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An effective approach of bacterial siderophore as nitrogen source triggering the desired biochemical changes in microalgae Chlorella variabilis ATCC 12198
Algal Research 43 (2019) 101610
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An effective approach of bacterial siderophore as nitrogen source triggering the desired biochemical changes in microalgae Chlorella variabilis ATCC 12198
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Soundarya Rajapitamahunia,b, Khushbu Bhayania, Pooja Bachania,b, Vamsi Bharadwaj S.Va,b, ⁎ Sandhya Mishraa,b, a b
Division of Biotechnology and Phycology, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, Gujarat, India Academy of Scientific & Innovative Research (AcSIR), CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, Gujarat, India
ARTICLE INFO
ABSTRACT
Keywords: Siderophores Nitrogen starvation Two stage cultivation Biomass Lipid
The present study was aimed to check whether catecholate siderophore can serve as a nitrogen source besides iron for microalgae. Catecholate siderophore from Idiomarina loihiensis RS 14 contains 13% nitrogen which can be a potential nitrogen source. Bacterial supernatant containing siderophores with different inoculum ratio was used to check the growth of Chlorella variabilis ATCC 12198. Nitrogen starvation with two stage cultivation was employed here to trigger the accumulation of lipid and carbohydrate. In continuous cultivation, the highest productivity of DCW was observed in 1%, 760 mg/L compared to culture grown at 0.5%, with an increase of 42.2% higher biomass productivity (21.66 mg/L/day). Similarly, in 1%, C. variabilis produced total 30% of lipid content which contains 75% of neutral lipid. Highest carbohydrate was (49.29%) found in cells grown under 9 days of stress in 1% followed by cells grown under 3 days of stress in 5% (46.52%) in stage II cultivation.
1. Introduction Microalgae is considered having pronounced potential in biodiesel production due to its enormous growth rate and lipid content [1]. Nitrogen is an essential element for the growth of microalgae and its deficiency has a huge impact in its metabolic activities [2]. This elusiveness of nitrogen can be satisfied by certain metabolites, helping microalgae to enhance its nutrient uptake [3]. For its sustainability, nitrogen inputs will be the fundamental concern in investigating microalgal cultivation strategy for achieving high nutrition valve and its productivity [4] Chlorella variabilis ATCC 12198 is a robust strain of unicellular green eukaryotic microalgae growing in marine water which after nitrogen starvation is capable of accumulating higher amount of lipids with prominent fatty acid profile suitable for biodiesel production [5]. There is a need of microbes for easy solubilisation of certain elements, for which chelators like siderophores are produced even under traces of iron amount in the environment for transportation and storage via specific membrane receptors [6,7]. It chelates by virtue of three chelating groups, hydroxamates, catecholates and hydroxyacids which are generally oxygenated forming complexes with great thermodynamic stability [8]. Idiomarina loihiensis RS 14 is equipped with such
siderophore production system and uses part of this nitrogen fixed to produce catechol siderophores for the uptake of metal ions [9]. This specific siderophore, has a short peptide chain, containing significant percentage of nitrogen for the proliferation of microalgae, besides iron as a biostimulant [10]. Siderophore containing N molecule in the structure has been characterized which is of specific interest to determine its potential as a nitrogen source for microalgae. Though there are various media present for micro-algal cultivation but, media optimized can stimulate nutrient requirement for the growth of distinct algal species by changing its effect on biomass, chlorophyll, lipid, carbohydrate and protein [11]. There are many reports mentioning advantage taken by siderophore-producing bacteria for metal uptake by utilizing foreign siderophores [12,13]. To discover, if this phenomenon can satisfy requirement of fixed nitrogen, we first isolated siderophores from I. loihiensis to collect the siderophores using ion-exchange resin as described in the methods [10]. The productivity of the biomass is crucial for algal biodiesel and industrialization [14]. Though there are number of factors mentioned earlier that influences lipid and carbohydrate in microalgae, nitrogen is one of them [15,16]. Two-stage cultivation strategy could be used, by providing good source of nutrients to obtain maximum cell density in stage I
⁎ Corresponding author at: Division of Biotechnology and Phycology, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, Gujarat, India. E-mail address: [email protected] (S. Mishra).
https://doi.org/10.1016/j.algal.2019.101610 Received 7 May 2019; Accepted 8 July 2019 Available online 21 August 2019 2211-9264/ © 2019 Elsevier B.V. All rights reserved.
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followed by nutrient-stress conditions in stage II to induce lipid and carbohydrate accumulation [17] which has been applied in this work. Using bacterial supernatant containing siderophore in nitrate free micro-algal medium was proposed for enhancing micro-algal biomass. This study highlights the use of bacterial siderophores as a nitrogen source for indigenous marine water green algal strain, Chlorella variabilis ATCC 12198. In the present study by contrast, a two-stage cultivation mode was applied using siderophore as a source of nitrogen without any additional pre-treatment to improve the growth and lipid content.
different inoculum for nitrate limitation experiment. At 6, 9 and 12 days of inoculation, harvesting was done by centrifugation at 10,000 rpm for 20 min and again inoculated in medium free of nitrate. The study in 500 mL of culture medium in 1000 mL conical flask was done in triplicates (50 mL of 10 day old culture as inoculum). 2.2.2. Bacterial growth conditions Bacteria was grown in a 2000 mL flasks under low iron conditions, for which CDLIM media (Chemically Defined Low Iron Media) was used for successful release of siderophores at 37°C, 180 rpm for 72 h. The culture centrifuged at 8000g for 15 min at 4 °C for the supernatant containing siderophores. Supernatant was then filtered through 0.25 μm filter to get devoid of cells. Samples from this batch mode were then used as the inoculum for the nitrogen stress experiments in microalgal medium [10]. The volume of the inoculum sample was used (i.e., 0.5, 1, 5, and 10% of the bacterial supernatant in the 1 L flasks).
2. Materials and methods 2.1. Identification of siderophores supporting micro-algal growth Prior to designing an experiment, it was mandatory to understand which class of siderophores can be replaceable of nitrogen in micro-algal medium to support its growth. The commercially available different functional group of siderophores like hydroxamate, carboxylate and catecholates were collected from Sigma Aldrich. The elemental composition of hydroxamate siderophore i.e. Deferoxamine mesylate C26H52N6O11, and catecholate siderophore i.e. Enterobactin C30H27N3O15. On the basis of molecular formula of purified siderophore from Idiomarina loihiensis RS14 i.e. C16H29N10O8 which constitutes a considerable number of nitrogen equivalents and when released to the environment even in smaller molar concentrations can account for a significant amount of available nitrogen. Based on previous reports of other organisms stealing foreign siderophores for their own benefit [12,13], it was of interest to demonstrate whether catecholate siderophore from Idiomarina loihiensis RS 14 would also serve to satisfy nitrogen requirements to support micro-algal growth following its enhanced lipid content.
2.3. Extraction and purification of siderophore Acidified (pH 2.5) bacterial culture's supernatant was added to the resin (Amberlite XAD-2,) for absorbing the siderophore [18] and poured into column. 100% methanol was used to obtain final elution and further concentrated by rotary evaporation (Buchi-R215) to yield the crude siderophore [19] and further purified on Sephadex G-10 [20]. Siderophore present in the fractions were recognised by CAS assay and for further purification RP-HPLC was carried out [21]. 2.4. Effect of extracted and purified siderophore on micro-algal growth C. variabilis grown in ZM was transferred in HCl washed flask having fresh media supplemented with purified siderophore. Positive and negative control was prepared as discussed earlier. Purified siderophore ranging 0 μg, 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg and 50 μg was added to the nitrate deficient medium. All the culture flasks were kept in triplicates.
2.2. Experimental set-up The CSIR-CSMCRI's isolated strain of C. variabilis ATCC PTA 12198 was studied for its growth enhancement by siderophore as a substitution of nitrogen. The experimental media was prepared similar to control except the nitrogen component, which was replaced with a bacterial supernatant containing siderophore (as Nitrogen source). Hence, the nitrogen component was the only volatile in each test. Preliminary experiment was carried by the adhesion of extracted and purified siderophore into the algal medium as a replacement of nitrate, later the bacterial supernatant containing siderophores were used in the algal media. Two stage nitrogen cultivation method is applied where microalgae is initially grown in medium containing nitrogen (bacterial supernatant containing siderophores) followed by transferring it into a nitrogen free medium (no bacterial supernatant). To provide similar concentration of nitrogen present in the controlled culture, standard chelate was added at an appropriate concentration from a stock solution. In two-stage cultivation algae is initially cultured in nutrient-sufficient conditions (0.5%) taken as control to obtain a maximum dry biomass followed by nitrogen starvation to trigger the accumulation of lipids.
