Valorisation of buttermilk in mixotrophic and heterotrophic conditions by using the acidophilic strain Galdieria sulphuraria

doi:10.21203/rs.3.rs-2762606/v1

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Valorisation of buttermilk in mixotrophic and heterotrophic conditions by using the acidophilic strain Galdieria sulphuraria

https://doi.org/10.21203/rs.3.rs-2762606/v1

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The growth of the polyextremophile red microalga Galdieria sulphuararia ACUF064 under mixotrophy and heterotrophy was evaluated in a lab-scale stirred photobioreactor, using buttermilk as carbon source. Beforehand, G. sulphuraria growth on glucose, galactose and lactose, was evaluated. The final biomass concentration of G. sulphuraria was about 2 and 1.5 times lower in mixotrophy and heterotrophy, respectively, when G. sulphuraria ACUF064 was grown on lactose rather than in glucose or galactose. No significant difference was observed in mixotrophy between growth in glucose and galactose. C-phycocyanin content was about 1.6 and 1.37 times higher in mixotrophy on lactose than on galactose and on glucose. In heterotrophy, C-phycocyanin content, on lactose was 1.57 and 1.56 times higher than values detected in glucose and galactose, respectively. The preliminary mixotrophic flask experiment with different buttermilk dilutions showed that the highest biomass production was obtained at 1:2.5 dilution rate, corresponding to 40% v/v of butter milk and to 2.5 gC L^− 1. On buttermilk, the highest biomass production was obtained in mixotrophy, at 1:2.5 dilution rate, in a 13 L photobioreactor where the algal productivity was 0.55 g L^− 1 d^− 1, and no contamination occurred at pH 1.6.

Polyextremophile microalgae

Galdieria sulphuraria

mixotrophy

buttermilk valorization

phycocyanin

dairy effluent valorisation

The dairy industry is among the most polluting agri-food activities due to their large water consumption and the consequently large volumes of wastewater produced (Raghunath et al., 2016). Reducing waste or adding value to by-products in milk processing is a pivotal strategy to enhance industrial food-related sustainability (Olabi et al. 2015).Buttermilk is one of the most important by-products of dairy industries and is made of the aqueous phase released during the churning of cream in butter processing. As recently reported (Ali, 2019), in 2015, the buttermilk production in the European Union was estimated in approximately 2 million tonnes and its consumption in the United States increased to 0.2 gallons (0.7571 L) per capita. Buttermilk is generally classified into two sub-products: sweet buttermilk (or cultured) and whey buttermilk (Ali, 2019). The chemical composition of sweet buttermilk is quite similar to that of skimmed milk for lactose (48.7–53.8%) and protein (31.5–33.1%) and presents a lower fat (5.7–13.1%) content compared to buttermilk from cheese (Lambert et al. 2016; Sodini et al., 2006).

Although several studies have largely reported the potential use of buttermilk in several food productions, such as in yogurt, bakery or cheese (Dewettinck et al. 2008; Vanderghem et al. 2010; Fuller et al. 2013; Gassi et al. 2016), its application still remains limited (Bahrami et al. 2015). In cheese, the increase of sweet-cream buttermilk content significantly modifies its composition, resulting in a softer and moister curd. Cheese containing more than 25% of sweet-cream buttermilk are not acceptable from a sensorial point of view. Moreover, the buttermilk is usually used as powder in food industry, which implies the application of evaporation and spray drying processes that increase the production means. Due to the large amount of organic compounds, buttermilk represents a valuable carbon source useful in bioprocessing. Results obtained by Vigliengo and Reinheimer (2009), revealed that whey or buttermilk are suitable medium ingredients for biomass production of thermophilic lactic acid bacteria.

Some microalgae can grow with or without light on such organic substrate, and their cultivation represents a valid strategy for the valorisation of this by-product (Jain et al. 2022). In this scenario, the red extremophile species belonging to Galdieria genus are among the most suitable microalgae. Galdieria sulphuraria is a polyextremophile unicellular microalga able to grow at temperatures up to 56°C and at a pH between 0.5 and 4. Additionally, G. sulphuraria presents a versatile metabolism, growing autotrophically, heterotrophically or mixotrophically (Abiusi et al. 2022b).

The mixotrophic metabolism consists in the combined use of an organic carbon and light for growth. In short, the carbon dioxide released from substrate catabolism can be reused as a carbon source for intracellular anabolic reactions. As recently observed (Curien et al. 2021; Abiusi et al. 2021, 2022a; Canelli et al. 2023), the mixotrophic strategy showed higher biomass productivity compared to autotrophy (doubled), and a higher pigmentation compared to heterotrophic conditions (Sloth et al. 2006). In fact, mixotrophic growth has been proposed to overcome autotrophy limits for biomass productivity (Tredici 2010). G. sulphuraria is being able to use several carbon sources (up to 50 different) (Gross and Schnarrenberger 1995), including lactose, showing a broad metabolic versatility (Tischendorf et al., 2007; Zimermann et al., 2020). The right combination between mixotrophic pathways and versatile metabolism results very interesting for the development of bioremediation processes. On the other side, heterotrophy is a tool to overcome the costly light-based processes of microalgae. The heterotrophic production results in higher biomass concentrations and higher growth rates, therefore yielding higher removal rates (Barros et al. 2019). However, contamination by bacteria and fungi is a notable issue when the medium contains sources of organic carbon. The cultivation of extremophilic microalgae is a strategy that can prevent the growth of bacteria or other predators, improving the efficacy of microalgae-based treatment (Abiusi et al. 2022b).

