Influence of Low Salt Concentration on Growth Behavior and General Biomass Composition in Lyngbya purpurem (Cyanobacteria)

Cyanobacteria are essential for the vast number of compounds they produce and the possible applications in the pharmaceutical, cosmetical, and food industries. As Lyngbya species’ characterization is limited in the literature, we characterize this cyanobacterium’s growth and biomass. L. purpureum was grown and analyzed under different salinities, culture media, and incubation times to determine the best conditions that favor its cell growth and the general production of proteins, carbohydrates, lipids, and some pigments as phycocyanin and chlorophyll a. In this study, each analyzed biomolecule’s highest content was proteins 431.69 mg g−1, carbohydrates 301.45 mg g−1, lipids 131.5 mg g−1, chlorophyll a 4.09 mg g−1, and phycocyanin 40.4 mg g−1. These results can provide a general context of the possible uses that can be given to biomass and give an opening to investigate possible biocompounds or bio metabolites that can be obtained from it.


Introduction
Currently, cyanobacteria have risen in importance due to the many compounds produced by them and their possible applications in the food, energy, and pharmaceutical industries. The genus Lyngbya is an excellent example of cyanobacterial strains, showing extraordinary diversity in the production of more than 200 secondary metabolites with biological activity [1,2]. Some Lyngbya strains appear to be halophilic, adapting to different salt concentrations in the medium. For instance, the strain Lyngbya contorta shows better cell growth and protein production while increasing the salt concentration from 5 ppt to 40 ppt [3,4].
Several Lyngbya strains can produce pigments and bio-compounds with potential applications as pharmaceuticals due to their properties, including neuroprotective, anti-oxidant, antibacterial, antifungal, anti-inflammatory, antileishmanial, antidiabetic, and anticancer activity (Table 1). However, these possible applications in L. purpureum are still largely unknown [5][6][7][8]. Examples of these pigments are chlorophyll and phycocyanin, enabling cyanobacteria to efficiently use the light spectrum they are exposed to [9]. Single-cell protein (SCP) from Lyngbya and other cyanobacterial species are an alternative source of protein and other biomolecules. SCP can be used in human nutrition as protein supplements, animal breeding and cattle fattening, paper and leather processing, and other industries [10,11]. Table 1. Beneficial properties and applications of Lyngbya biomolecules.

Biomolecule Beneficial Properties and Applications Reference
General biomolecules of Lyngbya species Chlorophyll a Potential applications in the cosmetic, food, and pharmaceutical industries due to its anticarcinogenic and antimutagenic activity, and inhibitory effect on genotoxicity.
It has been shown to reduce acne lesions and sebum levels, exhibiting antioxidant activity and protection against lipid oxidation at concentrations of 1mM L −1 .
Cyanobacterial strains are a potential source of carbohydrates. These carbohydrates consist mainly of glycogen and exopolysaccharide layers, which can be used for biofuels production [24]. Moreover, Lyngbya biomass has been used to obtain natural fibers with the same quality as the produced with jute and sisal fibers [44]. Regarding lipids, they are found widely in cyanobacteria's membranes. For example, L. limnetica has lipidic productivity of 1.565 g L −1 d −1 [45].
L. purpurem is one of the Lyngbya species that has not been characterized yet in its general biomass composition and cell growth. This strain was first described in 1845 by Hooker and Harvey, who named it after the purple color they perceived in it, even though it was later discovered that Lyngbya purpurem acquires different pigmentations depending on the environmental conditions [46,47], being blue-green its most common color. This species' lack of information and characterization allowed us to explore its general protein, carbohydrate, lipid, and pigments composition under low salt concentrations, different culture medium, and incubation time. All these experimentations to determine the best conditions to obtain higher biomass concentration and, subsequently, a vast amount of subproducts as expressed in Table 1 or otherwise not reported yet.

