Transcriptomic and metabolomic profiling of a Rhodotorula color mutant to improve its lipid productivity in fed-batch fermentation.

Lipids produced by oleaginous microorganisms enrich the supply of feedstock for bio-fuel. In this study, a mutant (Mut) obtained by UV-nitrosoguanidine mutagenesis turned its colony color from orange-red to light-yellow and showed higher lipid productivity at 37 °C than the original strain Rhodotorula sp. U13N3 (Rht) in the glycerol medium. The metabolic changes between Mut and Rht in batch fermentation were investigated by transcriptomic and metabolomic profiling at the biomass accumulation (30 h) and lipid production (96 h) stages. The average base number in each strain was 5.80 × 109 ± 0.38 × 109 bp (mean ± SD) with 62.43% ± 0.13% GC ratio, and 7499 unigenes were assembled after Illumina sequencing. Moreover, 33 metabolites were quantified by 1H NMR-based profiling. The multi-omics results demonstrated that Mut showed increased glycerol transport and utilization capabilities especially at the first stage (30 h). Then the carbon flux shifted from the TCA cycle to lipid production (96 h). The increased lipid productivity of Mut was partially attributed to the down-regulation of mannitol biosynthesis. However, the mechanism for color change was elusive. At 96 h, the low level of cytosol glycerol probably restricted the lipid production. As a result, supplementation of glycerol in fed-batch fermentation remarkably improved the biomass, lipid production, and lipid content to 34.60 g/L, 25.72 g/L, and 74.3% (w/w dcw), respectively. The cell morphology implied that excessively prolonging the fermentation time was detrimental to the final lipid yield due to cell breakage. In conclusion, the Rhodotorula mutant provided a candidate strain for lipid production with glycerol as the carbon source.

Metabolic coupling in the co-cultured fungal-yeast suite of Trametes ljubarskyi and Rhodotorula mucilaginosa leads to hypersecretion of laccase isozymes.

Trametes ljubarskyi produces multiple laccase isozymes under various physicochemical conditions. During co-cultivation condition Rhodotorula mucilaginosa showed inter-specific interactions with T. ljubarskyi and hypersecretion of laccases; however, the underlying molecular mechanism is less-known. The analysis of proteomics data of co-cultivated cultures revealed the mechanism of metabolic coupling during fungal-yeast interactions. The results suggested high score GO terms related to stimulus-response, protein binding, membrane components, transport channels, oxidoreductases, and antioxidants. The SEM studies confirmed the cellular communication and their inter-specific interactions. This study allows us to deepen and refine our understanding of fungal-yeast symbiotic interaction; further, it also establishes a mutual relation by metabolic coupling for 10-fold higher laccase isozyme secretion (6532 U/ml). The purified laccase isozymes showed acidic pH optima (pH 3-4), higher thermo-stability (60 °C), and broad enzyme kinetics (Km) values. Our study also provides an in-depth understanding of laccase isozymes and their potential to degrade synthetic dyes, which may help the fungi to survive in an adverse environment.

Biosorbents based on esterified starch carrying immobilized oil-degrading microorganisms.

The complex sorbents based on hydrophobized starch, which contain oil-degrading microorganisms, have been proposed for effective sorption and utilization of petroleum-related pollutants. The sorbents were made on the base of benzoic, lauric and stearic acid esters of starch with degrees of substitution of 0.4-1.1. The esterification of starch was carried out by the reaction with acyl chlorides of the corresponding acids in an aqueous-organic medium. The structure of the esters was studied by SEM, IR and NMR spectroscopy. As a result of porous hydrophobic structure, these sorbents are capable of binding and retention of petroleum products on the water surface, and keeping the flotation for at least 30days after the petroleum products sorption. The test of biodegradability of the obtained samples revealed that the modified starches can be degraded by microscopic fungi, therefore they do not cause secondary pollution. The cultures of yeast Rhodotorula glutinis VKM Y-2993D and bacteria Pseudomonas libanensis VKM B-3041D immobilized on the sorbent facilitate the rapid utilization of accumulated petroleum products.

Production, purification, and characterization of an extracellular acid protease from the marine Antarctic yeast Rhodotorula mucilaginosa L7.

