Biomass of Euglena sp.produced in Mineral Medium Supplemented With Craft Brewery Residue

doi:10.21203/rs.3.rs-3338867/v1

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Research Article

Biomass of Euglena sp.produced in Mineral Medium Supplemented With Craft Brewery Residue

https://doi.org/10.21203/rs.3.rs-3338867/v1

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published 07 Mar, 2024

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Industrial waste from craft breweries can be used to grow microorganisms due to its high nutrient content. Reusing waste and reducing its improper disposal are great economic and environmental strategies. Thus, the objective was to cultivate Euglena sp. in benchtop 1-L photobioreactors using different concentrations of NPK-enriched (0.5–6.0 g L^− 1) mineral medium with and without the supplementation with a mixture of brewing residues (BR-Mix) with increase of scale (30 L) and BR-Mix supplementation (30% v:v). Carbohydrates, lipids, proteins, fatty acids, and antioxidant potential) were analyzed. The BR-Mix-supplemented medium (0.5 g L^− 1 NPK) yielded 15.3 ± 1.4% lipids, 35.1 ± 0.1% proteins, 9.07 ± 0.001% carbohydrates, and 61.3% polyunsaturated totty acids. The antioxidant potential was 170.9 ± 26.3 µmol eq g^− 1 of Trolox. Supplementing with 25% BR-Mix resulted in a different biomass composition compared to control (without supplementation): increasing the content of carbohydrates (38.5%), paramylon (91%), and lipids (17.38%), and reducing proteins (26,6%) and antioxidant potential (41,8 µmol eq g^− 1).

Brewing residue

Euglena sp.

biomass

microalgae

paramylon

The clean industrial residues collected from craft breweries present an opportunity for cultivating microorganisms, in special Euglena sp., an interesting microalgal biomass. This potential not only addresses waste management issues but also contributes to economic and environmental sustainability by reducing improper disposal. We examined the influence of the brewery waste mixture on enhancing the Euglena sp. growth potential. The subsequent phase of our study involved scaling up the process to a 30 L photobioreactor. Throughout our investigation, we analyzed parameters such as carbohydrates, lipids, proteins, and antioxidant potential under these conditions with a mineral medium and brewery waste.

Microalgae are single-celled microorganisms that can quickly grow and accumulate high biomass yields. Their biomass is a nutritional source of proteins, carbohydrates, lipids, vitamins, pigments, and minerals [1, 2], relevant to food production. In addition, it can be applied to producing biofuels and as pharmaceutical and agricultural inputs [3].

These microorganisms are essential to sustainable processes [4], especially if the cultivation is total or partial in wastewater, leading to (1) the recovery of nutrients, (2) reduction of treated water supply and (3) less use of arable areas [5, 3].

Some industrial residues can be used as culture media for microalgae, as they have high nutrient loads, contributing to an economically viable production [6].

In order to achieve a higher performance of microalgal biomass production, it is important to control the growing conditions, such as aeration, medium composition, light intensity, pH and temperature. Microalgae can grow in open (ponds and raceway tanks) or closed (photobioreactors) systems. Using photobioreactors enables higher contaminant-free biomass yields and the use for nobler purposes, such as feeding, since it is possible to control better the cultivation parameters [7].

Large volumes of water are used in craft brewing industries, usually discharged without treatment [8]. This wastewater can come from various stages of the process. It may contain suspended solids, sugars, yeasts, and a high organic matter load [9]. In order to reduce improper disposal, reusing brewing wastewater can become a major economic and environmental strategy.

Microalgae have gained great prominence to value these residues by producing biomass with potential economic exploitation [10]. In addition, it is possible to reduce costs using conventional culture media to develop these microorganisms by reusing nutrient-riched residues [11].

In the last decade, several studies showed that microalgae could grow in brewing wastewater and obtain biomass for various uses [12, 13, 10, 14–16]. However, the biomass cannot be used as a food source since the medium is not clean. It is imperative to implement the use of highly concentrated brewing waste to improve the biotechnological biomass potential and avoid environmental pollution [17].

Euglena sp. is a flagellated protist microalga commonly found in freshwater with a high nutritional load that can grow photoautotrophically, heterotrophically, or mixotrophically [18]. Euglenophytes have a complex metabolic capacity and produce relevant biomolecules: carbohydrates, proteins, and amino acids [19].

According to Wu et al. [20], depending on the metabolic pathway, it is possible to promote changes in the composition of the biomass of Euglena gracilis, increasing the paramylon (β-1,3-glucan) production, the main carbohydrate. Another applicability is also highlighted by Eze et al. [21], who recognized significant effects on biological activities such as angiotensin-converting enzyme inhibition, hypocholesterolemia activity, and increased red blood cell count. In addition, Mahapatra et al. [22] obtained biomass of Euglena sp. with higher lipid content due to the medium containing high concentrations of ammonia nitrogen and organic material. However, studies with euglenophytes grown in brewing residue were not found, leaving a gap to know the quality and potential of biomass.