Cell Growth of L. purpurem at Different Low Salt Concentrations and Culture Media
By growing L. purpurem in different culture media, a clear difference in its growth is appreciated ( Figure 1). In BG11 medium, the obtained biomass ranged from 0.65 g L −1 to 1.18 g L −1 during the days 15-25, while in Bold 3N medium, growth ranged between 0.41 g L −1 and 0.68 g L −1 on the same incubation time; being clear that L. purpurem grew better and faster in the BG11 medium. The BG11 medium has a higher nitrogen concentration compared to Bold 3N medium (1.5 g L −1 and 0.75 g L −1 , respectively). Regarding BG11 medium (Figure 1a), the low and medium salt concentrations showed a better growth than that presented by the medium with high salt concentration, where the cyanobacterium starts showing an exponential behavior until day 25. The factorial design analysis shows that low and medium salt concentrations enhance cell growth at day 25. This growth observed in BG11 cultures was higher compared with previously reported in other studies [1]. On the other hand, L. purpurem produced 0.68 g L −1 of dry biomass in Bold 3N medium with a medium salt concentration on both 20th and 25th days ( Figure 1b). Furthermore, it was determined that the best salt concentration is with medium salt concentration.

Proteins, Lipids, and Carbohydrates Content in L. purpurem's Biomass at Different Low Salt Concentrations and Culture Media
In order to analyze the possible applications of L. purpureum biomass, a general biomass characterization was performed. For this purpose, the protein content in the culture media was analyzed. BG11 cultures produced a higher protein content than the cultures grown in Bold 3N ( Figure 2). In BG11 medium, the protein content ranged from 268.02 mg g −1 to 431.69 mg g −1 under different salinities between days 15 and 25. On the other hand, protein concentration in Bold 3N ranged from 162.22 mg g −1 to 253.71 mg g −1 during the same time length. The factorial design analysis illuminates that both, low and high salt concentrations, enhance protein content at day 20 in BG11 cultures. Also, it was discovered that low salinity enhances protein content at day 15 in Bold 3N cultures. It is interesting to observe that the highest protein content obtained from the biomass was 43.22% in BG11 medium and 27% in Bold 3N medium, which is higher than the observed in another study where L. aestuarii produced 13.5% DW [48]. Other cyanobacteria, such as Arthrospira maxima, have been reported to achieve a protein production of 50.7%. Also, Leptolyngbya sp. has produced 52.6% of proteins under salinities from 0 to 80 g/L [49]. Bold 3N cultures produced a higher lipid content than the cultures grown in BG11 ( Figure 3). In BG11 medium, the lipid content varied from 29.84 mg g −1 to 93.12 mg g −1 under different salinities at days 15-25, while in Bold 3N medium, lipid content ranged from 46.45 mg g −1 to 131.55 mg g −1 during the same period.
The factorial design analysis of the BG11 cultures determined that medium salt concentration enhances lipid content at day 20. This lipid content corresponded to 9.23% of the total biomass weight. In the case of Bold 3N cultures, it was determined that high salinity enhances lipid content at day 25. At this point, lipid concentration was 12% of the total DW of the biomass.
The total carbohydrate content was analyzed. The highest concentrations were found in the cultures grown at a high salt concentration in Bold 3N culture (271.83 mg g −1 ) and in BG11 in medium salt concentration (301.45 mg g −1 ) (Figure 4). The average carbohydrate production from days 15 to 25 in BG11 was 210.94 mg g −1 , while Bold 3N, carbohydrate production was 166.74 mg g −1 (Figure 4a,b). From the factorial design analysis of the BG11 cultures, medium salt concentration enhances carbohydrate content at day 20 (29% of the total DW of biomass). Regarding the Bold 3N cultures, low salt concentration enhances carbohydrates production at day 15 (27% of the total DW of biomass).

Pigments Behavior in L. purpurem's Biomass Different Low Salt Concentrations and Culture Media
One of the most common pigments produced by L. purpurem is chlorophyll a ( Figure 5). The highest contents were found in BG11 cultures in contrast to 3N Bold cultures. The highest concentrations were found in the cultures grown in the medium salt concentration on both culture media: 4.09 mg g −1 produced in BG11 on day 15, and 0.38 mg g −1 produced in Bold 3N on day 15. From the factorial design analysis of BG11 cultures, it was determined that neither of the two factors (salt concentration nor incubation time length) is essential for chlorophyll a production. For Bold 3N cultures, it was seen that low and medium salt concentrations enhance chlorophyll a content at day 25 and 15, respectively.
Even though previous studies show that Lyngbya sp. produces phycobiliproteins as the phycocyanin [25,50], neither the amount of phycocyanin produced nor the factors that enhance its production have been reported. From the two different culture media used, Bold 3N enhances a higher phycocyanin production ranging from 18.6 mg g −1 to 40.4 mg g −1 , whereas with BG11, phycocyanin ranged from 6.2 mg g −1 to 23.7 mg g −1 (Figure 6). Our results can be compared to other studies where 20.43 g of phycocyanin was produced by Lyngbya sp. A09DM at incubation day 30 [19].
N. muscorum and P. foveolarum were also exposed to different salt concentrations, producing phycocyanin in a range of concentration from 30 mg g −1 to 70 mg g −1 , approximately [51].

