Post on 29-Jun-2018
MICROALGAE CULTURE (3)
BIO301
Dr Navid Moheimani
n.moheimani@murdoch.edu.au
Macronutrients C CO2, HCO3
-, CO32-, organic molecules
O O2, H2O, organic molecules
H H2O, organic molecules, H2S
N N2, NH4+, NO3
-, NO2-, amino acids, purines, pyrimidines, urea, etc
Na Several inorganic salts, i.e. NaCl, Na2SO4
K Several inorganic salts, i.e. KC1, K2SO4, K3PO4
Ca Several inorganic salts, i.e. CaCO3, Ca2+ (as chloride)
P Several inorganic salts, Na or K phosphates, Na2 glycerophosphate
S Several inorganic salts, MgSO4, amino acids
Mg Several inorganic salts, CO32-, SO4
2- or Cl- salts
Cl As Na+ , K+ , Ca2+, or NH4- salts
Micronutrients
Fe FeCl3, Fe(NH4)2SO4, ferric citrate
Zn SO42¯ or Cl¯ salts
Mn SO42¯ or Cl- salts
Br As Na+, K+, Ca2+, or NH4- salts
Si Na5SiO59H2O
B H5BO5
Trace Elements
Mo Na+ or NH4+ molybdate salts
V Na5VO4.16H2O
Sr SO42-or Cl- salts
Al SO42-or Cl- salts
Rb SO42- or Cl- salts
Li SO42- or Cl- salts
Cu SO42- or Cl- salts
Co Vitamin B12, SO42- or Cl- salts
I As Na+, K+, Ca2+, or NH4- salts
Se Na2SeO3
CO2
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
Why algae need CO2? Because mass transfer from atmosphere into pond
culture too slow, factor of about ten (e.g.
productivity without CO2 addition only about 2-8
g/m2-day of biomass (limiting factor is diffusion of
CO2 across the “boundary layer” and the slow
reaction of CO2 with H2O to produced H2CO3 -,
which then equilibrates with the bicarbonate buffer
CO2 addition to algae cultures • Land plants take CO2
from air (0.04%).
• Algae use Ci from the
growth medium, either
CO2aqueous , or
bicarbonate, or both,
depending on strain, pH,
alkalinity, etc.
• If CO2 is not supplied in
sufficient amounts
culture pH increases,
photosynthesis slows
down, eventually stops
CO2 + H2O HCO3- + H+ CO3
2- + H+
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.
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 N2.
• Most algae can use nitrate (NO3-), nitrite (NO2
-) or ammonium (NH4
+) as an N source
• Urea (NH2)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).
Dunaliella salina cultures. ( = 1 g.l-1 KNO3; = 1 g.l-1 NH4NO3;
O = 0.5 g.l-1 NH4NO3
(a) growth (b) carotenogenesis
KNO3
NH4NO3 1 g.l-1
0.5
g.l-1
KNO3
NH4NO3
P
• The major form in which algae take up
phosphorous is as inorganic phosphate
(H2PO4- and HPO4
2-)
• Uptake is optimum at alkaline pH
• Can also use organic P compounds
• High concentrations of P may be toxic!
• Luxury phosphate uptake
S, Ca, Mg, Na, Cl
• Required by all algae to some degree
• Ca:Mg ratio generally more important than actual concentrations
Si
• Most (all?) algae have a low Si
requirement
• Diatoms have high Si requirement (added
as H4SiO4 (silicic acid)
• Germanium if added at a molar ratio of
Ge/Si of 0.1 to 0.2 inhibits diatom growth
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!
O2
• Algae require O2 (but some can survive
periods of anoxia)
• High O2 will inhibit photosynthesis
(photorespiration)
• Competes as substrate for Ribulose,bis-
phosphate carboxylase/oxygenase
Isolating Algae
• Enrichment Culture
• Serial Dilution
• Single cell isolation
• ‘streaking’ on agar plates and colony selection
• Density Centrifugation
• The ‘spray’ method
Ideally can release oil (Botryococcus braunii)
Rapid Growth
High lipid content
Temperature optimum
High photosynthetic
efficiency
Ability to tolerate
high irradiances
Salinity
Shear tolerance
Non-sticky
Grows in selective
environment
Tolerate high O2
Heavy and large
cells
Weak or
no cell wall
Lipid composition
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.)
