Studying the Effects of Different Phosphorous Concentrations on Biomass and β-carotene Production in Nitrogen Starved Dunaliella salina

 

Mohammad Hossein Morowvat1,2*, Younes Ghasemi1,2

1Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, P.O. Box 71345-1583, Shiraz, Iran.

2Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, P.O. Box 71468-64685, Shiraz, Iran.

*Corresponding Author E-mail: mhmorowvat@sums.ac.ir

 

ABSTRACT:

Dunaliella salina biotechnology is a fast-growing area relying mostly on biomass yield and β-carotene accumulation which might be reached up to 10% of the microalgal cell dry weights. Micro and macroelements concentration in the culture environment plays a great role in β-carotene and biomass productivity in D. salina. Here, we investigated the influences of different phosphorous, and nitrogen concentrations, on β-carotene and biomass production in D. salina. Besides, the concentration of other important macromolecules including proteins, lipids and also carbohydrates were measured in each cultivation mode in nutrient rich or nutrient starved media. Phosphorous and nitrogen limitation brought a significant reduction (48.49%) in biomass production after simultaneous phosphorous and nitrogen deprivation in D. salina culture during 28 days of experiment. Moreover, the β-carotene accumulation level was interestingly elevated (11.080 mg L-1 in nitrogen starvation, and 14.614 mg L-1 in double phosphorous and nitrogen depleted medium) in comparison with its preliminary levels (6.615 mg L-1) in the Johnson culture medium. The results confirmed the possibility of bioprocess engineering approach based upon nutrient rich media at the first cultivation stage and then nutrient depletion strategy at the second step to maximize the β-carotene accumulation amounts in D. salina with the minimum biomass reduction. The operational conditions should be optimized for scale up studies

 

KEYWORDS: Biomass, Dunaliella salina, Lipid, Nitrogen starvation, Phosphorous concentration.

 


INTRODUCTION:

Microalgae are considered as robust candidates for waste water treatment1, biofuel production2, steroid biotransformation3, and single cell protein production4. Dunaliella salina, is a unicellular photosynthetic green microalga without a rigid cell wall structure. It has been widely applied as a natural source for biomass, lipids, antioxidants and β-carotene production. Nowadays, carotenoids have attained various applications in different food, cosmetic and pharmaceutical industries as coloring agent, antioxidant, radical scavenger, and also feed and food supplement5,6.

 

It has been shown that lipid and β-carotene production enhances during nutrient starvation especially in late exponential growth phase7,8. The lipid and carotenoids accumulation phenomenon is occurred after phosphate and nitrogen starvation in microalgal culture medium. Based upon it, the medium composition plays a great role in microalgal growth and lipid production and subsequently impacts the total biomass and β-carotene productivity9. It has been indicated that nitrogen limitation has some significant effects on D. salina cell physiology, fatty acid metabolism and β-carotene production5,10. On the other hand, the synergistic effects of simultaneous nitrogen and phosphorous limitation on growth trend and β-carotene production in D. salina is remained unknown. This study focuses on the assessment of the effects of phosphorus concentration on enhancing the biomass and β-carotene production in a naturally isolated D. salina strain under nitrogen limitation to find the optimal culture composition for biomass, lipids and β-carotene production.

 

MATERIALS AND METHODS:

Strain, culture media and cultivation mode

D. salina was obtained from the Microalgal Culture Collection of Shiraz University of Medical Sciences (MCCS), Shiraz, Iran. Johnson medium11 was exploited for preservation and cultivation of D. salina. The microalgal strain was cultured for 28 days. In the first 10 days of experiment, the microalgal strain was cultivated in phosphorous and nitrogen rich Johnson medium to obtain the maximal growth capacity. In the second phase of the experiment, which was performed for 18 days of cultivation, D. salina cells were filtered through a filter membrane with pore size of 1 mm and washed twice using normal saline solution. In next step, 100 mL of freshly prepared Johnson culture medium with normal nitrogen (1 g L-1) and phosphorous (0.035 g L-1) concentrations and starved conditions including nitrogen starved (0 g L-1) medium with normal phosphorous concentration (0.035 g L-1), nitrogen starved (0 g L-1) medium with half (1/2) phosphorous concentration (0.0175 g L-1), and nitrogen starved (0 g L-1) medium with quarter (1/4) phosphorous concentration (0.00875 g L-1) were added to the medium. Each study was performed in triplicate using Erlenmeyer flasks (500 mL). the cultivation conditions were set at 130 rpm rate of agitation, 60 mol m-2 s-1 light intensity, at 25°C.

