Sustainable Pathways for Algal Bioenergy

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2 THE BIOREMEDIATION AND PHOTOPHYSIOLOGY EXPERIMENTS IN PILOT PBRs WITH AIMS OF MECHNISITIC MODELS DEVELOPMENT A Silkina, N Ginnever Centre for Sustainable Aquatic Research, Swansea University

3 The EnAlgae Project WP1 Cultivation & processing Data collection from 9 pilot facilities Best practice sharing Outreach WP2 Routes to market Economic Modelling Life Cycle Analysis Regulation and Policy WP3 Guiding industry and policy Smart information and data tools

4 Objectives of EnAlgae project - Reduce risk and accelerate implementation of pilots; - Implement algal biotechnologies within process chains; - Generate reliable data to inform the development of an ICT (modelling) decision support tool - Provide product and process descriptions - Develop and share best practice among algal biomass and bioenergy producers across NWE

5 EnAlgae Lead Partner Centre for Sustainable Aquatic Research (CSAR) Swansea University PBR capacity ~ 5L monitoring and (bio)chemical analysis. research for harvesting and processing modelling tool developement

6 EnAlgae Targets for remediation Test suitability of waste stream use : agricultural, fish farm and AD municipal waste as nutrients source Compare the Nitrogen and Phosphorus uptake by different species in different cultivation PBR Compare the productivity of species Provide the data set for modelling tool

7 Bioremediation Why use waste? A number of cost/efficiency advantages Environmental advantages Phosphate sources are scarcer and will be economically unviable to mine by 23 Waste nutrients source can help to reduce the mining of phosphorous and recycle this valuable mineral Difficulties of preparation (e.g. filtration) associated with using liquid and solid waste sources Liquid wastes- high in Ammonia -toxic to algae Not have an optimal nutrient profile Algae need adaptation to the waste source during initial cultivation

8 Species used Chlorella minutissima Scenedesmus sp. Isolate from steel industrial site

9 Experimental conditions days of cultivation in tubular PBR Batch and Semi continuous mode Close monitoring of biological parameters cells, biovolume, cellular C:N:P:Chl Water chemistry and biochemistry analysis DIN, DIP, ph, T, PFD; lipid, carbohydrates Log-in data- ph, T; light

10 Biovolume (µm 3 ml -1 ) Biovolume (µm3ml-1) Results of growth 3,E+9 3,E+9 2,E+9 2,E+9 Chlorella minutissima 3,E+9 3,E+9 2,E+9 2,E+9 Scenedesmus sp. 1,E+9 1,E+9 5,E+8 5,E+8,E Time (day -1 ),E Time (day-1) Cultures were able to grow using waste nutrients Cultures again entered a growth phase after partial harvest growth rate

11 Concentration (umol L -1 ) Concentration (umol L -1 ) Nutrient uptake by algae Nutrient uptake during semi-continuous cultivation of Scenedesmus sp. TON Phosphate Time (day -1) Nutrient uptake during semi-continuous cultivation of Chlorella minutissima TON Phosphate Time (Day-1) 5 Waste nutrients are gradually taken up by the algae during cultivation P and N uptake

12 Fv/Fm (relative units) Fv/Fm (relative units) PSII photoefficiency Fv/Fm is the efficiency of photosystem 2 (PSII), the main light harvesting and processing complex in microalgae Can be used as a measure of cell stress Fv/Fm Scenedesmus sp. Fv/Fm Chlorella minutissima,7,7,6,6,5,5,4,4,3,3,2,2,1, Time (day-1) Time (day-1)

13 Biochemical composition summary Diary waste remediation Scenedesmus sp. % Carb composition % Lipid composition % Protein 6 4 Diary waste remediation Chl. minutissima

14 Growth using fish waste Biovolume (x1 6 ) µm3 ml Biovolume Chlorella minutissima Time (day -1 ) CONTROL Trout waste The specific growth rate is similar to control sample Waste nutrients are gradually taken up by the algae during cultivation P is accumulated in cell Comcentration µmoll Ammonia µmol/l Phosphate µmol/l Time (day-1) Concentration umoll Phosphorus uptake and cell P Time (day-1)