Considering the demand for adequate disposal of waste from craft beer production in the southern region of Brazil, and the emerging residue application in biotechnological processes, the objective of this research was to evaluate the use of mineral medium (NPK) supplemented with brewing residue in the cultivation of Euglena sp. to obtain biomass with nutritional potential, characterized in proteins, carbohydrates, lipids and antioxidant activity.

2.1 Characterization of culture media and inoculum standardization

As a mineral culture medium, we chose to use a solution of agricultural fertilizer containing nitrogen, phosphorus and potassium (NPK) of the brand Yara Mila COMPLEX in concentrations of 12%, 11% and 18%, respectively. The NPK medium, in different concentrations (0.5 to 6.0 g L^− 1), was prepared using distilled water and autoclaved at 121°C for 20 min.

The residues of the malting, hop addition, and fermentation processes used in this study were collected in a craft brewery in Santa Cruz do Sul/RS, southern Brazil. For the experiments, each residue was filtered in cotton mesh for disposal of the larger solids and centrifuged at 3600 rpm. After centrifugation, the liquid fraction of each residue was transferred to polypropylene recipients, stored in a refrigerator, and kept at a temperature < 10°C.

For the formulation of the supplemented culture medium, Brewery Residue Mix (BR-Mix), the three filtrates were mixed in equal parts, and the medium was later analyzed for pH, amount of Ca, Co, Cu, Fe, Mg, Mn, Zn, K, N (NO₃-, NO₂⁻ and NH₃), biochemical oxygen demand (BOD₅), chemical oxygen demand (COD), hardness, total phosphorus (PO₄³⁻) and total suspended solids (TSS), following the analytical methodology of the Standard Methods for the Examination of Water and Wastewater [23].

The microalgae strain Euglena sp. was kindly provided by the Phycology Laboratory of the Federal University of Santa Maria, Rio Grande do Sul, Brazil (SISGEN A863DC8- UFSM). This strain was maintained in a solution of 0.5 g L^− 1 NPK (20% of inoculum and 80% of medium). The inoculum was kept under constant LED light (148 µmol photons m^− 2 s^− 1) and artificial aeration through mini air compressors (180 L s^− 1, Boyu Sc7500) and renewed every 7 days. The experiments started with an initial cell density of 2.0 x 10⁵ cells mL^− 1 according to Ambriz-Perez et al. [24].

2.2 Cultivation of Euglena sp.

Euglena sp. was grown in a medium with different NPK fertilizer concentrations (0.5, 1.0, 1.5, 3.0, and 6.0 g L^− 1), with and without BR-Mix. The cultures were triplicated in 2-L Erlenmeyer flasks for 12 days. BR-Mix supplementation was done by adding 5 mL every 2 days to a total final medium volume of 1 L, resulting in a final supplementation of up to 3% by the end of the experiment. The cultures were monitored every 2 days using optical density (OD) measurements (UV/Vis spectrophotometer ChromTech UV-1100) at 680-nm wavelength (λ). The cellular density (CellD) was determined through direct cell counting (Sedgwick-Rafter counting chamber). The cultures were carried out in a temperature-controlled room at 23°C, with artificial light from 20.5 W tubular LED lamps (average of 148 µmol m^− 2 s^− 1 photons) with a photoperiod of 12L:12D (light/dark cycles) controlled by a programmable digital timer (T01 Luuk Young). Temperature and luminosity were monitored using a datalogger recorder (HOBO Pendant® UA-002-64). Artificial aeration was done by mini air compressors (180 L s^− 1, Boyu Sc7500), and the initial pH was adjusted to 4 using 1 mol L^− 1 phosphoric acid solution. The cultures were interrupted before entering the stationary growth phase because biomass obtained in the growth phase may present interesting characteristics in terms of lipids and carbohydrates [25].

The cultures were centrifuged at 3200 rpm, and the biomass (pellet) was dried at 50°C for up to 72 h. The productivity was measured based on the dry biomass (g L^− 1 d^− 1) every two days of cultivation [26].

2.3 Upscaling of Euglena sp. cultivation

The scale-up in the culture volume and the increase in BR-Mix concentration were performed in three stages, as shown in Fig. 1. The first stage involved the adaptation of the microalgae Euglena sp. to the cultivation medium. This was conducted in quintuplicate with a volume of 1 L (20% inoculum and 70% of the base medium with fertilizer (0.5 g L^− 1). Additionally, 20 mL of BR-Mix was added every two days, totaling a 10% supplementation for 10 days.

In the second stage, the content of each flask was transferred to 2-L Erlenmeyer flasks, and 30% of the fertilizer medium at a concentration of 0.5 g L^− 1 NPK was added. 20% of BR-Mix (33 mL) was added every 2 days for 12 days. Due to the dilution in this stage and considering the amount of BR-Mix already added in the first stage, the total BR-Mix at the end was 25%.

