Mixotrophic cultivation of Spirulina platensis in dairy wastewater: Effects on the production of biomass, biochemical composition and antioxidant capacity.

Mixotrophic cultivation of microalgae provides a very promising alternative for producing carbohydrate-rich biomass to convert into bioethanol and value-added biocompounds, such as vitamins, pigments, proteins, lipids and antioxidant compounds. Spirulina platensis may present high yields of biomass and carbohydrates when it is grown under mixotrophic conditions using cheese whey. However, there are no previous studies evaluating the influence of this culture system on the profile of fatty acids or antioxidant compounds of this species, which are extremely important for food and pharmaceutical applications and would add value to the cultivation process. S. platensis presented higher specific growth rates, biomass productivity and carbohydrate content under mixotrophic conditions; however, the antioxidant capacity and the protein and lipid content were lower than that of the autotrophic culture. The maximum biomass yield was 2.98 ±0.07 g/L in growth medium with 5.0% whey. The phenolic compound concentration was the same for the biomass obtained under autotrophic and mixotrophic conditions with 2.5% and 5.0% whey. The phenolic compound concentrations showed no significant differences except for that in the growth medium with 10.0% whey, which presented an average value of 22.37±0.14 mg gallic acid/g. Mixotrophic cultivation of S. platensis using whey can be considered a viable alternative to reduce the costs of producing S. platensis biomass and carbohydrates, shorten cultivation time and produce carbohydrates, as it does not require adding expensive chemical nutrients to the growth medium and also takes advantage of cheese whey, an adverse dairy industry byproduct.

Introduction

Microalgae have great biotechnological potential and can be used in several segments of the chemical, food, pharmaceutical and cosmetic industries and for biofuel production [1]. These microorganisms are an abundant source of natural proteins, lipids, carbohydrates, vitamins, pigments and enzymes [2] [3]. Moreover, numerous species are reported to have a significant antioxidant effect attributed to their biocompounds [2]. Antioxidants are compounds that are capable of inhibiting or retarding the oxidation of an oxidizable substrate and have a relevant role in human health, protecting the body against the oxidizing action of free radicals [4]. The antioxidant activity of S. platensis has recently been studied, and many in vivo studies have shown that this microorganism significantly reduces oxidative stress [5]. Phenolic compounds, including simple phenols, flavonoids, tannins, lignins, and phenolic acids and their derivatives, are potential candidates for scavenging free radicals due to their redox properties [6]. Phenolic acid molecules consist of benzene ring carboxylic groups and one or more hydroxyl or methoxy group, and the donation of electron or hydrogen atoms stabilizes the free radicals, conferring their antioxidant activity [1].

Commercial-scale algal cultivation has been occurring for over a decade [7], mainly to produce Spirulina spp. for natural high protein supplements, Haematococcus spp. as a source of the antioxidant astaxanthin, and Dunaliella salina for provitamin A production. Astaxanthin has high commercial value, and the production of natural astaxanthin from Haematococcus spp. has significantly greater antioxidant capacity than that of synthetic astaxanthin. Synthetic astaxanthin is sold at a lower price than that of natural astaxanthin; thus, the improved production of natural astaxanthin has attracted strong research interest. [8]. Recent studies have evaluated various strategies to increase important biomolecule synthesis by microalgae, such as lipids for biodiesel [9] and carbohydrates for bioethanol [10] production. The high cost of microalgae cultivation has been a major obstacle for commercializing its products. One of the viable solutions to reduce the costs of microalgae cultivation is to couple the high-value biomass production system for commercialization with agroindustry waste treatment since microalgae are known to effectively eliminate a variety of pollutants in wastewater, such as nitrogen, phosphates and organic carbons, among others [11]. Mixotrophic cultivation is a preferable microalgae cultivation technique for biomass production [12]. However, mixotrophic cultures are susceptible to contamination, which substantially influences microalgae growth, suggesting that closed and sterilized photobioreactors instead of open ponds should be used for cultivation. Despite the higher cost of photobioreactors, biomass yields can reach 5–15 g/L, which is 3–30 times higher than those produced under autotrophic growth conditions [13].

A polluting byproduct of the dairy industry in Brazil, which has environmental repercussions, is cheese whey, which is the residue from manufacturing various types of cheese, yogurt, ice cream and butter through different processes. Cheese is one of the world’s leading agricultural products, with whey wastewater generation that is approximately four times the volume of processed milk [14]. It has been demonstrated that it is possible to produce microalgae biomass in mixotrophic cultures with whey since this waste provides important substrates, such as sugars, as a source of organic carbon for microalgae species [15].

Girard et al. [15] cultured Scenedesmus obliquus under mixotrophic conditions and demonstrated that this species presented higher specific growth rates and biomass yields when replacing 40% (v/v) of the standard medium with whey than when it was cultured with only standard medium. Salla et al. [2] studied the mixotrophic cultivation of S. platensis in diluted Zarrouk culture medium with the addition of concentrated whey protein residues and high lactose levels. They observed that there was an increase in biomass and carbohydrate productivity by the species under study. Tsolcha et al. [16] developed a biological (algal) secondary cheese whey wastewater treatment system to produce biodiesel while simultaneously removing the polluting nutrients and chemical oxygen demand. Recent studies have demonstrated that S. platensis is also a promising bioethanol producer due to the carbohydrate concentration in its biomass, which could potentially reach 50% [2] and up to 60% [10] by changing the growth medium. In addition, the species may contain a high antioxidant compound content, which is capable of adding value to the cultivation process.

The abovementioned potential of S. platensis has motivated developing research to produce high-value biomass of commercial interest under mixotrophic growth conditions to reduce costs in a sustainable production system. Since whey is a potential pollutant produced in large volumes by dairy industries around the world, the aim of this research was to cultivate S. platensis under mixotrophic culture conditions using Zarrouk medium supplemented with whey from the production of buffalo mozzarella cheese to study its influence on the biomass production, specific growth rates, biochemical composition and antioxidant activity of this species.

Materials and methods

Microorganisms and culture medium

The S. platensis D9Z strain was obtained from the Microalgae Bank of the Laboratório de Ambientes Recifais e Biotecnologia com Microalgas (LARBIM) of the Federal University of Paraíba, Brazil. A xenic strain of Spirulina platensis was maintained under sterile conditions in test tubes at a temperature of 25°C and irradiance of 238 μmol m-2 s-1 under a 12 h light/dark photoperiod in Zarrouk standard culture medium [17]. For the mixotrophic cultures, the complete Zarrouk medium was supplemented with 2.5% (v/v), 5.0% (v/v) and 10% (v/v) buffalo mozzarella cheese whey from the TAPUIO Agropecuária Ltd. cheese industry located in Rio Grande do Norte, Brazil. The cultivation conditions used in this work, such as temperature, irradiance and aeration, were based on previous studies that describe the optimum conditions for S. platensis cultivation [18].

Clarification of cheese whey for microalgae cultivation

The whey was collected and stored in plastic bottles in a freezer at -20°C until its preparation for inoculation. The whey was clarified for use in the experiments. For use in the growth medium, the previously clarified whey was thawed in a refrigerator at 5°C and then autoclaved (121°C, 15 min), filtered through a 20 μm screen and centrifuged in a 24-BT simplex II centrifuge for 15 min at 1500 rpm for removal of the precipitated material. The whey was then autoclaved again to avoid possible contamination prior to addition into the growth medium. (dx.doi.org/10.17504/protocols.io.4t5gwq6)

Experimental design

The inocula was obtained from cultures using Zarrouk medium [17]. Precultures (1 L) were made in Erlenmeyer flasks under the same conditions as that of the experimental apparatus to obtain the amount of cells needed to start the experiments. (dx.doi.org/10.17504/protocols.io.4txgwpn)

Cultures were prepared aseptically in a device with 5 fluorescent lamps of 45 W with a luminous intensity of 238 μmol m-2 s-1 measured on the outer surface of the vials using a Q201 quantum radiometer (Macam Photo-Metrics Ltd., Livingston, Scotland); a 12 h light/dark photoperiod was implemented under constant sterile aeration promoted by pumps (JAD Air Pump S-510) at a specific flow rate of 0.5 vvm (volume of air per volume of medium per minute). The initial S. platensis concentration ranged from 0.2 to 0.3 g/L. All assays were performed in triplicate. When the cultures reached the stationary phase, the biomasses were collected by filtration on 20 μm screens and washed in distilled water to remove salt remnants. Then, the biomass was lyophilized in a lyophilizer (LJJ02—JJ Scientific) and frozen in a freezer at -20°C until chemical characterization. The pH of the cultures was monitored every 24 hours throughout the duration of the experiment with a previously calibrated pH meter (K39-0014PA). (dx.doi.org/10.17504/protocols.io.4tygwpw)

Determination of cellular concentration

The cell concentration was monitored daily until the cultures reached the stationary phase, a period of approximately 17 days until reaching the early stationary phase of growth, which is when carbohydrates are accumulated in the microalgal biomass [2]. The growth monitoring of the S. platensis cultures was performed by measuring the absorbance at λ = 670 nm in a spectrophotometer (SP-22 Biospectro). A calibration curve was generated to relate the absorbance to the cell dry weight. To quantify the biomass, a known volume of culture was filtered on 40 μm pore glass fiber filters (47 mm, Sartorius), and the obtained biomass was oven dried at 80°C and quantified by weight difference according to Lourenço [19]. The filters used were previously treated in a muffle furnace at 400°C for 4 h.

