Microalgal carotenoids: Potential nutraceutical compounds with chemotaxonomic importance

Algal Research 15 (2016) 24–31

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Microalgal carotenoids: Potential nutraceutical compounds with chemotaxonomic importance Chetan Paliwal a,c, Tonmoy Ghosh a,c, Basil George a, Imran Pancha a,c, Rahulkumar Maurya a,c, Kaumeel Chokshi a,c, Arup Ghosh b,c, Sandhya Mishra a,c,⁎ a b c

Division of Salt and Marine Chemicals, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, India Division of Wasteland Research, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, India Academy of Scientific & Innovative Research (AcSIR), CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, India

a r t i c l e

i n f o

Article history: Received 4 June 2015 Received in revised form 20 November 2015 Accepted 28 January 2016 Available online xxxx Keywords: Microalgae Carotenoids Chemotaxonomy Biomarkers Antioxidants

a b s t r a c t There are more than 600 different carotenoids which perform a range of functions in various organisms including microalgae. In the present study, chemosystematics approach was followed to segregate 57 microalgal strains based on their carotenoid composition using principal component analysis (PCA) and hierarchical clustering. The present findings suggest that lutein and violaxanthin can be effective chemotaxonomic markers for Chlorophyta members with an average content of 1.26 mg g−1 and 0.14 mg g−1 dry cell weight (DCW), respectively. Similarly, myxoxanthophyll and echinenone can be used as markers for Cyanophyta members with average contents of 0.23 mg g−1 and 0.32 mg g−1 DCW, respectively. The total carotenoid content ranged from 0.23 to 7.2 mg g−1 DCW. Our method combining PCA and artificial hierarchical clustering has been proposed as an alternative method for identification of carotenoids as biomarkers for classifying unknown microalgal strains based on their pigment profiles. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Carotenoids are a group of diverse lipophilic pigments with over 600 members that play a central role in light harvesting as well as photoprotection in plants and microorganisms. However, given their diverse and ubiquitous nature, they have been used for many years as important tools for identifying the presence of certain microalgal groups in different aquatic ecosystems all over the world [2]. These microalgal groups in various aquatic habitats have been found to display a fixed pattern of carotenoids during specific growth stage, which is often useful for their identification [16,25]. The constituent pigments of these groups are considered excellent chemotaxonomic biomarkers due to their specificity. HPLC characterization of such pigments can lead to a wealth of information about the taxonomic composition and prevailing physiological conditions [15]. Often, these studies give an indication about the influence of climatic and anthropogenic activities on phytoplankton response on a large geographical area [5]. Several research groups have recorded their observations on the prevailing phytoplankton populations in specific areas based on such pigment profiles. Fietz and Nicklisch [5] studied the phytoplankton population in Lake Baikal using a rapid HPLC and CHEMTAX based method to identify the different groups. A similar strategy was utilized

by Madhu et al. [15] for the characterization of phytoplanktonic community structures in Gulf of Mannar and Palk Bay areas. Alternatively, Paerl et al. [20] utilized HPLC analysis followed by photodiode array spectrophotometry to identify areas of eutrophication in coastal areas. The usage of CHEMTAX algorithmic approach has been widespread for these kinds of studies as it utilizes data matrices for calculating the abundance of various algal classes based on the HPLC profiles of their pigments [14]. However, to the best of our knowledge, a statistical approach for identifying carotenoid biomarkers as representatives of specific phytoplankton groups represents a void that can be addressed. Statistical methods utilize a smaller dataset of pigment concentrations to predict representative carotenoid molecules as biomarkers of specific phytoplankton groups in an ecosystem. We have proposed a statistical analysis of the major pigments in 57 different strains of microalgae and cyanobacteria isolated from coastal waters of western India. Hierarchical clustering and principal component analysis (PCA) enabled us to identify certain representative carotenoid molecules by utilizing a far smaller dataset than utilizing CHEMTAX. 2. Materials and methods 2.1. Microalgae identification

⁎ Corresponding author at: Division of Salt and Marine Chemicals, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, India. E-mail address: [email protected] (S. Mishra).

http://dx.doi.org/10.1016/j.algal.2016.01.017 2211-9264/© 2016 Elsevier B.V. All rights reserved.

57 different microalgal species, belonging to the different phylum (Chlorophyta and Cyanophyta) were isolated from Indian waters. The

C. Paliwal et al. / Algal Research 15 (2016) 24–31

cultures were identified based on their morphological characteristics [10].

25

at 10,000g for 5 min. The pellet was lyophilized and stored at −20 °C until further use. 2.3. Extraction of carotenoids

2.2. Culture conditions All microalgae strains were cultured in a 100 ml Erlenmeyer flask with 50 ml of their respective culture medium (Table 1). The cultures were maintained in uniform conditions at 25 ± 2 °C under a light intensity of 60 μmol m2 s−1 and 12:12 h light dark period with manual shaking twice a day. Cultures were harvested after 30 days by centrifugation

The carotenoids were extracted from pre-weighed lyophilized microalgal biomass in 5 ml of 99.9% pure methanol. The cellular extracts were mixed thoroughly to increase the solvent contact time for maximum carotenoid extraction and were later incubated at 45 °C for 24 h in dark, as described in Pancha et al. [19]. After 24 h, the extract was centrifuged at 10,000g for 5 min to remove the cell debris and its

Table 1 Freshwater microalgal isolates with different medium of cultivation and their respective collection sites. Phylum