The third stage was carried out in a 45-L closed tank (transparent polypropylene) fed with 10 L of the cultures from the second stage. The final culture volume reached 30 L by adding 60% of fertilizer medium (0.5 g L^− 1 NPK) and 30% of BR-Mix (300 mL of 5%-BR-Mix every 2 days starting at the end of the experiment). Temperature, aeration, illumination sets and productivity analysis for the three stages were conducted as discussed in section 2.2, in a temperature-controlled room at 23°C, with artificial light from 20.5 W tubular LED lamps (average of 148 µmol m^− 2 s^− 1 photons) with a photoperiod of 12L:12D, temperature and luminosity monitored using a datalogger recorder and artificial aeration done by mini air compressors.

Figure 1

2.4 Characterization of biomass

Total lipids (%), fatty acid profile (%), total carbohydrates (%), monosaccharide profile (%), total proteins (%), and antioxidant potential (µmol eq g^− 1) were analyzed. The extraction of lipids was performed by the method of Bligh, Dyer [27], adapted by Souza et al. [28]. The lipid profile was determined by gas chromatography with mass spectrometry (GC/MS, 5977B, Agilent, USA) and gas chromatography with a flame ionization detector (GC/FID, QP2010plus, Shimadzu, Japan) for quantification.

The total carbohydrates were quantified by hydrolysis with concentrated acid [29]. The monosaccharide profile was determined by high-performance liquid chromatography with a refractive index detector (HPLC-RID, 10A, Shimadzu, Japan) [30].

The protein quantification was performed through elemental nitrogen analysis (Flash EA1112, Thermo Electron Corporation, Italy) using the nitrogen-to-protein conversion factor of 4.78 [31, 32]. The antioxidant potential was determined with the oxygen radical absorption capacity assay method in Trolox µmol eq g^− 1: the biomass was suspended in methanol to quantify the equivalent Trolox content, which is analogous to the antioxidant potential of tocopherol (ORAC) [30].

2.5 Statistical tests

Data analysis was performed using the PrismGraph 10.0 software (USA) for nonparametric samples with analysis of variance (ANOVA) followed by Tukey's test of multiple comparisons grouped by variance. Significance tests were performed with a 95% confidence level.

3.1 BR-Mix characterization

Table 1 presents the BR-Mix characterization, used as a supplement in the Euglena sp. cultivation. It is important to highlight the presence of organic carbon compounds in the culture medium, expressed by chemical oxygen demand (COD) and biochemical oxygen demand (BOD). These parameters indicate the amount of organic carbon in the medium, which microalgae can use as an energy source. In addition, it is relevant to consider the presence of nitrogen (N) in the medium in the forms of nitrate (NO₃⁻), nitrite (NO₂⁻) and ammonia (NH₃). Nitrogen is an essential nutrient for microalgae growth, and its adequate bioavailability is important to ensure a good performance of crop cultivation Gupta et al. [33].

Table 1

Characterization of the BR-Mix medium, composed by mixing in equal parts the liquid residues of the malting, hop addition and fermentation processes of a craft brewery.
Parameters	Concentration (mg L^− 1)
Ca	13.82
Co	0.001
Cu	0.26
Fe	0.07
Mg	214.10
Mn	0.83
Zn	0.29
K	466.40
P-PO₄³⁻	101.30
N-NO₃⁻	0.73
N-NO₂⁻	0.16
N-NH₃	31.23
BOD₅	9124.1
COD	91798.9
Hardness	376.7
TSS pH	252.1 5.5
BOD₅ = Biochemical Oxygen Demand; COD = Chemical Oxygen Demand; Total Suspended Solids (TSS).

Table 1

Phosphorus (P), often phosphate (PO₄³⁻), is also an essential nutrient for microalgae, crucial in synthesizing DNA, RNA, and energy compounds [34].

Therefore, evaluating these parameters (COD, BOD, NO₃⁻, NO₂⁻, NH₃ and PO₄³⁻) enables one to observe their availability and utilization by microalgae.

Concerning BR-Mix, it is still important to consider that the organic load may vary according to the brewing process [35]. The residues used in this research can be considered co-products of craft beer production since they were not collected as effluent at the end of the manufacturing process. Nutrient concentrations are naturally higher than commercial brewery wastewater that can contain wash water and other compounds of production processes. Choi [36] reports 38 to 89 mg L^− 1 and 34 to 57 mg L^− 1 of total nitrogen and phosphorus in brewery wastewater.

