Enhanced high β-carotene yeast cell production by Rhodotorula paludigena CM33 and in vitro digestibility in aquatic animals

Impact of micronutrient concentration in Feed Medium on R. paludigena CM33 high cell density fed-batch fermentation

The effectiveness of a growth-promoting and β-carotene-producing Feed Medium for fed-batch fermentation was explored further in this research, using an optimal medium for batch fermentation of R. paludigena CM334. In feed medium, the carbon and nitrogen source concentrations were elevated to 500 g/L and 77.2 g/L, respectively. Optimization of micronutrient concentrations to enhance cell density and β-carotene content, in sync with the elevated C-source and N-source, was the focus.

Fed-batch fermentation was executed in a 22-L bioreactor, starting with batch fermentation using optimal cultivation conditions and medium4. In the batch cultivation phase, glucose and ammonium sulfate were exhausted in 20 h (Fig. 1), indicating the cell’s response to the cultivation system. At this point, the average biomass reached 16.63 ± 1.02 g/L, and the average β-carotene concentration was 32.28 ± 1.63 mg/L for all experiments (Fig. 1a–d). Subsequently, the fed-batch cultivation phase began by replenishing the Feed Medium with varying concentrations of micronutrients, as described in Materials and Methods and Table 1. The fed-batch cultivation phase was run for 76 h using DOT stat feed control (total cultivation time of 96 h), leading to different biomass, β-carotene, yields, and productivities for each feed medium, as indicated in Table 2.

Figure 1
figure 1

Comparison of fed-batch fermentation profiles of R. paludigena CM33 in the 22L-bioreactor using different feed media (a) Feed Medium 1, (b) Feed Medium 2, (c) Feed Medium 3, and (d) Sucrose Feed Medium. Feed medium 1, 2, and 3 used glucose as a carbon source with a concentration of micronutrients of 1, 2, and 3 times, respectively, compared to the optimum concentration for batch fermentation. Sucrose Feed Medium uses sucrose as a carbon source with a concentration of micronutrients 3 times higher than the optimum concentration for batch fermentation.

Table 2 Comparison of the biomass and β-carotene concentrations of R. paludigena CM33 with different feed media in a 22-L bioreactor at 96 h.

After 96 h of fed-batch cultivation, it became evident that Feed Medium 1, characterized by the lowest concentration of micronutrients, resulted in the least favorable outcomes in terms of biomass and β-carotene production. The biomass and β-carotene levels were 72.00 ± 0.75 g/L and 184.13 ± 0.00 mg/L, respectively, as shown in Table 2. This low level of cell proliferation and growth can be attributed to potential constraints arising from the limited availability of some components in micronutrients19. As observed in Fig. 1a, there was a continuous accumulation of ammonium sulfate during the fed-batch cultivation with Feed Medium 1 until the end of the process, with the ammonium sulfate concentration reaching 5.17 ± 0.04 g/L at 96 h. The accumulation of ammonium sulfate might have occurred from the cultivation limitation under conditions of low micronutrient availability, causing the microorganism to experience nutrient imbalance18.

To address the challenges posed by micronutrient limitations, the concentration of micronutrients was doubled in Feed Medium 2. This adjustment yielded notably improved results, with biomass and β-carotene concentrations reaching 77.89 ± 0.77 g/L and 249.42 ± 1.33 mg/L, respectively. These findings indicate that increasing the concentration of micronutrients has an impact on nutrient balance, consequently boosting biomass and β-carotene output. Additionally, in pursuit of higher β-carotene yields, a state of nitrogen limitation was intentionally introduced, as supported by Cescut et al.30 and Saenge et al.31.

Although the increased in micronutrient concentration improved outcomes, an accumulation of ammonium sulfate reaching a concentration of 2.54 ± 0.03 g/L at 96 h, as shown in Fig. 1b. This persistent ammonium sulfate accumulation suggests that while the adjustment addressed certain nutrient imbalances, additional factors involving co-factors or micronutrients may require further investigation. It indicates that the mere presence of carbon and nitrogen sources is insufficient if specific co-factors or balanced nutrition is lacking. This deficiency can prevent the yeast R. paludigena CM33’s ability to effectively utilize these resources, impacting cell growth, metabolic processes, and biosynthesis, as noted in Li et al.18.