Class

Order

Genus species

Location

Chlorophyta

Chlorophyceae

Chlamydomonadales

Grasiella emersonni CCNM 1001a Grasiella emersonii CCNM 1011b Grasiella emersonii CCNM 1015b Chlorococcum sp. CCNM 1023b Chlorococcum sp. CCNM 1025b Chlorococcum sp. CCNM 1027a Desmodesmus subspicatus CCNM 1008b Scenedesmus sp. CCNM 1028b Anikistrodesmus sp. CCNM 1031b Monoraphidium minutum CCNM 1042c Acutodesmus dimorphus CCNM 1045 c Monoraphidium sp. CCNM 1046b Scenedesmus sp. CCNM1053b Scenedesmus sp. CCNM 1061a Bracteacoccus pseudominor CCNM 1018c Chlorella sp. CCNM 1002a Chlorella sp. CCNM 1004a Chlorella sp. CCNM1005a Chlorella sp. CCNM 1007b Chlorella sp. CCNM 1014b Chlorella variabilis CCNM 1017c Chlorella sp. CCNM 1019b Chlorella sp. CCNM 1021c Chlorella sp. CCNM 1030a Chlorella sp. CCNM 1036a Chlorella sp. CCNM 1040a Chlorella sp. CCNM 1043c Chlorella sp. CCNM 1052b Chlorella sp. CCNM 1074a Micractinium sp. CCNM 1006b Micractinium sp. CCNM 1041c Dictyosphaerium sp. CCNM 1047b Trentepohlia sp. CCNM 1073b Nannochloropsis sp. CCNM 1012b Geitlerinema CCNM 2010c Oscillatoria sp. CCNM 2007b Microcoleus sp. CCNM 2011b Oscillatoria sp. CCNM 2012b Phormidium sp. CCNM 2019c Phormidium sp. CCNM 2032b Phormidium sp. CCNM 2034c Phormidium sp. CCNM 2043b Phormidium sp. CCNM 2046b Phormidium sp. CCNM 2055 Plectonema sp. CCNM 2031b Lyngbya sp. CCNM 2050c Anabaena sp. CCNM 2016b Anabaena sp. CCNM 2029b Nostoc sp. CCNM 2017b Westiellopsis prolific CCNM 2030b Synechococcus lividus CCNM 2503b Synechococcus sp. CCNM 2508b Synechocystis sp. CCNM 2513b Synechococcus sp. CCNM 2514b Synechococcus sp. CCNM 2519b Synechocystis pevalekii CCNM 2520b Chroococcus sp. CCNM 2507b

Diu Bhad Road Bhavnagar Kumbarwada Gautameshwar Bhavnagar Gautameshwar Gautameshwar Diu Hazira Ankleshwar Chennai Gautameshwar Bagdana Mithapur Adri road Okha – Salt farm, Bhavnagar Bhavnagar – Diu Okha Tadd Bhavnagar Narayan sarovar Ankleshwar Gautameshwar Bagdana Diu Lakpath Chennai Calcutta Andhra pradesh Mithapur – Thalaja – Kodinar Diu Saltfarm, Bhavnagar Samakhyali Bridge Hazira – Anand Bhavnagar – Anand – Anand – Kutch Okha Salt farm, Bhavnagar Porbandar Calcutta –

Sphaeropleales

Cyanophyta

Trebouxiophyceae

Chlorellales

Ulvophyceae Eustigmatophyceae Cyanophyceae

Trentepohliales Eustigmatales Pseudanabaenales

Oscillatoriales

Nostocales

Synechococcales

Chroococcales a b c

Bold's Basal Medium. BG11 Medium. Zarrouk's Medium [7].

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C. Paliwal et al. / Algal Research 15 (2016) 24–31

volume was made up to 50 ml using pure methanol. It was then stored at −80 °C until further analysis.

literature suggests that TC can be as high as 100 mg g−1 DCW in some strains of Dunaliella sp. under stress conditions [18].

2.4. HPLC analysis for carotenoid profiling

3.2. HPLC analysis for carotenoid profiling

HPLC-PDA analysis of carotenoids and chlorophylls was done with slight modifications in a previously described method [21]. Each sample was analysed using a TSKgel ODS 120T column 4.6 mm × 250 mm (Tosoh Corporation, Japan) installed in a Shimadzu HPLC system with a diode array detector. The samples were eluted with a gradient binary solvent system consisting 0% B (0–2 min), 0–25% B (2–15 min), 25–60% B (15–17 min), 60–90% B (17–29 min), 90% B (29–39 min), 90–0% B (39–41 min), and 0% B (41–47 min) where Solvent A is a (v/v) mixture of 80% acetonitrile, 15% methanol and 5% dichloromethane while Solvent B is 30% acetonitrile, 20% methanol and 50% dichloromethane. The carotenoids and chlorophyll were detected at 437 nm and identified based on their retention time by comparing standards (DHI, Denmark). The concentrations were quantified based on the standard area.

The carotenoid composition of 57 microalgae strains was detailed using HPLC separation and their correlation was analysed through Spearman's correlation, presented as a heatmap (Fig. 1). We analysed 8 different xanthophylls (neoxanthin, violaxanthin, canthaxanthin, antheraxanthin, astaxanthin, lutein, zeaxanthin and myxoxanthophyll) and 3 different carotenes (echinenone, α-carotene and β-carotene) distributed in 2 phyla: Chlorophyta and Cyanophyta. The dataset generated was largely comparable to the dataset necessary for CHEMTAX classification.

2.5. Statistics All experimental analysis was carried out in triplicate. The carotenoid profiling (mg g−1) was compared by analysis of variance (ANOVA) using Infostat v2013d, with p ≤ 0.001. The multivariate analysis was carried out using Past v3.0 to establish the relationship between and within different orders and families belonging to the two phyla and to evaluate the distribution of carotenoids in the microalgal strains. A principal component analysis was performed on carotenoid composition (Table 2). In addition, a dendrogram was obtained by Ward hierarchical clustering using squared Euclidean distance [26] described by the first 8 principal components with eigen-values N 1 (Kaiser's rule of statistics). 3. Results and discussion 3.1. Total carotenoids 57 microalgal strains (Table 1) distributed among 8 orders of 5 classes of two phyla, Chlorophyta and Cyanophyta, were studied for their total carotenoid (TC) content. There was significant variation ranging from 0.23–7.2 mg TC g−1 dry cell weight (DCW) in the overall study. Chlorophyta had a variation ranging from 0.53–7.2 mg TC g− 1 DCW while Cyanophyta showed a variation of 0.23–2.72 mg TC g− 1 DCW. Among the Chlorophyceae members, Sphaeropleales had a higher TC content of 0.53–7.2 mg g−1 DCW, which confirms the observations of Aburai et al. [1] for different Scenedesmus sp. with reported TC contents between 2.6–10.45 mg g−1 DCW. Among the Chlorophyceae members of other orders like Chlorellales (Trebouxiophyceae), Trentepohliales (Ulvophyceae) and Eustigmatales (Eustigmatophyceae), a lower TC content (0.75–3.64 mg g−1 DCW) was observed with a few exceptions in Chlorellales, i.e. Chlorella sp. CCNM 1007 (4.85 mg g−1 DCW), Chlorella sp. CCNM 1052 (5.43 mg g−1 DCW) and Chlorella sp. CCNM 1019 (6.14 mg g−1 DCW). These exceptions have TC contents that are more than what has been reported for Micractinium sp. (3.9 ± 0.3 mg g−1 DCW) and Chlorella sp. (3.9 ± 0.2 mg g−1 DCW) [11]. If we focus on the TC content of Cyanophytes, Synechocystis sp. CCNM 2513 emerges as an exception with a total carotenoid content of 4.41 mg g−1. The variations in the TC of different strains of the same genus growing under similar conditions suggest that these variations exist between members of different species or of the same genera; the role of environmental factors in such variations can be satisfactorily ruled out (Table 1). There was no observed trend for the TC of strains belonging to the same genus that were isolated from the same geographical locations. It is also evident that the TC content can be as high as 7.2 mg g−1 DCW in Anikistrodesmus sp. CCNM 1031 (see Table 3). In general, according to previous reports on microalgae genera, the TC content may vary from 2–40 mg g− 1 DCW [6,9]. However, available