According to Arantes et al. [9], the COD of brewery wastewater can vary between 2,000 and 6,000 mg L^− 1 and is associated with high solids content and potential dilution by water. These concentrations are very different from those found in this research, where the mixture of residues comprising the BR-Mix culture medium presented a high COD (91,798.9 mg L^− 1) and a lower BOD (9,124.1 mg L^− 1). However, these values result from the combination of the three filtrates (residues of the malting, hop addition, and fermentation processes) in our experiments.

The TSS concentration (252.08 mg L^− 1) found in the BR-Mix was according to Kumar et al. [5] (50–6500 mg L^− 1). TSS is an important aspect of microalgae cultivation and was the reason for the gradual supplementation with BR-Mix. High TSS, primarily due to the presence of yeast residue, cereals, and inorganic additives, can affect the turbidity of the medium [37]. The wastewater used to cultivate microalgae may have high turbidity, blocking light penetration, reducing photosynthesis and cell growth [38]

The concentration of other elements, such as Mg, Ca, and Fe, can contribute to the development of microalgae. They are known to contain these elements that sequester from the environment [39].

3.2 Cultivation of Euglena sp. and supplementation of BR-Mix

Table 2 shows the yield (g L^− 1) at the end of 12 days for the experiments with and without BR-Mix supplementation. Yield was higher for NPK concentrations of 0.5 to 1.5 g L^− 1 supplemented with BR-Mix (p < 0.05) than the other cultivation conditions evaluated. It was possible to observe that 0.5 g L^− 1 NPK was the best condition to produce biomass, resulting in better environmental and economic performance.

Table 2

Average yield (g L^-1 day^-1) of *Euglena* sp. crops in medium with NPK, with and without supplementation of the brewing residue (BR-Mix).
NPK concentration	Average productivity every 2 days (g L^− 1 day^− 1)
(g L^− 1)	Without BR-Mix	With BR-Mix
0.5	0.06 ± 0.03a	0.16 ± 0.04b
1.0	0.07 ± 0.04a	0.16 ± 0.06b
1.5	0.08 ± 0.06a	0.19 ± 0.07b
3.0	0.08 ± 0.05a	0.07 ± 0.05a
6.0	0.09 ± 0.02a	0.08 ± 0.03a
* Equal letters do not differ significantly

Table 2

Using BR-Mix is relevant since it reduces the dependence on fertilizer to cultivate microalgae and valorizes the waste, which otherwise would negatively impact the environment. A similar result can be observed concerning the final biomass yield, in which an increase of over 50% was obtained by supplementing BR-Mix and a lower NPK concentration (Fig. 2).

Figure 2

The higher productivity with BR-Mix supplementation suggests that growth occurred in the mixotrophic mode. Although Euglena sp. can grow photoautotrophically and heterotrophically, it is possible to obtain biomass mixotrophically with bioproduct content of commercial interest [40].

Eze et al. [21] evaluated biomass production in mixotrophic culture using NPK medium (15:15:15) as an alternative medium for E. gracilis. These cultures were supplemented with ethanol as an organic carbon source and peptone as an organic nitrogen source: they achieved a maximum biomass yield of 2.6 g L^− 1, slightly higher than our highest yield (2.1 g L^− 1; 3% of BR-Mix). Although the NPK medium is an effective culture medium, craft beer wastewater as an alternative carbon source is relevant for better biomass yield.

3.3 Characterization of Euglena sp. biomass

Table 3 presents the mean concentrations (%) of proteins, lipids, and carbohydrates of the biomass from cultures with and without 3%-BR-Mix. Varying the NPK concentration for the cultures without BR-Mix resulted in a significant difference (p < 0.05) in the protein content, following the NPK concentration in the medium. The same behavior was observed for the carbohydrate content. Contrastingly, increasing the NPK concentration reduced total lipid content.

There was a difference in protein content among all NPK concentrations when comparing the experiments without and with 3% BR-Mix supplementation. Notably, there was a significant increase (about 2.5 times) in protein accumulation with the BR-Mix supplementation at the lowest NPK concentration. In this context, if the objective were to produce biomass of Euglena sp. with high protein content, the best choice among the proposed conditions would be cultivating with 6 g L^− 1 NPK supplemented with 3% BR-Mix.

It is understood that these microorganisms can accumulate large amounts of proteins and are considered an attractive alternative to meat and fish [41]. Some authors have reported that Euglena sp. can produce protein containing all the 20 proteinogenic amino acids [42, 43].