2.5. Effect of bacterial supernatant on micro-algal growth In stage I cultivation, cells were grown at three different inoculum of bacterial supernatant (containing siderophores as nitrogen source) viz. 0.5%, 1%, 5% and 10% and control was used with the regular nitrogen source i.e. NaNO3 till 18 days. Further, in two stage cultivation, cells initially grown as a control (NaNO3 as nitrogen source) were transferred to 1% and 5% of bacterial supernatant containing siderophore containing flasks. In stage I cultivation at 10% of bacterial supernatant, C. variabilis cells did not grow even after 7 days and therefore in both stage I and two stage cultivation, control, supernatant with 1% and 5% supernatant were used for further examination. Light intensity was (150 μmolm−2 s−1) and photoperiod was (12:12) hours of light: dark period. All the experiments were performed in triplicate in 1000 mL Erlenmeyer flasks containing 500 mL of Zarrouk's media inoculated with 50 mL of actively growing C. variabilis.
2.2.1. Micro-algal growth conditions Zarrouk's media was used for C. variabilis which consisted per litre of NaHCO3 16.8 g; K2HPO4 0.5 g; MgSO4.7H2O 0.2 g; NaCl 1.0 g; CaCl2. 2H2O 0.04 g; A5 1.0 mL; B6 1.0 mL; pH 10. EDTA and FeSO4.7H2O 0.01 g. NaNO3 constituent of media was omitted (nitrogen source). 50 mL of the young culture of C. variabilis was inoculated in the 1000 mL flasks (triplicates) containing 500 mL of medium. Culture was cultivated under 150 μmolm−2 s−1 of light intensity and 12:12 h of light: dark period at 37 °C and regularly shaken. The initial algal concentration was the same for all growth conditions with 4 × 118 cells/ mL or 0.2 optical density reading at 750 nm. Cells were grown with siderophores as nitrate source as positive control, without siderophores i.e. (without nitrate source) as negative control along with bacterial supernatant containing siderophores in
2.6. Estimation of biomass productivity (BP) The optical density at 750 nm was estimated for the growth of microalgae. According to following equation, BP (mg/L/day) was calculated. P = (X2 − X1) / (t2 − t1), where X2 and X1 are the dry cell weight concentration (mg/L) at time t2 and t1, respectively [22]. 2.7. Quantum yield of photosystem II (Fv/Fm) was measured with pulse-amplitude-modulated (PAM) fluorometry (AquaPen-C AP-C100, Photon System Instruments). Fv/Fm is calculated according to the following equation: [23] 2
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Fig. 1. A. Effect of siderophore on cell density of C.variabilis under nitrogen starvation All of the experiments were carried out in triplicate (n = 3) and the values are expressed as means ± standard deviations, analyzed with one-way analysis of variance (ANOVA) using InfoStat, 2012. The mean values were compared with LSD test and a significant difference was considered at p ≥ (0.05). B Effect of siderophore on Biomass productivity of C. variabilis under nitrogen starvation. All of the experiments were carried out in triplicate (n = 3) and the values are expressed as means ± standard deviations, analyzed with one-way analysis of variance (ANOVA) using InfoStat, 2012. The mean values were compared with LSD test and a significant difference was considered at p ≥ (0.05).
Fv /Fm = (Fm
suspended to the same. They were incubated under selected conditions for two stage cultivation, in (12:12) h light: dark period at 25 °C. Chlorella sp. lipid extracted by the method of [23] (a mixture of chloroform and methanol) and quantified gravimetrically with separation through column chromatography [24]. Biomass was vortexed thoroughly, ultrasonicated at room temperature in chloroform:methanol (1:2, v/v), centrifuged and measured gravimetrically. Lipids are mainly phospholipids (PL), glycolipids (GL) and neutral lipids (NL). All the above fractions are fractionated by sequential elution by chloroform:acetic acid (9:1, v/v) for neutral lipid, acetone:methanol (9:1, v/v) for glycolipids and 100% methanol for phospholipids after drying at 60°C [25]. Crude protein was estimated according to [26].
F0)/Fm
2.8. Estimation of pigments Pigment content was estimated in the pellet after centrifugation of 2.0 mL of culture at 10,000 rpm for 15 min +2.0 mL of 100% methanol, mixed well and incubated at 45 °C for 24 h in the dark. The total pigment content was calculated according to following equations:
Chlorophyll a; Chl
a (lµg/mL) = 16.72 (A665.2)–9.16 (A652.4)
Chlorophyll b; Chl
b (lµg/ml) = 34.09 (A652.4)–15.28 (A665.2)
2.10. Estimation of carbohydrate
Carotenoids (lµg/ml) = [1000 (A470)
1.63 (Chl
a)
104.9 (Chl
b)]/221 [22]
To determine carbohydrate, 100 mg of dried biomass with 10 mL of 2% H2SO4 was hydrolysed at 121 °C for 20 min, further CaCO3 used to neutralize and diluted to 100 mL distilled water. The total sugar content in supernatant was estimated after centrifugation at 10,000 rpm for 5 min [27].
2.9. Estimation of lipids and protein To optimize the maximum cell density, different inoculum of supernatant of varying siderophore amount were studied on micro-algal growth. Cultures grown with standard nitrogen source in the medium at (stage I) were stopped after reaching the late log phase. Cells were recovered through centrifugation at 10,000 rpm for 10 min. Cells collected were washed several times with nitrogen deficient ZM and again
2.11. Estimation of nitrate In the micro-algal culture medium total residual nitrate concentration was estimated using sodium hydroxide and salicylic acid. The algal 3
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Fig. 2. A (Stage I Cultivation) Effect of Siderophore containing supernatant on nitrate uptake by C. variabilis. All of the experiments were carried out in triplicate (n = 3) and the values are expressed as means ± standard deviations, analyzed with oneway analysis of variance (ANOVA) using InfoStat, 2012. The mean values were compared with LSD test and a significant difference was considered at p ≥ (0.05). B (Stage II Cultivation) Effect of Siderophore containing supernatant on nitrate uptake by C. variabilis in 1% inoculum All of the experiments were carried out in triplicate (n = 3) and the values are expressed as means ± standard deviations, analyzed with oneway analysis of variance (ANOVA) using InfoStat, 2012. The mean values were compared with LSD test and a significant difference was considered at p ≥ (0.05). C (Stage II Cultivation) Effect of Siderophore containing supernatant on nitrate uptake by C. variabilis in 5% inoculum All of the experiments were carried out in triplicate (n = 3) and the values are expressed as means ± standard deviations, analyzed with oneway analysis of variance (ANOVA) using InfoStat, 2012. The mean values were compared with LSD test and a significant difference was considered at p ≥ (0.05).
culture's filtrate was evaporated to dryness and 2 mL of PDA reagent was added and further diluted with water followed by the addition of 10 mL concentrated ammonium hydroxide, dark pink color formation occurred which was estimated at 410 nm [28].
2.12. Statistical analysis Experiments in triplicates presenting the data here are in mean values, analyzed with one-way analysis of variance (ANOVA) using InfoStat, 2012. The mean values were compared with LSD test and a significant difference was considered at p ≥ (0.005). 4
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Fig. 3. A (Stage I cultivation) Determination of Biomass productivity All of the experiments were carried out in triplicate (n = 3) and the values are expressed as means ± standard deviations, analyzed with one-way analysis of variance (ANOVA) using InfoStat, 2012. The mean values were compared with LSD test and a significant difference was considered at p ≥ (0.05). B (Stage II cultivation) Determination of Biomass productivity. All of the experiments were carried out in triplicate (n = 3) and the values are expressed as means ± standard deviations, analyzed with one-way analysis of variance (ANOVA) using InfoStat, 2012. The mean values were compared with LSD test and a significant difference was considered at p ≥ (0.05).
