Rhodosporidium Kratochvilovae Research Articles

Natural lycopene and β-carotene synthesis by Rhodosporidium kratochvilovae yeasts: Sustainable production, chemical characterization and antioxidative properties

Two yeast strains, Rhodosporidium kratochvilovae Y-42 and Y-43, were evaluated for their ability to synthesize natural carotenoids through submerged fermentations, using cheese whey (CW). Initially, CW was hydrolyzed with commercial β-galactosidase to yield glucose and galactose and the subsequent fermentations with R. kratochvilovae Y-42 and Y-43 led to the production of 1.87 and 1.64 mg/L of total carotenoids, respectively. HPLC-DAD analysis of the chemical profile indicated that β-carotene was the predominant carotenoid. Conversely, when CW hydrolysis occurred using fermented wheat bran, which also served as source of free amino nitrogen (FAN), both yeasts synthesized almost solely lycopene. Lycopene production was investigated under different C/FAN ratios (40 and 60 g/g) and incubation temperatures (23, 28 and 33 °C). Under the optimized culture conditions yeast strain Y-42 produced 36.4 mg/L carotenoids, composed of more than 97% of lycopene, highlighting the substantial role of media in carotenoids synthesis and composition. Moreover, the lycopene-rich carotenoid extract exhibited antioxidant activity (IC50 equal to 0.89 mg/mL), signifying also the possibility to be further used as a functional additive in novel food formulations. Evidently, this study showed for the first time that Rhodosporidium kratochvilovae can naturally produce lycopene using renewable resources, thus reinforcing the shift towards sustainable food production.

Comprehensive Response of Rhodosporidium kratochvilovae to Glucose Starvation: A Transcriptomics-Based Analysis.

Microorganisms adopt diverse mechanisms to adapt to fluctuations of nutrients. Glucose is the preferred carbon and energy source for yeast. Yeast cells have developed many strategies to protect themselves from the negative impact of glucose starvation. Studies have indicated a significant increase of carotenoids in red yeast under glucose starvation. However, their regulatory mechanism is still unclear. In this study, we investigated the regulatory mechanism of carotenoid biosynthesis in Rhodosporidium kratochvilovae YM25235 under glucose starvation. More intracellular reactive oxygen species (ROS) was produced when glucose was exhausted. Enzymatic and non-enzymatic (mainly carotenoids) antioxidant systems in YM25235 were induced to protect cells from ROS-related damage. Transcriptome analysis revealed massive gene expression rearrangement in YM25235 under glucose starvation, leading to alterations in alternative carbon metabolic pathways. Some potential pathways for acetyl-CoA and then carotenoid biosynthesis, including fatty acid β-oxidation, amino acid metabolism, and pyruvate metabolism, were significantly enriched in KEGG analysis. Overexpression of the fatty acyl-CoA oxidase gene (RkACOX2), the first key rate-limiting enzyme of peroxisomal fatty acid β-oxidation, demonstrated that fatty acid β-oxidation could increase the acetyl-CoA and carotenoid concentration in YM25235. These findings contribute to a better understanding of the overall response of red yeast to glucose starvation and the regulatory mechanisms governing carotenoid biosynthesis under glucose starvation.

Carotenoid biosynthesis is associated with low-temperature adaptation in Rhodosporidium kratochvilovae