Moreover, the growing interest in G. sulphuraria, lies on the production of the high-value blue phycobiliproteins C-phycocyanin (C-PC), allophycocyanin, and chlorophyll (Albertano et al. 2000). Phycocyanins are used as dyes in diagnostic histochemistry, as colorants in cosmetics and food industry and, as therapeutic agents due to their antioxidant properties in the pharma sector (Pagels et al. 2019). Commercially, C-PC is produced in phototrophic cultures of the cyanobacterium Spirulina (Arthrospira) platensis by using sunlight as an energy source, meeting all issues regarding to large scale production which are already overcome by mixotrophic Galdieria cultures. Due to its interesting chemical and nutritional characteristics, G. sulphuraria could potentially be used as an ingredient for human consumption (Graziani et al. 2013). The aim of the present study was to assess the valorisation of buttermilk by growing G. sulphuraria ACUF064 both mixotrophically and heterotrophically. Firstly, the effect of lactose, glucose and galactose on growth and C-PC accumulation was assessed both under mixotrophy and heterotrophy. Secondly, the optimal dilution rate of buttermilk in synthetic medium was evaluated in lab-scale batch experiments. Finally, the growth of G. sulphuraria and its phycocyanin content were evaluated both mixotrophically and heterotrophically in a 13 L photobioreactor.

Microalgal strain, media and cultivation conditions

G. sulphuraria ACUF064, kindly provided by “Federico II” Naples University, was cultivated photo-autotrophically in 250 mL Erlenmeyer flasks containing 100 mL of modified Allen’s medium (Allen and Stanier, 1968). The medium used for flask and reactor cultivation contained the following components (mol L^− 1): 12.2⋅10 ^− 3 H₃PO₄, 80.0⋅10^− 3 (NH₄)₂SO₄, 6.5⋅10^− 3 MgSO₄⋅7H₂O, 4.7⋅10^− 4 CaCl₂⋅2H₂O, 6.3⋅10^− 4 FeNaEDTA, 0.2⋅10^− 3 Na2EDTA⋅2 H₂O, 1.7⋅10^− 3 NaCl, 8.1⋅10^− 3 KCl, 8.0⋅10^− 4 H₃BO₃, 8.1⋅10^− 5 MnCl₂⋅4H₂O, 8.2⋅10^− 5 ZnCl₂, 3.2⋅10^− 5 CuSO₄⋅5H₂O, 1.7⋅10^− 5 Na₂MoO₄⋅2H₂O and 1.7⋅10^− 5 CoCl₂⋅6H₂O. pH was adjusted to 1.6 with 2 M H₂SO₄.

Axenic autotrophic stock cultures were incubated in 250 mL flasks containing 100 mL of culture, in an incubator (Multitron II, Infors HT, Switzerland) operated at 37°C, 2% v/v CO₂, 60% of humidity, 125 rpm, under a photon flux density of 100 µmol m^− 2 s^− 1 and with a photoperiod 16:8 (day:night). These cultures were used for the experiments described below.

Carbon Source Flask Experiments

G. sulphuraria ACUF064 cultures, containing either lactose, galactose or glucose (5 gC L^− 1) were grown mixotrophically and heterotrophically. In particular, a 10 day pre-adaptation period for each carbon source was conducted to adapt G. sulphuraria from autotrophy to mixotrophy and to heterotrophy. Mixotrophic flask experiment was carried out into 250 mL Erlenmeyer flasks containing 150 mL of modified Allen’s medium. Flaks were inoculated at a 0.2 OD₇₅₀ with the pre-acclimated culture and incubated at same conditions reported above. The same conditions were used for heterotrophic experiment, for which the flasks were wrapped in aluminium foil. For both experiments, OD₇₅₀ and OD₆₂₀ were measured in samples taken after 0, 22, 27, 46, 70, 75 and 94 hours from inoculation. Dry weight (gx L^− 1) and cell count (cells mL^− 1) determinations were performed in samples taken after 0, 22, 70 and 94 hours from inoculation. An aliquot of 15 mL of sample was aseptically taken after 0, 46, 75 and 94 hours from inoculation and centrifuged at 4700 rpm for 10 min. The supernatant fractions were stored at − 20°C and used for total organic carbon (TOC) and total nitrogen (TN) determinations, while the pellet, washed with demineralized water, was cooled to − 20°C, lyophilized and stored. The C-phycocyanin content was measured on lyophilized pellet obtained from the last sampling time for each flask. The specific growth rate (µ, d^− 1), during exponential growth phase, was calculated according to:

$$\mu = \frac{\text{ln}\left({C}_{x(n+1)}\right)-\text{l}\text{n}\left({C}_{xn}\right)}{{t}_{(n+1)}-{t}_{n}}$$

where C_xn and C_x(n+1) are biomass concentration at times t_n and t_n+1. Experiments were performed in duplicate. The heterotrophic and mixotrophic biomass yield per carbon consumed (Y _x/C) was calculated as follows:

$${Y}_{x/C}= \frac{{C}_{x(n+1)}-{C}_{xn}}{ {C}_{n}-{C}_{n+1}}$$

where C_n – C_n+1 stands for the carbon concentration (g L^− 1) at times t_n and t_n+1. The biomass yield on nitrogen consumed (Y _x/N) was calculated as described above but considering the nitrogen concentrations (g L^− 1).

