3.1 An examination of the growth profile of yeast Rhodotorula mucilaginosa
Rhodotorula mucilaginosa and Candida tropicalis were extracted from an oil refinery in Guwahati, Assam, India (Prabhu et al., 2019; Gedela et al., 2023). R. mucilaginosa is a common saprophytic fungus from the Basidiomycota phylum found in various environments, including soils, aquatic systems, and food products. Although the genus is well known for its saprophytic lifestyle, several species, including R. mucilaginosa, Rhodotorula glutinis, and Rhodotorula minuta, have shown pathogenic potential in humans, particularly in immunocompromised populations (Wirth and Goldani, 2012; Zaas et al., 2003).
A genus of unicellular yeast called Rhodotorula is distinguished by its characteristic orange/red color when grown on conditions intended for yeast cultivation. One orange yeast cell was found during the initial screening of R. mucilaginosa. These colonies clearly exhibit the distinctive orange/red hue of R. mucilaginosa (Sppl. Figure 3.1). The image provides a visual representation of the colonies that this type of yeast generates when grown on culture media (Kurtzman et al., 2011).
In the preliminary enquiry, yeast medium (YM) enriched with mineral salt medium (MSM) was used to conduct batch shake cultures. These studies on R. mucilaginosa's metabolic capacities have focused on its performance on yeast-based medium. R. mucilaginosa produced lipids, carotenoids, and β-carotene when grown under these conditions (Gedela et al., 2023).
Lipids are vital macromolecules with a wide range of applications, including biofuels and nutritional supplements. In yeast medium, R. mucilaginosa has been found to efficiently synthesis and accumulate lipids. This feature makes it a good option for biotechnological applications targeted at the long-term production of biofuels and other lipid-derived products.
Carotenoids are coloured chemicals with antioxidant capabilities that are commonly employed in the food and cosmetic sectors due to their health advantages and brilliant hues. R. mucilaginosa is known for its ability to produce carotenoids. These chemicals serve an important function in shielding cells from oxidative stress and can be used in a variety of industrial applications. β-Carotene, a carotenoid precursor of vitamin A, plays a crucial role in human health, especially for eyesight and immunity. R. mucilaginosa produces β-carotene in yeast medium, indicating its potential as a natural supply of this essential nutrient. This brings up the possibility of its usage in dietary supplements and food fortification to fight vitamin A deficiency.
Growing R. mucilaginosa in yeast medium results in the production of lipids, carotenoids, and β-carotene, which has major significances. This yeast species could be a versatile and sustainable source of these valuable compounds, meeting the needs of a variety of industries such as biofuel production, nutrition, and cosmetics. Future research and development could optimize culture conditions and increase production processes to fully realize R. mucilaginosa's biotechnological potential. The yeast, R. mucilaginosa will undoubtedly play an important part in producing eco-friendly and cost-effective solutions to fulfil comprehensive requirements as our understanding and capabilities in microbial biotechnology increase.
By examining the substrate utilization of yeast medium supplemented with Carbon sources, Nitrogen sources, Phosphate sources and Sodium acetate, more optimization studies were conducted.
R. mucilaginosa is the subject of study on optimizing culture conditions to boost lipid production, carotenoids, and β-carotene. Experiments were carried out to determine the ideal conditions that would lead to higher yields of these valuable chemicals. In the observed growth profile of R. mucilaginosa, glucose as a carbon source resulted in increased optical density (OD) biomass, carotenoid (57.50 ± 1.54 µgg− 1), and β-carotene (25.50 ± 0.77 µgg− 1) production, but lower lipid production (55.22 ± 2.14% w/w) than other carbon sources and, while the maximal yield of cell dry weight (CDW) was 3.35 ± 0.07 gL− 1 (Table 4.1, Fig. 3.2 A, B, C). When sucrose, maltose, lactose, and galactose were employed, OD, biomass, carotenoid, and carotene production decreased, while lipid production (56% w/w) increased slightly. Specifically, galactose produced somewhat less OD, biomass, carotenoid, and β-carotene than glucose, but more than sucrose, maltose, and lactose (Table 4.1, Fig. 3.2 A).
