Sustainable carotenoid production using amylaceous agro-industrial byproducts: Process efficiency and environmental assessment

doi:10.21203/rs.3.rs-7982452/v1

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Sustainable carotenoid production using amylaceous agro-industrial byproducts: Process efficiency and environmental assessment

https://doi.org/10.21203/rs.3.rs-7982452/v1

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The transition to production systems with sustainable design requires innovative biotechnological approaches to obtain high-value-added compounds from renewable resources. Microbial carotenoids, known for their antioxidants and antimicrobial properties, have promising applications in the food, pharmaceutical, and cosmetic industries. However, their large-scale production is constrained by high costs, limiting commercial viability. In this study, grain by-products were explored as sustainable low-cost feedstocks for microbial carotenoid production, enhancing both economic and environmental feasibility. Corn, soybean, rice, and wheat bran hydrolysates were evaluated as carbon and nutrient sources, supporting microbial growth and carotenoid synthesis without additional supplementation or detoxification. Despite variations in hydrolysate composition, carotenoid production was achieved across all substrates, with rice and soybean bran yielding 24.55 and 28.41 mg_carotenoids/L_{growth medium}, respectively. Life cycle assessment (LCA) identified rice bran as the most environmentally favorable option, reinforcing its potential as a sustainable bioprocess feedstock. This study highlights the valorization of agro-industrial residues as an efficient strategy to reduce production costs and environmental impact, contributing to the development of more sustainable biotechnologies for carotenoid synthesis and beyond.

Sustainable bioprocess

High-added-value compounds

Environmental feasibility

Renewable feedstocks

Life cycle Assessment

Interest in products from alternative and renewable sources has risen due to the environmental impacts of using fossil fuels. The urgency to find alternative solutions has escalated into a global concern, particularly considering the limited availability of petroleum-derived resources (Ashokkumar et al., 2022; Antar et al., 2021). In this context, biorefineries play a central role in transitioning toward a circular bioeconomy, optimizing the use of renewable resources through integrated processes to obtain valuable bioproducts (Mujtaba et al., 2022). A crucial aspect of biorefineries is the cost and availability of the substrate/feedstocks. The agro-industrial sector generates a substantial volume of by-products, approximately 140 billion metric tons of biomass annually worldwide. These biomasses can serve as low-cost materials in bioprocesses to enhance biorefineries' economic viability and environmental aspects (UNEP, 2019; Koul et al., 2022).

Grain and cereal crops such as wheat, rice, corn, and sugarcane are primary contributors to agro-industrial biomass production (FAO STAT, 2022). Biomass from the cultivation and processing of these grains and cereals, notably bran, is a rich source of carbohydrates, starch (a carbon source), proteins (a nitrogen source), and minerals (a source of micronutrients) (Anderson and Simsek, 2019; Balbino et al., 2023). These components make bran an excellent substrate for cultivating microorganisms to produce industrially valuable bioproducts, such as pigments, proteins, and enzymes (Balbino et al., 2023; Spaggiari et al., 2021).

Given its nutrient-rich composition, bran is an attractive feedstock providing the essential carbon, nitrogen, and nutrients for microbial cultivation in industrial bioprocesses. To effectively utilize these nutrients, efficient bioprocess methods are needed, making sugars, nutrients, and minerals available for microbial use in bioprocess. Biomass hydrolysis using diluted acid simplifies the process, making it readily applicable in the industry, and allows for the direct use of neutralized hydrolysate in fermentation processes, without the need for additional separation or acid recovery steps, which are necessary in hydrolysis with concentrated acid. By minimizing the generation of undesirable waste or by-products and simplifying processing steps, this strategy boosts the sustainability and economic viability of the bioprocess (Zhou et al., 2021).

In addition to substrate availability, producing a range of biomolecules is crucial for the success of biorefinery (Yuan et al., 2021). While biofuel production remains essential, the growing demand for sustainable solutions has driven the development of biotechnological compounds that can serve as alternatives to traditional natural and synthetic products. For instance, several high-value-added biomolecules offer lower environmental impact and enhanced process efficiency (Medeiros et al., 2022).

Natural pigments are promising compounds obtainable through the bioprocessing of agro-industrial by-products. Beyond providing color, these biopigments offer nutritional benefits and have valuable properties, such as cytotoxic, antioxidant, antimicrobial, antimalarial, anticancer, and antitumor activities (Sharma et al., 2024). Carotenoids, for example, are lipid-soluble pigments known for their high antioxidant capacity, useful in applications like skin care, food and feed colorants, and pharmaceutical additives (Foong et al., 2021; Díaz-Ruiz et al., 2023). The carotenoids market is projected to reach USD 1.84 billion by 2024, with a CAGR of 4.64% from 2024 to 2029 (Mordor Intelligence Research & Advisory, 2023). Yeast species are promising sources of diverse pigments like carotenoids due to their rapid growth, minimal production of toxic by-products, and ability to consume C5 and C6 carbon sources. These characteristics help minimize processing time and cost (Paul et al., 2023; Balbino et al., 2023).

