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Screening, isolation and identification of fresh water microalgae and factors influencing of polysaccharide production.

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 N[O.sub.3.sup.-] and S[O.sub.4.sup.2-] 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 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]. Industrially, 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 (3.12 x [10.sup.-5] M citric acid, 1.76 x [10.sup.-2] M NaN[O.sub.3], 3.04 x [10.sup.-4] M MgS[O.sub.4].7[H.sub.2]O, 1.89 x [10.sup.-4] M [Na.sub.2]C[O.sub.3], 2.79 x [10.sup.-6] M EDTA, 3 x [10.sup.-5] M [C.sub.6][H.sub.5]+4yFexNy[O.sub.7], 2.45 x [10.sup.-4] M Ca[Cl.sub.2].2[H.sub.2]O, 1.75 x [10.sup.-4] M [K.sub.2]HP[O.sub.4] and 1.5 % Agar) and cultured under a controlled temperature (30[degrees]C) 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[degrees]C 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.

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 to 1 x [10.sup.5] 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 1.76 x [10.sup.-2] M of [Na.sub.2]N[O.sub.3] and on BG-11 with reduced 1.76 x [10.sup.-4] M [Na.sub.2]N[O.sub.3]. 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 NaN[O.sub.3] with fluorescent light

Treatment D: BG-11 medium less of NaN[O.sub.3] with sunlight

Studied in a nitrogen source concentration, One hundred mL of starter microalgae (1 x [10.sup.5] cell/mL) was inoculated into 900 ml BG-11 medium with high and less NaN[O.sub.3] in a 2 L bottle. The culture was sparged with filtered air at 30[degrees]C 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[degrees]C for 18 h. Subsequently, the cell was baked at 55[degrees]C and then studied for optimization of polysaccharide extraction by precipitation as follow.

Polysaccharide extraction by acidified NaCl[O.sub.2] solution:

The extraction method was modified from Sui et al., 2012 [29]. Dried microalgae 0.5 g and NaCl[O.sub.2] 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[degrees]C 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[degrees]C and then centrifuged at 9,000 rpm, at 10[degrees]C for 20 min to collect the supernatant. The supernatant was precipitated with 3 volumes of ETOH and kept overnight at 4[degrees]C and then centrifuged at 9,000 rpm, at 10[degrees]C, for 10 min to collect the precipitated polysaccharide.

Polysaccharide extraction by alkaline [H.sub.2][O.sub.2] solution:

The polysaccharide extraction by alkaline [H.sub.2][O.sub.2] 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 [H.sub.2][O.sub.2] (30% [H.sub.2][O.sub.2] 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[degrees]C 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[degrees]C, polysaccharide was collected by centrifugation at 9,000 rpm, 10[degrees]C 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[degrees]C for 3 h and then centrifuged at 9,000 rpm, 10[degrees]C for 10 min. The supernatant was precipitated with ETOH, overnight at 4[degrees]C and centrifuged at 9,000 rpm, 10[degrees]C 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].

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[degrees]C for 10 min to separate algae cell from supernatant. Supernatant was concentrated by heating at 80[degrees]C 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[degrees]C for 24 h, RPS precipitated was collected by centrifugation at 9000 rpm, 10[degrees]C for 10 min.

Extraction of CPS:

The cell free medium was dried in the oven at 55[degrees]C for 24 h. For extraction, 0.3 g dried cell was mixed with 24 mL of 0.1 M NaCl[O.sub.2], then incubated at 75[degrees]C before adding 25 []L glacial acetic acid at hourly interval of 4 h. The mixture was centrifuged at 9000 rpm, 10[degrees]C for 10 min. CPS was then precipitated by adding 3 volumes of cold 99.9% ethanol to the supernatant. After standing at 4[degrees]C 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[degrees]C 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[degrees]C 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: [H.sub.2]O = 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% [H.sub.2]S[O.sub.4] in methanol and then incubated at 100[degrees]C 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, KK-LP4 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 (Table 1). 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.

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).

Comparison of polysaccharide extraction method:

Results of polysaccharide extraction from the three methods showed that extraction by NaCl[O.sub.2] gave a polysaccharide dry yield of 96.0 mg/g while extraction by [H.sub.2][O.sub.2] 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 NaCl[O.sub.2] would be selected to be used in this study.

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 NaN[O.sub.3] with sunlight) and C (BG-11 medium less of NaN[O.sub.3] 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.

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.

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 NaCl[O.sub.2] 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.

ARTICLE INFO

Article history:

Received 3 October 2015

Accepted 10 October 2015

Published Online 13 November 2015

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 Post-Harvest 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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Jintanart Wongchawalit and Jiraphan Premsuriya

Department of Microbiology, Faculty of Liberal Arts and Science, Kasetsart University Kamphaeng-Saen Campus, Nakhon Pathom, 73140, Thailand

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: faasjnw@ku.ac.th

Table 1: Resulting of total sugar analysis from polysaccharide

Samples    CPS    RPS

Chr-WD3    +++    ++
PR-KD2     ++     ++
KK-LP4     +      -
Np-KhR4    +      -
PK-P[H.sub.2]     +      -

* (+) = having reaction (-) = non-reaction
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Author:Wongchawalit, Jintanart; Premsuriya, Jiraphan
Publication:Advances in Environmental Biology
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Date:Oct 1, 2015
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