Appl Biochem Biotechnol DOI 10.1007/s12010-016-2128-2
A Multivariate Approach to Evaluate Biomass Production, Biochemical Composition and Stress Compounds of Spirulina platensis Cultivated in Wastewater Abuzer Çelekli 1 & Ali Topyürek 1 & Giorgos Markou 2 & Hüseyin Bozkurt 3
Received: 28 October 2015 / Accepted: 6 May 2016 # Springer Science+Business Media New York 2016
Abstract The study was performed to investigate the effects of using cow effluent for the cultivation of Spirulina platensis on its biomass production and cell physiology. S. platensis was cultivated in three different cow effluents (CE) used as cultivation medium during 15 days. CE was prepared using dry cow manures, and it was further modified with supplement of NaNO3 (CEN) and NaNO3 + NaCl (CENS). High nitrate value stimulated chlorophyll-a and total protein content of the cyanobacterium and also biomass production in standards medium (SM) and CEN media. Total carbohydrate content of S. platensis grown in CE media was found to be higher (p < 0.05) than that of SM. Productions of biomass and biochemical compounds by the cyanobacterium grown on the CE and SM media were evaluated by using multivariate approach. Conductivity, oxidation reduction potential (ORP), salinity, pH, and TDS played important role (p < 0.01) in the biochemical composition. As an effective explanatory factor, ORP had a significant positive correlation with H2O2, whereas negatively correlated with chlorophyll-α, biomass production, filament length, and proline. Canonical correspondence analysis proposed that biochemical compounds of S. platensis were not only affected by salinity and nutrition of media but also by pH and ORP. The present study indicated that CEN as a low cost model medium had high potential for the production of biomass by S. platensis with high protein content. Keywords Biomass . Biochemical compound . Cow effluent . Spirulina platensis
* Abuzer Çelekli
[email protected]
1
Department of Biology, Faculty of Art and Science, Gaziantep University, 27310 Gaziantep, Turkey
2
Department of Natural Resources Management and Agricultural Engineering, Agricultural University of Athens, 11855 Athens, Greece
3
Department of Food Engineering, Faculty of Engineering, Gaziantep University, 27310 Gaziantep, Turkey
Appl Biochem Biotechnol
Introduction Spirulina platensis has gained the interest for its biotechnological importance as a potential source of nutrition, pharmaceuticals, and other high value products [1]. S. platensis is a photosynthetic cyanobacterium, which is interesting industrially because it is a rich source of protein, essential amino acids, vitamins, fatty acids, and pigments. This cyanobacterium is considered as one of the most valuable sources of proteins, containing up to 72 % of its biomass of fatty acids [linoleic (19–26 %), gamma-linolenic (16–25 %), oleic (3–8 %), and palmitic (34–42 %)] acid, of pigments (chlorophyll, phycocyanin, etc.) of vitamins and phenolics [1, 2]. Besides these, biomass of Spirulina displays interesting capabilities; it can be used to remove undesired compounds such as heavy metals, dyes, excess nutrients (N, P, etc.), and pesticides from liquid wastewaters. It is pointed out from plenty researches that Spirulina is a very interesting bio-sorbent for wastewater remediation [3–5]. Nowadays, commercial Spirulina productions require the addition of inorganic chemical compounds, which contribute significantly to the production costs. It is highlighted that the nutrients in the effluents from animal manure are sufficient to support cell growth [1, 6]. Cultivation of cells in the effluent of animal manure treatment facilities presents an alternative to the current practice of land application of manure [7–9]. Production of Spirulina in effluents from animal manure offers several advantages, including a significant saving in the cost and solving disposal problems of waste. It was reported that animal wastes as a source of nutrients are utilized for the growth of Spirulina cultures [1]. Environmental conditions (e.g., nutrients, pH, salinity, light intensity, and temperature) can affect biomass production by S. platensis and also change its biochemical composition [10, 11]. Physico-chemical variables such as nitrate, salt, and pH strongly influence biomass production and biomass [11]. The pH regimes influence not only chemistry of media but also the physiology of organism and the production of biomass [1]. Nitrogen and NaCl have a significant effect on the biomass and protein production of Spirulina [1, 10]. High salt in the medium can affect photosystems I and II of Spirulina species due to its destructive effect on protein degradation [12]. However, Spirulina being an alkalophilic microorganism requires relative high concentrations of sodium ions for unhindered growth. Therefore, the addition of sodium to cultivation media with low salinity is required [13]. Stress conditions cause the generation of reactive oxygen species (ROS), mainly superoxide anion (O2•−), singlet oxygen (1O2), hydroxyl radical (OH•), and hydrogen peroxide (H2O2) [14, 15]. ROS could react with photosynthetic pigments, proteins, nucleic acids, and lipids, which cause lipid peroxidation, membrane and nucleic acid damages, enzymes inactivation, metabolite degradation, and even cell death. Defense systems of cellular mainly consist of enzymatic and non-enzymatic antioxidants to decrease stress of oxidative metabolisms. Compounds such as ascorbic acid, glutathione, carotenoids, and phenolics are formed to get rid of ROS [14]. Lipid peroxidation induces the formation of several breakdown products, such as malondialdehyde (MDA). High MDA accumulation can indicate excessive lipid peroxidation which causes to change membrane permeability. Under creating similar conditions, water stress can produce the synthesis of proline. Accumulation of proline may be an indicator of the stress response [16]. Several studies exist, in which animal wastes and wastewaters were used for the production of Spirulina [17, 18]. However, there is lack of information about the effect of the wastewaters on the physiology of cell and biomass production of S. platensis. The present study aimed to study the effect of the use of wastewaters as low cost and eco-friendly cultivation medium on biomass production and physiology of S. platensis. As wastewater cultivation medium model,
Appl Biochem Biotechnol
extracted nutrients from dried cow manure were used to supplement the cultivation media. The objective of this study was to investigate effects of the different media on the production of biomass, pigments, protein, carbohydrate, malondialdehyde, proline, –SH group, H2O2 content, and total phenolic compounds by Spirulina platensis by use of multivariate approach.
Materials and Methods Microorganism and Cultivation Spirulina platensis [10] was inoculated and cultivated in Schlösser’s medium. It was prepared by dissolving of following nutrients in distilled water (per liter): 13.61 g of NaHCO3, 4.03 g of Na2CO3, 0.50 g of K2HPO4, 2.50 g of NaNO3, 1.00 g of K2SO4, 1.00 g of NaCl, 0.20 g of MgSO4·7 H2O, 0.04 g of CaCl2·2 H2O. About 6 mL of metal solution (97 mg of FeCl3·6 H2O, 41 mg of MnCl2·4 H2O, 5 mg of ZnCl2, 2 mg of CoCl2·6 H2O, 4 mg of Na2MoO4·2 H2O), 1 mL of micronutrient solution (50.0 mg of Na2EDTA, 618 mg of H3BO3, 19.6 mg of CuSO4·5 H2O, 44.0 mg of ZnSO4·7 H2O, 20.0 mg of CoCl2·6 H2O, 12.6 mg of MnCl2·4 H2O, 12.6 mg of Na2MoO4·2 H2O), and 0.15 mg of B12 vitamin were added into prepared nutrient solution. Dried cow manure was mixed with tap water (20 g L−1) and holds about 72 h for extraction and dissolving of the nutrients. Cow effluent (CE) obtained from the mentioned mixture was filtered through Sartorius systems (0.45 and 0.80 μm mesh sizes of acetate filters) in order to eliminate unwanted matters and then was autoclaved. pH of medium was adjusted by use of dilute 0.1 M HCl and 2.0 M NaOH solutions and its value was determined by pH meter (Hanna, pH 211 microprocessor). Batch cultivations were performed in 500-mL Erlenmeyers containing 400 mL medium. The cyanobacterium cultivated in three cow effluent (CE) media and Spirulina medium (SM). The CE medium was prepared by using cow effluents without dilution. The CEN and CENS media were prepared by enrichment of CE medium with addition of 0.5 g L−1 NaNO3 and 0.5 g L−1 NaNO3 + 0.5 g L−1 NaCl, respectively. Initial Spirulina biomass of 0.02 g L−1 which was previously adapted to each specific media was inoculated into batch culture. Besides, each medium was also prepared without cyanobacteria. These cultures were incubated on an orbital shaker at 90 rpm for 12 days under 140 μmol photons m−2 s−1 continuous illumination using white fluorescent lamps. All cultures were conducted under the same environmental conditions (temperature, light intensity, etc.). Experiments were carried out in duplicates.
