to the treatment of certain diseases.Considering the above, the present PhD thesis aims to: a) biologically treat highly polluted wastewaters from the agroindustrial sector and the food industry employing mixed cultures of microalgae/cyanobacteria under non aseptic, aerobic conditions, with simultaneous recovery of the microbial biomass’s intracellular components, emphasizing mainly in lipids for bioenergy production, and b) the optimization of growth conditions of the microalgae Tetraselmis striata, a strain that presents high biotechnological interest in the field of aquaculture, as its biomass is a significant source of high added value products, such as lipids (with important profile of polyunsaturated fatty acids), carbohydrates, proteins and pigments, which can improve the health and fish growth. Initially the cyanobacterium Leptolyngbya sp. was used for the biotreatment of poultry, dairy, mixed poultry-dairy wastewaters, and out of date fruit juices. Two different production systems, those of suspended and attached growth mode were studied, taking into advantage the ability of the specific strain to form large aggregates. It should be noted that in a potential full scheme the attached growth system could significantly reduce the biomass harvesting costs by avoiding other expensive methods. At the same time, the ability of the cyanobacterium to accumulate lipids in the suspended or the attached biomass was examined for biodiesel production. Then, aiming to recover high added value products from microalgae, the microalga Tetraselmis striata was cultured in high salinity drilling water. The specific strain is of great interest for the purposes of nutrition, while using drilling seawater as growth substrate could avoid a potential degradation of biomass’s composition in case of cultivation using wastewaters. In all tested operational parameters extensive analyses of Tetraselmis striata’s biomass biochemical composition (lipids, carbohydrates, proteins, pigments) were carried out, aiming to find the optimal conditions for growth and accumulation of intracellular compounds. The experiments conducted with Leptolyngbya sp. and Tetraselmis striata took place not only at laboratory-scale (3.5-4 L) but also at pilot-scale (ranging from 9 to 280 L) bioreactors, aiming to examine the ability of the specific strains to escalate in larger working volumes, highlighting their potential for full-scale cultivation. Finally, the suitability of the recovered metabolic compounds was evaluated, for commercial applications such as biofuel production and incorporation into conventional fish feeds.Η περιβαλλοντική ρύπανση και η κρίση του γλυκού νερού είναι οι κύριες αιτίες που καθιστούν την επεξεργασία των υγρών αποβλήτων αναγκαία. Υγρά απόβλητα προκύπτουν κυρίως από διάφορες παραγωγικές διαδικασίες που σκοπό έχουν να καλύψουν κύριες ανάγκες του ανθρώπου, όπως τη διατροφή του, και αποτελούνται από υψηλές συγκεντρώσεις οργανικού φορτίου, θρεπτικών συστατικών και νερού. Κατά την επεξεργασία τους οι φυσικοχημικές ή/και βιολογικές διεργασίες με χρήση βακτηρίων αν και θεωρούνται πολύ αποτελεσματικές για την απομάκρυνση του οργανικού φορτίου, δεν οδηγούν σε πλήρη απομάκρυνση αζώτου και φωσφόρου, ενώ απαιτείται κατανάλωση ενέργειας (παροχή αερισμού) για τη διαχείρισή τους, παράγοντας δευτερογενώς λάσπη. Τα μικροφύκη προσφέρουν μια εναλλακτική και αποτελεσματική λύση στην επεξεργασία των αποβλήτων, καθώς μπορούν να αφομοιώσουν ταυτόχρονα διαφορετικά οργανικά και ανόργανα συστατικά, συμπεριλαμβανομένων και ορισμένων βαρέων μετάλλων, αξιοποιώντας τα για την ανάπτυξη και την παραγωγή πολύτιμης βιομάζας. Συνεπώς, η χρήση μικροφυκών στη βιολογική επεξεργασία υγρών αποβλήτων μπορεί να επιτύχει σημαντική αφαίρεση ρύπων σε συνδυασμό με την παραγωγή βιομάζας, η οποία στη συνέχεια μπορεί να αξιοποιηθεί στην παραγωγή βιοενέργειας ή/και την παραγωγή άλλων εμπορικών προϊόντων (π.