3.2.1. Phylum chlorophyta Carotenoids can be classified as primary or secondary carotenoids based on their functionality. Photoprotection of photosynthetic organisms from excessive sunlight is a primary function of the carotenoids in which the xanthophyll cycle plays a key role in higher plants and some algal genera. Xanthophyll cycle is the reversible conversion of violaxanthin to zeaxanthin via antheraxanthin for the thermal dissipation of excessive energy at the photosynthetic antenna [3]. This formation of zeaxanthin is directly correlated with the non-photochemical quenching of chlorophyll (Chl) fluorescence, which is a major photoprotective mechanism in various genera of algae [27]. Four classes (Chlorophyceae, Eustigmatophyceae, Trebouxiophyceae and Ulvophyceae) were studied in the Chlorophyta phylum for their variation in carotenoid profiling. Among these xanthophyll cycle components, antheraxanthin was found in lowest concentrations in Chlorophyta with an average of 0.03 mg g−1 DCW. Neoxanthin and violaxanthin were found to be present at an average of 0.83 mg g−1 DCW and 0.14 mg g−1 DCW, respectively. Violaxanthin content was 22.6% of the TC content, which is similar to what has been observed in Eustigmatophyceae [13]. However, we have not found proof of co-existence of neoxanthin and violaxanthin, which is different from what has been reported by Ston and Kosakowska [24], possibly due to the the limits of detection of both these pigments. Zeaxanthin is another component of the xanthophyll cycle that was found in considerable amounts with an average value of 0.37 mg g−1, whose presence in chlorophytes has been, previously reported [8]. Lutein was another major xanthophyll found in the chlorophytes which was absent in cyanophytes. These observations have been reported by Takaichi et al. [25]. Among the two orders (Chlamydomonadales and Sphaeropleales) of Chlorophyceae, Chlamydomonadales had higher lutein content (0.25–2.80 mg g−1 DCW) in comparison with Sphaeropleales, the exceptions being Scenedesmus sp. (CCNM 1053 and 1061) (Table 2). Among different classes, the lutein content was least in Trentepohliales (Ulvophycea) order (0.39 mg g−1 DCW). The Trebouxiophyceae members had a lesser lutein content as compared to Chlorophyceae with the exception of Chlorella sp. CCNM 1019 (3.13 mg g−1 DCW) and Chlorella sp. CCNM 1007 (2.53 mg g−1 DCW). Nannochloropsis sp. CCNM 1012 of Eustigmatophyceae contains 1.39 mg g−1 DCW lutein. Astaxanthin and canthaxanthin, considered secondary carotenoids not associated with the photosynthetic apparatus, averaged 0.12 mg g−1 and 0.05 mg g−1 DCW respectively. It is possible that the stress phase may lead to higher levels of these carotenoids than during log phase. The β-carotene content ranged from 0.04–0.59 mg g− 1 DCW in the entire Chlorophyta while α-carotene was in a range of 0.01–0.15 mg g−1 DCW. 3.2.2. Phylum cyanophyta Three orders (Pseudanabaenales, Synechococcales and Chroococcales) were studied in the Cyanophyta phylum. It was observed that echinenone (0.03–1.05 mg g−1 DCW) coexists with myxoxanthophyll (0.01–2.12 mg g−1 DCW) in all members of Cyanophyta while being absent in the entire Chlorophyta. In contrast, myxoxanthophyll was not detected in Chroococcus sp. CCNM 2507 of Chroococcales. β-carotene

Table 2 Microalgae strains with their carotenoid composition in mg g−1 DCW (n = 3) using HPLC. Neoxanthin

Violaxanthin

Astaxanthin

Myxoxanthophyll

Antheraxanthin

Lutein

Zeaxanthin

Canthaxanthin

Echinenone

α-carotene

β-carotene

Grasiella emersonni CCNM 1001 Chlorella sp. CCNM 1002 Chlorella sp. CCNM 1004 Chlorella sp. CCNM 1005 Micractinium sp. CCNM 1006 Chlorella sp. CCNM 1007 Desmodesmus subspicatus CCNM 1008 Grasiella emersonii CCNM 1011 Nannochloropsis sp. CCNM 1012 Chlorella sp. CCNM 1014 Grasiella emersonii CCNM 1015 Chlorella variablis CCNM 1017 Bracteacoccus pseudominor CCNM 1018 Chlorella sp. CCNM 1019 Chlorella sp. CCNM 1021 Chlorococcum sp. CCNM 1023 Chlorococcum sp. CCNM 1025 Chlorococcum sp. CCNM 1027 Scenedesmus sp. CCNM 1028 Chlorella sp. CCNM 1030 Ankistrodesmus sp. CCNM 1031 Chlorella sp. CCNM 1036 Chlorella sp. CCNM 1040 Micractinium sp. CCNM 1041 Monoraphidium minutum CCNM 1042 Chlorella sp. CCNM 1043 Acutodesmus dimorphus CCNM 1045 Monoraphidium sp. CCNM 1046 Dictyosphaerium sp. CCNM 1047 Chlorella sp. CCNM 1052 Scenedesmus sp. CCNM 1053 Scenedesmus sp. CCNM 1061 Trentepohlia sp. CCNM 1073 Chlorella sp. CCNM 1074 Oscillatoria sp. CCNM 2007 Geitlerinema sp. CCNM 2010 Microcoleus sp. CCNM 2011 Oscillatoria sp. CCNM 2012 Anabaena sp. CCNM 2016 Nostoc sp. CCNM 2017 Phormidium sp. CCNM 2019 Anabaena sp. CCNM 2029 Westiellopsis prolific CCNM 2030 Plectonema sp. CCNM 2031 Phormidium sp. CCNM 2032 Phormidium sp. CCNM 2034 Phormidium sp. CCNM 2043 Phormidium sp. CCNM 2046 Lyngbya sp. CCNM 2050 Phormidium sp. CCNM 2055 Synechococcus lividus CCNM 2503 Chroococcus sp. CCNM 2507 Synechococcus sp. CCNM 2508 Synechocystis sp. CCNM 2513 Synechococcus sp. CCNM 2514 Synechococcus sp. CCNM 2519 Synechocystis pevalekii CCNM 2520