Physico-chemical variables Environmental variables (temperature, conductivity, concentration of dissolved oxygen, salinity, pH, total dissolved solid (TDS), and oxidation-reduction potential (ORP) of media before inoculating culture and after harvesting of the biomass) were measured in situ using of oxygentemperature meter (YSI professional plus model).
Biomass Density and Filament Length Biomass density was calculated from absorbance value which was measured by use of UV-VIS spectrophotometer (Jenway 6305). Calibration curve was obtained from absorbance against dry weight (g L−1) of Spirulina biomass [10]. During absorbance measurement, dilution technique
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was used to prevent the tendency of clumping for dense culture. Besides, in order to determine biomass value, the cultivation medium was filtered through acetate membrane filters (0.45 μm pore size). The filter was hold at 70 °C through overnight, cooled, and weighed. Filament length was determined by measuring dimensions of at least 25 randomly chosen cells by the use of Olympus light microscope attached DP73 model digital camera with imaging software (Olympus BX53 and Olympus CellSens Vers. 1.6).
Biochemical Component Analyses Biochemical response of S. platensis was determined at the different media used. Pigment content was measured following a method proposed by Wellburn [19]. Pigments were extracted by using 80 % acetone, centrifuged, and then their absorbances were spectrophotometrically measured (UV/VIS Jenway 6305). Lipid peroxidation level in S. platensis biomass was determined by measuring MDA value following of the method proposed by Zhou [20]. About 0.1 g of dried biomass was mixed with 5 mL of 10 % trichloroacetic acid for homogenization after that centrifuged at 7826×g for 15 min. About 2 mL of supernatant with 2 mL of 1 % thio-barbituric acid reagent was heated at 95 °C for 30 min. This mixture was cooled and centrifuged at 1960×g for 5 min. Absorbance values were obtained by use of a spectrophotometer at 532 nm. Purchased MDA from Merck (Schuchardt CHG) was used for preparing a standard curve. Proposed method by Bates et al. [21] was used for proline determination. Briefly, homogenized 0.025 g of dried biomass with 5 mL of 3 % sulfo-salicylic acid was centrifuged at 2817×g for 10 min. About 2 mL of supernatant was taken and mixed with 2 mL of ninhydrin and 2 mL of acetic acid (glacial), after then, this mixture was heated at 100 °C for 1 h. After the cooling of this mixture, extraction was carried out with toluene. Thereafter, absorbance was determined by use of spectrophotometer at 520 nm by use of standard curve of L-proline obtained from Merck (Merck KGaA Darmstadt). Amount of total protein was analyzed by Folin–Ciocalteu (F-C) method [22]. Shortly, 0.025 g of dried biomass was mixed with 5 mL of 0.1 M phosphate buffer with pH 7.0. After centrifugation of this mixture at 11,269×g for 10 min, supernatant was treated with F-C reagent and solution of alkali copper and waited for 30 min. The absorbance value was measured by using a spectrophotometer at 750 nm. Obtained bovine serum albumin from Sigma-Aldrich (Sigma-Aldrich CHEMIE GmbH, Steinheim) was used for preparing a standard curve. The F-C method [23] was used to determine total phenolic compound in S. platensis. About 0.025 g of dried samples was homogenized by using 5 mL of 0.1 M phosphate buffer at pH 7.0. After centrifugation at 11,269×g for 10 min, 0.1 mL of supernatant was reacted with 0.5 mL of F-C reagent for 2 min. After that, 1.5 mL of 20 % Na2CO3 was added into the solution and solution volume was completed to 5 mL with distilled water. This mixture was hold at room temperature for 2 h and then absorbance values were determined by use of a spectrophotometer at 765 nm. Gallic acid from Sigma-Aldrich (Sigma-Aldrich CHEMIE GmbH, Steinheim) was used to prepare standard calibration curve. Hydrogen peroxide (H2O2) content was determined by following Sergiev et al. [24] study. About 0.1 g of biomass was mixed with 5 mL of 10 % TCA in an ice bath. After centrifugation at 3835×g for 10 min, 1.5 mL of 50 mM phosphate buffer and 1 mL of 1 M potassium phosphate was added into 2 mL of the supernatant. Absorbance value of this mixture at 390 nm was measured by spectrophotometer. H2O2 solution (Sigma-Aldrich CHEMIE GmbH, Steinheim) was used for the preparation of a standard curve.