χ. λιπασμάτων, ζωοτροφών) με τρόπο φιλικό προς το περιβάλλον. Τα μικροφύκη εξαιτίας της μεγάλης εξελικτικής φυλογενετικής ποικιλομορφίας τους, μπορούν να συσσωρεύουν ενδοκυτταρικά πληθώρα χημικών συστατικών αποτελώντας σημαντική πηγή για εκμετάλλευση των παραγόμενων βιομορίων σε εύρος εμπορικών εφαρμογών. Συγκεκριμένα η βιομάζα μικροφυκών περιέχει σε υψηλές ποσότητες πρωτεΐνες, λιπίδια, υδατάνθρακες και χρωστικές ουσίες. Τα λιπίδια μικροφυκών μπορούν να χρησιμοποιηθούν στον τομέα των βιοκαυσίμων και στην παραγωγή βιοντίζελ. Οι υδατάνθρακές τους μπορούν να αποτελέσουν πηγή άνθρακα (C) για τις βιομηχανίες ζύμωσης αντικαθιστώντας τα απλά σάκχαρα και τη λιγνοκυτταρινούχα βιομάζα ενώ, έχουν χρησιμοποιηθεί και στην παραγωγή βιοαιθανόλης. Επίσης, τα πολυακόρεστα μακράς αλυσίδας λιπαρά τους οξέα, παίζουν σημαντικό ρόλο στη διατροφή, προσδίδοντας ευεργετικές ιδιότητες στα συμπληρώματα διατροφής που προορίζονται για τον άνθρωπο (π.χ βιταμίνες) ή τα ζώα (π.χ. ιχθυοτροφές, ζωοτροφές). Επιπλέον, ερευνητικές εργασίες αναφέρουν πως οι πρωτεΐνες και οι χρωστικές ουσίες μικροφυκών παρουσιάζουν ιδιότητες επιθυμητές στη βιομηχανία φαρμάκων και καλλυντικών καθώς, συμβάλλουν στη θεραπεία ορισμένων ασθενειών. Με γνώμονα τα ανωτέρω, η παρούσα διδακτορική διατριβή έχει στόχο: α) τη βιολογική επεξεργασία επιβαρυμένων υγρών αποβλήτων του αγροτοβιομηχανικού τομέα και της βιομηχανίας των τροφίμων χρησιμοποιώντας μεικτές καλλιέργειες μικροφυκών/κυανοβακτηρίων υπό μη ασηπτικές, αερόβιες συνθήκες, με ταυτόχρονη ανάκτηση των ενδοκυτταρικών συστατικών της μικροβιακής βιομάζας, εστιάζοντας κυρίως στα λιπίδια για παραγωγή βιοενέργειας, και β) τη μελέτη βελτιστοποίησης των συνθηκών ανάπτυξης του μικροφύκους Tetraselmis striata το οποίο παρουσιάζει υψηλό βιοτεχνολογικό ενδιαφέρον και ιδιαίτερα στον τομέα των υδατοκαλλιεργειών, καθώς η βιομάζα του αποτελεί πηγή προϊόντων υψηλής προστιθέμενης αξίας, όπως λιπίδια (με σημαντικό προφίλ πολυακόρεστων λιπαρών οξέων), υδατάνθρακες, πρωτεΐνες και χρωστικές, τα οποία βελτιώνουν την υγεία και την ανάπτυξη των ιχθύων. Αρχικά, χρησιμοποιήθηκε το κυανοβακτήριο Leptolyngbya sp. για την επεξεργασία αποβλήτων ορνιθοτροφείου, τυροκομείου, μεικτών αποβλήτων τυροκομείου-ορνιθοτροφείου και ληγμένων εμπορικών χυμών. Εξετάστηκαν δυο διαφορετικά συστήματα παραγωγής, εκείνα της αιωρούμενης και προσκολλημένης ανάπτυξης, αξιοποιώντας με αυτό τον τρόπο την ικανότητα του εν λόγω είδους να σχηματίζει επιμήκη συσσωμάτια. Να επισημανθεί ότι σε μεγάλης κλίμακας παραγωγή, το σύστημα προσκολλημένης ανάπτυξης θα μπορούσε να μειώσει σημαντικά το κόστος συγκομιδής της βιομάζας, αποφεύγοντας άλλες κοστοβόρες μεθόδους. Παράλληλα, εξετάστηκε η ικανότητα του κυανοβακτηρίου να συσσωρεύει στην αιωρούμενη ή προσκολλημένη βιομάζα λιπίδια με στόχο την παραγωγή βιοντίζελ. Στη συνέχεια, με σκοπό την ανάκτηση προϊόντων υψηλής προστιθέμενης αξίας από τη βιομάζα μικροφυκών καλλιεργήθηκε το μικροφύκος Tetraselmis striata σε νερό γεώτρησης υψηλής αλατότητας. Το συγκεκριμένο είδος παρουσιάζει ιδιαίτερο ενδιαφέρον για τους σκοπούς της διατροφής, ενώ με τη χρήση αλμυρού νερού γεώτρησης ως μέσω ανάπτυξης αποφεύγεται η υποβάθμιση της σύστασης της βιομάζας σε περίπτωση που αυτό αναπτύσσονταν σε υγρά απόβλητα. Σε όλες τις λειτουργικές παραμέτρους που εξετάσθηκαν πραγματοποιήθηκε εκτενής ανάλυση της βιοχημικής σύστασης της βιομάζας του Tetraselmis striata (λιπίδια, υδατάνθρακες, πρωτεΐνες, χρωστικές ουσίες) με σκοπό να βρεθούν οι βέλτιστες συνθήκες για την ανάπτυξη αλλά και τη συσσώρευση των ενδοκυτταρικών συστατικών. Τα πειράματα που πραγματοποιήθηκαν τόσο με το Leptolyngbya sp. όσο και με το Tetraselmis striata έλαβαν χώρα όχι μόνο σε αντιδραστήρες εργαστηριακής κλίμακας (3.5 - 4 L) αλλά και σε αντιδραστήρες πιλοτικής κλίμακας (από 9 έως και 280 L), με στόχο να εξεταστεί η ικανότητα των εν λόγω ειδών να εγκλιματίζονται σε μεγαλύτερους λειτουργικούς όγκους, αναδεικνύοντας τη δυνατότητα εφαρμογής τους και σε μεγάλης κλίμακας παραγωγή. Τέλος, αξιολογήθηκε η καταλληλότητα των ανακτηθέντων μεταβολικών προϊόντων για την αξιοποίησή τους σε εμπορικές εφαρμογές, όπως αυτές της παραγωγής βιοκαυσίμων ή της ενσωμάτωσης σε συμβατικές ιχθυοτροφές

