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SN Applied Sciences

, 1:532 | Cite as

Influence of agriculture fertilizer for the enhanced growth and astaxanthin production from Haematococcus lacustris RRGK isolated from Himachal Pradesh, India

  • Ramamoorthy Karuppan
  • Anand Javee
  • Sreekala Kannikulathel Gopidas
  • Nagaraj SubramaniEmail author
Research Article
  • 133 Downloads
Part of the following topical collections:
  1. 2. Earth and Environmental Sciences (general)

Abstract

In the present study, Haematococcus lacustris RRGK which is the former name of H. lacustris HPI-001 isolated from Himachal Pradesh, India, and another strain H. lacustris SAG-19a retrieved from Gottingen Culture Collection, Germany, were used. The H. lacustris SAG-19a served as a control. The both strains were grown in a Bold basal medium in which the components of NaNO3, K2HPO4∙3H2O and KH2PO4 were replaced with agriculture fertilizers such as NPK (17:17:17), urea (CH4N2O) and DAP (diammonium phosphate; (NH4)2HPO4 + potash (K2CO3) in different concentrations. The isolate of H. lacustris HPI-001 showed maximum growth at 1.2 mM NPK, while the H. lacustris SAG-19a showed maximum growth at 1.5 mM. However, both the strains showed maximum astaxanthin content at 0.6 mM NPK. The H. lacustris HPI-001 showed a maximum growth and astaxanthin content with 3.3 mM and 4.9 mM of urea, respectively, while H. lacustris SAG-19a showed maximum growth and astaxanthin accumulation with 3.3 mM and 6.6 mM of urea, respectively. The H. lacustris HPI-001 showed maximum growth at 250 mg/L (NH4)2HPO4 + 200 mg/L K2CO3 and maximum astaxanthin content at 150 mg/L (NH4)2HPO4 + 100 mg/L K2CO3, while the maximum growth in H. lacustris SAG-19a was observed at 200 mg/L (NH4)2HPO4 + 150 mg/L K2CO3, and the maximum astaxanthin content at 150 mg/L (NH4)2HPO4 + 100 mg/L K2CO3. The basal medium was also amended with the addition of commercial NaHCO3 wherein the maximum growth and content of astaxanthin in H. lacustris HPI-001 was at 0.6 mM and 1.5 mM of NaHCO3, respectively. The H. lacustris SAG-19a showed maximum growth and astaxanthin content at 1.5 mM and 0.6 mM, respectively. Based on the investigation, a medium was formulated and named as modified HPI-001 medium. The results of the present study suggest that commercial agricultural fertilizers may be used as excellent substitutes to enhance cell growth and astaxanthin production in H. lacustris.

Keywords

Haematococcus lacustris Carotenoids Astaxanthin Agriculture fertilizer 

1 Introduction

Microalgae are photosynthetic microorganisms that can synthesize various organic biomolecules such as proteins, carbohydrates, lipids, pigments and vitamins from carbon dioxide (CO2) [1]. These microalgae products have attracted intensive academic and industrial application including cosmetics, nutraceuticals, pharmaceuticals, aquaculture, food and biofuels [2, 3, 4]. The natural astaxanthin can be produced from microalgae, yeast, bacteria and various seafood including salmon, lobster, trout, red sea bream, arctic shrimp, crawfish, krill and fish eggs [5, 6]. Various microorganisms such as Haematococcus lacustris, Chromochloris zofingiensis, red yeast Phaffia rhodozyma and marine bacterium Agrobacterium aurantiacum can produce astaxanthin [7]. The H. lacustris can accumulate a maximum astaxanthin of up to 4% dry weight under unfavorable environmental stress conditions [8, 9, 10].

The H. lacustris (Girod-Chantrans) Rostafinski 1875 is a green, motile, biflagellate microalga found in freshwater environments. It belongs to class Chlorophyceae, order Volvocales and family Haematococcaseae. Its life cycle consists of four stages such as macrozooids or zoospore, microzooids, palmella and aplanospore stages [11]. The macrozooid cells are between 8 and 20 μm long with a distinct gelatinous extracellular gelatinous matrix of variable thickness, and these may be divided into 2–32 daughter cells by mitosis [9]. They form amorphous multilayered structures in the inner regions of the extracellular matrix or the primary cell wall as they develop into non-motile palmella and become resting vegetative cells [12]. When the culture is exposed to stress conditions such as the decrease in nutrients and the increase in light intensity and high salinity, the palmella stage transforms into the hematocyst aplanospore stage [9, 13]. These microalgae can be usually found in temperate regions around the world and has been isolated from Europe, Africa, North America and Himachal Pradesh in India [14, 15] and also reported from Arctic region [16]. It is also found across various environmental and climatic conditions such as brackish water and on the rocks on seashore [16]. The astaxanthin-rich non-motile aplanospore coccoid cells have an exceptional tolerance to a wide range of adverse conditions [17, 18, 19]. The highly complex and dynamic composition of cell wall, photosynthetic apparatus reduction and cell dehydration allows H. lacustris to survive in hostile environment, but on the other hand presents an issue when H. lacustris biomass has to be processed to extract valuable intracellular compounds such as astaxanthin [20].