 

Microalgal Growth Monitoring:

To observe the microalgal growth pattern in each culture medium, dry cell weight method was employed every two days during 28 days of experiment as described before12with some modifications. Besides, the microalgal cell number also monitored through direct counting method using Neubauer haemocytometer under light microscope.

 

Analytical methods:

Total lipid content was extracted using chloroform and methanol solvent system (1:2) and quantified gravimetrically as described elsewhere11. The results of lipid determination assay were obtained and reported as % w/w from the total obtained biomass after 28 days of study. The carbohydrate content of the studied microalgal strain in each culture medium was measured using phenol-sulfuric acid method13. The total proteins content was assessed using Kochert method13 in triplicate. The total concentration of β-carotene was measured according to a previously adopted protocol11.

 

Statistical analysis

GraphPad prism version 6.00 provided by GraphPad Software, La Jolla California, USA and also IBM SPSS software version 22.0 provided by Armonk, NY: IBM Corp. were used to observe the significance of the results in this study. Statistical difference at 5% were regarded as significant for ANOVA analysis.

 

RESULTS AND DISCUSSION:

Impacts of N and P starvation on D. salina growth pattern

The composition of microalgal culture medium defines the chemical and nutritional conditions for growth and metabolism. Nitrogen, and sulfur starvation has proven14 to induce a vast range of cellular and biochemical response mechanisms such as biomass and lipid enhancement in Chlamydomonas reinhardtii. Besides, it has been shown that D. salina, aggregates β-carotene and lipids in the nutrient starved media15. On the other hand, optimizing a suitable ration of nitrogen and phosphorus concentration could be of importance to reach the best conditions for growth, biomass, lipid and β-carotene production. 

 

The studied D. salina strain was cultivated in two different stages, in which during the first stage a nutrient rich medium was added to the culture medium for 10 days and during the second stage which last for 18 days a nutrient deficient media was added to the culture media. The growth trend of D. salina strain was observed regularly and the sampling procedure was done every two days. The results of cell growing assessment is provided in Fig. 1. As it could be seen, in the first three days of study, D. salina cells exhibited the lag phase of growth pattern and after it the microalgal cells achieved the exponential growth phase from the third to tenth day of the cultivation experiment. After it, the microalgal cells reached to the stationary growth phase. The results of cell counting study indicated that the initial cell number of the studied D. salina strain was 1.384×106 cells mL-1. In the second stage of cultivation experiment, the cell number of the studied D. salina strain was increased to the maximum amount of 3.960×106 cell mL-1 in 19th day of experiment at the basic culture medium. On the other hand, the maximal number of D. salina cells was found to be 2.747 ×106 cells mL-1 in 23rd day of experiment at nitrogen starved medium. The nitrogen starved with half concentration of phosphorous experiment, the maximum cell number of 2.513 ×106 cells mL-1 in 23rd day of experiment was observed, whilst a final amount of 2.196 ×106 cells mL-1 was comprehend at the nitrogen starved with the quarter concentration of phosphorous levels at the 23rd day of experiment. Finally, in the double nitrogen and phosphorous starvation study, only 1.987 ×106 cells mL-1 was achieved in the 23rd day of experiment As it could be seen in the Fig. 1, at the second stage of cultivation, after the nutrient deficient regimen, D. salinagrowth trend exhibited a typical sigmoidal pattern in five different examined cultivation media. Nevertheless, the nutrient rich medium composed of appropriate concentrations of N and P exhibited higher slopes in comparison with the other four nutrient deprived media. During the second stage of microalgal cultivation, the cell growth pattern in the nutrient limited experiments slowly reduced probably because of nitrogen and phosphorous shortage in the cultivation medium. As indicated in Fig. 1, N and P double starvation brought more decrease (48.49%) in D. salina cell numbers in comparison with other starved media. The experienced reduction in the microalgal growth in nutrient limited media could be attributed to the inappropriate growth situations. The observed growth reduction phenomenon in the studied D. salina was in quite compliance with the provided data from other researches5,15.