Utilization of Biomass Derived from Cyanobacteria-Based Agro-Industrial Wastewater Treatment and Raisin Residue Extract for Bioethanol Production

Biofuels produced from photosynthetic microorganisms such as microalgae and cyanobacteria could potentially replace fossil fuels as they offer several advantages over fuels produced from lignocellulosic biomass. In this study, energy production potential in the form of bioethanol was examined using different biomasses derived from the growth of a cyanobacteria-based microbial consortium on a chemical medium and on agro-industrial wastewaters (i.e., dairy wastewater, winery wastewater and mixed winery–raisin effluent) supplemented with a raisin residue extract. The possibility of recovering fermentable sugars from a microbial biomass dominated by the filamentous cyanobacterium Leptolynbgya sp. was demonstrated. Of the different acid hydrolysis conditions tested, the best results were obtained with sulfuric acid 2.5 N for 120 min using dried biomass from dairy wastewater and mixed winery–raisin wastewaters. After optimizing sugar release from the microbial biomass by applying acid hydrolysis, alcoholic fermentation was performed using the yeast Saccharomyces cerevisiae. Raisin residue extract was added to the treated biomass broth in all experiments to enhance ethanol production. Results showed that up to 85.9% of the theoretical ethanol yield was achieved, indicating the potential use of cyanobacteria-based biomass in combination with a raisin residue extract as feedstock for bioethanol production

HuR-Regulated mRNAs Associated with Nuclear hnRNP A1-RNP Complexes

Post-transcriptional regulatory networks are dependent on the interplay of many RNA-binding proteins having a major role in mRNA processing events in mammals. We have been interested in the concerted action of the two RNA-binding proteins hnRNP A1 and HuR, both stable components of immunoselected hnRNP complexes and having a major nuclear localization. Specifically, we present here the application of the RNA-immunoprecipitation (RIP)-Chip technology to identify a population of nuclear transcripts associated with hnRNP A1-RNPs as isolated from the nuclear extract of either HuR WT or HuR-depleted (KO) mouse embryonic fibroblast (MEF) cells. The outcome of this analysis was a list of target genes regulated via HuR for their association (either increased or reduced) with the nuclear hnRNP A1-RNP complexes. Real time PCR analysis was applied to validate a selected number of nuclear mRNA transcripts, as well as to identify pre-spliced transcripts (in addition to their mature mRNA counterpart) within the isolated nuclear hnRNP A1-RNPs. The differentially enriched mRNAs were found to belong to GO categories relevant to biological processes anticipated for hnRNP A1 and HuR (such as transport, transcription, translation, apoptosis and cell cycle) indicating their concerted function in mRNA metabolism

