Department of Chemical Engineering

University of San Carlos Technological Center

Nasipit, Talamban, Cebu City

ChE 426 N
Introduction to Biotechnology

A Critique on a
Effects of Various Organic Carbon Sources on the Growth and Biochemical Composition
of Chlorella pyrenoidosa

Submitted to
Dr. Camila Flor Y. Lobarbio

by
Carl John Louie G. Navalta

February 3, 2016

Introduction
In this study, Chlorella pyrenoidosa was utilized, and the changes in its biochemical
composition by different carbon sources in different concentrations were investigated. Chlorella is
a single-celled algae that grows in fresh water and has high protein content which is the main
reason why these algae are commercially cultivated at a large scale to produce healthy food and
animal nutritional supplements (Metting, 1996; Spolaore et al., 2006). In cultivation, addition of
glucose to the medium has been reported to affect the lipid contents of Chlorella sp., Chlorella
sorokiniana, and Chlorella vulgaris compared with autotrophically grown cells. However, the
effects of more organic nutrients on the biochemical composition of more chlorella strains are also
studied in order to evaluate the biochemical potential of mixotrophic cultivation.
To carry out the metabolic processes necessary for growth, algae require carbon, hydrogen,
oxygen, nitrogen, phosphorous, sulfur, magnesium, iron, potassium, various other cations, and a
number of trace elements. In mixotrophic cultivation, the microorganisms carbon sources are
from organic compounds, thus monosaccharides (glucose, fructose, and galactose), disaccharides
(maltose, lactose and sucrose) and starch are used in the study. Nitrogen condition of the culture
was also altered, under normal nitrogen condition, low nitrogen condition and nitrogen-deficient
condition.

Summary of the Study

The study was carried out in four methods, (a) algal straining, (b) determination of growth
and (c) determination of biochemical composition of the algae and (d) statistical analysis of the
results.
In algal straining, the cells were grown axenically in light-dark cycle with direct exposure
to illumination and optimum cell growth conditions. At the exponential phase, the cells were
harvested and inoculated into a BBM medium containing the nutrients needed by the
microorganisms for growth. Sugar and starch were added to the medium for mixotrophic growth.
For the low nitrogen condition, the concentration of NaNO3 was reduced to 5% of the normal

condition and for nitrogen deficiency, the NaNO3 concentration is zero. The flasks were manually
shaken three times a day an all experiments were conducted in triplicate.
In the determination of the growth of the cell, the researchers used a haemocytometer,
monitoring the cell density of the algae under a microscope.
For the determination of the biochemical composition of the algae, a Fourier transform
infrared (FTIR) spectrometer was used. Before spectrometry, the algal culture was centrifuged for
10 minutes and cell pellets were washed twice with deionized water. Then the analysis was carried
(1)
out as described by Feng et al. (2013). The data obtained in the spectrometer
were processed with

OMNIC 6.0 software. The spectrum baseline was corrected by a rubber-band method using 64
baseline points with exclusion of CO2 bands. The characteristic peak areas of lipid (AL), protein
(AP) and carbohydrate (AC) were calculated by integration. The weights, in milligram, of lipid
(WL), protein (WP) and carbohydrate (WC) were calculated according to the formulas by Pistorius
et al. (2009):
AL = -2.30 + 78.96 x WL
AP = -.27 + 12.72 x WP

(2)

AC = 0.07 + 2.05 x WC

(3)

The percentages of the lipid, protein and carbohydrate content were calculated with the
following formulas by Feng et al. (2013) with the assumption that the algal cells contain only these
biochemical composition:
" =

"
100
" + & + '

(4)

& =

&
100
" + & + '

(5)

' =

'
100
" + & + '

(6)

Data gathered were presented as means with standard error, and the statistical significances
were assessed by analysis of variance (ANOVA) followed by post hoc analysis using a computer
program. A P value of less than 0.05 was considered as statistically significant.
Compared to a control medium, addition of sugars and starch to the medium increased the
growth of the algae. However, monosaccharides have greater observable effects on the growth
than the disaccharides and starch added. Among the tested monosaccharides, glucose turned out
that its is a best carbon source for the mixotrophic growth, followed by galactose and fructose.
Among the tested disaccharides, maltose was the best carbon source, followed by sucrose and
lactose. Least effects were observed for the addition of starch.
On the effect of the addition of sugars and starch to the biochemical composition of C.
pyrenoidosa, both the lipid and protein content decreased significantly. But the decrease in the
protein content is greater than the decrease in lipid content. But on the other hand, there is an
increase in carbohydrate content. Obvious change in the biochemical composition of the algae can
be observed at a concentration of 3.0 g L-1 of sugars and starch.
To further investigate the effects of sugars and starch on biochemical composition of the
algae during its growth, the lipid, protein and carbohydrate content is analyzed after incubation for
3, 6, and 10 days with the addition of 3.0 g L-1 sugars and starch. For these 3 sets of incubation
time, the change in the chemical composition is similar, where the lipid and protein content
decreased while the carbohydrate content increased. But the effect of the addition of disaccharides
and starch is not as remarkable as the addition of the three monosaccharides during cell growth. In
general, with the data gathered, the biochemical compositions of mixotrophic cells are similar to
those of the autotrophic cells in the addition of the three disaccharides and starch but mixotrophic
cells showed a remarkable change in its compositions in the addition of the three monosaccharides,
and also resulted to higher increase in the growth of C. pyrenoidosa but a decrease in the protein
content.
Under low nitrogen condition, the growth of the algae was inhibited. Addition of carbon
sources had stronger effects on normal condition compared with nitrogen stress condition. In the
study, low nitrogen condition led to a similar pattern of biochemical composition change in

