Valorization of Dairy Wastewater for the Production of Lipids, Carotenoids, and β-Carotene in Rhodotorula mucilaginosa

doi:10.21203/rs.3.rs-4837439/v1

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Valorization of Dairy Wastewater for the Production of Lipids, Carotenoids, and β-Carotene in Rhodotorula mucilaginosa

https://doi.org/10.21203/rs.3.rs-4837439/v1

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One of the leading causes of environmental pollution in India is the waste generated from the dairy sector. Significant amounts of organic and inorganic components are characteristics of the wastewater generated by this industry. To address this problem, sustainable and economical methods of lowering its ecological footprint are obligatory. The present study uses raw dairy wastewater (RDW) to synthesise lipids and carotenoids from Rhodotorula mucilaginosa. Batch and fed-batch experiments were used to scale up the process after shake flask experiments using mineral salt media with RDW. In batch fermentation studies with only RDW resulted in the generation of lipid content, carotenoids, and β-carotene at values of 52.02 ± 1.52 (% w/w), 30.63 ± 0.15 µg.g^-1, and 12.42 ± 0.06 µg.g^-1. Furthermore, the observed chemical oxygen demand (COD) removal efficiency was approximately 48.5±0.24%. Additionally, intriguing findings were obtained when RDW was supplemented with 15gL^-1 of sodium acetate, increasing the lipid content (% w/w), carotenoids, and β-carotene levels by 66.3 ± 0.3 (% w/w), 64.0 ± 0.32 µg.g^-1, and 19.7 ± 0.09 µg.g^-1, respectively. The maximum chemical oxygen demand (COD) removal efficiency was around 62.3±0.31%. When fed-batch reactor studies were conducted with RDW while maintaining 15gL^-1 sodium acetate, notable improvements over batch reactor studies were observed. The experimental outcomes revealed a lipid content of 72.14 ± 0.3 (% w/w), carotenoid levels of 67.16 ± 0.3 µg.g^-1, β-Carotene content of 31.7± 0.15 µg.g^-1, and a chemical oxygen demand (COD) removal efficiency of approximately 78± 0.3%. A comprehensive study of the lipid samples using gas chromatography revealed a wide range of fatty acid compositions. This thorough characterization underscores their significant potential for utilization in biodiesel production.

Environmental Engineering

Biotechnology and Bioengineering

Applied & Industrial Microbiology

Rhodotorula mucilaginosa

batch fermentation

fed-batch

lipid

carotenoids

β-carotene

In the intricate tapestry of modern industrial agro-processing, the dairy industry is an important colossus, satisfying the ever-increasing worldwide demand for dairy products. With an approximate 13.1% share of the global milk supply, India has emerged as a leading producer of milk globally. The dairy sector, with over 286 major and small-scale operations in India, is known to contribute significantly to water pollution, generating substantial amount of wastewater, a byproduct characterized by a complex blend of organic and inorganic materials. The dairy industry in India releases over 110 million tons of milk and 275 million tons of effluent per year [1] where the quantity and quality of dairy effluent varies greatly based on the specific production features of the dairy plant. Since these factories produce a wide range of products, each of which is made independently, the number of contaminants in the wastewater fluctuates over time as new technologies are applied to the manufacturing unit. This association affects the efficacy of wastewater treatment [2]. Although the contaminants are harmful to the environment, they may be exploited to make value-added products, like lipids, proteins, carbohydrates, etc. These may be recovered quantitatively from dairy effluent and used for a variety of purposes. One approach to convert wastewater into biodiesel involves lipid extraction, where lipids are extracted from the wastewater and subsequently processed into biodiesel [3]. Also, it is well-recognized that biorefineries and microbial cell factories have the ability to convert waste materials into high-value-added products. Biorefineries can efficiently degrade organic waste streams such as agricultural wastes, food waste, and industrial residue into their constituent components by harnessing the metabolic capabilities of microorganisms. The constituents may then be transformed into an extensive array of beneficial byproducts, such as pharmaceuticals, nutraceuticals, biofuels, and biochemicals. In this approach, microbial cell factories—which are designed to generate particular compounds of interest—are essential because they operate as versatile biocatalysts, enabling the synthesis of target molecules from sustainable feedstocks [4]. Thus, using similar principles, several studies have employed microorganisms to decontaminate dairy effluent before incorporating it into a synthesis of lipids and biofuels. The parameters of wastewater and the types of microorganisms present influence the removal of nutrients, the duration of culture, biomass productivity, and the generation of various products [5].

