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DIMITAR VALEV: Wastewater Treatment with Algae Doctoral Dissertation, 118 Pp ANNALES UNIVERSITATIS TURKUENSIS UNIVERSITATIS ANNALES AI 627 AI Dimitar Valev WASTEWATER TREATMENT WITH ALGAE Dimitar Valev Painosalama Oy, Turku, Finland 2020 Finland Turku, Oy, Painosalama ISBN 978-951-29-8094-9 (PRINT) – ISBN 978-951-29-8095-6 (PDF) TURUN YLIOPISTON JULKAISUJA ANNALES UNIVERSITATIS TURKUENSIS ISSN 0082-7002 (Print) SARJA – SER. AI OSA – TOM. 627 | ASTRONOMICA – CHEMICA – PHYSICA – MATHEMATICA | TURKU 2020 ISSN 2343-3175 (Online) WASTEWATER TREATMENT WITH ALGAE Dimitar Valev TURUN YLIOPISTON JULKAISUJA – ANNALES UNIVERSITATIS TURKUENSIS SARJA – SER. AI OSA – TOM. 627 | ASTRONOMICA – CHEMICA – PHYSICA – MATHEMATICA | TURKU 2020 University of Turku Faculty of Science and Engineering Department of Biochemistry / Molecular Plant Biology Doctoral programme in Molecular Life Sciences Supervised by Dr. Esa Tyystjärvi Dr. Taina Tyystjärvi Department of Biochemistry / Department of Biochemistry / Molecular Plant Biology, Molecular Plant Biology, University of Turku, FI-20014 University of Turku, FI-20014 Turku, Finland Turku, Finland Dr. Taras Antal Department of Botany and Plant Ecology Pskov State University Pskov 180000 Russia Reviewed by Professor Amit Bhatnagar Professor Koenraad Muylaert Water Chemistry & Microbiology Laboratory of Aquatic Biology University of Eastern Finland KU Leuven Kuopio, Finland Kortrijk, Belgium Opponent Professor Ondřej Prášil Centre Algatech Institute of Microbiology, The Czech Academy of Sciences Třeboň, Czech Republic The originality of this publication has been checked in accordance with the University of Turku quality assurance system using the Turnitin OriginalityCheck service. ISBN 978-951-29-8094-9 (PRINT) ISBN 978-951-29-8095-6 (PDF) ISSN 0082-7002 (Painettu/Print) ISSN 2343-3175 (Sähköinen/Online) Painosalama Oy, Turku, Finland 2020 UNIVERSITY OF TURKU Faculty of Science and Engineering Department of Biochemistry Molecular Plant Biology DIMITAR VALEV: Wastewater treatment with algae Doctoral Dissertation, 118 pp. Doctoral Programme in Molecular Life Sciences June 2020 ABSTRACT Microalgae have been under investigation as alternatives for wastewater treatment for the last 60 years. However, we are yet to see an algal based process to be trusted as a treatment strategy on a large scale. At present, recycling of the wastewaters and nutrient utilization have been solidified as main objectives in wastewater treatment in European Union. The metabolism of microalgae and cyanobacteria enables them to assimilate and store inorganic nutrients in their biomass. Therefore, under the new wastewater treatment objectives microalgae might present a promising solution. The present work investigates the possibility of using algal biomass grown in wastewater for the production of a high value compound eicosapentaenoic acid (EPA), a type of Omega-3 fatty acid. The heterokont microalgae Nannochloropsis oculata (Eustigmataceae) were successfully grown in wastewaters from a paper/pulp mill, and we show that EPA content stayed at the same level as in microalgae grown in control conditions. Furthermore, a stable wastewater treatment process was established in an up- scale tubular photobioreactor (PBR) with the microalgae Ettlia oleoabundans (Chlorophyceae), achieving 99.6 % and 75 % phosphate and ammonium removal efficiencies, respectively. The work points out the importance of wastewater pre- treatment and wastewater optimization in order to achieve a reliable wastewater treatment process with microalgae. Finally, the cyanobacterium Synechocystis sp. PCC6803 and its sigma factor mutants were investigated in order to evaluate potential stress induced by the wastewaters. The experiments revealed that sigma factor D (sigD) may play an important role in acclimation to wastewaters and its over expression might give an edge of the photosynthetic cells used for wastewater treatment in outdoor conditions. 3 TURUN YLIOPISTO Luonnontieteiden ja tekniikan tiedekunta Biokemian laitos Molekulaarinen kasvibiologia DIMITAR VALEV: Wastewater treatment with algae Väitöskirja, 118 s. Molekulaaristen biotieteiden tohtoriohjelma Kesäkuu 2020 TIIVISTELMÄ Levien käyttöä vaihtoehtoisena jätevedenpuhdistusmenetelmä on tutkittu jo 60 vuoden ajan, mutta suurimittakaavainen leväprosessi ei silti ole vielä missään käytössä. Euroopan Unioni pitää jätevesien kierrätystä ja niiden sisältämien ravinteiden talteenottoa jätevedenpuhdistuksen tärkeimpinä päämäärinä. Levät ja syanobakteerit ovat lupaava ratkaisuvaihtoehto jätevedenpuhdistukseen, koska niiden aineenvaihdunta mahdollistaa epäorgaanisten ravinteiden assimilaation ja varastoitumisen biomassaan. Käsillä olevassa työssä tutkittiin mahdollisuutta jätevedessä tuotettavan leväbiomassan käyttämiseen eikosapentaeenihapon (EPA) tuottamiseen. EPA on markkina-arvoltaan merkittävä omega-3-rasvahappo. Heterokonta-ryhmään kuulu- van Eustigmataceae-heimon Nannochloropsis oculata -levää kasvatettiin onnistuneesti selluloosa- ja paperitehtaan jätevedessä, ja EPA:n määrä biomassassa säilyi levissä samalla tasolla kuin kontrollioloissa kasvatetuissa levissä. Työssä rakennettiin stabiili jätevedenpuhdistusprosessi, jossa Ettlia oleoabundans -nimistä viherlevää (Chlorophyceae) kasvatettiin suuressa putki- fotobioreaktorissa. Prosessi poisti jäteveden fosfaatista 99.6 % ja ammoniumista 75 %. Työ osoitti, että jäteveden esikäsittely ja ravinnepitoisuuden optimointi ovat toimivan käsittelyprosessin edellytyksiä. Lopuksi työssä tutkittiin myös Synechocystis sp. PCC6803 -syanobakteerin ja erityisesti sen sigmatekijämutanttien toimivuutta jätevedenpuhdistuksessa erityisesti jäteveden aiheuttaman stressin kannalta. Kokeet osoittivat sigD-sigmatekijän näyttelevän tärkeää roolia syanobakteerin akklimaatiossa. Työn perusteella SigD:n ylituotto voisi parantaa syanobakteerin käyttömahdollisuuksia ulko-olosuhteissa. 4 Table of Contents Abbreviations ................................................................................... 7 List of Original Publications ......................................................... 10 1 INTRODUCTION ...................................................................... 11 1.1 Origins, and health and environmental impacts of wastewaters . 11 1.2 Constituents and characterization of wastewaters .................. 11 1.2.1 Physical characteristics ............................................... 11 1.2.2 Chemical characteristics .............................................. 12 1.2.2.1 Inorganic chemical characteristics ................ 12 1.2.2.2 Organic chemical characteristics................... 13 1.2.3 Biological characteristics ............................................. 14 1.3 Wastewater treatment ............................................................ 14 1.3.1 Physical methods ........................................................ 15 1.3.2 Chemical methods ....................................................... 15 1.3.3 Biological methods ...................................................... 16 1.4 Requirements define technology ............................................ 18 1.5 Photoautotrophic microalgae and cyanobacteria...................... 19 1.5.1 Oxygenic photosynthesis ............................................. 19 1.5.1.1 Light driven redox reactions of photosynthesis ... 19 1.5.1.2 Carbon fixation reactions .............................. 20 1.6 Nutrient requirements of photoautotrophic microbes .............. 21 1.6.1 Macronutrients............................................................. 21 1.6.1.1 Carbon (C) .................................................... 21 1.6.1.2 Nitrogen (N) .................................................. 22 1.6.1.3 Phosphorus (P) ............................................. 23 1.6.1.4 Sulphur (S) ................................................... 23 1.6.2 Metal micronutrients involved in photosynthesis .......... 24 1.7 Biotechnological application of microalgae and cyanobacteria ... 25 1.8 PBRs for suspended growth ................................................... 26 1.8.1 Open systems ............................................................. 26 1.8.2 Closed PBRs ............................................................... 27 1.9 Genetically modified photoautotrophs..................................... 27 1.10 Photoautotrophs in wastewater treatment .............................. 29 2 AIMS OF THE STUDY .............................................................. 31 3 METHODOLOGY ..................................................................... 33 3.1 Wastewaters .......................................................................... 33 3.2 Strains and growth conditions ................................................ 35 5 3.2.1 Nannochloropsis oculata ............................................. 35 3.2.2 Ettlia oleoabundans ..................................................... 36 3.2.3 Synechocystis sp. PCC6803 ....................................... 37 3.3 Growth measurements ........................................................... 37 3.4 Biomass Dry Weight Analysis................................................. 38 3.5 Nutrient content and removal efficiency .................................. 38 3.6 Lipid analysis ......................................................................... 39 3.7 Chlorophyll a and carotenoids ................................................ 39 3.8 Photosynthesis measurements .............................................. 39 3.9 Statistical analysis .................................................................. 40 4 OVERVIEW OF RESULTS
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