MICROALGAE CULTURE (3) BIO301 Dr Navid Moheimani

Transcription

1 MICROALGAE CULTURE (3) BIO301 Dr Navid Moheimani

2 Nutrients Macronutrients (g.l -1 ) Micronutrients (mg.l -1 ) Trace Elements (μg.l -1 )

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4 Macronutrients C O H N CO 2, HCO 3-, CO 2-3, organic molecules O 2, H 2 O, organic molecules H 2 O, organic molecules, H 2 S N 2, NH 4+, NO 3-, NO 2-, amino acids, purines, pyrimidines, urea, etc Na Several inorganic salts, i.e. NaCl, Na 2 SO 4 K Several inorganic salts, i.e. KC1, K 2 SO 4, K 3 PO 4 Ca P Several inorganic salts, i.e. CaCO 3, Ca 2+ (as chloride) Several inorganic salts, Na or K phosphates, Na 2 glycerophosphate S Mg Cl Several inorganic salts, MgSO 4, amino acids Several inorganic salts, CO 3 2-, SO 4 2- or Cl - salts As Na +, K +, Ca 2+, or NH 4 - salts

5 Micronutrients Fe Zn Mn Br Si FeCl 3, Fe(NH 4 ) 2 SO 4, ferric citrate SO 2 4 or Cl salts SO 2 4 or Cl - salts As Na +, K +, Ca 2+, or NH - 4 salts Na 5 SiO 5 9H 2 O B H 5 BO 5

6 Trace Elements Mo V Sr Al Rb Li Cu Co I Na + or NH + 4 molybdate salts Na 5 VO 4.16H 2 O SO 2-4 or Cl - salts SO 2-4 or Cl - salts SO 2-4 or Cl - salts SO 2-4 or Cl - salts SO 2-4 or Cl - salts Vitamin B 12, SO 2-4 or Cl - salts As Na +, K +, Ca 2+, or NH 4- salts Se Na 2 SeO 3

7 CO 2 a heavy colourless gas that does not support combustion, dissolves in water to form carbonic acid, is formed especially in animal respiration and in the decay or combustion of animal and vegetable matter, is absorbed from the air by plants in photosynthesis, and is used in the carbonation of beverages Precursor to chemicals Foods Beverages Inert gas Fire extinguisher Super critical solvent Agricultural applications Oil recovery Refrigerant Coal, methane recovery Niche uses

8 Why algae need CO 2? Because mass transfer from atmosphere into pond culture too slow, factor of about ten (e.g. productivity without CO 2 addition only about 2-8 g/m 2 -day of biomass (limiting factor is diffusion of CO 2 across the boundary layer and the slow reaction of CO 2 with H 2 O to produced H 2 CO 3 -, which then equilibrates with the bicarbonate buffer

9 CO 2 addition to algae cultures Land plants take CO 2 from air (0.04%). Algae use C i from the growth medium, either CO 2aqueous, or bicarbonate, or both, depending on strain, ph, alkalinity, etc. If CO 2 is not supplied in sufficient amounts culture ph increases, photosynthesis slows down, eventually stops CO 2 + H 2 O HCO H + CO H +

10 Organic C {hetrotrophs and/or mixotrophs} Main form used by algae is acetate (up to about 1 g.l -1 ) Some can also use glucose Other organic C sources ethanol, galactose etc.

11 N The usual nitrogen sources in algal media are (1) nitrate; (2) ammonium; or (3) urea The heterocystous blue-green algae (cyanobacteria), can also fix atmospheric N 2. Most algae can use nitrate (NO 3- ), nitrite (NO 2- ) or ammonium (NH 4+ ) as an N source Urea (NH 2 )CO is also potentially a good nitrogen source for almost all algal species. Urea is hydrolysed before its N is incorporated into the algal cells by the action of either the enzyme urease, or the enzyme urea amidolyase (UALase).