Particularly considering the global energy crisis, the search for new fossil fuel substitutes has become imperative. One of the most promising sources of bioenergy, biodiesel has gained popularity recently due to its ecologically sound, renewable, biodegradable, and nontoxic qualities [6]. Transesterification of vegetable oils is the primary process used to make biodiesel; nevertheless, the production of vegetable oils is fraught with difficulties due to seasonal variations in temperature, availability of arable land, labour shortages, and other factors. Due to its comparable fatty acid makeup, microbial oil, sometimes referred to as single cell oil (SCO), has been identified as a promising renewable oil that can potentially replace vegetable oils [7]. Oleaginous microorganisms like bacteria, yeasts, and microalgae may build up significant concentrations of lipids that make up above 20% (w/w) of the total dry biomass weight, and under some circumstances, up to 70%, in order to produce microbial lipids [8]. Certainly, yeast strains can be a prominent alternative due to specific benefits they have over other oleaginous microorganisms due to high amount of lipid synthesis. They are unicellular, have quick duplication times, and proliferate readily in intense, regulated environments [7]. Furthermore, one of yeast's most significant traits is its capacity to produce lipids from a wide range of source materials, like crude glycerol, waste from municipal and industrial wastewater including dairy effluent. The most well-known genera of oleaginous yeast are Yarrowia, Rhodotorula, Lipomyces, Candida, Cryptococcus, and Trichosporon [9]. There are, however, a few hurdles to overcome, such as the uncertain economic viability of microbial oils owing to the high cost of cultivation substrate along with the challenges associated with microorganisms' ineffective utilization of substrates due to differences in their characteristics and the fermentation processes that may affect lipid production as well as other by products. Thus, growing oleaginous microorganisms that have acclimated to that substrate in combination with low-cost organic compounds found in organic wastes, such as dairy wastewater, might be a potential strategy to achieve commercial viability [10].

The present research focuses on the culturing of Rhodotorula mucilaginosa yeast using dairy industry effluent as a growth medium. The principal objective is to harness the metabolic ability of R. mucilaginosa to produce value-added products like β-carotene, carotenoids, and lipids. Carotenoids being natural pigments are generated by the Rhodotorula genus and can be utilized in various industrial sectors in the form of food additives, supplements, animal feed, cosmetics etc. for their diverse antioxidant and health benefits. The study also assesses how acetate affects oleaginous yeast development and lipid metabolism. In addition to being a carbon substrate, acetate has observed to cause intracellular lipid accumulation. Nevertheless, excessive acetate usage acidifies the cytoplasmic membrane and prevents microorganisms from growing [11]; emphasizing that choosing the proper acetate concentration is crucial for high lipid accumulation and to verify that flask studies on R. mucilaginosa were carried out. The methodology was further widened, and batch fermentations were carried out for the yeast. This served as a comparative study, with raw dairy wastewater being utilized solely in one instance and supplemented with either glucose or sodium acetate in the other two. Following batch reactor research, fed-batch reactor studies were conducted to improve lipid, carotenoid, and β-carotene production. Analysing the fatty acid composition of the lipid samples not only broadens the understanding but also presents promising avenues for the production of cost-effective products like biodiesel.

2.1 Materials

All the analytical (AR) grade chemicals utilized in this investigation were acquired from HiMedia laboratories in Mumbai, India. Raw dairy wastewater (RDW) was collected from Purabhi Dairy Pvt. Ltd. in Guwahati, India, and kept at 4°C until later analysis. The properties of the raw dairy wastewater were determined using the established techniques defined by the American Public Health Association (APHA).

2.2 Microorganism

The strain R. mucilaginosa was isolated in a previous study [12] and was revived by culturing on Yeast Peptone Dextrose (YPD) media containing yeast extract (3 gL^− 1), peptone (5 gL^− 1), and glucose (10 gL^− 1) for 48 hours at 30°C. It was further sub-cultured periodically and glycerol stocks were prepared and stored at -80°C for later experiments.

2.3 Shake flask studies

To investigate the relative impact of RDW and mineral salt medium (MSM) on the growth of R. mucilaginosa and their effect on subsequent production of lipids, carotenoids, and beta-carotene, shake flask studies were conducted. In this investigation, 20 ml of MSM media was prepared, consisting of 0.8 gL^− 1 KCl, 0.1 gL^− 1 CaCl₂, 0.8 gL^− 1 NaCl, 0.1 gL^− 1 FeSO₄, 2.0 gL^− 1 NH₄Cl, 2.0 gL^− 1 Na₂HPO₄, and 0.2 gL^− 1 MgSO₄. This medium was then blended at varying ratios with RDW, having four different concentrations: flask 1 with RDW:MSM ratio of 4:0, followed by flask 2 at 1:1, flask 3 at 2:1, and finally, flask 4 at 4:1.

2.4 Bioreactor studies

2.4.1. Inoculum preparation and fermentation media

A single colony was taken from an overnight-grown culture plate and placed in 50 ml of YEPD medium containing yeast extract (10 gL^− 1), peptone (20 gL^− 1), and dextrose (20 gL^− 1), which was then incubated for 12 hours at 30°C with agitation at 180 rpm. The fermentation medium in the stirred tank reactor was inoculated using 8% (v/v) seed culture obtained from this incubation. The fermentation media for batch and fed-batch reactor studies included 1600 ml of RDW and 400 ml of MSM along with 15 gL^− 1 of sodium acetate. The pH of the culture was automatically adjusted to 6.0 by adding 0.5 M H₂SO₄ and 1 M NaOH. Each component was autoclaved separately before being combined in the reactor.