Cell Growth of L. purpurem at Different Low Salt Concentrations and Culture Media
BG11 medium contains an inorganic carbon source (Na 2 CO 3 20 mg L −1 ). This amount of nitrogen and the inorganic carbon source can positively affect the growth of the microalgae. Therefore, better growth of L. purpurem was obtained using the BG11 culture medium. The total dry biomass presented in this work resulted higher than the 0.642 g L −1 of Lyngbya's biomass previously reported in the literature, where the BG11 medium did not contain any variation in the salt concentration [52]. Moreover, some studies have reported the growth of L. aestuarii in f/2 medium with a salinity of 0.03 ppb, producing 0.0024 g L −1 at day 30 (Shruthi & Rajashekhar, 2014). The biomass growth results in BG11 and Bold 3N at different salt concentrations give the possibility of using these media to obtain an optimal growth of L. purpurem and marine Lyngbya species. Compared to other cyanobacteria, the obtained biomass of L. purpureum can be compared with the 1.45 g L −1 and 1.63 g L −1 obtained by Spirulina subsalsa BDU51591 and Oscillatoria sp. BDU51731, respectively [53]. Although the salt concentrations used in this study were deficient and not comparable to seawater, this cyanobacterium has the potential to be used at higher salinities, which can be explored in further studies.
Furthermore, cyanobacteria production in seawater can be an excellent option to produce biofuels and metabolites of interest [54]. Also, to counteract the salty environment's stress, cyanobacteria prevent the entry of Na + ions into the cell using Na + /H + antiporters and K + transporters or by accumulating solutes as sucrose, trehalose, glucosyl glycerol, proline, and glycine betaine. Additionally, exopolysaccharides (EPSs) protect cyanobacteria against osmotic and salt stress [55]. Therefore, the NaCl concentrations could influence the biomass content.

Protein, Lipid, and Carbohydrate Contents in L. purpurem's Biomass at Low Salt Concentrations and Culture Media
It can be observed that independently of the salt concentration evaluated; there was a higher protein production in the BG11 culture medium than in Bold 3N medium. The high amount of protein produced may result from the nitrogen concentration used in BG11, which was twice the concentration used in the Bold 3N medium (1.5 g L −1 and 0.75 g L −1 , respectively). It has been reported before that a higher level of nitrogen stimulates the production of proteins since nitrogen is one of the principal components in amino acids [56,57]. Their proteins also tend to have high lysine content, making them suitable for food supplements [58].
Regarding the total production of lipids, the highest concentration was obtained using the Bold 3N medium at the low salt concentration on days 20 and 25. Similar to our results, other studies have evaluated the response to nitrogen starvation in microalgae like Chlorella sp. [59], Chlorella vulgaris [60], Scenedesmus sp. [61], and P. tricornutum [62], finding a lipid accumulation. For instance, Chandra et al. reported a higher lipid and FAMES production performed by Lyngbya sp. using a culture media with low nitrogen concentration [63]. Also, our results show a more significant lipid production (131.5 mg g −1 ) compared to the produced by Chlorella sp. (85mg g −1 ) and Ankistrodesmus sp. (81 mg g −1 ) grown in the BG11 culture medium.
It is important to highlight that our lipids production was higher than most previous studies in the literature. For example, Lyngbya sp. showed a lipid production of 102.95 mg g −1 at day 19 [52], and L. dendrobia produced a total lipid content of 10.55% when it is cultured in BG11 medium. Similarly, L. limnetica, cultured in BG11, produced a lipidic content of 18.1% [64], and L. aestuarii produced 14.4% DW of lipids when it was grown in f/2 medium [48]. Among the lipids that have been found in Lyngbya's biomass, saturated and unsaturated FAME can be used as biofuels or for animal feed. For example, Chandra et al. reported palmitic and linoleic acid production. On the one hand, palmitic acid has antimicrobial properties; on the other hand, linolenic acid has been used as a dietary supplement. [63,65]. Compared to other cyanobacteria, the lipids produced in our study are comparable to the obtained by Chroococcus sp. BDU20201 (5.79%), Spirulina subsalsa BDU51591 (18.51%), Oscillatoria salina BDU50701 (11.9%), and Oscillatoria sp. BDU51731 (16%), making this cyanobacterium a good option for obtaining biofuels. Our findings are also consistent with the results obtained with other Lyngbya strains, where 4-12% DW belongs to lipids [53].
Our results show a higher carbohydrate production in comparison to other strains of Lyngbya sp. For instance, the total carbohydrate content of 172.89 mg g −1 in Lyngbya sp. [52] was obtained, which is similar to the 182 mg g −1 DW obtained in our study at day 25 in Bold 3N added with a high salt concentration. Also, L. aestuarii obtained an 18.4% of carbohydrates when it was grown in f/2 medium [48]. This result is similar to the obtained by L. purpurem in our study with BG11 medium (29% of the total DW of biomass). Large amounts of sucrose have been found in cyanobacteria; this carbohydrate is the most used for biofuels production. It was observed that the addition of optimal amounts of NaCl to enhance sucrose accumulation by Synechocystis sp. PCC6803. [66]. The total carbohydrate content from microalgae biomass varies between 3% and 40% [58], which supports our results (27% and 29%), which means that Lyngbya purpurem can produce one of the highest carbohydrate contents among different microorganisms.