Table 1A (Stage I cultivation) determination of pigments. All of the experiments were carried out in triplicate (n = 3) and the values are expressed as means ± standard deviations, analyzed with one-way analysis of variance (ANOVA) using InfoStat, 2012. (Different superscript letters within column indicates significant differences at p < 0.05.) Treatments
Chlorophyll a (μg/mL)
Positive control Negative control 0.5% 1% 5%
12.01 ± 0.32a 5.23 ± 0.24a 7.12 ± 0.36a 13.06 ± 0.25a 6.15 ± 0.31a
Chlorophyll b (μg/ mL) 7.02 ± 0.29a 2.56 ± 0.41b 3 0.23 ± 0.36b 7 0.24 ± 0.54a 3.19 ± 0.08b
Chlorophyll (a + b) (μg/ mL) 19.03 ± 0.61a 7.79 ± 0.65ab 10.35 ± 0.72ab 20.30 ± 0.79a 9.34 ± 0.39c
Chlorophyll (a/b) (μg/ mL) 1.71 ± 1.10a 2.04 ± 0.58a 2.20 ± 1.00a 1.80 ± 0.46a 1.92 ± 3.87ab
Carotenoids (μg/mL)
Carot/Chlo(a + b) (μg/mL)
14.01 ± 1.23a 8.02 ± 2.15a 10.01 ± 0.06a 9.02 ± 0.14a 9.01 ± 0.26ab
0.73 ± 2.01a 1.02 ± 3.30a 0.96 ± 0.08a 0.44 ± 0.17a 0.96 ± 0.66ab
Table 1B (Stage II cultivation) determination of pigments. All of the experiments were carried out in triplicate (n = 3) and the values are expressed as means ± standard deviations, analyzed with one-way analysis of variance (ANOVA) using InfoStat, 2012. (Different superscript letters within column indicates significant differences at p < 0.05.) Treatments 0.5% 3d-1% 6d-1% 9d-1% 3d-5% 6d-5% 9d-5%
Chlorophyll a (μg/mL) a
7.10 ± 0.45 6.15 ± 0.23ab 9.23 ± 0.52ab 14.21 ± 0.86a 6.25 ± 0.59a 6.45 ± 0.79a 6.29 ± 0.65a
Chlorophyll b (μg/mL) b
3.23 ± 1.23 2.15 ± 0.56ab 3.56 ± 0.78abc 7.43 ± 0.55a 3.21 ± 0.69b 2.56 ± 1.23a 1.59 ± 1.24b
Chlorophyll (a + b) (μg/mL) ab
Chlorophyll (a/b) (μg/mL) ab
10.33 ± 1.68 8.30 ± 0.79ab 12.79 ± 1.30abc 21.64 ± 1.41a 9.46 ± 1.28ab 9.01 ± 2.02a 7.88 ± 1.89ab
2.19 ± 0.36 2.86 ± 0.41ab 2.59 ± 0.66c 1.91 ± 1.56a 1.94 ± 0.85a 2.51 ± 0.64ac 3.95 ± 0.52ab
5
Carotenoids (μg/mL) c
10.01 ± 1.23 8.25 ± 0.59a 6.29 ± 0.75ab 8.59 ± 1.23ab 5.63 ± 0.89ab 5.42 ± 0.51ab 5.61 ± 0.42ac
Carot/Chlo(a + b) (μg/mL) 0.96 ± 0.73ab 0.99 ± 0.74ab 0.49 ± 0.57ab 0.39 ± 0.87a 0.59 ± 0.69ab 0.60 ± 0.25abc 0.71 ± 0.22ab
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Fig. 4. A (Stage I cultivation) Determination of Lipid, Carbohydrate and Protein content. All of the experiments were carried out in triplicate (n = 3) and the values are expressed as means ± standard deviations, analyzed with one-way analysis of variance (ANOVA) using InfoStat, 2012. The mean values were compared with LSD test and a significant difference was considered at p ≥ (0.05). B (Stage II cultivation) Determination of Lipid, Carbohydrate and Protein content. All of the experiments were carried out in triplicate (n = 3) and the values are expressed as means ± standard deviations, analyzed with one-way analysis of variance (ANOVA) using InfoStat, 2012. The mean values were compared with LSD test and a significant difference was considered at p ≥ (0.05).
3. Results and discussion
via effects of siderophore uptake by C. variabilis, Fig. 2a shows trend of nitrate consumption by C. variabilis during stage I cultivation experiment. Before the log phase, C. variabilis consumed around 21% of nitrate at 0.5% and 1%; while at 5%, it consumed around 30% nitrate. This data is accordance to [30], in which Chlorella zofingiensisp consumes around 30% of nitrate. Nitrate consumption in C. variabilis growing at two stage cultivation with 1% and 5% bacterial supernatant is shown in Fig. 2b and Fig. 2c, respectively. At 1% supernatant of the bacteria with C. variabilis having 9 days of stress consumed maximum amount of nitrate i.e. 55.8% compared to 5% under stage I cultivation. Cells with 3 and 6 days of stress at 1% consumed around 78.5 ppm of nitrate. At 5% cells with 3 and 9 days stress consumed around 74% and 69.3% nitrate, respectively. In stage two cultivation, having 9 days of stress C. variabilis consumed highest amount of nitrate. It has been observed that in both stage I and two stage cultivation, C. variabilis consumed more nutrients through nitrogen consumption. Though we could not detect any nitrate content in negative control but found 53% of nitrate was consumed in positive control in stage I cultivation.
3.1. Effect of extracted and purified siderophore on cell density, biomass productivity Fig. 1a shows the effect of different siderophore concentration on cell density (OD) at 750 nm of microalgae under nitrogen starvation. 30 μg of siderophore concentration showed maximum growth with increasing time. Prior to that with increase i.e. 40–50 μg cell growth started declining. Fig.1b clearly shows that biomass productivity of 30 μg added siderophore was similar to the control i.e. 19 ± 1.26 mg−1L−1D−1. The decrease of cell growth with increase concentration of siderophore may be because of the presence of a chelator and its slower uptake that leads to poor growth [29]. 3.2. Effects of supernatant on nitrate uptake by C. variabilis As siderophore contains N, nitrate concentration was determined 6
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Fig. 5. Determination of Neutral Lipid (NL), Glyco Lipid (GL) and Phospho Lipid (PL) in stage I and stage II cultivation. All of the experiments were carried out in triplicate (n = 3) and the values are expressed as means ± standard deviations, analyzed with one-way analysis of variance (ANOVA) using InfoStat, 2012. The mean values were compared with LSD test and a significant difference was considered at p ≥ (0.05). A – Positive control, B - Negative control, C- 0.5%, D- 1%, E- 5%, F- 3 days stress in 1%, G- 6 days stress in 1%, H- 9 days stress in 1%, I- 3 days stress in 5%, J- 6 days stress in 5%, K- 9 days stress in 5%. NL-neutral lipid, GL-glyco lipid, PL-phycolipid.
3.3. Effect of bacterial supernatant containing siderophore on dry cell weight and biomass productivity
all three supernatant (0.5%, 1% and 5%), optimum supernatant for higher biomass productivity of C. variabilis was observed in 1% which increased 15% of biomass productivity compared to positive control.