Growth parameters

The growth rate (μ d-1) of S. platensis was calculated as the slope of the natural log of the biomass concentration versus time during the exponential phase, when the correlation coefficients of these two variables were above 0.98 [20]. The duration of the exponential growth phase was different for each evaluated cultivation condition and was considered in the calculations.

The maximum biomass productivity PXmax (g.L.day-1) was evaluated according to Eq (1), where Xmax is the maximum biomass concentration at time t, Xo is the biomass concentration at time zero, and t is time.

The productivity was calculated for each day compared to that of day zero. The maximum value obtained was defined as the maximum biomass productivity.

Chemical analyses

Clarified buffalo cheese whey samples were analyzed for the percentage of total solids, total protein, fat, lactose, and moisture using a DairySpec (Bentley Instruments Inc., Chaska Minnesota, USA). The equipment was calibrated using buffalo cheese whey calibration samples with different concentrations.

The dry matter and the ash content were quantified according to the AOAC [21] methodology. The total protein content was determined by the Kjeldahl method [21]. Kjeldahl nitrogen was measured (TECNAL digester block, model TE-040; TECNAL Kjeldahl TE-0364 distillation unit) using 200 mg samples of the dry biomass. The sample was first digested using a concentrated sulfuric acid digester solution following a progressive heating ramp and then distilled using boric acid, sodium hydroxide and indicator solutions. After distillation, the obtained solution was titrated with 0.1 N hydrochloric acid (HCl). The total protein content was calculated by multiplying the value of total nitrogen by a conversion factor of 4.78 [22]. Total carbohydrate determination was performed by a method published by Korchet [23] and adapted by Derner [24]. The standard curve was prepared from an anhydrous glucose solution. The total lipid content was determined according to Bligh and Dyer [25]. Extraction and esterification of the fatty acids present in the biomass were carried out according to Menezes et al. [26] to identify the fatty acid profile.

A gas chromatograph (Thermo Scientific-CG/FID-FOCUS) with a flame ionization detector (FID) and a Supelco SP2560 capillary column (100 m × 0.25 mm, 0.2 μm) was used to separate the fatty acids. Nitrogen was used as the carrier gas (1.2 mL/min). The injector and detector temperatures were 230°C and 270°C, respectively. The column temperature programming was as follows: the temperature was held at 40°C for 3 min, then increased to 180°C for 5 min at a rate of 10°C/min; the temperature was increased again to 220°C for 3 min at a rate of 10°C/min, and, finally, the temperature was increased to 240°C at a rate of 20°C/min and was maintained for 25 min. Nitrogen was used as the carrier gas at 0.9 mL/min. The injected sample volume was 1 μL with a split ratio of 10:1. The peaks were integrated and compared to a SupelcoTM37 Fatty Acid Methyl Ester (FAME) standard mixture from Sigma Aldrich.

Antioxidant activity

The lyophilized microalgae biomass was extracted with methanol to obtain the antioxidant extracts. One gram of each sample was weighed, 40.0 mL of methanol was added, and the mixture was sonicated in an ice bath (Unique, model USC-1400A) for 20 min. For extraction, the sample was shaken for three hours at 25°C and centrifuged at 8000 rpm for 10 min. The extracts were dried in an air circulating oven (SOLAB, model SL102) at 35°C and stored in the dark at room temperature until they were used. Each extract was resuspended to 5.0 mg/mL with distilled water and stored in amber bottles under refrigeration until use. The entire extraction procedure was performed under in 4.8 lux illumination to avoid photodegradation.

Phenolic compounds

The total extractable phenolic contents were determined by the Folin-Ciocalteu colorimetric method [27]. The concentration of phenolic compounds was estimated using a standard calibration curve with gallic acid. The results were expressed as mg GAE/g (milligrams of gallic acid equivalent per gram of extract).

Antioxidant capacity via the ABTS method

The antioxidant capacity of the microalgae via the ABTS•+() method was determined according to the methodology described by Rufino et al. [28]. The standard curve was prepared using Trolox at μmol concentrations. The antioxidant capacity results were expressed in μmol Trolox/g extract (antioxidant capacity equivalent to that of Trolox).

Antioxidant capacity via the FRAP assay

The antioxidant capacity of the microalgae extracts was estimated by the iron reduction method (FRAP), which is based on the ability of an antioxidant to reduce Fe3+ to Fe2+ and was performed following the methodology described by Benzie and Strain [29]. Two standard curves were prepared, one using Trolox and one with ferrous sulfate. The antioxidant capacity results were expressed in μmol Trolox/g extract (antioxidant capacity equivalent to that of Trolox) or μmol ferrous sulfate/g extract (antioxidant capacity equivalent to that of ferrous sulfate).

Statistical analysis

The data were utilized to calculate the average value of three independent tests, and the values were expressed as the mean ± standard deviation (SD). Statistical analyses were performed with SAS software version 9.0. The data were analyzed using a general linear model (PROC MEANS and GLM). Duncan’s new multiple range test was used to compare treatment averages (whey inclusion levels) of the microalgae characteristics. Statistical significance occurred in all analyses when the calculated p-value was ≤0.05.

Results and discussion

Whey composition

The whey composition of buffalo mozzarella cheese may vary depending on several factors, including the quality, milk composition and technology of the manufacturing process [30]. According to Sales et al. [30], whey represents approximately 55% of the nutrients present in the milk, thereby constituting a waste product that is rich in organic matter with great potential for mixotrophic and heterotrophic microalgae cultivation. The basic characterization of the clarified buffalo cheese whey is shown in Table 1.

Table 1

Composition of clarified buffalo mozzarella cheese whey used for the cultivation of S. platensis.

Variables		Clarified cheese whey
Total solids (%)		6.77
		Fat (%)	0.02
		Total Protein (%)	0.60
		Lactose (%)	5.07
Moisture (%)		93.23

Lactose represented almost 75% of the total solid percentage in the whey composition, constituting the main source of available carbon, along with the presence of proteins and fats in smaller proportions.

Growth curve of S. platensis

S. platensis was grown under autotrophic and mixotrophic conditions with 2.5%, 5.0% and 10.0% cheese whey for 17 consecutive days until reaching the early stationary phase of growth to evaluate important kinetic parameters, such as maximum biomass concentration, maximum yield and specific growth rate. The whey concentrations utilized were baed on previous studies. Mouther [31] showed that the optimal cheese whey concentration for S. platensis mixotrophic cultivation was 3.0%, and inhibition occurs at higher whey concentrations. Moreover, heterotrophic cultivation was inappropriate for S. platensis, possibly due to some inhibiting factors for this species present in the whey. Fig 1 shows the growth curves of S. platensis in the cultures evaluated in this study:

Fig 1

Growth curve of S. platensis grown on autotrophic Zarrouk medium and mixotrophic medium supplemented with buffalo mozzarella cheese whey at concentrations of 2.5%, 5.0% and 10.0%.

The error bars represent the standard deviations (n = 3).

The lag phase for all cultures occurred within the first 24 hours. This is expected since inoculum was used in the standard growth medium. The maximum biomass production for all cultures was obtained at the early stationary phase of growth. The control culture presented a maximum biomass concentration of 1.75 g/L, which was the lowest value when compared to that of all the studied conditions (Table 2). It was observed that autotrophic Xmax was similar to the values found by Kumari et al. [32] (1.87 g/L) and superior to the values found by Andrade and Costa [33] (1.44 g/L) and Mourthé [31] (1.01 g/L). Mixotrophic cultivation of S. platensis with 5.0% whey reached the highest biomass yield (2.98 g/L). Similar values were observed by Salla et al. [2] in S. platensis cultures using whey protein concentrate. This value was also similar to that found by Andrade and Costa [33] (2.8 g/L) when cultivating S. platensis in diluted Zarrouk medium with added molasses, a byproduct from the sugarcane industry, and subtly higher than the values reported by Marquez et al. [34] (2.5 g/L) and Chen and Zhang [35] (2.4 g/L) when they used glucose as an organic substrate. The culture with 10.0% whey showed double the specific growth rate of the culture with 5.0%, and it had the highest biomass productivity on the 4th day (Fig 2).