Optimal Buttermilk Dilution Determination

Buttermilk samples, kindly provided by “Caseificio del Cigno SPA” located in Agnadello (CR) - Italy, were pre-treated as follows: frozen buttermilk samples were thawed and centrifuged at 4700 rpm, for 10 min at 7° C. After centrifugation, the liquid phase was separated from the solid upper organic phase (mainly fat), and then immediately used for trials. Three different buttermilk dilutions (as 1:5, 1:2.5 and 1:1.66), were obtained by adding 20, 40 and 60% (v/v) of buttermilk into modified Allen’s medium. Allen’s medium was added to prevent any inorganic nutrient limitation, was prepared on purpose at different strengths to supplement any lack of nutrients due to buttermilk dilution. The test was performed in flasks and in mixotrophy to assess the effect on G. sulphuraria ACUF064 growth. The pH of the resulting medium was adjusted to 1.6–1.8 and the cultivation was done as described in the previous section. A broad characterization of the buttermilk was carried out and is reported in Table 1.

Table 1

Physico-chemical traits of buttermilk samples used in the present study
Parameters	Measure unit	Values
pH	-	4.42
TC	g/L	3–5
TN	mg/L	225
N-NH₄	mg/L	12
PO₄	mg/L	423
BOD₅	mg/L	4510
COD	mg/L	8750

Photobioreactor Setup And Operation

Experiments in mixo - and heterotrophic conditions were conducted in batch mode in a 13 L stirred tank bioreactor (NW200, Infors HT, Switzerland), controlled through Labfors 4 benchtop (Infors HT, Switzerland), for 6 days. A picture of the photobioreactor setup is reported in Fig. 1. The bioreactor presents a cylindrical shape, with an inner diameter of 200 mm and a maximum height of 445 mm. The reactor was used at a working volume of 8 L. During mixotrophy, half of the lateral surface of the reactor was illuminated using a vertical light panel (ReaLight-24, Ontwikkelwekplaats WUR, NL) placed 8 cm far from the reactor. Incident light intensity on the reactor surface was calibrated by measuring 24 points equally distributed on the inner surface of the empty reactor with a light meter (LI-250A, LI-COR, USA). Light intensity was provided in a continuous mode starting from 100 µmol_ph m^-2 s^-1 and was adjusted according to the biomass growth by keeping a constant specific light supply rate (q_ph). The specific light supply rate was maintained between 5.8 and 1.8 µmol_ph gx^-1 s^-1 as referenced in previous studies of Abiusi et al., 2021). During heterotrophic cultivation, the reactor was kept in the darkness.

The reactor was equipped with a dissolved oxygen (DO) sensor (InPro 6800 Series, Mettler Toledo, USA) and a pH probe (EasyFerm Bio HB K8 325, Hamilton, USA). The DO probe was calibrated at 0% and 100% DO. Zero-point oxygen calibration was performed by immersing the probe into a 15 mL tube containing 2–5 mg of Na₂S₂O₄ dissolved in deionised water. The 100% saturation point was performed leaving overnight the probe inside the reactor and under maximal aeration (1 L min^− 1). The pH probe was calibrated by using the two standard buffer solutions at pH 2 and pH 4 (VWR Chemicals, USA). The pH was continuously measured and controlled at 1.6 by automatic base addition (2 M NaOH) with a cascade loop.

The temperature of the reactor was kept at 37°C by the heat exchange between the culture vessel and a water jacket, in which the temperature was regulated by an external water bath. To prevent evaporation, the reactor was equipped with a condenser (4°C). Stirring was controlled in cascade ranging from 100 to 250 rpm to keep a DO of 20%. Air enriched with 2% v/v carbon dioxide was provided at a flow rate of 0.5–1 L min − 1 (according to minimum DO of 20%) using mass flow controllers (Smart TMF 5850S, Brooks Instruments, USA). Both mixotrophic and heterotrophic experiments were performed for 6 days in reactor vessels set up as follows: once the empty reactor vessel was autoclaved at 121°C for 15 min, it was aseptically filled with medium filtered through a 0.22 µm pore size filter (Sartobran® Capsule 0.2µm, Sartorius, USA). Consequentially, the DO sensor was inserted in the reactor vessel and left overnight for 100% DO calibration. After calibration, an amount of buttermilk was added to reach a concentration of 2.5 gC L^-1, corresponding to about 40% of the total volume, and immediately inoculated with a fresh culture of G. sulphuraria ACUF064 pre-adapted on lactose (mixotrophically or heterotrophically, according to the experimental set) to OD₇₅₀ of 0.3. Daily sampling was done at the same time, except for the exponential phase, where multiple samplings were performed. Concurrently, a 15 mL aliquots of sample for TOC, TN and CP-C content determinations were treated and stored, as previously described. The heterotrophic and mixotrophic biomass yield per carbon unit Y _x/C and per nitrogen unit Y _x/N consumption, the specific growth rate (µ, d^− 1) and the productivity (r_x) during exponential growth were calculated as described above.

Offline Analysis

The photosystem II maximum quantum yield (QY, Fv/Fm) was measured at 455 nm with an AquaPen-C AP-C 100 instrument (Photon Systems Instruments, Czech Republic). Before the measurement, samples were adapted to darkness for 15 min at room temperature.