autotrophic and mixotrophic cells in the addition of three disaccharides and starch, where there is
a decrease of protein content, an increase in the carbohydrate content and less changes in lipid
content. But in the addition of three monosaccharides, there is a different change for autotrophic
and mixotrophic cells. Addition of monosaccharides significantly decreased the cellular protein
content and significantly increased the carbohydrate content, but there is no significant change in
the lipid content.
The changes to the biochemical composition of C. pyrenoidosa brought about by the
addition of different carbon sources were further assessed under nitrogen deficient condition. A
significant increase of carbohydrate content was induced by nitrogen deficiency in mixotrophic
and autotrophic cells in the addition of sucrose and starch, while there is no significant change in
the carbohydrate content observed in mixotrophic cells with the addition of glucose. In consistent
with the results under the normal nitrogen condition and low nitrogen condition, the addition of
sucrose and starch to mixotrophic cells, compared to autotrophic cells under nitrogen deficient
condition, yield no significant difference in the lipid, protein and carbohydrate contents.
The addition of monosaccharides to the mixotrophic culture of C. pyrenoidosa significantly
affects its biochemical composition compared to the addition of disaccharides and starch under
different nitrogen conditions. And the effects of monosaccharides on cellular biochemical
composition could be different from those of the disaccharides and starch when the alga is
mixotrophically cultivated for biotechnological applications. Both nitrogen stress under
autotrophic condition and the addition of monosaccharides significantly increased the
carbohydrate content of the algae, and further enhanced its carbohydrate accumulation through the
addition of monosaccharides.
For biotechnological applications, the productivity of C. pyrenoidosa will have different
patterns with the addition of different carbon sources. Disaccharides and starch may have a
potential to increase algal productivity while maintaining a high protein content for biomass.

Evaluation
The green alga C. pyrenoidosa was exenically grown in a good condition in which the cell
growth, represented as cell density, shows no lag phase after a 10-day culture. The inoculum was
given enough nutrients for growth and the nitrogen condition was also altered to determine also its
effect when various carbon sources were introduced to the medium. Although the researchers
didnt show a plot of cell density versus time, which enables the reader to see the trend of the cell
growth as to when the cell exhibits lag phase, exponential phase, stationary phase and death
phase. Because through this, and as an additional information to their study, the reader will be able
to know the specific growth rate, the doubling time and the number of doubling generations which
is helpful for future study of the biotechnological application of the algae.
The researchers used programs to accurately determine the biochemical composition of the
algae and also for data processing. In determining the concentration, in percentages, of lipid,
protein and carbohydrate, the researchers assumed that the cell only contains these, but there are
other biochemical component that are present inside the cell but they the assumption is just safe
because other components are negligible in amount compared to the amount of lipid, protein and
carbohydrate.
There are many useful facts that the researchers have written on their study, specially on
the comparison of autotrophic and mixotrophic cell culture. They have also presented a good
comparison between chlorella species that they are able to suggests that an increase or a decrease
on the biochemical composition of the algae can be strain specific. They have also cited that there
are still unknown studies on the utilization of disaccharides and complex sugars by chlorella
species. Moreover, the results of the experiment were shown in figures which are easy to
understand and their explanations on the results were clearly presented with facts that are compared
to their results and hypothesis were stated. Because of this supporting information, the study makes
it more interesting to read and the readers might get information and the interest to further study
C. pyrenoidosa and other chlorella species.
The study didnt only focus on the effect of carbon sources to the growth and biochemical
composition of the algae, but also the effect of low nitrogen concentration and nitrogen deficient
condition, where the increase and decrease of lipid, protein and carbohydrate content is altered. It

is good that this was also presented for biotechnological application. If it wished that the growth
of the algae increases while maintaining the high protein content, disaccharides and starch is used,
while on the other hand, if the use is solely for reproducing the algae, monosaccharides are used
for higher cell density.

Conclusion
The aim of the study, which is to investigate the effect of various organic carbon sources
to the growth and biochemical composition of algae, was achieved by the researchers through
addition of three monosaccharides, three disaccharides and starch to the medium. The effects were
shown in figures and trends were discussed thoroughly with current facts as their basis of
comparison that made their study more significant.
It was found that glucose is the most effective carbon source which stimulates cell
productivity and increasing carbohydrate content in the cell. However, disaccharides and starch,
although did not show significant effect on the biochemical composition, is effective when the
desired product is the increase in cell productivity and maintaining the content of the cell.
The results in this study is a great help to the biotechnological industry, mostly in the
production of food for humans and nutritional supplements for animals.