Table 2

Maximum biomass concentration (Xmax), maximum yield (Ymax) and specific growth rate (μ) of S. platensis.

Cheese Whey %	Xmax (g.L-1)	Pmax (g.L-1.day-1)	μ (day-1)
0.0	1.65c±0.18	0.1011b±0.003	0.183d±0.008
2.5	2.16b±0.02	0.1195b±0.001	0.258c±0.013
5.0	2.98a±0.07	0.1793a±0.005	1.024b±0.018
10.0	2.18b±0.16	0.1628a±0.015	2.048a±0.036

Different letters indicate significant differences for different treatments (p < 0.05).

Fig 2

Productivity of S. platensis grown on autotrophic Zarrouk medium and mixotrophic medium supplemented with buffalo mozzarella cheese whey on the 4th day of growth.

The error bars represent the standard deviations (n = 3).

Mixotrophic cultivation of S. platensis using 2.5% and 5.0% whey was favorable for biomass yield. This process could reduce the cost of producing microalgae while also consuming a waste that is discarded into the environment. The growth medium supplemented with 10.0% whey would be relevant in an industrial process since the biomass could be produced in the largest amount in a short time interval of approximately 4 days of growth. It is very important to evaluate the biochemical composition of the biomass in this period to check the viability of producing certain metabolites of commercial interest, which will be done at a later stage. Currently, many studies involving microalgae culture in effluents have been researched mainly as a strategy for lipid and carbohydrate production [2] [9]. The biomass yields and growth rates of S. platensis observed in this study were higher than those observed in the studies reported by Tsolcha et al. [16], Economou et al. [36] and Dourou et al. [37]. All of these microalgae cultivations were performed under non-aseptic conditions that, despite minimizing the cost of the process, led to lower biomass yields. Mixotrophic and heterotrophic cultures under aseptic conditions can be developed in closed photobioreactors, leading to high biomass and/or biocompound yields, which may justify the higher cost of making the process feasible. Mixotrophic cultures show reduced photoinhibition and higher growth rates in relation to those of autotrophic and heterotrophic cultures, having independence in assimilating both growth substrates and the ability to perform photosynthesis as an advantage since the acetyl-CoA pool is maintained for both carbon sources of CO2 fixation (Calvin cycle) and for the extracellular organic carbon [38].

pH control

The pH influences the CO2 and mineral solubility in the growth medium, directly or indirectly interfering with the algae metabolism; thus, it is one of the most important growth parameters. The pH of the cultures during growth was monitored every 24 h, as shown in Fig 3:

Fig 3

pH, which was monitored every 24 hours in S. platensis cultures grown in Zarrouk autotrophic medium and mixotrophic medium supplemented with buffalo mozzarella cheese whey.

The error bars represent the standard deviations (n = 3).

Mixotrophic cultures had lower pH ranges than that of the control culture. This is expected since the whey is acidic. The pH values in these cultures showed similar ranges from the beginning to the end of the experiment, where the lowest value was observed in the culture with 10.0% whey. In general, pH variation was more pronounced for the control (9.3–11.8) with a notable increase in the last 3 days. The autotrophic growth medium is usually rich in bicarbonate, and inorganic carbon may be in the form of CO2, H2CO3, HCO3- (bicarbonate) or CO3- (carbonate), and their proportions depend on pH. The proportions of HCO3- or CO2- increase at basic pH, but CO2 is the carbon source that is predominantly used by algae when the pH of the growth medium is low. The growth of the microalgal cultures was confirmed by the gradual increase in the pH of the medium, which is attributed to the inorganic carbon consumption for cell growth. Inorganic carbon consumption forces a displacement of the carbonate-bicarbonate equilibrium in the buffer system [39]. The optimal pH growth range of S. platensis is 9.5 to 10.5 [40]. Although the pH was not in the optimal working range, there was no growth limitation when comparing the biomass production and growth rate of S. platensis cultivated in this work with those of other studies reported in the literature, as discussed above.

Biochemical composition

S. platensis is one of the most studied microalgae species and is widely used as a food product and dietary supplement due to its nutritional profile and high nutrient bioavailability. According to Habib et al. [41], the basic chemical composition of S. platensis consists of proteins (50–70%), carbohydrates (15–25%), lipids (6–8%) and minerals (7–13%), and these percentages may vary depending on the type of culture. This species has also been studied in the production of biofuels, mainly bioethanol and bio-oil, because it is a low lipid species and is an attractive biomass for thermal conversion and fermentation processes [16] [42] [43]. The biochemical composition of the biomass obtained in this work is presented in Table 3.

Table 3

Biochemical composition of S. platensis biomass cultivated in autotrophic growth medium and mixotrophic growth medium with the addition of buffalo mozzarella cheese whey.

Cheese Whey (%)	Dry Matter (%)	Total Protein (%)	Fat (%)	Carbohydrates (%)	Ash (%)
0.0	93.65ab±1.60	65.55a±1.88	5.18a±0.38	27.17c±1.57	6.04b±0.38
2.5	91.77bc±0.6	60.62b±0.30	2.04d±0.03	23.29c±1.54	14.21a±0.03
5.0	90.57c±0.52	44.56c±0.74	2.56c±0.15	47.83a±0.78	14.03a±0.14
10.0	94.92a±0.99	39.62d±0.36	3.40b±0.18	40.65b±3.66	6.36b±012

Different letters indicate significant differences for different treatments (p < 0.05).

The biomass obtained from autotrophic cultivation presented a high total protein content and low carbohydrate, lipid and ash contents, similar to the results reported by Madkour et al. [44] and Salla et al. [2]. The ash content increased in the cultures with the addition of 2.5% and 5.0% whey. Madkour et al. [44] demonstrated that the protein and carbohydrate contents for S. platensis grown in Zarrouk growth medium were 52.95% and 13.20%, respectively. Salla et al. [2] obtained a carbohydrate content ranging from 20.0% to 60.0% and a protein content of 45.40%. The biochemical composition of microalgae is strongly influenced by the growth medium, temperature, aeration, irradiance, and culture volume, among other factors [45]. In this study, all variables were held constant, and only the growth medium composition changed, so this parameter was responsible for the observed differences in the biochemical composition. It is possible to notice a clear, direct relationship in which the whey content increased in the growth medium and the total protein synthesis decreased for all studied conditions, while the carbohydrate content increased in the cultures with whey additions above 2.5%, according to Fig 4.

Fig 4

Influence of the whey percentage on the synthesis of carbohydrates and proteins by S. platensis.

The error bars represent the standard deviations (n = 3).

There was an increase in carbohydrates and a decrease in protein content in the mixotrophic cultures. S. platensis produces high levels of protein, which justifies its wide commercial application as a protein-rich supplement, and this mainly occurs when the growth medium is rich in nitrogen. It has been shown that some types of microalgae accumulate carbohydrates and other non-nitrogenous biocompounds when an organic source is added to the growth medium, so these results are expected [2]. This study demonstrated the feasibility of applying mixotrophic cultivation to produce high-carbohydrate microalgae. Moreover, S. platensis is a species that has been studied in thermal degradation processes for producing bio-oil, biochar and gas. Some studies have shown that bio-oils produced from microalgae are more stable, have a lower oxygen content and a higher calorific value than that of lignocellulosic bio-oil [46]. However, bio-oil presents a high nitrogen content that is undesirable because it deactivates acid catalysts used for coprocessing crude oil in refineries and emits NOx during combustion [42]. In addition to increasing biomass production, the mixotrophic culture of S. platensis using cheese whey may decrease the processing cost and produce low protein biomass, thus being favorable for application in thermal degradation processes for producing biofuels.

S. platensis is a low lipid species when grown under standard conditions, but this percentage may increase as a function of culture conditions. It was observed that there was a decrease in lipid synthesis in the mixotrophic cultures of S. platensis when compared to that of the control culture, but there were no significant differences as a function of the added whey percentage. The fatty acid methyl ester (FAME) profiles of S. platensis are shown in Table 4.

Table 4

The fatty acid methyl ester (FAME) profiles (percentage of total FAMEs) present in S. platensis biomass cultured in autotrophic medium and mixotrophic medium with the addition of buffalo mozzarella cheese whey.

