Advances inEnvironmental Biology, 9(26) Special 2015, Pages: 29-37
AENSI Journals
Advances inEnvironmental Biology
ISSN-1995-0756
EISSN-1998-1066
Journal home page: http://www.aensiweb.com/AEB/
Screening, Isolation and Identification of Fresh Water Microalgae and Factors
Influencing of Polysaccharide Production
Jintanart Wongchawalit and Jiraphan Premsuriya
Department of Microbiology, Faculty of Liberal Arts and Science, Kasetsart University Kamphaeng-Saen Campus, Nakhon Pathom, 73140,
Thailand
ARTICLE INFO
Article history:
Received 3 October 2015
Accepted 10 October 2015
Published Online 13 November 2015
Keywords:
Microalgae, Polysaccharide,
18S rDNA, Bioresource,
Chlamydomonas debaryana
ABSTRACT
Biopolymer secreted from algae consisting of polysaccharide is recent becoming
important to several industrial fields. Several researches of polysaccahride have been
reported in a part of medicine benefit such as anticoagulant, antiviral, antioxidative and
anticancer. In this study 25 isolates of pure microalgae strain were screened for
polysaccharide production by morphological characteristics under a microscope using
negative staining. The isolates that had a capsule or clear zone surrounding the cell
were selected. Five selected isolates, Np-KhR4, PR-KD2, PK-PH2, SK-SK and ChrWD3 were cultivated in 200 ml BG-11 medium under fluorescent lamp condition. The
cellular polysaccharide (CPS) and released polysaccharide (RPS) were extracted and
determined total sugar by the phenol-sulfuric acid method. The isolate Chr-WD3 can
produce more polysaccharide than the other isolates and it was identified as
Chlamydomonas debaryana (99% identity) by 18S rDNA sequencing. C. debaryana
Chr-WD3 was selected for preliminary extraction of polysaccharide by treatments of
NaClO2, H2O2 and hot water. The result showed that the mildly acidic precipitation
gave the highest dry weight of polysaccharide production at 96.0 mg/g cell dry weight
(9.6% w/w), the alkaline solution of H2O2 gave a moderate polysaccharide production
at 45.0 mg/g cell dry weight (4.5% w/w), whereas hot water, a neutral solution,
treatment gave the lowest dry weight of polysaccharide. Two factors: concentration of
nitrogen source in BG-11 medium and light source, had an affected on polysaccharide
production. The results showed that at low concentration of nitrogen source (1.76x10-4
M NaNO3) C. debaryana Chr-WD3 produced more CPS but less RPS than at high
concentration (1.76x10-2 M NaNO3). Under natural sun light treatment (approx. 67,000
lux of light intensity), C. debaryana Chr-WD3 produced more CPS and RPS than in the
fluorescent lamp treatment (approx. 3,000 lux of light intensity). Preliminary
monosaccharides composition of CPS and RPS were elucidated by acid hydrolysis and
analyzed by thin layer chromatography (TLC). Both CPS and RPS were
heteropolysancharide containing the same monosaccharides composition including
galactose, mannose, arabinose, xylose, rhamnose and three unidentified
monosaccharides.
© 2015 AENSI Publisher All rights reserved.
To Cite This Article: Jintanart Wongchawalit and Jiraphan Premsuriya. Screening, Isolation and Identification of Fresh Water Microalgae
and Factors Influencing of Polysaccharide Production. Adv. Environ. Biol., 9(26), 29-37, 2015
INTRODUCTION
Microalgae are extremely small plant-like organisms, lacking leaves and roots, which occur in sea water
and fresh water[1]. They have been used as a food for a long time because they are a good protein source health
benefits. Several species of microalgae can produce useful organic compounds or secondary metabolites for
example; carotenoids, phycobiliproteins, polyunsaturated fatty acids, sterols, vitamins and important
polysaccharides [2]. Many researches have extensively investigated the polysaccharides from microalgae with
the purpose of discovering a new microalgae strain for the production of novel polysaccharide or genetically
engineering by plausible procedures [3]. The optimum conditions for polysaccharide production were also
studied, such as the influence of reducing NO3- and SO42- on polysaccharide production of Rhodella reticulate,
this showed more polysaccharide dry weight and the stationary phase is the best stage for producing
polysaccharides [4]. The effect of nitrogen sources and light intensity on secretion of extracellular
polysaccharide from Nostoc strains found that some strains increase total sugar production when increasing light
Corresponding Author: Jintanart Wongchawalit, Department of Microbiology, Faculty of Liberal Arts and Science,
Kasetsart University Kamphaeng-saen Campus, Nakhon Pathom, 73140, Thailand.
