Growth performance and biochemical composition of Tetradesmus obliquus (Turpin) M.J. Wynne in media with different nitrogen concentrations

Desempenho do crescimento e composição bioquímica de Tetradesmus obliquus (Turpin) M.J. Wynne em meio com diferentes concentrações de nitrogênio

ABSTRACT:

Microalgae have a great potential to change their metabolism pathway under stress condition, and nitrogen deficiency is an effective technique for maximizing lipids synthesis. This study focus on the growth performance, and biochemical composition of Tetradesmus obliquus (Turpin) M.J. Wynne cultured in different nitrogen concentrations (BG 11-Blue-Green Medium, 50% N+, 50% N-, 75% N+, 75% N-). Specific growth rate and protein content of T. obliquus decreased with nitrogen deficiency, but its lipid content was increased. Maximum specific growth rate (0.20-0.21) and protein content (47.82%, 47.62% DW-dried weight) values were obtained in 75% N+, 50% N+ medium. The highest lipid percentage was obtained in 50% N- medium. The biochemical composition of T. obliquus changes based on nitrogen concentration. Polyunsaturated fatty acids (PUFAs) are more abundant (49.69%) in this microalga under deficient nitrogen. To obtain more lipid and PUFAs in T. obliquus, N deficiency is an effective method, but it is not applicable to protein content. T. obliquus can be used as an alternative nutritional supplement with its high PUFAs content. Since different results are obtained from species to others in such studies, culture conditions should be determined for each species and according to the desired item.

Key words:
biochemical composition; Tetradesmus obliquus; microalgae culture; nitrogen

RESUMO:

As microalgas têm um grande potencial para alterar sua via metabólica sob condição de estresse, e a deficiência de nitrogênio é uma técnica eficaz para maximizar a síntese de lipídios. Este estudo enfoca o desempenho do crescimento e a composição bioquímica de Tetradesmus obliquus (Turpin) M.J. Wynne cultivado em diferentes concentrações de nitrogênio (BG 11-Blue-Green Medium, 50% N+, 50% N-, 75% N+, 75% N- ). A taxa de crescimento específico e o teor de proteína de T. obliquus diminuíram com a deficiência de nitrogênio, mas seu teor de lipídeos aumentou. Os valores máximos de taxa de crescimento específico (0,20-0,21) e teor de proteína (47,82%, 47,62% DW-peso seco) foram obtidos em meio 75% N+, 50% N+. O maior percentual lipídico foi obtido em meio 50% N-. A composição bioquímica de T. obliquus muda com base na concentração de nitrogênio. Os ácidos graxos poliinsaturados (PUFAs) são mais abundantes (49,69%) nesta microalga sob nitrogênio deficiente. Para obter mais lipídios e PUFAs em T. obliquus, a deficiência de N é um método eficaz, mas não é aplicável ao teor de proteína. T. obliquus pode ser usado como um suplemento nutricional alternativo com seu alto teor de PUFAs. Como são obtidos resultados diferentes de uma espécie para outra nesses estudos, as condições de cultivo devem ser determinadas para cada espécie e de acordo com o item desejado.

Palavras-chave:
composição bioquímica; Tetradesmus obliquus; cultivo de microalgas; nitrogênio

INTRODUCTION

With the increasing world population, the amount of energy needed by humanity is increasing and scientists are working on alternative energy sources and food. Microalgae, which are single-celled aquatic microorganisms, can be grown in fresh water, salt water and wastewater as they are tolerant to environmental stresses. This has caught the attention of scientists.

Different growing conditions, affects the microalgae growth rates, biomass yields, production of lipids and fatty acids in different ways to their nutritional values (ZARRINMEHR et al., 2019; ZIENKIEWICZ et al., 2020).

They can change their biochemical composition under different stress conditions and easily synthesize the target molecules (YAAKOB et al., 2021). Previous studies showed that changes in concentrations of nutrients, especially nitrogen (N) and phosphorus (P) create remarkable changes in microalgae growth, lipid and protein content, amino acids and fatty acid methyl esters (FAMEs) composition (ZHUANG et al., 2018).