Pigments Behavior in L. purpurem's Biomass Different Low Salt Concentrations and Culture Media
The chlorophyll-a production has been reported in other cyanobacteria such as Spirulina platensis, finding a production of 4.28 mg g −1 [67], 11.5 mg g −1 [68], and 12.3 mg g −1 [69]. These represent a higher production than those obtained in our study. The cyanobacteria N. muscorum and P. foveolarum have been exposed to different salt concentrations where chlorophyll a achieved a production range from 14 mg g −1 to 20 mg g −1 , approximately [51]. Furthermore, it has been shown that salt concentration can affect the chlorophyll-a structure negatively in different cyanobacteria such as Spirulina platensis [70].
It is interesting to observe that although a higher biomass growth and chlorophyll content were obtained in BG11 medium, in terms of phycobiliprotein production, the best culture medium was Bold 3N. From the factorial design analysis of Bold 3N cultures, it was determined that salt concentration is essential in phycocyanin production. Setyoningrum & Nur reported that an increase in salt concentration causes a significant increment in Spirulina maxima's phycocyanin production to answer stressful environmental conditions [71]. Table 2 summarizes all the optimal growth conditions found in the present study.

Reagents and Equipment
The reagents used for the culture medium, and the lipids and carbohydrates analysis were purchased from Sigma-Aldrich (Saint Louis, MO, USA). The Lowry Protein Assay Kit was purchased from Thermo Fisher Scientific (Rockford, lL, USA). The spectrophotometric measurements were taken in an absorbance microplate reader BGM Labtech (Fluorstar omega, 415-0470, Ortenberg, Germany).

Culture Conditions
The strain Lyngbya purpurem LB 2716 was purchased from UTEX (UTEX, Austin, TX, USA) (Figure 7). Erlenmeyer flasks were adapted in a shaker at 160 rpm (Scientific industries, SI1600 Orbital-genie, NY, USA) with a photoperiod of 12:12 using LED lamps (Hydro Grow LED, Los Angeles, CA, USA) with a light intensity of 100 µmol m −2 s −1 . The temperature of the incubation room was 21 • C. Two different culture media were used: modified BG11 and Bold 3N. The BG11 culture medium contained: NaNO 3

Cell Growth of L. purpurem at Different Salt Concentrations and Culture Medium
For each experiment, 0.30 g DW of wet biomass were used as seed culture and were inoculated into a 250 mL Erlenmeyer flask with the corresponding culture medium, resulting in 150 mL of total volume. A Factorial Design was used for this research and was applied to each set of experiment (Table 3). Cell growth and biomass composition of L. purpurem was evaluated in 2 different culture media (BG11 and Bold 3N) under 3 different salt concentrations: low (12.5 mg L −1 ), medium (25 mg L −1 ), high (50 mg L −1 ), during 3 different incubation time lengths (15, 20, and 25 days).
The culture medium Bold 3N is often used to grow L. purpurem. Its original composition contains 25 mg L −1 of NaCl, so this value was taken as a reference to set the different salinities used in this study. At incubation days 15, 20, and 25, the biomass of all salt concentrations (low, medium, and high) was dried at 4 • C for two days. The dry biomass (DB) was weighted and resuspended in 20 mL of bi-distilled water. The weight of DB was measured to obtain the cell growth behavior.