The choice of culture medium for microalgae growth depends on the nutritional requirements that contribute toward the efficient growth and biomass production [21, 22]. The alternative sources of nutrients in the composition of culture media are urea, ammonium sulfate, water plants, swine manure and inorganic fertilizers (NPK) [23]. Nutrients are crucial in the growth and development of microalgae, influencing the physiological adaptation and biochemical composition of microalgae [15]. Nitrogen and phosphorus are the main elements that limit growth of microalgae, which usually depends on the physiological requirements of each nutrient [24]. The low CO2 concentration in the air is also limiting factor of photosynthesis in plants and algae [25]. There are several types of basic elements for growth of microalgae such as carbon, hydrogen, nitrogen, oxygen, phosphorus, magnesium, iron, sulfur and trace elements [26]. Potassium is a macroelement which is required for growth-related metabolic activities [27]. The inorganic fertilizer is simple and may be used with alternative medium for microalgae, since they are widely available, dissolve easily, have a defined composition, high nitrogen and phosphorus rate, and maintain moderate pH in the medium [28, 29]. The inorganic fertilizers such as NPK result in an efficient alternative medium for large-scale laboratory growth of Chlorophyceaen members [30].

Astaxanthin (C40H52O4, 3, 3′-dihydroxy-β, β-carotene-4,4′-dione), a keto-carotenoid, is considered as a “super antioxidant” which spans the cell membrane bilayer and significantly reduces the free radicals and oxidative stress in human body, thereby helping to maintain a healthy state [5, 31, 32]. It has the most effective natural antioxidant activity and is known to be 10–65 times higher than that of β-carotene, canthaxanthin, zeaxanthin, lutein and vitamin C, and is 100 times more effective than α-tocopherol [11, 33]. It has wide range of applications in cosmetics, foods, nutraceuticals, aquaculture and pharmaceuticals industries [11, 34, 35]. Astaxanthin is also used as a promising therapeutic agent against atherosclerosis, cancers, hypertension, diabetes, cardiovascular diseases and several neurological diseases (e.g., Alzheimer’s disease) because it is known to cross the blood–brain barrier and enables to provide antioxidant benefits beyond that barrier [36, 37]. Astaxanthin is also currently used in the prevention and control of many pathological conditions involving low oxidation and inflammation [38, 39].

In the present study, a new strain of H. lacustris HPI-001 was isolated from Himachal Pradesh, India, and another strain H. lacustris SAG-19a culture was obtained from Sammlung von Kulturen, Pflanzen Physiologisches Institut, University of Gottingen, Gottingen, Germany. The latter was treated as control, and its adaptation to laboratory conditions was investigated. The two strains were grown in Bold basal medium (BBM) [40] amended with the addition of commercial fertilizers such as N:P:K (17:17:17), CH4N2O and (NH4)2HPO4 + K2CO3 and its influence on growth and astaxanthin production were evaluated.

2 Materials and methods

2.1 Isolation of H. lacustris HPI-001

The water samples were collected from Palampur in March 2013. Palampur (32°N latitude; 76°E longitude) is a town in the state of Himachal Pradesh in India and is 1325 ft. above sea level with average annual temperature of 19 °C and average annual rainfall of 250 cm and above. The water sample from each aquatic site was collected and stored in plastic bottles. The samples were inoculated in BBM at 25 ± 1 °C, under 30 μE m−2 s−1 light irradiations and a photoperiod of 12/12 (light/dark). The cultures were thoroughly mixed manually twice a day, and the experiment was conducted at laboratory conditions. The cells of H. lacustris HPI-001 were isolated and prepared unialgal cultures by serial dilution followed by streak plate technique on 2% agar in BBM. The cells were identified based on morphological and molecular studies. H. lacustris SAG-19a was retrieved from Gottingen Culture Collection, Germany.

2.2 Composition of culture medium

The two strains of H. lacustris were cultured in the BBM in which the components, sodium nitrate (NaNO3), dibasic potassium phosphate trihydrate (K2HPO4∙3H2O) and potassium phosphate monobasic (KH2PO4), were replaced with different concentrations of commercial fertilizers such as N:P:K (17:17:17), urea (CH4N2O), diammonium phosphate ((NH4)2HPO4) and potassium carbonate (K2CO3) (Shri Sai Gopal Agrotech Pvt. Ltd., Karnataka, India). The other chemicals of the medium were obtained from Sisco Research Laboratories Pvt. Ltd., Mumbai (Table 3). The growth and astaxanthin production in two test organisms were also recorded in basal medium added with different concentrations (0.3, 0.6, 0.9, 1.2, 1.5 and 1.8 mM) of commercial sodium bicarbonate (NaHCO3) fertilizers (Table 1). The growth and astaxanthin production of H. lacustris HPI-001 and the H. lacustris SAG-19a in modified Bold basal medium (3 N-BBM + V) and formulated modified HPI-001 medium was compared (Table 2).
Table 1

Medium composition

Chemical components

mg/L

N:P:K (17:17:17) a

2500

CH 4 N 2 O a

13,000

CaCl2∙3H2O

2500

MgSO4∙7H2O

2500

NaHCO 3 a

2500

(NH 4 ) 2 HPO 4 a

5000

K 2 CO 3 a

7500

NaCl

2500

EDTA (with Na2)

750

FeCl3∙6H2O

97

MnCl2∙4H2O

41

ZnCl2∙6H2O

5

CoCl2∙6H2O

2

Na2MoO4∙2H2O

4

All the medium components dissolved in 1 L of distilled water

pH of the medium is 7.5

aReplaced commercial fertilizers in basal medium (3N-BBM + V)