Tel: 0-3428-1105-7 ext. 7655, Fax: 0-3428-1057; E-mail: [email protected]
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Jintanart Wongchawalit and Jiraphan Premsuriya, 2015
Advances inEnvironmental Biology, 9(26) Special 2015, Pages: 29-37
intensity. Additionally, one strain could produce high levels of extracellular polysaccharide (EPS) in both
diazotrophic and non-diazotrophic conditions [5]. Numerous algae polysaccharide functions were researched in
several fields. For instance environmental, polysaccharide was used in helping oceanic organisms for reducing
the toxic in water by creating a complicated inorganic charge [6]. Synthesis of bioactive substances is a growth
promoter for other algae and plants [7]. Study of algae strains adaptation exhibited high polysaccharides in toxic
stress condition [8]. Heavy metal adsorption gave a positive result for Mn(II), Cu(II), Pb(II) and Hg(II)
adsorption of Anabaena spiroides by using released EPS [9] and Cd and Pb adsorption of Padina gymnospora
by using cell wall polysaccharides [10]. Pharmaceutically, it was reported that the polysaccharide from Nostoc
commune has an antioxidant activity on superoxide anions and hydroxyl radicals and increases antioxidant
enzyme activity [11]. Also, Zhang et al, (2010) found five genuses of polysaccharide producing microalgae that
gave a powerfully antioxidant capacity [12]. Moreover, sulfated polysaccharides from algae were reported to
have anti-HIV activity particularly, the brown seaweed group, red seaweed group and cyanobacteria group [13].
In addition, sulfated polysaccharides also have anticancer activities such as fucoidans and porphyran [14] which
correspond to Yamamoto et al (1986) works that porphyran from Porphyra yezoensis could suppress Sarcoma80 growing [15]. Furthermore, the sulfated polysaccharides from the Chlorophyta group have an anticoagulant
capability by restraining the thrombin enzyme [16], similar to Arthrospira platensis [17] and Monostroma
latissimum (cyanobacteria group) which have an efficient anticoagulation [18]. Industrically, several economical
algae polysaccharides are extensively used in industry and dairy farming namely agar from red algae; Gelidium
spp. and Gracilaria spp. [21, 24, 19], carrageenan from red algae; Kappaphycus alvarezii, Eucheuma
denticulatum [21, 19], Chondrus crispus [22, 23, 19], alginate from brown algae; Laminaria spp., Macrocystis
spp., Ascophyllum spp. and Fucus spp. [20, 19]. Moreover, there are other important polysaccharides from
algae for examples, fucoidans; Cladosiphon okamuranus, Laminaria digitata and Saccharina latissima [19],
laminaran; Laminaria spp., Saccharina spp. [19], ulvan; Ulva sp. [25, 19] and porphyran; Palmaria spp. [26,
19].
Due to their various advantageous properties of algae polysaccharides, Thailand is a tropical country where
there are plenty of fresh water bio-resources for collecting and sampling microalgae. Additionally, there is a
great interesting to discovery the new useful microalgae polysaccharides. Consequently, the objectives of this
research are isolation and identification of polysaccharide producing microalgae strain from fresh water, study
of factors influencing of growth and polysaccharide production conditions. Finally, we investigated the
preliminary analysis of monosaccharide composition.
MATERIALS AND METHODS
Microalgal cultures and media:
Twenty five pure fresh water microalgae were kindly obtained under collaboration with Post-Harvest and
Products Processing Research and Development Office, Department of Agricultural, Thailand. They were
collected on BG-11 agar slant (3.12x10-5 M citric acid, 1.76x10-2 M NaNO3, 3.04x10-4 M MgSO4.7H2O,
1.89x10-4 M Na2CO3, 2.79x10-6 M EDTA, 3x10-5 M C6H5+4yFexNyO7, 2.45x10-4 M CaCl2.2H2O, 1.75x10-4 M
K2HPO4 and 1.5 % Agar) and cultured under a controlled temperature (30 oC) with 3000 lux light intensity, 12
h per day until the green colure of microalgae appeared on the slant.