2.4.2. Batch and fed-batch studies

A 5 L stirred-tank bioreactor (Sartorius, B-LITE) with a 2 L working capacity was used for batch fermentation. The experiment was conducted with the following parameters: aeration set at 0.8 vvm (closely monitored dissolved oxygen content), temperature of 30°C, and agitation ranging from 300 to 400 rpm with pH 6. The air flow rate was adjusted between 1–3 vvm using a peristaltic pump (Miclins, India). 10% (v/v) of the inoculum size was used to inoculate the culture, and an antifoaming agent was added intermittently to control foaming. Samples were collected at every 24-hours interval to measure O.D., biomass concentration, lipid content, carotenoids, and β-carotene.

In the fed-batch fermentation study, by employing the pulse feeding strategy, sodium acetate was replenished each time when its concentration dropped below the set value of 15 gL^− 1. The feeding regimen occurred at specific intervals: 36 h, 48 h, 72 h, 96 h, 120 h, and 144 h, maintaining a consistent substrate concentration throughout the fed-batch studies.

2.5 Biomass productivity

To compute biomass productivity, the following formula was used:

$$\:Px=\frac{\begin{array}{c}{X}_{i}-{X}_{0}\end{array}}{\begin{array}{c}{T}_{i}-{T}_{0}\end{array}}$$

Biomass productivity [PX (g biomass L^− 1.h^− 1)] in relation with T = 0h.

X_i (gL^− 1) = biomass concentration at T = i;

X₀ (gL^− 1) = biomass concentration at T = 0h;

T_i = time at i (hours);

T₀ = initial time (T = 0h).

The specific growth rate (µd^− 1) was calculated as following equation [13]:

$$\:\mu\:=(\text{ln}{x}_{1}-ln{x}_{0})/{t}_{1}-{t}_{0}$$

where x₁ and x₀ are the biomass concentrations (gL^− 1), and t₁ and t₀ is the time correspondingly.

2.6 Sodium acetate estimation using high-performance liquid chromatography (HPLC)

To determine the precise concentration of sodium acetate in the samples of batch reactor and fed-batch reactor studies, High-Performance Liquid Chromatography (HPLC) method was used with the initial concentration of 15 gL^− 1 of sodium acetate. The HPLC column temperature was maintained at 60^oC throughout the study with 9 mM sulfuric acid as a solvent at a rate of 0.5 millilitres per minute. Sodium acetate peak identification in the chromatogram was aided by a comparison to peaks produced by valid standards [14–16]. Since each component has a distinct retention time, it is possible to accurately identify and measure the amount of sodium acetate in our samples.

2.7 Determination of lipid content

Yeast cell contents were determined using an altered Bligh and Dyer technique [17], which included extraction, drying, and further weighing. In this methodology, yeast cells were rinsed twice with 50 mL of distilled water after being centrifuged at 5000 g for five minutes with a 50 mL sample. After adding 10 mL of 4M HCL, the mixture was incubated for one to two hours at 60°C. The acid-hydrolysed mass was then combined with a Chloroform/Methanol mixture (1:1, 20 mL) and incubated at room temperature for around 2–3 hours. Phase separation was aided by centrifugation at 2000 g for five minutes at room temperature. Following the retrieval of the lower phase containing lipids, the lipids were dried and weighed after evaporating for ten minutes under a low-pressure condition. This procedure guarantees efficient and complete lipid extraction [18].

2.8 Total carotenoids estimation using a spectrophotometer

The sample was centrifuged to extract the supernatant, followed by washing of cell pellet with deionized water thrice. The intracellular carotenoids were then extracted into the acetone phase by suspending the cells in it and agitating them with a bead grinder. Following centrifugation, the supernatant was collected, and the pellet was then centrifuged again until it became colourless. After that, acetone extracts having carotenoids were collected and measured at 455 nm [19]. A reference standard of β-Carotene (Sigma) was used [20].

The formula to determine the total carotenoid content [21] is:

Carotenoids content (µg/g) =

$$\:\frac{A\:x\:V\left(ml\right)x{10}^{4}}{\begin{array}{c}{A}^{1\%}\\\:\:\:\:\:\:\:1cm\end{array}x\:\:P\left(g\right)\:}$$

where A = absorbance, V = Total extract volume, P = sample weight, and

$\:\begin{array}{c}{A}^{1\%}\\\:\:\:\:\:\:\:\:1cm\end{array}$ =2500 (β-carotene extinction coefficient in acetone) specific extinction 2500 in acetone at 454 nm;

$\:\begin{array}{c}{E\:}^{1\%}\\\:\:\:\:\:\:\:\:1cm\end{array}$ (100 ml g^− 1cm ^− 1) [22].