Table 2

Formulated commercial modified HPI-001 medium

Chemical components

mg/L

N:P:K (17:17:17)

50

CH4N2O

300

CaCl2∙3H2O

2500

MgSO4∙7H2O

2500

NaHCO3

125

(NH4)2HPO4

150

K2CO3

100

NaCl

2500

EDTA (with Na2)

750

FeCl3∙6H2O

97

MnCl2∙4H2O

41

ZnCl2∙6H2O

5

CoCl2∙6H2O

2

Na2MoO4∙2H2O

4

All the medium components dissolved in 1 L of distilled water

pH of the medium is 7.5

2.3 Maintenance and study of algal culture

The two strains of H. lacustris were grown under autotrophic condition, in a photoperiod of 12/12 h (light/dark) in liquid BBM and maintained at 25 ± 1 °C, under 30 µEm-2 s-1 light intensity provided by warm white Philips set of lamps (36 W; 4ft Philips Trulite, made in India), the light intensity was measured by using lux meter (TES 1331, Taiwan), and the culture parameters periodically were measured for 30 days. The experiments were conducted with sterilized 250-mL Erlenmeyer conical flasks. Ten milliliters each of pure culture was taken at exponential growth phase (7 days), and this initial culture density of 2.0 × 104 cells/mL was inoculated into 90 mL of sterilized BBM medium under aseptic condition. The growth parameters such as cell number, pigments such as chlorophyll a (Chl a) and chlorophyll b (Chl b), total carotenoids and astaxanthin contents were recorded at every 5-day interval during the study period. Monoalgal culture of the algae was used in the following experiments.

2.3.1 Cell count

“Neubauer” hemocytometer (REF: 0303 212, Neubauer Improved Bright-Line, HBG, Germany) was used for the purpose. The mean of the cell numbers recorded in four chambers was calculated and expressed as multiples of 104 cells/mL.

2.3.2 Growth curve

Growth curves were plotted with cell count values (multiples of 104 cells/mL) against respective days on which the cell count was measured.

2.3.3 Determination of specific growth rate (µ), division rate (K) and generation time (DT)

The specific growth rate implies the number of generation or the number of doublings that occur per unit of time in an exponential growth culture. The specific growth rate was determined using the following equation [41]: µ = ln (Nt/N0)/(T − t); Nt = no of cells at the end of the log phase; N0 = no of cells at the start of log phase; T = final day of log phase; t = starting day of log phase, if T expressed in days from the growth rate (µ) can be converted to K = µ/1n (2).

2.3.4 Extraction and estimation of pigments

Five milliliters of culture was centrifuged at 5000 rpm for 10 min, and the supernatant was discarded. The algal pellet was added with 5 mL of 100% acetone and macerated using pestle and mortar wrapped with black paper and kept overnight at 4 °C. The sample was centrifuged (R-8C; Remi Instruments Ltd, Mumbai, India) at 5000 rpm for 10 min, supernatant was collected, and the absorbance was measured at 661.6 nm, 644.8 nm, 470 nm and 490 nm wavelengths in Ultrospec 1100 pro UV–visible spectrophotometer (Amersham Bioscience, Germany) in standard quartz cuvette (190–2500 nm), path length 10 mm. The chlorophyll and total carotenoid contents were calculated using Lichtenthaler equations [42]. The amount of astaxanthin was determined from the acetone extract measured at 490 nm. The per unit volume of astaxanthin concentration (mg/L) was calculated by using the methods [43].
$$\begin{aligned} & {\text{Chl}}\,a\left( {\text{mg/L}} \right) = 11.24 \times A_{661.6} - 2.404 \times A_{644.8} \\ & {\text{Chl}}\,b\left( {\text{mg/L}} \right) = 20.13 \times A_{644.8} - 4.19 \times A_{661.6} \\ & {\text{Total}}\,{\text{carotenoids}}\,\left( {\text{mg/L}} \right) = \frac{{1000 \, \times \, A_{470} {-} \, 1.9 \, \times {\text{ Chl}}\,a{-}63.14 \, \times {\text{Chl}}\,b}}{214} \\ & {\text{Astaxanthin}}\,\left( {\text{mg/L}} \right) = 4.5 \times A_{490} \times {\text{ Va}}/{\text{Vb}} \\ \end{aligned}$$
where A = absorbance; Va = volume of extracts; Vb = volume of culture sample.

2.3.5 Statistical analysis

All the experiments were carried out in triplicate and expressed as mean ± standard errors. The graphs were prepared by Graph Pad Prism 6 software.

3 Results

3.1 Identification of isolates

The different growth phase of the microalga H. lacustris HPI-001 was identified through microscopic examination and observed the cell size and shapes such as spherical, ellipsoidal or pear shape, in addition to which the cells clearly showed two flagella of equal length emerging from the anterior end, and a cup-shaped chloroplast with numerous, scattered pyrenoids. Further the culture was confirmed through molecular studies (18S rRNA) and identified as H. lacustris HPI-001, and the molecular data were submitted in GenBank and received accession number (KT285940). Under unfavorable conditions, the macrozooids get transition into the coccoid vegetative cell state by loosing flagella (LABOMED VISION 2000, 40 × magnifications). When the cultures were exposed to stress conditions, the volume of the cell increased with a diameter of over 40 µm and the cell wall became resistant to the harsh conditions in which it was present. The maturation cyst aplanospore cells were accompanied by the enhancement of carotenoids biosynthesis and a gradual change in cell color to red. During optimal growth conditions, the daughter cells were released from the cystic cells and then vegetative cells regenerated from the daughter cells. Both the strains were maintained at Algal Culture Collection, Centre for Advanced Studies in Botany, University of Madras, Tamil Nadu, India (Figs. 1, 2).
Fig. 1