The production, purification, and characterization of an extracellular protease released by Rhodotorula mucilaginosa L7 were evaluated in this study. This strain was isolated from an Antarctic marine alga and previously selected among others based on the capacity to produce the highest extracellular proteolytic activity in preliminary tests. R. mucilaginosa L7 was grown in Saboraud-dextrose medium at 25 °C, and the cell growth, pH of the medium, extracellular protease production and the glucose and protein consumption were determined as a function of time. The protease was then purified, and the effects of pH, temperature, and salt concentration on the catalytic activity and enzyme stability were determined. Enzyme production started at the beginning of the exponential phase of growth and reached a maximum after 48 h, which was accompanied by a decrease in the pH as well as reductions of the protein and glucose concentrations in the medium. The purified protease presented optimal catalytic activity at pH 5.0 and 50 °C. Finally, the enzyme was stable in the presence of high concentrations of NaCl. These characteristics are of interest for future studies and may lead to potential biotechnological applications that require enzyme activity and stability under acidic conditions and/or high salt concentrations.

Interplay between carotenoids, hemoproteins and the "life band" origin studied in live Rhodotorula mucilaginosa cells by means of Raman microimaging.

Raman microimaging of live Rhodotorula mucilaginosa cells, cultured under aerobic and anaerobic conditions, showed striking differences in the composition and distribution of cell components. The analysis of these differences and recovery of oxidative phosphorylation upon environmental changes enabled the interrelation of carotenoids, hemoproteins and the unknown species considered as the "Raman signature of life".

Enhanced lipid production with undetoxified corncob hydrolysate by Rhodotorula glutinis using a high cell density culture strategy.

In recent years, energy crisis and environmental issues such as greenhouse effect, global warming, etc. has roused peoples' concern. Biodiesel, as renewable energy, has attracted much attention to deal with such problems. This work studied the lipid production by Rhodotorula glutinis with undetoxified corncob hydrolysate. The results indicated that R. glutinis had high tolerance to the inhibitors in corncob hydrolysate and it could utilize undetoxified corncob hydrolysate directly for lipid production. The cell grew well with undetoxified hydrolysate in the batch culture of 5L fermentor with the optimized C/N ratio of 75, lipid titer and lipid content reached 5.5g/L and 36.4%, respectively. High cell density culture with two-stage nitrogen feeding strategy was studied to enhance the lipid production, biomass, lipid concentration and lipid content of 70.8, 33.5g/L and 47.2% were obtained. The results indicated the potential application for lipid production by R. glutinis with corncob hydrolysate directly.

Highly enantioselective production of (R)-halohydrins with whole cells of Rhodotorula rubra KCh 82 culture.

Biotransformation of ten α-haloacetophenones in the growing culture of the strain Rhodotorula rubra KCh 82 has been carried out. Nine of the substrates underwent an effective enantioselective reduction to the respective (R)-alcohols according to Prelog's rule, with the exception of 2-chloro-1,2-diphenylethan-1-one that was not transformed by this strain. The expected reduction proceeded without dehalogenation, leading to the respective (R)-halohydrins in high yields. The use of this biocatalyst yielded (R)-2-bromo-1-phenyl-ethan-1-ol (enantiomeric excess (ee) = 97%) and its derivatives: 4'-Bromo- (ee = 99%); 4'-Chloro- (ee > 99%); 4'-Methoxy- (ee = 96%); 3'-Methoxy- (ee = 93%); 2'-Methoxy- (ee = 98%). There were also obtained and characterized 2,4'-dichloro-, 2,2',4'-trichloro- and 2-chloro-4'-fluoro-phenyetan-1-ol with >99% of enantiomeric excesses.

Assessment of ß-carotene content, cell physiology and morphology of the yellow yeast Rhodotorula glutinis mutant 400A15 using flow cytometry.

Flow cytometry was used to assess ß-carotene content, cell membrane permeability, cell size and granularity in Rhodotorula glutinis mutant 400A15 grown under different oxygen transfer coefficients (k L a) and carbon to nitrogen ratios (C/N). A Doehlert distribution was used in order to select the best conditions that induced the highest carotenoids production. The highest ß-carotene content (0.79 mg g(-1) DCW) at the lowest k L a and C/N (5 × 10(-3) s(-1) and 11.3 respectively). Under these conditions, the biomass concentration attained 18.60 g L(-1). The highest ratio of cells with permeabilised membranes (2.6 %), and the highest cell size and granularity were also obtained under these conditions. It was observed that C/N showed a stronger influence than the k L a on the measured cell parameters.

Monitoring of real changes of plasma membrane potential by diS-C(3)(3) fluorescence in yeast cell suspensions.