Fatty Acid	Whey %
	0.0	2.5	5.0	10.0
C10:01	nd	nd	0.70–0.73	0.62–0.69
C12:02	0.97–1.11	0.73–0.75	0.60–0.64	0.62–0.65
C13:03	1.43–1.51	1.01–1.03	0.74–0.92	0.92–0.93
C14:04	0.62–1.39	0.71–0.72	0.59–0.59	0.23–0.24
C14:15	0.89–0.96	0.69–0.69	0.48–0.48	0.50–0.51
C15:06	0.23–0.55	0.32–0.33	0.28–0.30	0.30–0.31
C15:17	0.66–0.68	0.62–0.63	0.58–0.60	0.50–0.67
C16:08	40.60–42.15	45.72–45.75	44.87–44.91	44.29–43.68
C16:19	1.67–1.76	2.04–2.23	2.29–2.31	2.82–2.87
C17:010	0.16–0.27	0.49–0.49	0.18–0.35	0.16–0.35
C17:111	0.25–0.28	0.22–0.23	nd	2.41–2.51
C18:012	2.31–4.42	2.28–2.39	2.45–2.55	0.24–0.25
C18:1n9c13	nd	0.63–0.67	069–0.74	0.68–5.53
C18:1n9t14	9.78–11.38	10.92–10.92	12.00–12.06	3.25–3.26
C18:2n6c15	nd	0,34–0,36	nd	nd
C18:2n6t16	10.99–11.59	10.46–10,45	8.84–8.87	10.68–11.24
C20:017	14.54–15.15	16.09–16.13	17.25–17.29	19.88–19.89
C20:218	0.16–0.19	nd	nd	nd
C20:3n619	0.57–0.60	0.26–0.21	0.43–0.44	nd

1Capric acid

2Lauric acid

3Tridecanoic acid

4Myristic acid

5Myristoleic acid

6Pentadecanoic acid

7Cis-10-pentadecanoic acid

8palmitic acid

9Palmitoleic acid

10Heptadecanoic acid

11Cis-10-heptadecanoic acid

12Stearic acid

13Oleic acid

14Elaidic acid

15Linoleic acid

16Linolelaidic acid

17Arachidic acid

18Cis-11,14-eicosadienoic acid

19Cis-8,11,14-eicosatrienoic acid

nd: not detected

(These numbers are the lowest and highest FAME contents).

There were no significant changes in the FAME profile, except for the culture with 10.0% whey, in which the percentage of C18:1n9c decreased by 70%. The fatty acid ester found at the greatest percentage was C16:0 (40.60–45.75%), followed by C20:0, C18:2n6c and C18:1n9c. Prates et al. [47] reported that palmitic acid was predominant in the fatty acid profile of S. platensis. This acid is an important source of energy in infant feeding since breast milk contains 20% to 30% of this fatty acid. However, saturated fatty acids have been associated with an increased risk of cardiovascular disease in adults [48]. Palmitic acid is widely used in the pharmaceutical, cosmetic and surfactant industries and can be successfully applied industrially [49].

The total phenolic content and antioxidant activity of the S. platensis biomass were quantified as shown in Tables 5 and 6:

Table 5

Total extractable phenolic content.

Biomass	% Whey
	0.0	2.5	5.0	10.0
Phenolic compounds (mg gallic acid/g)	33.20a±0.92	30.60b±0.43	33.23a±0.36	22.37c±0.14

Different letters indicate significant differences for different treatments (p < 0.05).

Table 6

Antioxidant activity of S. platensis by the ABST•+ (2,2'-azino-bis (3-ethylbenzothiazolin)-6-sulfonic acid) free radical scavenging method and by FRAP ferric reduction.

Whey %	FRAPsf (μmol FS/g)	FRAPtx (μmol Tx/g)	ABTS•+ (μmol Tx/g)
0.0	35.35a±0.87	20.68a±0.51	17.67a±0.52
2.5	15.25b±0.08	8.95b±0.05	14.73b±0.14
5.0	15.39b±0.10	9.03b±0,06	14.62b±0.45
10.0	14.54b±0.14	8.54b±0,08	11.64c±0.14

FRAP (ferric reducing antioxidant power; ABTS (2,2'-azino-bis(3-ethylbenzothiazolin)-6-sulfonic acid). Different letters indicate significant differences for different treatments (p < 0.05).

S. platensis presented a high phenolic compound content (22.37–33.23 mg gallic acid/g) when compared to that reported in other studies by Sousa and Silva [50] (1.65 mg/g), Parisi et al. [51] (4.22–4.41 mg/g), and Colla et al. [52] (0.0024–0.0049 mg/g). There were no significant differences in the phenolic compound concentrations when autotrophic and mixotrophic cultures were compared, except for a reduction of 33.0% in the culture with 10.0% whey. The phenolic compound production in this study was probably potentiated as a function of the irradiance of the cultures (238 μmol m-2 s-1), according to a study conducted by Kepekçi and Saygideger [53]. Although there are no previous studies reporting the influence of mixotrophic cultivation on microalgae antioxidant synthesis, some studies have reported that environmental stresses such as exposure to metals [45] or stress by exposure to UV light [54] increased the synthesis of phenolic compounds.

The production of antioxidants depends on the cultivation conditions and the extraction method for quantification. In general, the antioxidant activity of the mixotrophic cultures decreased when compared to that of the autotrophic culture. The antioxidative activity in the autotrophic culture increased in the following order: FRAPsf>FRAPTx>ABTS•+, while the same performance was observed for all assays in the mixotrophic cultures, regardless of the whey content in the growth medium; therefore, the antioxidant activity was similar when evaluated by ABTS•+ and FRAPsf and less when evaluated by the FRAPtx method. The antioxidant activity of S. platensis obtained by the ABTS•+ method (11.68–17.67 μmol Tx/g) was lower than the values reported by Kepekçi & Saygideger [53] (31.41–54.16 μmol Trolox g−1). The value observed with the FRAPtx method from the autotrophic culture (20.68 μmol Tx/g) was higher than that found by Hossain et al. [55] (8.81 μmol Tx/g). It is likely that the inhibition of oxidative stress resulted in the generation of free radical species in the cells, which reacted and decreased antioxidant synthesis, possibly because the medium provides favorable conditions for the organisms and has no influence on promoting antioxidant production [3]. Goiris et al. [3] observed a reduction in the antioxidants of microalgae biomass when nitrogen-limited medium was used. They concluded that nutritional stress is not an effective strategy for improving the overall antioxidant content in microalgae.

The results of the present study support that the mixotrophic cultivation of S. platensis with 5% whey could be a viable strategy for the production of phenolic compounds and/or antioxidant compounds with a relatively low cost-optimized process since the biomass productivity was higher in this condition than at lower whey cultivation conditions. No antioxidant activity data were found in the literature in similar culture conditions as performed in this study. Many studies have evaluated the antioxidant capacity of foods supplemented with S. platensis, such as wheat bread dough [48], cookies [49] and yogurt [56], among others. They observed that the antioxidant capacity of all foods was higher when supplemented with S. platensis.

Conclusions

The biomass production, biochemical composition and antioxidant capacity of the mixotrophic S. platensis microalgae cultured in growth medium with different cheese whey contents were evaluated. In general, the mixotrophic cultivation increased the biomass and carbohydrate productivity and decreased the antioxidant capacity, protein and lipid productivity; however, there were no significant differences in phenolic compound content between the cultivation conditions, except for that of the culture with 10.0% whey. The maximum cell concentration (2.98 g/L) was obtained in the experiment with the addition of 5.0% whey. There was a 70% and 76% increase in biomass production and carbohydrate content, respectively when compared to those of the control. Under these culture conditions, the increase in carbohydrate production in S. platensis indicated that this species is a potential biomass source for the production of bioethanol. These data help to explain the microalgal growth and its chemical composition and enhance our understanding of how to improve the performance of mixotrophic cultivation. Overall, the results support the mixotrophic cultivation of S. platensis as a viable strategy to reduce the production costs of biomass and carbohydrates and simultaneously contribute to mitigating the environmental problems caused by eliminating whey in the dairy industry. If the cost of microalgae production can be optimized, bioethanol might be obtained in large-scale production combined with wastewater treatment and carbon sequestration.

Supporting information.

(DOCX)

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12 Jun 2019

PONE-D-19-15520

Mixotrophic cultivation of Spirulina platensis in dairy wastewater: effect on the production of biomass, biochemical composition and antioxidant capacity

PLOS ONE

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The manuscript has no page neither line numbers. Reviewers have difficulties to track and manage the manuscript if page and line numbers do not exist. The text has too many paragraphs, especially in the Introduction section. Can you please compact it? I also noticed excessive number of references.

It is a common practice to use abbreviation of the species genus, such as S. platensis, once you already give the full species binomial name in the first mention.

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10 Jul 2019

3. Additional Editor Comments:

3.1. The manuscript has no page neither line numbers. Reviewers have difficulties to track and manage the manuscript if page and line numbers do not exist.

We agree with no page neither line numbers in the manuscript may be confusing for the reviewers. We added page and line numbers to the manuscript.

3.2. The text has too many paragraphs, especially in the Introduction section. Can you please compact it? I also noticed excessive number of references.

We agree with the text has too many paragraphs and references in the manuscript. We reduced paragraphs from the Introduction and Results and Discussion. The references were reduced from 75 to 59.