Culturing the microalgae in N deficiency medium is one of the most efficient factors for increasing the lipid concentration of microalgae and many studies have been done for this purpose (NAYAK et al., 2019). N deficiency increases the amount of lipid, changes biomass productivity and growth performance may not result in parallel. If the lipid yield is decreased by reducing the amount of nitrogen, the number of cells may not increase sufficiently and this may cause the lipid yield to decrease (SHARMA et al., 2012). Recent studies are needed to clarify this issue and to understand the metabolic mechanism by working with different species under different conditions. Also, it should be determined which medium is more effective for increasing C16/C18 fatty acids that can improve the combustion performance of biodiesel and saturated fatty acids which are important for oil and biodiesel production (AN et al., 2020).

We had an accession number for Tetradesmus obliquus from NCBI and we cultured this microalga easily before like a preliminary study; so, in this research it was chosen as material. Beside this, there are lots of novel study with Tetradesmus that aim the same question of this research and had attractive results (KIM et al., 2019).

The fact that microalgae change their biochemical composition under varying environmental conditions, the number of studies that aim to obtain more lipid and protein and change the amino acid and fatty acid composition via exposing to nutrient stress is increasing. Based on this idea, in the present research, T. obliquus was cultured in media with different N concentrations, and the effects of N amount on growth parameters of the culture and biochemical composition of T. obliquus were investigated. With this study, a species that has not been studied in this field before will be brought to the literature.

MATERIALS AND METHODS

Isolation and culturing of microalgae

T. obliquus was isolated from Ergene River Basin (Turkey) and characterized by molecular approach phylogenetically as Acutodesmus obliquus (NCBI Accession Number: KF470790) in 2014. But in 2015, Wynne reclassified this microalga as T. obliquus (Turpin) M.J. Wynne, so we used the current name in this study (WYNNE & HALLAN, 2015). It was obtained from the Algal Biotechnology Laboratory, Mehmet Akif Ersoy University (Burdur, Turkey). Light and scanning electron micrographs of T. obliquus are shown in figure 1.

Figure 1
Scanning electron microscope of T. obliquus (B).

For cultivation, the first step was to culture the microalgae in BG11 (Blue-green medium) (RIPPKA et al., 1979), until it reaches its exponential phase under 16/8h light/dark photoperiod (100 µmolphoton m⁻²s⁻¹), at 24±3 °C and pH 7.5.

The second step entails harvesting and transferring microalgae into media with different N concentrations (BG 11 (Control), 50% N+, 50% N-, 75% N+, 75% N-), the BG 11 that is control medium has 1.5 g/L (NaNO₃) N concentration (Table 1) [N+: contains more nitrogen than BG11; N-: contains less nitrogen than BG11].

Table 1
Recipe of BG 11 medium (RIPPKA et al., 1979).

Compound	Amount (mg L-1H2O)
Na₂CO₃	20
Na₂ EDTA	1
NaNO₃	1500
K₂HPO₄	40
MgSO4.7H₂O	75
CaCl.2H₂O	36
Citric acid	6
Ferric ammonium citrate	6
Trace metal mixa	1 mL L-1

• ^a
  H₃BO₃, 2.86 g/L; MnCl₂.4H₂O, 1.81 g/L; ZnSO₄.7H₂O, 0.222 g/L; Na₂MoO₄.2H₂O, 0.39 g/L; CuSO₄·5H₂O, 0.079 g/L; Co(NO₃)₂·6H₂O, 0.049 g/L.

For this purpose, the cells were inoculated into cultures in 2000 mL flasks under the conditions mentioned above. The cultured flasks were bubbled with air gas (1 L/minute). For adjusting the initial cell number value as 3.6x10⁵ cell/mL; optical density (OD, 680 nm) of all cultures was measured about 0.09.

Growth performance analysis

Growth performance was analyzed by the number of whole cells in colonies (CN, cell/mL) via a thoma hemocytometer; the OD value was measured with spectrophotometer (Shimadzu UV-1650) at 680 nm on the same day when the culture was sampled (WANG et al., 2009).

Dried weight (DW) values were measured by harvested of culture (30 mL) through glass fiber filter (Whatman GF/C, 1.2 μm, UK), which were left to dry in oven at 105 °C for two hours (BOUSSIBA et al., 1992). Harvesting was made by centrifuging the cultures while in their stationary phase flowed by drying.

For calculating the specific growth rate of each replicate, cell number and DW values during exponential phase (exp.) were used (GUILLARD, 1973).

