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Experiment 3: Microalgae Cultivation in Photo Bioreactor and Cell

Harvesting by Centrifugation

The optimal temperature for phytoplankton cultures is generally between 20

and 24C, although this may vary with the composition of the culture
medium, the species and strain cultured. Most commonly cultured species of
micro-algae tolerate temperatures between 16 and 27C. Temperatures
lower than 16C will slow down growth, whereas those higher than 35C are
lethal for a number of species. If necessary, algal cultures can be cooled by a
flow of cold water over the surface of the culture vessel or by controlling the
air temperature with refrigerated air - conditioning units.

The pH range for most cultured algal species is between 7 and 9, with the
optimum range being 8.2-8.7. Complete culture collapse due to the
disruption of many cellular processes can result from a failure to maintain an
acceptable pH. The latter is accomplished by aerating the culture (see
below). In the case of high-density algal culture, the addition of carbon
dioxide allows to correct for increased pH, which may reach limiting values of
up to pH 9 during algal growth.

Mixing is necessary to prevent sedimentation of the algae, to ensure that all

cells of the population are equally exposed to the light and nutrients and to
improve gas exchange between the culture medium and the air. The latter is
of primary importance as the air contains the carbon source for
photosynthesis in the form of carbon dioxide. For very dense cultures, the
CO2 originating from the air (containing 0.03% CO2) bubbled through the
culture is limiting the algal growth and pure carbon dioxide may be
supplemented to the air supply (e.g. at a rate of 1% of the volume of air).
CO2 addition furthermore buffers the water against pH changes as a result of
the CO2/HCO3- balance
The growth of the culture is globally seen as a large complex of biochemical
or chemical reactions, which use substrates and yield products in the form of
the desired product or/and cell mass.

Cell growth always produces CO2 which is acidic and to keep the pH constant,
this acid (which partially escapes) has to be compensated by the addition of
another acid by a pH controller with an acid pump. In balanced regular
growth the production of CO2 and the need of pH correcting acid is
proportional to the growth. Therefore, the amount of added pH correction
solution (acid or base) is a precise indication of the growth extent of the
culture. Thus, ph factor is remain stable in six days.

Optical density (OD) of the culture is measured to estimate the growth

and metabolic activity of the cells. It can be seen from the result that the
optical density went up from 0.044 in day 1 to 0.151 day 5 however in the
final day it went down to 0.116

1. Lag or induction phase

This phase, during which little increase in cell density occurs, is relatively
long when an algal culture is transferred from a plate to liquid culture.The lag
in growth is attributed to the physiological adaptation of the cell metabolism
to growth, such as the increase of the levels of enzymes and metabolites
involved in cell division and carbon fixation.

2. Exponential phase

During the second phase, the cell density increases as a function of time
according to a logarithmic function. The specific growth rate is mainly
dependent on algal species, light intensity and temperature.

3. Phase of declining growth rate

Cell division slows down when nutrients, light, pH, carbon dioxide or other
physical and chemical factors begin to limit growth.

4. Stationary phase

In the fourth stage the limiting factor and the growth rate are balanced,
which results in a relatively constant cell density.

5. Death phase

During the final stage, water quality deteriorates and nutrients are depleted
to a level incapable of sustaining growth.

Production sugar increased up to day 3 then gradually dropped to 0.005 in

day 5. It means that sugar as energy source for cell had decreased over

The last is dry cell weight or CDW, the weight or mass of organic matter or
soil after removal to constant weight. From the graph the result of CDW is
following the growth curve phase. The weight climbed until day 3 and then
fell until day 6. This is because, the cell has lack of nutrient thus it started to
decrease of cell weight.
Contamination with bacteria, protozoa or another species of algae is a
serious problem for monospecific/axenic cultures of micro-algae. The most
common sources of contamination include the culture medium (sea water
and nutrients), the air (from the air supply as well as the environment), the
culture vessel, and the starter culture.

Seawater used for algal culture should be free of organisms that may
compete with the unicellular algae, such as other species of phytoplankton,
phytophagous zooplankton, or bacteria. Sterilization of the seawater by
either physical (filtration, autoclaving, pasteurization, UV irradiation) or
chemical methods (chlorination, acidification, ozonization) is therefore
required. Autoclaving (15 to 45 min. at 120C and 20 psi, depending on the
volume) or pasteurization (80C for 1-2 h) is mostly applied for sterilizing the
culture medium in test tubes, erlenmeyers, and carboys.