BackgroundLow temperatures greatly limit the growth of microorganisms. Low-temperature adaptation in microorganisms involves multiple mechanisms. Carotenoids are naturally occurring lipid-soluble pigments that act as antioxidants and protect cells and tissues from the harmful effects of free radicals and singlet oxygen. However, studies on the regulation of carotenoid biosynthesis at low temperatures in microorganisms are limited. In this study, we investigated the correlation between carotenoids and low-temperature adaptation in the cold-adapted strain of Rhodosporidium kratochvilovae YM25235.ResultsCarotenoid biosynthesis in YM25235 was inhibited by knocking out the bifunctional lycopene cyclase/phytoene synthase gene (RKCrtYB) using the established CRISPR/Cas9 gene-editing system based on endogenous U6 promoters. The carotenoids were extracted with acetone, and the content and composition of the carotenoids were analyzed by spectrophotometry and HPLC. Then, the levels of reactive oxygen species (ROS) and the growth rate in YM25235 were determined at a low temperature. The results indicated that the carotenoid biosynthesis and ROS levels were increased in the YM25235 strain at a low temperature and inhibition of carotenoid biosynthesis was associated with higher ROS levels and a significant decrease in the growth rate of YM25235 at a low temperature.ConclusionsThe regulation of carotenoid biosynthesis was associated with low-temperature adaptation in YM25235. Our findings provided a strong foundation for conducting further studies on the mechanism by which YM25235 can adapt to low-temperature stress.

Exploring the potential of novel R. kratochvilovae red yeasts towards the sustainable synthesis of natural carotenoids

The development of microbial cell factories is nowadays a main route for the production of safe and environmentally friendly bioactive compounds. Rhodosporidium kratochvilovae yeasts constitute an unexplored source for the production of natural carotenoids, thus further insights on their metabolism and fermentative behavior would contribute towards the sustainable synthesis of bioactive compounds. The aim of this study was to investigate the ability of two R. kratochvilovae isolates (FMCC Y-42 and Y-43) to produce carotenoids under different experimental conditions. Cell growth, carotenoid synthesis and lipid accumulation were evaluated using synthetic media containing either glucose or mixtures of glucose:galactose (1:1), under different carbon-to-nitrogen (C/N) ratios. Carotenogenesis was highly affected by C/N ratio in both strains, while a competitive trend with lipid accumulation was also observed. Yeast strain Y-42 reached the highest concentration of total carotenoids (2.59 mg/L) when grown on glucose-based medium at a C/N ratio of 80 after 39 h of fermentation. Likewise, the strain Y-43 produced approximately 2 mg/L of total carotenoids from both glucose (C/N of 80) and its mixture with galactose (C/N 80 and 120), with similar biomass concentration (9.3, 10.3 and 10.9 g/L, respectively). HPLC-DAD analysis of carotenoid extracts indicated that the main carotenoid was β-carotene (60–75%) followed by γ-carotene (9–18%), torulene (6–13%) and torularhodin (5–17%), regardless the fermentation conditions or the strain-employed. This is the first study to reveal γ-carotene synthesis by R. kratochvilovae strains. The obtained results provided new knowledge on the production of natural carotenoids by novel red yeast strains and could pave the way towards their application in sustainable functional food products and nutraceuticals.

Hybrid histidine kinase HisK2301 modulates carotenoid production to counteract cold-induced oxidative stress in Rhodosporidium kratochvilovae YM25235 under low temperature.

Hybrid histidine kinases (HHKs) are major sensor proteins for fungi that contribute to stress tolerance. In the present work, we investigated the roles and mechanisms of the HHK HisK2301 in cold-adapted Rhodosporidium kratochvilovae strain YM25235. The HisK2301 deletion strain was constructed by homologous recombination method and arranged for multiple stress tests. We analysed the content of carotenoid using UV-Vis and HPLC. Relative transcript levels of genes phytoene desaturase (RKCrtI) and phytoene synthase and lycopene cyclase (RKCrtYB) were analysed by RT-qPCR. Intracellular reactive oxygen species (ROS) generation was measured using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Our results clearly indicated that YM25235 produces γ-carotene, torulene, β-carotene and torularhodin, with the latter two components strongly related to adapt to cold. HisK2301 is crucial for YM25235 adaptation to different types of stress such as cold, salt, osmotic and oxidative stress. Growth at low temperature clearly induced oxidative stress in YM25235, as more ROS accumulated at cold. During cold stress, HisK2301 is suggested to sense cold-induced ROS signals and then promote carotenoid production partially by RKCrtI and RKCrtYB to scavenge excessive ROS production. Such an inducible protective system may confer YM25235 fast response and better adaptation to cold stress. To conclude, our findings give the first insight into the effect of HisK2301 on carotenoid biosynthesis and cold-induced oxidative stress in fungi under low temperature and suggest the potential use of the cold-adapted HHK HisK2301 in industrial production of carotenoid.

