Identification of microalgae cultured in Bold’s Basal medium from freshwater samples, from a high-rise city

This study aimed at exploring microalgal heterogeneity from fresh water samples collected from inland water bodies in the heavily built city of Singapore. Culturable pure isolates (n = 94) were subject to an in-house microalgal DNA extraction method and LSU rDNA sequencing. Isolates were analysed for their predominance and distribution. A total of 17 different algal genera were identified (H = 2.8, EH = 0.6), of which Scenedesmus spp. and Chlorella spp. constituted 27.5% and 21.3% of isolates respectively, followed by Micractinium spp. (18.8%) and Chlamydomonas spp. (12.5%). We also report 16 new microalgal strains from this region. The data is important from an ecological and biotechnological perspective.

DNA extraction, PCR and sequencing. DNA was extracted from pure isolates by a slight modification of the protocol of Martin-Laurent et al 14 . Briefly 5-6 microalgal pure colonies were added to 500 µl of lysis buffer [(100 mM Tris-HCl pH8, 100 mM EDTA, 100 mM NaCl, 1% (w/v) Polyvinylpyrrolidone, 2%(w/v) sodium dodecyl sulphate)] in a microcentrifuge tube. The tube was dipped in liquid nitrogen for 5 s (4 times) and sonicated at 7 watts for 6 s (5 times). A volume of 500 µl of phenol:chloroform:isoamylalcohol (25:24:1) was added to the suspension and then centrifuged at 14,000 rpm for 5 min. The supernatant was combined with 1/10 volume of 5 M sodium acetate and placed on ice for 10 min, after which it was centrifuged at 14,000 rpm for 5 min. One volume of ice-cold 100% ethanol was added and DNA pelleted by centrifugation (13,000 rpm for 10 min, 4 °C).
The pellet was air-dried, and the DNA was stored in 50 µl of TE buffer. The PCR reaction mixture was performed in a total volume of 50 µl. Five microliter of genomic DNA in TE buffer pH 8.0, 1 µl each of forward and reverse primer, 25 µl of PCR master mix (Promega, P119A, USA) and 18 µl of nuclease free water. The protocol for PCR amplification was initial denaturation (94 °C, 3 min, 1 cycle), 35 cycles of denaturation (95 °C, 45 s), annealing (47.5 °C, 1 min) and extension (72 °C, 1.3 min); followed by final extension (72 °C, 5 min, 1 cycle). The D1-D2 LSU rDNA sequences for each microalga was amplified with universal primers 19 . On agarose gel electrophoresis, bands in between 200 and 1000 bp were excised and purified using a gel extraction kit. The PCR products were outsourced to AIT Biotech Company Singapore for DNA sequencing services.
Analysis. The LSU rDNA sequences obtained were blasted against available sequences from GenBank data base for identification. Shannon diversity index (H) and Evenness (E H ) was calculated as a measure of diversity of both genera and species in the locations and zones. Menhinick's index (D) was calculated for the number of species per zone 20 . Poisson regression and Poisson regression allowing for over-dispersion was used to analyse whether significant associations could be made between zones, locations and identified strains.

Results
With LSU rDNA sequencing, a total of 94 cultivable isolates could be identified. Fourteen isolates which were found to be similar in species identity for particular locations were not included in subsequent analysis. Eighty isolates were further analysed (  Table 2, genus richness (D) was slightly higher for CZ(4.5) followed by WZ(3.7), EZ(3.3) and least in the NZ(2.7). When compared for uni and multi algal heterogeneity in each sample, 7 locations had three or more genera. Two locations CL (in the WZ) and location BG (CZ) had 6 genera. CL and BG also had higher strain diversity in our study (H = 1.7), followed by CG (H = 1.5) and USR, BG (H = 1.29). Zone and location associations with strains in this study were not found to be significant.