 

Fig. 1: Growth trend of the studied D. salina in basic and starved culture media during 28 days of experiment

 

Impacts of N and P starvation on D. salina biomass composition:

Dried cell weight method was employed to observe the growth trend of D. salina in five distinct culture media. After the first stage of growth, at the end of the 10th day the microalgal cell dry weight revealed to be 0.098 g L-1. At the second stage of cultivation which lasts for 18 days, a maximum amount of 1.064 g L-1 was observed in nutrient rich Johnson medium (Fig. 2). Beginning the N and P limitation study, after addition of appropriate culture media with previously defined concentrations of N and P for each cultivation experiment, D. salina cells continued their typical growth with a normal sigmoid-like trend in all the five studied conditions. At the end of 28 days of cultivation experiment, the final biomass content was found to be 0.874 g L-1 in N starved medium, 0.693 g L-1 in N and P 1/2 starved medium, 0.638 g L-1 in N and P 1/4 starved medium and also 0.516 g L-1 in double N and P starved medium. The studied microalgal strain showed a higher biomass production and cell growth rate in comparison with the starvation conditions. On the other hand, it was found that biomass production under nitrogen deficient condition with sufficient phosphorus supply was more similar to that of the control (with sufficient nutrition).D. salina strain is known to be a promising microalgal candidate for biomass production as the single cell protein (SCP). Regarding the biomass production results of this study, reliability of using this microalgal strain for SCP production is warranted.

 

In the other hand, the ultimate lipid content of D. salina was found to be 0.234 g L-1 (21.99% in the total obtained biomass) in nutrient rich medium, 0.305 g L-1 (34.90%) in N starved medium, 0.258 g L-1 (37.23%) in N and P 1/2 starved medium, 0.242 g L-1 (37.93%) in N and P 1/4 starved medium, and finally 0.210 g L-1 (40.70%) in double N and P starved medium. On the other words, after 28 days of microalgal growth, the obtained lipid amount was increased in N and/or P starvation experiments and the double N and P starvation showed the highest increase in the lipid levels. Moreover, this data suggested the crucial importance of N and P concentrations for biomass, pigments and lipid production in D. salina. Essential elements limitation is considered to recruit the lipid aggregation in different microalgal strains16, 17. In this study, it was also revealed that the N and P starved regimen could elevate the lipid biosynthetic pathways in D. salina cells. Moreover, a combination of nitrogen starvation and moderate phosphorous limitation improved the lipid and β-carotene accumulation. The obtained data could be employed to increase the growth rates and also improve the β-carotene production process. The obtained lipids could be exploited for food or feed application and also for biodiesel production.

 

The total concentrations of protein and carbohydrates in the ultimate biomass was also quantified. It was discovered that the final concentration of the proteins and carbohydrates in the microalgal cells grown in nutrient rich medium to be 0.347 g g-1 (32.62%) and 0.362 g g-1 (34.02%), respectively (Fig. 2). Moreover, a cumulative amount of 0.121 g g-1 comprising the 11.37% of the final biomass (1.064 g L-1) was considered as nucleic acids, other impurities and probable errors. The results of the same experiments in starved culture media indicated that the final protein and carbohydrates concentration at the end of N starvation was 0.218 g g-1 (24.94%) and 0.269 g g-1 (30.78%), respectively. The quantity of remained ingredients and compounds was 0.082 g g-1 that is regarded to be 9.38% of the final attained biomass (0.874 g L-1). Furthermore, in N and P 1/2 starvation condition, the final amounts of proteins and carbohydrates were found to be 0.159 g g-1 (22.94%) and 0.216 g g-1 (31.17%), namely. Notably, 0.060 g g-1 (8.66%) of the remaining materials were considered as nucleic acids and impurities, whilst the ultimate amount of microalgal biomass was detected as 0.693 g L-1. Findings ofD. salina culture in N and P 1/4 starvedJohnson medium were reported to be 0.141 g g-1 (22.11%) proteins and 0.201 g g-1 (31.50%) carbohydrates. Additionally, a complete levels 0.054 g g-1 (8.46%) of the final microalgal biomass (0.638 g L-1) was regarded as the nucleic acids, impurities and probable mistakes. In double N and P starved medium, the final levels of proteins (0.099 g g-1, 19.19%) and carbohydrates (0.166 g g-1, 31.17%) were reported. Besides, 0.041 g g-1, (7.94%) was considered as the residuals, and the total biomass concentration was shown to be 0.516 g L-1. The maximum amounts of protein and carbohydrate production was occurring in nutrient rich medium. The observed changes in D. salina biomass ingredients revealed the variations of microalgal cell metabolism and physiology during the different experiment with various concentrations of N and P in the culture medium. Based on the biomass composition data, D. salina strain might be regarded as a robust platform for biomass, lipids and carotenoids production. Nutrient starvation approach could be employed to achieve higher carotenoids and lipid contents.

