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# Deciphering nutrient stress for production of biofuel precursors in Selenastrum capricornutum UTEX 1648

Niranjana Roy

A1 Flats, MIT Quarters, MIT Manipal, Udupi 576104, Karnataka

Dr. Pannaga Pavan Jutur

Omics of Algae Group, International Center for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110067, Delhi

## Abstract

Making algal biofuels commercially viable and sustainable means decreasing greenhouse gas (GHG) emissions to attain higher yields of biomass along with biofuel precursors and other high-value bioproducts. Selenastrum capricornutum UTEX 1648 is an oleaginous green microalga belonging to the class Chlorophyceae is considered as most promising renewable feedstocks to synthesize biofuel precursors. Understanding the molecular imprints is important for elucidating the physiological mechanisms of lipid biosynthesis in this microalga when subjected to nutrient stress. Algal Oils are rich in the triacylglycerols (TAGs) that serve as material for conversion to biofuels. Studies on the biosynthetic pathways and rate limiting steps of triacylglycerol formation in microalgae are still infancy. Our present research aims to study the overall TAG biosynthesis pathway i.e., either altering the lipid content with increase in overall lipid production or shift the balance of lipid production when subjected to nutrient stress. In this context, our focus was to study S. capricornutum under nutrient deprivation. We have initiated our experiments to find out their molecular responses on growth and lipid profiles under different nutrient limitation (nitrogen, phosphorous, and/or sulfur) conditions. Our preliminary data (as per 4-week work plan) shows that the microalgae was grown for 10 days at 24°C, under 60 µmol photons m-2s-1with continuous illumination, and with shaking at approximately 100 rpm. Under these conditions, the doubling time is 59.9 h; the specific growth rate, 0.27 ; the biomass concentration, 235.0 mg L-1; and the biomass productivity, 23.5 mg L-1. We have also analysed the dry cell weight of cells employing standard protocols and was found to be 13.21 µg per 106cells. Currently, we are growing cells to determine the effects of nutrient deprivation on the growth of S. capricornutum at 24°C, 60 µmol photons m-2s-1with continuous illumination as per these following conditions: BG-11 as control, BG-11 lacking nitrate [N-], BG-11 lacking phosphate [P-], BG-11 lacking sulphate [S-]. In conclusion, the best condition among all with better biomass and higher lipid productivity in S. capricornutum, may be a critical step toward making algae-derived biofuels economically competitive for industrial production.

Keywords: biofuels, gas chromatography, microalgae, Selenastrum capricornutum, triacylglycerols

## Background

Decreasing fossil fuels and the impact of its use on global warming has led to an increasing demand for its replacement by sustainable renewable biofuel. Microalgae are photosynthetic organisms that offer a potential feedstock for both the food and energy sectors. These are diverse group of organisms and are able to tolerate a wide range of abiotic stresses and can utilize even waste water/leachates with high biomass yield. Microalgae have a tendency to alter their physiology under certain stress and accumulate either lipid or carbohydrate. Microalgal fuel offer considerable advantages over other fuel sources as it is renewable, clean, sustainable and can also sequester greenhouse gases like CO2. However, this is often accompanied with retarded growth, which is a major obstacle towards commercialization. Apart from biofuels, they are also capable of producing high value precursors like omega-3-fatty acids (Eicosapentaenoic acid and Docosahexaenoic acid), carotenoids, phycobiliproteins etc. However, this is often accompanied with retarded growth, which is a major obstacle towards commercialization. Various microalgae are being used for the production of triacylglycerol (TAGs) under nutrient starvation condition such as Nannochloropsis and Parachlorella.

Selenastrum sp. is a crescent-shaped, freshwater green microalgae belonging to Chlorophyceae family. It is an oleaginous microalga which has the potential to be used as an ecotoxicological bioindicator and bioremediation. It can be used for the removal of aromatic hydrocarbons, phenolics, dissolved organic nitrogen [3, 5, 6, 7]. In this study, the rationale is to understand the effects of nutrient deprivation i.e. nitrogen (N), phosphorus (P) and sulfur (S) on the growth profiles and physiology of Selenastrum. This preliminary analysis will help us to gain insights into the lipid metabolic pathways for further understanding the deviation in the carbon flux upon different nutrient stress. Further, we might be able to generate a model which can be a potential feedstock for both bioremediation and biofuel production.