Appl Biochem Biotechnol
Thiol (–SH) group content in biomass extract was estimated by following Ellman [25] study. Briefly, 0.1 g of biomass and 5 mL of 5 % metaphosphoric acid was homogenized and centrifuged at 17,608×g for 15 min. The supernatant (0.5 mL), 2.5 mL of 150 mM phosphate buffer (pH 7.4), and 0.5 mL of 6 mM dithio(bis)nitrobenzoic acid (DTNB) were homogenized by the vortex. After incubation for 20 min, the absorbance at 412 nm was measured by use of a spectrophotometer. Reduced L-glutathione (Sigma-Aldrich, CHEMIE GmbH, Steinheim) was used as a calibration curve preparation.
Fourier Transform Infrared Spectrometric Analysis A FTIR equipped with an attenuated total reflection (FTIR–ATR, Perkin Elmer, Spectrum 100) was used for the infrared analyses of the biomass from different media.
Statistical Analyses SPSS (SPSS version 15.0, SPSS Inc., Chicago, IL, USA) was used for one-way ANOVA to compare data between studied groups. Duncan’s multiple range test was also performed to distinguish groups. To determine the relationship among the tested variables, Pearson correlation test was carried out. Canonical correspondence analysis (CCA) was used to discover the relationship between predictor variables (physico-chemical factors) and the response variable (biochemical contents) of S. platensis. To reduce skewness, variables of media were ln-transformed except for pH [26]. For explanation of response data variance in CCA, Monte Carlo simulations with forward selection were tested to find the significance of predictor variables. The package CANOCO program was applied for the analyses of ordination [26].
Results and Discussion Physico-chemical Variables of Media Environmental variables of media before the inoculation of algal culture and after S. platensis harvesting are summarized in Table 1. S. platensis was cultivated in wastewater-based media (extracted dry cow manure was used as the model wastewater; see BMaterials and Methods^) to be compared with a synthetic medium (SM) that is frequently used for biomass production. In this study, the effect of the different media (wastewater supplemented with N and NaCl or the synthetic one) on the growth, biomass composition, and stress compounds of S. platensis were investigated. S. platensis grown on CE-based media and SM gave different responses. Length of filament, production of biomass, pigments, MDA, proline, proteins, carbohydrates, –SH group, H2O2, and phenolic compounds of the cyanobacterium were affected by environmental variables of these media. The cyanobacterium was cultivated at 25 °C under alkaline condition. Before the cultivation, SM had significantly (p < 0.05) higher conductivity, salinity, TDS, and oxidation-reduction potential (ORP) than that of CE-based media. Dissolved oxygen (DO) and ORP were significantly changed (p < 0.05) before the inoculating and after the harvesting of S. platensis. This was due to photosynthesis and the oxygen generation and probably due to the change of the other variables of the cultivation media due to uptake of nutrients or the degradation of organic substances [27].