Discussion
The isolation, cultivation and identification of microalgae indigenous to an environment is primarily of ecological, biotechnological and commercial interest 1 . BBM, a traditional chemically defined medium was used to isolate microalgae in our study. We were able to successfully grow and isolate 17 different microalgal genera in this medium. We are aware that several genera may not be easily cultivable in this medium, hence we do not claim that this study is representative of the total microalgal biodiversity in Singapore. While some microalgae were easy to identify based on cellular morphology, coccoid forms were generally difficult to distinguish based on microscopy. In this regard, species identification using LSU rDNA sequencing was a powerful technique. One of the greatest challenges was to standardize a method that would allow for extraction of DNA from different types of algae. This is probably because cell wall compositions of microalgae vary widely and may include cellulose, pectins, hemicelluloses, arabinogalactan proteins (AGPs), extensin, lignin, β-mannans, β-xylans, complex sulfated polysaccharides and glycoproteins 12 . Eventually, an in-house modification of the technique by Martin-Laurent et al 14 led to a freeze-sonication based extraction method that was useful in extracting all the isolates we chose to study.
Microalgae are referred to as green gold because of their commercial value. They are cultivable throughout the year, have a low land demand and are a rich source of organic compounds. The three major uses of algae are biofuels (biochar, bioethanol, oil, biohydrogen), direct use (food and supplements for humans and animals), bioproducts (fatty acids, antioxidants, coloring agents, vitamins, anticancer and antimicrobial drugs) 1,21 . Scenedesmus obliquus, the most common microalga in our study, is reported to be used in effluent treatment, fish feed, biodiesel and pharmaceutical industries [22][23][24][25] , Scenedesmus pectinatus, Scenedesmus acuminatus and Scenedesmus acutus are also biofuel candidates [26][27][28] . Scenedesmus bajacalifornicus has pharmaceutical potential 29 . Chlorella vulgaris is widely used in nutrition and biodiesel 30 . Additional to these commercial uses, Chlorella sorokiniana has pharmaceutical and fish feed uses 31 www.nature.com/scientificreports/ potential 34 . Micractinium reisseri is useful in waste water treatment 35 . Chlamydomonas reinhardtii is used as a molecular model and host organism for algal manipulation studies 36 , Chlamydomonas incerta is reported in pollutant removal 37 and Chlamydomonas peterfii is used in chemical and radiation toxicity testing 38 . Fasciculochloris boldii and Ourococcus multisporus also have biofuel potential 39,40 . Coelastrum sp. and Asterarcys quadricellulare have nutrition and pharmaceutical potential 41,42 . Parachlorella beijerinckii is currently employed in the cosmetics industry 43 . Chlorella spp. and Scenedesmus spp. has high removal rates for nitrates, ammonia, nitrites and phosphates 44 . Due to the presence of poly unsatuarated fatty acids, Ankitrodesmus, Chlorella, Chlamydomonas and Scenedesmus spp are known to have cardioprotective value 45 . A large group of our microalgal genera have no prior biotechnological studies on them, such as Desmodesmus sp., Ascochloris sp., Chloromonas sp., Dictyosphaerium sp., Eudorina sp. and Mychonastes sp. Future knowledge of this untapped potential could pave way for further scientific research.
Microalgae are unique to ecological sites of isolation. While our isolates were from fresh water; a past study on algae from bark in this city reported green algae Dictyochloropsis spp. and Pseudomarvania aerophytica among others which were not found in our aquatic sources 46 . Scenedesmus obliquus was the most common (27.5%) among our isolates. It was interesting to observe that studies on algal diversity in various countries showed different predominant genera. For example, studies from India showed Oscillatoria sp. and Lynbygia sp 47 ; studies www.nature.com/scientificreports/ from South Africa showed Chlorella sp., Neochloris sp. and Chlamydomonas sp 48 ; those from America showed Chlorella sp. and Chlorococcum sp. 49 and studies from the Baltic showed Synechococcus and Synechocystis as most common 50 . The presence of a wide variety of carbon-capturing photosynthetic microorganisms in the aqueous habitats interspersed through the high-rise city adds to the natural bio-diversity. Our study is important as it shares the most common microalgal genera present in inland fresh waters. Some of them are known to be of established commercial importance, while others are yet to be explored. We contribute additional knowledge of new microalgal genera and species. With the advent of algae occupying an important place in the pipeline of next generation fuels, foods and nutraceuticals, this study opens avenues for research in the biotechnology sector.