Light microscopic image of isolate H. lacustris HPI-001 cells in life cycle in laboratory conditions. a Biflagellated motile cells (macrozooids); b non-motile green vegetative cells (palmella); c astaxanthin accumulating palmella cell in transition to aplanospores; d astaxanthin accumulated aplanospore cell. Scale bar: 10 µm

Fig. 2

Light microscopic image of H. lacustris SAG-19a cells in life cycle in laboratory conditions. a Biflagellated motile cells (macrozooids); b non-motile green vegetative cells (palmella); c astaxanthin accumulating palmella cell in transition to aplanospores; d astaxanthin accumulated aplanospore cell. Scale bar: 10 µm

3.2 Effect of different concentrations of commercial N:P:K (17:17:17)

The two strains of H. lacustris were grown at different concentrations of commercial NPK (17:17:17). The H. lacustris HPI-001 showed maximum cell number of 47 × 104 cells/mL in 1.2 mM of commercial NPK on 15th day of culture. At 1.2 mM, a specific growth rate of 0.117 day−1, division rate of 0.169 day−1 and generation time of 5.931 day were recorded. The maximum concentration of Chl a (7.13 mg/L) and Chl b (3.57 mg/L) was recorded on 20th day at 1.2 mM NPK. The H. lacustris SAG-19a showed a maximum cell number of 42 × 104 cells/mL, and a maximum concentration of Chl a (6.25 mg/L) and Chl b (3.13 mg/L) on 20th day of 1.5 mM NPK. The increase in growth was 11%, and pigments Chl a 14% and Chl b were 10% which was more than that of H. lacustris SAG-19a. The alga H. lacustris HPI-001 accumulated maximum total carotenoids (22.33 mg/L) and astaxanthin content (17.94 mg/L) in 0.6 mM NPK on 30th day (Fig. 3). The H. lacustris SAG-19a exhibited maximum content of total carotenoids (20.60 mg/L) and astaxanthin (15.37 mg/L) in 0.6 mM of commercial NPK on 30th day (supplementary data S1). In the H. lacustris HPI-001, the increase in content of total carotenoids (10%) and astaxanthin (16%) was more than that of H. lacustris SAG-19a.
Fig. 3

Effect of different concentrations of commercial agriculture fertilizer N:P:K (17:17:17) (0.3, 0.6, 0.9, 1.2, 1.5 and 1.8 mM) on a cell number (a), chlorophyll a (b), total carotenoids (c) and astaxanthin content (d) isolate of H. lacustris HPI-001 at different intervals in laboratory conditions

3.3 Effect of different concentrations of CH4N2O

The alga H. lacustris HPI-001 showed maximum cell number of 41 × 104 cells/mL on 15th day in 3.3 mM of CH4N2O. At 3.3 mM of CH4N2O, the specific growth rate, division rate and generation time were 0.197 day−1, 0.284 day−1 and 3.515 day, respectively. The maximum synthesis of pigments Chl a (8.26 mg/L) and Chl b (4.13 mg/L) was on 15th day in 3.3 mM CH4N2O. The H. lacustris SAG-19a exhibited maximum growth of 32 × 104 cells/mL in 3.3 mM CH4N2O on 15th day. The increase in growth (28%), pigments Chl a (14%) and Chl b (13%) was more than that of H. lacustris SAG-19a. The maximum accumulation of total carotenoids in H. lacustris HPI-001 was 18.50 mg/L, and astaxanthin content was 15.19 mg/mL at 4.9 mM of CH4N2O on 25th day (Fig. 4). The H. lacustris SAG-19a showed a maximum concentration of 15.97 mg/L total carotenoids and 12.84 mg/L astaxanthin content at 6.6 mM CH4N2O on 25th day (supplementary data S2). The increment of total carotenoids (15%) and astaxanthin content (18%) was more than H. lacustris SAG-19a.
Fig. 4

Effect of different concentration of commercial agriculture fertilizers CH4N2O on a cell number (a), chlorophyll a (b), total carotenoids (c) and astaxanthin content (d) isolate of H. lacustris HPI-001 at different intervals in laboratory conditions

3.4 Effect of different concentrations of (NH4)2HPO4 and K2CO3

Under different concentrations of (NH4)2HPO4 + K2CO3, the alga H. lacustris HPI-001 exhibited the maximum cell number of 35 × 104 cells/mL in the medium with 250 mg/L (NH4)2HPO4 + 200 mg/L K2CO3 on 20th day of culture. At 250 mg/L (NH4)2HPO4 + 200 mg/L K2CO3, a specific growth rate of 0.178 day−1, division rate of 0.257 day−1 and generation time of 3.893 day were observed. The maximum synthesis of pigments recorded was 7.11 mg/L Chl a and 3.56 mg/L Chl b on 20th day at 250 mg/L (NH4)2HPO4 + 200 mg/L K2CO3. The maximum growth in H. lacustris SAG-19a was recorded as 30 × 104 cells/mL in the medium with 200 mg/L (NH4)2HPO4 + 150 mg/L K2CO3 on 20th day. Similarly, the maximum concentration of Chl a was 6.76 mg/L and Chl b was 3.39 mg/L at 200 mg/L (NH4)2HPO4 + 150 mg/L K2CO3 on 20th day. The increase in growth (16%) and pigments Chl a (5%) and Chl b (5%) was more than that of H. lacustris SAG-19a. The H. lacustris HPI-001 accumulated a maximum total carotenoids of 15.99 mg/L and astaxanthin content of 12.69 mg/L at 150 mg/L (NH4)2HPO4 + 100 mg/L K2CO3 on 25th day (Fig. 5), while the H. lacustris SAG-19a accumulated a maximum total carotenoids of 13.88 mg/L and astaxanthin content of 11.99 mg/L at 150 mg/L (NH4)2HPO4 + 100 mg/L K2CO3 on 30th day (supplementary data S3). The increase in total carotenoids (15%) and astaxanthin content (10%) was more than control of H. lacustris SAG-19a.
Fig. 5