0.13 ± 0.05 0.12 ± 0.08 0.00 0.17 ± 0.11 0.83 ± 0.32 0.82 ± 0.46 2.73 ± 1.23 0.09 ± 0.03 0.00 0.96 ± 0.11 0.40 ± 0.12 0.30 ± 0.19 0.23 ± 0.05 0.39 ± 0.16 0.61 ± 0.27 1.37 ± 0.83 1.89 ± 0.25 1.17 ± 0.98 0.73 ± 0.29 0.56 ± 0.31 3.10 ± 1.53 0.10 ± 0.06 0.07 ± 0.04 0.37 ± 0.11 0.59 ± 0.05 0.27 ± 0.13 4.13 ± 2.45 2.64 ± 0.76 0.25 ± 0.16 2.82 ± 1.01 1.37 ± 0.52 0.02 ± 0.01 1.85 ± 0.39 0.12 ± 0.09 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.18 ± 0.01 0.02 ± 0.01 0.00 0.10 ± 0.08 0.00 0.26 ± 0.02 0.00 0.08 ± 0.04 0.62 ± 0.22 0.05 ± 0.01 0.14 ± 0.05 0.08 ± 0.03 0.09 ± 0.04 0.82 ± 0.15 0.12 ± 0.09 0.00 0.31 ± 0.11 0.00 0.26 ± 0.02 0.00 0.25 ± 0.04 0.00 0.01 ± 0.01 0.12 ± 0.04 0.43 ± 0.11 0.10 ± 0.03 0.02 ± 0.01 0.23 ± 0.09 0.07 ± 0.04 0.00 0.20 ± 0.05 0.04 ± 0.03 0.02 ± 0.01 0.08 ± 0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.03 ± 0.01 0.11 ± 0.03 0.15 ± 0.05 0.01 ± 0.01 0.10 ± 0.02 0.17 ± 0.07 0.37 ± 0.06 0.03 ± 0.01 0.00 0.00 0.08 ± 0.02 0.00 0.00 0.34 ± 0.12 0.00 0.00 0.08 ± 0.06 0.00 0.15 ± 0.09 0.06 ± 0.1 0.98 ± 0.24 0.03 ± 0.01 0.00 0.00 0.27 ± 0.04 0.00 0.07 ± 0.01 0.30 ± 0.12 0.10 ± 0.07 0.58 ± 0.11 0.20 ± 0.09 0.02 ± 0.01 0.00 0.04 ± 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.05 ± 0.02 0.01 ± 0.01 0.19 ± 0.06 0.15 ± 0.04 0.03 ± 0.01 0.24 ± 0.07 0.02 ± 0.01 0.10 ± 0.03 0.30 ± 0.06 0.55 ± 0.19 0.06 ± 0.03 0.04 ± 0.02 0.14 ± 0.09 0.18 ± 0.04 0.05 ± 0.02 0.21 ± 0.11 0.02 ± 0.01 0.00 0.23 ± 0.06 2.12 ± 0.64 0.08 ± 0.03 0.16 ± 0.05 0.29 ± 0.08

0.00 0.00 0.01 ± 0.01 0.00 0.02 ± 0.01 0.07 ± 0.03 0.07 ± 0.01 0.00 0.03 ± 0.01 0.02 ± 0.01 0.00 0.07 ± 0.02 0.00 0.09 ± 0.04 0.00 0.02 ± 0.01 0.09 ± 0.03 0.00 0.09 ± 0.02 0.02 ± 0.01 0.10 ± 0.05 0.01 ± 0.01 0.00 0.04 ± 0.01 0.16 ± 0.06 0.00 0.02 ± 0.01 0.10 ± 0.03 0.04 ± 0.02 0.04 ± 0.01 0.02 ± 0.01 0.01 ± 0.01 0.00 0.03 ± 0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.89 ± 0.24 0.80 ± 0.42 0.60 ± 0.40 0.52 ± 0.08 2.36 ± 1.83 2.53 ± 1.42 1.98 ± 1.39 0.56 ± 0.05 1.39 ± 0.77 1.93 ± 0.58 1.34 ± 0.19 1.13 ± 1.02 1.16 ± 0.42 3.13 ± 0.74 1.56 ± 0.06 1.01 ± 0.36 0.82 ± 0.41 0.57 ± 0.15 2.80 ± 1.83 1.81 ± 0.64 2.07 ± 0.16 0.45 ± 0.15 0.40 ± 0.09 1.06 ± 0.70 2.20 ± 1.11 0.65 ± 0.38 2.00 ± 1.42 1.32 ± 0.29 1.00 ± 0.45 1.35 ± 0.83 0.65 ± 0.37 0.25 ± 0.02 0.39 ± 0.18 0.34 ± 0.13 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.34 ± 0.11 0.30 ± 0.13 0.35 ± 0.15 0.19 ± 0.11 0.18 ± 0.09 0.65 ± 0.35 0.23 ± 0.09 0.17 ± 0.09 0.47 ± 0.15 0.40 ± 0.13 0.59 ± 0.19 0.36 ± 0.12 0.38 ± 0.21 0.88 ± 0.33 0.47 ± 0.23 0.41 ± 0.13 0.35 ± 0.15 0.36 ± 0.12 0.98 ± 0.47 0.54 ± 0.28 0.46 ± 0.26 0.18 ± 0.1 0.12 ± 0.09 0.21 ± 0.15 1.11 ± 0.65 0.09 ± 0.03 0.54 ± 0.26 0.61 ± 0.21 0.48 ± 0.15 0.36 ± 0.19 0.18 ± 0.05 0.09 ± 0.05 0.17 ± 0.09 0.14 ± 0.02 0.14 ± 0.06 0.11 ± 0.08 0.37 ± 0.16 0.21 ± 0.09 0.00 0.18 ± 0.15 0.12 ± 0.09 0.03 ± 0.02 0.08 ± 0.01 0.29 ± 0.13 0.37 ± 0.15 0.20 ± 0.13 0.60 ± 0.21 0.09 ± 0.03 0.13 ± 0.08 0.27 ± 0.15 0.10 ± 0.05 0.05 ± 0.02 0.21 ± 0. 0.25 ± 0.07 0.91 ± 0.36 0.73 ± 0.17 0.65 ± 0.21