Screening and isolation of polysaccharide producing microalgae:
Polysaccharide producing microalgae were screened and isolated by morphological characteristic under
light microscopy using the wet mount technique. Nigrosin staining (Negative staining) was performed and then
characterized as a capsule or clear layer loosely enclosing the cells under light microscopy. Isolated microalgae
were cultured in 200 mL of BG-11 medium in a 250 mL erlenmeyer flask, at 30 oC with continuous 3000 lux of
cool white fluorescent light and aeration by using an air compressor for 15 days. After culturing, released
polysaccharide (RPS) and cellular polysaccharide (CPS) were extracted and analyzed for total sugar by phenolsulfuric method [27].
Identification of polysaccharide producing microalgae:
The isolated microalgae were identified by amplifying ribosomal RNA gene sequences using 18S rDNA
gene sequencing. DNA was extracted using DNA Isoplant II kit (Wako Pure Chemical Industrial Ltd., Japan)
with isolated microalgae grown on BG-11 medium for 10 days and centrifuged for cell harvesting. The extracted
DNA was amplified 18S rDNA by polymerase chain reaction (PCR) with Taq polymerase (Toyobo, Tokyo,
Japan) using universal eukaryotic primers (5’-GTCAGAGGTGAAATTCTTGGATTTA-3’as a forward primer
and 5’-AGGGCAGGGACGTAATCAACG-3’ as a reverse primer) [28]. PCR products were approximately 700
bp. Following sequencing by ABI PRISM 310 Genetic Analyzer, the nucleotide sequences of 18S rRNA gene
were compared to 18S rRNA gene sequence database of GeneBank by using the NCBI BLAST program.
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Jintanart Wongchawalit and Jiraphan Premsuriya, 2015
Advances inEnvironmental Biology, 9(26) Special 2015, Pages: 29-37
Study on factors influencing of polysaccharide production:
Preparing EPS producing microalgae starter:
One loop of seed stock was streaked on 100 mL BG-11 agar slant in a bottle and incubated at room
temperature under controlled light intensity of 3000 lux fluorescent lamp for 18 h per day. After 14 days, the
colony on the cultivated agar slant was dissolved by transferring 100 mL of BG-11 broth to a bottle. Then, cell
suspension as a starter was prepared by continuously culturing under an air compressor until the cell
concentration reached to1x105 cell/mL.
Optimize culture condition for microalgae growth:
Experiments were assigned to study two factors for growth conditions. The first, we investigated the effect
of the nitrogen source in the medium on the growth rate and EPS production. The selected EPS producing
microalgae was cultivated on BG-11 with 1.76x10-2 M of Na2NO3 and on BG-11 with reduced 1.76x10-4 M
Na2NO3. In addition, both of cultivation conditions were also controlled with the light source, one was cultured
outdoor under sun light and the other was cultured under fluorescent light. In brief for the culture conditions,
there were 4 treatments as following;
Treatment A: BG-11 medium with fluorescent light
Treatment B: BG-11 medium with sunlight
Treatment C: BG-11 medium less of NaNO3 with fluorescent light
Treatment D: BG-11 medium less of NaNO3 with sunlight
Studied in a nitrogen source concentration, One hundred mL of starter microalgae (1×105 cell/mL) was
inoculated into 900 ml BG-11 medium with high and less NaNO3 in a 2 L bottle. The culture was sparged with
filtered air at 30 oC and illuminated with cool white fluorescent light intensity at 3000 lux for 12 h per day
(Treatment A and C). Growing under sunlight, a small outdoor greenhouse was constructed for this experiment.
The culture was prepared the same as in fluorescent light cultivation but under natural light conditions
(Treatment B and D). Temperature and light intensity was measured and recorded every day at 9.00, 12.00 and
15.00 o’clock. Additionally, the microalgae culture was collected for measuring rate of growth by monitoring
absorbance at 430 nm and then the cell amount was calculated from the standard curve between A430 nm and
cell concentration.
Comparison methods for polysaccharide extraction:
An isolated microalga was cultured in BG-11 medium with 3000 lux of fluorescent, at 30 oC for 18 h.