Effect of different concentration of commercial agriculture fertilizers (NH4)2HPO4 + K2CO3 on a cell number (a), chlorophyll a (b), total carotenoids (c) and astaxanthin content (d) isolate of H. lacustris HPI-001 at different intervals in laboratory conditions

3.5 Effect of different concentrations of NaHCO3

The H. lacustris HPI-001 exhibited a maximum cell number of 51 × 104 cells/mL at 0.6 mM commercial NaHCO3 in 20 days. At 0.6 mM of NaHCO3, a specific growth rate of 0.184 day−1, division rate of 0.266 day−1 and generation time of 3.759 day were observed. The maximum synthesis of pigments, Chl a (9.52 mg/L) and Chl b (4.76 mg/L), was in 0.6 mM of NaHCO3 on 20th day. The H. lacustris SAG-19a showed a maximum growth of 45 × 104 cells/mL at 1.5 mM NaHCO3 on 20th day. The maximum concentration of Chl a (8.81 mg/L) and Chl b (4.40 mg/L) was in 0.6 mM NaHCO3 on 20th day. The increment of growth (13%) and pigments Chl a (8%) and Chl b (8%) was more than that of H. lacustris SAG-19a. The maximum accumulation of total carotenoids in the isolate of H. lacustris HPI-001 was 19.24 mg/L, and astaxanthin content was 15.53 mg/L in 1.5 mM of NaHCO3 on 25th day of culture (Fig. 6). The strain H. lacustris SAG-19a synthesized maximum total carotenoids of 16.89 mg/L and astaxanthin content of 13.71 mg/L in 0.6 mM of NaHCO3 on 30th day (supplementary data S4). The increase in total carotenoids (13%) and total astaxanthin (13%) was more compared to H. lacustris SAG-19a.
Fig. 6

Effect of different concentration of commercial NaHCO3 on a cell number (a), chlorophyll a (b), total carotenoids (c) and astaxanthin content (d) isolate of H. lacustris HPI-001 at different intervals in laboratory conditions

3.6 Comparative study of the two strains of H. lacustris grown in modified 3 N-BBM + V medium and formulated modified HPI-001 medium

The isolate H. lacustris HPI-001 grown in two different media tested showed a maximum growth of 43 × 104 cells/mL in formulated modified HPI-001 medium on 20th day. The alga grown in formulated modified HPI-001 medium showed a specific growth rate of 0.162 day−1, division rate of 0.233 day−1 and generation time of 4.284 day. The alga synthesized 8.68 mg/L Chl a and 5.12 mg/L Chl b on 20th day, and 25.51 mg/L total carotenoids and 22.21 mg/L astaxanthin content on 30th day in formulated modified HPI-001 medium (Fig. 7). The H. lacustris HPI-001 showed a maximum growth of 37 × 104 cells/mL in modified 3N-BBM + V medium on 20th day. The maximum synthesis of pigments Chl a (7.44 mg/L), Chl b (3.72 mg/L), total carotenoids (22.88 mg/L) and astaxanthin content (20.45 mg/L) was on 20th and 30th day in modified 3N-BBM + V medium. In case of H. lacustris SAG-19a, the maximum cell number of 40 × 104 cells/mL in formulated modified HPI-001 medium was on 20th day. The alga grown in formulated modified HPI-001 medium showed a specific growth rate of 0.184 day−1, division rate of 0.266 day−1 and generation time of 3.763 day. The maximum concentration of pigments Chl a (7.22 mg/L) and Chl b (4.36 mg/L) was on 20th day, and maximum accumulation of total carotenoids (23.59 mg/L) and astaxanthin content (20.10 mg/L) was on 30th day in formulated modified HPI-001 medium (Table 3). The H. lacustris SAG-19a showed maximum growth of 35 × 104 cells/mL on 20th day of modified 3N-BBM + V medium. The maximum synthesis of Chl a (6.50 mg/L), Chl b (3.25 mg/L), total carotenoids (20.55 mg/L) and astaxanthin content (18.25 mg/L) was on 20th and 30th day in modified 3N-BBM + V medium (supplementary data S5). The increase in growth (8%), Chl a (20%), Chl b (17%), total carotenoids (8%) and astaxanthin content (10%) was more than that of H. lacustris SAG-19a.
Fig. 7

Comparative study of algae grown in modified 3N-BBM + V medium and formulated commercial modified HPI-001 medium on a cell number (a), chlorophyll a (b), total carotenoids (c) and astaxanthin content (d) isolate of H. lacustris HPI-001 at different intervals in laboratory conditions