In this study, we explored the potential of corn, soybean, rice, and wheat bran hydrolysates (CBH, SBH, RBH, and WBH, respectively) as a low-cost substrate for carotenoid production using Rhodotorula mucilaginosa. The proposed process underwent an environmental performance using the life cycle assessment (LCA) methodology, which covered impacts on global warming, ozone formation, terrestrial acidification, mineral resource scarcity, and fossil resource scarcity. This approach aims to enhance greenness and sustainability by using renewable feedstocks and eliminating intermediate processing, specifically the need for acid recovery during hydrolysis. Additionally, it avoids the need for supplementation or detoxification of the hydrolysate before fermentation, thus preventing the generation of undesirable waste or by-products and reducing the consumption of reagents and process time. This alignment with the principles of green chemistry is expected to contribute to the economic and environmental sustainability of future pigment biorefineries and other bioprocesses based on grain and cereal by-products.

2.1 Dilute-acid hydrolysis of grains by-products

The corn, soybean, rice, and wheat bran were generously donated by local farmers in Guaratinguetá, SP, Brazil. The brans were subjected to dilute-acid hydrolysis under the standard conditions established by Ayadi et al. (2019) and Martiniano et al. (2022). Briefly, hydrolysis was performed in an autoclave with 2% (v/v) sulfuric acid and 15% (w/v) total solids loading at 121°C for 60 min. After hydrolysis, the liquid and solid fractions were separated via vacuum filtration. The liquid fractions (corn bran hydrolysate, CBH; soybean bran hydrolysate, SBH; rice bran hydrolysate, RBH; and wheat bran hydrolysate, WBH) were recovered and stored at -4°C for further carotenoid production studies. The concentrations of sugars, furan derivatives, and phenolic compounds in the four bran hydrolysates were analyzed using high-performance liquid chromatography (HPLC).

2.2 Yeast-derived carotenoid production

2.2.1 Microorganism and inoculum preparation

Yeast Rhodotorula mucilaginosa was used to produce carotenoids. To activate the yeast, cells from the stock culture were transferred to Erlenmeyer® flasks (125 mL) containing 30 mL of medium with commercial glucose (YM) (composition (g/L): glucose (30), peptone (5), yeast extract (3), and malt extract (3)). The flasks were incubated in an orbital shaker at 30°C, 300 rpm for 18 h. The cells were recovered by centrifugation at 3000 xg for 15 min, washed, and resuspended in 0.1% (w/v) peptone aqueous solution. The cells were utilized as inoculum for the subsequent fermentation processes.

2.2.2 Hydrolysates as a growth medium for carotenoid production

Considering the different concentrations of sugar in the four hydrolysates, the concentration of sugars (sum of glucose, xylose, and arabinose) in CBH, RBH, and WBH was standardized to approximately 10–12 g/L by simply adding the right amount of water, guaranteeing the minor concentration obtained after soybean bran hydrolysis. This “sugar standardization” aimed to obtain comparable results between hydrolysates. The initial pH of each hydrolysate was adjusted to 5.5 by adding sodium hydroxide micro-pearls. Erlenmeyer® flasks (250 mL) containing 60 mL of CBH, SBH, RBH, or WBH were sterilized at 121°C for 15 min in an autoclave. After sterilization, the flasks were inoculated with activated yeast cells and incubated at 30°C, 300 rpm for 72 h. Samples were collected every 2 h up to 10 h and after 24, 48, and 72 h to determine the cellular biomass concentration and sugar consumption.

2.3 Cell disruption and carotenoid extraction

Because carotenoids are produced intracellularly, a proper cell disruption process and biopigment extraction were applied to the samples. The harvested pigmented cell pellets were washed three times, resuspended in 2 mL distilled water, and dried at 60°C for 24 h. 0.1 g of dry cells were resuspended in 10 mL of 2 mol/L NaOH aqueous solution and incubated in a water bath at 65°C for 10 min. The cells were centrifuged (3000 xg for 15 min) and frozen overnight. The pellets were resuspended in 10 mL methanol: acetone solution (7:3, v/v) and homogenized by ultrasonication with cycles of 20 s ON/5 s OFF for 10 min to achieve complete cell disruption and the release of intracellular carotenoids. The cell debris was separated by centrifugation (3000 xg for 15 min) and the colored supernatants (carotenoid-rich extracts) were collected.