Subsequently, the cell was baked at 55 oC and then studied for optimization of polysaccharide extraction by
precipitation as follow.
Polysaccharide extraction by acidified NaClO2 solution:
The extraction method was modified from Sui et al., 2012 [29]. Dried microalgae 0.5 g and NaClO2 0.375 g
were added to 40 mL of DI water. The solution was mixed and 25 L of acetic acid was added. Then the
mixture was cooled in water and shaken in a water bath at 75 oC for 4 h, also adding 25 mL of acetic acid at h 1,
2 and 3. After that, the solution was incubated overnight at 24 oC and then centrifuged at 9,000 rpm, at 10 oC for
20 min to collect the supernatant. The supernatant was precipitated with 3 volumes of ETOH and kept overnight
at 4 oC and then centrifuged at 9,000 rpm, at 10 oC, for 10 min to collect the precipitated polysaccharide.
Polysaccharide extraction by alkaline H2O2 solution:
The polysaccharide extraction by alkaline H2O2 solution was modified from Sui et al., 2012 [29]. Baked
microalgae 0.5 g in 5 mL DI water was added 30% of 0.165 mL H2O2 ( 30% H2O2 was diluted to 1%). The pH
was adjusted to 11.5 with 1M NaOH. The solution was mixed at room temperature for 18 h and then centrifuged
at 9,000 rpm, at 10 oC for 10 min. The supernatant was adjusted pH to 6.5 by adding 1M HCl and the
precipitated polysaccharide was mixed with 4 volumes of cold ethanol. After standing the mixture overnight at 4
o
C, polysaccharide was collected by centrifugation at 9,000 rpm, 10 oC for 10 min. The precipitated
polysaccharide was kept for the next experiment.
Polysaccharide extraction by heat and neutral solution:
The polysaccharide extraction method was modified from Hye-Jeong et al., 2008 [30]. Dried microalgae
0.5 g was dissolved in DI water at 80 oC for 3 h and then centrifuged at 9,000 rpm, 10 oC for 10 min. The
supernatant was precipitated with ETOH, overnight at 4 oC and centrifuged at 9,000 rpm, 10 oC for 10 min.
After that, the precipitated polysaccharide was kept. All the extracted polysaccharides were analyzed for total
sugar by phenol-sulfuric acid method [27].
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Jintanart Wongchawalit and Jiraphan Premsuriya, 2015
Advances inEnvironmental Biology, 9(26) Special 2015, Pages: 29-37
Extraction of extracellular polysaccharide:
Polysaccharides from microalgae were extracted as 2 parts; released polysaccharide (RPS) and cellular
polysaccharide (CPS) [29].
Extraction of RPS:
After culturing microalgae under the 4 treatments, the culture from each treatment was centrifuged at 9000
rpm, 10 oC for 10 min to separate algae cell from supernatant. Supernatant was concentrated by heating at 80 oC
until the volume decreased to 10 times of the initial volume. RPS was precipitated by adding 3 volume of cold
99.9% ethanol to concentrated supernatant. After standing the mixture at 4 oC for 24 h, RPS precipitated was
collected by centrifugation at 9000 rpm, 10 oC for 10 min.
Extraction of CPS:
The cell free medium was dried in the oven at 55 oC for 24 h. For extraction, 0.3 g dried cell was mixed
with 24 mL of 0.1 M NaClO2, then incubated at 75 oC before adding 25 L glacial acetic acid at hourly interval
of 4 h. The mixture was centrifuged at 9000 rpm, 10 oC for 10 min. CPS was then precipitated by adding 3
volumes of cold 99.9% ethanol to the supernatant. After standing at 4 oC for 24 h, CPS precipitated was
harvested by centrifugation with the same conditions as above.
All the extracted polysaccharides were analyzed for total sugar by using the phenol-sulfuric acid method
[27].
Preliminary purification of polysaccharide:
The polysaccharide was deprotinated by adding 2 volumes of Sevag’s reagent (chloroform: 1-butanol = v/v
= 4:1) [31]. The mixture was shaken on a stirrer with 200 rpm at 25 oC overnight. After that, the mixture was
kept standing at room temperature until 2 layers occurred. The lower layer was discarded and the upper layer of
the solvent was collected by the following method. Polysaccharide solution was dialyzed against 2 L of tap
water in a dialysis bag molecular weight cut off 30 kDa, following by stirring for 48 h. After dialysis, the
polysaccharide solution was lyophilized and weighted to record the dry weight of CPS and RPS.