Table 3

Comparative study on growth and astaxanthin content of H. lacustris HPI-001 and H. lacustris SAG-19a using agriculture fertilizers

Different parameters conditions

H. lacustris HPI-001 cell number (104 cells/mL)

H. lacustris SAG-19a cell number (104 cells/mL)

H. lacustris HPI-001 Astaxanthin (mg/L)

H. lacustris SAG-19a Astaxanthin (mg/L)

N:P:K (17:17:17) (mM)

 0.3

33 ± 0.344

33 ± 0.444

17.49 ± .0.414

14.66 ± 0.410

 0.6

40 ± 0.420

35 ± 0.350

17.94 ± 0.509

15.37 ± 0.455

 0.9

43 ± 0.527

37 ± 0.325

17.57 ± 0.503

15.02 ± 0.326

 1.2

47 ± 0.524

40 ± 0.515

17.15 ± 0.303

14.72 ± 0.510

 1.5

37 ± 0.422

42 ± 0.520

16.81 ± 0.419

14.50 ± 0.420

1.8

30 ± 0.324

38 ± 0.210

16.36. ± 0.286

14.16 ± 0.222

CH4N2O (mM)

    

 1.6

37 ± 0.525

25 ± 0.447

13.20 ± 0.407

11.35 ± 0.467

 3.3

41 ± 0.338

32 ± 0.557

13.85 ± 0.481

13.83 ± 0.448

 4.3

35 ± 0.520

29 ± 0.407

14.11 ± 0.532

12.13 ± 0.435

 4.9

31 ± 0.544

27 ± 0.348

15.19 ± 0.509

12.47 ± 0.539

 6.6

28 ± 0.244

24 ± 0.532

14.74 ± 0.409

12.84 ± 0.285

 8.3

25 ± 0.243

22 ± 0.546

14.53 ± 0.307

12.81 ± 0.372

(NH4)2HPO4 + K2CO3 (mg)

    

 100 + 50

25 ± 0.419

22 ± 0.510

12.21 ± 0.528

11.54 ± 0.450

 150 + 100

31 ± 0.533

26 ± 0.425

12.69 ± 0.307

11.99 ± 0.525

 200 + 150

33 ± 0.300

30 ± 0.520

12.28 ± 0.206

11.76 ± 0.310

 250 + 200

35 ± 0.267

28 ± 0.310

11.96 ± 0.406

11.51 ± 0.520

 300 + 250

28 ± 0.164

24 ± 0.255

11.67 ± 0.553

11.33 ± 0.425

NaHCO3 (mM)

    

 0.3

37 ± 0.444

31 ± 0.515

13.93 ± 0.374

9.85 ± 0.455

 0.6

51 ± 0.342

34 ± 0.325

14.37 ± 0.521

12.71 ± 0.420

 0.9

46 ± 0.536

36 ± 0.310

14.73 ± 0.345

10.10 ± 0.510

 1.2

35 ± 0.233

41 ± 0.215

14.91 ± 0.463

9.84 ± 0.525

 1.5

33 ± 0.144

45 ± 0.420

15.53 ± 0.370

9.73 ± 0.420

 1.8

30 ± 0.443

39 ± 0.443

15.07 ± 0.455

9.51 ± 0.510

 Modified HPI-001 medium

43 ± 0.351

40 ± 0.425

22.21 ± 0.357

20.10 ± 0.429

 3N-BBM + V medium (Control)

37 ± 0.320

35 ± 0.442

20.45 ± 0.549

18.25 ± 0.425

Each value is the means of three experiments with triplicate each (n = 3). Statistically the means of three experiments not significantly different (P  < 0.05)

4 Discussion

H. lacustris is a photosynthetic green microalga that can produce astaxanthin in cells. In general, astaxanthin production from H. lacustris is accomplished through a two-stage cultivation process including vegetative (green) and aplanospore (red) stages [44, 45]. The accumulation of astaxanthin is affected by environmental stress factors such as light, temperature, pH, salt concentration and nutritional stresses [11]. However, some factors limit outdoor production such as light intensity and temperature. Therefore, the induction of high astaxanthin accumulation with nutrition is a significant option. Though, including the neutral pH, neutral to the moderately basic range was recognized as the most favorable and suitable condition for the fungal contaminant to infect the algae cells [46], the present cultures were found to be devoid of any fungal contamination while grown at a pH of 7.5–7.8. In the present study, the commercial agriculture fertilizers such as NPK (17:17:17), CH4N2O, (NH4)2HPO4, K2CO3 and NaHCO3 were used for the enhancement of growth and astaxanthin production in two strains of H. lacustris.

In common, nitrogen has a marked positive effect on growth and a negative effect on lipid accumulation due to the fact that microalgae can assimilate several nitrogen sources like nitrate, nitrite, ammonium and urea [47]. However, the nitrogen is a crucial element for microalgae’s growth development, reproduction and other physiological activities [48]. Dominguez-Bocanegra et al. [49] reported that the H. lacustris culture in test BBM obtained a maximum cell density of 3.5 × 104 cells/mL, whereas Goksan et al. [47] obtained a maximum cell density of 2.6 × 104 cells/mL with BG-11 medium plus different nitrogen sources. Similarly, Dalay et al. [21] reported that the commercial fertilizers (N:P:K 20:20:20) maximized cell concentration to 0.90 g/L with growth rate of 0.150 day−1. Since the microalga H. lacustris grow better in culture media with low N:P, high phosphorus levels favored the accumulation of biomass content [50]. However, when compared to nitrogen, phosphorus concentration over 50% caused a decrease in growth of culture [51]. In fact, differences in N:P concentrations may reduce algal growth for its adaptation to stress conditions [52].