Cell concentrations were determined by using a Coulter Multisizer III (Beckman Coulter Inc., USA) with a 50 µm aperture tube. Samples set at an OD₇₅₀ of 0.3–0.8 were 100 times diluted in ISOTON II diluent and the number of cells was analysed in 1 mL, counting particles measured in the range between 2 and 10 nm in order to count only cells belonging to G. sulphuraria species.

Dry weight concentration (DW) was calculated by measuring the weight difference between pre-weighted empty filters and filters containing biomass.. Shortly, an aliquot of the culture (2 − 5 mL) was diluted into 25 mL of deionised water and filtered over a pre-weighted Whatman GF/F glass microfiber filter (diameter of 55 mm, pore size of 0.7 µm). Pre-weighted filters and filters with biomass were washed with deionised water (25 mL) and dried at 105°C overnight, in a desiccator with silica for at least 2 h, and finally weighed on a scale (Cubis MCE225S-2S00-I, Sartorius Lab Instruments, Germany). DW measurements were performed in duplicate.

The total organic carbon (TOC) and total nitrogen (TN) content in the supernatant were measured by using a TOC-L analyzer (Shimadzu, Japan). The supernatant was diluted in demineralized water to reach a carbon content of 100–1000 ppm and a nitrogen content of 10–100 ppm. The optical density was measured with a spectrophotometer (DR6000, Hach-Lange, USA) at 620 and 750 nm. The samples were diluted with modified Allen’s medium until an OD₇₅₀ of 0.2–0.8.

The average absorption cross-section (a_x, m² gx^− 1) in the PAR region (400–700 nm) of the spectrum was determined as described by de Mooij et al. 2015. Briefly, the absorbance was measured with a UV-VIS/double beam spectrophotometer (Shimadzu, Japan) equipped with an integrating sphere (ISR-2600) and using cuvettes with an optical path of 2 mm. The absorbance from 740 to 750 nm was subtracted to the whole spectrum, and the average absorbance was normalised to the DW concentration of the sample. The ratio (Chl/Car) was calculated by dividing the light absorbed at 600 − 700 nm (chlorophylls) and the light absorbed at 400 − 500 nm (carotenoids).

Phycocyanin Extraction And Quantification

Phycocyanin from G. sulphuraria ACUF064 was quantitatively extracted by bead beating (Precellys 24, Bertin Technologies, France) 10 mg of lyophilised biomass as described by Abiusi et al.(2022). The C-phycocyanin (C-PC) was calculated by measuring the absorbance at 620 nm and at 652 nm of the supernatant and converting it into concentration using the Kursar and Alberte equation (Kursar & Alberte, 1983). The concentration of C-PC was then referred to the DW of G. sulphuraria.

Statistical analysis

One-way analysis of variance (ANOVA) and Tukey’s HSD post hoc test for means separation were performed using the STATISTICA ETL software (version 10, StatSoft. inc., Tulsa, OK, USA). The significance level was set at p ≤ 0.01.

Carbon sources flask experiments

Prior to the experiments with buttermilk, we evaluated the growth of Galdieria sulphuraria individually in glucose, galactose and lactose. In Fig. 2, dry weight measured at 0, 1, 2, 3 and 4 days after inoculum in mixotrophy (panel A) and after 0, 1, 3 and 4 days after inoculum in heterotrophy (panel B) are shown. G. sulphuraria ACUF064 registered the highest values of biomass dry weight (8.43 ± 0.70 on galactose and 7.95 ± 0.07 g_x L^− 1 on glucose) in mixotrophy. Growth on lactose 4 days after inoculation was lower leading to a biomass dry weight concentration of 4.28 ± 0.12 g_x L^− 1 in mixotrophy. In heterotrophy, values of DW were 6.21 ± 0.20 and 5.6 ± 0.21 g_x L^− 1 with glucose and galactose, respectively, being slightly lower than in mixotrophy. For lactose, the DW obtained was the lowest (3.58 ± 0.89 g_x L^− 1).

Besides the DW, the specific growth rate (µ), the cell concentration, the biomass yield on nitrogen and carbon and the C-PC content were also monitored and the results are reported in Table 1. In mixotrophy, the µ estimated from DW measurements was 1.12 ± 0.03 d^− 1 for glucose, 1.03 ± 0.16 d^− 1 for galactose and 1.02 ± 0.01 d^− 1 for lactose. From the cell counts (expressed as cellsּ 10⁸ mL^− 1),the highest concentration (3.03 ± 0.12) was observed on glucose, compared to galactose (2.51 ± 0.02) and lactose (2.21 ± 0.01). In heterotrophy, the highest specific growth rate was also observed with glucose (1.06 ± 0.01 d^− 1), followed by galactose (1.02 ± 0.01 d^− 1), and lactose (0.94 ± 0.02 d^− 1). In that case, the growth rate observed in lactose was significantly lower from all the different conditions tested. Also in heterotrophy, the highest cell concentration was observed for the cultures grown with glucose (1.97 ± 0.07) and in a decreasing order, galactose (1.87 ± 0.15) and then lactose (1.43 ± 0.12). The cultures grown in heterotrophy showed a smaller cell concentration than the cultures grown in mixotrophic conditions, most likely due to the additional fixation of CO₂.

