Journal of Environmental Treatment Techniques                              PDF
2017, Volume 5, Issue 2, Pages: 124-131
J. Environ. Treat. Tech.
ISSN: 2309-1185
Journal weblink: http://www.jett.dormaj.com
Experimental Identifications of Fresh Water Microalgae Species
and Investigating the Media and pH Effect on the Productions of
Microalgae
Tadele Assefa Aragaw 1*, Abraham M. Asmare 2
1- Faculty of Chemical and Food Engineering, Bahir Dar Institute of Technology, Bahir Dar University, Ethiopia
2- Institute of disaster Risk management and Food Security Studies, Bahir Dar University, Ethiopia
Received: 25/07/2017
Accepted: 16/09/2017
Published: 30/10/2017
Abstract
Identifying predominant species around freshwater system is essential to propose a project for harvesting those species which
can be used for colored waste treatment, biofuel production, for cosmotics and carbon sequestration. The objective of this study
was to investigate the co-culture microalgae species and identifying the predominant fresh water co-cultured microalgae
species.The production of microalgae (mixed culture) in photobioreactor configurations wasinvestigated, using different media
formulations in batch operation. In the present study the predominant co-cultured freshwater microalgae Scenedesmussp.,
Chlorellasp., Synedra sp. and Achnanthidium sp. were investigated in batch culture system and Effect of culturing media( BB
Medium and BG-11 Medium) for effective algal growth ware determined. The Bold basalt medium has maximum optical density
value than the BG-11 medium and the maximum biomass concentration was found 0.608 g/L in the Bolt basalt medium and
0.5624 g/L in BG-11 medium for 15 day cultivation time.The study revealed that amount of time required to adapt the
environment differed significantly in both growth medium type. Also pH range has an effect on the mass productions of algae.
From this study, the high productions of mixed culture microalgae were investigated at pH 8.
Keyword: Identification, Microalgae, Mixed culture, media, photobioreactor, Phycology
1 Introduction1
materials used for the set-up, source of light, how the algae
Microalgae
are
microscopic
photosynthetic
will circulate through the reactor, how to provide CO2, and
microorganisms. They are eukaryotic cell and they contain
how to control other parameters such as pH, temperature
and nutrient concentration in the media [3, 4]. A study on
similar organelles such as chloroplasts, nucleus, etc.
Microalgae are generally more efficient than land based
Chlorella vulgaris by [5] found that the most adequate
plants in utilizing sun light, CO2 water and other nutrients
growth was under indoor conditions and utilizing inorganic
nutrient media.
rates. They can also grow in a variety of aquatic
environments the use of fertilizers and pesticides which
Algae are most commonly cultivated in batch cultures
results in less waste and pollution [1, 2].
and harvesting is done before the next batch is started. It is
easier to control the environmental conditions when the
Different approaches can be taken when looking to
grow microalgae in large volumes. Mainly outdoor ponds,
algae are grown in batches. The algal culture is mixed by
with light supplied by the sun, and photo-bioreactors,
shaking or impeller mixing to promote the nutrient and
gaseous exchange. Carbon dioxide gas is supplied to the
which can be outdoor or indoor and light, can be supplied
by electric lights. Species control is better achieved under
culture based on the pH. Light is provided to the algae
closed conditions that are very common in laboratory
cultures by either natural or artificial light sources [2].
setting. Closed systems provide a better opportunity meet
Ideal growth conditions for microalgal cultures are
specific demands and in the control and optimization cell
strain specific, and the biomass productivity depends upon
many factors. These include abiotic factors like
growth parameters. In the case of photo-bioreactors, many
design considerations need to be made depending on what
temperature, pH, water quality, minerals, carbon dioxide,
the end goal is. There are basic design features that should
light cycle and intensity, and biotic factors like cell fragility
and cell density. Mechanical factors include mixing, gas
be considered regardless of the configuration; like the
bubble size and distribution, and mass transfer; these are of
particular concern in photo bioreactors. Algae are grown
heterotrophically
using organic compounds and
Corresponding author: Tadele Assefa Aragaw: Faculty of
autotrophically with CO2 supply. Culture productivity
Chemical and Food Engineering, Bahir Dar Institute of
increases with optimal mineral nutrition. Nitrogen and
Technology, Bahir Dar University, Ethiopia; E-mail:
taaaad82@gmail.com.