When studying the growth profile of R. mucilaginosa using multiple carbon sources, it is critical to understand the microbe's varied metabolic routes for each substrate. The different growth rates and efficiency in utilizing various sugars can be ascribed to their structural complexity and metabolic accessibility. Glucose, as a simple sugar and monosaccharide, is quickly absorbed by microbial cells and enters the glycolytic pathway. This immediate entrance into central metabolism causes rapid energy production and biomass formation. The rapid growth rate reported using glucose as a carbon source could be due to its ease of transport across the cell membrane and the lack of enzymatic degradation before consumption (Berg, 2015; Nelson and Cox, 2017; Madigan et al., 2018). Sucrose, maltose, and lactose are disaccharides that must be hydrolyzed to form monosaccharides before being digested. Enzymes like sucrase, maltase, and lactase hydrolyze these disaccharides, converting them into glucose and fructose (sucrose), glucose and glucose (maltose), and glucose and galactose (lactose). Because of the need for this enzymatic breakdown, it enters metabolic pathways later than glucose, resulting in slower initial growth rates (Berg, 2015; Nelson and Cox, 2017; Madigan et al., 2018).
Although galactose is a monosaccharide, it is not as easily processed as glucose. Galactose must be transformed into glucose-1-phosphate via the Leloir route before entering glycolysis. This conversion needs several biochemical steps, slightly postponing the commencement of energy production when compared to straight glucose utilization (Timson, 2007). The desire for glucose can be traced back to evolutionary adaptation, in which microorganisms refined their metabolic machinery for the fastest possible energy production to outcompete other organisms in nutrient-rich settings. This mechanism, known as catabolite repression, guarantees that when glucose is present, enzymes required for the metabolism of alternative carbon sources are suppressed. This regulatory mechanism selects the most efficient energy source to maximize growth and survival (Berg, 2015; Nelson and Cox, 2017; Madigan et al., 2018).
3.2 Examine the alive cell structure of lipids and carotenoids in R. mucilaginosa
The potential of R. mucilaginosa, a specific species of yeast, as a viable option for producing lipids, carotenoids, and β-carotene has garnered increasing attention in recent research (Gedela et al., 2022). An accumulation of lipids within the yeast cells was further shown by microscopic observations made utilizing sophisticated techniques including Field Effect Scanning Electron Microscopy (FESEM) (Fig. 3.3) and Fluorescence and Phase Contrast Microscopy with Nile red labeling (Fig. 3.4). These measurements also showed that the yeast cells had an ellipsoid shape and had a diameter of roughly 3–4 µm (Elle et al., 2010). Notably, R. mucilaginosa has demonstrated promising findings in both its consumption and the assessment of its capacity for lipid accumulation. There is great promise for the sustainable production of lipids for biodiesel application, carotenoids, and β-carotene from this research (Gedela et al., 2023).
The FESEM pictures, obtained at a magnification of 50,000X and an electron high tension (EHT) of 2.0 kV, provide important information on the cellular structure of the cells under investigation. Images (A) and (B) show depictions of cells that seem to be under stress. The findings from the examination of R. mucilaginosa for lipid droplets using FESEM offer important new information about the distribution and morphology of lipid storage in this organism. FESEM is a potent imaging method that enables the viewing of surface structures at high resolution, which makes it especially useful for researching subcellular elements like lipid droplets and microorganisms.
FESEM would have been used in the examination analysis to investigate the yeast Rhodotorula sp., including R. mucilaginosa samples at a high magnification, allowing the discovery of lipid droplets at the cellular level. The size, shape, and distribution patterns of these lipid droplets within the R. mucilaginosa cells would have been visible in the collected photos.
The broad interpretation of these findings includes their importance for comprehending R. mucilaginosa's lipid metabolism and storage processes. Lipid droplets are essential for energy homeostasis and cellular function because they are intracellular organelles engaged in lipid metabolism and storage. The FESEM pictures show that R. mucilaginosa has lipid droplets, and their quantity and presence provide information on the organism's physiological modifications and metabolic processes.