The MAP-Kinase HOG1 Controls Cold Adaptation in Rhodosporidium kratochvilovae by Promoting Biosynthesis of Polyunsaturated Fatty Acids and Glycerol.

The aim of this study was to investigate the role of RKHog1 in the cold adaptation of Rhodosporidium kratochvilovae strain YM25235 and elucidate the correlation of biosynthesis of polyunsaturated fatty acids (PUFAs) and glycerol with its cold adaptation. The YM25235 strain was subjected to salt, osmotic, and cold stress tolerance analyses. mRNA levels of RKhog1, Δ12/15-fatty acid desaturase gene (RKD12), RKMsn4, HisK2301, and RKGPD1 in YM25235 were detected by reverse transcription quantitative real-time PCR. The contents of PUFAs, such as linoleic acid (LA) and linolenic acid (ALA) was measured using a gas chromatography-mass spectrometer, followed by determination of the growth rate of YM25235 and its glycerol content at low temperature. The RKHog1 overexpression, knockout, and remediation strains were constructed. Stress resistance analysis showed that overexpression of RKHog1 gene increased the biosynthesis of glycerol and enhanced the tolerance of YM25235 to cold, salt, and osmotic stresses, respectively. Inversely, the knockout of RKHog1 gene decreased the biosynthesis of glycerol and inhibited the tolerance of YM25235 to different stresses. Fatty acid analysis showed that the overexpression of RKHog1 gene in YM25235 significantly increased the content of LA and ALA, but RKHog1 gene knockout YM25235 strain had decreased content of LA and ALA. In addition, the mRNA expression level of RKD12, RKMsn4, RKHisK2301, and RKGPD1 showed an increase at 15°C after RKHog1 gene overexpression but were unchanged at 30°C. RKHog1 could regulate the growth adaptability and PUFA content of YM25235 at low temperature and this could be helpful for the cold adaptation of YM25235.

Genome-Wide Transcriptional Changes of Rhodosporidium kratochvilovae at Low Temperature.

Rhodosporidium kratochvilovae strain YM25235 is a cold-adapted oleaginous yeast strain that can grow at 15°C. It is capable of producing polyunsaturated fatty acids. Here, we used the Nanopore Platform to first assemble the R. kratochvilovae strain YM25235 genome into a 23.71 Mb size containing 46 scaffolds and 8,472 predicted genes. To explore the molecular mechanism behind the low temperature response of R. kratochvilovae strain YM25235, we analyzed the RNA transcriptomic data from low temperature (15°C) and normal temperature (30°C) groups using the next-generation deep sequencing technology (RNA-seq). We identified 1,300 differentially expressed genes (DEGs) by comparing the cultures grown at low temperature (15°C) and normal temperature (30°C) transcriptome libraries, including 553 significantly upregulated and 747 significantly downregulated DEGs. Gene ontology and pathway enrichment analysis revealed that DEGs were primarily related to metabolic processes, cellular processes, cellular organelles, and catalytic activity, whereas the overrepresented pathways included the MAPK signaling pathway, metabolic pathways, and amino sugar and nucleotide sugar metabolism. We validated the RNA-seq results by detecting the expression of 15 DEGs using qPCR. This study provides valuable information on the low temperature response of R. kratochvilovae strain YM25235 for further research and broadens our understanding for the response of R. kratochvilovae strain YM25235 to low temperature.

Carotenoids produced by yeast biomass protect mechanisms regulating endothelial barrier function from lipopolysaccharide-induced damage in the rat heart.