The fluorescent dye 3,3'-dipropylthiadicarbocyanine, diS-C(3)(3), is a suitable probe to monitor real changes of plasma membrane potential in yeast cells which are too small for direct membrane potential measurements with microelectrodes. A method presented in this paper makes it possible to convert changes of equilibrium diS-C(3)(3) fluorescence spectra, measured in yeast cell suspensions under certain defined conditions, into underlying membrane potential differences, scaled in the units of millivolts. Spectral analysis of synchronously scanned diS-C(3)(3) fluorescence allows to assess the amount of dye accumulated in cells without otherwise necessary sample taking and following separation of cells from the medium. Moreover, membrane potential changes can be quantified without demanding calibration protocols. The applicability of this approach was demonstrated on the depolarization of Rhodotorula glutinis yeast cells upon acidification of cell suspensions and/or by increasing extracellular K(+) concentration.

The effects of irradiation and microfiltration on the cells growing and total lipids production in the cultivation of Rhodotorula glutinis.

The results of this study indicate that the irradiation could enhance the cells growing of Rhodotorula glutinis to 54.2 ± 1.6g/L as compared to the control (without irradiation) of 38.3 ± 1.2g/L. However, different wavelength of LEDs' (red, green, blue and white) had no significant impacts on the growth and on the lipid content. The accumulation of potential inhibitive metabolic products probably impedes growth, which restricts more biomass accumulated in the fed-batch operation with irradiation. The combining of the fed-batch operation with irradiation and microfiltration can successfully improve the growth of R. glutinis to the maximum of 72.4 ± 0.6g/L and 51.2 ± 4.9% of lipid content obtained. Conclusively, the integration process of a fed-batch operation, irradiation and microfiltration can effectively enhance cell growth in R. glutinis, without any reimbursement of lipid contents. This finding might be useful when applied to the commercialized cultivation of R. glutinis for biodiesel production.

Selection of lipase producing yeasts for methanol-tolerant biocatalyst as whole cell application for palm-oil transesterification.

Methanol-tolerant lipase producing yeast was successfully isolated and selected thorough ecological screening using palm oil-rhodamine B agar as one step-approach. All 49 lipase-producing yeasts exhibited the ability to catalyze esterification reaction of oleic acid and methanol at 3 molar equivalents. However, only 16 isolates catalyzed transesterification reaction of refined palm oil and methanol. Rhodotorula mucilagenosa P11I89 isolated from oil contaminated soil showed the strongest hydrolytic lipase activity of 1.2U/ml against palm oil. The production of oleic methyl ester and fatty acid methyl ester (FAME) of 64.123 and 51.260% was obtained from esterification and transesterification reaction catalyzed by whole cell of R. mucilagenosa P11I89 in the presence of methanol at 3 molar equivalents against the substrates, respectively. FAME content increased dramatically to 83.29% when 6 molar equivalents of methanol were added. Application of the methanol-tolerant-lipase producing yeast as a whole cell biocatalyst was effectively resolved major technical obstacles in term of enzyme stability and high cost of lipase, leading to the feasibility of green biodiesel industrialization.

Effects of dissolved oxygen level on cell growth and total lipid accumulation in the cultivation of Rhodotorula glutinis.

The total amount of lipids produced in Rhodotorula glutinis is a subject which has attracted increasing attention due to the potential biodiesel conversion from these microbial oils. The effects of the dissolved oxygen (DO) level in lipid accumulation were examined in this study. Variations of different medium volumes (30, 40 and 50ml) and shaking speed (60, 150 and 210rpm) in the flask trials were adopted to explore the DO effects on lipid production. All of the results revealed that a low DO could retard cell growth, while enhancing lipid accumulation. The 5l-fermentor results also confirm that a low DO (25 ± 10%) batch could have higher lipid content than that of high DO batch (60 ± 10%). Nevertheless, the DO level would not obviously affect the lipid composition profile. Oleic acid (C18:1) was the primary fatty acid in both batches. Due to the slow biomass growth rate resulting from the low DO, a two-stage DO controlled strategy (consisting of a high DO stage and following a low DO stage) was performed to improve the cell growth and lipid accumulation simultaneously. However, the strategy was not successful on the enhancement of total lipid production as compared to other batches. Conclusively, even a low DO could retard cell growth; the total production of lipids in the batch with low DO was higher that of the high DO batch due to the enhancement of lipid accumulation. Therefore, the batch operation of R. glutinis at the low DO was suggested for the purpose of lipid production.