12 Ammonia & Ammonium NH 4 + NH 3 + H + pka in FW at 20 o C = 9.23

13 (a) growth (b) carotenogenesis KNO g.l -1 1 g.l -1 NH 4 NO 3 KNO 3 NH 4 NO 3 Dunaliella salina cultures. ( = 1 g.l -1 KNO 3 ; = 1 g.l -1 NH 4 NO 3 ; O = 0.5 g.l -1 NH 4 NO 3

14 P The major form in which algae take up phosphorous is as inorganic phosphate (H 2 PO 4 - and HPO 4 2- ) Uptake is optimum at alkaline ph Can also use organic P compounds High concentrations of P may be toxic! Luxury phosphate uptake

15 S, Ca, Mg, Na, Cl Required by all algae to some degree Ca:Mg ratio generally more important than actual concentrations

16 Si Most (all?) algae have a low Si requirement Diatoms have high Si requirement (added as H 4 SiO 4 (silicic acid) Germanium if added at a molar ratio of Ge/Si of 0.1 to 0.2 inhibits diatom growth

17 Fe Essential for ALL algae Needs to be added in chelated form (i.e. FeCl2 with EDTA or citrate) to be able to be take up High concentrations are toxic!

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19 Other bits & Pieces Vitamins (mainly B 12 and other B series) Selenium

20 O 2 Algae require O 2 (but some can survive periods of anoxia) High O 2 will inhibit photosynthesis (photorespiration) Competes as substrate for Ribulose,bisphosphate carboxylase/oxygenase

21 Isolating Algae Enrichment Culture Serial Dilution Single cell isolation streaking on agar plates and colony selection Density Centrifugation The spray method

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24 Rapid Growth High lipid content Temperature optimum High photosynthetic efficiency Ability to tolerate high irradiances Salinity Ideally can release oil (Botryococcus braunii) Shear tolerance Non-sticky Grows in selective environment Tolerate high O 2 Heavy and large cells Weak or no cell wall Lipid composition

25 Microalgal cultivation techniques

26 General Types of Microalgae Cultivation systems (partial list) Open ponds Lagoons (unmixed ponds) Inclined shallow systems Circular central-pivot ponds Raceway type mixed ponds Covered ponds, various types Attached growth reactors Hybrid systems, various types Pond + PBR (including PBRs for inoculum) Closed photobioreactors (PBRs) Flat panel reactors: vertical, horizontal, inclined panels Tubular reactors: horizontal, vertical, helical, etc. Hanging bag type reactors. Floating, submerged reactors Reactors with light diffusers Artificial lighting (LEDs, etc.) Attached growth reactors And many, many more All have positives and negatives. Bottom line: cost of biomass and products & use of limited resources (land, water, nutrients, etc.)

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29 Productivity with CO 2 addition: Open unmixed ponds: Yield = (g.l -1 ) = (d -1 ) [DT = d] Pr = (g.l -1.d -1 ) = (g.m -2.d -1 ) Open Raceway Pond: Yield = (g.l -1 ) = (d -1 ) [DT = 10 3 d] Pr = (g.l -1.d -1 ) = (g.m -2.d -1 ) Some closed photobioreactor (Maybe): Yield = (g.l -1 ) = (d -1 ) [DT = 10 3 d] Pr = (g.l -1.d -1 ) = Varied (g.m -2.d -1 )

30 Arguments for PBRs vs. Ponds PBRs have: 1. Higher algae concentration / easier harvesting 2. No or little invasions by algae weeds, grazers; 3. Higher total biomass productivities (g/l/d) 4. Better process control (T, CO2, O2, etc.). 5. Little or no evaporative water losses 6. More efficient use of CO2, nutrients etc. Ponds have: 1. Larger unit scales (hectares vs. <100 m2) 2. Lower capital ( times lower) costs 3. Lower operating costs and energy consumption 4. Self-cooling (allows evaporative cooling) 5. Pond gas exchanger (reduces O2 level) WHAT IS THE BALANCE OF PROS AND CONS? Depends on what we are trying to do: for large-scale, low cost production, only can use ponds. Aquaculture needs large-scale low cost algae production; for that PBRs not feasible. ALSO: advantages of PBRs are mostly illusionary, not real! Best way is to learn from reality: commercial production experience.

31 GENERAL COMPARISON OF ALGAE PRODUCTION SYSTEMS Capital Cost Running Cost Yield (g/m2-d) Reliability Unmixed Ponds Mixed Raceways Cascade System * (1) * * ** (2) ** ** ** *** (2) **** ** *** *** (2) Tubular Photobioreactor ****** **** *** **** Fermenter ****** **** (3) ****** ***** 1 Depends on land & water cost as very large pond area required; 2 The range of species which can be cultured is very limited; 3 Expensive as it requires sugars and sterility, but does not require light.

