MICROALGAE CULTURE (3)

BIO301

Dr Navid Moheimani

n.moheimani@murdoch.edu.au

Nutrients

• Macronutrients (g.l-1)

• Micronutrients (mg.l-1)

• Trace Elements (μg.l-1)

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).

Ammonia & Ammonium

NH4+ NH3 + H+

pKa in FW at 20oC = 9.23

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!

Other bits & Pieces

• Vitamins (mainly B12 and other B series)

• Selenium

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

Microalgal cultivation

techniques

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!!!!

Fact or fiction?

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

Why Botryococcus braunii

• Excrete oil

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.

Can B. braunii be

milked for a long term?

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