Table 3

Table 3

Comparative mean values (%) of biomolecules of biomass obtained without and with BR-Mix and different concentrations of NPK (g L^-1).
NPK (g L^− 1)	Average Proteins (%)		p	Average Lipids (%)		p	Average Carbohydrates (%)		p
NPK (g L^− 1)	Without BR-Mix	With BR-Mix	p	Without BR-Mix	With BR-Mix	p	Without BR-Mix	With BR-Mix	p
0.5	13.9	35.1	< 0.000001	14.0	15.3	0.47839	31.4	9.1	0.000005
1.0	25.5	35.6	< 0.000001	11.6	16.0	0.12588	27.2	8.8	< 0.000001
1.5	29.2	35.4	< 0.000001	14.2	15.2	0.24152	31.6	9.7	< 0.000001
3.0	36.3	35.9	0.00805	8.4	8.8	0.78770	53.6	66.5	0.000001
6.0	36.2	37.3	0.00017	6.8	6.8	> 0.9999	57.4	35.7	0.000002

Table 3 shows no significant difference (p < 0.05) in lipid content for cultures with and without BR-Mix, regardless of the NPK concentration. Thus, if the objective were to accumulate lipids, the best choice of culture medium is the NPK concentration of 0.5 g L^− 1 supplemented with 3% of BR-Mix, reducing production costs and improving sustainability. It is noteworthy that, for commercial purposes, biomass with low lipid content is still valuable as long as the fatty acid profile possesses differentiated functions and applicability.

The lipid and protein results in this research are similar to other studies using Euglena sp. Eze et al. [21] cultivated Euglena sp. in different NPK concentrations: the biomass showed lipid and protein contents of 16.4% and 33.33%, respectively. Mondal et al. [44] showed Euglena sp. reached 39 to 61% protein and 14 to 38% lipid content. However, the biomass yield and lipid content change according to the nutrient bioavailability [45]. Ikaran et al. [46] demonstrated that various microalgae can adjust their metabolic pathways to store large amounts of lipids without nitrogen.

There was no difference in carbohydrate content (p < 0.05) between the biomass produced without and with 3%-BR-Mix supplementation. The highest carbohydrate content (66.46%) was found with 3 g L^− 1 NPK and BR-Mix (3%).

The results for the biomass profile of Euglena sp. align with the content reported by other authors, for instance, Feregrino-Mondragón et al. [47], who cultivated Euglena sp. in liquid residues generated in a biochemistry laboratory and obtained 36–42% protein, 56–62% carbohydrate (paramylon), and 2–3% lipid content. Biomass composition is linked to the metabolic pathway that the microalgae use. This study stimulated the mixotrophic cultivation of the Euglena sp. using residue containing several nutrients and rich in organic carbon.

3.4 Lipid profile of Euglena sp. biomass.

The lipids synthesized by microalgae are directly linked to growing conditions, such as nutrient supply, light, and temperature. Under N-starving conditions, C produces molecules such as lipids [48]. The maximum lipid content in our studies was 16% (Table 3; 1,0g L^− 1 NPK and 3% BR-Mix) indicating that the tested media did not foster lipid overaccumulation. However, as can be seen in Table 4 (without BR-Mix supplementation) and Table 5 (with BR-Mix supplementation), the lipid fraction presented a high content of polyunsaturated.

Additionally, fatty acids with 20 carbons and different amounts of unsaturation were observed. BR-Mix supplementation led to better results in the total unsaturated fatty acids. These values are comparable to the results found by Reza et al. [49], who produced E. gracilis in a modified Cramer–Myers medium. They found a maximum of 19.03% of total lipids but with ∼30.66% of polyunsaturated fatty acids of 20 to 22 carbon chains. Studies conducted by Mata et al. [50] with Euglena sp. showed a lipid accumulation of around 20%, containing eicosapentaenoic acid (EPA C20:5 ω -3) and (C22:6) docosahexaenoic acid (DHA C:226 ω-3), which are commercially important omega-3 fats (long-chain polyunsaturated fatty acids).

Tables 4 and 5

Table 4

Lipid profile (%) of *Euglena* sp. biomass grown without BR-Mix supplementation.
FAs	Lipid Profile (%) in different NPK concentrations (g L^− 1)
FAs	0.5	1.0	1.5	3.0	6.0
C13:0	1.2 ± 0.1	-	-	-	-
C14:0	12.4 ± 0.8	2.4 ± 0.2	1.3 ± 0.1	7.3 ± 0.5	8.6 ± 1.4
C15:0	2.1 ± 0.1	-	-	-	-
C16:0	26.7 ± 0.8	31.1 ± 0.3	46.8 ± 1.9	38.2 ± 1.3	40.6 ± 1.6
C16:1	0.9 ± 0.3	1.1 ± 0.1	-	-	4.6 ± 1.5
C16:2	6.8 ± 0.2	5.3 ± 0.4	-	3.7 ± 0.1	3.3 ± 0.4
C16:3	-	4.6 ± 0.1	-	3.4 ± 0.3	2.7 ± 0.7
C18:0	3.0 ± 0.5	3.4 ± 0.3	10.2 ± 0.2	3.7 ± 0.1	4.6 ± 0.4
C18:1	11.8 ± 1.2	14.8 ± 0.6	29.8 ± 0.7	5.7 ± 0.3	6.3 ± 0.1
C18:2	17.4 ± 0.6	22.1 ± 0.4	1.3 ± 0.8	17.2 ± 0.7	16.0 ± 0.5
C18:3	6.8 ± 2.4	15.2 ± 0.4	1.9 ± 1.1	17.0 ± 0.6	13.2 ± 3.2
C20:2	1.6 ± 2.2	-	-	1.0 ± 0.1	-
C20:4	3.7 ± 1.6	-	-	1.3 ± 0.2	-
C20:5	5.7 ± 1.0	-	-	1.6 ± 0.6	-
Saturated	45.3	36.9	57.4	49.3	53.8
Unsaturated	54.7	63.1	42.1	50.7	46.2
Polyunsaturated	41.8	47.2	12.3	45.0	35.2
FAs- fatty acids, C12:0 (Lauric acid); C14:0 (Myristic acid); C16:0 (Palmitic acid); C16:1(Palmitoleic acid); C16:2 (palm-linoleic acid); C16:3 (palm-linolenic); C18:0 (Stearic Acid); C18:1 (Oleic acid); C18:2 (Linoleic acid); C18:3 (Y-linolenic acid); C20:2 (Eicosadienoic acid); C20:3 (Di-homo-y-linolenic acid); C20:4 (Arachidonic acid); C20:5 (Eicosapentaenoic acid-EPA).