Study of monosaccharide compositions by thin layer chromatography (TLC):
EPS 3 mg was hydrolyzed with 1 mL of 2 M trifluoroacetic acid (TFA) at 100 oC for 0, 0.5, 1, 2, 4, 8, 12,
24 and 32 h. The hydrolyzed polysaccharide was co-concentrated with 99.9% methanol five times to evaporate
TFA from the sample. The derivable monosaccharides were analyzed by TLC. Polysaccharide acid hydrolysate
was spotted on TLC plate and compared with standard sugars. Eighty five percent of acetronitrile was used as a
solvent 2 times and then isopropanol: 1-butanol: H2O = 12: 3: 4) was used as the second solvent 1 time for
separation of monosaccharide composition. The TLC plate was stained with 0.03% -napthol and 5% H2SO4 in
methanol and then incubated at 100 oC until the TLC occur color spot.
RESULTS AND DISCUSSION
Isolation of microalgae strains for polysaccharide production:
The result from negative staining of microalgae found that 5 isolates, Chr-WD3, PR-KD2, Np-Khr4, KKLP4 and PK-PH2 from all 25 isolates can produce polysaccharides by characterization of capsule production or
clear zone surrounding the cell as see in Figure 1. Preliminary screening polysaccharide producing microalgae
was performed by total sugar analysis with the phenol-sulfuric acid method showing that CPS from Chr-WD3
had the darkest color compared to RPS of Chr-WD3 and other microalgae (Table1). Chr-WD3 was selected for
polysaccharide production because of the wide clear zone on negative staining and the best reaction of total
sugar using the phenol-sulfuric acid method.
(A)
(B)
(C)
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Jintanart Wongchawalit and Jiraphan Premsuriya, 2015
Advances inEnvironmental Biology, 9(26) Special 2015, Pages: 29-37
(D)
(E)
Fig. 1: Photomicrographs of negative stained microalgae strains with capsule. A: Chr-WD3; B: PR-KD2; C:
KK-LP4; D: Np-KhR4; E: PK-PH2
Table 1: Resulting of total sugar analysis from polysaccharide
Samples
Chr-WD3
PR-KD2
KK-LP4
Np-KhR4
PK-PH2
*(+) = having reaction (-) = non-reaction
CPS
+++
++
+
+
+
RPS
++
++
-
Identification of polysaccharide producing microalgae
The PCR amplification of genomic DNA showed a single band of DNA product, about 700 bp on gel
electropholysis as show in Figure 2. The result of BLASTX showed 99% closet identity by 18S rRNA gene of
Chlamydomonas debaryana strain SAG 26.72 (Genbank accession no. JN903975.1).
Fig. 2: Agarose gel electrophoresis analysis of amplification of 18S rRNA gene product; the first lane: DNA
marker (10,000 bp DNA ladder), the second lane: PCR product 700 bp of Chr-WD3
Comparison of polysaccharide extraction method:
Results of polysaccharide extraction from the three methods showed that extraction by NaClO2 gave a
polysaccharide dry yield of 96.0 mg/g while extraction by H2O2 gave polysaccharide dry yield of 45.0 mg/g and
extraction by heat found only a few polysaccharide were precipitated (could not weight) as show in Figure 3.
These polysaccharides were analyzed using the phenol-sulfuric acid method showing yellow and orange color
which is represented as a total sugar on the polysaccharides. Therefore, the extraction method by NaClO2 would
be selected to be used in this study.