In both, the enhanced micronutrient concentration and nitrogen limitation scenarios, the importance of cultivation conditions and nutrient balance is underscored. These factors are crucial in determining the performance of R. paludigena CM33 in terms of biomass and β-carotene production. The findings highlight the intricacies involved in bioprocess optimization and the need for a finely tuned nutrient equilibrium to maximize yields in biotechnological applications. Moreover, the improvements in growth parameters and carotenoid production, detailed in Table 2, further reinforce these conclusions.

Despite the similar initial concentrations of ammonium sulfate across all feed media, in Feed Medium 3 a complete depletion of ammonium sulfate was observed, indicating a balanced utilization rate with glucose. It yielded the highest biomass and β-carotene concentrations (89.84 ± 0.41 g/L and 251.64 ± 1.06 mg/L, respectively) when compared to Feed Medium 1 and 2. While the yeast extract provided additional nitrogenous compounds, its higher concentration in Feed Medium 3 (1.5 g/L) did not necessarily translate to increased nitrogen availability for growth, as evidenced by the complete depletion of ammonium sulfate which is 51.67 times higher concentration to yeast extract. This observation implies a potential shift in the yeast’s metabolic focus from growth to secondary metabolite production, such as carotenoids, under conditions where nitrogen sources were exhausted (Fig. 1c), aligning with the findings of Cescut et al.30 and Saenge et al.31. This phenomenon of metabolic shift, rather than straightforward nitrogen limitation, may contributed to the enhanced carotenoid accumulation observed in Feed Medium 3. Since the batch phase was consistent across all experiments, regardless of the feed medium used, the observed differences in each experiment can primarily be attributed to variations in the micronutrient concentration. This in-depth analysis of the effect of micronutrients shows the potential of Feed Medium 3 for further development in larger scale processes, notably for industrial applications.

In the Rhodotorula yeast, the nitrogen limitation led to the decreasing in growth rate and carotenoid accumulation through residual carbon source utilization. Changes in carbon source to citrate increased citrate accumulation, enhancing carotenoid synthesis32,33. The inclusion of inorganic salts, specifically disodium hydrogen phosphate (Na2HPO4) and potassium dihydrogen phosphate (KH2PO4), serves a dual purpose in the culture medium. These salts function as buffers, maintaining the medium’s pH, which is crucial for optimal cellular pH balance during the growth and carotenoid synthesis. This role of pH in influencing both growth and carotenoid production in Rhodotorula yeast has been reported by Aksu et al.34 and Kot et al.35. Their research underscores the significant impact that pH has on the overall efficacy of the bioprocess, particularly in terms of both yeast growth and carotenoid yield. Increasing the concentration of magnesium ions can have a significant impact on both growth and carotenoid synthesis36, as magnesium ions act as essential cofactors in many cellular processes37. Elevated levels of magnesium boost the synthesis of carotenoids by stimulating the function of acetyl CoA carboxylase38, while the lack or excessive quantity of magnesium results in metabolic changes39. This might potentially have an adverse impact on the process of carotenoid production.

Comparison of the effect of Feed Medium with glucose and sucrose on cell growth and β-carotene production

This study evaluates the use of sucrose as an alternative carbon source to glucose in the fed-batch cultivation of R. paludigena CM33, primarily to reduce production costs. The data presented in Table 2 demonstrates that substituting glucose with sucrose in the feed medium resulted in comparable biomass production (87.78 ± 0.30 g/L) but significantly enhanced β-carotene levels (285.00 ± 1.84 mg/L). The increase in β-carotene production during the fed-batch phase, when transitioning from glucose to sucrose, can be attributed to a metabolic shift in R. paludigena CM33, as evidenced by research from Lee et al.40 and Sánchez et al.41. This shift involves the enzymatic breakdown of sucrose into glucose and fructose, a process catalyzed by invertase,detailed in studies by Park et al.42 and Gong et al.43. The presence of both glucose and fructose alters the yeast’s metabolic flux, with fructose entering glycolysis at a point that bypasses the regulatory phosphofructokinase step. This variation allows for a more efficient flow through the pentose phosphate pathway, essential for producing NADPH, a vital cofactor in carotenoid biosynthesis. The dual availability of these monosaccharides enhances metabolic efficiency and NADPH availability, crucial for the increased synthesis of β-carotene. This phenomenon emphasizes the importance of substrate diversity in regulating metabolic pathways and enhancing secondary metabolite production in yeasts43.