Expression of occludin and junctional adhesion molecule A (JAM-A), transmembrane proteins of tight junctions (TJs), was analysed to characterize endothelial paracellular permeability in the heart of rats subjected to a bolus of bacterial lipopolysaccharide (LPS) at a dose of 1 mg/kg. Potential protective effects of natural carotenoids (10 mg/kg/day) produced by yeast biomass Rhodosporidium kratochvilovae on expression of occludin and JAM-A also examined in LPS-injected rats (n = 6 per group). LPS decreased expression of occludin by 40% (P < 0.01), JAM-A by 36% (P < 0.001) and increased plasma levels of malondialdehyde (MDA) and lysosomal N-acetyl-b-D-glucosaminidase (NAGA) compared to controls. Ten-day diet rich in yeast biomass containing carotenoids (torularhodin, torulene, β-carotene) attenuated LPS-induced changes in expression of TJ proteins as observed by increased expression of occludin by 30% (P < 0.05) and JAM-A by 61% (P < 0.001) to the control values. Carotenoids also reduced oxidative stress and cellular injury indicated by decreased levels of MDA and NAGA. The results show that diet rich in yeast biomass containing natural carotenoids could protect mechanisms regulating paracellular endothelial barrier function from LPS-induced damage in the heart.

Utilization of Clarified Butter Sediment Waste as a Feedstock for Cost-Effective Production of Biodiesel.

The rising demand and cost of fossil fuels (diesel and gasoline), together with the need for sustainable, alternative, and renewable energy sources have increased the interest for biomass-based fuels such as biodiesel. Among renewable sources of biofuels, biodiesel is particularly attractive as it can be used in conventional diesel engines without any modification. Oleaginous yeasts are excellent oil producers that can grow easily on various types of hydrophilic and hydrophobic waste streams that are used as feedstock for single cell oils and subsequently biodiesel production. In this study, cultivation of Rhodosporidium kratochvilovae on a hydrophobic waste (clarified butter sediment waste medium (CBM)) resulted in considerably high lipid accumulation (70.74% w/w). Maximum cell dry weight and total lipid production were 15.52 g/L and 10.98 g/L, respectively, following cultivation in CBM for 144 h. Neutral lipids were found to accumulate in the lipid bodies of cells, as visualized by BODIPY staining and fluorescence microscopy. Cells grown in CBM showed large and dispersed lipid droplets in the intracellular compartment. The fatty acid profile of biodiesel obtained after transesterification was analyzed by gas chromatography-mass spectrometry (GC–MS), while its quality was determined to comply with ASTM 6751 and EN 14214 international standards. Hence, clarified sediment waste can be exploited as a cost-effective renewable feedstock for biodiesel production.

Innovative screening approach for the identification of triacylglycerol accumulating oleaginous strains

Currently, triacylglycerides (TAG) accumulation in the form of lipid droplets (LDs) in oleaginous microorganisms is of immense importance due to their ability to get transesterified into value-added products in the form of biodiesel. Hence, in order to search for oleaginous microorganisms having high lipid content among a wide range of samples from different niches, there is a compulsive need to develop simple, reliable and rapid methods for screening of TAG accumulating strains. Conventional methods require multistep processes for the isolation, cultivation, extraction and estimation of lipids to identify oleagenic strains. To overcome these challenges, we are proposing an easy, live cell imaging technique for the estimation of lipids via visualization of TAG accumulation in probable strains at the single cell level that gives real-time monitoring of intracellular lipid accumulation in yeasts. In this screening technique, only 100 μl of specific neutral lipid accumulating medium was used to grow the isolated culture in the microtiter plate. The harvested cells were stained with LipidTOX™ Green and visualized by a LED based digital inverted fluorescence microscope. Among 446 yeast colonies screened, maximum lipid producing yeast strains Rhodosporidium kratochvilovae HIMPA1 and Rhodotorula minuta, having supersized lipid body of 5.05 ± 0.87 μm and 4.46 ± 0.61 μm, respectively, were identified as potential candidates for biodiesel production. To the best of our knowledge, this is the first report of using LipidTOX™ Green for the staining of lipid droplets present in yeast cells as per the literature.