## Objective of the Research

To study the effect of major macronutrient deprivation i.e. Nitrogen, Phosphorus and Sulfur on growth and biomolecular composition (lipids, carbohydrate, protein) of Selenastrum capricornutum

## LITERATURE REVIEW

Usage of fossil fuels have serious consequences towards health and environment; extraction process can wreak havoc on ecosystems and communities, transportation can lead to serious accidents and spillage, burning of these fuels will release toxic substances and contribute to global warming. Rapid urbanization will increase the global energy demand by 53% at the end of 2035. Therefore, there is an increasing pressure for the development of renewable and sustainable fuels. The 2nd generation biodiesel from oil crops like corn, soyabean, jatropa are unfortunately not able to replace fossil fuels as they complete with agricultural land and do not produce economically sufficient biofuel.

Microalgae comparatively have a lot of advantages in being used to produce biofuels. Microalgae are highly diverse, photosynthetic autotrophs, that have rapid growth with high biomass and are found in marine/freshwater systems thus eliminating the need to complete with arable land [2]. According to the US department of energy, microalgae have the capability to produce 100 times more oil per acre land than any other terrestrial plant. [1] They are also capable of growing in wastewater and can be used for bioremediation. Since they are photosynthetic, they could also be used to sequester the greenhouse gas CO2 while simultaneously being used for the production of biofuel precursors. Some microalgae can also produce certain high value precursors like omega-3fatty acids (Eicosapentaenoic acid and Docosahexaenoic acid), carotenoids, phycobiliproteins etc. Microalgae can be used to produce different types of renewable fuels such as biodiesel, ethanol, hydrogen, methane etc.

Despite the advantages of using microalgae as a potential source for biofuels, they are not yet commercialized as there is a need for selection of the most economical strain, developing bioreactors for large scale production, optimizing culture, harvesting and downstream conditions. Culturing conditions such as nutrient content, salinity, pH, temperature, light exposure are some factors which affects the biochemical composition of a cell as well as its biomass accumulation. Microalgae have the ability to produce neutral TAG lipid bodies during stress conditions. These TAG depositions are then trans esterified and converted to biofuels. Nutrient deprivation is the most commonly used strategy as it has a significant effect on TAG content and fatty acid composition. However, a major drawback to nutrient deprivation is that the growth rate slows down which ultimately will make the process less economical [8]. High cellular lipid content and biomass concentration are the most important criteria towards selection of a strain for commercialization. Hence, different strains have to be tested under nutrient deprivation to find out the best strain for commercialization which can produce high TAG content without drastic decrease in growth rate [8].

Selenastrum capricornutum is a sickle shaped freshwater green microalgae belonging to the class Chlorophyceae. It is most commonly used for bioremediation of pollutants such as polycylic aromatic hydrocarbons, phenolics, heavy metals and certain pharmaceutical compounds. The rapid growth of industrialization, urbanization and agriculture has led to increased generation of toxic waste products which are dumped in the nearby water bodies. These pollutants can have disastrous effects on the ecosystem. Some pollutants can bioaccumulate and can directly be harmful to our health. Microalgae are capable of bioadsorption, bioaccumulation and/or biodegradation of pollutants. S. capricornutum has been effectively used for biodegradation of nonlyphenols, a type of phenolic compound used in surfactants. It has also been used for treatment of polycyclic aromatic compounds such as benzo(a)pyrene, phenanthrene, fluoranthene, [7] pyrene etc. Some pharmaceuticals which can be degraded by S. capricornutum include finasteride [5]; estradiol and 17-alpha ethynylestradiol with efficiency of 88-100% and 60-95% respectively [6]; cyclophosphamide and ranitidine, ciprofloxacin and sulfamethoxazole [3]. S. capricornutum has also been used as an ecotoxicological bio-indicator for detection of heavy metal such as Cu2+, Pb2+, and Cr3+ with 67 to 98% efficiency.