The carbon and nitrogen at the beginning and at the end of the experiment were measured to estimate the yields of biomass. Overall, the yields of biomass on carbon consumed were higher in mixotrophic conditions compared to heterotrophic conditions for each substrate. Under mixotrophy, the highest yield were observed under lactose (1.93 ± 0.04 gx g_C⁻¹) and galactose (1.90 ± 0.24 gx g_C⁻¹), compared to glucose (1.63 ± 0.05 gx g_C⁻¹). An opposite trend was observed in heterotrophy, where the highest yield observed for glucose (1.41 ± 0.18 gx g_C⁻¹) was decreased by 13%, while the yields for galactose and lactose were also reduced 33% and 47%, respectively. Concerning the yield of biomass per nitrogen, the highest yield under mixotrophy was observed for galactose (9.88 ± 0.34 g_x g_N⁻¹), followed by glucose (8.18 ± 0.02 g_x g_N⁻¹) and then lactose (7.55 ± 0.01 g_x g_N⁻¹). The yields in heterotrophy were generally higher (46% higher for glucose and 5% higher for lactose), with the exception of galactose, where the yield decrease by 12%.

The accumulation of C-PC differs depending on the substrate used. The C-PC content was highest when G. sulphuraria was grown on lactose and under mixotrophic conditions. The C-PC content was 27% and 37% lower when G. sulphuraria was grown in glucose and galactose, respectively. In heterotrophic conditions, the C-PC content was generally reduced by more than double. Still, the highest accumulation occurred after using lactose (2.34% w/w), while the yield equally decreased by 36% for both glucose and galactose. Overall, the results emphasise thatG. sulphuraria ACUF064, when cultivated on lactose, showed a significant better behaviour in mixotrophy than in heterotrophy in terms of C-PC accumulation.

Table 1

Overview of the offline measurements (C_x, C-PC content and cell counts) process parameters (specific growth rate) and stoichiometric yields (Y_x/C, Y_x/N) of *Galdieria sulphuraria* ACUF 064 growth in heterotrophic and mixotrophic conditions in flask. Glucose, galactose and lactose concentration was 5 gC L^-1. Values are expressed as averages ± standard deviation.
	Glucose		Galactose		Lactose
	Heterotrophy	Mixotrophy	Heterotrophy	Mixotrophy	Heterotrophy	Mixotrophy
µ (d^− 1)	1.06 ± 0.01^a	1.12 ± 0.03^a	1.02 ± 0.01^a	1.03 ± 0.16^a	0.94 ± 0.02^b	1.02 ± 0.01^a
Y _x/C (g_x g_C⁻¹₎	1.41 ± 0.18 ^a	1.63 ± 0.05 ^a	1.28 ± 0.12 ^a	1.90 ± 0.24 ^a	1.08 ± 0.51 ^a	1.93 ± 0.04 ^a
Y _x/N (g_x g_N⁻¹₎	12.8 ± 0.18	8.18 ± 0.02	8.72 ± 0.35	9.88 ± 0.34	7.89 ± 0.03	7.55 ± 0.01
C-PC (% w_C−PC w_x⁻¹)	1.49 ± 0.22	4.3 ± 0.33	1.50 ± 0.06	3.7 ± 0.17	2.34 ± 0.07	5.9 ± 0.18
C_x (g_x L^− 1)	6.21 ± 0.20	7.95 ± 0.07	5.6 ± 0.21	8.43 ± 0.70	3.58 ± 0.89	4.28 ± 0.12
Cells count (cells∙10⁸ mL^− 1)	1.96 ± 0.07	3.03 ± 0.12	1.87 ± 0.15	2.51 ± 0.02	1.43 ± 0.12	2.21 ± 0.01

Among the rows the same letter indicates no significant differences (p > 0.01).

Optimal Buttermilk Dilution Determination

Three different buttermilk dilutions (1:5, 1:2.5 and 1: 1.66) were obtained by adding of buttermilk into modified Allen’s medium at 20, 40 and 60% v/v. The different dilutions were tested in flasks and in mixotrophy to assess the effect on the growth of G. sulphuraria ACUF064. The final biomass concentration (C_x), the overall productivity (r_x) and the total organic carbon (TOC) measured at the beginning of the trial, are reported in Table 2. The C_x measured 7 days after inoculum was 4.26 ± 0.16 g_x L^− 1 at 40% v/v dilution, while values of 3.85 ± 0.06 and 1.56 ± 0.28 g_x L^− 1 were registered at 20 and 60% v/v dilution rate, respectively. The productivity (r_x), expressed as g_x L^− 1 d^− 1, showed the highest rate in the experiment containing 40% buttermilk, while values of 0.50 and 0.17 were detected in the experiments at 20% and 60%, respectively. The TOC, measured immediately after inoculation, reveals values of 1.6 ± 0.2, 2.5 ± 0.4 and 4.5 ± 0.5 g_c L^− 1 in cultures enriched with 20%, 40% and 60% v/v of buttermilk, respectively. The best dilution ratio of buttermilk for biomass production was 40% v/v.

Table 2

Summary of *Galdieria sulphuraria* ACUF 064 growth in flask at different buttermilk dilution ratios in mixotrophy. Comparison of final biomass concentration (C_x), productivity (r_x) and total organic carbon content at the beginning of the trial. Values are expressed as averages ± standard deviation (n = 2).
Buttermilk dilution ratio (% v/v)	TOC (gC L^− 1)	C_x (g_x L^− 1)	r_x (g_x L^− 1 d^− 1)
20	1.7 ± 0.2	3.85 ± 0.06	0.50 ± 0.01
40	2.5 ± 0.4	4.26 ± 0.16	0.54 ± 0.02
60	4.5 ± 0.5	1.56 ± 0.28	0.17 ± 0.04

Growth on buttermilk of G. sulphuraria ACUF064 in stirred tank photobioreactor.