Novel Histidine Kinase Gene HisK2301 from Rhodosporidium kratochvilovae Contributes to Cold Adaption by Promoting Biosynthesis of Polyunsaturated Fatty Acids and Glycerol.

Hybrid histidine kinase (HHKs) are widespread in fungi, but their roles in the regulation of fungal adaptation to environmental stresses remain largely unclear. To elucidate this, we cloned HisK2301 from Rhodosporidium kratochvilovae strain YM25235, characterized HisK2301 as a novel HHK, and further investigated the role of HisK2301 by overexpressing it in YM25235. Our results revealed that HisK2301 can promote adaptation of YM25235 to cold, osmotic, and salt stresses. During cold stress, HisK2301 significantly enhanced the biosynthesis of polyunsaturated fatty acids (PUFA) and intracellular glycerol. HisK2301 also augmented the expression levels of Δ12/Δ15 fatty acid desaturase ( RKD12) and glycerol-3-phosphate dehydrogenase1 ( GPD1), which are responsible for PUFA and glycerol biosynthesis, respectively. To conclude, our findings give the first insight into the defense and mechanisms of HisK2301 in fungi against cold stress and suggest the potential use of the novel cold-adapted HHK HisK2301 in industrial processes, such as large-scale production of PUFA.

Production of single cell oil from cane molasses by Rhodotorula kratochvilovae (syn, Rhodosporidium kratochvilovae) SY89 as a biodiesel feedstock

BackgroundSingle cell oil has long been considered an alternative to conventional oil sources. The oil produced can also be used as a feedstock for biodiesel production. Oleaginous yeasts have relatively high growth and lipid production rates, can utilize a wide variety of cheap agro-industrial wastes such as molasses, and can accumulate lipids above 20% of their biomass when they are grown in a bioreactor under conditions of controlled excess carbon and nitrogen limitation.ResultsIn this study, Rhodotorula kratochvilovae (syn, Rhodosporidium kratochvilovae) SY89 was cultivated in a nitrogen-limited medium containing cane molasses as a carbon source. The study aims to provide not only information on the production of single cell oil using R. kratochvilovae SY89 on cane molasses as a biodiesel feedstock, but also to characterize the biodiesel obtained from the resultant lipids. After determination of the sugar content in cane molasses, R. kratochvilovae SY89 was grown on the optimized cane molasses for 168 h. Under the optimized conditions, the yeast accumulated lipids up to 38.25 ± 1.10% on a cellular dry biomass basis. This amount corresponds to a lipid yield of 4.82 ± 0.27 g/L. The fatty acid profiles of the extracted yeast lipids were analyzed using gas chromatography, coupled with flame ionization detector. A significant amount of oleic acid (58.51 ± 0.76%), palmitic acid (15.70 ± 1.27%), linoleic acid (13.29 ± 1.18%) and low amount of other fatty acids were detected in the extracted yeast lipids. The lipids were used to prepare biodiesel and the yield was 85.30%. The properties of this biodiesel were determined and found to be comparable to the specifications established by ASTM D6751 and EN14214 related to biodiesel quality.ConclusionsBased on the results obtained, the biodiesel from R. kratochvilovae SY89 oil could be a competitive alternative to conventional diesel fuel.

The Effect of Zn(II) Ions and Reactive Oxygen on the Uptake of Zinc and Production of Carotenoids by Selected Red Yeasts.