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Journal of Environmental Treatment Techniques
2017, Volume 5, Issue 2, Pages: 124-131
phosphorus are essential nutrients in all algae growth
Basal Medium (BBM) inorder to obtain stock algal cultures
Medias [6].
to be used in the experiments and the PH was adjusted (7.5
The two most important factors that affect algae
±0.5) with 0.1N sodium hydroxide and hydrochloric acid
biomass productivity are light and temperature. The energy
[9, 10].BBM and BG-11 macronutrients and micronutrients
for growing algae is provided by light through
are used at the rate of 10 mLs and 1mLfor each liter of
photosynthesis. Light energy must be effectively utilized to
algae cultured respectively. Algal growth could be
achieve higher biomass productivity. Temperature
increased by bubbling in a mixture of air and carbon
influences the rates of all chemical reactions related to algal
dioxide instead of air alone. Fast growing cultures could be
growth and metabolism [7].
enhanced with carbon dioxide pulsed in several times a
To estimate growth rates, one must have a series of
day, keeping the pH below 10.
measurements, at different times, that will permit the
Algae were cultured first with
250ml conical flask
calculation of the rate of change in biomass concentration.
photobioreactor (PBR) in BBM and BG-11 media without
Cell number should be counted, either through a direct
inoculums and sterilization with continuous air bubble
method, as through light microscopy with ahemacytometer,
feeding and with several times a day CO2 flashing (this
or indirectly through biomass concentration (as dry weight)
keeping the pH below 10) until the medium turns green,
or optical density, as long as this measurements correlate
signaling adequate algae growth. Once maximum density is
linearly with the number of cells
[8] .Under a typical
attained, the predominant species were identified according
homogenous batch regime (in a closed system).
to its morphology and microscopic observations as
Identifying predominant species around marine system
prominent genera using digital camera equipped
is essential to propose a project for harvesting those species
microscopy (Olympus DP 73).
which can be used for colored waste treatment, biofuel
Table 2.1 Bold’s Basal Medium (BBM) (Bischoff and
production, for cosmotics and carbon sequestration. The
Bold, 1963). Once obtained the small volume growth algae,
objective of this study was to investigate the co-culture
by scale up several new cultures were used progressively
microalgae speciesand identifying the predominant fresh
for larger vessels for enough culture for mass production
water co-cultured microalgae species.
tanks and some of were kept as stocks (All the media
compositions were sterilized using autoclave at121ºC for
2 Materials and Methods
15 minutes at 15 psi). Harvesting with large vessel were
taken to a batch PBR (500ml and then 1000ml) conical
2.1 Experimental Setup
flask medium used for higher biomass production with
250ml,
500ml and
1000ml conical flask
photobioreactor were used as batch reactors in temperature
50ml and 100ml inoculums from the previously cultured
incubation chamber and sealed with cap stoppers and glass
microalgae respectively.
Under favorable conditions, a newly inoculated culture
tubes through which air fed, exhausted and screwed with
plastic covers. Continuous Air flow
(with
2.0 L/min
in 1 L bottle takes 10-14 days to reach peak density. Once
volumetric air flow rate) from the air pump through
maximum density is attained, healthy algae bottle cultures
are stable for two additional weeks. sterilized water was
polyvinylchloride pipes connected with the glass tubes on
the top of photobioreactor. The air feed tube immersed at
added to make up for any evaporation loss, the PBR was
the bottom of the growth container to allow mixing, to
left undisturbed for about 5 to 10 minutes, then the sample
prevent sedimentations of the algae, to ensure that all cell
was taken.
of the populations are equally exposed to light and
2.3 Algae Cell Density Determination
nutrients, and to improve gas exchange between the culture
medium and the air.
Monitoring algal growth in mass tank cultures is
Two red, two blue and one cool white fluorescent
essential for successful production. The cell density of a
given sample can be measured with the different methods:
lamps were employed as the light source for growth
microalgae with an average light intensity of 1,450 Lux
wet weight, dry weight, optical density, direct cell
with 12/12 light-dark photoperiod with negligible external
counting with a chamber, and successive dilutions followed
by plating. For this study, the optical density method was
light interference. The temperature incubation chamber was
monitored 25±2 0c to optimize the growth conditions of
used. Standard routines to estimate algal concentration
microalgae.The carbon dioxide feed were used several
include direct cell
counts, chlorophyll content
measurement, and absorbance or turbidity numerical
times per day as a carbon source of the species.
correlations. When spectrophotometrical absorbance is the
chosen method, a reading.