Cells grown at 1% (760.24 mg/L) in stage I cultivation showed highest DCW, compared to the cultures grown at 0.5% (530.62 mg/L) and 5% (404.72 mg/L) (Fig. 3a). DCW was observed increasing in two stage cultivation with the increase in supernatant from 0.5% to 1% and 5% (Fig. 3b). It is observed that in 1% stressed cultures, highest DCW was found in 6 days stressed culture (620 mg/L). Similar trend was also observed in 5% stressed cultures, with highest DCW (578 mg/L) in 6 days stress culture. At 0.5% and 1%, DCW of culture with 3 days of stress was 578 mg/L, and (580 mg/L) respectively. In stage I cultivation, culture grown at 1% showed 42.2% enhanced BP (21.66 mg/L/ day) whereas the culture grown at 0.5% (14.72 mg/L/day) and 5% started declining with decrease in the BP (11.22 mg/L/day). In two stage cultivation, maximum BP was found at 1% supernatant (17.2 mg/ L/day) and 5% supernatant (16.05 mg/L/day) under 6 days of stress which was 25–28% more as compared to that of 0.5% supernatant grown culture. According to [31] different concentration of siderophore in bacterial supernatant increased the biomass of green algae Neochloris oleoabundans and Scenedesmus sp. BA032. In the present study, among
3.4. Effect of bacterial supernatant containing siderophore on pigments Photosynthetic pigments content was measured in C. variabilis. (Table 1A) showing effect on pigments content at stage I cultivation. At 1% supernatant, chlorophyll-a (13.06 μg/mL), chlorophyll-b (4.11 μg/ mL) and carotenoids (9.02 μg/mL) content showed highest that was significantly higher (P < 0.05) than cultures grown at 0.5% and 5% (Table 1A). At 0.5% chlorophyll a/b and carotenoids/chlorophyll (a + b) ratio were (2.33). Culture grown in 1% supernatant had higher pigment content and chlorophyll a/b ratio compared to 5%. (Table 1B) shows effects on pigments content in two stage cultivation. When nitrogen is starved, photosynthetic rate decreases with simultaneous effect on the pigment composition, leading to the decrease in chlorophylls and increase of total carotenoid [32]. At 1% supernatant, cultures grown in 9 days of stress observed (21.64 μg/mL) content of chlorophyll (a + b) which was highest compared to ratio of carotenoids/chlorophyll (a + b) (0.39). 7
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3.5. Effect of bacterial supernatant containing siderophore on lipid, carbohydrate and protein
Declaration of author's contribution Dr. Sandhya Mishra and Soundarya Rajapitamahuni designed the experiment and written the manuscript. Experiments were performed by Khushbu bhayani, Pooja Bachani, and Vamsi Bharadwaj S.V. Manuscript was spell checked by Dr. Sandhya Mishra. “No conflicts, informed consent, human or animal rights applicable”.
Nitrogen has an important role on biomass and lipid productivity [16]. According to [33] nitrogen deficient has increased the cell lipid content to 10% of the dry weight of C. vulgaris. Combination of lipid and carbohydrate using two stage cultivation provides an alternative to biodiesel and bioethanol-production system. However, the enhancement of lipid is correlated with lower biomass and lipid productivity [34]. Two stage cultivation under nutrient sufficient followed by nitrogen starvation facilitates both higher growth rate and higher lipid content [17]. The effects on lipid, carbohydrates and crude protein content of C. variabilis. is shown in (Fig. 4a and b). To characterize the lipids, it was again fractionated into neutral lipid, glycolipid and phospholipid (Fig. 5). In stage I cultivation, at 1%, supernatant C.variabilis produced total lipid content of 30% containing 75% of neutral lipid. Increasing the supernatant from 0.5% to 1% didn't show any significant change in biochemical composition of C. variabilis. Although, total lipid content decreased to 15% at higher addition of supernatant 5%, which was significantly lower (P < 0.05) than lipid produced at 0.5% (35.22%) and 1% (30.35%) but this did not affect the neutral lipid content. During two stage cultivation, the neutral lipid content was increased (Fig. 4b). C. variabilis with 3, 6 and 9 days of stress at 1% showed 64%, 69% and 68%, neutral lipid resulted in 11%, 10% and 12% increase in neutral lipid content compared to control (0.5%) culture and at 5%. This is in well agreement with the report of [35] where lipid content decreased above 50 μg siderophore. In stage I, culture grown at 5% showed highest carbohydrate accumulation (44.49%) compared to 0.5% (39.56%) and 1% (41.23%) (Fig. 4a). During two stage cultivation, culture grown under 9 days of stress at 1% showed maximum carbohydrate accumulation (49.29%) followed by cells grown under 3 days of stress at 5% (46.52%). This shows production of carbohydrate was higher in cultures at 1% than 5% (Fig. 4b). During stress conditions, carbohydrate production presides compared to lipid production in microalgae [36]. At 5% supernatant, in both the stages lipid content decreased, but at 0.5% and 1%, carbohydrate content increased.
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Mishra, Co-cultivation of siderophore-producing bacteria Idiomarina loihiensis RS14 with Chlorella variabilis ATCC 12198, evaluation of micro-algal growth, lipid, and protein content under iron starvation, J. Appl. Phycol. (2018) 1. [11] K. Lee, M.L. Eisterhold, F. Rindi, S. Palanisami, P.K. Nam, Isolation and screening of microalgae from natural habitats in the Midwestern United States of America for biomass and biodiesel sources, J. Nat. Sci. Biol. Med. 5 (2) (2014) 333. [12] S. Amin, Review on biofuel oil and gas production processes from microalgae, Energy Convers. Manag. 50 (7) (2009) 1834–1840. [13] A. D'Onofrio, J.M. Crawford, E.J. Stewart, K. Witt, E. Gavrish, S. Epstein, K. Lewis, Siderophores from neighboring organisms promote the growth of uncultured bacteria, Chem. Biol. 17 (3) (2010) 254–264. [14] P.S. Nigam, A. Singh, Production of liquid biofuels from renewable resources, Prog. Energy Combust. Sci. 37 (1) (2011) 52–68. [15] C. Yeesang, B. Cheirsilp, Effect of nitrogen, salt, and iron content in the growth medium and light intensity on lipid production by microalgae isolated from freshwater sources in Thailand, Bioresour. Technol. 102 (3) (2011) 3034–3040. [16] S.H. Ho, C.Y. Chen, J.S. Chang, Effect of light intensity and nitrogen starvation on CO2 fixation and lipid/carbohydrate production of an indigenous microalga Scenedesmus obliquus CNW-N, Bioresour. Technol. 113 (2012) 244–252. [17] G. Mujtaba, W. Choi, C.G. Lee, K. Lee, Lipid production by Chlorella vulgaris after a shift from nutrient-rich to nitrogen starvation conditions, Bioresour. Technol. 123 (2012) 279–283. [18] S.A. Amin, D.H. Green, D. Waheeb, A. Gärdes, C.J. Carrano, Iron transport in the genus Marinobacter, Biometals 25 (1) (2012) 135–147. [19] J.M. Vraspir, P.D. Holt, A. Butler, Identification of new members within suites of amphiphilic marine siderophores, Biometals 24 (1) (2011) 85–92. [20] M.J. Fujita, K. Nakano, R. Sakai, Bisucaberin B, a linear hydroxamate class siderophore from the marine bacterium Tenacibaculum mesophilum, Molecules 18 (4) (2013) 3917–3926. [21] J.B. Neilands, K. Konopka, B. Schwyn, M. Coy, R.T. Francis, B.H. Paw, A. Bagg, Comparative biochemistry of microbial iron assimilation, Iron Transport in Microbes, Plants and Animals, VCH Publishers, New York, 1987, pp. 3–33. [22] I. Pancha, K. Chokshi, B. George, T. Ghosh, C. Paliwal, R. Maurya, S. Mishra, Nitrogen stress triggered biochemical and morphological changes in the microalgae Scenedesmus sp. CCNM 1077, Bioresour. Technol. 156 (2014) 146–154. [23] E.G. Bligh, W.J. Dyer, J.L. Luque-Garcıa, M.L. De Castro, A rapid method of total lipid extraction and purification, Can. J. Biochem. Physiol. 37 (8) (1959) 911–917Ultrasound-assisted soxhlet extraction: an expeditive approach for solid sample treatment: application to the extraction of total fat from oleaginous seeds, J. Chromatogr. A 1034 (1–2) (2004) 237–242. [24] J.L. Luque-Garcıa, M.L. De Castro, Ultrasound-assisted soxhlet extraction: an expeditive approach for solid sample treatment: application to the extraction of total fat from oleaginous seeds, J. Chromatogr. A 1034 (1–2) (2004) 237–242. [25] K. Chokshi, I. Pancha, A. Ghosh, S. Mishra, Nitrogen starvation-induced cellular