2.9 Fatty Acids Methyl esters (FAME) analysis

Fatty acid methyl esters (FAME) were produced using the Gas Chromatography (GC) analysis as reported by Sinanoglou et al. [23] in order to determine the composition of fatty acids. Quantitative and qualitative analyses were conducted using an Agilent 6890 Series Gas Chromatograph (Agilent Technologies, Palo Alto, CA, USA). The analysis utilized a SLB-IL100 column (30 m x 0.25 mm, I.D. × 0.20 µm film thickness) and a flame ionization detector (GC-FID, Varian 450, Netherlands). During the initial phase of direct transesterification, 30 mg of lyophilized biomass was treated with 1.0 mL of 2% (w/v) NaOH in methanol, in conjunction with glass beads. This mixture was incubated at 90°C for 20 minutes in a shaking water bath at a speed of 150 rpm. In the second phase, 1.0 mL of 5% (v/v) H₂SO₄ in methanol was blended to the mixture, which was then incubated under the similar conditions for another 20 minutes. After completing the first and second incubation stage, the reaction mixture was allowed to cool to the room temperature. The cooled transesterified mixture was then mixed with 1.0 mL of deionized water and hexane to help promote the production of FAME in the hexane layer. In order to ensure purity, the hexane layer was filtered using a 0.2 µm filter, rinsed twice with distilled water, and then immediately put through GC analysis. Helium was used as the carrier gas, with a flow rate of 1.5 mL/minute. Throughout the study, the temperatures of the injector and detector were kept at 250°C. An injection volume of 1 µL was used to inject the sample. For GC-FID calibration, the FAME mix C14-C22 (Supelco 37, USA) was employed as the standard [24].

2.10 Evaluation of biodiesel properties based on FAME profiles

Two critical standards for measuring biodiesel quality are the ASTM D6751 standard in the United States and the EN 14214 standard in Europe. These regulations set forth a number of requirements and testing procedures that are essential for assessing the integrity of biodiesel. These standards examine properties like viscosity (ƞ), cetane number (CN), pour point (PP), cloud point (CP), flash point (Tf), iodine value (IV), saponification value (SV), degree of unsaturation (DU), and highest heating value (HV). These variables, which are closely related to the length of the carbon chain and the existence of unsaturated fatty acids produced by microbial systems, together define the properties of biodiesel. Therefore, using experimentally determined equations based upon these criteria, the characteristics of biodiesel were evaluated.

$$\:\text{ƞ}\:=0.235\:\text{W}\text{C}\:-0.468\text{W}\text{d}\text{b}$$

$$\:\text{C}\text{N}\:=\:3.930\text{W}\text{C}\:-15.936\text{W}\text{d}\text{b}$$

$$\:\text{T}\text{f}\:=23.362\text{W}\text{C}\:+\:4.854\text{W}\text{d}\text{b}$$

$$\:\text{C}\text{P}=18.134\text{W}\text{C}\:-0.790\text{W}\text{U}\text{S}$$

PP $\:=$ 18.880W_C $\:-$ 1.00W_US (5)

SV $\:=$ $\:\sum\:($560) $\:\times\:$ P_FA)$\:/$MW (6)

IV $\:=$ $\:\sum\:($254 $\:\times\:$ P_FA $\:\times\:$ N_D) /MW (7)

DU $\:=$(W_MUFA) + (2 $\:\times\:$ W_PUFA) (8)

HV $\:=$46.19 $\:-\left(1794/MWi\right)-$0.21 $\:\times\:$ N_D (9)

The equations 1–5 was described by Su et al. [25] and equations 6–8 was obtained from Francisco et al. [26]. The empirical equation for heating value was taken from Ramírez-Verduzco et al. [27].

2.11 Analytical methods

Sodium acetate concentration in media was estimated by HPLC equipped with sophisticated solvent delivery system (210), a refractive index (RI) detector (355) (Varian, the Netherlands), and a specialized Meta Carb-87^H carbohydrate column (300 x 6.5 with particle size 8 µm). All the water parameters including COD, BOD, total suspended solids and total dissolved solids were assessed using the APHA, Standard Methods for the Examination of Water and Wastewater 1995 [28]. The Folin-Lowry and Dubois methods were used to detect protein and carbohydrates, respectively, using a UV-Vis spectrophotometer (119Systronics, Mumbai, India) [29] which provided crucial information about the composition of dairy wastewater as shown in Table 1. All tests were carried out in triplicates, and the mean values were considered and represented as error bars in the graphical representation.