Table 5

Lipid profile (%) of *Euglena* sp. biomass cultivated with BR-Mix supplementation.
FAs	Lipid Profile (%) in different NPK concentrations (g L^− 1)
FAs	0.5			1.0		1.5		3.0		6.0
C12:0		-	0.3 ± 0.0		-		-		-
C14:0		3.0 ± 0.1	8.1 ± 0.1		5.6 ± 0.1		3.5 ± 0.2		3.4 ± 0.2
C16:0		19.6 ± 1.4	21.0 ± 0.4		25.8 ± 0.8		48.7 ± 1.8		47.4 ± 2.4
C16:1		1.5 ± 0.1	1.8 ± 0.0		1.3 ± 0.0		0.6 ± 0.1		0.5 ± 0.1
C16:2		7.5 ± 0.4	8.1 ± 0.1		6.0 ± 0.2		1.7 ± 0.3		2.0 ± 0.5
C16:3		6.0 ± 0.1	5.7 ± 0.2		0.7 ± 0.2		2.4 ± 0.4		-
C18:0		2.5 ± 0.1	3.4 ± 0.3		4.7 ± 0.2		6.8 ± 0.4		2.2 ± 0.0
C18:1		12.2 ± 0.5	14.2 ± 1.1		20.9 ± 0.7		12.2 ± 0.5		8.2 ± 0.0
C18:2		23.2 ± 0.7	21.1 ± 0.1		23.2 ± 0.0		14.2 ± 0.8		13.8 ± 0.4
C18:3		18.9 ± 0.9	14.6 ± 0.1		11.5 ± 0.9		9.4 ± 0.8		9.7 ± 1.6
C20:2		0.5 ± 0.3	0.6 ± 0.1		0.2 ± 0.2		0.5 ± 0.0		0.5 ± 0.1
C20:3		0.4 ± 0.3	-		-		-		0.4 ± 0.1
C20:4		0.1 ± 0.0	-		-		-		-
C20:5		3.1 ± 1.3	1.1 ± 0.9		-		-		-
C22:6		1.8 ± 1.2	-		-		-		-
Saturated		25.1	32.8		36.2		59.1		59.0
Unsaturated		75.0	67.2		63.8		40.9		41.0
Polyunsaturated		61.3	51.3		41.6		28.1		28.6
FAs – fatty acids, C12:0 (Lauric acid); C14:0 (Myristic acid); C16:0 (Palmitic acid); C16:1(Palmitoleic acid); C16:2 (palm-linoleic acid); C16:3 (palm-linolenic); C18:0 (Stearic Acid); C18:1 (Oleic acid); C18:2 (Linoleic acid); C18:3 (Y-linolenic acid); C20:2 (Eicosadienoic acid); C20:3 (Di-homo-y-linolenic acid); C20:4 (Arachidonic acid); C20:5 (Eicosapentaenoic acid-EPA); C22:6 (Docosahexaenoic acid-DHA).

According to Ogawa et al. [51], Euglena sp. triacylglycerols have an abundance of myristic acid (C14:0). In our results, it was possible to obtain the same saturated fatty acid content; however, its content was not higher than palmitic acid (C16:0). Overall, the most abundant fatty acids were C16:0, C18:1, C18:2 and C18:3.

We highlight that the microalgae were in a favorable condition of growth since they tend to accumulate more lipids under stressful situations (higher light intensity or low nutrient concentration) [52].

Notably, the total unsaturated fatty acids are higher at lower NPK concentrations (Table 5). Additionally, the long-chain polyunsaturated fatty acids (EPA, DHA), arachidonic acid (ARA, 20:4 ω-6), and γ-linolenic acid (GLA, 18:3 ω-6) are the most studied lipid compounds in microalgae. They are essential fatty acids for humans [53].