The optimal dilution rate (40% v/v), found in preliminary flask experiments, was evaluated in a 13 L stirred tank reactor, and the effect on growth was studies in mixotrophy and heterotrophy. Temperature, oxygen, carbon dioxide, pH, stirring and light (when needed) were constantly monitored and kept at optimal levels (as described above). The biomass dry weight concentration (g_X L^− 1) and total organic carbon content (g_c L^− 1) were measured daily in both mixotrophic and heterotrophic experiments as reported in Fig. 3. After 6 days, the biomass dry weight concentration was highest in mixotrophy, reaching a value of 1.22 ± 0.02 g_x L^− 1, compared to the 0.78 g_x L^− 1 found in heterotrophy. The growth was also followed by cell count measurements. After 6 days from inoculation, cell concentration was 2.85ּ 10⁷ and 1.90ּ 10⁷ cells mL^− 1 were observed for mixotrophy and heterotrophy, respectively. Concurrently with the carbon source experiments, the µ was also higher in mixotrophic conditions, although the growth rates observed were notably lower (more

As reported in Table 3, the yield of biomass on carbon was higher in mixotrophy, although the difference is as well as the specific growth rate (µ) and productivity (r_x). Furthermore the C-PC content, as expected, was higher in mixotrophy than in heterotrophy, reaching 2.43 ± 0.34 and 0.57 ± 0.17 w_C−PC w_x⁻¹%, respectively. The absorption spectra recorded to calculate the average dry-weight-specific optical cross section were also used to detect changes in pigment ratios when shifting from mixotrophy to heterotrophy. Indeed, the ratios Chl/Car registered value of 0.65 ± 0.08 and 0.38 ± 0.05 for mixotrophy and heterotrophy, respectively

Overall, G. sulphuraria ACUF064 showed a slower growth in heterotrophy compared to mixotrophy.

Table 3

Overview of the offline measurements (Cx, C-PC and Chl/Car), process parameters (r_x and µ) and stoichiometric yield (Y_x/C) of *Galdieria sulphuraria* ACUF 064 grown in heterotrophic and mixotrophic conditions in a stirred tank reactor containing 40% v/v of buttermilk (2.5 gC L^-1) as carbon source. Values are expressed as averages ± standard deviation when technical replicates were available (n = 2).
	r_x	Y _x/C	C-PC	µ	Chl/Car	C_x
	g_x L^− 1 d^− 1	g_x g_c⁻¹	% w_C−PC w_x⁻¹	d^− 1	ABS (600–700) (400–500)⁻¹ nm	g_x L^− 1
Mixotrophy	0.55	0.79	2.43 ± 0.34	0.36	0.65 ± 0.08	1.22 ± 0.02
Heterotrophy	0.41	0.70	0.57 ± 0.17	0.29	0.38 ± 0.05	1.04 ± 0.14

In the last years, the cultivation of G. sulphuraria on several organic wastes derived from food industry has been explored. G. sulphuraria 074G growth on food waste from restaurants and bakeries was evaluated (Sloth et al. 2017), or, on granular starches derived from potato (Rahman et al. 2020). Zimermann et al. (2020) tested whey permeate as a carbon source for G. sulphuraria SAG 107.79. Corn stover hydrolysates have been recently supplied to G. sulphuraria UTEX 29.19 for growth (Portillo et al. 2022), while digestate and hydrolyzed straw was investigated for the strain SAG 21.92 (Pleissner et al. 2021). With a few exceptions, the growth of G. sulphuraria on carbon sources containing disaccharides, such as lactose had not been deeply explored before. The use of complex carbohydrates (di-, tri, and polysaccharides) depends on the presence of hydrolytic enzymes. While polysaccharides are generally converted into simpler sugars in the extracellular environment, di- and trisaccharides can only be metabolized in the cytosol. Therefore, the presence of a specific carrier is required (Tischendorf et al., 2007). Most of the previous studies about heterotrophic and mixotrophic cultivation of G. sulphuraria have been performed on hexoses, such as glucose.

In the present study, the growth performance observed for G. sulphuraria ACUF064 on lactose was lower but comparable to the one observed on glucose or galactose. Despite the known variability among species from the same genus, and even among strains of the same species, G. sulphuraria ACUF064 showed a similar growth on glucose and galactose, as observed for other strains. A similar heterotrophic growth was observed for G. sulphuraria 74G when sucrose (disaccharides), glucose and galactose were the used carbon sources (Gross & Schnarrenberger, 1995). The specific growth rates (µ) observed during the heterotrophic experiment for G. sulphuraria ACUF064 were similar to those reported by Schmidt et al., (2005) for G. sulphuraria 74G who calculated µ values of 1.10, 1.08, 1.01 for glucose, fructose and sucrose, respectively. In the present research, the µ was similar for the three carbon sources, both in mixotrophy and in heterotrophy, as also reported by Sloth et al., (2006) for G. sulphuraria 74G on glucose, fructose or glycerol. The µ was 1.2 ± 0.1 d^− 1 in all the three cases, independent of the light intensity, tested in the range between 0 and 117 µmol photons m^− 2 s^− 1 Also Portillo et al., (2022), using G. sulphuraria UTEX2919, did not report significant differences in µ values comparing heterotrophic and mixotrophic conditions (0.97 ± 0.04 d^− 1 and 1.10 ± 0.08 d^− 1, respectively), using xylose as a carbon source. When glucose was used, they registered µ of 1.46 ± 0.03 d^− 1 in heterotrophy and 1.49 ± 0.00 d^− 1 in mixotrophy, a significantly higher growth rate than the ones obtained in this study.