 

 

Fig. 2: Total biomass production and composition in basic and starved culture media

 

β-carotene content of D. salina:

The total β-carotene contents obtained from five different experiments including nitrogen and/or phosphorous concentration media was determined after 28 days of study. Fig. 3 depicts the mean values for β-carotene concentration with error bars obtained from the D. salina biomass after each experiment. As it could be seen, the total β-carotene concentration was increased from 6.615 mg L-1 in the basic Johnson medium as the nutrient rich medium to 11.080 mg L-1 in N starved medium, 12.082 mg L-1 in N and P 1/2 starvation, 12.415 mg L-1 in N and P 1/4 starvation, and 14.614 mg L-1 in N and P starved culture media, respectively. Based upon the provided data, it could be concluded that the nutrient limitation approach elevated the β-carotene accumulation phenomenon in the studied microalgal strain up to 167.50% (N starvation), 182.64% (N and P 1/2 starvation), 187.68% (N and P 1/4 starvation) and 220.92% (N and P starvation) in comparison with its primary levels in basic Johnson medium with physiologic concentrations of nitrogen and phosphorous. Hence, it could be suggested that N and P deprivation strategy could be exploited as a process engineering strategy to maximize the β-carotene biosynthesis pathway. Besides, it could be implied that the available levels of nitrogen and phosphorous elements in the culture medium for growing the microalgal cells are in inverse relation with β-carotene producing metabolic networks. This observation is in agreement with other studies reporting more β-carotene accumulation in presence of nitrogen, sulfur, iron and manganese starvation5,15. Although it should not be ignored that maximizing the β-carotene levels in D. salina strains due to essential nutrient strategy is a species-specific procedure with a general repetitive pattern18. The current experiment, indicates the effects of different phosphorous concentration in presence of nitrogen starvation on the growth pattern, biomass production and composition and also more importantly the amount of β-carotene in D. salina strain. The highest β-carotene accumulation level was detected as 14.614 mg mL-1 in double N and P deficient culture medium. Nitrogen and phosphorous deprivation situation is assumed to hamper the microalgal cell division and also reduce the rate of many biomass biosynthetic pathways, hence it could be quite rational that the cells division rate is reduced during N and P limitation.

 

Fig. 3: β-carotene concentration (mg L-1) obtained from D. salina after 28 days of cultivation in nutrient rich and different N and P starved media

 

CONCLUSION:

To sum up, the applicability of nutrient starvation strategy to increase β-carotene and lipid production for carotenoids and biodiesel production was confirmed. Nitrogen and phosphorous were indicated as crucial nutritional elements involving in β-carotene biosynthesis, D. salina growth and lipid overexpression. Due to its notable growth trend, amounts of β-carotene production contents and higher lipid contents; the studied D. salina strain might be explored as a robust producer microalga for industrial purposes. Besides, nutrient limitation approach as a bioprocess engineering-based approach should be evaluated for optimization and scale up studies

 

ACKNOWLEDGEMENT:

This work was supported by Research Deputy of Shiraz University of Medical Sciences, Shiraz, Iran (Grant no. 95-01-36-11911).

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 24.11.2017          Modified on 18.12.2017

Accepted on 24.12.2017        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(2):494-498.

DOI: 10.5958/0974-360X.2018.00090.2