3.3. It is a common practice to use abbreviation of the species genus, such as S. platensis, once you already give the full species binomial name in the first mention.

The name Spirulina platensis was abbreviated to S.platensis in the text body of the manuscript.

3.4. While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

We uploaded our figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool as suggested.

Submitted filename: Response to Reviewers.doc

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30 Jul 2019

PONE-D-19-15520R1

Mixotrophic cultivation of Spirulina platensis in dairy wastewater: effect on the production of biomass, biochemical composition and antioxidant capacity

PLOS ONE

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Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Reviewers raised major concerns about the general manuscript structure as well as the statistical significance of the presented differences in the results section. More comments are listed below.

==============================

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Reviewer #1: (No Response)

Reviewer #2: (No Response)

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Reviewer #2: Yes

**********

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Reviewer #2: No

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Reviewer #2: No

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Highlight: Not provided

Graphical abstract: Not provided

General remarks: This manuscript focus on the growth of S. platensis microalgae using a pollutant by-product called cheese whey for high biomass and carbohydrate production. The used of English in this manuscript is poor and require to further amend the language. There is a lot of work needed to further improve this manuscript (e.g., structure arrangement, language, unnecessary introduction) My recommendation is not to accept this paper for now as there is still lack of assurance and required amendment in this manuscript. I hope my following comments world improve the following manuscript.

COMMENTS:

The used of English and grammar were poor. This section is not constructive enough in this section

- Page 3, Line 63 – 65. Please read and cite some suggested paper for these biomolecules.

1. Khoo, K. S., Lee, S. Y., Ooi, C. W., Fu, X., Miao, X., Ling, T. C., & Show, P. L. (2019). Recent advances in biorefinery of astaxanthin from Haematococcus pluvialis. Bioresource technology, 121606.

2. Ren, H. Y., Xiao, R. N., Kong, F., Zhao, L., Xing, D., Ma, J., ... & Liu, B. F. (2019). Enhanced biomass and lipid accumulation of mixotrophic microalgae by using low-strength ultrasonic stimulation. Bioresource technology, 272, 606-610.

Also, please remove “bioactive compounds”.

- Page 4. Line 90 – 104, Please combine all into one paragraph.

-Why is such concentration (2.5%, 5.0% and 10%) of cheese whey deployed for the culture medium? Why not (2.5%, 5.0% 7.5% and 10%?). Please give clarification for this. By doubling up the concentration is difficult for optimization.

- Page 9, Line 266 – 269, the English used is too poor, please rephrase.

- Based on Figure 1, why did the author stop at Day 17 and state it’s the maximum growth? Why not increase to Day 20 to see if there is any growth?

- There is a lot of new paragraph for each section. Advise author to keep 1-3 paragraph is sufficient enough.

Page 10, Line 295 – 298, This paragraph does not make any sense and clueless on discussion. Please revise.

- I realize that the discussion part is too long and will get lost during reading. Advise author to revise overall of this manuscript. Keep it short and simple for the reasoning.

- Page 13, Line 376, what species? Please state it.

Page 17, Line 462 – 488. Combine all into one paragraph or remove it. It is too lengthy and not necessary for this result and discussion section. Much more to introduction.

- Please revise the structure of the overall manuscript. There are still too many new opening paragraphs which does not seem to be important.

- For Figure 2, why is the error bar for 10% whey cheese is so large. Some were small and some were large. This shows the inconsistency for the data collected.

Reviewer #2: The authors used an experimental approach and determined the biochemical composition of the algae, in addition to biomass yields, specific growth rates, and antioxidant activity. The authors compared their results to those found in other studies, providing plenty of context. They found that Spirulina platensis can reach higher biomass yields and specific growth rates when clarified cheese whey is added to the growth medium, and that carbohydrate content increases. Protein, lipid, and antioxidant contents decreased, however, which affects which products can be made from the algae. Overall the cheese whey would be effective for improving biomass production and reducing cultivation costs for algae products that need high carbohydrate costs.

One major way the study could be improved is by performing statistical analyses, which are not described in the methods section. However, results throughout the abstract, results, and discussion sections are often described as significant, even though it appears statistical tests were not performed. If statistical analyses are not going to be performed, the authors should be transparent about this, and only use qualitative descriptors to present the results. It appears that all data underlying results of the study are not available in the supplementary information (e.g., data for each of the control and treatment replicates that are used to provide the average and standard deviations in tables and figures). The authors should further clarify how their current manuscript complies with the journal’s data availability policy, or should include more of the experimental data in supporting documents or ideally in an online public data repository (e.g., Figshare). Lastly, the intended meaning of the manuscript writing is mostly understandable, but further editing is needed to improve readability.

Minor and specific comments are described below:

Abstract - It would be helpful to state in the abstract what the intended product from this alga is, and what characteristics are necessary (e.g. high carbohydrate content). This would help the reader evaluate whether the summarized results (e.g., increased carbohydrates and decreased lipids under mixotrophic conditions) are advantageous or disadvantageous for the intended product. Since the authors found that protein and antioxidants decreased under mixotrophic conditions, perhaps there should also be a statement about what specific types of products the algae can be used for after growing with cheese whey (i.e., products that just need carbohydrates).

Line 48: This sentence could be improved for clarity. Does the description “relative to the culture” mean “relative to the autotrophic culture”?

Line 73: Please clarify what “light invasion” means. Does this mean photoinhibition?

Line 102: It may be helpful to specify in the Introduction which biochemical components are advantageous for which algae products. For example, which products would need high carbohydrates and antioxidants, and which products would need high lipids. Please also define phenolic compounds and what product they are advantageous for.

Line 119: Please state if the culture was axenic (did not contain bacteria) or not.

Line 122: Please specify what type of percentage this means (e.g., volume per volume or mass per volume). Were nutrient concentrations the same across treatments and the control, even if different percentages of cheese whey were added?

Line 161: Does the added cheese whey affect optical density of the medium? Please explain how you used different standard curves because of this fact, and that they are available in the Supporting Information. (I believe Figure S2 is for the 2.5% whey treatment, but the caption says 5% whey).

Line 169: Here and in the Results section, the specific growth rate is often referred to as the maximum specific growth rate, which could be incorrect. Based on the description in the methods section, the authors are calculating the specific growth rate based on data measured in the experiment, not the maximum specific growth rate, which would require measuring many specific growth rates at different substrate concentrations.

Line 172: Writing out these equations for specific growth rate seems unnecessary, the authors could simply that the specific growth rate was calculated as the slope of the natural log of biomass concentration versus time during exponential phase, when the correlation coefficients of these two variables was above 0.98. Please also state which days were used to calculate the specific growth rate, since based on Figure 1 it does not look like the natural log of biomass versus time would be linear for the entire duration of the experiment.

Line 183-184: The description of the polynomial function seems unclear. Did the authors just calculate the productivity for each day compared to day zero using Eq. 2, or did they use another function to compare productivity between all different days?

Line 191: Could the authors briefly state how total nitrogen was measured?

Line 225: Please explain why two antioxidant methods were used. Was it to cross-check the results?

Table 1: How were these variables measured for the whey? Should they be included in the methods section?

Table 2: As stated previously, this variable is the specific growth rate, not the maximum specific growth rate, based on the calculation method described in the methods section. This table should also include standard deviations based on the triplicates.

Line 318: Authors are referring to a figure of productivity, so should clarify that they mean productivity and not specific growth rate.

Figure 4: Please include a y-axis label.

Table 4: Please explain these numbers, are they the lowest and highest fatty acid content (% of total fat?) of the three replicate cultures?

Please discuss the implications of having a higher ash content and lower antioxidant activity when whey is added to the medium at optimal percentages (5%). Does this change which industries would be able to use the algae?

**********

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Reviewer #1: No

Reviewer #2: Yes: Sarah E. Loftus

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13 Sep 2019

Dear Reviewers,

Response to reviewers

We would like to thank the reviewers for careful and thorough reading of this manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of this manuscript. Our response follows (the reviewer’s comments are in italics and blue color).

Reviewer #1:

Highlight: Not provided

Graphical abstract: Not provided

General remarks: This manuscript focus on the growth of S. platensis microalgae using a pollutant by-product called cheese whey for high biomass and carbohydrate production. The used of English in this manuscript is poor and require to further amend the language. There is a lot of work needed to further improve this manuscript (e.g., structure arrangement, language, unnecessary introduction) My recommendation is not to accept this paper for now as there is still lack of assurance and required amendment in this manuscript. I hope my following comments world improve the following manuscript.

We agree with the reviewer that sufficient care was not taken in the original manuscript vis-à-vis the English and interpretations. In the revised manuscript, the English and grammatical errors are corrected and the interpretations and flow have been significantly improved. The translation of the manuscript to English has been corrected and revised by ECB – English Consulting Brazil (Annex A).