Appl Biochem Biotechnol Table 1 Environmental variables (± standard deviation) of media before inoculating Spirulina platensis and after harvesting of the biomass
Before
Environment
Unit
SM
Temperature
°C
24.82 ± 0.20a,A
24.76 ± 0.18a,A
24.72 ± 0.21a,A
24.96 ± 0.18a,A
a,A
a,A
a,A
9.89 ± 0.43a,A
b,A
10.28 ± 0.42b,A
b,A
5.44 ± 0.21b,A
b,A
5.49 ± 0.20b,A
a,A
5.12 ± 0.19a,A
ab,A
21.8 ± 0.98b,A
pH Conductivity TDS Salinity DO ORP After
NO3 Temperature
9.47 ± 0.40 −1
mS cm
−1
mg L
TDS Salinity DO ORP NO3
a,A
21.09 ± 0.89
a,A
14.40 ± 0.63
a,A
ppt
13.37 ± 0.58 −1
mg L
a,A
5.57 ± 0.20
a,A
SHE
25.8 ± 1.19
−1
gL o C
pH Conductivity
CE
−1
mS cm
−1
mg L mg L SHE
−1
gL
b,A
10.11 ± 0.28
b,A
5.41 ± 0.19
b,A
4.81 ± 0.22
a,A
5.24 ± 0.22
ab,A
24.1 ± 1.01
9.81 ± 0.39 10.21 ± 0.33
5.42 ± 0.27 4.87 ± 0.25 5.04 ± 0.23 23.6 ± 0.99
1.86 ± 0.05 24.82 ± 0.19a,A
b,A
1.46 ± 0.06 24.82 ± 0.22a,A
a,A
1.88 ± 0.11 24.85 ± 0.23a,A
1.87 ± 0.10a,A 24.86 ± 0.21a,A
10.15 ± 0.44a,A
9.79 ± 0.41a,A
9.90 ± 0.43a,A
9.94 ± 0.39a,A
a,A
b,A
b,A
9.83 ± 0.38b,A
b,A
6.39 ± 0.27b,A
b,A
5.55 ± 0.21b,A
a,B
7.08 ± 0.30a,B
a,A
23.8 ± 0.94b,A
a,A
1.42 ± 0.07a,A
20.64 ± 0.93
a,A
12.69 ± 0.56 12.65 ± 0.52
−1
9.68 ± 0.42
CENS
a,A
a,A
ppt
CEN
a,B
7.57 ± 0.35
a,B
19.1 ± 0.88
a,A
1.20 ± 0.07
9.53 ± 0.41
b,A
5.53 ± 0.20
b,A
4.86 ± 0.20
a,B
7.23 ± 0.27
b,A
23.9 ± 0.98
b,A
1.03 ± 0.04
9.64 ± 0.45 5.61 ± 0.25 4.95 ± 0.23 7.24 ± 0.34 20.0 ± 0.89 1.28 ± 0.08
Different lowercase letters show statistical difference in physico-chemical variables at α = 0.05 level in each lines. Different capital letters show a statistical difference in physico-chemical variables at α = 0.05 level in each columns before inoculating of culture and after the harvesting of S. platensis. Values with the same letters in the same parameters show that the values did not differ by the Duncan test at 0.95 confidence interval TDS total dissolved solid, DO dissolved oxygen, ORP oxidation-reduction potential
Biomass Production and Cell Length Biomass production by S. platensis was found to be 1.47 ± 0.08 g L−1 in the SM, which was higher than those in CE medium. The lower biomass production in CE-based media was due to a series of differences in their composition, such as macro- and micro-nutrient concentration and bioavailability, the presence of growth inhibitors originating in the manure, or the presence of colored substances. The fawn color of CE may had a negative impact causing the reduction of photosynthetic activity of S. platensis. Depraetere et al. [17] reported that the growth rate of Arthrospira platensis in piggery effluent was limited due to the dark color by reducing light penetration and so limiting the rate of nutrient recovery. For this reason, they removed the color of piggery wastewater before the cultivation to enhance the productivity of A. platensis in the pig effluent [17]. The lower biomass concentration observed in CE-based media shows that the potential for biomass growth is lower in that media than in the synthetic SM. Between the CE-based media, CEN medium displayed the best biomass concentration, while CENS displayed the lowest one. Addition of nitrate into CE medium could have positive influence on the biomass production. On the other hand, biomass production by the alga gradually decreased (p > 0.01) from 1.26 to 0.99 dw g L−1 for CEN and CENS, respectively. This could be due to addition of salt into CE. In any case, the N content was quite enough to support biomass growth, so any difference between the three CEbased media should not be due to the addition of N but rather due to the increase of dissolved salts. Length of S. platensis filament significantly varied from 499.4 ± 55.3 μm to 273.2 ± 35.81 μm obtained from SM and CE groups, respectively (Table 2). The highest
Appl Biochem Biotechnol Table 2 Biomass production, filament length, and biochemical composition (± standart deviation) of S. platensis in different media SM Protein Carbohydrate Chlorophyll-a Carotene MDA Proline H2O2
(mg g−1) −1
(mg g ) −1
(mg g ) −1
(mg g ) −1
(μg g ) −1
(μg g ) −1
(μg g ) −1
Phenolic compounds –SH group
(mg g ) (mg g−1)
Biomass
(g L−1)
Filament length
(μm)
CE
CEN
632.4 ± 8.91a
572.9 ± 5.83b
a
b
157.5 ± 3.62
673.3 ± 10.44c b
179.4 ± 3.41
a
a
9.20 ± 0.87
a
7.63 ± 0,89 65.21 ± 2.80
