Fig. 1. Map of location of the htw saar, University of Applied Sciences, in Saarbrücken, Germany

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1 Micro-algae Pilot fact sheet 1. htw saar Name Operating organisatio n Mini location map Mass production of microalgae integrated in a marine zero-exchange recirculation aquaculture system Hochschule für Technik und Wirtschaft des Saarlandes, University of Applied Sciences, htw saar, Saarbruecken, Germany Fig. 1. Map of location of the htw saar, University of Applied Sciences, in Saarbrücken, Germany Germany Htw saar next to MFV htw saar France Target of activity Fig. 2. Map of locations of htw saar in Saarbrücken and Marine Fishfarm Völklingen (MFV), where the htw saar operates an experimental RAS with integrated PBRs Assess the technical feasibility and economic potential of : - Recovery of nutrients from process water of a marine zeroexchange recirculation aquaculture (RAS) system by integration of photobioreactors (PBRs) for the production microalgae

2 Summary of facilities Team members Involved companies/ research center - Mass production of microalgae based on the bioremediation of process water from a RAS - Production of microalgae as live food and green water for a marine hatchery to be built in support of the nearby Marine Fishfarm Völklingen (MFV). - Two experimental RAS, each with a 7m 3 production tank and 3 m 3 in the core water purification system consisting of drum filter, ozon-fortified flotation and biofilters for nitrification and denitrification; - a 5 litre flat panel airlift PBR (Subitec GmbH, Germany) for culture maintenance ; - a 120 litre indoor tubular PBR (Sander Elektroapparatebau, Germany) adjacent to the RAS; - 3 x 25 litre flat panel airlift PBR (Subitec) located in a greenhouse; - a 120 litre tubular PBR (IGV GT 100, Germany) located in a greenhouse and connected with the RAS; - experimental flotation apparatus. - Prof. Rainer Eisenmann, chemical analytics (IPP) - Prof. Benendikt Faupel, process automation (IPP) - Prof. Barbara Grabowski, statistics - Prof. Klaus Kimmerle, physical process technology (IPP) - Prof. Uwe Waller, marine biology (IPP) - Dr. Anneliese Ernst, plant physiology and microbiology - Hong-Phuc Bui, statistics - Alex Finck, Dipl. Eng., analyst/programmer - Andreas Kulakowski, MS bioprocess engineering - Sven Spaniol, MS automation - Kai Wagner, MS automation - Stefan Weisskircher, Dipl. Eng., programmer - Rebecca Berger/Hosang, bio-technical assistant - Philip Champiomont - Želimir Difković - Stefan Feichtinger (Uni Rostock) - Daniel T. Lang - André Rodriguez - Christian Steinbach - Friedrich Tietze Institute of Physical Process Technology (IPP) of the htw saar

3 Description Rationale Growth equipment At the htw saar biologists and engineers are working together to improve process control in recirculation aquaculture systems (RAS). RAS are closed loop production systems for aquatic animals. State of the art RAS operate with an almost completely closed water circulation. The production of animals in a confined environment requires a continuous water treatment which, however, cannot avoid accumulation of dissolved excretion products in the process water. Algae can use sunlight as source of energy and the excretion of fish (N,P, CO2) as nutrients for growth. These nutrients in the process water can be recovered by harvesting algae. The integration of microalgae in the water treatment system of RAS is a step towards a smaller CO2 footprint of aquaculture. It is also a promising resource for the sustainable production of valuable ingredients of fish feed such as omega-3 fatty acids and pigments. Thus the production of microalgae based on nutrients from the bioremediation of process water in RAS can create additional income in aquaculture. The htw saar operates four experimental PBRs litre tubular PBR (Sander). This system comprises horizontal transparent polymethylmethacrylate (PMMA) tubes and a transparent unit for gas-exchange. The system is located indoor and illuminated with artificial light for 24 hours. It is connected to the RAS via filtering devices that allow supply of algae with dissolved nutrient from RAS and retain algae in the PBR. The system is equipped with probes for the measurement of ph, temperature and oxygen. CO2 is supplied from bottles via a ph controlled valve litre tubular PBR GT 100 (IGV GmbH). This mobile device with horizontal glass tubes is assembled on a steel platform and located in the glass house. It is operated with natural light only. It is connected to the RAS via a sump and similar filtering devices as the indoor PBR. The system is equipped with probes to measure ph, temperature, and optical density. It contains a level - and a pressure control system for automatic operation. Temperature is controlled using a water dripping system. CO2 is supplied from bottled CO2 via a ph controlled valve -3 x 25 litre flat panel airlift PBR (Subitec GmbH). The transparent plastic panels are located in the greenhouse. They are connected with a sump for the preparation of artificial seawater. The panels are not yet integrated in the RAS. The culture is aerated with a mix of air and CO2. Panels are equipped with a ph and temperature sensor. Temperature is controlled with water sprayers; CO2 is supplied from bottled CO2 via a ph-controlled valve. - 5 litre flat panel airlift PBR (Subitec GmbH). As a starter culture for the larger PBR, a 5-litre high cell density culture is maintained in a semi-automated fed-batch mode. The culture is aerated with a mix of air and CO2. CO2 is supplied from bottled CO2 via a ph-controlled