0.06 ± 0.02 0.10 ± 0.05 0.05 ± 0.02 0.02 ± 0.01 0.00 0.04 ± 0.03 0.04 ± 0.02 0.13 ± 0.05 0.05 ± 0.03 0.03 ± 0.01 0.11 ± 0.07 0.03 ± 0.01 0.00 0.09 ± 0.04 0.00 0.04 ± 0.01 0.11 ± 0.07 0.00 0.12 ± 0.03 0.04 ± 0.01 0.00 0.07 ± 0.01 0.06 ± 0.02 0.01 ± 0.01 0.15 ± 0.06 0.00 0.06 ± 0.02 0.05 ± 0.03 0.06 ± 0.05 0.00 0.00 0.06 ± 0.04 0.00 0.02 ± 0.01 0.04 ± 0.02 0.02 ± 0.01 0.11 ± 0.04 0.06 ± 0.03 0.06 ± 0.01 0.08 ± 0.03 0.03 ± 0.02 0.02 ± 0.01 0.05 ± 0.03 0.07 ± 0.02 0.11 ± 0.03 0.05 ± 0.02 0.14 ± 0.08 0.03 ± 0.01 0.04 ± 0.01 0.08 ± 0.02 0.03 ± 0.02 0.00 0.06 ± 0.03 0.20 ± 0.12 0.24 ± 0.09 0.19 ± 0.11 0.18 ± 0.03

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.20 ± 0.07 0.12 ± 0.03 0.67 ± 0.08 0.68 ± 0.11 0.00 0.30 ± 0.15 0.23 ± 0.07 0.00 0.00 0.07 ± 0.02 0.10 ± 0.05 0.54 ± 0.15 0.00 0.03 ± 0.02 0.00 1.05 ± 0.11 0.11 ± 0.04 0.00 0.58 ± 0.24 1.01 ± 0.45 0.70 ± 0.34 0.42 ± 0.19 0.55 ± 0.26

0.00 0.03 ± 0.01 0.002 0.02 ± 0.01 0.03 ± 0.0.01 0.04 ± 0.01 0.04 ± 0.02 0.01 ± 0.01 0.02 ± 0.01 0.09 ± 0.02 0.15 ± 0.05 0.04 ± 0.01 0.04 ± 0.01 0.07 ± 0.02 0.05 ± 0.02 0.04 ± 0.02 0.15 ± 0.03 0.03 ± 0.01 0.10 ± 0.02 0.05 ± 0.01 0.15 ± 0.06 0.00 0.02 ± 0.01 0.04 ± 0.01 0.12 ± 0.03 0.06 ± 0.02 0.10 ± 0.15 0.07 ± 0.02 0.03 ± 0.01 0.03 ± 0.01 0.02 ± 0.01 0.01 ± 0.01 0.04 ± 0.02 0.01 ± 0.01 0.00 0.00 0.00 0.37 ± 0.08 0.39 ± 0.07 0.00 0.28 ± 0.09 0.10 ± 0.04 0.26 ± 0.11 0.20 ± 0.07 0.31 ± 0.15 0.30 ± 0.15 0.65 ± 0.18 0.00 0.23 ± 0.12 0.64 ± 0.19 0.00 0.00 0.30 ± 0.09 0.00 0.00 0.00 0.68 ± 0.21

0.10 ± 0.03 0.11 ± 0.04 0.11 ± 0.02 0.07 ± 0.01 0.15 ± 0.03 0.27 ± 0.09 0.26 ± 0.07 0.08 ± 0.01 0.15 ± 0.04 0.10 ± 0.02 0.59 ± 0.17 0.17 ± 0.04 0.17 ± 0.06 0.33 ± 0.09 0.22 ± 0.07 0.14 ± 0.02 0.04 ± 0.01 0.11 ± 0.01 0.34 ± 0.11 0.19 ± 0.02 0.09 ± 0.01 0.05 ± 0.01 0.07 ± 0.01 0.14 ± 0.03 0.53 ± 0.13 0.09 ± 0.01 0.32 ± 0.05 0.17 ± 0.04 0.10 ± 0.02 0.25 ± 0.08 0.12 ± 0.02 0.03 ± 0.01 0.04 ± 0.01 0.04 ± 0.02 0.26 ± 0.09 0.45 ± 0.14 0.62 ± 0.16 0.24 ± 0.09 0.21 ± 0.07 1.03 ± 0.65 0.11 ± 0.03 0.03 ± 0.01 0.11 ± 0.02 0.12 ± 0.02 0.26 ± 0.08 0.19 ± 0.04 0.54 ± 0.16 0.28 ± 0.07 0.17 ± 0.03 0.36 ± 0.11 0.14 ± 0.04 0.18 ± 0.05 0.22 ± 0.06 0.83 ± 0.12 0.76 ± 0.13 0.62 ± 0.19 0.37 ± 0.10