2.2 Growth Media and Algae Harvesting
Wavelength of
750 nm is usually recommended,
Algal biomass: Water sample was collected from Lake
Tana, with different sampling location. All the chemicals
although values of 680 and 687 nm have also been used
used were of analytical grad for the media stock solutions
[12, 13, 14, and 15]. These values are correlated to the light
absorbance of chlorophyll, which could be best determined
preparation as shown from table2.1 and 2.2 and stored in
the refrigerator at approximately -4ºC until the final media
at wavelength around 625 nm (PerkinElmer, Lambda 35).
preparation. There are numerous recipes available for the
preparation of algal nutrients. The one used depends on
2.4 Pigments production and Biomass concentrations
nutrient levels in local, ambient water and the algal species
The analysis of profile pigments produced by microalga
cultured. Routine cultivation was carried out at 25± 2°C
cells after culture on nutrient medium showed two
under incubation chambers and light intensity of 1,300 lux
maximum peaks obtained at corresponding to the Peaks of
for 12 days. Many fresh water algae grow well with Bold
absorbance were between 400-500 nm and 600-700 nm.
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Journal of Environmental Treatment Techniques
2017, Volume 5, Issue 2, Pages: 124-131
This showed that chlorophyll a and chlorophyll b,
700 nm,
625nm, the subsequent analysis for growth
respectively. Since the maximum absorption peaks and
determination was 625nm throughout the experiment
clear band width were observed between the ranges of 600-
Table 2.1 Bold’s Basal Medium (BBM) (Bischoff and Bold, 1963)
Stock Solution (g/l
Component
dH O)
Quantity Used
Macronutrients
NaNO3
25.00
10mL
CaCl2 ·2H2O
2.50
10mL
MgSO4 ·7H2O
7.50
10mL
K2HPO4
7.50
10mL
KH2PO4
17.50
10mL
NaCl
2.50
10mL
AlkalineEDTASolution
1mL
EDTA
50.00
KOH
31.00
AcidifiedIronSolution
1mL
FeSO4 ·7H2O
4.98
H2SO4
1mL
BoronSolution
1mL
H3BO3
11.42
TraceMetalsSolution
1mL
ZnSO4 ·7H2O
8.82
MnCl2 ·4H2O
1.44
MoO3
0.71
CuSO4 ·5H2O
1.57
Co(NO3)2 ·6H2O
0.49
Table 2.2.BG- 11 (Allen,1968, Allen and Stanier, 1968, Rippkaetal,1979)
Stock Solution (g/l
Component
Quantity Used
dH2O)
Ferric Citrate solution
6
1mL
Citrica cid
6
1mL
Ferricammoniumcitrate
6
1mL
NaNO3
1.5g
K2HPO4 ·3H2O
40
1mL
MgSO4 ·7H2O
75
1mL
CaCl2 ·2H2O
36
1mL
Na2CO3
20
1mL
MgNa2EDTA·H2O
1.0
1mL
Trace metal solución
(See following recipe)
1mL
H3BO3
----
2.86g
MnCl2 ·4H2O
----
1.810 g
----
0.220 g
ZnSO4 ·7H2O
79
1 ml
ZnSO4 ·7H2O
49.4
0.39 g
Na2MoO4 ·2H2O
1 ml
Co(NO3)2 ·6H2O
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Journal of Environmental Treatment Techniques
2017, Volume 5, Issue 2, Pages: 124-131
Data of the cell density using optical density versus
where, OD625 is the optical density at 625 nm and B is the
culture times were plotted and submitted to polynomial
algae biomass concentrations (g/L).
adjusts. The polynomial equations obtained were used to
calculate the maximum optical density. The maximum
3. Result and Discussion
specific growth rate
(max d-1) was obtained through
3.1. Effect of Culture Medium on the Growth Curve and
exponential adjustment in the logarithmic phase of growth.
Biomass Concentration
Therefore, the growth of algae and biomass concentration
As can be seen from Figure
3.1, the zero day
was monitored by measuring optical density at a
cultivation time was simply clear marine water, but after
wavelength 625 nm. The optical density of the sample was
five day the culture become a little green color. Finally, as
measured at 625 nm using a spectrophotometer and optical
can be seen cultivation were in deep green color (algae).
path length of 10 cm. Algae biomass concentration was
The daily OD of microalgae was recorded by absorbance at
estimated using the following equation (Eq. 1).