4. Conclusion The growth of microalgae for biofuel production is nitrogen intensive. C. variabilis under nitrogen starvation using iron chelating siderophores as a nitrogen source also in media for enhancement of micro-algal growth could significantly reduce external N requirements. The idea of this approach is that the nitrogen can be provided in terms of siderophore to the micro-algal growth and two stage cultivation strategies can be applied for enhancing the lipid, carbohydrate content. Indicating further fate of siderophores, these studies could be useful to evaluate the biofuel potential of the isolated C. variabilis strain that can further be applied for pilot scale production. Declaration of Competing Interest The authors declare no conflict of interests. Acknowledgments CSIR-CSMCRI Registration Number: PRIS–144/2018 has been assigned to the manuscript. RS acknowledge DST for her INSPIRE funding support. RS, acknowledges AcSIR for Ph.D. enrolment. KB and PB are grateful to CSIR-HRDG, New Delhi for the financial support in the form of CSIR-SRF fellowship. RS, PB and VB acknowledge AcSIR for PhD registration. 8
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crosstalk of ROS-scavenging antioxidants and phytohormone enhanced the biofuel potential of green microalga Acutodesmus dimorphus, Biotechnol. Biofuels 10 (1) (2017) 60 Dec. E.W. Becker, Microalgae: Biotechnology and Microbiology, vol. 10, Cambridge University Press, 1994. Yemm, Willis, The estimation of carbohydrates in plant extracts by anthrone, Biochem. J. 57 (3) (1954) 508. D.A. Cataldo, M. Maroon, L.E. Schrader, V.L. Youngs, Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid, Commun. Soil Sci. Plant Anal. 6 (1) (1975) 71–80. R. Popat, F. Harrison, A.C. da Silva, S.A.S. Easton, L. McNally, P. Williams, S.P. Diggle, Environmental modification via a quorum sensing molecule influences the social landscape of siderophore production, Proc. R. Soc. B Biol. Sci. 284 (1852) (2017) 20170200, , https://doi.org/10.1098/rspb.2017.0200. P. Feng, Z. Deng, L. Fan, Z. Hu, Lipid accumulation and growth characteristics of Chlorella zofingiensis under different nitrate and phosphate concentrations, J. Biosci. Bioeng. 114 (4) (2012) 405–410. J.A. Villa, E.E. Ray, B.M. Barney, Azotobacter vinelandii siderophore can provide
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nitrogen to support the culture of the green algae Neochloris oleoabundans and Scenedesmus sp. BA032, FEMS Microbiol. Lett. 351 (1) (2014) 70–77. L. Simon, F. Kiss, A. Bakó, F. Hajdu, T.Z. Hörcsik, Á. Balogh, I. Pais, Effect of gallium on photosynthetic pigments and peroxidase activity of Chlorella pyrenoidosa, J. Plant Nutr. 12 (10) (1989) 1123–1140. Y. Liang, N. Sarkany, Y. Cui, Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions, Biotechnol. Lett. 31 (7) (2009) 1043–1049. J. Klok, P.P. Lamers, D.E. Martens, R.B. Draaisma, R.H. Wijffels, Edible oils from microalgae: insights in TAG accumulation, Trends Biotechnol. 32 (10) (2014) 521–528. D.K. Singh, N. Mallick, Accumulation potential of lipids and analysis of fatty acid profile of few microalgal species for biodiesel feedstock, J. Microbiol. Biotechnol. Res. 4 (1) (2017) 37–44. S. Cordeiro, C.D. Vaz Raquel, M.S. Magalhães Izabela, Barbosa Sérgia, A.R. Francisco, Effects of nutritional conditions on lipid production by cyanobacteria, An. Acad. Bras. Cienc. 89 (3, Suppl) (2017) 2021–2031 Epub July 24, 2017 https://doi.org/10.1590/0001-3765201720150707.
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An effective approach of bacterial siderophore as nitrogen source triggering the desired biochemical changes in microalgae Chlorella variabilis ATCC 12198
T
Soundarya Rajapitamahunia,b, Khushbu Bhayania, Pooja Bachania,b, Vamsi Bharadwaj S.Va,b, ⁎ Sandhya Mishraa,b, a b
Division of Biotechnology and Phycology, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, Gujarat, India Academy of Scientific & Innovative Research (AcSIR), CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, Gujarat, India
ARTICLE INFO
ABSTRACT
Keywords: Siderophores Nitrogen starvation Two stage cultivation Biomass Lipid
The present study was aimed to check whether catecholate siderophore can serve as a nitrogen source besides iron for microalgae. Catecholate siderophore from Idiomarina loihiensis RS 14 contains 13% nitrogen which can be a potential nitrogen source. Bacterial supernatant containing siderophores with different inoculum ratio was used to check the growth of Chlorella variabilis ATCC 12198. Nitrogen starvation with two stage cultivation was employed here to trigger the accumulation of lipid and carbohydrate. In continuous cultivation, the highest productivity of DCW was observed in 1%, 760 mg/L compared to culture grown at 0.5%, with an increase of 42.2% higher biomass productivity (21.66 mg/L/day). Similarly, in 1%, C. variabilis produced total 30% of lipid content which contains 75% of neutral lipid. Highest carbohydrate was (49.29%) found in cells grown under 9 days of stress in 1% followed by cells grown under 3 days of stress in 5% (46.52%) in stage II cultivation.
1. Introduction Microalgae is considered having pronounced potential in biodiesel production due to its enormous growth rate and lipid content [1]. Nitrogen is an essential element for the growth of microalgae and its deficiency has a huge impact in its metabolic activities [2]. This elusiveness of nitrogen can be satisfied by certain metabolites, helping microalgae to enhance its nutrient uptake [3]. For its sustainability, nitrogen inputs will be the fundamental concern in investigating microalgal cultivation strategy for achieving high nutrition valve and its productivity [4] Chlorella variabilis ATCC 12198 is a robust strain of unicellular green eukaryotic microalgae growing in marine water which after nitrogen starvation is capable of accumulating higher amount of lipids with prominent fatty acid profile suitable for biodiesel production [5]. There is a need of microbes for easy solubilisation of certain elements, for which chelators like siderophores are produced even under traces of iron amount in the environment for transportation and storage via specific membrane receptors [6,7]. It chelates by virtue of three chelating groups, hydroxamates, catecholates and hydroxyacids which are generally oxygenated forming complexes with great thermodynamic stability [8]. Idiomarina loihiensis RS 14 is equipped with such
siderophore production system and uses part of this nitrogen fixed to produce catechol siderophores for the uptake of metal ions [9]. This specific siderophore, has a short peptide chain, containing significant percentage of nitrogen for the proliferation of microalgae, besides iron as a biostimulant [10]. Siderophore containing N molecule in the structure has been characterized which is of specific interest to determine its potential as a nitrogen source for microalgae. Though there are various media present for micro-algal cultivation but, media optimized can stimulate nutrient requirement for the growth of distinct algal species by changing its effect on biomass, chlorophyll, lipid, carbohydrate and protein [11]. There are many reports mentioning advantage taken by siderophore-producing bacteria for metal uptake by utilizing foreign siderophores [12,13]. To discover, if this phenomenon can satisfy requirement of fixed nitrogen, we first isolated siderophores from I. loihiensis to collect the siderophores using ion-exchange resin as described in the methods [10]. The productivity of the biomass is crucial for algal biodiesel and industrialization [14]. Though there are number of factors mentioned earlier that influences lipid and carbohydrate in microalgae, nitrogen is one of them [15,16]. Two-stage cultivation strategy could be used, by providing good source of nutrients to obtain maximum cell density in stage I
⁎ Corresponding author at: Division of Biotechnology and Phycology, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, Gujarat, India. E-mail address: [email protected] (S. Mishra).
https://doi.org/10.1016/j.algal.2019.101610 Received 7 May 2019; Accepted 8 July 2019 Available online 21 August 2019 2211-9264/ © 2019 Elsevier B.V. All rights reserved.