In this study, the rationale is to understand the effects of nutrient deprivation i.e. nitrogen (N), phosphorus (P) and sulfur (S) on the growth profiles and physiology of S. caprinornutum. This preliminary analysis will help us to gain insights into the lipid metabolic pathways for further understanding the deviation in the carbon flux upon different nutrient stress. Further, we might be able to generate a model which can be a potential feedstock for both bioremediation and biofuel production

## Culture Conditions

Selenastrum capricornutum UTEX 1648 was obtained from Culture Collection of Algae, University of Texas, Austin and maintained at International Center of Genetic Engineering and Biotechnology, New Delhi. Cultures were incubated under continuous illumination (~30 μmol m−2 s−1 photosynthetically active radiation [PAR]) on an orbital shaker (100rpm) at 25˚C. They were grown for 10 days in BG-11 medium. The composition of media components for BG-11 medium (g/L): K2HPO4—4; MgSO4.7H2O—7.5; CaCl2.2H2O—3.6; citric acid—0.6; FeC6H5O7NH4OH—0.6; EDTA—0.1; Na2CO3—2.0; NaNO3--150; Trace metals—1ml/L. These precultured cells were collected by centrifugation and resuspended in regular BG-11 medium, BG-11 medium lacking nitrogen, phosphorus and sulfur for control, N-, P-, S- conditions respectively. The cell density of the inoculum was 5 x 106 cells/ml. Samples were taken out immediately after resuspension (0 day) and at intervals of 2, 4, 6, 8, 10 days for biochemical analysis. All the experiments were done in biological replicates. Cell growth was monitored by counting cells with hemocytometer and absorbance reading at 750nm. The growth rate was calculated by the following equation.

${K}^{\text{'}}=\left(lnN2/N1\right)/\left(t2-t1\right)$

The doubling time was also calculated as per the following equation once the growth rate was known,

composition of BG-11 medium
 Components of BG-11 medium Amount (g/L) K2HPO4 4 MgSO4.7H2O 7.5 CaCl2.2H2O 3.6 citric acid 0.6 FeC6H5O7NH4OH 0.6 EDTA 0.1 Na2CO3 2 NaNO3 150 Trace metals 1 ml/L

## Dry Weight Analysis

A known biomass of cells was centrifuged at 5000 rpm for 10 mins and 2-3 washes were given first with milliQ water and then with 500 μl of 0.5 (NH4)HCO3. The cells were resuspended in 500 μl of milliQ and added to pre-weighted dry crucibles. The weight was noted down and the crucibles were kept in the hot air oven at 80˚C for 4 hours. These crucibles were then kept in a muffled furnace at 600˚C for 1hr for measuring the ash free dry weight.

## Lipid Analysis by Sulfo-Phospho-Vanillin (SPV) assay

The standard lipid stock used was canola oil (2 mg/ml) in chloroform. Dilutions of the stock were prepared and heated at 60˚C for 10 mins to evaporate the chloroform and 100 μl of water was added to the standard solutions. A known biomass of cultures was centrifuged at 8000 rpm for 10mins and resuspended in 100 μl of water. 2ml of concentrated H2SO4 was added to the samples and heated at 100˚C for 10 mins and cooled for 5 mins in an ice bath. 5ml of freshly prepared SPV reagent was added and incubated at 37˚C for 15mins. The absorbance was taken at 530nm.

## Chlorophyll and Carotenoid Estimation

A known biomass of cultures was centrifuged at 8000 rpm for 10 mins and resuspended in 1ml 100% methanol and heated at 60˚C for 1hr. The absorbance was taken at 470, 652, 665 nm for carotenoids, chlorophyll b and chlorophyll a respectively.