625 nm with the help of (PerkinElmer, Lambda 35 UV/VIS
spectrophotometer). The OD value was determined in both
B = 0.38 ∗ OD625
(1)
media (Figure 3.2).
Figure 3.1: The photobioreactor systems
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Journal of Environmental Treatment Techniques
2017, Volume 5, Issue 2, Pages: 124-131
within 10 days and after that cells growth was very slow as
BB Medium
BG-11 Medium
shown in Figure 3.2. As each microalgal species shows
2
different biochemical composition
(e.g., protein, amino
acids, carbohydrate, lipid, fatty acids, chlorophyll and
1.5
carotenoids) in different media which is reflected by
different optical density (OD) of samples.
1
3.2. Algal biomass concentration
The algal biomass concentration was determined by the
0.5
equation (g/l): B=0.38OD625. As can be seen from figure
3.3 exponential phase, stationary phase and dead phase
0
were done to confirm that how long already harvested
0
2
4
6
8
10
12
14
microalgae can maintain. As a result, the corresponding
Cultivation Time( days)
algae color for each phase has been shown from green to
yellowish. This reveal that once the exponential growth
(a)
phase finished, the species can maintain for 9-10 days in
BBM BG-11
conditioned environment.
0.62
BB Medium
0.52
2
0.42
1.5
0.32
0.22
1
0.12
0.02
0.5
1
2
3
4
5
6
7
8
9
10
0
Cultivation Time (day)
0
3
6
9
12
15
18
21
24
27
(b)
Cultivation Time(day)
Figure 3.2: (a) Effect of two growths media in the same
cultivation date (b) microalgal biomass concentration
Figure 3.3: BB medium growth curve with 10% inoculums
by previously cultured species
As shown from Figure 3.2, the different media have a little
bit different optical density values. This is due the effect of
3.3. Identify the predominant fresh water co-cultured
amount and type of medium nutrients. The BB medium has
species
somehow higher optical density value that the BG-11
Microalgae species was identified from the untreated
medium. Even though the media in not significantly affect
co-cultured water sample collected in Lake Tana. The
the optical density value (Table 3) the BB medium was
specific characteristics found for algal strains under light
used for the subsequent studies.
microscopy were assessed and compared with Wehr and
Growth of the algal species on the two media is
Sheath (2002) to find the respective algal genera. The
compared in Figure
3.2. Importantly, the algal growth
obtained results were also confirmed on online algal
exhibited a significant difference in growth rate between
database
called
“Micrographia”
available
at
the two media. Maximum growth rates of algae were more
(http://www.micrographia.com/index.html ).
in BBM than BG-11. The amount of time required to adapt
Alga cells cultivated on nutrient medium gave different
the environment differed significantly in both growth
species. The size of the microalgal species was different
medium type. The lag phase for BG-11 medium is longer
with circular shapes, road shapes and spherical shapes. The
than BBM. BB medium was devised to provide a defined
observation by transmission electronic microscopy
freshwater medium suitable for culturing microalgae. This
confirmed the predominant species was dominantly green
is due to Concentrations and type of nutrients of the major
algae. The identification of the species was done on the
elements in BB medium are suitable for culturing of these
basis of morphology of microalgae.
microalgae species.
The Predominant consortium of microalgae species was
The results revealed that microalgal species had
found in this study was: Scenedesmus sp., Chlorella sp.,
approximately
4 days lag period and reached the
Synedra sp., Achnanthidium sp.
exponential phase within 12 days in BBM medium, but 8
days lag period and reached rapidly the exponential phase
within 12 days. Microalgae cells achieved stationary phase
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Journal of Environmental Treatment Techniques
2017, Volume 5, Issue 2, Pages: 124-131
As can be seen from Figure 3.4 chlorela sp. dominantly
grown but Synedra sp. and Achnanthidium sp. was found in
small amount. This revealed that, mostly the fresh water
microalgae species are green algae.
3.4. Effect of pH on the productions of microalgal species
As can be seen from figure 3.5, the optimum growth of
the fresh water algae was at PH 8 for both media. The
absorbance of bold basalt medium is higher than the BG-11
medium but the significant difference level inadequate.
From this it can be deduce that for the growth of
microalgae is effected at far basic and acidic condition.