3.1 Shake flask studies for the synthesis of lipids, carotenoids, and β-carotene

In the initial investigation, employing a 4:1 ratio of raw dairy wastewater (RDW) to mineral salt medium (MSM) in flask 4 via batch shake culture technique exhibited higher levels of cell dry weight, lipids, carotenoids, and β-carotene production in R. mucilaginosa, yielding 0.34 ± 0.06 gL^− 1, 46.31 ± 1.52%, 15.16 ± 0.08 µg.g^− 1, and 3.91 ± 0.12 µg.g^− 1, respectively, surpassing those achieved with other three flasks. Upon investigating sodium acetate at varying concentrations of 0.5%, 1%, 1.5%, and 2% with RDW, it became evident that the optimal production occurred at a concentration of 1.5%. This concentration yielded 62.28% lipids, 41.16 µg.g^− 1 carotenoids, and 28.03 µg.g^− 1 β-carotene, respectively, highlighting its effectiveness in enhancing overall product yield (Fig. 1). At a concentration of 2% sodium acetate, there was a significant reduction observed in both lipid and carotenoid contents. Mandal et al.[30] also reported that concentrations exceeding 2% of sodium acetate exhibited toxicity towards the cells. Thus, the noticeable result may be attributed to the deliberate addition of sodium acetate, which functions as a strong inducer for acetyl CoA, an essential precursor molecule involved in the pathways leading to the production of lipids and carotenoids. As a basic component of cellular metabolism, acetyl CoA starts the synthesis of several important molecules [31]. As a result, supplementing with sodium acetate increases lipid and carotenoid levels significantly, highlighting the critical function of acetyl CoA regulation in modifying the production of these significant molecules.

3.2 Batch reactor studies for the synthesis of lipids, carotenoids, and β-carotene

A 5-L stirred-tank bioreactor (Sartorius, B-LITE) with a 2L working capacity was used for a batch culture. The observations from this study showed that R. mucilaginosa produced cell dry weight (CDW) of 3.21 ± 0.01 gL^− 1 and lipid content of 52.02 ± 1.52% (w/w) at 96 hours with carotenoids content of 30.63 ± 0.15 µg.g ^− 1, and β-carotene content of 12.42 ± 0.06 µg.g ^− 1 at 108 hour, when using raw dairy wastewater (RDW) as a substrate (Fig. 2) utilizing rich organic compounds, including lactose, proteins, and other nutrients in it. The biomass productivity and lipid productivity here were 0.06 gL^− 1 h^− 1 and 0.017 gL^− 1h^− 1, respectively, and the specific growth rate (µ_max) was found to be 0.012 h^− 1. Following the addition of 1.5% sodium acetate to raw dairy effluent in the batch bioreactor, significant improvements were seen in terms of growth, biomass, lipid, carotenoids, and β-carotene productivity and yield. In particular, R. mucilaginosa contained 5.92 ± 0.02 gL^− 1 of cell dry weight (DCW), 66.3 ± 0.3% (w/w) of lipids at 84 hours and 64.0 ± 0.32 µg.g^− 1 of carotenoids, and 19.7 ± 0.09 µg.g ^− 1 of β-carotene at 120 hours (Fig. 3). As the yeast metabolizes organic compounds present in the raw dairy wastewater, it efficiently decreases the organic load, resulting in a reduction in COD levels with a removal efficiency of 62.3 ± 0.31%. Notably, R. mucilaginosa in this setup used sodium acetate as an additional carbon source in alongside the dairy effluent, where sodium acted as an ion channel and acetate triggered the acetyl pathway for activities involved in the manufacture of lipids reaching its maximum at 84 hours [32]. After this, the culture may enter the stationary phase, which is characterized by slower growth rates caused by nutritional constraint or metabolic changes. While lipid synthesis may still occur during this phase, it is likely to be less prominent than in the exponential phase due to the prioritization of other cellular activities. Simultaneously, the observed buildup of carotenoids, including β-carotene, at 120 hours in the same batch reactor might be attributed to metabolic changes in the yeast. Metabolic fluxes are redirected towards the synthesis of secondary metabolites, such carotenoids, since lipid synthesis declines after 84 hours. Carotenoids and β-carotene accumulation at 120 hours may be regulated by variables such as nutrient availability, ambient circumstances, and metabolic pathway interactions [11].

Also, sodium acetate degradation efficiency was checked where the initial sodium acetate concentration was maintained at 15 gL^− 1 and samples were collected from the reactor at 12-hour intervals for sodium acetate quantification using HPLC. After 144 hours, sodium acetate concentration decreased significantly, from 15 gL^− 1 to 0.036 gL^− 1, indicating a remarkable 99.8% degradation efficiency. Further the results for biomass productivity and lipid productivity in this case were 0.159 gL^− 1 h^− 1 and 0.04 gL^− 1h^− 1, respectively, and the specific growth rate (µ_max) was found to be 0.028 h^− 1.