The C-PC content measured in mixotrophy for each carbon source supplied was far from the maximum (10% w_C−PC w_x⁻¹) observed in a previous study with the same strain (Abiusi et al., 2022). This divergence is explained by differences in the cultivation conditions. Experiments from Abiusi et al. (2022) were performed in chemostat operation mode in a stirred reactor where the illumination was optimized. Interestingly, in heterotrophic conditions, the C-PC content, both on glucose and galactose, were similar to those registered by Graverholt and Eriksen (2007) with G. sulphuraria 74G in fed-batch with sufficient glucose concentration (1.6% w_C−PC w_x⁻¹), while lower compared to glucose depleted or glucose-limited cultures (2.7% w_C−PC w_x⁻¹). G. sulphuraria ACUF064 supplied with lactose showed a C-PC content of 2.34 ± 0.07% w_C−PC w_x⁻¹, comparable to C-PC accumulation registered when glucose depleted or glucose-limited conditions occurred. As largely explained in previous studies (Graverholt and Eriksen, 2007b; Portillo et al., 2022; Sloth et al., 2006; Sørensen et al., 2013), phycocyanin accumulation is a response triggered under abiotic stress such as glucose limitation (Graverholt and Eriksen, 2007b; Portillo et al., 2022; Sloth et al., 2006b;rensen et al., 2013). Furthermore, Sloth et al., (2006) reported that the substrate structure could affect the growth rate and C-PC content. It seems that carbon in lactose is less available, due to the structural complexity of the molecule, and this causes a carbon limitation for G. sulphuraria. This results in a lower growth rate and a higher C-PC content. In particular, G. sulphuraria 74G showed a higher growth when supplied with glycerol (Triol with three carbon atoms) than with glucose, but, at the same time, glycerol fed cultures had a lower C-PC content than glucose fed ones.

Such observations are supported by the biomass production per carbon or nitrogen unit. The low Y_x/C indicates the lower efficiency of microalgae in converting lactose into biomass, compared to simpler sugars, such as glucose and galactose. At the same time, the low Y_x/N proves that nitrogen is accumulating and redirected to the synthesis of phycocyanin. In mixotrophic conditions, C-PC content was more than double, indicating stress induced by lactose, but not by the other carbon sources. The high Y_x/C observed in mixotrophy compared to heterotrophy implied the additional use of light as energy source for biomass synthesis. This results into higher nitrogen accumulation (Low Y_x/N) both in the form of chlorophylls, phycobiliproteins and of proteins. The phycocyanin content in lactose fed cultures in heterotrophy disagrees with the results of previous studies by Tischendorf et al., (2007) and Zimermann et al., (2020), who observed an almost complete loss of pigmentation when the strain was grown in the same conditions. Finally, a direct comparison could be made with data from Zimermann et al., (2020) between two strains in heterotrophic conditions fed on lactose. In particular, in our heterotrophic flask experiment, the growth of G. sulphuraria ACUF064 was lower on lactose than on glucose but the pigmentation was greater, while in the case of G. sulphuraria SAG 107.79 the opposite results were obtained.

Focusing on biomass production on buttermilk as carbon source, it is interesting to highlight that no experiment with any Galdieria species had been performed before. Hence, it is not possible to derive information about buttermilk performance as a substrate. The inhibitory effect of buttermilk on G. sulphuraria ACUF064 growth was pre-tested in flasks. Despite lactose is the main carbon source in such substrate, other organic molecules are present which can hamper growth. Dairy glycerophospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol are the phospholipids commonly found in buttermilk. Within the sphingolipid class, sphingomyelin, glucosylceramide, and lactosylceramide are found (de Freitas Mascarello et al. 2019).

Buttermilk proteins were reported to exhibit bioactive characteristics, such as antibacterial activity (Hancock et al. 2002). Whey proteins and caseins are the most abundant macromolecules, even though other three proteins, such as xanthine, butyrophilin and periodic acid Schiff6/7 have also been found (Holzmüller and Kulozik 2016).

Contaminant populations in buttermilk were entirely inhibited because of the low pH of the cultivation medium (pH 1.6) (data not shown). Therefore, the problem of contamination, which could negatively affect the culture, as reported by Di Caprio, (2020), seems negligible. Due to the lack of information about organic compounds limiting the growth of Galdieria (Ternes 2015; Pleissner et al. 2021), and considering the complexity and heterogeneity of buttermilk (Sodini et al. 2006; Ali 2019), further investigations are necessary to identify which molecules could negatively affect Galdieria growth and their concentrations.