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followed by nutrient-stress conditions in stage II to induce lipid and carbohydrate accumulation [17] which has been applied in this work. Using bacterial supernatant containing siderophore in nitrate free micro-algal medium was proposed for enhancing micro-algal biomass. This study highlights the use of bacterial siderophores as a nitrogen source for indigenous marine water green algal strain, Chlorella variabilis ATCC 12198. In the present study by contrast, a two-stage cultivation mode was applied using siderophore as a source of nitrogen without any additional pre-treatment to improve the growth and lipid content.
different inoculum for nitrate limitation experiment. At 6, 9 and 12 days of inoculation, harvesting was done by centrifugation at 10,000 rpm for 20 min and again inoculated in medium free of nitrate. The study in 500 mL of culture medium in 1000 mL conical flask was done in triplicates (50 mL of 10 day old culture as inoculum). 2.2.2. Bacterial growth conditions Bacteria was grown in a 2000 mL flasks under low iron conditions, for which CDLIM media (Chemically Defined Low Iron Media) was used for successful release of siderophores at 37°C, 180 rpm for 72 h. The culture centrifuged at 8000g for 15 min at 4 °C for the supernatant containing siderophores. Supernatant was then filtered through 0.25 μm filter to get devoid of cells. Samples from this batch mode were then used as the inoculum for the nitrogen stress experiments in microalgal medium [10]. The volume of the inoculum sample was used (i.e., 0.5, 1, 5, and 10% of the bacterial supernatant in the 1 L flasks).
2. Materials and methods 2.1. Identification of siderophores supporting micro-algal growth Prior to designing an experiment, it was mandatory to understand which class of siderophores can be replaceable of nitrogen in micro-algal medium to support its growth. The commercially available different functional group of siderophores like hydroxamate, carboxylate and catecholates were collected from Sigma Aldrich. The elemental composition of hydroxamate siderophore i.e. Deferoxamine mesylate C26H52N6O11, and catecholate siderophore i.e. Enterobactin C30H27N3O15. On the basis of molecular formula of purified siderophore from Idiomarina loihiensis RS14 i.e. C16H29N10O8 which constitutes a considerable number of nitrogen equivalents and when released to the environment even in smaller molar concentrations can account for a significant amount of available nitrogen. Based on previous reports of other organisms stealing foreign siderophores for their own benefit [12,13], it was of interest to demonstrate whether catecholate siderophore from Idiomarina loihiensis RS 14 would also serve to satisfy nitrogen requirements to support micro-algal growth following its enhanced lipid content.
2.3. Extraction and purification of siderophore Acidified (pH 2.5) bacterial culture's supernatant was added to the resin (Amberlite XAD-2,) for absorbing the siderophore [18] and poured into column. 100% methanol was used to obtain final elution and further concentrated by rotary evaporation (Buchi-R215) to yield the crude siderophore [19] and further purified on Sephadex G-10 [20]. Siderophore present in the fractions were recognised by CAS assay and for further purification RP-HPLC was carried out [21]. 2.4. Effect of extracted and purified siderophore on micro-algal growth C. variabilis grown in ZM was transferred in HCl washed flask having fresh media supplemented with purified siderophore. Positive and negative control was prepared as discussed earlier. Purified siderophore ranging 0 μg, 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg and 50 μg was added to the nitrate deficient medium. All the culture flasks were kept in triplicates.
2.2. Experimental set-up The CSIR-CSMCRI's isolated strain of C. variabilis ATCC PTA 12198 was studied for its growth enhancement by siderophore as a substitution of nitrogen. The experimental media was prepared similar to control except the nitrogen component, which was replaced with a bacterial supernatant containing siderophore (as Nitrogen source). Hence, the nitrogen component was the only volatile in each test. Preliminary experiment was carried by the adhesion of extracted and purified siderophore into the algal medium as a replacement of nitrate, later the bacterial supernatant containing siderophores were used in the algal media. Two stage nitrogen cultivation method is applied where microalgae is initially grown in medium containing nitrogen (bacterial supernatant containing siderophores) followed by transferring it into a nitrogen free medium (no bacterial supernatant). To provide similar concentration of nitrogen present in the controlled culture, standard chelate was added at an appropriate concentration from a stock solution. In two-stage cultivation algae is initially cultured in nutrient-sufficient conditions (0.5%) taken as control to obtain a maximum dry biomass followed by nitrogen starvation to trigger the accumulation of lipids.
2.5. Effect of bacterial supernatant on micro-algal growth In stage I cultivation, cells were grown at three different inoculum of bacterial supernatant (containing siderophores as nitrogen source) viz. 0.5%, 1%, 5% and 10% and control was used with the regular nitrogen source i.e. NaNO3 till 18 days. Further, in two stage cultivation, cells initially grown as a control (NaNO3 as nitrogen source) were transferred to 1% and 5% of bacterial supernatant containing siderophore containing flasks. In stage I cultivation at 10% of bacterial supernatant, C. variabilis cells did not grow even after 7 days and therefore in both stage I and two stage cultivation, control, supernatant with 1% and 5% supernatant were used for further examination. Light intensity was (150 μmolm−2 s−1) and photoperiod was (12:12) hours of light: dark period. All the experiments were performed in triplicate in 1000 mL Erlenmeyer flasks containing 500 mL of Zarrouk's media inoculated with 50 mL of actively growing C. variabilis.
2.2.1. Micro-algal growth conditions Zarrouk's media was used for C. variabilis which consisted per litre of NaHCO3 16.8 g; K2HPO4 0.5 g; MgSO4.7H2O 0.2 g; NaCl 1.0 g; CaCl2. 2H2O 0.04 g; A5 1.0 mL; B6 1.0 mL; pH 10. EDTA and FeSO4.7H2O 0.01 g. NaNO3 constituent of media was omitted (nitrogen source). 50 mL of the young culture of C. variabilis was inoculated in the 1000 mL flasks (triplicates) containing 500 mL of medium. Culture was cultivated under 150 μmolm−2 s−1 of light intensity and 12:12 h of light: dark period at 37 °C and regularly shaken. The initial algal concentration was the same for all growth conditions with 4 × 118 cells/ mL or 0.2 optical density reading at 750 nm. Cells were grown with siderophores as nitrate source as positive control, without siderophores i.e. (without nitrate source) as negative control along with bacterial supernatant containing siderophores in
2.6. Estimation of biomass productivity (BP) The optical density at 750 nm was estimated for the growth of microalgae. According to following equation, BP (mg/L/day) was calculated. P = (X2 − X1) / (t2 − t1), where X2 and X1 are the dry cell weight concentration (mg/L) at time t2 and t1, respectively [22]. 2.7. Quantum yield of photosystem II (Fv/Fm) was measured with pulse-amplitude-modulated (PAM) fluorometry (AquaPen-C AP-C100, Photon System Instruments). Fv/Fm is calculated according to the following equation: [23] 2
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Fig. 1. A. Effect of siderophore on cell density of C.variabilis under nitrogen starvation All of the experiments were carried out in triplicate (n = 3) and the values are expressed as means ± standard deviations, analyzed with one-way analysis of variance (ANOVA) using InfoStat, 2012. The mean values were compared with LSD test and a significant difference was considered at p ≥ (0.05). B Effect of siderophore on Biomass productivity of C. variabilis under nitrogen starvation. All of the experiments were carried out in triplicate (n = 3) and the values are expressed as means ± standard deviations, analyzed with one-way analysis of variance (ANOVA) using InfoStat, 2012. The mean values were compared with LSD test and a significant difference was considered at p ≥ (0.05).
Fv /Fm = (Fm
suspended to the same. They were incubated under selected conditions for two stage cultivation, in (12:12) h light: dark period at 25 °C. Chlorella sp. lipid extracted by the method of [23] (a mixture of chloroform and methanol) and quantified gravimetrically with separation through column chromatography [24]. Biomass was vortexed thoroughly, ultrasonicated at room temperature in chloroform:methanol (1:2, v/v), centrifuged and measured gravimetrically. Lipids are mainly phospholipids (PL), glycolipids (GL) and neutral lipids (NL). All the above fractions are fractionated by sequential elution by chloroform:acetic acid (9:1, v/v) for neutral lipid, acetone:methanol (9:1, v/v) for glycolipids and 100% methanol for phospholipids after drying at 60°C [25]. Crude protein was estimated according to [26].
F0)/Fm
2.8. Estimation of pigments Pigment content was estimated in the pellet after centrifugation of 2.0 mL of culture at 10,000 rpm for 15 min +2.0 mL of 100% methanol, mixed well and incubated at 45 °C for 24 h in the dark. The total pigment content was calculated according to following equations:
Chlorophyll a; Chl
a (lµg/mL) = 16.72 (A665.2)–9.16 (A652.4)
Chlorophyll b; Chl
b (lµg/ml) = 34.09 (A652.4)–15.28 (A665.2)
2.10. Estimation of carbohydrate
Carotenoids (lµg/ml) = [1000 (A470)
1.63 (Chl
a)
104.9 (Chl
b)]/221 [22]
To determine carbohydrate, 100 mg of dried biomass with 10 mL of 2% H2SO4 was hydrolysed at 121 °C for 20 min, further CaCO3 used to neutralize and diluted to 100 mL distilled water. The total sugar content in supernatant was estimated after centrifugation at 10,000 rpm for 5 min [27].