COMMENTS:

1) The used of English and grammar were poor. This section is not constructive enough in this section:

- Page 3, Line 63 – 65. Please read and cite some suggested paper for these biomolecules.

1. Khoo, K. S., Lee, S. Y., Ooi, C. W., Fu, X., Miao, X., Ling, T. C., & Show, P. L. (2019). Recent advances in biorefinery of astaxanthin from Haematococcus pluvialis. Bioresource technology, 121606.

2. Ren, H. Y., Xiao, R. N., Kong, F., Zhao, L., Xing, D., Ma, J., & Liu, B. F. (2019). Enhanced biomass and lipid accumulation of mixotrophic microalgae by using low-strength ultrasonic stimulation. Bioresource technology, 272, 606-610.

Also, please remove “bioactive compounds”.

Response: The entire paragraph has rewritten as per the reviewer’s suggestion including Line 63-65 (page 3) and these references have been added (current references 8 & 9) and briefly discussed in the text on p. 12

We have removed “Bioactive compounds”.

2) - Page 4. Line 90 – 104, please combine all into one paragraph.

Response: The entire section is now revised. The text has rearranged as per the reviewer’s suggestion as follows:

“Girard et al. [15] cultured Scenedesmus obliquus under mixotrophic conditions and demonstrated that this species presented higher specific growth rates and biomass yields in replacing 40% (v/v) of the standard ‘Bold’s basal medium’ (BBM) by whey. Salla et al. [2] studied the mixotrophic cultivation of S.platensis cyanobacterium in diluted Zarrouk growth medium with the addition of concentrated whey protein residues and high lactose levels. They observed that there was an increase in biomass and carbohydrate productivity by the species under study. Tsolcha et al. [16] developed a biological (algal) second cheese whey wastewater treatment system to generate renewable energy in the form of biodiesel, while simultaneously removing polluting nutrients and chemical oxygen demand. Recent studies demonstrate that S.platensis is also a promising bioethanol producer due to carbohydrate concentration in its biomass may reach 50% [2] and up to 60% [10] by changing the growth medium. In addition, the species may contain high content of antioxidant compounds being capable to add value to the process.”

3) Why is such concentration (2.5%, 5.0% and 10%) of cheese whey deployed for the culture medium? Why not (2.5%, 5.0% 7.5% and 10%?). Please give clarification for this. By doubling up the concentration is difficult for optimization.

Response: We appreciate the suggested from the reviewer. We agree with the reviewer that by doubling up the concentration is difficult for optimization in this study case and the optimization process has been one of the hot topics for the future of work since it was not the focus of this preliminary work. To examine the effect of whey addition on growth medium of S.platensis, the selected microalgae were triplicate cultured in mixotrophic conditions with 2.5%, 5.0% and 10.0% of cheese whey. The experimental logic for defining whey addition concentrations in the microalgae cultures was based on previous studies involving microalgae mixrotrophic cultivation (Salla et al. 2017; Mouther (2010)). Mouther (2010) showed that optimal cheese whey concentration for S. platensis mixotrophic cultivation was 3.0% and that concentration above 6% caused growth inhibition. Salla et al. 2017 evaluated S.platensis mixotrophic cultivation with residues obtained through the processes of ultra- and nanofiltration of whey at concentrations of zero, 1.25% and 2.5% (v/v). In this sense, it was observed that they evaluated the S.platenis mixotrophic cultivation by doubling up the concentration of the organic carbon source. Gao et al. 2019 examined the effect of initial TOC/TN (ratio of organic carbon to nitrogen) ratio of wastewater on the cultivation of microalgae, the selected microalgae were triplicate cultured in simulated wastewater with TOC/TN ratio of 0, 1, 3, 6, 12, 24 and 30, respectively.

References:

Salla ACV, Margarites AC, Seibel FI, Holz LC, Brião VB, Bertolin TE, et al. Increase in the carbohydrate content of the microalgae Spirulina in culture by nutrient starvation and the addition of residues of whey protein concentrate. Bioresour Technol. 2016;209: 133-141.

Mourthé K. Obtenção de biomassa de Arthrospira platensis (Spirulina platensis) utilizando do soro de leite. Tese de doutorado, Universidade Federal de Minas Gerais. 2010. Available from: http://www.bibliotecadigital.ufmg.br/dspace/handle/1843/BUOS-8EJQW3.

Feng Gao, Hong-Li Yang, Chen Li, Yuan-Yuan Peng, Yuan-Ming Guo. Effect of organic carbon to nitrogen ratio in wastewater on growth, nutrient uptake and lipid accumulation of a mixotrophic microalgae Chlorella sp. Bioresource Technology. 2019; 282:118-124.

4) Page 9, Line 266 – 269, the English used is too poor, please rephrase.

Response: The text (pag 9, line 266-269) has rewritten as per the reviewer’s suggestion as follows:

“The whey concentrations were based on previous studies: Mouther (2010) [26] showed that optimal cheese whey concentration for S. platensis mixotrophic cultivation was 3.0% and inhibition occurs at higher whey concentrations. Moreover, heterotrophic cultivation was inappropriate for S. platensis, possibly due to some inhibiting factor for this species present in the whey.”

5) Based on Figure 1, why did the author stop at Day 17 and state it’s the maximum growth? Why not increase to Day 20 to see if there is any growth?

Response: All cultures reached stationary phase after 17 days of cultivation. The microalgae were cultivated until the early stationary phase of growth, which is when carbohydrates are accumulated in the microalgal biomass according to Moura et al. 2006 and Salla et al. 2017. The stationary phase is often due to a growth-limiting factor such as the depletion of an essential nutrient, and/or the formation of an inhibitory product. Stationary phase results from a situation in which growth rate and death rate are equal. The number of new cells created is limited by the growth factor and as a result, the rate of cell growth matches the rate of cell death.

References:

Moura, A.M., Bezerra Neto, E., Koening, M.L., Leça, E.E.Chemical composition of microalgae in semi-intensive culture: Chaetoceros gracilis Schutt, Isochrysi sgalbana Parke and Thalassiosira weiss flogii (Grunow). Revista Ciência Agronômica. Volume 37, Issue 2, Pages 142-148

.Salla ACV, Margarites AC, Seibel FI, Holz LC, Brião VB, Bertolin TE, et al. Increase in the carbohydrate content of the microalgae Spirulina in culture by nutrient starvation and the addition of residues of whey protein concentrate. Bioresour Technol. 2016;209: 133-141.

6) There is a lot of new paragraph for each section. Advise author to keep 1-3 paragraph is sufficient enough.

Response: The entire manuscript is now revised. We have now completely rewritten this manuscript and have removed the superfluous and irrelevant discussions and related references as per the reviewer’s suggestion.

7) Page 10, Line 295 – 298, This paragraph does not make any sense and clueless on discussion. Please revise.

Response: (Page 10, “line 295 – 298”) This paragraph has been deleted as it was considered to be superfluous.

8) I realize that the discussion part is too long and will get lost during reading. Advise author to revise overall of this manuscript. Keep it short and simple for the reasoning.

Response: The entire manuscript is now revised. We have now completely rewritten this manuscript and have removed the superfluous and irrelevant discussions and related references as per the reviewer’s suggestion.

9) Page 13, Line 376, what species? Please state it.

Response: The name of specie has been mentioned as follows:

“…the basic chemical composition of S. platensis consists of proteins (50 - 70%), carbohydrates (15 - 25%)…”

10) Page 17, Line 462 – 488. Combine all into one paragraph or remove it. It is too lengthy and not necessary for this result and discussion section. Much more to introduction.

Response: In Page 17, the text (“Line 462 – 488”) has been deleted as per reviewer’s suggestion and part of the text was moved from results and discussion to introduction.

11) Please revise the structure of the overall manuscript. There are still too many new opening paragraphs which does not seem to be important.

Response: The entire manuscript is now revised. We have now completely rewritten this manuscript and have removed the superfluous and irrelevant discussions and related references as per the reviewer’s suggestion.

12) For Figure 2, why is the error bar for 10% whey cheese is so large. Some were small um erro experimental maior and some were large. This shows the inconsistency for the data collected.

Response: The monitoring of the growth of S. platensis cultures was performed by measuring the absorbance at λ = 670 nm in spectrophotometer (SP-22 Biospectro). A calibration curve was generated to relate the absorbance to the cell dry weight. The opacity of growth medium using cheese whey as organic carbon source increases with increasing whey concentration. The higher opacity of samples can make absorbance determination by spectrophotometry difficult increasing the experimental error but within acceptable limits. Moreover, the culture with 10% whey grew faster than the other cultures in the early days thus the high density of the cell is another factor that could increase experimental error by spectrophotometry requiring samples dilutions to maintain the linearity range of method. In Fig 2 appears that culture with 5% whey also showed a considerable standard deviation possibly for the same reasons. In this sense, is was expected that mixotrophic cultivation with 10% whey may lead to large experimental errors. Curves of dry weight biomass determined from optical density measurements from relevant study (Poddara et al. 2018) show that the standard deviation obtained from date of heterotrophic and mixotrophic growth were higher than those obtained with autotrophic growth.