Furthermore, the FESEM investigation offers both qualitative and quantitative data regarding the characteristics of lipid droplets in R. mucilaginosa. This includes information on their intracellular location, density, and size distribution, all of which may provide crucial cues on the organism's strategies for storing lipids and its responses to its surroundings.
The lipid droplet study conducted using FESEM on R. mucilaginosa provides significant insights into the cellular architecture and lipid metabolism of the organism.
A fluorescent microscope can be used to take pictures that reveal important details about the properties of the cells under study. Image (A) illustrates cells under bright-field light and the fluorescence produced by the Nile red-neutral lipid combination at the same time. In a similar vein, cells are shown in image (B) under bright-field illumination, together with fluorescence from the Nile red-neutral lipid complex. Images from R. mucilaginosa lipid and carotene morphometric identification and fluorescence spectroscopy provide important visual information about the distribution and composition of these chemicals throughout the organism. A sensitive analytical method called fluorescence spectroscopy detects the amount of fluorescent light molecules emit after being excited by a particular wavelength of light. Morphometric identification is the quantitative use of imaging techniques to analyze morphological traits, such as size and shape (de Carvalho et al., 2012; Pacia et al., 2016; Elfeky et al., 2019). Fluorescence spectroscopy would have been used in the research investigation to identify and measure the fluorescence signals released by the carotene and lipid molecules in the R. mucilaginosa samples. Fluorescence emissions from lipids and carotenes can be identified and evaluated by exciting the samples with light at the right wavelengths, which reveals details about their relative quantities and patterns of distribution (de Carvalho et al., 2012; Pacia et al., 2016; Elfeky et al., 2019).
Morphometric identification, on the other hand, examines the morphological features of lipid and carotene structures within R. mucilaginosa cells using imaging methods like microscopy. Quantitative information on the quantity and distribution of lipid droplets and carotenoid bodies within the cells can be obtained by measuring their size, shape, and spatial structure. These photos' broad description captures their importance in comprehending the physiological roles and metabolic routes of lipid and carotene molecules in R. mucilaginosa. The complementing data provided by morphometric identification and fluorescence spectroscopy techniques about the location and abundance of these chemicals shed light on their functions in cellular metabolism and environmental adaptation. Moreover, a thorough investigation of the dynamics of lipids and carotenoids in R. mucilaginosa is made possible by the combination of fluorescence spectroscopy and morphometric identification (de Carvalho et al., 2012; Pacia et al., 2016, Elfeky et al., 2019).
The visual data acquired from fluorescence spectroscopy and morphometric identification of R. mucilaginosa lipid and carotene offer significant insights into the organism's metabolic processes and adaptation mechanisms.
The figure includes distinct spectra representing different components within the yeast. The Raman spectra of lipids are specifically shown in panel (A), which shows a single spectrum taken from the cross-marked regions. Lipids are present as indicated by the marker bands at 1136 cm− 1 and 1441 cm− 1. Similarly, panel (B) shows the Raman spectra of carotenoids with a single spectrum taken from the pink indicated line. Carotenoids are verified by the marker bands at 1000 cm− 1, 1190 cm− 1, 1560 cm− 1, 2190 cm− 1, and 2330 cm− 1. The final set of photos in panel (C) shows the combination of lipids and carotenoids, emphasizing the marker bands for each.
Additionally, sophisticated analytical methods were used to characterize the lipids and carotenoids generated in this investigation. The lipids' characteristics were examined using Raman spectroscopy, and the results showed good lipid properties. Unique peaks in the Raman spectra verified the accumulation of carotenoids and lipids within R. mucilaginosa (Fig. 3.5) (de Carvalho et al., 2012; Pacia et al., 2016; Elfeky et al., 2019).