In conclusion, it is noted that the crops obtained 14 different fatty acids with 75% of unsaturation with 3% BR-MIX (0,5 g L^− 1 NPK). In comparison, 13 fatty acids with 63% unsaturation were obtained without supplementation (1,0 g L^− 1 NPK).

3.5 Carbohydrate profile of Euglena sp. biomass

Table 6 shows the monosaccharide content: glucose, galactose, rhamnose, fucose and ribose. Other strains of Euglena sp. [54] and E. gracilis [45] presented the same monosaccharides.

It was observed that there was a higher glucose concentration, a paramylon monomer characteristic of euglenophyte biomass, in the highest NPK concentrations. Gissibl et al. [55] reported that the heterotrophic cultivation can improve paramylon accumulation; mixotrophic cultivation under light hindered paramylon accumulation and conservation. This indicates that paramylon degradation might be triggered by a photoreceptor. Gulk et al. [45] tested butanol, ethanol, glucose, glycine, and glycerol as carbon sources, reaching better carbohydrate yields with ethanol. Sun et al. [56] and Kim et al. [57] showed that there was also a great potential to produce paramylon heterotrophically. In our results, the effect of the carbon source on carbohydrate concentration was not observed, as there was the co-effect of increased nitrogen in the medium, which can lead to a higher paramylon content [58].

Table 6

Carbohydrate profile (%) with and without BR-Mix supplementation in different concentrations of NPK.
Monosaccharide		Monosaccharide profile (%) in different NPK concentrations (g L^− 1)
Monosaccharide		0.5		1.0		1.5		3.0	6.0
		No addition of BR-Mix
Glucose	10.8		16.4		19.6		57.1			67.0
Galactose	58.3		64.1		65.3		40.1			24.5
Ramnose	17.2		10.8		9.0		0.3			2.0
Ribose	-		-		-		-			-
Fucose	11.3		4.21		3.1		0.2			2.7
	With the addition of BR-Mix
Glucose	10.9		13.8		14.5		48.8			54.2
Galactose	60.2		53.3		59.5		48.0			42.1
Ramnose	10.0		8.5		10.4		2.5			0.3
Ribose	5.2		5.9		4.9		-			0.5
Fucose	2.0		2.8		1.9		0.6			-

Table 6

Recently, in a study by Kim et al. [59] with E. gracilis grown in beer bagasse, the paramylon content reached 32.3% (w/w). In our results, glucose content varied from 10 to 67% (w/w) indicating the potential of paramylon production with or without BR-Mix supplementation.

3.6Antioxidant potential

The antioxidant potential by ORAC test was presented in Fig. 3. Higher antioxidant potential (p < 0.05) was found in the cultures with BR-Mix supplementation than without, especially in the 0.5 and 1.0 g L^-1 NPK concentrations, which reached 170 .97 ± 26.3 and 172.79 ± 24.2 µmol eq g^-1, respectively. Ahmed et al. (2014) analyzed the profile of 12 microalgae regarding the industrial applicability: the results showed that the antioxidant potential obtained by the ORAC method ranged from 45 to 577 µmol eq g^-1, demonstrating the potential use of these microalgae for human health, as food or as dietary supplements. In addition, Mahapatra et al. [22] reported a high α-tocopherol content in Euglena gracilis biomass.

Figure 3

The antioxidants found in microalgae are mainly pigments such as chlorophylls, carotenoids, phenolic compounds and tocopherols [60]. Tocopherols, chlorophylls, and carotenoids comprise the fat-soluble antioxidant, and polyphenols, phycobiliproteins, and vitamin C represent the water-soluble compounds [61]. Previous studies reported that some physical factors could favor the accumulation of antioxidants in the microalgae cell, and the greater availability of light increases the cellular carotenoids [62–64].

Considering our data for carbohydrate, lipid, and protein content and the antioxidant potential, it is observed that depending on the mixotrophic conditions, it generates biomass with potential for different uses. A photoperiod with more light intensity may increase the antioxidant capacity of Euglena sp. due to its pigment content. On the other hand, reducing light time may favor paramylon production. 12:12 photoperiod (L:D) may be altered to achieve greater antioxidant potential. These factors can be controlled according to the target bioproduct and application. Under the analyzed conditions, 0.5 g L^-1 NPK with BR-Mix (3%) improved lipid production and antioxidant activity.

3.7 Upscaling of Euglena sp. cultivation and BR-Mix supplementation

One of the biggest obstacles to enabling microalgae production commercially is related to biomass yield [65]. Cultivating Euglena sp. with 0.5 g L^− 1 NPK and 3% BR-Mix produced the best biomass productivity and composition. Thus, the BR-Mix supplementation was increased in a three-step manner until reaching a final culture medium volume of 30 L to test the system's scalability. Figure 4 shows biomass's average yield (g L^− 1) and cell growth (cell mL^− 1).