Figure 3.4: Microscopic photogram of co-cultured
microalgae species
0.9
BBM
BG-11
BBM
BG-11
0.8
1.4
0.7
1.2
0.6
1
0.5
0.8
0.4
0.6
0.3
0.4
0.2
0.2
0.1
0
0
4
6
8
10
4
6
8
10
pH Range
pH Range
(a)
(b)
BBM
BG-11
BBM
BG-11
2
2.5
1.8
1.6
2
1.4
1.2
1.5
1
0.8
1
0.6
0.5
0.4
0.2
0
0
4
6
8
10
4
6
8
10
pH Range
pH Range
(c)
(d)
Figure 3.5 pH Effect on the algal growth (a) three day, (b) six day, (c) nine day and (d) twelve day cultivation time
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Journal of Environmental Treatment Techniques
2017, Volume 5, Issue 2, Pages: 124-131
3.5. ANOVA Analysis
p-value = 0.435> 0.05. This shows that, at
0.05 level of
Assume that the population variances are not equal, so
significance, there is no significant difference between the
the non-pooled t-test (“Equal variances not assumed”) can
two growth media types, that is the two growth media
be used. From the independent t-test table 4.1, with 27.897
performance of optical density means are almost the same
degrees of freedom, P-value = Sig. (2-tailed) =0.307. Since
(both media type have the same optical density value.
Table 3 independent t-test analysis of the two growth media for optical density value (BB media and BG-11 media).
Levene's Test for
Equality of
T-test for Equality of Means
Variances
95% Confidence
Sig. (2-
Mean
Std. Error
Interval of the
F
Sig.
t
df
tailed)
Difference
Difference
Difference
Lower Upper
Equal variances
0.216
0.646
1.040
28
0.307
0.234060
0.224983
-0.2267960.694916
assumed
Absorbance
Equal variances not
1.040
27.897
0.307
0.234060
0.224983
-0.2268730.694993
assumed
4 Conclusion
of1,450 Lux with
12/12 light-dark photoperiod with
negligible external light interference. For this condition,
The findings revealed that Bold Basalt Medium is the
optimal suitable medium for culturing marine microalgal
BB medium was devised to provide a defined freshwater
species in a mixed culture system due to the comparatively
medium suitable for culturing microalgae. The observation
by transmission electronic microscopy confirmed that the
high productivity of biomass at 25±20C, free air volumetric
flow rate 2.0 L/min, and under continuous light intensity
predominant species was dominantly green algae.
laboratory assistance in the Department of Chemical and
Acknowledgement
Engineering at Bahir Dar University, Bahir Dar Institute of
My sincere gratefulness goes to my supervisor Dr.
Technology.
Abraham Mebrat. we would like to thank Faculty of
Dr. Abraham .M Asmare was a Ph.D. in the Department of
Chemical and Food Engineering, Bahir Dar Institute of
disaster Risk management and Food Security Studies at
Bahir Dar University, Institute of disaster Risk
Technology staffs for their help financially and accessing
all materials to successful completion of my research work.
management and Food Security Studies and a director in
We would like to thank Dr. Bizuayehu and Dr.
the Institute of disaster Risk management and Food
Security Studies
AtikiltAbebe for them guidance and great support for
making this work possible and for all the support along the
work by reading microscope and spectroscopy results
Funding information
respectively.
Since we are from Ethiopia which is categorized under
low-income countries list, we need to kindly request the
waiver.
Abbreviations
pH: numerical scale used to specify the acidity or
basicity;
0C:
degree
centigrade
Compliance with Ethical Standards
;%:Percentage
;UV: Ultra violet
;BBM:bold basal
Conflict of interest: The authors declare that they have no
medium;BG-11:Green algae medium;PBR:photobioreactor;
conflict of interest.
B :algae biomass concentration; nm: nanometer;OD625:
Ethical approval: This article does not contain any studies
optical density at
625nm; cm:centimeter; λmax: lambda
with human participants or animals performed by any of
max.
the authors.
Open Access: This article is distributed under the terms of
Authors’ Contribution Statement
the Creative Commons Attribution
4.0 International
Tadele Assefa Assefa carried out the completion of
License
(http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted use, distribution, and
harvesting, identification, microagal biomass analysis
spectroscopic data collection and interpretation and
reproduction in any medium, provided you give appropriate
completed the manuscript drafting and final writing.
credit to the original author(s) and the source, provide a
Abraham M Asmare participated in the data analysis and
link to the Creative Commons license, and indicate if
identification of the species experiments and assisted in the
changes were made.
manuscript drafting.
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