2.4 Life cycle assessment (LCA)

The life cycle analysis (LCA) was conducted to compare the potential environmental impacts associated with yeast-derived carotenoid production using hydrolysates from corn, soybean, rice, and wheat bran, to identify the scenarios with the lowest impacts.

The environmental profile of the procedure proposed in this study was evaluated using LCA methodology according to the ISO 14040 and ISO 14044 standards (ISO, 2006a, 2006b). Data on the impacts of producing the materials and electricity consumed were mostly sourced from the Ecoinvent database version 3.9.1. (Wernet et al., 2016). However, as data were not available for corn bran and soybean bran production, they were obtained from the World Food LCA database version 3.5 and Agribalyse version 3.1, respectively (Nemecek et al., 2019; Asselin-Balençon et al., 2022). The SimaPro software version 9.5.0.2 was used to model the process (Table S1) and to calculate the environmental impacts. The system boundary adopted was cradle-to-gate, from bran hydrolysate preparation to fermentation. The impacts were estimated by applying the ReCiPe 2016 method from the hierarchist perspective (Huijbregts et al., 2017). Based on data availability, the following impact categories were selected: global warming (equivalent to the carbon footprint), ozone formation - human health, terrestrial acidification, mineral resource scarcity, and fossil resource scarcity.

2.5 Analytical methods

2.5.1 Cell biomass concentration

Yeast cell concentrations were determined using a spectrophotometer. Absorbances were converted to concentration (g/L) using a previously established standard curve. The maximum specific growth rate (µmax, h^− 1) was determined by linear regression between the optical density (600 nm) obtained from the exponential growth phase and time (h).

2.5.2 Determination of sugar, furan derivate, and phenolic compounds

Glucose, xylose, and arabinose were quantified using an HPLC Agilent 1200 series (Agilent Technologies Inc., USA) equipped with a refractive index detector (RID-6A) and HPX-87H (300 × 7.8 mm) column (Bio-Rad, USA). The analysis was performed under the following conditions: 45°C column temperature, 0.01 N H₂SO₄ as the mobile phase, 0.6 mL/min flow rate, and 20 µL injection volume.

Furan derivatives (furfural and 5-hydroxymethylfurfural (5-HMF)) and phenolic compounds (4-hydroxybenzoic acid, ferulic acid, gallic acid, p-coumaric acid, vanillic acid, pyrocatechol, syringaldehyde, and vanillin) were analyzed according to Skendi et al. (2017) using a Zorbax C18 column (Agilent, Santa Clara, CA, USA) at 30°C with a gradient of 1% (v/v) acetic acid in water (A), acetonitrile (B), and methanol (C) at a flow rate of 1.3 mL/min. The respective analytes were detected using a UV detector at wavelengths of 260, 280, and 320 nm.

2.5.3 Determination of the total soluble protein concentration

The concentration of total soluble proteins was determined according to the method described by Lowry et al. (1951). Approximately 5 ml of reagent A (48 ml of 2% sodium carbonate in 0.1 n sodium hydroxide, 1 ml of 0.5% copper sulfate, and 1 ml of 1% sodium potassium tartrate) was added to the sample and kept aside for 15 min. 0.5 ml of freshly prepared reagent B (Folin-Ciocalteu solution: water, 1:1) was added and mixed. The test tubes were then incubated for 30 min in the dark. Measurements were taken at 660 nm, according to the absorption spectrum of the colored reaction product, using an Eppendorf Biospectrometer® fluorescence. A standard reference curve was prepared using bovine serum albumin (Sigma-Aldrich) at 40 to 400 µg/mL concentrations.

2.5.4 Carotenoids analysis and quantification

The total carotenoid concentration was quantified as β-carotene equivalents using calibration curves of a β-carotene (Sigma-Aldrich) standard solution ranging from 1 to 60 µg/mL, prepared in methanol: acetone (7:3, v/v), and analyzed by spectrophotometric scans. Spectrophotometric scans were performed on a Thermo Scientific® UV-Vis spectrophotometer (model Genesis 10S, China). Carotenoid quantification was performed by identifying peaks in the region between 350 and 700 nm.

The total carotenoid values (mg_carotenoids/L_solvent) were used to calculate the specific concentration of total carotenoids, expressed in mg_carotenoids/g_cells, and the concentration of total carotenoids per volume of the growth medium, expressed in mg_carotenoids/L_{growth medium}, calculation formulas of total carotenoids concentration are described in Supplementary Information.