Additionally, batch culture dynamics were examined to clarify the impact of glucose as a carbon substrate on lipid, carotenoids, and β-carotene synthesis with respect to growth and biomass parameters. As the primary carbohydrate, glucose serves as an essential carbon source for the growth of microorganisms and the accumulation of biomass. The results of the batch experimentation were impressive, using glucose as a fundamental substrate, R. mucilaginosa showed a cell dry weight (CDW) of 8.98 ± 0.04 gL^− 1and lipid content of 59.6 ± 0.29% (w/w) at 84 hours and carotenoids content of 55.66 ± 0.27 µg.g^− 1, and β-carotene content of 21.3 ± 0.10 µg.g^− 1 at 120 hours with a COD removal efficiency of 59.8 ± 0.22% (Fig. 4). R. mucilaginosa effectively uses glucose for lipid synthesis during exponential growth, achieving maximal lipid content at 84 hours. As glucose becomes limited during the stationary phase, metabolic priorities shift toward secondary metabolite production, particularly carotenoids and β-carotene, indicating adaptive responses to changing environmental conditions. Here the results for biomass productivity and lipid productivity in were 0.117 gL^− 1 h^− 1 and 0.037 gL^− 1h^− 1, respectively, and the specific growth rate (µ_max) was found to be 0.016 h^− 1.

These results highlight how R. mucilaginosa responded to sodium acetate availability with marked growth, carotenoid, and β-carotene biosynthesis, lipid productivity and yield when compared to results obtained with glucose-supplemented dairy wastewater. Based on a thorough evaluation of the data, all the obtained results support the effectiveness of sodium acetate, which has also been verified by Gong et al. [33], as a better carbon source for the microbial synthesis of lipids, carotenoids, and β-carotene by the oleaginous yeast R. mucilaginosa. A possible reason for the observed outcomes is that sodium acetate functions as a direct precursor of acetyl CoA, which is an essential step in the pathways involved in lipid production. This direct method allows for more effective conversion of substrates into lipids than glucose, which requires additional steps for conversion to acetyl CoA [34]. Furthermore, sodium acetate with RDW could offer a better carbon-to-nitrogen ratio than glucose, since acetate metabolism is intricately related to lipid production pathways, which may activate critical enzymes and transcription factors associated with lipid accumulation [35]. The balance of carbon and nitrogen availability is crucial for microbial growth and metabolite production, and sodium acetate addition may improve this ratio for lipid and carotenoid biosynthesis [19].

3.2 Fed-batch reactor studies for the synthesis of lipids, carotenoids, and β-carotene

In fed-batch fermentation, determining the right feeding strategy is crucial for microorganisms since it is a key factor in determining increased productivity and the best possible yield of the target product [36]. After the initial batch phase is over, the fed-batch culture methodology involves continuously adding substrate to maintain cellular metabolism until the fermentation process is finished. Studies comparing fed-batch and batch culture techniques revealed that the fed-batch model produced much more of lipids and carotenoids. Particularly, the utilization of fed-batch culture methods enabled the attainment of substantially higher levels of R. mucilaginosa CDW, lipid content (% w/w), carotenoids, β-carotene, quantified at 8.33 ± 0.04 gL^− 1, 72.14 ± 0.3% (w/w) at 108 hours, 67.1 ± 0.3 µg.g^− 1, and 31.7 ± 0.12 µg.g ^− 1 at 120 hours, respectively (Fig. 5). Moreover, sodium acetate degradation efficiency was analysed where a substantial reduction in sodium acetate concentration was noted after 72 hours. Employing a pulse feed strategy, the sodium acetate concentration was restored to 15 gL^− 1. This approach was reiterated after 132 hours, ensuring the maintenance of a constant sodium acetate concentration of 15gL^− 1 which was subsequently stabilized after 144 hours, indicating saturation in the culture. The COD level decreased for 72 hours followed by an increase when sodium acetate levels were replenished, achieving a maximum removal efficiency of 78 ± 0.3% approximately.

As stated by Pereira et al.[37], a substantial increase in the production of lipids and carotenoids may have resulted from the significant amount of carbon introduced into the medium in form of sodium acetate at regular intervals, which raised the C/N ratio and favoured the accumulation of lipids in the cells. In comparison to batch reactors, fed-batch reactors allowed for a larger buildup of lipids due to the higher carbon supply and longer feeding duration. According to research by Beopoulos et al.[38], the reason for the reduction in lipid synthesis after 108 hours is a nitrogen constraint, which causes the growth rate of R. mucilaginosa to decline rapidly while the rate of carbon assimilation decreases more gradually. Consequently, carotenoid production peaks around 120 hours, which is also the stationary period, where metabolic priorities majorly shift to secondary metabolite synthesis and stress response mechanisms. Beyond this the yeast enters a decline phase which impacts the carotenoid production pathway, thereby making it less active and henceforth reducing the carotenoids production. The production profiles of lipids and carotenoids in the fed-batch reactor system are shaped by the complex interactions among substrate availability, metabolic fluxes, and temporal control of metabolic pathways, all of which influence lipid and carotenoids production patterns in the fed-batch reactor [36].