Using multi-parameter flow cytometry to monitor the yeast Rhodotorula glutinis CCMI 145 batch growth and oil production towards biodiesel.

Multi-parameter flow cytometry was used to monitor cell intrinsic light scatter, viability, and lipid content of Rhodotorula glutinis CCMI 145 cells grown in shake flasks. Changes in the side light scatter and forward light scatter were detected during the yeast batch growth, which were attributed to the different yeast growth phases. A progressive increase in the proportion of cells stained with PI (cells with permeabilized cytoplasmic membrane) was observed during the yeast growth, attaining 79% at the end of the fermentation. A high correlation between the Nile Red fluorescence intensity measured by flow cytometry and total lipid content assayed by the traditional gravimetric lipid analysis was found for this yeast, making this method a suitable and quick technique for the screening of yeast strains for lipid production and optimization of biofuel production bioprocesses. Medium growth optimization for enhancement of the yeast oil production is now in progress.

Asymmetric reduction of substituted acetophenones using once immobilized Rhodotorula glutinis cells.

The asymmetric reductions of acetophenone and its analogues using once immobilized Rhodotorula glutinis cells were studied. The performance and reaction parameters of the immobilized cells were also investigated and it was determined that the cells could be used 15 times in batch processes. All chiral alcohols obtained using purification procedures were of sufficient enantiopurity (>99%) of the (S)-enantiomer. The applicability of the optimized process for a preparative scale bioreduction was shown. Under the optimum conditions, 35mM (4.3g) of the product ((S)-1-phenylethanol) was produced from 45mM (5.4g) of the substrate (acetophenone) with one time immobilized R. glutinis EBK-2 cells (6g wet weight). The yield was calculated as 77%. In this study, it was found that the buffer level had a very significant effect on the reaction activity. Our results demonstrate that the optimized process can be implemented on a preparative scale.

A voltammetric Rhodotorula mucilaginosa modified microbial biosensor for Cu(II) determination.

It is the first report about the usage of Rhodotorula mucilaginosa as a biomaterial to construct a microbial biosensor based on carbon paste for determination of copper. Cu(II) was preconcentrated electrode surface at open circuit and then detected with electrochemical techniques, including Cyclic Voltammetry (CV) and Differential Pulse Stripping Voltammetry (DPSV). Some parameters such as pH of preconcentration solution, preconcentration time, scan rate and effect of interfering heavy metal ions were carried out for optimum responses. The best defined cathodic peak was obtained at pH 5 with 0.05 M NaNO(3) and a scan rate of 100 mV/s. The linear range for the developed microbial biosensor was found in the range of 1.0 x 10(-7) and 1.0 x 10(-5)M (0.0064 and 0.64 mg/L) at the response time of 15 min (R(2)=0.98). The easy fabrication, sensitivity, low cost and fast response time showed the advantages of the biosensor to conventional techniques.

[Study on biosorption of uranium by Rhodotorula glutinis].

Characteristic of uranium biosorption in water solution by Rhodotorula glutinis was investigated in the present study and the optimal pH for uranium adsorption was found to be 6-7. At the same time, maximum adsorption capacity of 149.4 mgU/(g dry cell) was identified, and Langmuir adsorption models can be used to simulate the isothermal biosorption process with high correlation coefficient of 0.99. According to Fourier transform infrared spectra, a new peak at wave number of 904 cm(-1), which can be assigned to the stretch vibration of UO2, was detected in the cell which was contacted by the uranium, indicated that uranium was really absorbed by Rhodotorula glutinis. Changes in the uranium-exposed yeast biomass were in the stretching vibrations of amino or hydroxyl groups, which shift from 3309 to 3287 cm(-1), and in the stretching vibrations of C--O band, which shift from 1068 to 1080 cm(-1), and these are all attributed to the important role that they may played in the binding of uranium. Hardly any changes can be found in the characteristic IR adsorbing peaks of protein at wave numbers of 1653, 1540 and 1237 cm(-1) before and after uranium adsorption, making it clear that the major component and the structure of the biomass remained intact. 96% of the absorbed uranium can be easily desorbed by 0.1 mol x L(-1) NaHCO3. Obviously, the application potential of this yeast in the uranium wastewater treatment was very wide and expansive, and more more work should be done to realize its industrial use.