2.6 Statistical Analysis

Data were analyzed using STATISTICA (StaSoft, Inc., Oklahoma, USA) and are presented as mean ± standard deviation (SD). Means were tested for significant differences with 95% confidence through a one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. The level of significance was set at p < 0.05.

3.1 Bran hydrolysates characterization

Cereals and grain cultivation, such as corn, soybean, rice, and wheat, are global practices in the agricultural sector that generate a substantial number of by-products when processed on an industrial scale (Kaur et al., 2023). Therefore, it is essential to investigate the potential of agro-industrial by-products as low-cost sources of carbon and nutrients to obtain high-value-added products. Despite this potential, releasing sugars and nutrients from by-products biomass is necessary.

Initially, dilute-acid hydrolysis was applied to corn, soybean, rice, and wheat bran to release monomeric sugars, resulting in the production of corn bran hydrolysate (CBH), soybean bran hydrolysate (SBH), rice bran hydrolysate (RBH), and wheat bran hydrolysate (WBH). Dilute-acid hydrolysis is an efficient and well-established method for breaking down lignocellulosic and amylaceous materials due to its ability to penetrate plant cell walls and weaken the intermolecular bonds between cellulose, hemicellulose, and lignin, making sugars and nutrients in biomass more accessible for microbial consumption (Lv et al., 2024). These four hydrolysates are rich in pentoses, hexoses, proteins, and compounds that can inhibit microbial growth. It is suggested that the proportions of starch, cellulosic, and hemicellulosic fractions and the structural differences between each type of bran interfered with the sugar release during hydrolysis.

The sugar content (sum of glucose, xylose, and arabinose) ranged from 14 to 105 g/L in all hydrolysates, with CBH, SBH, RBH, and WBH containing 104.33 ± 0.36 g/L, 11.04 ± 0.34 g/L, 23.85 ± 0.45 g/L, and 43.37 ± 0.66 g/L, respectively. According to the literature, corn bran may contain approximately 12–77% carbohydrates, 11–32% starch, 10–28% cellulose, and 1–9% lignin (Philippini et al., 2021; Probst and Vadlani, 2015; Rose et al., 2010). The lower lignin content and higher carbohydrate, starch, and cellulose contents of corn bran likely contributed to the release of glucose during acid hydrolysis, resulting in a hexose-rich hydrolysate (Zhou et al., 2021).

To obtain comparative results on cell growth, sugar consumption, and biopigment production, the sugar content (sum of glucose, xylose, and arabinose) was adjusted to a range of 10–12 g/L by adding water in all four hydrolysates, which corresponds to the lowest sugar concentration found in SBH. The standardized bran hydrolysates were characterized for glucose, xylose, arabinose, total soluble protein content, and microbial growth-inhibiting compounds (organic acids, furan derivatives, and phenolics), as respective results presented in Fig. 1.

The standardized bran hydrolysates exhibited variation in glucose concentration, with CBH > RBH > SBH > WBH (Fig. 1a). In CBH, glucose was found at 10.85 ± 0.75 g/L, representing approximately 88% of sugar content (sum of glucose, xylose, and arabinose), with trace amounts of xylose and arabinose. Similarly, glucose was the main sugar in RBH and SBH, accounting for 63.7% and 49.7% of the sugar content, respectively, but displayed relatively smaller differences in glucose/xylose and glucose/arabinose ratios compared to CBH. Arabinose concentrations exceeded those of xylose in both RBH and SBH, with a more pronounced difference in SBH (32.1% arabinose and 18.0% xylose) than in RBH (20.9% arabinose and 15.3% xylose). In WBH, however, xylose was the primary sugar (4.07 ± 0.4 g/L) with similar concentrations of glucose, xylose, and arabinose and a higher xylose/glucose ratio compared to the other hydrolysates, which could be due to the higher hemicellulose content in wheat bran biomass (Ayadi et al., 2019).

Similar to the sugar profiles, the total soluble protein content in all hydrolysates varied in the order SBH > RBH > WBH > CBH (Fig. 1a). However, the ratio between the concentration of sugars (sum of glucose, xylose, and arabinose - carbon sources) and proteins (total soluble protein – nitrogen sources) in the standardized hydrolysates displayed relatively smaller variations between SBH (1.46 ± 0.50), WBH (2.16 ± 0.54), and RBH (1.99 ± 0.18), while CBH exhibited a higher carbohydrate/protein ratio (8.83 ± 0.89).