4 Analytical equipment DSP equipment valve. This indoor FPA-PBR is illuminated with natural light supplemented by artificial light during the darker halve of the year. - Spectrometer for manual control of optical density, chlorophyll and nutrients (Hach-Lange chemistry for nitrate, nitrite, ammonium and phosphate); Autoanalyzer AA3 (Seal, Germany) for nitrate, nitrite, ammonium and phosphate in the growth medium; Multi C/N 3100 (Analytik Jena) for elemental analysis of inorganic and organic carbon and total nitrogen in liquid samples; Muffel furnace Sensor for global solar radiation (on the roof of the greenhouse) Sensor for photosynthetic active radiation (inside the greenhouse) Flotation Table centrifuge (4 x 50 ml) Species cultured Nannochloropsis salina (marine Eustigmataceae) Nutrient sources The algae are cultivated in process water prepared from tap water and artificial sea water (Seequasal GmbH, Germany); The process water is conditions by the dissolved excretion of fish, which supply CO2, N and P for algae. If required, N and P of process water is supplemented by technical grade nitrate and analytical phosphate. The addition of a trace element solution comprising Fe (Lebosol, Germany), and salts of Co, Cu, Mo, Mn, and Zn (analytical grade) proved to be essential for algae growing in high cell densities. Pictures Fig. 3. Tubular PBR (GT 100, IGV) in greenhouse; on the right side: SPS (Siemens) for automatic controls, a container for chemicals for cleaning of the PBR, and the filtration/cell retention unit.

5 Design diagram(s) Fig. 4. Flat panel PBR (Subitec GmbH). The compressor for airlift and Siemns SPS for process control are placed in the back of the greenhouse. Processwater In Photobioreactor Processwater Out Sterilisation Cell Retention K-1 Harvest > 30 g/l dry mass Flotation Fig. 5. Integration of PBR in RAS. Process water of the RAS pass a membrane for sterilization to supply the microalgae in the PBR with new media. The algae are retained by a membrane in the cell retention unit. A flotation system is used to dewater the cells after harvest.

6 Biomass [g/l] Biomass [g/l], ph Temperature [ C] Graphs on productivity Tubular PBR, automatic recording ph Temperatur Biomass Time [day] Fig. 6. Biomass production in tubular PBR. Cells were harvested after 22 days. Total harvest 320 g (dry weigh of 100 litre of algae). Growth period 7 Sept to 2 Oct Flat panel airlift PBR, manual operation A l/day B l/day C l/day A B C Time [day] Fig.. Biomass production in flat panel airlift PBR. The algae were allowed to grow for 16 days. Then constant volumes (A, B, and C) were replaced with fresh medium on a daily bases over a period of 17 days. The biomass (dry weight) harvested during daily harvesting was 32.2 g, 172 g and 256 g in A, B and,

7 Modelling outputs? respectively. The total harvest (including the standing stock at the end of the experiment was 191g, 266g and 299 g (dry weight) from 25 litre in each panel. Growth period: 24 June to 27 July We uploaded automatically collected data from the tubular PBR to the Enalgae database (test version). Environmental management i.e. how does this align with Priority 2 project RESULTS and project OUTPUTS? Energy management strategies/applications Water management strategies/applications Waste management strategies/applications None to date None to date. We measured demand of cooling water of flat panel airlift PBR in thegrowth period June and Juli 2014 None to date