NPK fertilizer is a low-cost nitrogen fertilizer and a relevant tool in microalgal culture, mainly for Chlorophyceae. In the current study, the Bold basal medium minus NaNO3, K2HPO4∙3H2O and KH2PO4 was added with commercial agricultural fertilizer (N:P:K 17:17:17) and used to grow two different strains of H. lacustris. The isolate of H. lacustris HPI-001 showed a maximum growth of 47 × 104 cells/mL with specific growth rate of 0.117 day−1. The growth attained here is higher than that reported by Dominguez-Bocanegra et al. [49] and Goksan et al. [47]. The pigment Chl a was increased to 7.13 mg/L at 1.2 mM of commercial (N:P:K 17:17:17) in 15th and 20th day cultures. At 0.6 mM (N:P:K 17:17:17), the total carotenoids increased to 22.35 mg/L and astaxanthin content to 17.94 mg/L on 30th day. In strain H. lacustris SAG-19a exhibited a maximum cell number increased up to 42 × 104 cells/mL specific growth of 0.122 day−1. The isolate H. lacustris HPI-001 increased in growth (11%), pigment Chl a (14%), Chl b (10%), total carotenoids (10) and astaxanthin content (16%) was more than control of H. lacustris SAG-19a.

It has been reported that nitrate is an important nutrient supplement, affecting the growth and biomass accumulation in the microalgae. It is also able to significantly change the rates of cell metabolism through the transformation among different forms [53, 54]. Moreover, nitrate was demonstrated to have a vital role for haematocyst germination in H. lacustris [55]. Nitrogen depletion in the culture medium is a stress factor of H. lacustris, while high phosphorus concentration foregrounds algal growth in the vegetative stage which normally lasts between 9 and 20 days according to the ratio between biomass and cell activity [56]. Chlorophyll as a nitrogen-rich compound can be utilized as an intracellular nitrogen supporter to continue the growth of cells once nitrogen sources are decreased in culture. However, chlorophyll degradation may occur due to the reduced level of chlorophylls a and b with reutilizing of nitrogen for cells [57]. These strategies are also employed in the H. lacustris cultures, and a remarkable enhancement of astaxanthin production is achieved due to nitrogen deficiency. For example, Fabregas et al. [55] reported the combined effects of light intensity and nutrient deficiency on astaxanthin synthesis by H. lacustris. In the present investigation, when grown in BBM devoid of NaNO3 and added with the commercial fertilizer CH4N2O, the H. lacustris HPI-001 showed maximum growth of 41 × 104 cells/mL with specific growth rate of 0.197 day−1 and H. lacustris SAG-19a exhibited the maximum cell number of 32 × 104 cells/mL with specific growth rate of 0.205 day−1. The increase in growth (28%) was higher than that of H. lacustris SAG-19a. H. lacustris HPI-001 produced maximum concentration of Chl a (8.26 mg/L) and Chl b (4.13 mg/L) at 3.3 mM CH4N2O on 15th day. The 4.9 mM of CH4N2O supported the highest accumulation of total carotenoids (18.49 mg/L) and astaxanthin (15.19 mg/L) on 25th day. The increase in pigments such as Chl a (14%), Chl b (13%), total carotenoids (15%) and astaxanthin content (18%) was higher compared to that of H. lacustris SAG-19a.

Phosphorus is an important nutrient for algal growth. It is responsible for the energy transfer of cells and the formation of cell membranes and nucleic acids. Besides being a structural element in nucleic acid and phospholipids, it plays crucial roles in various biological functions such as energy transformation, activation of metabolic intermediates, signal transduction cascades and regulation of enzymes [58, 59]. When compared to nitrate, phosphate has received less attention in the optimization approach and has long been considered to promote the growth at moderate or low concentration approximately 0.5 mM [60, 61], while at the same time it can promote the carotenogenesis at higher concentration up to 0.9 mM [60]. Harker et al. [62] reported that the carotenoids accumulation has been shown to be reduced when phosphate supply was increased above 0.85 mM. There are some few reports on H. lacustris growth in N/P ratio close or below 1, but in all cases it was identified that the low N/P conditions were favorable for growth [20, 21, 62]. Brinda et al. [63] reported that the high biomass and astaxanthin accumulation were achieved in H. lacustris under phosphorus deficiency conditions. In our study, the K2HPO4.3H2O and KH2PO4 in basal medium was replaced with commercial agricultural fertilizer (NH4)2HPO4 + K2CO3 and both the strains were grown in it. In the H. lacustris HPI-001 exhibited maximum cell number of 35 × 104 cells/mL with a specific growth rate of 0.178 day−1, in which the growth (16%) was more than that of H. lacustris SAG-19a. Similarly, the photosynthetic pigment also increased as compared to the control on 20th days. The maximum accumulation of total carotenoids (15.99 mg/L) and astaxanthin content of (12.69 mg/L) in isolate H. lacustris HPI-001 was on 25th days. Ping et al. [64] reported that only 11 mg/L carotenoids were obtained in phosphate deficiency conditions. In the isolate, the increase in carotenoids (15%) and astaxanthin content (10%) was more than that of H. lacustris SAG-19a.