In addition to monomeric sugars, compounds that inhibit microbial growth and metabolism can also be formed during the hydrolysis of lignocellulosic and amylaceous biomass. For instance, acetic acid forms due to the deacetylation of acetylated pentosane and hydrolysis of the acetyl groups in hemicellulose (Świątek et al., 2020). At the same time, 5-HMF can form through a series of chemical reactions between cellulose and starch, including glucan hydrolysis, isomerization of glucose to fructose, and dehydration of fructose into HMF (Iris and Tsang, 2017; Zhao et al., 2019). Concerning standardized bran hydrolysates, SBH contained 80.50 ± 3.53 mg/L of acetic acid, which is about two-fold higher than the concentration presented in RBH and WBH (43.00 ± 3.46 and 37.66 ± 3.35 mg/L, respectively), and about six-times higher than CBH (12.50 ± 3.50 mg/L). Whereas, 5-HMF concentration ranged from 12 to 20 mg/L in SBH, RBH, WBH, and a trace concentration was found in CBH (0.57 ± 0.06 mg/L) (Fig. 1b).

Phenolic compounds can also be formed during the acid hydrolysis of lignocellulosic and amylaceous biomass from lignin degradation (Luo et al., 2021). Among phenolic compounds quantified in standardized bran hydrolysates, pyrocatechol was detected at higher levels in SBH (14.73 ± 0.31 mg/L), but not in CBH (Fig. 1c). Ferulic acid was present in all hydrolysates, ranging from 1.7 to 4.5 mg/L, with RBH containing the highest content (Fig. 1c). Vanillic acid, vanillin, and p-coumaric acid were present in minor concentrations in all hydrolysates, ranging from 0.4 to 1.8 mg/L, with SBH displaying the highest concentration. The concentration of inhibitory compounds in bran hydrolysates (shown in Figs. 1b and 1c) is influenced by the dilution required to standardize the sugar concentration to 10–12 g/L. The CBH was diluted with a larger volume of water due to its higher sugar concentration, resulting in a standardized hydrolysate with lower concentrations of inhibitory compounds.

Therefore, given that the composition of the growth medium can significantly impact the microbial growth rate, substrate consumption, and secondary metabolite production, these four standardized hydrolysates were evaluated as growth media for R. mucilaginosa yeast cells and carotenoid production.

3.2 Rhodotorula mucilaginosa cultivation and carotenoid production using standardized bran hydrolysates

3.2.1 Sugar consumption

R. mucilaginosa is known for assimilating different carbon sources (Hamidi et al., 2020). The consumption of glucose, xylose, and arabinose by the yeast R. mucilaginosa grown in standardized hydrolysates was evaluated (Fig. 2). Despite variations in consumption patterns based on hydrolysate composition, glucose was the first sugar consumed under all conditions. However, distinct observations were made for a specific case of CBH (Fig. 2a). Glucose was not entirely consumed after 72 h, as observed in experiments with the other hydrolysates. After 72 h of cultivation, there was still circa 3.0 g/L of glucose to be consumed. Moreover, arabinose remained unutilized, whereas xylose consumption occurred between 10 and 72 h (Fig. 2a). These results suggest that SBH, WBH, and RBH enable more efficient sugar consumption by yeast cells than CBH. In all three hydrolysates, R. mucilaginosa was shown to have the ability to fully consume glucose, xylose, and arabinose after 72h of cultivation.

Using WBH, as shown in Fig. 2d, glucose remained the preferred sugar, but xylose and arabinose metabolization began only after glucose was depleted (approximately 10 h of growth). In this case, xylose was consumed at a higher rate than arabinose. These differences in sugar consumption patterns may be attributed to variations in sugar concentration present in the hydrolysates, to their total soluble protein content, and the presence of microbial growth inhibitory compounds, all of which may influence yeast metabolism.

Indeed, the results demonstrated the capacity of R. mucilaginosa to metabolize multiple sugars found in amylaceous hydrolysates. These distinct sugar consumption patterns highlight the importance of carefully choosing suitable by-product-based growth media to optimize conditions for yeast cultivation and biopigment production, thereby enhancing the overall efficiency and bioprocess yield.

3.2.2 Cell biomass and carotenoid production

Similar to the variations in sugar consumption, differences in cell biomass and carotenoid production may occur due to hydrolysate compositions, which in turn influence growth and biomolecule production. Regarding the production of cellular biomass by R. mucilaginosa, similar patterns were observed when cultivated in the SBH, RBH, or WBH (Figs. 2b, 2c, and 2d, respectively). Using these three hydrolysates, the final cell biomass concentration ranged from 25 to 30 g/L, and the specific maximum growth rate (µmax.) was approximately 0.25 to 0.27 (h^− 1) (Table 1). On the other hand, using CBH, the yeast exhibited a lower final cellular biomass production (9.75 g/L) and µmax. (0.16 h^− 1). Furthermore, it was noted that the yeast growth in CBH slowed down after 10 h of cultivation compared to growth in the other standardized bran hydrolysates (Figure S1).