15 Summary & Conclusion Species N uptake rate P rate uptake Max Productivity Producti on rate C. minutissima Control C. minutissima experimental Scenedesmus sp. Control Scenedesmus sp. Experimental mg(n)l -1 d -1 mg(p)l -1 d -1 (g L -1 ) (g L -1 d -1 ) 5.27 ±.8.41± ± ±.4.43± ± ±.5.39± ± ±.4.37± ±.5.9 Cultures were successfully grow using waste nutrients Nutrient uptake related regulatory standards of waste release Potential exploitation of algal biomass

16 Design of PBR comparison Vertical and horizontal Tubular reactors were compared In control and waste remediation condition, productivity is higher in vertical system with Ø 11 mm(causerma et al, 211) The specific biomass (e.g. reach on lipids) quickly achieved on horizontal tubular PBRØ 43 mm

17 Lighting and Harvesting: The effect on the growth and photophysiology of Nannochloropsis oculata Dr Naomi Ginnever, Dr Alla Silkina, Professor Kevin Flynn

18 Experiment and Rationale In order to develop the photosynthetic and lighting portion of the Enalgae model experiments were undertaken to establish the effect of photoperiod (at a consistent photodose) and harvesting on the growth, photophysiology, physiology and biochemistry. Experiments were undertaken in a 4L tubular bioreactor, which was artificially lit using metal halide lights. The photodose ( mol photons m -2 ) was maintained over 4 experimental runs. 2 had a photoperiod of 11 hours and 2 had a photoperiod of 7 hours and 2 minutes. Supplied nutrient levels were calculated based on the known average cellular N content and the desired cell density

19 Methodology Samples were taken daily for cell counts and PAM fluorescence analysis PAM fluorescence measurements were made using a Walz PhytoPAM and these data were analysed using an iterative curve fitting solution (Eilers and Peeters (1988) This provided several photosynthetic parameters which are useful indicators of cell health, stress and light acclimation The quantum efficiency of photosystem II (Fv/Fm) is a very valuable measure of the overall cell health/stress Light saturation coefficient (Ek) indicates the light acclimation state of the cells

20 Cells/ ml Cells/ ml Initial Results The cultures were not harvested until 4 million cells were reached A 25% harvest was performed 3 times reducing the cell numbers to 3 million cells 5.E+7 4.5E+7 4.E+7 3.5E+7 3.E+7 2.5E+7 2.E+7 1.5E+7 1.E+7 5.E+6 5.E+7 4.5E+7 4.E+7 3.5E+7 3.E+7 2.5E+7 2.E+7 1.5E+7 1.E+7 5.E+6.E E Day (-1) Day (-1) N=2, Experimental replicates Max div.day -1 =.733,.711

21 Fv/Fm 15 (relative units) Fv/Fm 15 (relative units) Initial Results The Fv/Fm dropped dramatically after inoculation and after the initial partial harvest There then appeared to be an acclimation as the dramatic drop was not observed post partial harvest again Day (-1) Day (-1) The carotenoid content in the cultures of both experimental photoperiods increased significantly during the run From.5 ng (1 6 cells) to 1.75 ng (1 6 cells) and.65 ng (1 6 cells) to 2.1 ng (1 6 cells) (both P= <.5)

22 Ek (relative units) Ek (relative units) Initial Results The Ek increase increased post innoculation The Ek curve then saturated, and did not increase further This is likely due to the reduction in light per cell as the culture density increased meaning the cells do not need to become acclimated to a higher light level Day (-1) Day (-1) The ratio of Chl a to carotenoids decreased during the growth curve in both cultures

23 Discussion and Conclusions The analysis of the biomass of is at an early stage and will be analysed in detail to fully understand the effect of different photoperiods and light levels on the photosystems and cellular composition Work is also being completed to investigate the effect of photoperiod using different LED colour mixes Data from outdoor experimental runs at different times of year are also being incorporated into the model to complete this component of the model

24 Discussion and Conclusions The data suggests that despite the photodose being the same, the increased light level results in a longer lag phase After the 3 rd harvest there is also a slower recovery Therefore it is recommended that a lower light level be used for longer photoperiod The higher light level did result in a nonsignificantly higher carotenoid content

25 Discussion and Conclusions The initial partial harvesting had a negative effect on the photophysiology and resulted in a slight reduction in div.day However this effect was not observed after the second harvest At the 3 rd harvest only the higher light level was negatively effected Therefore it is clear harvesting may have a negative effect if a large harvest is performed and at high light levels.

26 The Enalgae model and decision support tool will be available for use by industry in NW Europe after the project is complete THANK YOU FOR YOUR ATTENTION