Additionally, detailed investigation of the kinetic factors regulating growth, biomass accumulation, lipid, carotenoids, and β-carotene production, elucidated using dairy wastewater treated with sodium acetate, provided relevant insights. Notably, the results for biomass productivity and lipid productivity were 0.257 gL^− 1 h^− 1 and 0.054 gL^− 1h^− 1, respectively, and the specific growth rate (µ_max) was found to be 0.053 h^− 1. The combined results of these studies highlight the effectiveness of fed-batch fermentation techniques in increasing microbial productivity and product yield, especially when dairy effluent containing sodium acetate is used as a growth medium for R. mucilaginosa. The findings of this study demonstrate that using sodium acetate in dairy wastewater instead of sole raw dairy wastewater, R. mucilaginosa exhibited greater growth, biomass production, lipids, carotenoids, and β-carotene synthesis, where sodium acetate functions as an additional carbon source [19].

3.3 Fatty acids methyl ester (FAME) by GC-FID analysis and evaluation for biodiesel properties

FAME analysis is particularly important in evaluating fatty acid content and its significance to biodiesel production. This analytical approach allows for precise quantification and characterization of fatty acids found in yeast lipids, giving important information about their potential for biodiesel production [39]. From the batch and fed-batch studies, the lipids generated by R. mucilaginosa were trans methylated, and the fatty acid composition profiles were examined directly in gas chromatography. The results of this study showed that long-chain fatty acids with carbon chain lengths ranging from C16 to C18 made up the majority of the lipid samples in both the batch and fed-batch configurations. It is interesting to note that the fed-batch lipid sample had a greater fatty acid content than the batch lipid sample. Specifically, the FAME analysis of the fed-batch sample revealed the presence of various fatty acids, including 0.48 ± 0.01% lauric acid (C12:0), 1.04 ± 0.10% myristic acid (C14:0), 32.05 ± 1.32% palmitic acid (C16:0), 1.85 ± 0.18% palmitoleic acid (C16:1), 0.55 ± 0.01% margaric acid (C17:0), 0.71 ± 0.04% ginkgolic acid (C17:1), 5.42 ± 0.7% stearic acid (C18:0), 50.18 ± 3.12% oleic acid (C18:1), 9.15 ± 1.79% linoleic acid (C18:2), and 1.70 ± 0.01% linolenic acid (C18:3). These findings are summarized in Table 2. The lipid composition in R. mucilaginosa shows a prevalence of certain fatty acids, especially palmitic acid (C16:0), stearic acid (C18:0), and oleic acid (C18:1), which combined constitute a larger percentage of total fatty acids. Notably, methyl palmitate (C16:0) and methyl oleate (C18:1), which together make up around 72% of the total FAME content, are the principal fractions of FAME obtained via the transesterification of R. mucilaginosa lipids. Furthermore, significant concentrations of methyl stearate (C18:0), methyl linoleate (C18:2), methyl palmitoleate (C16:1), and methyl linolenate (C18:3) have also been detected. The fatty acid profile also shows minor portions of unsaturated and saturated fatty acids, such as methyl laurate (C12:0), methyl palmitoleate (C16:1), methyl heptadecanoate (C17:0), and methyl heptadecenoic (C17:1). Remarkably, gas chromatography study shows that R. mucilaginosa has more unsaturated fatty acids than saturated fatty acids. These findings provide valuable insights into the lipid composition of R. mucilaginosa and its potential applications.

The qualities of the biodiesel evaluated in this study show good compliance with American Society for Testing and Materials (ASTM D6751-12) and European (EN 14214 − 2012) requirements and have been presented in Table 3. Notably, viscosity, cloud point, and pour point readings were all within acceptable levels, with viscosity at 3.917 mm² s ^− 1, cloud point at 1.08°C, and pour point at 2.58°C. Fuel performance can be hindered by high viscosity, which is a sign of poor flow characteristics and lower fuel atomization quality. Nonetheless, the measured results point to the good flow qualities of biodiesel and sufficient low-temperature performance.

In the realm of biodiesel production, the cetane number is a key indicator that represents the fatty acids composition in the feedstock [40]. Significantly, esters of saturated fatty acids, specifically those of palmitic (C16:0) and stearic (C18:0) acids, are linked to higher cetane numbers; conversely, esters of unsaturated fatty acids, such as those of linoleic (C18:2) and linolenic (C18:3) acids, are linked to lower cetane numbers [41]. Moreover, a crucial factor in the characterisation of biodiesel is the saponification value (SV), which represents the amount of potassium hydroxide needed to fully saponify one gram of oil [42, 43]. The SV value in our investigation was around 203.3 mg KOH g^− 1 (Table 3), which is within the range that the ASTM-D6751 standard considers acceptable. The examination of lipids and FAME obtained from R. mucilaginosa shows intriguing characteristics to produce biodiesel, highlighting its possibility to serve as a competitive alternative fuel source.