2.9. Estimation of lipids and protein To optimize the maximum cell density, different inoculum of supernatant of varying siderophore amount were studied on micro-algal growth. Cultures grown with standard nitrogen source in the medium at (stage I) were stopped after reaching the late log phase. Cells were recovered through centrifugation at 10,000 rpm for 10 min. Cells collected were washed several times with nitrogen deficient ZM and again
2.11. Estimation of nitrate In the micro-algal culture medium total residual nitrate concentration was estimated using sodium hydroxide and salicylic acid. The algal 3
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Fig. 2. A (Stage I Cultivation) Effect of Siderophore containing supernatant on nitrate uptake by C. variabilis. All of the experiments were carried out in triplicate (n = 3) and the values are expressed as means ± standard deviations, analyzed with oneway analysis of variance (ANOVA) using InfoStat, 2012. The mean values were compared with LSD test and a significant difference was considered at p ≥ (0.05). B (Stage II Cultivation) Effect of Siderophore containing supernatant on nitrate uptake by C. variabilis in 1% inoculum All of the experiments were carried out in triplicate (n = 3) and the values are expressed as means ± standard deviations, analyzed with oneway analysis of variance (ANOVA) using InfoStat, 2012. The mean values were compared with LSD test and a significant difference was considered at p ≥ (0.05). C (Stage II Cultivation) Effect of Siderophore containing supernatant on nitrate uptake by C. variabilis in 5% inoculum All of the experiments were carried out in triplicate (n = 3) and the values are expressed as means ± standard deviations, analyzed with oneway analysis of variance (ANOVA) using InfoStat, 2012. The mean values were compared with LSD test and a significant difference was considered at p ≥ (0.05).
culture's filtrate was evaporated to dryness and 2 mL of PDA reagent was added and further diluted with water followed by the addition of 10 mL concentrated ammonium hydroxide, dark pink color formation occurred which was estimated at 410 nm [28].
2.12. Statistical analysis Experiments in triplicates presenting the data here are in mean values, analyzed with one-way analysis of variance (ANOVA) using InfoStat, 2012. The mean values were compared with LSD test and a significant difference was considered at p ≥ (0.005). 4
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Fig. 3. A (Stage I cultivation) Determination of Biomass productivity All of the experiments were carried out in triplicate (n = 3) and the values are expressed as means ± standard deviations, analyzed with one-way analysis of variance (ANOVA) using InfoStat, 2012. The mean values were compared with LSD test and a significant difference was considered at p ≥ (0.05). B (Stage II cultivation) Determination of Biomass productivity. All of the experiments were carried out in triplicate (n = 3) and the values are expressed as means ± standard deviations, analyzed with one-way analysis of variance (ANOVA) using InfoStat, 2012. The mean values were compared with LSD test and a significant difference was considered at p ≥ (0.05).
Table 1A (Stage I cultivation) determination of pigments. All of the experiments were carried out in triplicate (n = 3) and the values are expressed as means ± standard deviations, analyzed with one-way analysis of variance (ANOVA) using InfoStat, 2012. (Different superscript letters within column indicates significant differences at p < 0.05.) Treatments
Chlorophyll a (μg/mL)
Positive control Negative control 0.5% 1% 5%
12.01 ± 0.32a 5.23 ± 0.24a 7.12 ± 0.36a 13.06 ± 0.25a 6.15 ± 0.31a
Chlorophyll b (μg/ mL) 7.02 ± 0.29a 2.56 ± 0.41b 3 0.23 ± 0.36b 7 0.24 ± 0.54a 3.19 ± 0.08b
Chlorophyll (a + b) (μg/ mL) 19.03 ± 0.61a 7.79 ± 0.65ab 10.35 ± 0.72ab 20.30 ± 0.79a 9.34 ± 0.39c
Chlorophyll (a/b) (μg/ mL) 1.71 ± 1.10a 2.04 ± 0.58a 2.20 ± 1.00a 1.80 ± 0.46a 1.92 ± 3.87ab
Carotenoids (μg/mL)
Carot/Chlo(a + b) (μg/mL)
14.01 ± 1.23a 8.02 ± 2.15a 10.01 ± 0.06a 9.02 ± 0.14a 9.01 ± 0.26ab
0.73 ± 2.01a 1.02 ± 3.30a 0.96 ± 0.08a 0.44 ± 0.17a 0.96 ± 0.66ab
Table 1B (Stage II cultivation) determination of pigments. All of the experiments were carried out in triplicate (n = 3) and the values are expressed as means ± standard deviations, analyzed with one-way analysis of variance (ANOVA) using InfoStat, 2012. (Different superscript letters within column indicates significant differences at p < 0.05.) Treatments 0.5% 3d-1% 6d-1% 9d-1% 3d-5% 6d-5% 9d-5%
Chlorophyll a (μg/mL) a
7.10 ± 0.45 6.15 ± 0.23ab 9.23 ± 0.52ab 14.21 ± 0.86a 6.25 ± 0.59a 6.45 ± 0.79a 6.29 ± 0.65a
Chlorophyll b (μg/mL) b
3.23 ± 1.23 2.15 ± 0.56ab 3.56 ± 0.78abc 7.43 ± 0.55a 3.21 ± 0.69b 2.56 ± 1.23a 1.59 ± 1.24b
Chlorophyll (a + b) (μg/mL) ab
Chlorophyll (a/b) (μg/mL) ab
10.33 ± 1.68 8.30 ± 0.79ab 12.79 ± 1.30abc 21.64 ± 1.41a 9.46 ± 1.28ab 9.01 ± 2.02a 7.88 ± 1.89ab
2.19 ± 0.36 2.86 ± 0.41ab 2.59 ± 0.66c 1.91 ± 1.56a 1.94 ± 0.85a 2.51 ± 0.64ac 3.95 ± 0.52ab
5
Carotenoids (μg/mL) c
10.01 ± 1.23 8.25 ± 0.59a 6.29 ± 0.75ab 8.59 ± 1.23ab 5.63 ± 0.89ab 5.42 ± 0.51ab 5.61 ± 0.42ac
Carot/Chlo(a + b) (μg/mL) 0.96 ± 0.73ab 0.99 ± 0.74ab 0.49 ± 0.57ab 0.39 ± 0.87a 0.59 ± 0.69ab 0.60 ± 0.25abc 0.71 ± 0.22ab
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Fig. 4. A (Stage I cultivation) Determination of Lipid, Carbohydrate and Protein content. All of the experiments were carried out in triplicate (n = 3) and the values are expressed as means ± standard deviations, analyzed with one-way analysis of variance (ANOVA) using InfoStat, 2012. The mean values were compared with LSD test and a significant difference was considered at p ≥ (0.05). B (Stage II cultivation) Determination of Lipid, Carbohydrate and Protein content. All of the experiments were carried out in triplicate (n = 3) and the values are expressed as means ± standard deviations, analyzed with one-way analysis of variance (ANOVA) using InfoStat, 2012. The mean values were compared with LSD test and a significant difference was considered at p ≥ (0.05).
3. Results and discussion
via effects of siderophore uptake by C. variabilis, Fig. 2a shows trend of nitrate consumption by C. variabilis during stage I cultivation experiment. Before the log phase, C. variabilis consumed around 21% of nitrate at 0.5% and 1%; while at 5%, it consumed around 30% nitrate. This data is accordance to [30], in which Chlorella zofingiensisp consumes around 30% of nitrate. Nitrate consumption in C. variabilis growing at two stage cultivation with 1% and 5% bacterial supernatant is shown in Fig. 2b and Fig. 2c, respectively. At 1% supernatant of the bacteria with C. variabilis having 9 days of stress consumed maximum amount of nitrate i.e. 55.8% compared to 5% under stage I cultivation. Cells with 3 and 6 days of stress at 1% consumed around 78.5 ppm of nitrate. At 5% cells with 3 and 9 days stress consumed around 74% and 69.3% nitrate, respectively. In stage two cultivation, having 9 days of stress C. variabilis consumed highest amount of nitrate. It has been observed that in both stage I and two stage cultivation, C. variabilis consumed more nutrients through nitrogen consumption. Though we could not detect any nitrate content in negative control but found 53% of nitrate was consumed in positive control in stage I cultivation.