Three strains of red yeast Rhodosporidium kratochvilovae, Rhodotorula glutinis and Sporidiobolus salmonicolor were studied for their responses to the presence metal stress, oxidative stress and a combination of these stress factors. For all yeast strains, the production of β-carotene increased in stress conditions. The combination of H2 O2 and Zn2+ significantly activated the pathways for the production of torularhodin in the strain R.glutinis (from 250 to 470μgg-1 DCW) as well as β-carotene (from 360 to 1100μgg-1 DCW) and torulene (from 100 to 360μgg-1 DCW) in Sp.salmonicolor. Strains of R.glutinis and Rh.kratochvilovae bound the majority of Zn(II) ions to the fibrillar part of the cell walls, whereas the strain Sp.salmonicolor bound them to both extracellular polymers and the fibrillar part of the cell walls. A decrease in the ability of yeasts to tolerate higher concentrations of Zn(II) in the presence of free radicals (hydrogen peroxide) was also found.

A novel malic enzyme gene, Mime2, from Mortierella isabellina M6-22 contributes to lipid accumulation.

This study was aimed at cloning and characterizing a novel malic enzyme (ME) gene of Mortierella isabellina M6-22 and identifying its relation with lipid accumulation. Mime2 was cloned from strain M6-22. Plasmid pET32aMIME2 was constructed to express ME of MIME2 in Escherichia coli BL21. After purification, the optimal pH and temperature of MIME2, as well as Km and Vmax for NADP+ were determined. The effects of EDTA or metal ions (Mn2+, Mg2+, Co2+, Cu2+, Ca2+, or Zn2+) on the enzymatic activity of MIME2 were evaluated. Besides, plasmid pRHMIME2 was created to express MIME2 in Rhodosporidium kratochvilovae YM25235, and its cell lipid content was measured by the acid-heating method. The optimal pH and temperature of MIME2 are 5.8 and 30°C, respectively. The act ivity of MIME2 was significantly increased by Mg2+, Ca2+, or Mn2+ at 0.5mM but inhibited by Cu2+ or Zn2+ (p < 0.05). The optimal enzymatic activity of MIME2 is 177.46 U/mg, and the Km and Vmax for NADP+ are 0.703mM and 156.25μg/min, respectively. Besides, Mime2 transformation significantly increased the cell lipid content in strain YM25235 (3.15 ± 0.24 vs. 2.17 ± 0.31g/L, p < 0.01). The novel ME gene Mime2 isolated from strain M6-22 contributes to lipid accumulation in strain YM25235.

Synchronized nutrient stress conditions trigger the diversion of CDP-DG pathway of phospholipids synthesis towards de novo TAG synthesis in oleaginous yeast escalating biodiesel production

In this study oleaginous yeast, Rhodosporidium kratochvilovae HIMPA1 grown in glucose synthetic medium containing different nitrogen (1 g/l and 0.1 g/l) and phosphorus (0.05 g/l and 0.1 g/l) limited conditions. Among various N and P-limited conditions, the highest lipid content (60.34± 0.69%) obtained under synchronized limitation of N and P (0.1 g/l N and 0.05 g/l P). Live fluorescent cell imaging of yeast cells after BODIPY505-515 nm staining endorses the results of triacylglycerol (TAG) accumulation in lipid droplets. The cells grown in synchronized limitation of N and P exhibited boosted cell size (6.76 ± 0.39 μm) and lipid droplet size (5.62 ± 0.28 μm). Under synchronized limitation of N and P, supersized irregular shaped lipid droplets (LD) coalesced to form big lobules in the cellular compartment of oleaginous yeast having 87.14% enhanced TAG accumulation as depicted by TLC. Synchronized nutrient limitation diverts the CDP-DG pathway of phospholipids synthesis towards de novo TAG synthesis. The maximum increment of oleic acid (C18:1) was reported in synchronized limitation of N and P that improve the biodiesel properties like oxidative stability, viscosity, cetane number and cold filter plugging point.