Figure 4

In the third stage of the experiment (up to 30% of BR-Mix supplementation), it was possible to observe a decrease in cell density and lower biomass yield. Biomass production was better in the first stage (2.98 g L^-1, 0.29 g L^-1 d^-1; up to 10% BR-Mix). In the second step, the final biomass was 4.56 g L^-1 in 22 days (0.22 g L^-1 d^-1; up to 25% BR-Mix supplementation). Jung et al. [66] obtained 5.16 g L^-1 or 0.74 g L^-1 d^-1 of E. gracilis biomass grown mixotrophically in Modified Huntner's medium.

The biomass reduction and cell density decline in the last cultivation stage may be associated with low luminosity inside the tank due to turbidity. The luminosity of the first and second steps remained at 148 µmol photons m^-2 s^-1: it was impossible to measure the luminosity with the datalogger inside the tank in the third step. The increase in the volume up to 30 L requires a new production system with higher light incidence and lower depth.

Euglena sp. presented 26.6% protein, 38.5% carbohydrate, and 17.38% lipid (m/m) content at the end of the second step. Carbohydrates also had a differentiated profile due to their high glucose (paramylon) content (91.4 ± 2.3%).

The lipid content was 17.38% (w/w): it was identified 10 fatty acids, mainly myristic acid (C14:0), palmitic acid (C16:0), and oleic acid (C18:1) and a content of unsaturated fatty acids of 41.1% (Table 7). It was observed that the antioxidant capacity obtained for biomass was 41.8 ± 13.1 µmol eq g^-1; this potential was lower than that found in the experiments with 3% BR-Mix.

Table 7

Main fatty acids found in biomass after *upscaling* with BR-Mix and final volume of 30 L of production.
Fatty Acid	%
C13:0	2.9
C14:0	20.7
C15:0	1.6
C16:0	25.3
C16:1	2.4
C18:0	6.2
C18:1	17.0
C18:2	9.5
C18:3	3.1
C20:4	2.1
Where: C13:0 - isomyristic acid, C14:0 - myristic acid, C15:0 - pentadecylic acid, C16:0 - palmitic acid, C16:1 - palmitoleic acid, C18:0 - stearic acid, C18:1 - oleic acid, C18:2 - linoleic acid, C18:3 - (α-) linolenic acid, C20:4 - arachidonic acid.

By comparing the results obtained on the scale of 1 L with 3%-BR-Mix supplementation, to the scale of 30 L with 30%-BR-Mix supplementation, it was observed that the stress caused by the wastewater and the hampered light incidence inside the tank reduced the antioxidant potential.

Using 0.5 g L^− 1 NPK and supplementing 25% BR-Mix to cultivate Euglena sp. showed the maximum biomass yield of 4.56 g L^− 1 in 22 days of cultivation. This best condition presented 26.6% protein, 17.3% lipid, 38.5% carbohydrate content and antioxidant potential of 41.8 ± 13.1 µmol eq g^− 1 Trolox. The high content of unsaturated fatty acids (41.1%) and paramylon (91.4%) was plausible. NPK plus craft brewery residue is a clean alternative to Euglena sp. production.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Funding

This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq – protocol 306216/2022-1, Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul 23/2551-0000161-6 and Secretaria de Inovação, Ciência e Tecnologia do RS process 22/2500-0000201-0.

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Giséle Alves, Maria A. O. Linton, Adriana Dupont, Leonardo Ritzel³ and Vitória C.Rocha. The first draft of the manuscript was written by Giséle Alves, Michele Hoeltz, Tiele M. Rizzetti and Rosana C. S. Schneider and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Data Availability

The datasets generated during and analysed during the current study are available in the paper.

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Biomass of Euglena sp.produced in Mineral Medium Supplemented With Craft Brewery Residue

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Abstract

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Statement of novelty

1. INTRODUCTION

2. METHODOLOGY

2.1 Characterization of culture media and inoculum standardization

2.2 Cultivation of Euglena sp.

2.3 Upscaling of Euglena sp. cultivation

2.4 Characterization of biomass

2.5 Statistical tests

3. RESULTS AND DISCUSSIONS

3.1 BR-Mix characterization

3.2 Cultivation of Euglena sp. and supplementation of BR-Mix

3.3 Characterization of Euglena sp. biomass

3.4 Lipid profile of Euglena sp. biomass.

3.5 Carbohydrate profile of Euglena sp. biomass

3.6Antioxidant potential

3.7 Upscaling of Euglena sp. cultivation and BR-Mix supplementation

4. CONCLUSION

Declarations

Competing Interests

Funding

Author Contributions

Acknowledgements

Data Availability

References

Status:

Journal Publication

Version 1