Efficient enantioselective reduction of 4'-methoxyacetophenone with immobilized Rhodotorula sp. AS2.2241 cells in a hydrophilic ionic liquid-containing co-solvent system.

The biocatalytic enantioselective reduction of 4'-methoxyacetophenone to (S)-1-(4-methoxyphenyl)ethanol was successfully conducted in a hydrophilic IL-containing co-solvent system using immobilized Rhodotorula sp. AS2.2241 cells. Of all the tested ILs, the best results were observed with the novel IL 1-(2'-hydroxy)ethyl-3-methylimidazolium nitrate (C(2)OHMIM.NO(3)), which showed a good biocompatibility with the cells and increased the cell membrane permeability moderately, thus improving the efficiency of the bioreduction. To better understand the bioreduction, several crucial influential variables were also examined. The optimal C(2)OHMIM.NO(3) content, buffer pH, reaction temperature and substrate concentration were 5.0% (v/v), 8.5, 25 degrees C and 12 mM, respectively. Under the optimized conditions, the initial reaction rate, the maximum yield and the product e.e. were 9.8 micromol/hg(cell), 98.3% and >99%, respectively, which are much better than the results previously reported. The established biocatalytic system has proven to be highly effective for the reduction of other aryl ketones. Also, the cells exhibited excellent operational stability in the presence of C(2)OHMIM.NO(3). Moreover, the ILs can accumulate within the cells, suggesting that ILs are likely to interact with the related enzymes within the cells.

Characterization of three endophytic, indole-3-acetic acid-producing yeasts occurring in Populus trees.

Three endophytic yeast, one isolated from stems of wild cottonwood (Populus trichocarpa), two from stems of hybrid poplar (P. trichocarpaxPopulus deltoides), were characterized by analyzing three ribosomal genes, the small subunit (18S), internal transcribed spacer (ITS), and D1/D2 region of the large subunit (26S). Phenotypic characteristics of the yeast isolates were also obtained using a commercial yeast identification kit and used for assisting the species identification. The isolate from wild cottonwood was identified to be closest to species Rhodotorula graminis. The two isolates from hybrid poplar were identified to be species Rhodotorula mucilaginosa. In addition, the three yeast isolates were observed to be able to produce indole-3-acetic acid (IAA), a phytohormone which can promote plant growth, when incubated with l-tryptophan. To our knowledge, the yeast strains presented in this study were the first endophytic yeast strains isolated from species of Populus.

Studies on lipid production by Rhodotorula glutinis fermentation using monosodium glutamate wastewater as culture medium.

Microbial lipid, as a raw material for biodiesel, can be produced by Rhodotorula glutinis with the monosodium glutamate (MSG) wastewater. The effect of adding glucose to MSG wastewater on lipid production was studied in this paper. Three different strategies, including initial addition, fed-batch addition and glucose feedback addition were attempted. The results show that addition of glucose was found favorable not only for cell growth but also for lipid synthesis. Of the three adding methods glucose feedback addition was the most effective one: about 25 g/L of biomass, 20% of lipid content and 45% of COD degradation were obtained respectively. And the components of the resulted lipid using different addition strategies were further studied.

Use of Rhodotorula minuta live cells hosted in water-in-oil macroemulsion for biotrasformation reaction.

A lecithin/water/isooctane water-in-oil (w/o) macroemulsion was used as a host system for biotransformation reactions. In particular, the hydrolytic activity of the yeast Rhodotorula minuta toward (+/-)-succinic acid bis-2-isopropyl-5-methylcyclohexyl ester and p-nitrophenyl butyrate is reported. Evidence that R. minuta entrapped in w/o macroemulsion is able to hydrolyze the p-nitrophenyl butyrate ester is presented. By performing the yeast-catalyzed hydrolysis of (+/-)-succinic acid bis-2-isopropyl-5-methylcyclohexyl ester, the synthesis of (-)-menthol was obtained, indicating that R. minuta retains its high stereoselectivity in the macroemulsion system. In addition, no significant differences were observed among the hydrolysis rates and yields obtained using yeast cells hosted into w/o macroemulsions containing different amounts of water. Optical microscopy studies on the distribution of diameters of the dispersed phase indicate that the macroemulsion system is stable in terms of polydispersity. The diameter of the w/o macroemulsion droplets is indeed constant irrespective of the addition of water and/or chemicals (involved in the biotransformation reaction) to the system hosting yeast cells. The reactor devised here might be applied to other interesting bioconversion processes.