Specific growth rate (µ)= ln (N_t/N₀)/T_t-T₀

N_t: CN at the end of exp. phase; N_0: CN at the start of exp. phase; Tt: the last day of exp. phase; T₀: initial day of exp. phase.

Doubling time: Tt = 0.6931/µ

Biomass productivity (BP): as the dry biomass produced per day (g L^-1 day^-1).

Volumetric BP P _Biomass was calculated by P_Biomass(gL^-1day^-1)=(X₂-X₁).(t₂-t₁)^-1. X ₁: DW (gL⁻¹) of t₁ (starting point of cultivation); X₂: DW (gL⁻¹) of t₂ (endpoint of cultivation).

Detection of protein and lipid content, amino acids and fatty acids profiles

The total N content of microalgae was measured according to Dumas method (SHEA & WATTS, 1939). For crude protein amount, this equation was used: “protein amount=nitrogen content x 4.44” (LOPEZ et al., 2010). The calculations were given as percentage of dried weight.

Amino acid compositions were specified by using HPLC method (KÖSE et al., 2011). For detecting crude lipid content Bligh and Dyer’s method was used with 100 mg dried biomass (BLIGH & DYER, 1959). Fatty acid methyl esters (FAMEs) were analyzed chromatographically using the sample preparing method (METCALF et al., 1966).

Statistical analysis

All experiment groups replicated 3 times (n = 3). The reported values are the mean ± standard deviation (SD) of three values. Data were analyzed using one-way analysis of variance (ANOVA) using Minitab Statistical Software 2016 2 (Microsoft, USA). A difference was considered significant at the level of P < 0.001.

RESULTS AND DISCUSSION

In this research, cell number (CN), optical density (OD) and dried weight (DW) values were used for detecting T. obliquus growth. Figure 2 shows the CN; figure 3 shows OD values and figure 4 represents DW of T. obliquus in different culture media.

Figure 2
Cell numbers (CN) of T. obliquus in media with different N concentrations. Error bars show the SD of three replicates for each N concentration (n = 3).

Figure 3
Optical density (OD) values of T. obliquus in media with different N concentrations. Error bars show the SD of three replicates for each N concentration (n = 3).

Figure 4
Dry weight (DW) values of T. obliquus in media with different N concentrations. Error bars show the SD of three replicates for each N concentration (n = 3).

Maximum cell number value (2433x10⁴ cell/mL) was obtained in the control medium at the 40th day and OD value (1.0 nm) and DW value (1.32 g/L) were obtained at the same day. The highest CN values were 50% N+, 75% N-, 75% N+, 50% N-, respectively. Also, it was observed that other data indicating growth performance such as OD, DW and specific growth rate values are in parallel with CN values.

R² correlation values between optical density-cell number and optical density-dry cell weight is given in table 2. Specific growth rate, doubling time and biomass productivity in media with different N concentrations is given in table 3.

Table 2
R² correlation values between optical density-cell number and optical density-dry cell weight.

Media	OD-CN	OD-DW
BG 11	0.949	0.9715
50% N+	0.895	0.9461
50% N-	0.899	0.9435
75% N+	0.885	0.8804
75% N-	0.955	0.9498

Table 3
Specific growth rate, doubling time and biomass productivity of T. obliquus in media with different N concentrations.

Media	Specific growth Rate (μ)	Doubling Time (Tt)	Biomass productivity gL-1day-1
BG 11 (Control)	0.2135 ± 0.001	3.2455 ± 0.009	0.00089 ± 0.00001
50% N+	0.2115 ± 0.009	3.2771 ± 0.010	0.00051 ± 0.00001
50% N-	0.1897 ± 0.007	3.6541 ± 0.006	0.00046 ± 0.00001
75% N+	0.2016 ± 0.008	3.4372 ± 0.010	0.00047 ± 0.00001
75% N-	0.2014 ± 0.001	3.4413 ± 0.014	0.00065 ± 0.00001

• Data were expressed as mean ± SD, n=3.

In this study, total protein and lipid content were showed in figure 5. As for protein amounts, from much to less, it is as follows: 50% N+ (47.82%); 75% N+ (47.62%), 75% N- (45.69%), 50% N- (44.31%); Control (42.03%).

