Microalgae culture

Overview

1. Interest and applications

2. Isolation and perpetual cultures
3. Batch and continuous cultures
4. Photobioreactors and outdoor growth
5. Measuring culture growth
6. Key factors for microalgae culturing

Bibliography:
Andersen R. (2005) Algal culturing techniques. Elsevier
‘Algae’: protists and cyanobacteria
 ‘Algae’: cyanobacteria and protists
Brown algae Red algae
Cyanobacteria

Dinoflagellates
Diatoms Green
algae
Historical notes

• Many of the methods and basic culture medium concepts that are used today
were developed in the late 1800s and early 1900s

• Beijerinck (1890) - The first report of pure (axenic) cultures of algae; first to isolate
free-living Chlorella and Scenedesmus in allegedly bacteria-free cultures

• Miquel (1892) – The first to isolate and establish pure (axenic) cultures of
freshwater and marine diatoms

• Warburg (1919) - Pioneering work to grow microalgae in dense laboratory cultures

(especially Chlorella)

• Ketchum and Redfield (1938) - Described a method for maintaining continuous

cultures of marine diatoms in large supplies for chemical analyses
Applications
• Biodiesel
• Bioethanol
• Bioactive compounds
• Added-value compounds
• Carbon sequestration
Applications
Added-value compounds
Added-value compounds
Bioactive compounds
Biofuels
Biofuels
Biofuels
Biofuels: algae advantages
 Isolation: heat sterilization
• Heat sterilization - the most common form; usually through
autoclaving: the most effective and popular way to sterilize
heat-resistant materials and liquids

• High temperatures (>100°C) - materials to be sterilized must

resist high temperatures (e.g., glassware, metallic
instruments, aluminum foil)

• Autoclave - heavy-walled closed chamber, high steam

pressure producing a high sterilizing temperature without
boiling liquids
 Isolation: filter sterilzation
• Liquids are filter-sterilized when the liquid contains fragile or volatile components
that are destroyed by high temperature

• Also used for convenience and rapidity when only a small volume of liquid is to be
sterilized.

• Membrane filters (i.e., different pore sizes, composition, color, and size), can be
autoclaved and used for stereilizing medium
 Isolation: important factors
• Variability: some species, often called
“weeds”, are easy to isolate and cultivate,
whereas others are difficult or seemingly
impossible to grow

• Understanding and mimicking the naturally

occurring environmental conditions

• Taxonomic knowledge of the target species

is important, e.g. diatoms require silica, and
euglenoids often require ammonia

• Elimination of contaminants, especially

those that can outcompete the target
species
Isolation: methods

Single-Cell Isolation by Micropipette

• The most common method
• Usually performed with a Pasteur pipette or
a glass capillary

Isolation with Use of Agar

• The preferred isolation method for many
coccoid algae and most soil algae
• Ease of use
• Axenic cultures can often be directly
established without further treatment
• Not all species grown on agar
 Isolation: methods
Dilution Techniques
• Effective for organisms that are abundant in the
sample
• Largely ineffective for rare organisms

Automatic isolation
Flow Cytometry with Cell Sorting -
counting and analyzing optical properties
of single cells suspended in a fluid
 Perpetual cultures
• Culture collections: repositories of diversity

• Continuous subculturing

• Loss of morphological, behavioural features

• Important factors:
Choice of culture medium
Light and temperature
Transfer interval
Culture containers
Perpetual cultures
 Cryopreservation
• storage of a living organism, or a portion thereof, at an ultralow temperature
(typically < -130°C) such that it remains capable of survival upon thawing

• largely an empirical science because the underlying biological mechanisms of

cell injury during freezing and thawing are not fully understood

• hundreds of species of cyanobacteria and eukaryotic microalgae have been

successfully cryopreserved
 Cryopreservation
• Important to minimize stress while algae are cooling to their freezing temperature

• An aqueous suspension of microalgae cooled to subzero temperatures is not frozen

homogenously
 Batch cultures
• Culture in which a base medium supports initial cell culture, and in which the
products remain in the bioreactor until the end of the run

• It is initiated by the transfer of a small portion of a culture into a new culture

medium, resulting in growth and an increase in biomass

• Advantages:
Low cost
Ease of manipulation
Small volume of media
Easy manipulations
Batch cultures: growth curve
Subculture
Batch cultures: growth curve

Lag phase

• Very slow growth

• Cells introduced into the fresh medium takes some time to
adjust with the new environment
Batch cultures: growth curve

Exponential phase

• Fastest growth rate – exponential growth

• Cells are adapted to the growth medium and conditions
• Metabolic activity is highest and DNA replication by binary fission
occurs at a constant rate
Batch cultures: growth curve

Stationary phase

• Cell division decreases, equals cell death rate; cell numbers remains
constant
• Nutrients in the growth medium are used up; accumulation of waste
materials, toxic metabolites and inhibitory compounds
• Subculturing required (replacing medium: semi-continuous culturing)
Batch cultures: growth curve

Decline phase

• Cell numbers decrease: growth < death rates

• Depletion of nutrients and accumulation of metabolic waste
products promotes cell death
 Continuous cultures
• Cell growth is maintained continuously, through regular addition of fresh medium
and removal of excess culture (effluent)