32 Raceway pond VS Biocoil 200L raceway pond 40L Biocoil Raes et al Comparison of growth of Tetraselmis in a tubular photobioreactor (Biocoil) and a raceway pond /s

33 Tetraselmis sp grown with CO 2 in raceway pond and Biocoil Open pond Biocoil Post-Harvesting cell density (cells.ml -1 ) 40 x x 10 4 Specific growth rate (d -1 ) 0.11± ±0.04 AFDW per cell (pg.cell -1 ) 333±87 489±42 Biomass concentration (mg AFDW.L -1 ) 152±6 500±60 Volumetric productivity (mgafdw.l -1.d -1 ) 15±1 85±11 Lipid productivity (mg AFDW.L -1.d -1 ) 7±1 28±4 BUT!!!!

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35 Fact or fiction?

36 Why microalgae for CO 2 capture, utilization, abatement? Fast growth rates, potentially high productivity No need for agricultural land Can grow on saline waters, use waste nutrients

37 For significant algal biomass productivity (> 10 t.ha -1.y -1 ), CO 2 must be supplied How can it be done? CO 2 pipe The devil is in the details: piping, transfer, outgassing

38 Current processes for fossil fuel utilization and CO 2 emissions vs. CO 2 utilisation/ recycling by microalgae Fuel Carbon (100%) Open Cycle Carbon Clean Gases Fuel Carbon (60%) Gross Calorific Value measures 27 MJ/kg Algae Biomass as Fuel Source (40% Fuel Carbon) Closed Cycle Carbon Management

39 Challenge remains to be resolved Commercial microalgae production for over 50 years Current cost of production > $4.kg -1 Scale required for biofuels ~ 100x or more BASF, Hutt Lagoon, Western Australia, Dunaliella salina production plant.

40 Milking of Algae a Novel method Milking- extract hydrocarbons without killing the algae Botryococcus braunii A green microalga Lives in colonies Has high oil contents Botryococcus braunii has two distinct features; Produces long chain hydrocarbons Stores the hydrocarbons outside of the cell wall 40

41 Repetitive milking of Botryococcus braunii Botryococcus braunii can produce the similar amount of hydrocarbons after 5 days of extraction when 1% CO 2 is aerated through the culture and after 11 days when no extra CO 2 is supplied to the culture, without any extra nutrients. Extraction can be repeated several times. Reference: Moheimani, N., R. Cord-Ruwisch, E. Raes and M. Borowitzka (2013). "Non-destructive oil extraction from Botryococcus braunii (Chlorophyta)." Journal of Applied Phycology 25: Moheimani, N., H. Matsuura, M. Watanabe and M. Borowitzka (2013). "Non-destructive hydrocarbon extraction from Botryococcus braunii BOT-22 (race B)." Journal of Applied Phycology:

42 Extraction pathways Conventional dry extraction Growth (CO 2, light, water, nutrients) Harvesting (Primary, Secondary) Drying Cell disruption Extraction Wet extraction Growth (CO 2, light, water, nutrients) Harvesting (Primary, Secondary) Cell disruption Extraction In situ extraction Growth (CO 2, light, water, nutrients) Harvesting (Primary) Cell disruption Extraction 42

43 Milking Growth (CO 2, sunlight, water, nutrients) Harvesting Let algae produce hydrocarbons again (CO 2, sunlight) Oil Extraction & recovery 43

44 Excrete oil Why Botryococcus braunii

45 Solvent based non-destructive extraction Solvent Solvent + HC Algae Mixing for 5, 10, 15, 20, min Algae Stop mixing

46 Can solvents be used for non-destructive oil extraction of Botryococcus braunii? 1. Hexane 2. Heptane 3. The optimum contacttime is 20 min

47 Does B. braunii de-novo the external hydrocarbon? 1. Yes 2. CO 2 additional significantly reduce the recovery type from 11 days to 5.

48 Can B. braunii be milked for a long term?

49 Productivity of conventional growth VS milking Productivity Nutrient rate Biomass Total oil Total HC External HC Usage Uptake.L -1.d -1.L -1.d -1 mg.l -1.d -1 mg.l -1.d -1 mg.l -1.d -1 mg.l -1.d -1 ng.cell -1.d -1 mgn mgp. mgn mgp Conv growth -CO CO milking - CO CO