Thereby, a comprehensive comparative analysis of various oleaginous yeasts is presented in Table 4. Strains such as Yarrowia, Rhodotorula, Apiotrichum, among others, are known for their proficiency in producing lipids from diverse substrates, each having distinct metabolic capabilities and potential applications. A thorough examination of the existing literature indicates that semi-synthetic media are predominantly employed, often supplemented with different enhancers, to augment lipid production. In a study conducted by Santamauro et al. [44] on Metschnikowia pulcherrima, the use of waste lignocellulose as a substrate in conjunction with complex media having multiple sugar source resulted in the synthesis of roughly 40% lipids in a fed-batch reactor. In another investigation using Rhodosporidium toruloides, several carbon sources, such as glucose, xylose, and arabinose, were supplemented in a defined media within a fed-batch reactor, resulting in the formation of approximately 50–75% lipid [45]. While limited studies have explored the use of RDW for cultivating R. mucilaginosa, this investigation demonstrates substantial lipid yield with a high fatty acid composition along with carotenoids synthesis without using any complex media or multiple carbon sources making the whole process more economical. These lipids can further serve as a valuable precursor for the synthesis of diverse high-value products pertaining to their inherent versatility and abundance making them indispensable in many industries spanning from pharmaceuticals, nutraceuticals to biofuels and many more.

In a broader perspective, dairy wastewater emerges as a crucial nutrient reservoir, critical for supporting microbial life and promoting ecological balance via nutrient recycling processes. Using this resource in conjunction with sodium acetate supplementation in a fed-batch reactor to cultivate R. mucilaginosa marks a significant step towards increasing lipid and carotenoid synthesis. Our study highlights the many benefits that come with this strategy, offering a sustainable solution to manage dairy effluent while also paving the road for the production of high-value by-products. Further, by introducing 1.5% sodium acetate to optimize carbon utilization alongside dairy effluent, we not only substantially enhanced the C/N ratio but also introduced a critical precursor for acetyl CoA, a pivotal intermediary in the pathways leading to lipid and carotenoid production. Additionally, our research shows that fed-batch culture produces 72.14% lipids and 67.1 µg.g^-1 of carotenoids and also removes a remarkable 78% of COD, indicating a significant decrease in organic contaminants and highlighting the environmental remediation potential of this approach. In terms of future potential, this work offers a rich landscape for additional investigation and development. The efficiency and scalability of lipid and carotenoid synthesis might be significantly increased by adjusting process factors such nutrient content, feeding strategy, and reactor design. Delving deeper into the metabolic pathways of R. mucilaginosa and using genetic engineering approaches may unlock novel avenues for improving lipid and carotenoid biosynthesis, which would increase yield and productivity.

Declaration of Ethics Statement.
This study does not make use of human or animal participants. This study's analysis and conclusions are purely based on theoretical models, simulations, and/or publicly available data. As a result, no ethical approval or permission were required for this study.

Declaration of Consent for Publication
All authors engaged in this study agree to have their work published in Bioresources and Bioprocessing. The contents of this paper have not previously been published and are not being considered for publication anywhere.

Declaration of Competing Interest
The authors state that they have no known competing financial interests or personal relationships that could have influenced the work presented in this study.

Author’s contribution

Ravi Gadela: methodology, analysis, writing (original draft),
Kannan Pakshirajan: Conceptualization, supervision, review, and editing.

Veeranki Venkata Dasu: handles conceptualization, supervision, resource management, review, and editing. All authors reviewed and approved the final manuscript.

Declaration of No Funding.
This study received no grants from public, commercial, or non-profit funding entities.

Declaration of Availability of Data and Materials
The data and materials used and analyzed in the current investigation are available from the corresponding author upon on request.

Acknowledgment

The authors are thankful to the Indian Institute of Technology Guwahati for providing the required facilities to conduct this research work and the Central Instrument Facility (CIF), IIT Guwahati.

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The authors declare no competing interests.

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Valorization of Dairy Wastewater for the Production of Lipids, Carotenoids, and β-Carotene in Rhodotorula mucilaginosa

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Abstract

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1. Introduction

2. Materials and Methods

2.1 Materials

2.2 Microorganism

2.3 Shake flask studies

2.4 Bioreactor studies

2.4.1. Inoculum preparation and fermentation media

2.4.2. Batch and fed-batch studies

2.5 Biomass productivity

2.6 Sodium acetate estimation using high-performance liquid chromatography (HPLC)

2.7 Determination of lipid content

2.8 Total carotenoids estimation using a spectrophotometer

2.9 Fatty Acids Methyl esters (FAME) analysis

2.10 Evaluation of biodiesel properties based on FAME profiles

2.11 Analytical methods

3. Results and Discussions

3.1 Shake flask studies for the synthesis of lipids, carotenoids, and β-carotene

3.2 Batch reactor studies for the synthesis of lipids, carotenoids, and β-carotene

3.2 Fed-batch reactor studies for the synthesis of lipids, carotenoids, and β-carotene

3.3 Fatty acids methyl ester (FAME) by GC-FID analysis and evaluation for biodiesel properties

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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