1.

cultivation techniques
Large-scale, open use of light to cultivate microorganisms, using the amount of CO2 injected
into the water as a variable to affect the growth of microorganisms, is simple to operate and
low cost, but it occupies a large area, has unstable culture conditions, and is difficult to
operate and control (will be discussed later) Mentioning the difficulty of injecting CO2 into
water) and easy pollution Light, carbon, temperature, and pH value have a great impact on
the growth of algae and can also be used as operating variables. For example, we can use
light or dark cycles to affect their growth rate and gas emissions in the water. Also can
consider allowing algae to survive with other bacteria, fungi, and yeast to improve efficiency
There is another microalgae called Nannochloropsis gaditana, it can be used to assess the
response of microalgae to light density. Cells were routinely kept in pre-culture at 22 ± 1 °C
in a growth chamber. During the exponential growth, counting the cells with a cell counter
and then centrifuging to remove the medium. Using the fresh medium at multiple cellular
concentrations to connect with the micro-photobioreactor. Illumination is not changed, and
no extra CO2 was supplied to this system so all the carbon dioxide the system needs comes
from the atmosphere.

2. biosynthesis
Algae can photosynthesize dissolved inorganic carbon (DIC) in water to obtain the energy
they need. This process is called Auto-phototrophic assimilation of inorganic carbons.
Heterotrophic assimilation of organic carbons means that algae do not obtain energy or
carbon through the absorption of light, but convert organic matter in the medium into the
required energy. In addition, we can also use genetic modification technology to improve the
CO2 absorption efficiency of algae. It also can convert light and CO2 to chemical energy
stored as organic carbon sources like carbohydrates and lipids, this is one of the most
effective ways to produce biomass from sunlight.

3. mechanism
CO2 accounts for a very important part in the photosynthesis of algae, and its opposite is
O2. We usually use O2 to limit the growth of algae. Different concentrations of O2 also have
different effects on the photosynthetic efficiency of algae. We can increase the concentration
of CO2 or gas flow in the water to change the photosynthetic efficiency of algae, and also
change the light or dark cycle, thereby affecting the carbon fixation efficiency. Random
mutagenesis is a simple and stable way to develop mutants with desired characteristics,
such as carbon fixation efficiency, photosynthetic efficiency, and CO2 absorption, thereby
affecting the growth of algae. No specific random mutation results are affected. Called
Random mutagenesis. Nuclear radiation mutagenesis, gamma rays are radiation emitted
during the transition and return of nuclear energy levels, destroying macromolecules in cells,
such as proteins, nucleic acids and carbohydrates. These damages may increase the
productivity or synthesis of algae Efficiency. Compared with UV mutagenesis, Nuclear
radiation mutagenesis has a higher energy density and mutation rate, and can interact with
molecules in substances to lead to more diverse phenotypes of mutant strains.

The first-order dynamic equation can model microalgae growth:

dX/dt=μX (1)
μ is the specific growth rate (day-1), X is the biomass concentration (gL-1) and t is the number
of days respectively. The specific growth rates of the cultures can be calculated by Eq.(2):
μ=[ln(Xt/X0)]/(t-t0) (2)
The biomass productivity rate (g L-1day-1) which is also known as linear growth rate can be
estimated by Eq.(3):
P=(Xt-X0)/(t-t0) (3)

4. pathway
Carbon can limit the growth of microalgae in many ways. The concentration of carbon is too
low to maintain the growth of microalgae. However, the cost of dissolving enough carbon
dioxide in water may be very high, so we may change other defecation factors, such as O2
concentration, temperature, and pH value, to achieve the same effect but at a lower cost. In
addition, different algae have different responses to different concentrations of CO2. Algae
have an intoxicating effect on high concentrations of carbon dioxide, resulting in inhibition of
photosynthetic efficiency and growth. An appropriate concentration of CO2 is better for the
growth of algae. Yes, the cost is also lower.

Each type of cultivation for microalgae is discussed in the following paragraphs.

1. Phototrophic cultivation:
Phototrophic cultivation occurs when microalgae use light, and use sunlight as energy and
use inorganic carbon (such as CO2) as the carbon source to generate chemical energy from
photosynthesis.
2. Heterotrophic cultivation:
Some species of microalgae can not only grow under light but also in dark conditions using
organic carbon. The situation when microalgae use organic carbon as energy and carbon
source can be called Heterotrophic cultivation.
3. Mixotrophic cultivation:
Mixotrophic cultivation means doing photosynthesis and using organic compounds and
inorganic carbon to be the carbon source for growth.
4. Photoheterotrophic cultivation:
Photoheterotrophic cultivation means microalgae needs light when it uses organic
compounds as carbon sources. The main difference between Mixotrophic and
Photoheterotrophic is that Mixotrophic needs light as its energy source and
Photoheterotrophic can complete this purpose by using organic compounds. Therefore,
Photoheterotrophic needs sugars and light simultaneously.
Cultivate conditions Carbon source Energy Cell Reactor to Cost
source density scale-up