The detection of lipid and carotene in R. mucilaginosa using Raman spectroscopy research yielded useful information about the molecular structure and chemical makeup of these substances within the organism. Vibrational spectroscopy, or Raman spectroscopy, studies how molecules scatter laser light to reveal details about their functional groups and chemical interactions (de Carvalho et al., 2012; Pacia et al., 2016; Elfeky et al., 2019). Raman spectroscopy would have been used in the study analysis to describe the R. mucilaginosa samples and determine whether lipid and carotene molecules were present based on their distinctive Raman spectra. Whereas carotenes show characteristic Raman peaks linked to their conjugated double bond system, lipids usually show Raman bands corresponding to CH stretching vibrations (de Carvalho et al., 2012; Pacia et al., 2016; Elfeky et al., 2019). Raman spectroscopy's ability to identify lipid and carotene molecules reveals their existence in R. mucilaginosa cells and clarifies their functions in cellular metabolism and environmental adaptability. Raman spectroscopy has respective benefits for the examination of biological samples, such as high sensitivity, specificity, and non-destructive, label-free detection. The method makes it possible to identify and measure lipid and carotene molecules in intricate biological matrices, which makes it easier to conduct in-depth investigations on the distribution and dynamics of these molecules within cells.
The standard correlation graph-which was devised by Aksu and Eren, 2007; Bhosale, 2001 is an essential tool for quantitative analysis when estimating β-carotene at 455 nm by spectrophotometry. The absorbance values of known β-carotene concentrations are usually plotted against their respective quantities in this graph, creating a linear connection that makes it possible to determine unknown amounts from their absorbance values. This probably involved making standard β-carotene solutions in different concentrations and then using a spectrophotometer to measure the absorbance at 455 nm. A statistical analysis of the obtained data would have been performed to determine a linear relationship between absorbance and concentration.
The standard correlation graph described by Aksu and Eren, 2007; Bhosale, 2001 for spectrophotometry-based estimation of β-carotene at 455 nm is a fundamental tool for quantitative analysis in analytical chemistry. After extensive testing and statistical analysis, it is established and provides a dependable and effective way to measure the amount of β-carotene in different samples.
The mass-to-charge ratio (m/z)-based chromatographic separation of lipids is depicted in the figure. The chromatogram may be seen in the figure, which displays several co-eluting peaks that indicate various lipid species. For each peak, the precise m/z values 200, 356.21, 316.12, 437.11, and 475 are given.
In addition, an examination using LC-MS was conducted to offer a thorough comprehension of the composition and structure of the lipids (Fig. 3.7). The R. mucilaginosa lipid profile study results from LC-MS provide comprehensive details regarding the variety and makeup of lipids found in the organism. LC-MS is a potent analytical method that combines mass spectrometry's sensitivity detection and molecular identification with liquid chromatography's separation capabilities.
Lipids can be identified and quantified using this technique by utilizing their mass-to-charge ratios in the mass spectrometer and their retention durations in the chromatographic column. This offers detailed information regarding the lipid profile of R. mucilaginosa, including the existence of various lipid classes and their molecular species (phospholipids, glycolipids, and neutral lipids, for example). The types and quantities of lipids found in R. mucilaginosa cells can be determined using the LC-MS analysis, which also provides important details on the organism's metabolic processes and defence mechanisms.
Moreover, the identification of lipid molecular species, such as headgroup compositions and fatty acid chains, is made possible by LC-MS. Furthermore, R. mucilaginosa's lipid profile can be examined using LC-MS analysis to learn more about how it responds to environmental stimuli and experimental manipulations.
Specific lanes in the figure correspond to various samples. Lane C is the reference lane for comparison, and it contains the triolein standard. The lipid sample from the zero-hour batch culture is represented by lane (1), the lipid sample from the 24-hour batch culture by lane (2), the lipid sample from the 72-hour batch culture by lane (3), and the lipid sample from the 96-hour batch culture by lane (4). The various samples can be divided using TLC according to their migration distances and lipid composition. This makes it possible to evaluate the neutral lipids in each sample qualitatively and provide information about their distribution and abundance across the cultures.
TLC study was carried out to further verify that the chosen microbe, R. mucilaginosa, could produce significant levels of intracellular neutral lipids relative to cell weight. The substantial lipid accumulation potential of R. mucilaginosa (Fig. 3.8) was confirmed by the TLC analysis results (Knittelfelder and Kohlwein, 2017). TLC was used to separate the neutral lipids from R. mucilaginosa. The qualitative analysis of the resulting material sheds light on the existence and variety of neutral lipid species inside the organism. TLC is a flexible chromatographic method that divides substances according to how well they migrate through a mobile phase and how well they bind to a stationary phase. When it comes to lipid analysis, TLC makes it possible to separate and see several lipid classes, including neutral lipids like free fatty acids, cholesterol esters, and triglycerides (Knittelfelder and Kohlwein, 2017).