Figure 5
Total protein and lipid contents of T. obliquus biomass harvested from media with different N concentrations. (P < 0.05).

Results of amino acid compositions and amounts (Table 4) show that essential amino acids (EAAs) -expect arginine- are higher in the control medium, generally. Arginine (47.95 mg/g DW) is the most important amino acid found in 75% N- medium. Other EAAs such as leucine (16.2 mg/g DW) and isoleucine (2.80 mg/g DW) in 75% N+ medium; histidine (3.32 mg/g DW) and lycne (5.22 mg/g DW) in 50% N+ medium; phenyl alanine (8.41 mg/g DW) in Control medium have the highest amount. Besides these, valine (19.23 mg/g DW) has the max. amount in 75% N+ medium. Arginine, leucine and valine are the dominant amino acids in T. obliquus.

Table 4
Amino acids profiles and amounts (mg/g DW) of T. obliquus biomass harvested from media with different N concentrations.

Amino acids	BG11	%50 N+	%50 N-	%75 N+	%75 N-
Arginine	15.09	15.11	14.23	11.11	47.95
Serine	20.48	0.242	0.263	0.339	0.246
Glycine	10.57	0.381	0.448	0.425	0.17
Alanine	10.82	0.497	0.808	0.52	0.402
Proline	24.35	0.673	1.851	1.253	0.148
Valine	8.982	1.443	5.567	19.23	2.411
Threoline	2.209	2.081	3.52	3.432	1.264
Methionine	4.214	1.491	2.286	3.251	0.359
Isoleucine	16.46	1.261	1.337	2.802	0.316
Leucine	26.11	7.078	11.23	16.2	0.411
Phenylalanine	8.415	1.728	5.421	3.095	0.121
Tyrosine	3.301	0.148	0.518	1.086	0.115
Aspartic acid	0.361	1.4	0.352	0.514	0.862
Glutamic acid	3.799	3.087	0.155	0.148	0.97
Histidine	10.09	3.328	0.124	0.252	0.785
Lysine	10.05	5.227	0.129	0.177	1.364
Total	175.301	45.175	48.239	63.834	57.894

Fatty acid results are shown in table 5; they are grouped as Saturated fatty acids (SFAs), Monounsaturated fatty acids (MUFAs), Polyunsaturated fatty acids (PUFAs). The highest level of total SFAs (23.33%) and palmitic acid (C16:0) (19.95%) values are in 75% N+ medium. The maximum total MUFAs (20.0%) and oleic acid (C18:1) (13.41%) amounts are in 75% N-. The highest percentage of PUFAs (49.69%) and linolenic acid (C18:3) (37.67%) are observed in 50% N- medium.

Table 5
FAMEs percentages (% of total fatty acids) of T. obliquus biomass harvested from media with different N concentrations.

Name of Fatty Acid	BG 11	50% N+	50% N-	75% N+	75% N-
----------------------------------------------------------------Saturated fatty acids (SFAs)-------------------------------------------------------------------
C14:0 (myristic acid)	2.208	2.118	2.097	2.277	2.185
C16:0 (palmitic acid)	19.342	18.555	18.371	19.953	19.141
C18:0 (stearic acid)	1.073	1.029	1.019	1.107	1.062
Total SFAs	22.623	21.702	21.487	23.337	22.388
-----------------------------------------------------------Monounsaturated fatty acids (MUFAs)-----------------------------------------------------------
C16:1 (9-hexenoic acid)	2.156	2.068	2.048	2.224	2.134
C18:1 (oleic acid)	12.513	13.004	11.885	12.908	13.415
C18:1 t10, t11e 12	4.498	4.315	5.111	4.64	4.451
Total MUFAs	19.167	19.387	19.044	19.772	20.00
------------------------------------------------------------Polyunsaturated fatty acids (PUFAs)-------------------------------------------------------------
C18:2 (linoleic acid, c9 c12)	12.348	11.845	12.015	11.234	12.219
C18:3 (linolenic acid, c9, c12, c15)	36.291	36.678	37.678	36.121	37.061
Total PUFAs	48.639	48.523	49.693	47.355	49.28
Others	7.789	7.473	7.40	8.033	7.707
Total FAMEs Amounts	98.218	97.085	97.624	98.497	99.375