• Constant chemical environment: all cells in a steady state, constant growth rate

• Two forms: turbidostat and chemostat

Continuous vs discontinous cultures

Discontinuous: variations in
nutrient level and cell density over
each growth cycle

Chemostat: fixed dilution rate; cells

experience constant nutrient limitation
akin to that seen in batch cultures just
before nutrient exhaustion

Turbidostat: cultures grow at their

maximal growth rate; dilution rate
based on culture density, generating
constant population size
Turbidostats vs chemostats
 Turbidostats
Commercially-available

Open source – DIY

Chemostat vs turbidostat
Photobioreactors
• Closed vessel for large-scale, indoor algal
phototrophic production under highly
controlled conditions and where energy is
supplied via artificial light

• Ability to control and optimize culture

parameters

• A multitude of photobioreactors have been

designed, built, and described; design
depend on the ultimate goal

• Fermentor – without light, microalgae are

grown heterotrophically
 Photobioreactors: light
• The most important parameter in the design of a photobioreactor

• Growth is limited by too little light, but too much light can cause photoinhibition
• Optically-thin containers are preferable to minimize light attenuation and self-
shading - the light intensity transmitted through a culture drops very quickly with
distance from the light source because of the high absorption by chlorophyll

• Adequate mixing “evens out” the light intensity while providing efficient gas exchange
and better pH and temperature control
Photobioreactors: other factors
• Gas exchange
• Photobioreactors can be bubbled with
air, but the low CO2 concentration in air
(0.033%) will often limit phototrophic
growth
• Care must be taken to ensure that the
CO2 input does not adversely lower the
pH level of the culture
• Removal of excess O2 – may lead to
photoxidative damage and increasing
rates of photorespiration

• pH and temperature - the next most important parameters to measure, but

simple to control

• Contamination by bacteria and fungi is not a significant problem because there is

generally very little free organic carbon
Types of photobioreactors
Types of photobioreactors
Types of photobioreactors
Photobioreactors under natural light
 Outdoor ponds
• Commercial-scale culture of microalgae
generally requires ton quantities of biomass -
volumes of +1,000,000 liters, thus done in
open outdoor ponds

• Open pond culture is cheaper than culture in

closed photobioreactors but is limited to a
relatively small number of algae species

• Even thought, species like: Chlorella spp.,

Spirulina spp., Dunaliella salina,
Haematococcus spp. Nannochloropsis spp.,
Phaeodactylum tricornutum, Scenedesmus
obliquus

• There is little literature on actual commercial

culture systems because of commercial
sensitiveness
 Outdoor ponds
• Deep tanks (aerated)
• Center-pivot
• Raceway ponds
• Important: mixing-depth relationship
Growth rates
• During exponential growth, the rate of increase in cell number per unit time is
proportional to the number of cells present in the culture at any time:

N0 - population size at the beginning

Nt - population size at time t
r - instantaneous rate of increase

• If t is expressed in days, then r can be converted to doublings per day (k) by

dividing r by the natural log of base 2:

• Doubling time, T2 (same units of time as r), can be calculated by:

Carrying capacity
• Exponential growth is limited to initial, limitation-free conditions

• Competition for limiting nutrients causes the culture to reach a maximum

concentration K

N0 - population size at the beginning

Nt - population size at time t
r - instantaneous rate of increase
K – Carrying capacity
Growth rates
• In continuous cultures, the continuous supply of fresh medium allows cultures
to remain in exponential growth indefinitely

• At steady state the specific growth rate of the population (µ) is determined by
the dilution rate:

µ = F/V =D

F - medium flow rate to and from the culture vessel (usually liters per day)
V - volume of the culture vessel (usually liters)
D - dilution rate
 Measuring growth rates

• Any estimate of growth rates requires a time series of measurements that allow
an estimate of the rate of change in biomass

• To estimate population growth rates, cell numbers (concentration) must be

counted – cell counting on the microscope

• Proxies, if linearly correlated with cell numbers:

• optical density (turbidity)

• in vivo fluorescence
• biomass (dry weight, particulate organic material)
• concentration of chlorophyll a, protein, carbohydrate, or lipids
Counting chambers
Growth media and growth conditions
Light spectra

• Different algal groups have different

photosyntehtic pigments, absorbing on different
regiosn of the visible spectrum

• Illumination must consider these specific

requirements
 Growth media
• Many culture media have been
developed and used for isolation and
cultivation of freshwater and marine
algae

• Media are generally composed of three

components:
• Macronutrients
• trace elements
• vitamins

• All three are often prepared as stock

solutions
 Marine culture media
• Natural seawater (NW) is a complex medium containing more than 50 elements
and a large and variable number of organic compounds

• For algal culture, NW is not sufficient, and enrichment with nutrients and trace
metals is required

• Macronutrients are generally considered to be nitrogen, phosphorus, and silicon -

required only for diatoms, silicoflagellates, and some chrysophytes

• pH buffers - mantain pH constant over time durign growth of the culture

• Artificial or synthetic seawater: basal (main) salts, enrichment

solution (often the same added to NW)
• Advantages:
• avoid variations in the quality of NW throughout the year
• avoid the need to control nutrient and trace element
concentrations
• limited availability of seawater at inland locations