Reference:

1.Nature Poddara, Ramkrishna Senb, Gregory J.O. Martin. Glycerol and nitrate utilisation by marine microalgae Nannochloropsis salina and Chlorella sp. and associated bacteria during mixotrophic and heterotrophic growth, Algal Research 33 (2018) 298–309.

Reviewer #2:

The authors used an experimental approach and determined the biochemical composition of the algae, in addition to biomass yields, specific growth rates, and antioxidant activity. The authors compared their results to those found in other studies, providing plenty of context. They found that Spirulina platensis can reach higher biomass yields and specific growth rates when clarified cheese whey is added to the growth medium, and that carbohydrate content increases. Protein, lipid, and antioxidant contents decreased, however, which affects which products can be made from the algae. Overall the cheese whey would be effective for improving biomass production and reducing cultivation costs for algae products that need high carbohydrate costs.

One major way the study could be improved is by performing statistical analyses, which are not described in the methods section. However, results throughout the abstract, results, and discussion sections are often described as significant, even though it appears statistical tests were not performed. If statistical analyses are not going to be performed, the authors should be transparent about this, and only use qualitative descriptors to present the results. It appears that all data underlying results of the study are not available in the supplementary information (e.g., data for each of the control and treatment replicates that are used to provide the average and standard deviations in tables and figures). The authors should further clarify how their current manuscript complies with the journal’s data availability policy, or should include more of the experimental data in supporting documents or ideally in an online public data repository (e.g., Figshare). Lastly, the intended meaning of the manuscript writing is mostly understandable, but further editing is needed to improve readability.

We agree with the reviewer that the study could be improved is by performing statistical analyses and it has been included in the work. All data underlying results of the study are now available in an online public data repository (Figshare)(DOI:10.6084/m9.figshare.9820181) and also in the supplementary information . In the revised manuscript, the English and grammatical errors are corrected and the interpretations and flow have been significantly improved. The translation of the manuscript to English has been corrected and revised by ECB – English Consulting Brazil (Annex A).

Minor and specific comments are described below:

1) Abstract - It would be helpful to state in the abstract what the intended product from this alga is, and what characteristics are necessary (e.g. high carbohydrate content). This would help the reader evaluate whether the summarized results (e.g., increased carbohydrates and decreased lipids under mixotrophic conditions) are advantageous or disadvantageous for the intended product. Since the authors found that protein and antioxidants decreased under mixotrophic conditions, perhaps there should also be a statement about what specific types of products the algae can be used for after growing with cheese whey (i.e., products that just need carbohydrates).

Response: Necessary correction has been incorporated as per reviewer’s suggestion as follows:

“Abstract: Mixotrophic cultivation of microalgae provides a very promising alternative for producing carbohydrate-rich biomass to convert into bioethanol and chemicals. It has been demonstrated that S. platensis may present high yields of biomass and carbohydrates when it is grown under mixotrophic conditions using cheese whey. However, there are no previous studies of this species to evaluate the influence of this culture system on the profile of fatty acids and the production of antioxidant compounds being extremely important for food and pharmaceutical applications as adding value to the process. S. platensis presented higher specific growth rates, biomass productivity and carbohydrate content under mixotrophic conditions, however the antioxidant capacity, protein and lipid content were lower than that of the autotrophic culture. The maximum biomass yield was 2.98 ±0.07 g/L in the growth medium with 5.0% whey. The phenolic compound concentration was the same for the biomass obtained in the autotrophic and mixotrophic conditions with 2.5% and 5.0% whey. The phenolic compound concentration no showed significant differences except in the growth medium with 10.0 % whey, which presented average values of 22.37±0.14 mg gallic acid/g. Mixotrophic cultivation of S. platensis using whey can be considered a viable alternative to reduce the costs of producing S. platensis biomass, shorten cultivation time and produce carbohydrates, as it does not require adding expensive substrates to the growth medium, while also taking advantage of cheese whey considered as a pollutant. Cultivation for S.platensis biomass production is the most widespread due to high biomass production capacity and it might be a promising bioethanol producer”.

2) Line 48: This sentence could be improved for clarity. Does the description “relative to the culture” mean “relative to the autotrophic culture”?

Response: This sentence has rewritten and the description “relative to the culture” was replaced by “relative to the autotrophic culture”.

3) Line 73: Please clarify what “light invasion” means. Does this mean photoinhibition?

Response: “Light invasion” has been deleted as per reviewer’s suggestion.

4) Line 102: It may be helpful to specify in the Introduction which biochemical components are advantageous for which algae products. For example, which products would need high carbohydrates and antioxidants, and which products would need high lipids. Please also define phenolic compounds and what product they are advantageous for.

Response: Thank you for your comment. This is a fair point. We have now included a new paragraph in the Introduction section, which addresses these issues.

4) Line 119: Please state if the culture was axenic (did not contain bacteria) or not.

Response: The culture was xenic and this information has been added in Material and methods section as per reviewer’s suggestion (Page X, Line 2-3) as follows:

“A xenic strain of Spirulina platensis was maintained under sterile conditions in test tubes with a temperature of 25°C and irradiance of 238 μmol.”

5) Line 122: Please specify what type of percentage this means (e.g., volume per volume or mass per volume). Were nutrient concentrations the same across treatments and the control, even if different percentages of cheese whey were added?

Response: Necessary correction has been incorporated as per reviewer’s suggestion as follows:

“…. For the mixotrophic cultures, the complete Zarrouk medium was supplemented with 2.5%(v/v), 5.0%(v/v) and 10%(v/v) buffalo mozzarella cheese whey….”

6) Line 161: Does the added cheese whey affect optical density of the medium? Please explain how you used different standard curves because of this fact, and that they are available in the Supporting Information. (I believe Figure S2 is for the 2.5% whey treatment, but the caption says 5% whey).

Response 1: The monitoring of the growth of S. platensis cultures was performed by measuring the absorbance at λ = 670 nm in spectrophotometer (SP-22 Biospectro). A calibration curve was generated to relate the absorbance to the cell dry weight. The opacity of growth medium using cheese whey as organic carbon source increases with increasing whey concentration. The higher opacity of samples may make absorbance determination by spectrophotometry difficult increasing the experimental error but within acceptable limits. In this sense, different standard curves were used for all cultures with a specific blank for each composition of growth medium.

Response 2: Figure S2 is for the 2.5% whey treatment and the caption has been corrected.

7) Line 169: Here and in the Results section, the specific growth rate is often referred to as the maximum specific growth rate, which could be incorrect. Based on the description in the methods section, the authors are calculating the specific growth rate based on data measured in the experiment, not the maximum specific growth rate, which would require measuring many specific growth rates at different substrate concentrations.

Response: We replaced the term (the maximum specific growth) for the correct one (the specific growth rate) as per reviewer’s suggestion.

8) Line 172: Writing out these equations for specific growth rate seems unnecessary, the authors could simply that the specific growth rate was calculated as the slope of the natural log of biomass concentration versus time during exponential phase, when the correlation coefficients of these two variables was above 0.98. Please also state which days were used to calculate the specific growth rate, since based on Figure 1 it does not look like the natural log of biomass versus time would be linear for the entire duration of the experiment.

Response 1: The equation for specific growth rate has been deleted and the topic has rewritten as follows:

“The growth rate (μ d-1) of S. platensis was calculated as the slope of the natural log of biomass concentration versus time during exponential growth phase, when the correlation coefficients of these two variables was above 0.98 [20].

Response 2: We agree that the natural log of biomass versus time was not linear for the entire duration of the experiment. The specific growth rates were calculated only in the exponential phase of growth. The duration time of exponential growth phase was different for each evaluated cultivation condition and it was considered in the calculations.

9) Line 183-184: The description of the polynomial function seems unclear. Did the authors just calculate the productivity for each day compared to day zero using Eq. 2, or did they use another function to compare productivity between all different days?

Response: We just calculated the productivity for each day compared to day zero using Eq. 2. Necessary changes have been made and this part is now rewritten as follows:

“The maximum biomass productivity PXmax (g.L.dia-1), were evaluated according to Eq. (1), where Xmax is biomass maximum concentration at time t, Xo is the biomass concentration at time zero, and t is time.

P_(Xmax=) (X_max-Xo)/t (1)

The productivity was calculated for each day compared to day zero. The maximum value obtained was defined as the maximum biomass productivity.”