Regarding biopigment production, specific concentrations of total carotenoids exhibited the following pattern: RBH ≈ CBH > SBH ≈ WBH (Table 1). Nevertheless, considering the concentrations of total carotenoids per volume of the growth medium, the use of RBH and SBH proved to be more favorable, yielding 28.41 and 24.55 mg_carotenoids/L_{growth medium}, respectively, compared to WBH and CBH (Table 1). Although the specific concentration of total carotenoids was lower when using SBH (0.83 mg_carotenoids/g_cells) compared to RBH (1.11 mg_carotenoids/g_cells), the higher cell biomass production by the yeast R. mucilaginosa cultivated in SBH resulted in the second-highest concentration of total carotenoids per volume of growth medium under these conditions (Table 1). These findings suggest that, when cultivated in SBH, the yeast diverted more energy to metabolic pathways for cell biomass production, thereby reducing the energy available for biopigment synthesis.

Table 1
Cell biomass and carotenoid concentrations produced by yeast *R. mucilaginosa* cultivated in corn bran hydrolysate (CBH), soybean bran hydrolysate (SBH), rice bran hydrolysate (RBH), and wheat bran hydrolysate (WBH).
	CBH	SBH	RBH	WBH
Cell biomass concentration (g/L)	9.75 ± 0.52	29.74 ± 0.18	25.69 ± 0.16	25.11 ± 0.78
µmax. (h^− 1)	0.16 ± 0.01	0.26 ± 0.02	0.27 ± 0.02	0.25 ± 0.04
Specific concentrations of total carotenoids (mg_carotenoids/g_cells)	1.08 ± 0.08	0.83 ± 0.09	1.11 ± 0.05	0.81 ± 0.08
Concentrations of total carotenoids per volume of growth medium (mg_carotenoids/L_{growth medium})	10.50 ± 0.31	24.55 ± 0.25	28.41 ± 0.23	20.28 ± 0.51

When CBH was employed, lower cell biomass production was observed (9.75 g/L) (Table 1). Although CBH allowed for achieving the second-highest specific concentrations of total carotenoids (1.08 mg_carotenoids/g_cells), similar to values obtained using RBH, the total carotenoid concentration per volume of growth medium was comparatively lower compared to RBH. This reduction was due to the limited cell biomass production in CBH. In other words, despite the cells accumulating higher concentrations of total carotenoids, using CBH as a substrate may result in low productivity due to reduced cell biomass production - a critical factor for industrial bioprocess implementation.

The most promising results were obtained using RBH as substrate, in which the yeast produced 25.69 g/L of cell biomass and 1.11 mg_carotenoids/g_cells, indicating balanced metabolic activities for both growth and biopigment production (Table 1). The highest concentration of total carotenoids per volume of growth medium was achieved using RBH (28.41 mg_carotenoids/L_{growth medium}), highlighting the potential of rice bran as a feedstock for biopigment production.

Despite the varying sugar metabolism, cellular biomass, and biopigment production of R. mucilaginosa, the feasibility of using different types of bran for biopigment production has been successfully demonstrated. Manimala and Murugesan (2017) previously evaluated various alternative biomass sources as substrates for carotenoid production by R. mucilaginosa, including rice bran and flour, wheat bran, coconut oil cake, sesame oil cake, tamarind seed powder, peanut oil cake, cassava bagasse, and sugarcane bagasse (Manimala and Murugesan, 2017). Using cassava bagasse yielded the highest carotenoid production, whereas other substrates like rice bran did not favor biopigment production (Manimala and Murugesan, 2017). In this study, rice bran in natura was mixed with a synthetic growth medium for subsequent submerged fermentation. When compared with our findings, the higher productivity using RBH as a growth medium suggests that the pre-treatment of rice bran with diluted acid, along with other by-products, may be crucial for the release of sugars, making them more accessible to microbial metabolism and, consequently, improving bioprocess productivity. Furthermore, dilute-acid hydrolysis allows the neutralized hydrolysate to be directly used in fermentation without additional steps for acid separation or recovery (Zhou et al., 2021). The strategy also avoids the need for medium detoxification or supplementation, reducing the use of extra reagents/nutrients and preventing the generation of waste or undesirable by-products. These modifications significantly improve the economic and environmental aspects associated with the process, enhancing its overall sustainability and the greenness of yeast-based biorefineries.