b
4.68 ± 0.468
b
a
66.51 ± 1.12a
b
80.50 ± 1.61b
b
846.9 ± 30.1b
b
11.51 ± 0.70a 24.21 ± 1.70d
22.20 ± 1.12 a
182.0 ± 15.51 a
63.60 ± 1.30
c
13.26 ± 1.22
79.30 ± 1.30
c
75.2 ± 2.39
1014.5 ± 19.3
a
839.9 ± 23.6
c
12.41 ± 0.70 3.90 ± 0.50a
3.50 ± 0.42 1.90 ± 0.30b
1.47 ± 0.08a
9.90 ± 0.50 16.80 ± 1.21c
1.05 ± 0.07b a
499.5 ± 55.10
11.33 ± 0.93b
18.80 ± 1.14
c
1.95 ± 0.33
a
661.1 ± 9.52d 177.5 ± 1.72b
172.4 ± 4.74
b
20.93 ± 1.31
CENS
1.26 ± 0.07a b
273.2 ± 35.81
5.75 ± 0.963a,b
0.99 ± 0.06b b
331.3 ± 32.52
202.3 ± 24.10c
Different letters indicate statistical difference in biochemical compounds among different media at α = 0.05 level
filament length was observed in SM, while in CE-based media, the length was significant lower. The data suggest that low values of salt, nitrate, electrical conductivity, TDS, and other factors have an adverse effect on the filament length of the cyanobacterium grown on CEbased media. The cyanobacterium cultured in CEN had remarkably higher filament length (331.4 ± 32.6 μm) than the other CE media. However, there was no clear relationship between the CE-based medium type and the filament length was observed. Shorter and straight filaments were observed especially in the CENS. In general, the variation of filaments length in relation to the culture conditions is rare, so there is lack of relative information.
Biomass Main Biochemical Composition Biochemical contents of S. platensis biomass in different media are given in Table 2. Chlorophyll-a content of biomass cultivated at CE and SM varied (p < 0.01) from 9.20 to 20.93 mg g−1, respectively. Between the CE-based media, no clear trend was observed. High nitrate value stimulated chlorophyll-a content in the cyanobacterium in SM medium. Increment of chlorophyll-a content was also found for S. platensis at different nitrate sources [28]. Piorreck et al. [29] reported that increment of chlorophyll in S. platensis was found with the increase of nitrogen concentration in the cultivation medium. Carotenoids content was higher in SM medium. The higher salt concentration should have a significant effect on carotenoids, because carotenogenesis could be enhanced by ROS which are generated by stress conditions, like high salt [30]. CEbased media displayed lower carotenoids content compared to SM. Between the three CE-based media types, increasing the addition of salt amounts resulted to a higher carotenoid content. Total protein content of S. platensis biomass cultivated in SM was higher (p < 0.05) than those in CE-based media. This could be due to CE contained not adequate available nitrogen. The protein production (572.9–632.4 mg g−1) by S. platensis cultivated in SM and CE-based media was very close to the protein content of same species in Zarrouk medium [31]. However, between the CE-based media, no clear trend of protein content was observed. Total carbohydrate content of S. platensis grown on CE-based media (179.4 ± 3.41 mg g−1) was found to be higher (p < 0.05) than that of SM. It is known that accumulation of carbohydrates takes place as a response to an
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immediate NaCl shock. However, in the CE-based media, the salt concentration was not high to induce salinity shock. The slightly higher carbohydrates content should be due to the overall differences on the media compositions. In the present study, in contrast to increment of carbohydrate content, there was a decrease in protein production with the increase of salinity, which agrees with Mary Leema et al. [31]. S. platensis grown on a medium with increasing salt concentration does not only increase the carbohydrate content but also decrease protein content [31]. Compounds stored in algal cell can be easily converted into osmo-regulative compounds as a response to osmotic pressure. Major osmotic carbohydrates such as glucosyl-glycerol and trehalose are accumulated under salinity stress [32]. The response to salinity stress is an energy consuming process due to the enhanced respiration [33] but the mechanisms have not yet been elucidated. It is probable that salinity stress not only affects light utilization but also affects osmoregulation (particularly metabolism of carbohydrates) to counteract ionic and osmotic stresses [34].