In the Sucrose Feed Medium, an unexpected accumulation of ammonium sulfate was noted, reaching 1.92 ± 0.01 g/L at 96 h (Fig. 1d). This accumulation, contrasting with the glucose feed medium, may indicate a differential carbon utilization strategy by R. paludigena CM33. Notably, despite this availability of ammonium sulfate, β-carotene accumulation in the sucrose medium was the highest among the tested media by the experiment’s end.

This finding calls for a reassessment of the previously held assumption, as mentioned in Cescut et al.30 and Saenge et al.31, which proposed that low levels of ammonium sulfate were critical for boosting carotene production. It now appears that the link between ammonium sulfate concentration and β-carotene synthesis is more intricate than originally thought. This complexity might be attributed to changes in metabolic pathways induced by sucrose, potentially channeling the metabolic flux towards an enhanced β-carotene synthesis, as previously discussed43. Furthermore, it is important to consider that β-carotene is synthesized as a secondary metabolite22. Therefore, the growth rate of the yeast is another crucial factor that must be taken into account. This factor could significantly influence the overall metabolic activity and hence the production of β-carotene, underscoring the multifaceted nature of this bioprocess.

Interestingly, the metabolic shift induced by using sucrose instead of glucose initially led to a temporary slowdown in microbial growth. However, this was accompanied by an increase in β-carotene production, potentially as a response to cellular stress44. The underlying biochemical process involves sucrose and glucose enhancing the synthesis of acetyl coenzyme A (CoA), a precursor for mevalonic acid, which is a key substrate in carotenoid production45. Kilian et al.46 reported that disaccharides such as sucrose, maltose, and cellobiose can significantly boost carotenoid production up to 12-fold, while high glucose concentrations might inhibit it.

Considering the titer, rate, and yield (TRY) metrics, the sucrose feeding system showed 13.26%, 13.36%, and 5.70% higher β-carotene concentration, volumetric productivity, and yield based on substrate consumed as glucose equivalent compared to the glucose feeding system, respectively. Additionally, using sucrose as the carbon source in the feed medium presents significant benefits over conventional glucose-based media, particularly for enhancing β-carotene production and reducing costs. One of the key advantages of using sucrose is its cost-effectiveness, as it is more than twice as affordable compared to glucose. This economic benefit, combined with its effectiveness in boosting β-carotene yields, makes sucrose an attractive alternative for feed media in fermentation processes. In the context of large-scale industrial production of microbial carotenoids, the selection of low-cost carbon sources is crucial. These findings hold significant implications for the commercial and industrial bioprocessing sectors, suggesting that sucrose is a viable alternative for the development and optimization of bioprocesses for protein and carotenoid production.

Comparative analysis of β-carotene production techniques

Table 3 presents a comparative analysis with other literature reviews focusing on fed-batch cultivation47,48,49,50,51,52. Among these, R. paludigena CM33 stands out for its superior β-carotene production, particularly notable when employing a DOT stat feed control strategy. This strain outperformed others, such as S. roseus51 and R. mucilaginosa48, in terms of biomass yields and β-carotene production. Specifically, it achieved biomass yields of 89.84 ± 0.41 g/L with glucose and 87.78 ± 0.30 g/L with sucrose, and β-carotene yields of 2.80 ± 0.02 mg/g cell with glucose and 3.25 ± 0.03 mg/g cell with sucrose. The productivity rates were also remarkable higher than that reported in all literature reviewed47,48,49,50,51,52 in Table 3. The biomass production rates (Qx) of 0.94 ± 0.00 g/L.h for glucose and 0.91 ± 0.01 g/L.h for sucrose, and β-carotene production rates (Qp) of 2.62 ± 0.01 mg/L.h for glucose and 2.97 ± 0.02 mg/L.h for sucrose were achived in this present work.

Table 3 Comparison of the performance of fed-batch culture conditions for biomass, β-carotene production, and productivity in red yeast.

The study further emphasized the natural capabilities and efficiency of R. paludigena CM33, a strain isolated directly from nature8. Its ability to thrive under various conditions and efficiently utilize sucrose as a substrate not only enhanced cost-effectiveness but also reduced overall cultivation time53. These attributes make sucrose-based media particularly promising for future development in fermentation processes, especially for applications in the animal feed, food, and pharmaceutical industries54. The high yields of protein biomass and β-carotene associated with this strain highlight its potential for commercial-scale applications.