Phototrophic Inorganic Light Low Open pound Low

system

Heterotrophic Organic Organic Medium Open cultivation Low

carbon systems

Mixotrophic Organic and light High Closed systems High

inorganic

Photoheterotrophic Organic Organic High Closed systems High

compounds and light
The type of light will also affect
Comparison of advantages and disadvantages of separation methods of
biomass and algae
1. Physical methods
(a)Centrifugation: Use the centrifugal force of gravity acceleration to separate algae
and biomass.
(b)Gravity sedimentation method: Use gravity to separate the algae in the solid
phase from the biomass in the liquid phase.
(c)Filtration: special filtration instruments or filter paper, membranes, etc. can be
used for separation. Pressure, temperature, and concentration can also be used as
control variables.
(d)Flotation: It is an "anti-sedimentation process" (algae particles move upward
during flotation and downward during sedimentation).

Method Advantage Disadvantage

(a)Centrifugation It has a wide range of uses It cannot be operated

and can separate most continuously, requires
algae and biomass. breaks, and requires time-
consuming cleaning and
cost.

(b)Gravity sedimentation Because of their different According to the

method densities, different algae can
be separated, and the top
clear liquid is biomass
energy.
experimental results, the
density of algae needs to be
considered. If low-density
algae cannot be separated
smoothly, high-density ones
can be applied, so it is not
widely applicable.

(c)Filtration Low cost, no chemical For specialized filtration

additives, and can be used instruments, clogging or
on a large scale. scaling will occur quickly, so
expensive filters need to be
replaced repeatedly.

(d)Flotation Simple operation, short time Chemical additives need to

consumption, small space be added and the cost is
required and high flexibility, high.
making it a better fresh
water separation method.
2. Chemical methods
(a)Inorganic flocculants: the negative surface charge conferred by COOH terminal on
the surface of the microalgal cells would be reduced or neutralized by adding
aluminum or iron-based chemical flocculants . More explicitly, dissolution of salts in
the medium liberates its respective cations (Al3+, Fe3+ or Zn2+), which reduces
electrostatic repulsive force between the cells, and enables large floc formation.
(b)Organic flocculants: natural or synthetic, or polyelectrolyte flocculants of
either anionic, cationic, or non-ionic

Method Advantage Disadvantage

(a)Inorganic flocculants High efficiency, short time Metal-related issues

(b)Organic flocculants consumption, no pollution to increase the cost of disposal
biomass, allowed to be and are not environmentally
reused, low cost and friendly and cannot be used
environmentally friendly. for food.

3. Biological methods
(a)Auto flocculation or (b)Bioflocculation: Add alkaline Auto flocculation or
Bioflocculation to increase the pH value to achieve carbonate precipitation and co-
precipitation of magnesium and calcium ions by reducing CO2 supply.

Method Advantage Disadvantage

(a)Auto flocculation low cost, eco-friendly, The recovery rate is low in

energy efficient. acidic conditions, but it can
be solved by using alkaline
solutions.

(b)Bioflocculation Environmentally friendly, low Limitations: Different

Bioflocculation consumption,
no chemical additives
required, and no pollution to
biomass energy.
Bioflocculation requires
different environmental
isolation. Bioflocculation of
bacteria and fungi may have
contamination risks and is
more expensive. It may
require higher organic
carbon and cannot be
applied on a large scale.
4. Electrical based harvesting:
Negatively charged algae cells move in the positively charged direction, and the
anode is charged due to electrophoretic movement. The cells lose their charge, and
form flocs or aggregates by the charge neutralization, and may settle at the bottom
depending on the density. or floating on the surface

Method Advantage Disadvantage

Electrical based harvesting
Rapid and strain
independent method,
environmental protection,
low price, simple operation,
and will not pollute biomass.
When used on a large scale,
it may not be possible to
observe algae at a
microscopic level, it
consumes high energy and
the equipment is easily
damaged.

5. Magnetic particle facilitated separation:

Utilize suspended cells to be labeled or adsorbed to micron-sized or nanometer-sized
magnetic particles, and achieve separation using external magnetic separation
labeled composites due to inherent paramagnetism

Method Advantage Disadvantage

Magnetic particle facilitated
separation
Short time consuming,
simple operation, cost-
effective and energy
efficient, efficient harvesting,
reusable, high stability.
The biomass energy in the
process or result may be
contaminated, which is
difficult to solve with current
technology, and investing
more magnetic
nanoparticles may increase
costs.