The carbon rate characterizes algal nutrition rates and constitutes 40–50% of algal biomass, although biomass is 47–50% in H. lacustris [65]. Moreover, based on the recent study, carbon mass fraction in H. lacustris can range from 46 to 55% [66]. The enrichment of CO2 is a requirement for achieving high microalgal culture productivity. At the same time, high CO2 levels are frequently stressful to microalgae, particularly for their photosynthetic apparatus [67, 68]. The recent research reported that the cultivation of H. lacustris under high CO2 demonstrated that increasing CO2 percentage in the gas mixture used for the culture sparging to 5% is favorable for astaxanthin accumulation [69, 70]. Ding et al. [71] reported that under steady-state conditions, astaxanthin content in H. lacustris was 0.41%. The previous studies on the cultivation of H. lacustris have showed that acetate appears to be an important carbon source, enhancing both growth and carotenogenesis [45, 72, 73]. However, the effect of acetate was concentration dependent, higher concentrations inhibiting growth but markedly increasing astaxanthin content per cell [74]. Bicarbonate is widely used as a primary carbon source in suspended culture of photosynthetic microorganisms [68]. The present investigation used basal medium added with commercial NaHCO3 to grow H. lacustris HPI-001 which showed a maximum growth of 51 × 104 cells/mL with specific growth rate of 0.184 day−1 and maximum chlorophyll production at 0.6 mM of NaHCO3 on 20th day, whereas the increase in total carotenoids (13%) and astaxanthin content (13%) was at 1.5 mM NaHCO3 on 25th day.

In batch culture processes, the optimal Haematococcus media produced a cell number of 6.25 × 104 cells/mL after 14 days of culture with no astaxanthin being accumulated [65]. A report by Domìnguez-Bocanegra et al. [49] states that the maximal growth of H. lacustris obtained was 3.5 × 104 cells/mL in the BBM medium under continuous illumination (177 µmol photons m−2 s−1) with continuous aeration (1.5 vvm). Based on the above observations, a modified medium was formulated and designed, named as formulated commercial modified HPI-001 medium. The algal strains grown in the above medium were compared with the modified 3N-BBM + V. The isolate H. lacustris HPI-001 showed maximum growth of 43 × 104 cells/mL with a specific growth rate of 0.162 day−1. The maximum synthesis of pigments was 8.68 mg/L Chl a, 5.12 mg/L Chl b on 20th day, while the maximum accumulation of total carotenoids (25.51 mg/L) and astaxanthin content (22.21 mg/L) was on 30th day in cultures using formulated modified HPI-001 medium. The H. lacustris SAG-19a culture serving as H. lacustris SAG-19a exhibited maximum growth of 40 × 104 cells/mL. The maximum concentration of pigments Chl a (7.22 mg/L) and Chl b (4.36 mg/L) was on 20th day, while the maximum accumulation of total carotenoids (23.59 mg/L) and astaxanthin content at (20.10 mg/L) was on 30th day, when cultured in the formulated modified HPI-001 medium. A comparatively high growth rate and pigment production was observed in the indigenously isolated H. lacustris HPI-001 than in the H. lacustris SAG-19a, when both were cultured using the formulated modified HPI-001 medium. This indicates that a promising alternative, cheaper and reproducible medium can be formulated using commercial agricultural fertilizers for achieving higher biomass and pigment astaxanthin production from H. lacustris.

5 Conclusion

From the present study, it is evident that the indigenous isolate of H. lacustris HPI-001 exhibited increased growth and astaxanthin production in the formulated modified HPI-001 medium when compared to the H. lacustris SAG-19a. This indicates that a promising alternative, cheaper and reproducible medium can be formulated using commercial agricultural fertilizers for achieving higher biomass and pigment astaxanthin production from H. lacustris. It is important that the modified HPI-001 medium which was formulated in this study for the first time is a very cost-effective medium compared to the control modified Bold basal medium (3N-BBM + V). Currently, the algal-based biotechnology companies and industries are looking for cost-effective solution for mass cultivation of commercially important microalga like H. lacustris for maximum biomass production. Hence, the present investigations recommend the formulated modified HPI-001 medium as a best source for maximum content of biomass and astaxanthin production. The outdoor studies are under progress with Bayir Extracts Pvt. Ltd., Bangalore, India.

Notes

Acknowledgements

The author Dr. SNR would like to thank M/s. Bayir Extracts Pvt. Ltd., Bangalore-560085, for providing the financial support. I would also like to thank Prof. R. Rangasamy for his valuable suggestion in this research work. Mr. KR, AJ, Ms. SKG are thankful to The Director, Centre for Advanced Studies in Botany, University of Madras for providing laboratory facilities.

Funding

The author Dr. SNR grateful to the Bayir Extracts Pvt. Ltd., Banglore – 560085, India, (Grand No. ARC/UICIC/CAS/Dr.S.N./2014/77) for sponsored financial support through University Industry Community Interaction Centre, (UICIC) University of Madras, Chennai.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

42452_2019_543_MOESM1_ESM.docx (1.1 mb)
Supplementary material 1 (DOCX 1089 kb)

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© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Ramamoorthy Karuppan
    • 1
  • Anand Javee
    • 1
  • Sreekala Kannikulathel Gopidas
    • 1
  • Nagaraj Subramani
    • 1
    Email author
  1. 1.Centre for Advanced Studies in BotanyUniversity of MadrasChennaiIndia

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