Nutritional composition

To investigate their feasibility for commercial development in animal feed supplement, dried R. paludigena CM33 cells were nutritionally analyzed after fed-batch cultivation in a 22L-bioreactor. Table 4 offers a comprehensive view of the nutritional composition of R. paludigena CM33 under various cultivation techniques and mediums, which is critical for evaluating its potential for commercial development in animal feed supplement. The data reveals several essential insights regarding the yeast’s nutritional profile. The analysis of key compounds, carotenoids accumulation, conformed the potential health-promoting and biotherapeutic properties of R. paludigena CM33. Directly, accumulation of carotenoids in the cells possibly makes R. paludigena CM33 as a colorant for promoting pigmentation in ornamental and consumed aquatic animals. Additinally, this indicates its potential to enhance the growth, disease resistance, and meat quality of aquatic animals when used as a probiotic supplement in their diet, as demonstrated by Sriphuttha et al.5.

Table 4 Comparative analysis of nutritional compositions of R. paludigena CM33 cultivation in different cultivation techniques and carbon sources.

Proteins constitute significant dietary needed for animal growth, and are the dominant organic material in animal tissue55. In contrast to carbohydrates (nitrogen-free extract), proteins constitute a major cost for aquafeed development due to it’s the costliest ingredient of dietary preparation56. In the present study, cultivating R. paludigena CM33 with sucrose feed demonstrated a negligible alteration in crude protein content (42.47%) compared to the glucose feed (42.96%), highlighting its suitability as a feed supplement for animals. Both variants, RPG and RPS, contain substantial protein content and essential nutrients necessary for animal feed utilization, positioning them as valuable resources in animal nutrition. For crude lipid, under optimized conditions, this strain is promising for lipid production as it accumulates lipids to > 20% of dry cell weight, and long chain fatty acids (C16 and C18) are predominant, especially oleic acid (C18:1) and palmitic acid (C16:0)8. However, fatty acid profile was not emphasized in the present study due to relatively low amounts of detected crude lipid contents. This is also for the effects from other nutritional components (crude fiber, ash, calcium, and phosphorus) since the inclusion level of supplement is relatively low in practical feed.

In vitro digestibility of protein and carbohydrate

Protein digestibility acts as a primary indicator in assessing feed ingredients for aquaculture purpose57. In general, yeast cells, such as S. cerevisiae, contained high amounts of essential nutrients, including proteins and carbohydrates58. As illustrated in Fig. 2, yeast samples and aquatic animal species had significant effects on IVDP and IVDC (P < 0.05). The commercially available yeast brands (CSC and CSCB) provided relatively low IVDP relative to SB or three alternative R. paludigena CM33 (RPO, PRG, and RPS) (P < 0.05). Using the digestive enzymes from Nile tilapia, better protein digestibility was observed in RPG and RPS samples relative to RPO. The different findings found in striped catfish and whiteleg shrimp indicate that RPO and RPS treatments achieved superior IVDP relative to RPG (P < 0.05). For IVDC, the yeast RPO was suitable for use as a feed supplement in three tested animal species. They provided moderate IVDC when screening by digestive enzymes from Nile tilapia but provided relatively high IVDC in the case of enzymes from striped catfish and whiteleg shrimp.

Figure 2
figure 2

The in vitro digestibility of protein (mmol DL-alanine equivalent/g sample, gray bars) and carbohydrate (mmol maltose/g sample, white bars) in yeast samples using digestive enzyme extracted from Nile tilapia (a,b), striped catfish (c,d), and whiteleg shrimp (e,f). Data are expressed as means ± SD (n = 4). Significant differences between treatments are indicated by different superscripts (P < 0.05). SB = S. bouladii; RPO = R. paludigena CM33 cultivated in optimal conditions; RPG = R. paludigena CM33 cultivated in glucose feed; RPS = R. paludigena CM33 cultivated in sucrose feed; CSC = commercially available yeast S. cerevisiae; and CSCB = commercially available yeast S. cerevisiae blended with multi-strain probiotics.

Since in vitro digestibility correlates with nutrient bioavailability59,60,61, our investigations suggest the potential of R. paludigena CM33 as an animal feed supplement, especially for aquatic animals. However, detailed composition, such as amino acid profiles, should be clarified before being used as an alternative feed supplement. Not only nutritive values but also yeast cells contain a number of active ingredients, which may promote the growth and health status of reared animals review by Mahdy et al.62. Before use as feed supplement, the level of R. paludigena CM33 in a practical feed, in comparison with a control unsupplemented feed, should be optimized. In vivo trials, observing growth, feed utilization, whole-body composition, and health status would be assessment criteria for clarifying aquaculture outcome.