All these results showed that high nitrogen concentration could accelerate the algal growth because nitrogen is a macro element for cell metabolism. Besides this, N is the most important component for chlorophyll and photosynthesis, cellular respiration, proteins and all enzymes. Similar to our results, XIE et al. (2017) reported that low N amount caused low cell growth of Chlorella vulgaris. ZHU et al. (2014) specified specific growth rate and BP values of Chlorella zofingiensis under N starvation/repletion; they stated that the values in the presence of nitrogen were almost twice the values in the absence of nitrogen. SONKAR & MALLICK (2017) stated that the algae density increased with increasing nitrogen. Similar to these studies, it is mentioned in many studies that high N concentration resulted in higher algal biomass (YODSUWAN et al., 2017).

Beside this result, some studies show that high nitrogen causes toxic affect because of the activity of nitrate reductase. This activity converts nitrate into nitrite and ammonia and these molecules create toxicity to reduce biomass production (JEANFILS et al., 1993). Our results suppored this item because the growth performance in the control medium is higher than 50% N+ and 75% N+ media.

Conversely, in our result, the second highest OD value was recorded in 75% N- medium. This situation could be explained by the increase in lipid accumulation and cell size in N deficiency. Anyway, the relation between CN and OD depends on culture media and cell age etc declared that (FAKHRY & MAGHRABY, 2015).

With the increasing population and energy demand, the search for raw materials that can be used for energy production has also accelerated. For this purpose, microalgae have begun to draw attention because of their ability to produce lipid and fatty acids that can be used in biodiesel production.

A lot of microalgae species when exposed to stress are known to accumulate large amount of lipid that causes high yields of oil (YADAVALLI et al., 2012). Microalgae have a defense mechanism under stress. Accumulating polysaccharides and/or neutral lipids in this way leads to the production of various metabolites such as carotenoids, neutral lipids, important FAMEs (YU et al., 2015).

So, nutrient starvation is the most effective and preferred way for this purpose. But it is seen in previous studies that under stress conditions growth performance rates are lower usually in the time that obtaining high lipid content (HUERLIMANN et al., 2010).

Beside this; in some species nutrient deficiency does not accelerate lipid accumulation. For example, under nutrient starvation Dunaliella salina decreased lipid content from 25 to 9% but increased carbohydrate from 16 to 56% (SPOLAORE et al., 2006). So, each species can react differently to the same conditions.

Microalgae are an important protein source with their high protein and amino acid synthesis capacity and they can be used as alternative/supportive nutrients and not only protein and amino acids; especially polyunsaturated fatty acids are so valuable as a supplement nutrient (KHAN et al., 2018). As they can be used as a nutritional supplement; it is important to detect the biochemical profiles will change depending on the change of culture conditions.

Results in our own study are not surprising because nitrogen is the basic building block of proteins. So, the amount of protein showed a direct proportion to the amount of nitrogen. BAJWA et al. (2018) in Chlorella, Scenedesmus, Nannochloropsis and Chlorococcum algal strains and MUTLU et al. (2011) found high protein content in N and P rich media for Chlorella vulgaris.

These results showed that N deficiency increased arginine amount and excess N increased valine amount. It could be understood from these results that N concentration is quite effective in modifying the amino acid composition. T. obliquus can be used as animal feed, as it contains most of the EAAs at high rates (LIM et al., 2018). HEMPEL et al. (2012) found the similar results for Chlorella vulgaris 132 to ours as 4.5% DW (Arginine).

WHO (2007) declared that lysine, leucine, isoleucine, valine, threonine, aromatic amino acids (phenylalanine and tyrosine), tryptophan, sulphur amino acids (methionine and cysteine) and histidine as indispensable amino acids. These amino acids are abundant in our samples as shown in the results which culture conditions increase these amino acids.

Maximum total lipid content (16.46 %DW) was detected in 50% N- medium and then 13.71%, 12.55%, 9.60% and 9.18% in Control, 75% N-, 75% N+, 50% N+, respectively. This is clear that N deficiency triggered lipid accumulation and it is compatible with many studies that stated N deficiency changed lipid metabolism and converted membrane lipids to neutral lipid storage (DOMINGUEZ et al., 2015). Physical and chemical parameters such as salinity, temperature, pH and nutrient levels are quite effective on fatty acid composition. Especially N concentration leads to obtain different results of same species (CHEN et al., 2011).