Biomass Physiological Status MDA, H2O2, proline, total thiol group, and total phenolic compounds of S. platensis grown on CEbased media and SM were determined to evaluate potential stress on the cyanobacterium (Table 2). The concentrations of MDA, proline, and phenolic compounds show that Spirulina cultivated with SM were higher, showing that the cells were more stressed than in CE-based media. With regard to CE media, increasing salt concentrations had a positive influence on the production of aforementioned biochemical compounds. Only H2O2 showed that Spirulina was more stressed in CE-based media than in SM. Under stress conditions, accumulated proline in a few plants had significant roles such as in scavenging of free radicals, protection of enzymes, and singlet oxygen quencher [16, 35, 36]. ROS are insufficiently detoxified by the organisms and can cause peroxidation of lipid, which results in the formation of different breakdown products, such as MDA. Abiotic factors could cause the generation of ROS from S. platensis such as singlet oxygen, hydroxyl radical, superoxide anion, and hydrogen peroxide [14, 15, 37]. Accumulations of proline and MDA in S. platensis under the stress conditions could be used as natural biomarker for the characterization of detection of free amino acids and lipid peroxidation, respectively. ROS could react with photosynthetic pigments, nucleic acids, lipids, and proteins, which cause nucleic acid, lipid peroxidation, membrane damages, enzymes inactivation, metabolite degradation, and even cell death. It was observed that accumulation of proline was more in SM than that of CE. This could be due to the higher salt, conductivity, and TDS contents of SM as it was in Verbruggen and Hermans [38] study. Besides, accumulation of proline is a common physiological response to various stresses and consensus has not achieved on the exact roles of proline accumulation. Moreover, CE medium had lower conductivity and TDS than those in SM. These and other factors could affect the growth and biochemical responses of S. platensis. Also, CE is the natural medium; therefore, adaption of S. platensis in this medium could be easier than that of SM [38].
Biomass Characteristics by FTIR-ATR Studies A change in characteristics of the biomass is one of the important points to understand effect of changes in abiotic factors of media. FTIR–ATR studies had been carried out analyzing in comparison with biomasses grown in different media. Results of FTIR–ATR are given in Fig. 1a–d for SM, CEN, CE, and CENS, respectively. In the spectrum of biomasses, major peaks could be assigned to stretching of –OH and –NH2 groups (3271–3280 cm−1), –CH vibration (2920–2925 cm−1), –NH2
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Fig. 1 FTIR–ATR spectra of S. platensis grown on (a) SM, (b) CEN, (c) CE, and (d) CENS media
or –C-N (amide) and –C = O bending groups (1627–1643 cm−1), –COO−, –CH3 wagging –P = O, and antisymmetric stretching groups (1394–1404 cm−1), –C–O and –C–N stretchings (1236– 1240 cm−1) and –C-N, –C-O, and –C-C stretching vibrations (1018–1023 cm−1) [39]. Remarkable differences in peaks were observed on the biomass of S. platensis cultured in the different media. These results showed that differences of environmental conditions cause changes in surface and in composition of biomass due to the different response of S. platensis to the different conditions. There was no differences on SM and CEN, but a difference in the spectra of CE and SM media was observed, especially, 3273, 2920, 1642, 1536, 1404, 1049cm−1. These results could be mainly from the formation of amino and carboxyl compounds. It was previously reported that algae cultivated in different environmental condition caused changes in biomass surfaces [39].