Laboratory- and Pilot-Scale Cultivation of <i>Tetraselmis striata</i> to Produce Valuable Metabolic Compounds

Marine microalgae are considered an important feedstock of multiple valuable metabolic compounds of high biotechnological potential. In this work, the marine microalga Tetraselmis striata was cultivated in different scaled photobioreactors (PBRs). Initially, experiments were performed using two different growth substrates (a modified F/2 and the commercial fertilizer Nutri-Leaf (30% TN—10% P—10% K)) to identify the most efficient and low-cost growth medium. These experiments took place in 4 L glass aquariums at the laboratory scale and in a 9 L vertical tubular pilot column. Enhanced biomass productivities (up to 83.2 mg L−1 d−1) and improved biomass composition (up to 41.8% d.w. proteins, 18.7% d.w. carbohydrates, 25.7% d.w. lipids and 4.2% d.w. total chlorophylls) were found when the fertilizer was used. Pilot-scale experiments were then performed using Nutri-Leaf as a growth medium in different PBRs: (a) a paddle wheel, open, raceway pond of 40 L, and (b) a disposable polyethylene (plastic) bag of 280 L working volume. Biomass growth and composition were also monitored at the pilot scale, showing that high-quality biomass can be produced, with important lipids (up to 27.6% d.w.), protein (up to 45.3% d.w.), carbohydrate (up to 15.5% d.w.) and pigment contents (up to 4.2% d.w. total chlorophylls), and high percentages of eicosapentaenoic acid (EPA). The research revealed that the strain successfully escalated in larger volumes and the biochemical composition of its biomass presents high commercial interest and could potentially be used as a feed ingredient

Laboratory- and Pilot-Scale Cultivation of Tetraselmis striata to Produce Valuable Metabolic Compounds

Marine microalgae are considered an important feedstock of multiple valuable metabolic compounds of high biotechnological potential. In this work, the marine microalga Tetraselmis striata was cultivated in different scaled photobioreactors (PBRs). Initially, experiments were performed using two different growth substrates (a modified F/2 and the commercial fertilizer Nutri-Leaf (30% TN&mdash;10% P&mdash;10% K)) to identify the most efficient and low-cost growth medium. These experiments took place in 4 L glass aquariums at the laboratory scale and in a 9 L vertical tubular pilot column. Enhanced biomass productivities (up to 83.2 mg L&minus;1 d&minus;1) and improved biomass composition (up to 41.8% d.w. proteins, 18.7% d.w. carbohydrates, 25.7% d.w. lipids and 4.2% d.w. total chlorophylls) were found when the fertilizer was used. Pilot-scale experiments were then performed using Nutri-Leaf as a growth medium in different PBRs: (a) a paddle wheel, open, raceway pond of 40 L, and (b) a disposable polyethylene (plastic) bag of 280 L working volume. Biomass growth and composition were also monitored at the pilot scale, showing that high-quality biomass can be produced, with important lipids (up to 27.6% d.w.), protein (up to 45.3% d.w.), carbohydrate (up to 15.5% d.w.) and pigment contents (up to 4.2% d.w. total chlorophylls), and high percentages of eicosapentaenoic acid (EPA). The research revealed that the strain successfully escalated in larger volumes and the biochemical composition of its biomass presents high commercial interest and could potentially be used as a feed ingredient

Biotreatment of Poultry Waste Coupled with Biodiesel Production Using Suspended and Attached Growth Microalgal-Based Systems