These results showed that N deficiency leads to increase in MUFAs and PUFAs amounts. Similar to our results, the amount of MUFAs increased because of nitrogen deficiency in some studies (SHIFRIN & CHISHOLM, 1981). DHUP & DHAWAN (2014) found that the percentage of MUFAs is much higher in the lowest N concentration medium in Monoraphidium sp.. Also, high cetane number indicates that biodiesel quality is associated with high amount of SFAs and MUFAs. According to our results, 75% N- medium with highest MUFAs (20.0%) is suitable for obtaining high cetane number.

Total lipid and FAMEs profiles could be used to estimate cetane number to determine the quality of biodiesel (ISLAM et al., 2013). So, studies that investigate the total lipid amount and fatty acid composition in varying culture conditions are very important in this regard. PUFAs are essential fatty acids, are important in animal and human nutrition and play serious role in cardiovascular health and baby growth. Thus, high PUFAs make algae more valuable (VOIGT et al., 2000). Some microalgae can produce a huge quantity of PUFA such as C22:5 + C22:6 (39.4%) in Schizochytrium limacinum, and C20:5 (25%) in Porphyridium cruentum (SAJJADI et al., 2018). Present study show that N deficiency causes an increase in PUFAs. In contrast to our results, ÖRDÖG et al. (2016) detected in their study with Chlorella species that higher SFAs and MUFAs values are 1% N medium and high PUFAs values are 10% N medium. It was declared that a marine diatom Phaeodactylum tricornutum cultured with higher nitrogen concentration accumulated more PUFA (YODSUWAN et al., 2017).

These results vary from species to species and other conditions of culture except the studied factor. So, to obtain clearer results on species basis, more studies should be done for this aim on this subject.

Lipid of microalgae contains SFAs, MUFAs and PUFAs; SFAs and MUFAs are more suitable for producing biodiesel and PUFAs are important in human and animal nutrition (OCHSENREITHER et al., 2016). In this study, the percentage of PUFAs is much higher than those of SFAs and MUFAs. Similar to our results, ABBA et al. (2017) reported that the ratios of SFAs, MUFAs and PUFAs were 38.7%, 16.7% and 44.9%, respectively.

Besides this, in a lot of studies SFAs concentrations were much higher than those of PUFAs (RINNA et al., 2017). Also, GONZALEZ et al. (2019) found high SFAs and total lipid value at low nitrate concentration in optimum temperature for Chaetoceros gracilis.

The proportions of myristic acid, palmitic acid, palmitic acid, and stearic acid in SFAs are very important for biodiesel production. Percentages of palmitic acid are between 18.37%-19.95% in our results. YASIN et al. (2019) found that palmitic acid amount is 27.21% and 23.84%, respectively, but higher results could be found in literature (YASIN et al., 2019). It means that T. obliquus is not suitable for biodiesel production. This microalga can best be used in nutrition because of its high PUFAs value.

CONCLUSION

The maximum population growth rate of T. obliquus was found in the control medium and then 50% N+ medium. High nitrogen concentration speeds up cell division and number. Maximum protein content was detected in 50% N+ and then 75% N+. Our results showed that the nitrogen is the basic molecule for synthesizing protein, and it causes an increase in protein content. The highest percentage of lipid was seen in 50% N-, Control, 75% N-, respectively. It can be understood that N deficiency causes an increase in lipid storage. And amino acid and FAMEs compositions change according to nitrogen amount. PUFAs concentration increase because of N deficiency.

From our study, to obtain beneficial biomass by modifying nutrient concentration from T. obliquus, nitrogen is a good choice for this aim. Due to high protein content in all media and high PUFAs concentration in N deficiency, this microalga can be used as a nutrition supplement for humans and animals. This study is also very important in terms of providing data for unstudied species and future studies in this field.

ACKNOWLEDGEMENT

This research was funded by Mehmet Akif Ersoy University, grant number 0455-MP-17.

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• CR-2023-0269.R1

Edited by

Editors: Leandro Souza da Silva (0000-0002-1636-6643) Corina L. V. A. F. Carranca

Publication Dates

• Publication in this collection
  22 Apr 2024
• Date of issue
  2024

History

• Received
  15 May 2023
• Accepted
  13 Dec 2023
• Reviewed
  28 Feb 2024