Rui Gomes Co-authors: Ana Marques; Dinis Maurício; Jaime Páramo; Ana Barros; Joana Silva

Transcription

1 The use of heterotrophy as a tool to overcome the long and costly autotrophic scale up process for large scale production: a case study for Chlorella vulgaris Rui Gomes Co-authors: Ana Marques; Dinis Maurício; Jaime Páramo; Ana Barros; Joana Silva EABA - Madrid

2 The Company in a Glance CMP-Allmicroalgae is a SECIL Group company 21 people, 15 of which have an university degree 1ha of installed tubular PBRs 1350m 3 of PBR volume 320km of piping 50ha available for expansion Fermentation volumes up to 5000L ISO9001, ISO14001, ISO22000, OHSAS18001, Halal certified 2

3 Reliably scale from vial to production scale assuring constant product quality Overcome the long time required to scale processes from vial to production scale Overcome scale process issues. pintrest.com Scale Challenges 3

4 Industry Constraints Meeting deliverable quantities at the required dates Meeting several types of quality requirements for different customers Maintaining homogeneity of the end product Assuring all certification requirements Ability to deliver taylor made products and to work with a variety of organisms. 4

5 Rethinking the Paradigm 5

6 Rethinking the Paradigm Although microalgae are photoautotrophic organisms, there is a number of them that retain the capacity to use different carbon sources to grow heterotrophically We have to look at microalgae as units to produce an end result, and use every tool we have to reach that goal at the lower possible cost 6

7 Preliminary Tests Metabolic Matabolic pathway shift stress tests Flasks Flask tests tests Fermentation tests Three main process tests 7

8 OD 600 nm Flask Tests ºC High (ammonia+nitrate) N 21 ºC Low (ammonia) N ºC High (ammonia+nitrate) N 26 ºC Low (ammonia) N ºC High (ammonia+nitrate) N 28 ºC Low (ammonia) N Culture time (h) Heterotrophic growth curves of C. vulgaris with nitrogen rich and nitrogen deficient media at 21, 26 and 28 ºC 8

9 Dry weight (g L -1 ) Flask Tests ºC 26 ºC 28 ºC 2 0 ammonia+nitrate High N ammonia Low N Final dry weight concentration of heterotrophic C. vulgaris growth with nitrogen rich and nitrogen deficient media at 21, 26, and 28 ºC 9

10 Flask Tests Nitrogen rich media Nitrogen deficient media Lipid accumulation as function of nitrogen availability (Nile Red staining negative image) 10

11 Fermentation Tests Media development Required Nitrogen N:P ratio Micronutrients; vitamins; others Ferm. optimization Carbon source choice Feed strategy Fermentation metrics Ferm process scale up Theoretical scale up criteria Verification of the theoretical scale up criteria Fermentation metrics 11

12 DCW (g.l -1 ) Fermentation Tests Fermentation development tests made on a 7L stirred tank reactor using fed batch strategy Final biomass DCW , , Date 12

13 Fermentation Tests Benchtop fermenter metrics DCW = g.l -1 m max = h -1 Productivity = 27.2 g L -1 d -1 13

14 Fermentation Tests Tip speed Correlates with mechanical stress Scale up parameter choice Power input Eddy size Impacts, OTR and mixing time Correlates with mechanical stress Reflects mechanical stress Related with the cell size Mixing time Reflects mass transfer constraints OTR Reflects the mass transfer for the oxygen 14

15 Fermentation Scale up Process > 30 cell lines in our culture collection 15

16 Fermentation Process Scalability SB Oils Plant in Orindiuva SP Brazil 16

17 Metabolic Pathway shift Stress Tests Heterotrophy Autotrophy Growth lags? Heterotropy Autotrophy Growth improves? Heterotrophy Autotrophy No change? 17

18 Metabolic Pathway shift Stress Tests A comparison was done using 7 autotrophic green walls that were seeded with autotrophically grown inoculum and 11 green walls that were seeded with heterotrophically grown inoculum, during the period from 7/6/2016 to 26/7/2016. Autotrophic grown culture presented a decrease of around 6% for the doubling time, and an increase of around 5% on the biomass productivity A side by side comparison made on June 9 th with the same inoculum concentration led to no significant differences between growth rate and productivities of the green walls seeded with these two types of inoculum. 18

19 Metabolic Pathway shift Stress Tests Heterotrophy Autotrophy Growth lags? Heterotropy Autotrophy Growth improves? Heterotrophy Autotrophy No change? 19

20 Production Pathway Options Culture collection vial Autotrophic inoculum phase Heterotrophic inoculum phase Autotrophic scale up Heterotrophic scale up Autotrophic production Heterotrophic production Autotrophic production 20

21 Autotrophic Pathway for CO 2 Capture Vial GW PBR S PBR M PBR L Raceway Flask scale up (3 to 6 weeks) Green wall scale up (1 week) PBR S scale up (1 week) PBR M scale up (1 week) PBR L inoculum build up (1 week) Production raceway inoculation TOTAL time to harvest: 8 to 11 weeks 21

22 Combined Pathway for CO 2 Capture Vial Flask scale up (3 days) GW Pre seed fermenter (5 days) PBR S Seed fermenter (5 days) PBR M Inoculum producing fermenter scale up (5 days) PBR L PBR L inoculum build up (1 week) Raceway Production raceway inoculation TOTAL time to harvest: 4.2 weeks 22

23 Comparison combined PW vs. autotrophic PW For the proposed combined pathway the carbon capture balance is up to 1.7% lower than the strictly autotrophic pathway For the autotrophic pathway, the area required for scale up from the production vial to the inoculum build up PBR represents 45% of the area required for the inoculum build up PBR whereas for the combined pathway that area drops down to 1.5% Time to harvest can be reduced 48% to 62% using the combined pathway which has the advantage of a faster reseed, if necessary 23

24 Present Operating Mode Presently CMP Allmicroalgae unit located in Pataias Portugal is doing both production and R&D. At this stage, roughly half of the unit is dedicated to R&D and the rest is dedicated to production. R&D supported by P2020 projects and specific Business Partners. 24

25 Take Home Messages Look at microalgae as tools to reach an end goal, and use every tool available to make that process cost effective Very high cell concentrations are possible Heterotrophy on a very large production scale is proven Metabolic pathway shift has negligible effect on C. vulgaris (UTEX) growth Collaboration is key for success. 25

26 Portuguese Academia Collaboration Univ Algarve IPL Univ Minho Univ Aveiro Ciimar FCL MARE ibet UPorto CCMAR CBQF UTAD UC ISA 26

27 Thank You The authors are extremely grateful to COMPETE 2020 that support the heterotrophic assays by Project POCI FEDER titled FermALG: Generating technology of fermentation inoculum for industrial production of microalgae. 27