TLC would have been used in the research analysis to separate the neutral lipid fraction that was recovered from the samples of R. mucilaginosa. An appropriate solvent system would have been used to spot the separated lipids onto a TLC plate and subject them to chromatographic separation. After separation, the existence of neutral lipid bands would have been observed on the TLC plate under UV light or with the use of a staining reagent.
The broad interpretation of these findings includes their importance for comprehending R. mucilaginosa's lipid composition and metabolic processes. In addition to being crucial components of membrane structure and cellular signalling, neutral lipids are molecules that store energy. The TLC analysis offers insights into the types and abundance of neutral lipids present in R. mucilaginosa cells, providing valuable information about its lipid metabolism and physiological adaptations (Knittelfelder and Kohlwein, 2017).
TLC also enables the qualitative evaluation of R. mucilaginosa's neutral lipid diversity. The organism's lipid biosynthesis pathways and capacity for environmental adaptation can be better understood with the help of this information. The qualitative examination of neutral lipids extracted from R. mucilaginosa using TLC analysis advances our knowledge of the lipid metabolism and cellular physiology of this organism.
3.3 FAME analysis by Gas chromatography
Gas chromatography using a flame ionization detector (GC-FID) and an SLB-IL100 column was used to examine the fatty acid methyl esters (FAMEs) contained in the lipids synthesized from yeast medium fed with sodium acetate by R. mucilaginosa. Before analysis, lipid samples from both fed-batch and batch processes were trans methylated. The FAME analysis yielded fatty acid composition profiles that demonstrated the lipids were primarily composed of long-chain fatty acids, with carbon chain lengths varying between C16 and C18.
Numerous fatty acids were identified by the analysis of the fed-batch sample: ginkgolic acid (C17:1), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), lauric acid (C12:0), myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (C16:1), margaric acid (C17:0), ginkgolic acid (C17:1), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3) (Table 3.1). It was discovered that of these fatty acids, R. mucilaginosa contained larger proportions of oleic, palmitic, and stearic acids overall. The two main FAME fractions, methyl oleate (C18:1) and methyl palmitate (C16:0), made up almost 72% of the overall FAME content (Table 3.2). There were notable concentrations of other fatty acids, including methyl stearate (C18:0), methyl linoleate (C18:2), methyl palmitoleate (C16:1), and methyl linolenate (C18:3).
Table 3.1
Gas chromatography analysis of FAME composition (%) profile in R. mucilaginosa
IUPAC Name | Fatty acids | | FAME (%, w/w) |
Methyl laurate | Lauric | C12:0 | 0.31 ± 0.02 |
Methyl tetradecanoate | Myristic | C14:0 | 0.94 ± 0.07 |
Methyl palmitate | Palmitic | C16:0 | 21.05 ± 1.02 |
Methyl palmitoleate | Palmitoleic | C16:1 | 1.10 ± 0.11 |
Methyl heptadecanoate | Margaric acid | C17:0 | 0.15 ± 0.06 |
Methyl heptadecenoic | Ginkgolic acid | C17:1 | 0.32 ± 0.01 |
Methyl octadecanoate | Stearic | C18:0 | 5.02 ± 0.16 |
Methyl oleic | Oleic | C18:1 | 51.18 ± 2.41 |
Methyl linoleate | Linoleic | C18:2 | 7.25 ± 1.61 |
Methyl linolenate | Linolenic | C18:3 | 1.03 ± 0.31 |
The fatty acid profile also included trace quantities of unsaturated fatty acids (methyl heptadecenoic) and saturated fatty acids (methyl laurate, methyl palmitoleate, and methyl heptadecanoate). According to the GC analysis, R. mucilaginosa has a higher concentration of unsaturated fatty acids than saturated fatty acids.