3.1. Effect of extracted and purified siderophore on cell density, biomass productivity Fig. 1a shows the effect of different siderophore concentration on cell density (OD) at 750 nm of microalgae under nitrogen starvation. 30 μg of siderophore concentration showed maximum growth with increasing time. Prior to that with increase i.e. 40–50 μg cell growth started declining. Fig.1b clearly shows that biomass productivity of 30 μg added siderophore was similar to the control i.e. 19 ± 1.26 mg−1L−1D−1. The decrease of cell growth with increase concentration of siderophore may be because of the presence of a chelator and its slower uptake that leads to poor growth [29]. 3.2. Effects of supernatant on nitrate uptake by C. variabilis As siderophore contains N, nitrate concentration was determined 6
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Fig. 5. Determination of Neutral Lipid (NL), Glyco Lipid (GL) and Phospho Lipid (PL) in stage I and stage II cultivation. All of the experiments were carried out in triplicate (n = 3) and the values are expressed as means ± standard deviations, analyzed with one-way analysis of variance (ANOVA) using InfoStat, 2012. The mean values were compared with LSD test and a significant difference was considered at p ≥ (0.05). A – Positive control, B - Negative control, C- 0.5%, D- 1%, E- 5%, F- 3 days stress in 1%, G- 6 days stress in 1%, H- 9 days stress in 1%, I- 3 days stress in 5%, J- 6 days stress in 5%, K- 9 days stress in 5%. NL-neutral lipid, GL-glyco lipid, PL-phycolipid.
3.3. Effect of bacterial supernatant containing siderophore on dry cell weight and biomass productivity
all three supernatant (0.5%, 1% and 5%), optimum supernatant for higher biomass productivity of C. variabilis was observed in 1% which increased 15% of biomass productivity compared to positive control.
Cells grown at 1% (760.24 mg/L) in stage I cultivation showed highest DCW, compared to the cultures grown at 0.5% (530.62 mg/L) and 5% (404.72 mg/L) (Fig. 3a). DCW was observed increasing in two stage cultivation with the increase in supernatant from 0.5% to 1% and 5% (Fig. 3b). It is observed that in 1% stressed cultures, highest DCW was found in 6 days stressed culture (620 mg/L). Similar trend was also observed in 5% stressed cultures, with highest DCW (578 mg/L) in 6 days stress culture. At 0.5% and 1%, DCW of culture with 3 days of stress was 578 mg/L, and (580 mg/L) respectively. In stage I cultivation, culture grown at 1% showed 42.2% enhanced BP (21.66 mg/L/ day) whereas the culture grown at 0.5% (14.72 mg/L/day) and 5% started declining with decrease in the BP (11.22 mg/L/day). In two stage cultivation, maximum BP was found at 1% supernatant (17.2 mg/ L/day) and 5% supernatant (16.05 mg/L/day) under 6 days of stress which was 25–28% more as compared to that of 0.5% supernatant grown culture. According to [31] different concentration of siderophore in bacterial supernatant increased the biomass of green algae Neochloris oleoabundans and Scenedesmus sp. BA032. In the present study, among
3.4. Effect of bacterial supernatant containing siderophore on pigments Photosynthetic pigments content was measured in C. variabilis. (Table 1A) showing effect on pigments content at stage I cultivation. At 1% supernatant, chlorophyll-a (13.06 μg/mL), chlorophyll-b (4.11 μg/ mL) and carotenoids (9.02 μg/mL) content showed highest that was significantly higher (P < 0.05) than cultures grown at 0.5% and 5% (Table 1A). At 0.5% chlorophyll a/b and carotenoids/chlorophyll (a + b) ratio were (2.33). Culture grown in 1% supernatant had higher pigment content and chlorophyll a/b ratio compared to 5%. (Table 1B) shows effects on pigments content in two stage cultivation. When nitrogen is starved, photosynthetic rate decreases with simultaneous effect on the pigment composition, leading to the decrease in chlorophylls and increase of total carotenoid [32]. At 1% supernatant, cultures grown in 9 days of stress observed (21.64 μg/mL) content of chlorophyll (a + b) which was highest compared to ratio of carotenoids/chlorophyll (a + b) (0.39). 7
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3.5. Effect of bacterial supernatant containing siderophore on lipid, carbohydrate and protein
Declaration of author's contribution Dr. Sandhya Mishra and Soundarya Rajapitamahuni designed the experiment and written the manuscript. Experiments were performed by Khushbu bhayani, Pooja Bachani, and Vamsi Bharadwaj S.V. Manuscript was spell checked by Dr. Sandhya Mishra. “No conflicts, informed consent, human or animal rights applicable”.
Nitrogen has an important role on biomass and lipid productivity [16]. According to [33] nitrogen deficient has increased the cell lipid content to 10% of the dry weight of C. vulgaris. Combination of lipid and carbohydrate using two stage cultivation provides an alternative to biodiesel and bioethanol-production system. However, the enhancement of lipid is correlated with lower biomass and lipid productivity [34]. Two stage cultivation under nutrient sufficient followed by nitrogen starvation facilitates both higher growth rate and higher lipid content [17]. The effects on lipid, carbohydrates and crude protein content of C. variabilis. is shown in (Fig. 4a and b). To characterize the lipids, it was again fractionated into neutral lipid, glycolipid and phospholipid (Fig. 5). In stage I cultivation, at 1%, supernatant C.variabilis produced total lipid content of 30% containing 75% of neutral lipid. Increasing the supernatant from 0.5% to 1% didn't show any significant change in biochemical composition of C. variabilis. Although, total lipid content decreased to 15% at higher addition of supernatant 5%, which was significantly lower (P < 0.05) than lipid produced at 0.5% (35.22%) and 1% (30.35%) but this did not affect the neutral lipid content. During two stage cultivation, the neutral lipid content was increased (Fig. 4b). C. variabilis with 3, 6 and 9 days of stress at 1% showed 64%, 69% and 68%, neutral lipid resulted in 11%, 10% and 12% increase in neutral lipid content compared to control (0.5%) culture and at 5%. This is in well agreement with the report of [35] where lipid content decreased above 50 μg siderophore. In stage I, culture grown at 5% showed highest carbohydrate accumulation (44.49%) compared to 0.5% (39.56%) and 1% (41.23%) (Fig. 4a). During two stage cultivation, culture grown under 9 days of stress at 1% showed maximum carbohydrate accumulation (49.29%) followed by cells grown under 3 days of stress at 5% (46.52%). This shows production of carbohydrate was higher in cultures at 1% than 5% (Fig. 4b). During stress conditions, carbohydrate production presides compared to lipid production in microalgae [36]. At 5% supernatant, in both the stages lipid content decreased, but at 0.5% and 1%, carbohydrate content increased.
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4. Conclusion The growth of microalgae for biofuel production is nitrogen intensive. C. variabilis under nitrogen starvation using iron chelating siderophores as a nitrogen source also in media for enhancement of micro-algal growth could significantly reduce external N requirements. The idea of this approach is that the nitrogen can be provided in terms of siderophore to the micro-algal growth and two stage cultivation strategies can be applied for enhancing the lipid, carbohydrate content. Indicating further fate of siderophores, these studies could be useful to evaluate the biofuel potential of the isolated C. variabilis strain that can further be applied for pilot scale production. Declaration of Competing Interest The authors declare no conflict of interests. Acknowledgments CSIR-CSMCRI Registration Number: PRIS–144/2018 has been assigned to the manuscript. RS acknowledge DST for her INSPIRE funding support. RS, acknowledges AcSIR for Ph.D. enrolment. KB and PB are grateful to CSIR-HRDG, New Delhi for the financial support in the form of CSIR-SRF fellowship. RS, PB and VB acknowledge AcSIR for PhD registration. 8
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