## Protein Estimation by Biuret Test

The standard protein stock used was BSA (2 mg/ml) in NaOH-MeOH. Dilutions of the stock were prepared and 50 μl of biuret reagent was added to 100 μl of dilutions and incubated for 15 mins at room temperature. A known biomass of culture was centrifuged at 8000 rpm for 10 mins and resuspended in 1 ml of 1N NaOH in 25% methanol and heated at 80˚C for 15 mins. The samples were then cooled and centrifuged at 8000 rpm for 10 mins. 100 μl of supernatant was taken and 50 μl of biuret reagent was added and incubated for 15 mins at room temperature. The absorbance was taken at 310 nm.

## Carbohydrate Estimation by Anthrone Test

The standard carbohydrate stock used was α-D-glucose (2 mg/ml) in milliQ water. Dilutions of the stock were prepared and 400 μl of chilled 75% H2SO4 and 800 μl of freshly prepared anthrone reagent was added to 200 μl of the dilutions. Anthrone reagent was prepared by initially dissolving 100mg of anthrone in 2ml absolute ethanol and then making up the volume to 50ml with 75% H2SO4. A known biomass of culture was centrifuged at 8000 rpm for 10 mins and resuspended in 200 μl of milliQ water. 400 μl of 40% (w/v) KOH was added to the samples and heated at 90˚C for 1 hr. After the samples had cooled down, 1.2 ml of cold absolute ethanol was added and stored at -20˚C overnight. The samples were centrifuged at 8000 rpm for 10 mins and resuspended in 1.5 ml milliQ. To 200 μl aliquots, 400 μl of chilled 75% H2SO4 and 800 μl of freshly prepared anthrone reagent was added. The samples were heated in a dry bath at 100˚C for 15 mins. The absorbance was taken at 578nm.

## BODIPY Staining to Visualize Lipid Bodies

The working concentration of BODIPY used was 1 μl/ml in 20% DMSO. A known biomass of cells was centrifuged at 8000 rpm for 10 mins and washed once with milliQ. The cells were resuspended in 1ml of BODIPY solution and incubated for 15 mins in dark at room temperature. The samples were centrifuged at 8000 rpm for 10 mins and washed once with milliQ. The cells were resuspended in 100 μl of milliQ and 10 μl was loaded onto slides for fluorescence microscopy. The excitation and emission wavelengths are 488 and 510 nm respectively.

## RESULTS AND DISCUSSION

Nitrogen, phosphorus, sulfur are all major macronutrients required for normal cell metabolism and growth. Thus under nutrient starvation, the growth rate will slow down and more inorganic carbon will be used for production of lipids. Biomass production and lipid biosynthesis both compete for inorganic carbon. Hence, lipid is known as the storage body. The growth of Selenastrum caprinornutum subjected to nutrient replete (control) and nutrient deprived conditions of nitrogen, phosphorus, sulfur in BG-11 medium is shown in figure 1. under all conditions, there was an initial lag phase followed by an exponential phase. The cell growth under deplete conditions was greatly reduced as compared to the control. Cell growth under nitrogen starvation was affected the most. The biomass productivity and dry cell weight of S.capricornutum under control conditions is 23.5 mg/L/day and 13.36 (μg/106cells). The specific growth rate and doubling time for all the conditions are given in table3.

Growth profiles of S.caprinornutum under nutrient replete and deplete [N-,P-,S-] conditions in BG-11 media
specific growth rate and doubling time of S.capricornutum under nutrient replete (control) and deplete [N-,P-,S-] conditions in BG-11 medium
 specific growth rate (day-1) doubling time (hours) control 0.19 63.9 nitrogen starvation 0.06 277.3 phosphorus starvation 0.11 151.4 sulfur starvation 0.12 143.4

## Pigment Analysis

The photosynthetic system is severely affected during stress conditions. There is drastic reduction in all the pigments during nitrogen starvation condition. Chlorophyll a content has reduced ~6 times from day 2 to day 10. Since chlorophyll is a protein and nitrogen is essential in protein synthesis, this reduction is justified. There is a slight reduction of pigments under phosphorus and sulfur starvation. Chlorophyll a content has reduced ~2.25 times from day 2 to day 10 under sulfur starvation.

