Laboratory based improvement of growth and yields in Chlorella using photobioreactors

Transcription

1 Laboratory based improvement of growth and yields in Chlorella using photobioreactors Stuart Rigby. Sustainable Processing Centre for Process Innovation CPI All rights reserved.

2 Technology Focus CPI work across a distinct set of technologies that offer the largest potential impact on the future of manufacturing: Sustainable Processing Printable Electronics Biologics Formulations

3 AlgaeCAT project aims To develop innovative, algae based solution for significant reduction of large scale industrial CO2 emissions. i.e. The development of a flexible system to use algae for the capture of CO2 on an industrial scale. Process intensification (reduced footprint) and optimization (reducing both capital and operating costs) to make algae based technology commercially viable. Development of a route to sustainable biomass production, thereby supporting fuel switching to non-fossil fuel sources (conversion of algal biomass to energy via anaerobic digestion, gasification, incineration & pyrolysis). Development of a low carbon, resource efficient "closed loop" solution.

4 Initial laboratory work to underpin AlgaeCat project Chlorella species chosen at an early stage because; Relative fast growth rate Robust Track record of successful use for flue gases Initially C vulgaris then later C kessleri

5 What is good growth and yields(g/l) in microalgae? Compare with other microbes. Dry cell weights and growth times Bacterial; Recombinant fedbatch E.coli BL g/l in 24-48hr Ps putida continuous 20g/l dilution rate of 0.1h -1 Lactic acid bacteria continuous 12g/l dilution rate 0.1h -1 Filamentous fungi; N. crassa biotransformation 14g/l 24-48hr Yeast; Pichia and Saccharomyces fed batch g/l 2-5 days

6 First PBR tests 4 litre cylindrical polycarbonate bioreactor light via tube in centre No temperature control ph control only by buffer Gas supplied via sparger.only means of agitation All gasses controlled by standard glass flowmeters

7 Starting medium used MBL-Woods Hole Component grams/l CaCl MgSO 4 7H 2 O NaHCO K 2 HPO NaNO Na 2 SiO 3.9H 2 O Na 2 EDTA FeCl 3 6H 2 O Trace Cu Zn Co Mn Mo plus Vit B12 B1 and TRIS buffer ph

8 Issues with first PBR 1. No Temperature control. Major fluctuations in facility used. 2. ph measured but not controlled 3. Poor control over CO 2 addition and measurement in off-gas. Sample taken from headspace 4. No off-gas condenser 5. Data collection limited 6. Agitation via bubbles; Essentially a bubble column

9 Issues with first PBR (cond) 7. Poor DCW after 14days 0.29g/l ; previous data had suggested up to 0.6g/l 8. Unable to sterilise 9. TRIS good substrate for Bacterial contamination

10 Measurement of CO 2 uptake Not possible by measuring gas CO 2 in inlet and outlet For CO 2 uptake; measure dry cell weight using relationship 1.0g of algae is produced from 1.8g CO 2 Confirmed by analysis from Stirling University; 50% carbon Oil content 21% Protein 45% Carbohydrate 36%

11 Measurement of CO 2 uptake Final volume in PBR was 3.42 litres about 0.5 litre lost due to evaporation At 0.29g/l DCW = 1g Total DCW in PBR 1.8g CO 2 removed from 1675g CO 2 added during 350hours (2% of 2 litres /min total gas flow) 0.1% of total CO 2 added was removed

12 Alternative medium S2; Bacterial simple salts Ammonium sulfate, Phosphate buffer, Magnesium sulfate, Iron sulfate and trace metals Ca Cu Mn Zn S2 varied for fermenters. Phosphoric acid used to maintain acid ph during autoclaving and ph brought up to 7 with ammonia solution Eventually concentration of medium reduced to ¼ strength as algal growth yield did not require full strength

13 Illuminated incubator

14 Incubation of flask in CO 2 atmosphere

15 C.protothecoides in MBL and EG JM medium

16 OD 600nm Growth rate of for Chlorella on MBL medium DCW=0.8g/l DCW=0.6g/l DCW=0.75g/l DCW=0.34g/l Time days C.vulgaris MBL C sorokinana MBL C protothecoides MBL C kessleri MBL