To further understand the influence of each hydrolysate on cell growth and biopigment production, these two parameters were plotted as a function of the total soluble protein content of hydrolysates and the carbon/nitrogen ratio of hydrolysates, respectively. As shown in Fig. 3a, a correlation was observed between the concentration of cellular biomass produced by R. mucilaginosa in the different hydrolysates and the total soluble protein content of the hydrolysates.

Considering that the sum of the main carbon sources (glucose, xylose, and arabinose) was standardized to 10–12 g/L in the four hydrolysates, the lower production of cellular biomass using CBH may be attributed to its lower nitrogen source (protein) concentration and, consequently, its high C_carbohydrate/N_protein rate. Varied levels of carbon and nitrogen sources can influence the carbon flux, potentially directing it away from biomass or carotenoid production (Gedela et al., 2023; Elfeky et al., 2019). Hydrolysates with C_carbohydrate/N_protein rates close to 2 (SBH, WBH, and RBH) exhibited higher carotenoid production than those with C/N ratios close to 8 (CBH) (Fig. 3b). It should be highlighted that achieving a balance between carotenoid production and microorganism growth is a key challenge in the biotechnological production of biopigments using yeast (Li et al., 2022). Under these conditions, RBH and SBH supported better cell growth and carotenoid production than the other hydrolysates. However, the use of RBH stands out because it enables a process with higher specific concentrations of total carotenoids than SBH.

In summary, these results demonstrate that R. mucilaginosa can grow and produce biopigments using CBH, SBH, RBH, and WBH as the sole nutritional source, without requiring additional detoxification steps or medium supplementation. This may increase the bioprocess costs, confirming the potential of these by-products for sustainable pigment biorefineries in the next generation.

3.3 Life cycle assessment

While life cycle assessments (LCA) are frequently conducted to evaluate the environmental impact of carotenoid production from microalgae, particularly for compounds like astaxanthin and β-carotene, studies focusing on producing yeast-derived carotenoids using renewable feedstocks remain limited (de Oliveira et al., 2024). A comprehensive LCA considering five impact categories (global warming, ozone formation – human health, terrestrial acidification, mineral resource scarcity, fossil resource scarcity) was conducted to compare four bioprocesses and to confirm the sustainability of converting agro-industrial by-products into hydrolysates for biopigment production in biorefineries.

This study evaluated four scenarios using different types of bran: corn, soybean, rice, and wheat. In each process, approximately 58 grams of bran were used. The bran underwent dilute-acid hydrolysis, yielding 300 mL of concentrated hydrolysate with varying total sugar content (sum of glucose, xylose, and arabinose). Following hydrolysis, the hydrolysates were standardized by adding water to ensure consistent sugar concentrations, after which they were used as a growth medium in fermentation processes. Before fermentation, R. mucilaginosa cells were activated in a pre-cultivation using YM medium and then concentrated by centrifugation. The standardized bran hydrolysates were subsequently inoculated with the activated yeast cells, and fermentation was conducted for 72 hours. The LCA results, expressed per mg of carotenoids, are shown in Fig. 4, enabling a direct comparison between CBH, SBH, RBH, and WBH.

Overall, the fermentation step had the most significant contribution to the environmental impacts in most categories, primarily due to the electricity consumed in this step. Our findings align with those of Mussagy et al. (2020), who reported that the fermentation stage was the main contributor to the environmental impacts in LCA of carotenoid production using R. glutinis (Mussagy et al., 2020). Despite the impact of the fermentation step, an exception was observed in mineral resource scarcity, where the main environmental hotspot was diluted acid hydrolysis, which is attributed to sulfuric acid consumption, impacting the availability of critical minerals like copper (Cu) and molybdenum (Mo). Bran production and cell activation also contribute up to approximately 30% in certain impact categories.

Among the scenarios, RBH demonstrated superior environmental performance across all impact categories. Despite not having the lowest impacts associated with hydrolysate preparation, RBH allows for the highest concentration of carotenoids per volume of growth medium. Consequently, this reduces the environmental impacts of fermentation - typically the largest contributor to the overall environmental impacts - and cell activation.

SBH and WBH exhibited intermediate environmental performance, with similar outcomes. However, in the mineral resource scarcity category, SBH performed worse than the other analyzed brans due to the extensive impact of acid hydrolysis, which requires a larger volume of concentrated hydrolysate. Additionally, the production of soybean bran is also more impactful due to the phosphorus scarcity, a result of intensive fertilization practices in soybean crops.