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Strains

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C. Paliwal et al. / Algal Research 15 (2016) 24–31

was found in Cyanophyta members in the range of 0.03–1.03 mg g−1 DCW, whereas zeaxanthin, the second most important carotenoid found in Cyanophyta, was in a range of 0.03–0.91 mg g−1 DCW. Interestingly, it was not detected in Anabaena sp. CCNM 2016 of Pseudanabaenales due to its very low concentration. It has been previously reported that the xanthophyll cycle is not present in the Cyanophyta while zeaxanthin is produced from β-carotene [23]. Therefore, due to the absence of the xanthophyll cycle, its associated pigments like antheraxanthin and violaxanthin are also absent in Cyanophyta. The absence of xanthophyll cycle is responsible for alternate mechanisms to cope with nonphotochemical quenching, like the presence of phycobilisomes. Also

noteworthy is the absence of astaxanthin among Cyanophyta members. One of the reasons may be their inability to accept zeaxanthin as substrate for astaxanthin biosynthesis [17]. Other carotenoids that are present are α-carotene and canthaxanthin whose contents vary in a range of 0.1–0.68 and 0.02–0.24 mg g−1 DCW, respectively. 3.3. Principal component and cluster analysis The principal component analysis provided a clear distinction between Chlorophyta and Cyanophyta; it showed a 71.23% variation, which reveals little correlation due to taxonomical implications. Green

Table 3 Microalgae strains with their respective Chl a, Chl b, Total Carotenoid content (in mg g−1 DCW) using HPLC (n = 3). Strains

Grasiella emersonni CCNM 1001 Chlorella sp. CCNM 1002 Chlorella sp. CCNM 1004 Chlorella sp. CCNM 1005 Micractinium sp. CCNM 1006 Chlorella sp. CCNM 1007 Desmodesmus subspicatus CCNM 1008 Grasiella emersonii CCNM 1011 Nannochloropsis sp. CCNM 1012 Chlorella sp. CCNM 1014 Grasiella emersonii CCNM 1015 Chlorella variablis CCNM 1017 Bracteacoccus pseudominor CCNM 1018 Chlorella sp. CCNM 1019 Chlorella sp. CCNM 1021 Chlorococcum sp. CCNM 1023 Chlorococcum sp. CCNM 1025 Chlorococcum sp. CCNM 1027 Scenedesmus sp. CCNM 1028 Chlorella sp. CCNM 1030 Anikistrodesmus sp. CCNM 1031 Chlorella sp. CCNM 1036 Chlorella sp. CCNM 1040 Micractinium sp. CCNM 1041 Monoraphidium minutum CCNM 1042 Chlorella sp. CCNM 1043 Acutodesmus dimorphus CCNM 1045 Monoraphidium sp. CCNM 1046 Dictyosphaerium sp. CCNM 1047 Chlorella sp. CCNM 1052 Scenedesmus sp. CCNM 1053 Scenedesmus sp. CCNM 1061 Trentepohlia sp. CCNM 1073 Chlorella sp. CCNM 1074 Oscillatoria sp. CCNM 2007 Geitlerinema sp. CCNM 2010 Microcoleus sp. CCNM 2011 Oscillatoria sp. CCNM 2012 Anabaena sp. CCNM 2016 Nostoc sp. CCNM 2017 Phormidium sp. CCNM 2019 Anabaena sp. CCNM 2029 Westiellopsis prolific CCNM 2030 Plectonema sp. CCNM 2031 Phormidium sp. CCNM 2032 Phormidium sp. CCNM 2034 Phormidium sp. CCNM 2043 Phormidium sp. CCNM 2046 Lyngbya sp. CCNM 2050 Phormidium sp. CCNM 2055 Synechococcus lividus CCNM 2503 Chroococcus sp. CCNM 2507 Synechococcus sp. CCNM 2508 Synechocystis sp. CCNM 2513 Synechococcus sp. CCNM 2514 Synechococcus sp. CCNM 2519 Synechocystis pevalekii CCNM 2520

Pigments Chla

Chlb

Total Car

(mg g−1)

(mg g−1)

(mg g−1)

1.88 ± 0.39 1.59 ± 0.83 1.54 ± 0.08 1.43 ± 0.42 8.28 ± 1.17 8.54 ± 1.34 6.60 ± 3.69 0.97 ± 0.64 3.89 ± 0.91 5.58 ± 2.08 4.46 ± 2.37 2.40 ± 0.19 2.85 ± 0.38 8.83 ± 1.67 3.27 ± 0.00 3.90 ± 3.48 5.26 ± 1.29 2.81 ± 0.43 8.74 ± 1.92 4.81 ± 1.25 4.81 ± 1.25 10.90 ± 2.68 1.18 ± 0.78 2.71 ± 1.02 0.84 ± 0.11 2.71 ± 1.02 2.37 ± 1.85 1.97 ± 0.43 9.86 ± 2.38 1.83 ± 1.67 3.37 ± 1.65 0.42 ± 0.18 6.43 ± 1.02 6.06 ± 1.39 1.28 ± 2.33 2.72 ± 0.52 2.56 ± 0.50 2.13 ± 0.24 2.39 ± 0.24 1.35 ± 0.44 2.56 ± 0.41 1.40 ± 0.17 1.97 ± 0.43 2.58 ± 0.69 2.46 ± 0.03 3.48 ± 0.05 2.85 ± 1.06 1.06 ± 0.09 1.50 ± 0.03 3.03 ± 1.01 0.53 ± 1.18 0.37 ± 1.42 4.07 ± 0.06 3.47 ± 1.80 1.45 ± 0.16 1.34 ± 0.53 2.49 ± .37

2.06 ± 0.24 1.92 ± 0.53 1.48 ± 0.99 1.73 ± 0.39 9.70 ± 1.42 9.58 ± 1.04 7.21 ± 1.31 1.18 ± 0.93 4.80 ± 0.58 6.44 ± 1.74 4.60 ± 1.24 2.43 ± 0.42 2.86 ± 0.45 9.76 ± 1.45 3.22 ± 0.56 4.55 ± 0.83 5.35 ± 1.47 2.68 ± 0.79 9.79 ± 1.61 5.44 ± 1.08 11.06 ± 2.20 0.83 ± 0.13 0.98 ± 0.12 2.63 ± 0.67 6.66 ± 1.13 2.39 ± 0.38 10.79 ± 1.10 6.06 ± 1.59 2.17 ± 1.78 6.98 ± 1.25 3.78 ± 0.59 0.50 ± 0.42 4.74 ± 1.31 0.69 ± 0.19 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