Open unmixed ponds:
Yield = 0.01 – 0.05 (g.L-1)
= 0.035 – 0.0693 (d-1) [DT = 20 – 10 d]
Pr = 0.00035 – 0.0035 (g.L-1.d-1) = 0.35 – 3.5 (g.m-2.d-1)
Open Raceway Pond:
Yield = 0.2 – 0.4 (g.L-1)
= 0.0693 – 0.231 (d-1) [DT = 10 – 3 d]
Pr = 0.014 – 0.0924 (g.L-1.d-1) = 4.2 – 27.72 (g.m-2.d-1)
Productivity with CO2 addition:
Some closed photobioreactor (Maybe):
Yield = 0.5 – 1.0 (g.L-1)
= 0.0693 – 0.231 (d-1) [DT = 10 – 3 d]
Pr = 0.034 – 0.231 (g.L-1.d-1) = Varied (g.m-2.d-1)
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 (10 – 100 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.
Capital
Cost
Running
Cost
Yield
(g/m2-d)
Relia-
bility
Unmixed Ponds *
(1) * * **
(2)
Mixed Raceways ** ** ** ***
(2)
Cascade System **** ** *** ***
(2)
Tubular
Photobioreactor ****** **** *** ****
Fermenter ****** ****
(3) ****** *****
1Depends on land & water cost as very large pond area required; 2The range of species which can be cultured is very limited; 3Expensive as it requires sugars and sterility, but does not require light.
GENERAL COMPARISON OF ALGAE PRODUCTION SYSTEMS
Raceway pond VS Biocoil 200L raceway pond
40L Biocoil
Raes et al. 2013 Comparison of growth of
Tetraselmis in a tubular photobioreactor (Biocoil)
and a raceway pond . 10.1007/s10811-013-0077-5
Open pond Biocoil
Post-Harvesting cell density (cells.mL-1) 40 x 104 120 x 104
Specific growth rate (d-1 ) 0.11±0.02 0.31±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
Tetraselmis sp grown with CO2 in
raceway pond and Biocoil
BUT!!!!
Why microalgae for CO2 capture,
utilization, abatement? • Fast growth rates, potentially high productivity
• No need for agricultural land
• Can grow on saline waters, use waste
nutrients
For significant algal biomass productivity
(> 10 t.ha-1.y-1), CO2 must be supplied
How can it be done?
CO2
pipe
The devil is in the details: piping, transfer, outgassing
Fuel Carbon
(60%)
Fuel Carbon
(100%) Open Cycle Carbon
Closed Cycle Carbon
Management
Current processes for fossil fuel utilization and CO2
emissions vs. CO2 utilisation/ recycling by microalgae
Clean
Gases
Algae Biomass as Fuel Source (40% Fuel Carbon) Gross Calorific Value
measures ≈ 27 MJ/kg
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.
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
Milking of Algae – a Novel method
40
• Botryococcus braunii can produce the similar amount of hydrocarbons after 5 days of extraction when 1% CO2 is aerated through the culture and after 11 days when no extra CO2 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: 1653-1661.
• 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: 1-11.
Repetitive milking of Botryococcus
braunii
41
Extraction pathways
• Conventional dry extraction
• Wet extraction
• In situ extraction
Growth (CO2, light, water,
nutrients)
Harvesting (Primary, Secondary)
Drying Cell
disruption Extraction
Growth (CO2, light, water,
nutrients)
Harvesting (Primary, Secondary)
Cell disruption Extraction
Growth (CO2, light, water,
nutrients)
Harvesting (Primary)
Cell disruption Extraction
42
Harvesting
Oil Extraction & recovery
Let algae produce hydrocarbons again (CO2,
sunlight)
Milking
Growth
(CO2, sunlight,
water, nutrients)
43
Solvent
Algae
Mixing
for 5, 10, 15, 20, …
min
Solvent + HC
Algae
Stop mixing
Solvent based non-destructive
extraction
Can solvents be used for non-destructive oil
extraction of Botryococcus braunii?
1. Hexane
2. Heptane
3. The optimum contacttime is 20 min
Does B. braunii de-novo the external
hydrocarbon?
1. Yes
2. CO2 additional significantly reduce the
recovery type from 11 days to 5.
Productivity Nutrient rate
Biomass Total oil Total HC External HC Usage
.L-1.d-1
Uptake
.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 -CO2 32 6.9 2.8 1.9 0.6 9.2 0.9 2.2 0.32
CO2 80 24 14.4 9.3 3.1 18.1 1.8 5.6 0.81
milking - CO2 2.7 0.7
CO2 11.7 3.1
Productivity of conventional growth VS milking