Poultry litter extract (PLE) was treated using a microbial consortium dominated by the filamentous cyanobacterium Leptolyngbya sp. in synergy with heterotrophic microorganisms of the poultry waste. Laboratory- and pilot-scale experiments were conducted under aerobic conditions using suspended and attached growth photobioreactors. Different dilutions of the extract were performed, leading to different initial pollutant (nitrogen, phosphorus, dissolved chemical oxygen demand (d-COD), total sugars) concentrations. Significant nutrient removal rates, biomass productivity, and maximum lipid production were determined for all the systems examined. Higher d-COD, nitrogen, phosphorus, and total sugars removal were recorded in the attached growth reactors in both laboratory- (up to 94.0%, 88.2%, 97.4%, and 79.3%, respectively) and pilot-scale experiments (up to 82.0%, 69.4%, 81.0%, and 83.8%, respectively). High total biomass productivities were also recorded in the pilot-scale attached growth experiments (up to 335.3 mg L&minus;1d&minus;1). The produced biomass contained up to 19.6% lipids (w/w) on a dry weight basis, while the saturated and monounsaturated fatty acids accounted for more than 70% of the total fatty acids, indicating a potential biodiesel production system. We conclude that the processing systems developed in this work can efficiently treat PLE and simultaneously produce lipids suitable as feedstock in the biodiesel manufacture

Optimization of Cultivation Conditions for <i>Tetraselmis striata</i> and Biomass Quality Evaluation for Fish Feed Production

The marine microalgae Tetraselmis striata was cultivated in drilling waters with different salinities. Growth substrate optimization was performed while the effects of different pH, temperature, photoperiod and CO2 flow rate on biomass productivity and its composition were studied. Results showed that the strain grew better in 2.8% drilling waters employing the fertilizer Nutri-Leaf together with ΝaHCO3. A pH value of 8 resulted in high biomass productivity (79.8 mg L−1 d−1) and biomass composition (proteins 51.2% d.w., carbohydrates 14.6% d.w., lipids 27.8% d.w. and total chlorophylls 5.1% d.w.). The optimum cultivation temperature was found to be 25 ± 1 °C which further enhanced biomass productivity (93.7 mg L−1 d−1) and composition (proteins 38.7% d.w., carbohydrates 20.4% d.w., lipids 30.2% d.w., total chlorophylls 5.1% d.w.). Photoperiod experiments showed that continuous illumination was essential for biomass production. A 10 mL min−1 flow rate of CO2 lead to biomass productivity of 87.5 mg L−1 d−1 and high intracellular content (proteins 44.6% d.w., carbohydrates 10.3% d.w., lipids 27.3% d.w., total chlorophylls 5.2% d.w.). Applying the optimum growth conditions, the produced biomass presented high protein content with adequate amino acids and high percentages of eicosapentaenoic acid (EPA), indicating its suitability for incorporation into conventional fish feeds. In addition, this study analyzed how functional parameters may influence the uptake of nutrients by Tetraselmis

Optimization of Cultivation Conditions for Tetraselmis striata and Biomass Quality Evaluation for Fish Feed Production

The marine microalgae Tetraselmis striata was cultivated in drilling waters with different salinities. Growth substrate optimization was performed while the effects of different pH, temperature, photoperiod and CO2 flow rate on biomass productivity and its composition were studied. Results showed that the strain grew better in 2.8% drilling waters employing the fertilizer Nutri-Leaf together with &Nu;aHCO3. A pH value of 8 resulted in high biomass productivity (79.8 mg L&minus;1 d&minus;1) and biomass composition (proteins 51.2% d.w., carbohydrates 14.6% d.w., lipids 27.8% d.w. and total chlorophylls 5.1% d.w.). The optimum cultivation temperature was found to be 25 &plusmn; 1 &deg;C which further enhanced biomass productivity (93.7 mg L&minus;1 d&minus;1) and composition (proteins 38.7% d.w., carbohydrates 20.4% d.w., lipids 30.2% d.w., total chlorophylls 5.1% d.w.). Photoperiod experiments showed that continuous illumination was essential for biomass production. A 10 mL min&minus;1 flow rate of CO2 lead to biomass productivity of 87.5 mg L&minus;1 d&minus;1 and high intracellular content (proteins 44.6% d.w., carbohydrates 10.3% d.w., lipids 27.3% d.w., total chlorophylls 5.2% d.w.). Applying the optimum growth conditions, the produced biomass presented high protein content with adequate amino acids and high percentages of eicosapentaenoic acid (EPA), indicating its suitability for incorporation into conventional fish feeds. In addition, this study analyzed how functional parameters may influence the uptake of nutrients by Tetraselmis