1.73 ± 0.47 1.59 ± 0.77 1.27 ± 0.65 1.10 ± 0.42 3.64 ± 2.31 4.85 ± 2.48 5.72 ± 2.89 1.15 ± 0.29 2.73 ± 1.23 3.58 ± 0.89 3.40 ± 0.86 2.18 ± 1.44 2.07 ± 0.79 6.14 ± 1.69 3.03 ± 0.74 3.03 ± 1.38 3.84 ± 1.12 2.24 ± 1.27 5.57 ± 2.88 3.27 ± 1.38 7.20 ± 2.35 0.89 ± 0.35 0.75 ± 0.27 1.99 ± 1.06 5.56 ± 2.24 1.26 ± 0.6 3.13 ± 4.38 5.49 ± 1.59 2.13 ± 0.97 5.43 ± 2.24 2.76 ± 1.12 0.53 ± 0.19 2.51 ± 0.7 0.82 ± 0.33 0.69 ± 0.26 0.71 ± 0.27 1.96 ± 0.5 1.71 ± 0.44 0.69 ± 0.16 1.83 ± 1.05 0.79 ± 0.31 0.28 ± 0.11 0.8 ± 0.23 1.30 ± 0.45 1.21 ± 0.49 1.32 ± 0.51 2.07 ± 0.72 0.61 ± 0.17 0.62 ± 0.26 2.61 ± 0.69 0.40 ± 0.16 0.23 ± 0.07 1.60 ± 0.48 4.41 ± 1.4 2.69 ± 0.95 2.12 ± 0.71 2.72 ± 0.89

TC/TChl 0.44 0.45 0.42 0.35 0.20 0.27 0.41 0.53 0.31 0.30 0.38 0.45 0.36 0.33 0.47 0.36 0.36 0.41 0.30 0.32 0.45 0.08 0.35 0.37 0.74 0.25 0.24 0.68 0.18 0.62 0.39 0.58 0.22 0.12 0.54 0.26 0.77 0.80 0.29 1.36 0.31 0.20 0.41 0.50 0.49 0.38 0.73 0.57 0.41 0.86 0.76 0.62 0.39 1.27 1.86 1.58 1.09

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were conserved within the respective phyla. Based on these observations, myxoxanthophyll and echinenone can be considered as strong chemotaxonomic biomarkers for Cyanophyta while lutein and violaxanthin can be indicative of Chlorophyta phylum. The identification and classification of microalgae follows the same basic principles in PCA as well as CHEMTAX [14]. However, the chief difference between CHEMTAX and our method lies in the size of the databases. CHEMTAX is mainly a mathematical treatment of the pigment profiles of an ecosystem based on how it discerns the members of the phytoplankton community whereas, in our method PCA and classical hierarchical clustering need an accurately predicted pigment analysis, which requires the isolation and culture of microorganisms under controlled conditions. Although different approaches, both these methods are an attempt to identify key carotenoid markers in a population of microalgae with the pigment profiles of actual strains as a reference. The dendrogram generated from Ward hierarchical clustering to assess the phylogenetic relationship between the different microalgal species has grouped them into 2 clusters. The six different classes of microalgae i.e. Chlorophyceae (Orders: Chlamydomonadales, Sphaeropleales), Trebouxiophyceae (Order: Chlorellales), Ulvophyceae (Order: Trentepohliales), Eustigmatophyceae (Order: Eustigmatales) and Cyanophyceae (Orders: Pseudanabaenales, Oscillatoriales, Nostocales, Synechococcales and Chroococcales) were clustered based on their pigment compositions. However, due to insufficient sampling in some of the taxa, a clear segregation of species cannot be made on the basis of the dendrogram (Fig. 3). On the basis of the carotenoid profiles obtained, we have achieved separation between the two phyla – Chlorophyta and Cyanophyta – which were also verified by the PCA bi-plot analysis. We also observed a broad classification of the different orders under these 2 phyla with certain exceptions. 3 orders – Chlorellales, Sphaeropleales and Oscillatoriales – were clearly defined with minimum overlapping; the larger sample size in all of them could be the deciding factor. 4. Conclusion This study produces statistical evidence for conservation of carotenoids across different phyla. PCA and hierarchical cluster analysis show that lutein and violaxanthin can be considered as chemotaxonomic markers for Chlorophyta while echinenone and myxoxanthophyll can be used as markers for Cyanophyta. Green algae are a potential source of lutein while cyanobacteria can be exploited for zeaxanthin and myxoxanthophyll. The lutein content in Chlorophyta is comparable with that of previously reported literature. β-carotene is present in all classes and orders of microalgae. Acknowledgements

Fig. 1. Heat map of the carotenoids profiling in the different fresh water strains of microalgae.

algae and cyanobacteria are clearly segregated in the PCA bi-plot (Fig. 2). The most discriminant variables along axis-I were neoxanthin and lutein while across axis-II they were astaxanthin, zeaxanthin and violaxanthin. The PCA bi-plot indicates the vast diversity present in the carotenoid data matrix in microalgae while the carotenoid trends

CSIR-CSMCRI assigned PRIS number for this manuscript is 226/2014. CP, TG, IP, and RM gratefully acknowledge CSIR, New Delhi for awarding Senior Research Fellowship. All authors acknowledge CSIR for providing the financial support through projects CSC0203, CSIR-MoES-NMITLI multi-institutional project TLP0096 and OLP0071. Authors also acknowledge Department of Science of Technology for providing finances through project no. GAP2006. The authors would also like to thank Dr. Arvind Kumar (CSIR-CSMCRI, Bhavnagar) for his constant encouragement. The authors would like to thank Dr. Parimal Paul (CSIRCSMCRI, Bhavnagar) and Dr. Harshad Brahmabhatt (CSIR-CSMCRI, Bhavnagar) for their help during the analysis. CP, TG, IP, RM and KC wish to acknowledge AcSIR-CSMCRI for Ph.D. enrolment. References [1] N. Aburai, S. Ohkubo, H. Miyashita, K. Abe, Composition of carotenoids and identification of aerial microalgae isolated from the surface of rocks in mountainous districts of Japan, Algal Res. 2 (2013) 237–243, http://dx.doi.org/10.1016/j.algal. 2013.03.001.

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Fig. 2. Bi-plot of microalgal strains obtained after PCA analysis of carotenoids profile (with first two principal components). The microalgae strains are indexed according to their CCNM numbers in the Table 1.

Fig. 3. Dendrogram obtained from hierarchical cluster analysis of microalgae strains are indexed according to their CCNM numbers in the Table 2.