10) Line 191: Could the authors briefly state how total nitrogen was measured?

Response: We briefly stated how total nitrogen was measured in material and methods section as follows:

“Kjeldahl nitrogen was measured (TECNAL digester block, model TE-040; TECNAL Kjeldahl TE-0364 distillation unit) using 200 mg samples of the dry biomass. The sample was first digested using concentrated sulfuric acid digester solution following a progressive heating ramp, and then distilled using boric, sodium hydroxide and indicator solutions. After distillation, the obtained solution was titrated with 0.1 N chloridic acid (HCL). The total protein content was calculated by multiplying the value for total nitrogen by the conversion factor of 4.78 [21].”

11) Line 225: Please explain why two antioxidant methods were used. Was it to cross-check the results?

Response: Antioxidants are compounds capable to either delay or inhibit the oxidation processes which occur under the influence of atmospheric oxygen or reactive oxygen species. They are used for the stabilization of polymeric products, of petrochemicals, foodstuffs, cosmetics and pharmaceuticals. The various analytical methods of evaluation of the antioxidant capacity fall into distinct categories. Due to the different types of free radicals and their different forms of action in living organisms, there is no a simple and universal method to quantify antioxidant activity. In this sense, different antioxidant methods were tested in this work. The ABTS method: The ABTS cation radical (ABTS•+) is formed by the loss of an electron by the nitrogen atom of ABTS (2,2’-azino-bis(3- ethylbenzthiazoline-6-sulphonic acid)). In the presence of Trolox (or of another hydrogen donating antioxidant), the nitrogen atom quenches the hydrogen atom, yielding the solution decolorization. The FRAP (ferric reducing antioxidant power) method: The FRAP (ferric reducing antioxidant power) method relies on the reduction by the antioxidants, of the complex ferric ion-TPTZ (2,4,6-tri(2-pyridyl)- 1,3,5-triazine). The binding of Fe2+ to the ligand creates a very intense navy blue color.

These references show different methods to evaluate antioxidant activity of biomass:

References:

Zhendong Yang, Weiwei Zhai. Identification and antioxidant activity of anthocyanins extracted from the seed and cob of purple corn (Zea maysL.) Innovative Food Science and Emerging Technologies 11 (2010) 169–176.

2. Sonia Milla, Edgar Uquiche. Antioxidant activity of supercritical extracts from Nannochloro psisgaditana: Correlation with its content of carotenoid sand to copherols. J.of Supercritical Fluids 111 (2016) 143–150.

12) Table 1: How were these variables measured for the whey? Should they be included in the methods section?

Response: These methods have been included in the in the methods section

“Clarified buffalo cheese whey samples were analyzed for percentage of total solids, total protein, fat, lactose, and moisture using the DairySpec (Bentley Instruments Inc, Chska Minnesota, USA). The equipment was calibrated using buffalo cheese whey calibration sample with different concentration ranges.”

13) Table 2: As stated previously, this variable is the specific growth rate, not the maximum specific growth rate, based on the calculation method described in the methods section. This table should also include standard deviations based on the triplicates.

Response: We replaced the term (the maximum specific growth) for the correct one (the specific growth rate). We included standard deviations based on the triplicates (Xmax(g.L-1), Pmax(g.L-1.day-1), µ (day-1)) in Table 2 as per reviewer’s suggestion.

14) Line 318: Authors are referring to a figure of productivity, so should clarify that they mean productivity and not specific growth rate.

Response: Necessary changes have been made and this part is now rewritten as follow:

“The culture with 10.0% whey showed double the specific growth rate of the culture with 5.0% and it had the highest biomass productivity on the 4th day (Fig 2).”

15) Figure 4: Please include a y-axis label.

Response: Necessary changes have been made and y-axis label is included in Figure 4.

16) Table 4: Please explain these numbers, are they the lowest and highest fatty acid content (% of total fat?) of the three replicate cultures?

Response: A gas chromatograph (Thermo Scientific-CG/FID-FOCUS) with flame ionization detector (FID) and Supelco SP2560 capillary column (100m x 0.25mm x 0.2μm) was used to determine the fatty acid profile. This method is accurate but it is costly so we chose to do these analyzes in duplicate. These numbers are the lowest and highest (FAMEs) content.” (Percentage of total FAMEs).

This section has been rewritten as follows:

‘’ The fatty acid methyl esters (FAMEs) profiles of S. platensis are shown in Table 4.

Table 4 - The fatty acid methyl esters (FAMEs) profile (percentage of total FAMEs) present in S. platensis biomass cultured in control medium and mixotrophic medium with the addition of buffalo cheese whey (These numbers are the lowest and highest (FAMEs) content).”

17) Please discuss the implications of having a higher ash content and lower antioxidant activity when whey is added to the medium at optimal percentages (5%). Does this change which industries would be able to use the algae?

Response: The maximum cell concentration (2.98 g/L) was obtained in experiment with addition of %5 whey. There has been a 70% and 76% increase in biomass production and carbohydrates content, respectively. With this culture conditions, the increase in carbohydrate production in S.platensis indicated this specie as potential biomass for production of bioethanol. These data would help to explain the microalgal growth and chemical compositions, and enhance the knowhow on improving the performance of mixotrophic cultivation. Moreover, the results of present study support that mixotrophic cultivation of S.platensis with 5% whey could be a viable strategy to the production of phenolic compounds and/or antioxidant compounds by lower cost optimized process since the biomass productivity was higher in this condition.

S. platensis is a species that has been studied in thermal degradation processes for producing bio-oil, biochar and gas. Some studies have shown that bio-oil produced from microalgae are more stable, have lower oxygen content and higher calorific value than lignocellulosic bio-oil. However, bio-oil presents high nitrogen content that is undesirable because it deactivates acid catalysts used for co-processing crude oil in refineries and emits NOx during combustion. The mixotrophic culture of S. platensis using cheese whey in addition to increasing biomass production may decrease the processing cost and produce low protein biomass, thus being favorable to applications in thermal degradation processes for producing biofuels. Huang et al. 2016 evaluated the bio-oil production from hydrothermal liquefaction (HTL) of high-ash microalgae. This work focused on high-ash microalgae, a representative of algae with practical importance which were widely cultivated in wastewater or other nitrogen unlimited environment. The microalgae feedstocks had relatively high ash contents (28.92 wt%, 35.23 wt%). This study demonstrated the feasibility of applying HTL to produce bio-oil from high-ash microalgae, and the findings on bio-oil properties and transfer behavior of carbon and nitrogen supplied useful information for downstream utilization. Liu et al. 2019 evaluated the hydrothermal carbonization (HTC) of natural microalgae containing a high ash content. The char generated from HTC is called as hydrochar. The hydrochar derived from microalgae has large irregular porosity aggregates and higher cation exchange capacity, which is different from the lignocellulose biochar. The higher nutrient content of nitrogen, ash and inorganic elements are beneficial in agriculture. Furthermore, hydrochar containing high aromaticity structures has the potential to be solid fuel. Many researches show that mineral salt as catalysts have been proven to play important roles in the yield and quality of products. The results of this study revealed that natural microalgae can be utilized by hydrothermal carbonization to generate renewable fuel resources.

References:

Yanqin Huang, Yupeng Chen, Jianjun Xie, Huacai Liu, Chuangzhi Wu. Bio-oil production from hydrothermal liquefaction of high-protein high-ash microalgae including wild Cyanobacteria sp. And cultivated Bacillariophyta sp. Fuel. 2016;183:9-19.

Huihui Liu, Yingquan Chen, Haiping Yang, Francesco G. Gentili, Hanping Chen. Hydrothermal carbonization of natural microalgae containing a high ash content. Fuel. 2019;249: 441-448.

Submitted filename: Response to reviewers (Revised Manuscript).docx

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19 Sep 2019

PONE-D-19-15520R2

Mixotrophic cultivation of Spirulina platensis in dairy wastewater: effect on the production of biomass, biochemical composition and antioxidant capacity

PLOS ONE

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8 Oct 2019

Response to Editor

Editor’s Comment: 1..Please upload a copy of Figures 1,2,3 which you refer to in your text . Or if the figure is no longer to be included as part of the submission please remove all reference to it within the text.

The copy of Figures 1, 2 and 3 have been uploaded.

Best,

Bruna Chagas

Submitted filename: Response to reviewers (Revised Manuscript).docx

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10 Oct 2019

Mixotrophic cultivation of Spirulina platensis in dairy wastewater: effect on the production of biomass, biochemical composition and antioxidant capacity

PONE-D-19-15520R3

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17 Oct 2019

PONE-D-19-15520R3

Mixotrophic cultivation of Spirulina platensis in dairy wastewater: effects on the production of biomass, biochemical composition and antioxidant capacity

Dear Dr. Chagas:

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