Biodegradation of phenol via meta cleavage pathway triggers de novo TAG biosynthesis pathway in oleaginous yeast

Phenol is reported to be one of the most toxic environmental pollutants present in the discharge of various industrial effluents causing a serious threat to the existing biome. Biodegradation of phenol by oleaginous yeast Rhodosporidium kratochvilovae HIMPA1 was found to degrade 1000mg/l phenol. The pathways for phenol degradation by both ortho and meta-cleavage were proposed by the identification of metabolites and enzymatic assays of ring cleavage enzymes in the cell extracts. Results suggest that this oleaginous yeast degrade phenol via meta-cleavage pathway and accumulates a high quantity of lipid content (64.92%; wt/wt) as compared to control glucose synthetic medium (GSM). Meta-cleavage pathway of phenol degradation leads to formation of pyruvate and acetaldehyde. Both these end products feed as precursors for de novo triacylglycerols (TAG) biosynthesis pathway which causes accumulation of TAG in the lipid droplets (LD) of 6.12±0.78μm grown on phenol while 2.38±0.52μm observed on GSM. This was confirmed by fluorescence microscopic images of BODIPY505-515nm stained live yeast cells. GC–MS analysis of extracted total lipid showed enhanced amount of monounsaturated fatty acid (MUFA) which was as 51.87%, 58.33% and 62.98% in presence of 0.5, 0.75 and 1g/l of phenol.

Inhibition of Polyunsaturated Fatty Acids Synthesis Decreases Growth Rate and Membrane Fluidity of Rhodosporidium kratochvilovae at Low Temperature.

The intention of this study was to investigate the role of polyunsaturated fatty acids (PUFA) in the cold adaptation of Rhodosporidium kratochvilovae YM25235 by knockout of the Δ12/Δ15-fatty acid desaturase gene (RKD12) to inactivate Δ12/Δ15-fatty acid desaturase. Polymerase chain reaction (PCR) amplification was used to detect the genomic structure of RKD12 gene in YM25235. The RKD12 gene was knocked out by DNA homologous recombination to inhibit the biosynthesis of PUFA. Then, the contents of linoleic acid (LNA) and α-linolenic acid (ALA) after gene knockout were investigated using a gas chromatography-mass spectrometer, followed by determination of the growth rate and membrane fluidity of YM25235 at low temperature. After PCR amplification, a 1611bp genomic fragment was amplified from YM25235. When the RKD12 gene was knocked out, the contents of LNA and ALA in YM25235 significantly decreased. The growth rate and membrane fluidity of YM25235 decreased significantly at low temperature. Inhibition of PUFA biosynthesis by RKD12 gene knockout influenced cold adaptation of YM25235 by decreasing the PUFA content in cell membranes and reducing the growth rate and membrane fluidity of YM25235 at low temperature.

Optimization of cultivation conditions for biotechnological production of lipid by Rhodotorula kratochvilovae (syn, Rhodosporidium kratochvilovae) SY89 for biodiesel preparation

Rhodotorula kratochvilovae (syn, Rhodosporidium kratochvilovae) SY89, an oleaginous yeast, isolated from Ethiopian soil, was grown under nitrogen-limited media. The capacity this with respect to biomass production, lipid yield and lipid content was evaluated. The influence of inoculum size, carbon sources, variations in glucose concentration, nitrogen sources, C/N ratio, pH, temperature, agitation, and aeration rate and incubation period were investigated. Inoculum size of 10% v/v, glucose as a carbon source at 50 g/L glucose, 0.50 g/L yeast extract and 0.31 g/L (NH4)2SO4, C/N ratio of 120, pH 5.5, incubation temperature of 30 °C, 225 rpm, 0.2 as aeration ratio and 144 h of incubation were found to be optimum conditions for lipid production. Then the yeast was grown in a batch bioreactor by combining the different optimized parameters together. Under the optimized conditions, the yeast gave maximum biomass (15.34 ± 1.47 g/L), lipid yield (8.60 ± 0.81 g/L) and lipid content (56.06 ± 1.70%). The dominant fatty acids exhibited in order of their relative abundance (%w/w), were oleic, palmitic, linoleic, stearic, linolenic and palmitoleic acids. The concentration of saturated and monounsaturated fatty acids adds up 78.63 ± 2.19%. This suggests that this strain could be used as a good feedstock for biodiesel production.