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Advances inEnvironmental Biology, 9(26) Special 2015, Pages: 29-37
Fig. 3: Dry weight of extracted polysaccharides from C. debaryana Chr-WD3 by NaClO2, H2O2 and Hot water
Fig. 4: Growth curve of C. debaryana Chr-WD3 in difference conditions; ( ) BG-11 fluorescence, (■) N-less
fluorescence, ( ) BG-11 sunlight and (×) N-less sunlight
Effect of nitrogen concentration and light source on growth and EPS production:
The results of the comparison between two difference concentrations of nitrogen showed that C. debaryana
Chr-WD3 grew better in high nitrogen concentration media (Fig. 4). Higher CPS was produced in lower
nitrogen condition while higher RPS was produced for both cultivated under the fluorescent light and outdoor
sunlight. The effect of light sources, all data including growth, CPS and RPS production showed a higher level
in outdoor sunlight condition than fluorescence light condition. Figure 5A shows the valued of dried weight of
RPS in the four treatments, treatment B (BG-11 with sunlight) gave the highest RPS dried weight following by
treatment A (BG-11 medium with fluorescent light), treatment D (BG-11 medium less of NaNO3 with sunlight)
and C (BG-11 medium less of NaNO3 with fluorescent light). While, the secretion of CPS capsuling the cell was
determined by mg dried weight as shown in Figure 5B. Highest CPS could be produced and secreted out of the
cell when cultivated C. debaryana Chr-WD3 under the treatment D, followed by treatment B, C and A,
respectively. Generally, microalgae can be grown better in the conditions having sufficient nitrogen and light,
whereas EPS production seems to be increased in lower nitrogen condition. In this study the growth results were
common but the EPS production results had some exception. RPS was increased in higher nitrogen condition
contrasting with CPS and the hypothesis. In this study EPS was extracted as crude extract, CPS and RPS could
be different saccharides even though they had the same monosaccharide composition, if not they could be the
same but the response of the algae cell are different like such as keeps EPS surrounding the cell when nitrogen
was insufficient and released more EPS when nitrogen was abundant.
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Jintanart Wongchawalit and Jiraphan Premsuriya, 2015
Advances inEnvironmental Biology, 9(26) Special 2015, Pages: 29-37
(A)
(B)
Fig. 5: Effect of nitrogen concentration and light source on RPS (5A) and CPS (5B) production
Study of monosaccharide compositions by thin layer chromatography (TLC):
The result of monosaccharide composition analysis by TLC is shown in Figure 6. The results show that
both CPS and RPS are heteropolysaccharide composing of the same monosaccharide components including
galactose, mannose, arabinose, xylose, rhamnone and 3 unidentified in the molecule. Monosaccharide
compositions of EPS from other Chlamydomonas species were reported in previous studies. Chlamydomonas
reinhardtii EPS contains glucose, galactose, rhamnose, arabinose and mannose [32] and Chlamydomonas
mexicana has glucose, galactose, arabinose, mannose, xylose, ribose and fucose [33]. The results suggest that
EPS from C. debaryana are different from other Chlamydomonas species.
Fig. 6: Thin layer chromatography of acid hydrolysis of CPS and RPS from C. debaryana Chr-WD3
Conclusions:
From 25 isolates of pure microalgae were screened for polysaccharide production. Only five isolates were
able to synthesize the polysaccharide. The isolate Chr-WD3 showed the highest polysaccharide production and
it was identified as Chlamydomonas debaryana (99% identity) by 18s rDNA sequencing. Three difference
extraction methods were used in this study. The most effective method for extraction was in acidified NaClO2
solution. The cellular polysaccharide (CPS) and release polysaccharide (RPS) were extracted. The result
showed that at low concentration of nitrogen source C. debaryana Chr-WD3 produced more CPS but less in
RPS production than at high concentration of nitrogen source. In sun light condition C. debaryana Chr-WD3
produced both CPS and RPS more than in fluorescent lamp condition. Monosaccharide composition of both
CPS and RPS consist the same monosaccharides moiety including galactose, mannose, arabinose, xylose,
rhamnose and three unidentified monosaccharides which are difference from the other Chlamydomonas species.
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Advances inEnvironmental Biology, 9(26) Special 2015, Pages: 29-37
ACKNOWLEDGEMENTS
This research was funded by grant from Department of Microbiology, Faculty of Liberal Arts and Science,
of Kasetsart University of the year 2015. I would like to express my sincere to Mr. Prayoon Enmak from PostHarvest and Products Processing Research and Development Office, Department of Agricultural, Thailand who
kindly supported microalgae isolates and kindly giving a skill of isolation technique. The authors are grateful to
Mr. Jiraphan Premsuriya and Mr. Phuttipong Pornpatcharawat for helping in a part of isolation, extraction and
optimize culture condition experiments. Additionally, the author thanks for Ms. Nadanong Seeoob for her kindly
encourage writing this manuscript.
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