Quantification of chlorophyll a, chlorophyll b, carotenoids in S.capricornutum subjected to nutrient replete (control) and deplete [N-,P-,S-] conditions in BG-11 medium

## Lipid Analysis

Under stress conditions, the ratio of reduced to oxidized metabolites cannot be maintained and there is a shortage of NADP+ and ADP. The biosynthesis of lipids utilizes NADPH and ATP to produce NADP+ and ADP and thus replenishes the pool. Hence, during stress conditions, the lipid content increases gradually. Among all the nutrient starvation conditions, nitrogen deplete showed the highest accumulation of lipid bodies in the cells. Under this condition, inorganic carbon directed towards production of lipids. However, under phosphorus and sulfur deprivation, the lipid content remained constant.

Quantification of total lipid content using SPV assay in S.capricornutum subjected to nutrient replete (control) and deplete [N-,P-,S-] conditions in BG-11 medium

## Protein Analysis

Nitrogen plays a fundamental role in protein synthesis. Hence under nitrogen depletion, the protein content decreases significantly. The protein content for control condition remains constant over the period of 10 days. Also under stress conditions, the cells will only produce essential proteins, those needed for survival which accounts for the decrease in protein content for all the conditions. Similarly, there is a decline in protein content under phosphorus and sulfur deprivation.

Quantification of total protein content using biuret test in S.capricornutum subjected to nutrient replete (control) and deplete [N-,P-,S-] conditions in BG-11 medium

## Carbohydrate Analysis

As mentioned above, under nitrogen deplete conditions, the photosynthetic fixed carbon is directed towards synthesis of carbohydrates and lipids. Hence, we can observe the increase in carbohydrate concentration along 10 days under nitrogen starvation. The carbohydrate content under phosphorus and sulfur starvation did not increase much.

Quantification of total carbohydrate content using anthrone test in S.capricornutum subjected to nutrient replete (control) and deplete [N-,P-,S-] conditions in BG-11 medium

## BODIPY Staining to Visualize the Lipid Droplets

Lipid bodies are dynamic organelles which store neutral lipids. Neutral lipids consist of TAGs (triacylglycerols) which can be transesterified and processed to biofuels. BODIPY (4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene) is a fluorescent neutral lipid dye which bind to the neutral lipid bodies and show fluorescence. BODIPY staining is preferred over nile red staining because BODIPY dye is insensitive to environmental polarity. solvents like DMSO, ethylene glycol etc can be used to improve the staining. At excitation wavelength of 488nm, the lipid bodies can be visualized as green droplets. The dye also stains the chloroplast red. Under nitrogen deprivation condition, lots of lipid bodies can be visualized compared to the control. Cells grown in phosphorus and sulfur deprivation conditions show lesser lipid bodies than nitrogen but more than the control. This is verifiable by the SPV assay data.

microscopy images of S.caprinorutum on day 10 grown in BG-11 medium subject to nutrient replete(A-B), nitrogen starvation (C-D), phosphorus starvation (E-F), sulfur starvation (G-H) conditions. The lipid bodies and chloroplast can be observed in green and red respectively.

## CONCLUSION

In the present study, Selenastrum caprinornutum was grown in minimal BG-11 medium for 10 days under continuous illumination under nitrogen, phosphorus and sulfur starvation. The control condition showed normal growth rate consistent with literature. Under nitrogen starvation the growth rate was highly retarded, reducing by ~31.5% from that on control. However, the lipid content of the cells increased tremendously. Under stress conditions, the cells start directing most of the inorganic carbon towards lipid biosynthesis rather than protein synthesis. Hence, under nitrogen starvation, the protein content reduces. The growth rate under phosphorus and sulfur starvation also reduced but not as much as under nitrogen starvation. The cells under these conditions did not have increased production of lipids. This preliminary data will allow us to further understand the lipid metabolic pathway under nitrogen deprivation though omics approaches.

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## ACKNOWLEDGEMENTS

I would like to thank my supervisor, Dr. Pavan P. Jutur and the Omics of Algae group, ICGEB. I would also like to thank Indian National Science Academy (INSA) for the fellowship.

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