Biological treatment of pulp and paper industry effluent by oleaginous yeast integrated with production of biodiesel as sustainable transportation fuel

Worldwide pulp and paper industry is recognized as a serious threat due to the discharge of various toxic contents present in its effluent to the surrounding environment. To combat this challenge, the role of oleaginous yeast to utilize the pulp and paper industry effluent for the integrated toxic contents removal and sustainable biodiesel production were undertaken. Oleaginous yeast Rhodosporidium kratochvilovae HIMPA1 was used as a model organism for its unique ability to utilize pulp and paper industry effluent as a culture medium and accumulate high quantity of triacylglycerol or neutral lipids as large size lipid droplets of 4.56 ± 0.24 μm within its cellular compartment. The maximum cell dry weight (13.87 g/L) with total lipid yield of 8.56 g/L was obtained after 144 h of cultivation. Data showed significant reduction in effluent toxic components i.e. color, 89%; lignin, 94.27%; phenol, 99.60%; biochemical oxygen demand, 77.36%; total dissolved solids, 84.59%; and chemical oxygen demand, 94.22%. Fatty acid methyl esters profile revealed high quantity of long chain monounsaturated fatty acids (45.43%) and polyunsaturated fatty acids (15.91%) that improves biodiesel quality under low temperature condition in terms of low cold filter plugging point along with good oxidative stability and cetane number as per American Society for Testing and Materials D6751-02 and European Committee for Standardization - EN14214 guidelines.

Oleaginous yeasts from Ethiopia.

Oleaginous microorganisms can produce high amounts of oil (>20 % of their biomass) under suitable cultivation conditions. In this research work 200 samples were collected from soil, plant surfaces (leaves, flowers and fruits), waste oils from traditional oil milling houses and dairy products (cheese, milk and yoghurt) in Ethiopia. Three hundred and forty yeast colonies were isolated from these samples. By applying Sudan III staining tests, 18 strains were selected as possible oleaginous yeasts. The 18 strains were identified and characterized for their lipid production as a feedstock for biodiesel production in the future. They were identified using morphological and physiological methods as well as sequencing the 3′end of the small-subunit rRNA gene, the internal transcribed spacer regions (ITS; ITS 1, ITS 2 and the intervening 5.8S rRNA gene), and the D1/D2 domain of the 26S rRNA gene. The 18 yeasts were identified as Cutaneotrichosporon curvatus (syn, Cryptococcus curvatus) (PY39), Rhodotorula kratochvilovae (syn, Rhodosporidium kratochvilovae) (SY89), Rhodotorula dairenensis (SY94) and Rhodotourula mucilaginosa (SY09, SY18, SY20, PY21, PY23, PY25, SY30, PY32, SY43, PY44, SY52, PY55, PY61, SY75 and PY86). Under nitrogen-limited cultivation conditions, R. mucilaginosa PY44 produced the highest biomass (15.10 ± 0.54 g/L), while R. mucilaginosa PY32 produced the lowest biomass (10.32 ± 0.18 g/L). The highest lipid yield of 6.87 ± 0.62 g/L and lipid content of 46.51 ± 0.70 % were attained by C. curvatus (syn, C. curvatus) PY39. On the other hand, R. mucilaginosa PY61 gave the lowest lipid yield (2.06 ± 0.52 g/L) and R. mucilaginosa SY52 gave the lowest lipid content of 16.99 ± 0.85 %. The results in this research work suggest that much more oleaginous yeasts can be isolated from Ethiopian environment. On the basis of their substantial lipid production abilities, the three oleaginous yeast strains PY39, SY89 and SY18 were selected and recommended for further optimization processes.