Since the best buttermilk dilution ratio, found in shake flasks, was 40% v/v (corresponding to 2.5 gC L^− 1), experiments at larger scale were conducted at such dilution. Even though heterotrophy is a better way to scale up industrial process (Barroso et al., 2019), mixotrophy in controlled conditions resulted to be a better option for G. sulphuraria cultivation on buttermilk. Indeed, the µ values registered were 0.36 and 0.29 d^− 1 in mixotrophy and heterotrophy, respectively. The growth rates found in this study were lower than those measured for G. sulphuraria 74G in heterotrophy, with food and bakery wastes (when properly hydrolyzed) (Sloth et al., 2017). The authors found µ values of 0.69 and 0.60 d^− 1 in restaurant waste and bakery waste, respectively. However, in both cases, the trials were supplemented with 5 g L^− 1 of glucose and 0.5 g L^− 1 of ammonium. In a recent study, Pleissner et al., (2021), using G. sulphuraria SAG21.92, found a µ of 1.0 d^− 1 in heterotrophy in the presence of 100% (v/v) of hydrolysed digestate supplemented with glucose in excess (50 g L^− 1). In experiments carried out by Rahman et al., (2020), instead, G. sulphuraria 74G was grown in the presence of corn and potato starch and similar growth rate values to our study were found (0.3–0.4 d^− 1). The lower growth rate observed in our experiments with buttermilk might reinforce the fact that cultures experienced carbon limitation.

In the present study, G. sulphuraria ACUF064 grown in buttermilk and in heterotrophy showed a lighter green colour and, at the same time, phycocyanin was still present (confirming our previous lactose experiment). These results contrast with the ones of Zimermann et al., 2020 using whey permeate (rich in lactose), where the cultures completely lost their pigmentation.

The low biomass yield per carbon unit measured both in mixotrophy and heterotrophy indicates difficulties for G. sulphuraria ACUF064 to use buttermilk as feed. The slightly higher value in mixotrophy than in heterotrophy, confirms that light is a useful energy source for Galdieria when the organic substrate is difficult to be used. Recently, Julius Pahmeyer et al. (2022) evaluated the design and the sustainability of a modular system for G. sulphuraria growth on organic waste estimating that by this set-up the production cost of dried biomass could be reduced to 4.38 € per kg.

The results of the present study revealed the suitable dilution ratio of buttermilk for biomass production.

Lactose proved to be a useful carbon source for G. sulphuraria ACUF064 growth, despite its complexity compared to simpler sugars such as glucose and galactose. Lactose induced a decrease in biomass production, but an increase of C-PC content, compared to glucose or galactose. These observations prove the possibility to use lactose-enriched effluents to support the G. sulphuraria ACUF064 growth.

The cultivation of G. sulphuraria ACUF064 proved to be an interesting approach to valorise buttermilk, especially in mixotrophy. However, further research is needed to improve biomass production and phycocyanin content through optimization of light supply rate or other operational strategies, including the use of chemostats or fed-batch approaches. Such performances should be analysed in the light of their cost effectiveness, taking into account the income resulting from microalgal biomass.

DO Dissolved oxygen concentration (% air saturation)

TN Total Nitrogen (g_N·L⁻¹)

TOC Total Organic Carbon (g_C·L⁻¹)

QY photosystem II maximum quantum yield measured at 455 nm

OD₇₅₀ Optical density at 750 nm

OD₆₂₀ Optical density at 620 nm

C_x biomass dry weight concentration (gx·L−1)

Y_x/C Biomass yield per carbon unit (g_x g_C^-1)

Y_x/N Biomass yield per nitrogen unit (g_x g_N^-1)

μ Specific growth rate (d⁻¹)

r_xVolumetric biomass production rate (gx·L⁻¹·d⁻¹)

a_xAverage- specific optical cross section (m2·g⁻¹)

Chl/Car Ratio between light absorbed at 600−700 nm (chlorophylls) and at 400−500 nm (carotenoids)

Subscript

_x Biomass

_C Carbon

_NNitrogen

Authorship contribution statement

Conceptualization, V.M., N.F.L., S.C.; Methodology, N.F.L., F.D.S.; Formal analysis, P.S.O., F.D.S. and N.F.L.; Investigation, P.S.O., F.D.S. and N.F.L.; Resources, S.C.; Data curation, P.S.O. and N.F.L.; writing—original draft preparation, P.S.O.; Writing—review and editing, V.M., N.F.L., S.C. and C.C.; Visualization, P.S.O. and N.F.L.; Supervision, N.F.L., S.C. and C.C.; Project administration, V.M., S.C.; Funding acquisition, S.C. All authors have read and agreed to the published version of the manuscript.

Acknowledgements

We would like to thank Michele Locatelli from Caseificio del Cigno S.P.A. for the supply of buttermilk. And our most sincere thanks to Prof. Antonino Pollio from University of Naples Federico II for his guidance and fruitful discussions on Galdieria cultivation.

Conflict of interests

We declare that the authors have no competing interests, as defined by Springer, or other interests that might be perceived to influence the results and/or discussion reported in this manuscript.

Data availability statement

The raw data generated during this research is available by the corresponding author upon request without reservation.

Funding statement

The contents of this work were financially supported by Algreen B.V.

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Valorisation of buttermilk in mixotrophic and heterotrophic conditions by using the acidophilic strain Galdieria sulphuraria

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Abstract

Figures

Introduction

Materials And Methods

Microalgal strain, media and cultivation conditions

Carbon Source Flask Experiments

Optimal Buttermilk Dilution Determination

Photobioreactor Setup And Operation

Offline Analysis

Phycocyanin Extraction And Quantification

Statistical analysis

Results

Carbon sources flask experiments

Optimal Buttermilk Dilution Determination

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

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Version 1