[2] R. Barlow, M. Kyewalyanga, H. Sessions, et al., Phytoplankton pigments, functional types, and absorption properties in the Delagoa and Natal Bights of the Agulhas ecosystem, Estuar. Coast. Shelf Sci. 80 (2008) 201–211, http://dx.doi.org/10.1016/j. ecss.2008.07.022. [3] M. Bertrand, B. Schoefs, P. Siffel, et al., Cadmium inhibits epoxidation of diatoxanthin to diadinoxanthin in the xanthophyll cycle of the marine diatom Phaeodactylum tricornutum, FEBS Lett. 508 (2001) 153–156. [5] S. Fietz, A. Nicklisch, An HPLC analysis of the summer phytoplankton assemblage in Lake Baikal, Freshw. Biol. 49 (2004) 332–345, http://dx.doi.org/10.1111/j.13652427.2004.01183.x.

[6] J.C. García-Cañedo, C.M. Flores-Ortiz, R.O. Cañizares-Villanueva, Carotenoid profile and content of the green microalgae Scenedesmus incrassatulus, J. Am. Diet. Assoc. 111 (2011) A94, http://dx.doi.org/10.1016/j.jada.2011.06.348. [7] B. George, I. Pancha, C. Desai, et al., Effects of different media composition, light intensity and photoperiod on morphology and physiology of freshwater microalgae Ankistrodesmus falcatus — a potential strain for bio-fuel production, Bioresour. Technol. 171 (2014) 367–374, http://dx.doi.org/10.1016/j.biortech. 2014.08.086. [8] T. Ghosh, C. Paliwal, R. Maurya, S. Mishra, Microalgal rainbow colours for nutraceutical and pharmaceutical applications, in: B. Bahadur, V.R. Manchikatla, L. Sahijram,

C. Paliwal et al. / Algal Research 15 (2016) 24–31

[9]

[10]

[11] [13]

[14]

[15]

[16]

[17]

K.V. Krishnamurthy (Eds.), Plant Biology and Biotechnology, Springer India 2015, pp. 777–791, http://dx.doi.org/10.1007/978-81-322-2286-6. K. Goiris, K. Muylaert, I. Fraeye, et al., Antioxidant potential of microalgae in relation to their phenolic and carotenoid content, J. Appl. Phycol. 24 (2012) 1477–1486, http://dx.doi.org/10.1007/s10811-012-9804-6. M.D. Guiry, G.M. Guiry, AlgaeBase, World-wide electronic publication, National University of Ireland, Galway, 2015 http://www.algaebase.org; Accessed 7 January 2015. H. Johnson, Co-utilisation of Microalgae for Wastewater Treatment and the Production of Animal Feed Supplements, Rhodes University, 2010. L. Lubián, O. Montero, I. Moreno-Garrido, et al., Nannochloropsis (eustigmatophyceae) as source of commercially valuable pigments, J. Appl. Phycol. 12 (2000) 249–255, http://dx.doi.org/10.1023/A:1008170915932. M.D. Mackey, D.J. Mackey, H.W. Higgins, et al., CHEMTAX-a program for estimating class abundances from chemical markers: application to HPLC measurements of phytoplankton, Mar. Ecol. Prog. Ser. 144 (1996) 265–283. N.V.V. Madhu, N. Ullas, R. Ashwini, et al., Characterization of phytoplankton pigments and functional community structure in the Gulf of Mannar and the Palk Bay using HPLC–CHEMTAX analysis, Cont. Shelf Res. 80 (2014) 79–90, http://dx. doi.org/10.1016/j.csr.2014.03.004. I.T. Marlowe, J.C. Green, A.C. Neal, et al., Long chain (n-C37–C39) alkenones in the prymnesiophyceae. Distribution of alkenones and other lipids and their taxonomic significance, Br. Phycol. J. 19 (1984) 203–216, http://dx.doi.org/10.1080/ 00071618400650221. J.F. Martín, E. Gudiña, J.L. Barredo, Conversion of beta-carotene into astaxanthin: two separate enzymes or a bifunctional hydroxylase-ketolase protein? Microb. Cell Factories 7 (2008) 3, http://dx.doi.org/10.1186/1475-2859-7-3.

31

[18] M. Mojaat, A. Foucault, J. Pruvost, J. Legrand, Optimal selection of organic solvents for biocompatible extraction of beta-carotene from Dunaliella salina, J. Biotechnol. 133 (2008) 433–441, http://dx.doi.org/10.1016/j.jbiotec.2007.11.003. [19] I. Pancha, K. Chokshi, B. George, et al., Nitrogen stress triggered biochemical and morphological changes in the microalgae Scenedesmus sp. CCNM 1077, Bioresour. Technol. 156 (2014) 146–154, http://dx.doi.org/10.1016/j.biortech.2014.01.025. [20] H.W. Paerl, L.M. Valdes, J.L. Pinckney, et al., Phytoplankton photopigments as indicators of estuarine and coastal eutrophication, Bioscience 53 (10) (2003) 953 Available from: http://bioscience.oxfordjournals.org/content/53/10/953.full. [21] C. Paliwal, T. Ghosh, K. Bhayani, et al., Antioxidant, anti-nephrolithe activities and in vitro digestibility studies of three different cyanobacterial pigment extracts, Mar. Drugs 13 (8) (2015) 5384–5401 Available from: http://www.mdpi.com/16603397/13/8/5384/. [23] M. Schagerl, B. Müller, Acclimation of chlorophyll a and carotenoid levels to different irradiances in four freshwater cyanobacteria, J. Plant Physiol. 163 (2006) 709–716, http://dx.doi.org/10.1016/j.jplph.2005.09.015. [24] J. Ston, A. Kosakowska, Qualitative and quantitative analysis of Baltic phytoplankton pigments*, Oceanologia 42 (2000) 449–471. [25] S. Takaichi, Carotenoids in algae: distributions, biosyntheses and functions, Mar. Drugs 9 (2011) 1101–1118, http://dx.doi.org/10.3390/md9061101. [26] J.H. Ward, Hierarchical grouping to optimize an objective function, J. Am. Stat. Assoc. 58 (1963) 236–244. [27] A. Young, H. Frank, Energy transfer reactions involving carotenoids: quenching of chlorophyll fluorescence, J. Photochem. Photobiol. B 36 (1996) 3–15, http://dx.doi. org/10.1016/S1011-1344(96)07397-6.