CBH had the lowest impact among the steps associated with hydrolysate preparation, as a smaller amount of concentrated hydrolysate is required in the preparation of standardized hydrolysate. Nonetheless, except for mineral resource scarcity, as mentioned above, CBH exhibited the worst overall environmental performance because the impacts derived from the remaining steps are higher than those of the other brans. Ultimately, it presents the lowest concentration of carotenoids per volume of growth medium, resulting in much higher effects from fermentation and cell activation operations.

In summary, using RBH emerges as the most environmentally sustainable option for carotenoid production in biorefineries. These findings emphasize the importance of selecting appropriate feedstocks to enhance the sustainability of bioprocesses. This approach improves resource efficiency, promotes sustainable production systems, and reduces environmental impacts.

This study demonstrated, for the first time, the potential of soybean, wheat, rice, and corn bran hydrolysates as low-cost substrates for carotenoid production using R. mucilaginosa, with a particular focus on RBH and SBH. The findings highlight the significant influence of bran hydrolysate composition on microbial metabolism, emphasizing the importance of studying the bioprocess conditions to stimulate growth and biopigment production. RBH exhibited the best environmental profile, while CBH tends to have the highest overall environmental impact, except in the category of mineral resource scarcity, where SBH had the highest impact. This approach prioritizes sustainability by reusing renewable feedstocks and avoiding additional processing steps, such as acid recovery from hydrolysates, medium supplementation, or detoxification. This method not only prevents the generation of undesirable waste or by-products but also reduces the use of reagents and process time. These insights contribute to the development of environmentally and economically sustainable bioprocesses for producing high-value compounds from renewable resources, opening new possibilities for further research on microbial carotenoid production, and promoting advancements in sustainable resource management.

Acknowledgments

This work was financially supported by Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior- Brazil (CAPES) [Finance Code 001, grant number 88887.495320/2020-00 + 88887.716897/2022-00], Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) [grant number 2023/097898], Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) [grant number 304166/2022-7]. This work is part of the project “INCT Yeasts: Biodiversity, preservation, and biotechnological innovation” funded by CNPq, Brasília, Brazil, [grant number #406564/2022-1]. CERES is supported by the Fundação para a Ciência e Tecnologia (FCT) through the projects UIDB/EQU/00102/2020 and UIDP/EQU/00102/2020. J.F.B. Pereira funding the project DRI/India/0044/2020 and project HIPERBIOTEX (COMPETE2030-FEDER-00708800), and Fundação Calouste Gulbenkian for funding the project DYELOOP. A.C. Dias also acknowledges FCT for financial support to CESAM [grant number UIDB/50017/2020 + UIDP/50017/2020 + LA/P/0094/2020], through national funds, and for the contract 10.54499/CEECIND/02174/ 2017/CP1459/CT0019.

Availability of Data and Materials

All data and materials will be available upon request.

Author Contributions

TRB, SSM, JCS, and JFBP conceived and designed research. TRB, SCI, and GCA conducted experiments. ACD, JCS, JFBP, and SSS contributed new reagents or analytical tools. TRB, SCI, ACD, and JFBP analyzed data. TRB, SSM, ACD, JFBP, JCS, and SSS wrote the manuscript. All authors read and approved the manuscript.

Declaration of Interest Statement

The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this study.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this study, the authors used the free version of CHATGPT (provided by OpenAI, San Francisco, California, U.S.; available at https://openai.com/blog/chatgpt) to partially aid with language editing. After using this tool, the authors reviewed and edited the content as needed, and took full responsibility for the publication's content.

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Sustainable carotenoid production using amylaceous agro-industrial byproducts: Process efficiency and environmental assessment

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Abstract

Figures

1. Introduction

2. Material and Methods

2.1 Dilute-acid hydrolysis of grains by-products

2.2 Yeast-derived carotenoid production

2.2.1 Microorganism and inoculum preparation

2.2.2 Hydrolysates as a growth medium for carotenoid production

2.3 Cell disruption and carotenoid extraction

2.4 Life cycle assessment (LCA)

2.5 Analytical methods

2.5.1 Cell biomass concentration

2.5.2 Determination of sugar, furan derivate, and phenolic compounds

2.5.3 Determination of the total soluble protein concentration

2.5.4 Carotenoids analysis and quantification

2.6 Statistical Analysis

3. Results and discussion

3.1 Bran hydrolysates characterization

3.2 Rhodotorula mucilaginosa cultivation and carotenoid production using standardized bran hydrolysates

3.2.1 Sugar consumption

3.2.2 Cell biomass and carotenoid production

3.3 Life cycle assessment

4. Conclusions

Declarations

References

Supplementary Files

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