Multivariate Approach Studies CCA was applied to elucidate relationship between environmental conditions and biochemical composition of S. platensis biomass. A total 91.8 % of cumulative variance in biochemical compounds was explained by the first two CCA axes, with over 99.8 % of correlations between predictor variables (environmental factors) on the response variable (biochemical contents). Application of forward selection using the Monte Carlo test indicated significant of the first two axes (p = 0.002, F = 44.798) (Table 3). Conductivity, ORP, salinity, pH, and TDS were the most effective explanatory factors, played significant roles (p < 0.01) on the response compounds (Fig. 2). Protein and carbohydrate were located close to the center of CCA ordination seemed a wide range of tolerance to the changes in environmental conditions in the media (Fig. 2). S. platensis grown on SM and CEN had high values of biomass, chlorophyll-a, and filament length, which was also supported by CCA. Aforementioned three variables were found close to the arrow of DO. In the ordination, ORP was located on the negative site of the first axis, while pH, TDS, conductivity, and salinity were laid on the positive site.
Appl Biochem Biotechnol Table 3 Main results of canonical correspondence analysis using Monte Carlo permutation test for biochemical composition of Spirulina platensis—environment variables relationship Axes
λ1
λ2
λ3
λ4
Eigenvalues
0.139
0.078
0.052
0.044
Species-environment correlations
0.998
0.960
0.883
0.819
Total inertia 0.313
Cumulative percentage variance Of species data
91.8
97.1
98.7
97.6
Of species-environment relation
93.0
98.4
99.3
78.1
Sum of all eigenvalues
0.313
Sum of all canonical eigenvalues
0.291
Test of significance of first canonical axis: eigenvalue = 0.354 F = 44.798
p = 0.002
Pearson correlation test (Table not shown) indicated that ORP had negative correlations with pH (p < 0.01, r = −0.886), conductivity (p < 0.05, r = −0.693), TDS (p < 0.05, r = −0.665), and salinity (p < 0.05, r = −0.679). In the ordination, H2O2 content of S. platensis was found to be close to the arrow of ORP and far away from pH, conductivity, TDS, and salinity (Fig. 2). On the other hand, proline, MDA, and phenolic compounds were relatively far away from ORP arrow. Furthermore, ORP was positively correlated with H2O2 (p < 0.05, r = 0.714) and total carbohydrate (p < 0.01, r = 0.693) of the cyanobacterium. Contrariwise, this factor was negatively correlated with chlorophyll-a (p < 0.01, r = −0.922), biomass (p < 0.05, r = −0.755), length of filament (p < 0.01, r = −0.770), and proline (p < 0.01, r = −0.785). CCA showed that biochemical compounds of S. platensis were not only affected by salinity of media but also by other explanatory factors such as pH, ORP, and DO, which however could be affected in turn by the growth pattern of S. platensis in each cultivation medium.
Conclusion The cyanobacterium grown on CEN had the highest protein content. On the other hand, CENS stimulated the production of MDA, proline, total phenolic, carbohydrate, and total thiol Fig. 2 Ordination diagram of canonical correspondence analysis showing biochemical compounds (circle) of S. platensis and environmental variables (arrows) in different media (up-triangular)
Appl Biochem Biotechnol
compounds. Amount of proline, MDA, and phenolic compounds show that Spirulina cultivated with SM was more stressed than in CE-based media. Composition of biomass was changed by the use of different media conditions, which was characterized by FTIR–ATR. CCA showed that biochemical compounds of S. platensis was not only affected by salinity of media but also by pH, ORP, and DO. Acknowledgments This research was founded by TUBITAK project no. 112Y054. We are grateful to Council of Scientific Research Projects Executive in Gaziantep University.
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