17 OD600nm Growth rate of four Chlorella species on EG:JM medium; Shake flasks DCW = 1.2g/l DCW=1.3g/l DCW=1.1g/l DCW=1.2g/l Time days C.vulgaris EG:JM C sorokinana EG:JM C protothecoides EG:JM C kessleri EG:JM

18 OD600/750nm Comparison of OD600nm vs OD750nm for C kessleri time hrs OD600 OD750

19 Moving to more controlled fermenter like system Tight control over temperature 30 C Axenic culture of Chlorella kessleri easily achieved. Sample very easy to take and QC check for contamination. Dissolved oxygen measured with DOT probe ph measured and controlled with NH 4 OH Condensor on offgas Air typically 0.4vvm (2 litres/min in 5 litre fermenter) CO 2 controlled with mass flow controller usually 5%v/v %CO 2 and %O 2 measured in offgas

20 PBR Fermenter

21 PBR/Fermenter

22 OD 600nm DCWg/l Algaecat: Inoculum run 13th March 2012 C.kessleri. Gasflow= 2 litre/min (0.4vvm) 5%CO time hrs OD600nm DCWg/l

23 Maintaining sterile conditions

24 Light in EC PBR/fermenter Light measured in 5 litre lab stirred tank PBR on inside wall Top 192 μmol m -2 sec -1 Middle 466 μmol m -2 sec -1 Bottom 143 μmol m -2 sec -1 6 X 15 watt 2700K Bulbs used. Good approximation to daylight

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26 Lab bioreactor with 20% starting level of CO 2 reduced to 5%; Struvite issues After 2 days OD600nm had dropped from to and the green culture had become colourless. The CO 2 level was reduced to 5% to see whether the culture could be revived After 7 days the culture was still colourless confirming it had died. A total of 50ml 7M NH 4 OH was added after 12hr and no further addition occurred during the run. This ammonia addition was used to buffer ph against the CO 2 being added.

27 Lab bioreactor with 20% starting level of CO 2 reduced to 5% ; Struvite issues CO 2 level reduced to 5%; the ph increased to 8 as the acidic buffering was perturbed due to the reduction of CO 2. The increase in ph produced increased precipitation of Struvite Magnesium ammonium phosphate NH 4 MgPO 4 6H 2 O. Gas sparger blocked preventing anymore CO 2 entering the PBR. This caused a further increase in the ph to 9. Any algal recovery was made impossible by these conditions

28 PBR with LED lighting

29 Comparison of PBR with normal white light and PBR with LED light OD600nm 40 DCW=9.1g/l LED lights DCW g/l DCW=5.5g/l OD600nm White light PBR OD600nm LED PBR DCWg/l of LED PBR Time days

30 Summary Poorly controlled PBR very low dry cell weight of 0.29g/l after 14 days Better control in shake flasks, improved medium DCW =1g/l in 7 days Tightly controlled white light PBR/fermenter system DCW = 5g/l in 37days Tightly controlled LED light PBR/fermenter system DCW = 9g/l in 20days

31 Summary (cond) Exponential growth very short probably <0.5g/l Light limitation; growth proceeds linearly Care needed with CO 2 /ph control

32 C prothecoides heterotophic growth on glycerol or glucose Can easily achieve >50g/l Slow growth helps mitigate O2 limitation Very amenable to large scale production Can compare favourably to bacterial fermentations

33 C prothecoides heterotophic growth on glycerol

34 Freeze dried C protothecoides algal biomass

35 OD Comparison Glucose and Glycerol Hetrotrophic and mixotrothic growth for Chlorella kessleri DCW=8.4g/l 20 DCW=6.0g/l DCW=1.6g/l Time hrs 25g/l Glycerol 25g/L Glucose 25g/L Glucose 25g/L Glycerol DCW=0.7g/l