Proteins, Lipids, and Carbohydrates Content in L. purpurem's Biomass at Different Salt Concentrations and Culture Medium
The DB was resuspended in 20 mL of bidistilled water and was used to quantify the proteins, lipids, carbohydrates and pigments present in the biomass. Each sample (20 mL) was sonicated in a Sonifier Cell Disruptor (Branson Model # 102 C) to achieve cellular lysis. After this, the biomass protein content was quantified using the Lowry Protein Assay Kit (Lot # RB230458A), the lipid content was quantified using the sulfo-phospho-vanillin assay described by Mishra et al. [72] and the carbohydrate content was quantified using the sulfuric acid-UV assay described by López-Legarda et al. [73].

Pigments Behavior in L. purpurem Biomass at Different Salt Concentrations and Culture Medium
Each sonicated sample was also used to determine the pigments concentration. 5 mL of each sample were dried at 40 • C for two days, followed by a resuspension in 5 mL of methanol (95%) and incubation at 60 • C for 5 min. The samples were centrifuged for 10 min at 4000 rpm and 15 • C. The chlorophyll a concentration was obtained by measuring the supernatant's absorbance at 652 and 665 nm wavelengths by spectrophotometry. The concentration was calculated with the following formula described by [74,75]: To evaluate the phycocyanin concentration, the DB was resuspended in 20 mL of bidistilled water after sonication. The phycocyanin concentration was obtained by measuring the supernatant's absorbance at 592, 618, and 645 nm wavelengths by spectrophotometry. The concentration was calculated with the following formula described by [76]: 15 (2)  20  12  2  Low  25  13  2  Medium  15  14  2  Medium  20  15  2  Medium  25  16  2  High  15  17  2  High  20  18  2  High  25  19  3  Low  15  20  3  Low  20  21  3  Low  25  22  3  Medium  15  23  3  Medium  20  24  3  Medium  25  25  3  High  15  26  3  High  20  27  3  High  25 Salinity: Low: 12.5 mg L −1 , Medium: 25 mg L −1 , High: 50 mg L −1 . Factorial Design was run per type of culture medium (BG11 and Bold 3N).

Data Analysis
All data were analyzed in Minitab 18 (Minitab Inc., State College, PA, USA). A Multi-Factor ANOVA with a Tukey pairwise comparison per type of culture media was used in order to analyze the factorial design data. The coated values were taken from the triplicate samples (n = 3) and expressed as +/− SD in Figures and Tables.

Conclusions
This study reports the first characterization of the biomass of Lyngbya purpurem under different culture conditions. This study was conducted in order to elucidate the optimal conditions to produce several high-value biomolecules. Total biomass production of 1.18 g L −1 was achieved at low salinity in BG11 medium on the 25th incubation day. The highest production of each analyzed biomolecule in this study was: protein 431.69 mg g −1 (BG11, low salinity at the 20th day), carbohydrate 301.45 mg g −1 (BG11, medium salinity at the 20th incubation day), lipid 131.5 mg g −1 (Bold 3N, low salinity at the 25th incubation day), chlorophyll a 4.09 mg g −1 (BG11, medium salinity at the 15th incubation day), and phycocyanin 40.4 mg g −1 (Bold 3N, medium salinity at the 20th day).
Lyngbya purpurem has shown to be an adequate protein and carbohydrates source, which can be used in the food industry as a food supplement for humans or animal feed. L. purpurem's biomass has high nutritional value due to its protein, carbohydrate, and lipid content. Furthermore, it is important to highlight the antioxidant and anticancer properties conferred by chlorophyll a, namely the anti-inflamatory, anticancerous, hepatoprotective, and neuroprotective activities of phycocyanin. As a natural bio-factory, L. purpurem's carbohydrates and lipids can also be used for biofuels and bioplastic production. Other applications, such as biofertilizer, nutraceutical, and biomaterials, can be evaluated as part of a future study to impact the pharmaceutical, nutraceutical, agricultural, and cosmetic industries.