Potential application values of a marine red yeast, Rhodosporidiums sphaerocarpum YLY01, in aquaculture and tail water treatment assessed by the removal of ammonia nitrogen, the inhibition to Vibrio spp., and nutrient composition.

In recent years, marine red yeasts have been increasingly used as feed diets for larviculture of aquatic animals mainly due to their rich nutrition and immunopotentiation, however little attention is given to their other probiotic profits. In this study, a marine red yeast strain YLY01 was isolated and purified from farming water and it was identified as a member of Rhodosporidiums sphaerocarpum by the phylogeny based on 18S rDNA sequence. The strain YLY01 could effectively remove ammonia nitrogen from an initial 9.8 mg/L to 1.3 mg/L in 48 h when supplemented with slight yeast extract and glucose in water samples and the removal rate of ammonia nitrogen was up to 86%. Shrimps (Litopenaeus vannamei) in experimental group incubated with the yeast YLY01 exhibited a higher survival rate than those in blank control group and positive control group challenged by Vibrio harveyi, and it manifested that the strain has high biosecurity to at least shrimps. The strain YLY01 could inhibit the growth of Vibrio cells when a small quantity of carbon source was added into farming water. In addition, a nutrition composition assay showed the contents of protein, fatty acids, and total carotenoids of the yeast YLY01 were 30.3%, 3.2%, and 1.2 mg/g of dry cell weight, respectively. All these results indicated that the marine red yeast YLY01 has a great potential to be used as a versatile probiotic in aquaculture and to be developed as a microbial agent for high-ammonia tail water treatment.

Biosurfactant production from newly isolated Rhodotorula sp.YBR and its great potential in enhanced removal of hydrocarbons from contaminated soils.

One of the very promising methods in the field of bioremediation of hydrocarbons is the application of biosurfactant- producing microorganisms based on the use of wastewater as renewable substrates of culture media, contributing to the reduction of costs. With this aim, the production, characterization and properties of the yeast strain YBR producing a biosurfactant newly isolated from an oilfield in Algeria, using wastewater from olive oil mills (OOMW) as a substrate for a low-cost and effective production, have been investigated. Screening of biosurfactant production was carried out with different tests, including emulsification index test (E24), drop collapse test, oil spreading technique and measurement of surface tension (ST). The isolated yeast strain was found to be a potent biosurfactant producer with E24 = 69% and a significant reduction in ST from 72 to 35 mN m-1. The study of the cultural, biochemical, physiological and genetic characteristics of the isolate allowed us to identify it as Rhodotorula sp. strain YBR. Fermentation was carried out in a 2.5 L Minifors Bioreactor using crude OOMW as culture medium, the E24 value reached 90% and a reduction of 72 to 35 mN m-1 in ST. A biosurfactant yield = 10.08 ± 0.38 g L-1 was recorded. The characterization by semi-purification and thin layer chromatography (TLC) of the crude extract of biosurfactant showed the presence of peptides, carbohydrates and lipids in its structure. The crude biosurfactant exhibited interesting properties such as: low critical micellar concentration (CMC), significant reduction in ST and strong emulsifying activity. In addition, it has shown stability over a wide range of pH (2-12), temperature (4-100 °C) and salinity (1-10%). More interestingly, the produced biosurfactant has proven to be of great potential application in the remobilization of hydrocarbons from polluted soil with a removal rate of greater than 95%.

Overexpression and repression of key rate-limiting enzymes (acetyl CoA carboxylase and HMG reductase) to enhance fatty acid production from Rhodotorula mucilaginosa.

Implementing two-way strategies to enhance the lipid production in Rhodotorula mucilaginosa with the help of metabolic engineering was focused on the overexpression of acetyl coenzyme A carboxylase (ACC1 carboxylase) gene and repression of 3-hydroxy 3-methylglutaryl reductase (HMG-CoA reductase). Using an inducer (sodium citrate) and inhibitor (rosuvastatin), the amounts of biomass, lipid, and carotenoid were estimated. In the presence of inhibitor (200 mM), 62% higher lipid concentration was observed, while 44% enhancement was recorded when inducer (3 mM) was used. A combination of both inhibitor and inducer resulted in a 57% increase in lipid concentration by the oleaginous yeast. These results were again confirmed by real-time polymerase chain reaction by targeting the expression of the genes coding for ACC1 carboxylase and 13-fold increase was recorded in the presence of inducer as compared with control. This combined strategy (inducer and inhibitor use) has been reported for the first time as far as the best of our knowledge. The metabolic engineering strategies reported here will be a powerful approach for the enhanced commercial production of lipids.

Immobilization of Laccase on Magnetic Nanoparticles and Application in the Detoxification of Rice Straw Hydrolysate for the Lipid Production of Rhodotorula glutinis.

The production of microbial lipid using lignocellulosic agroforestry residues has attracted much attention. But, various inhibitors such as phenols and furans, which are produced during lignocellulosic hydrolysate preparation, are harmful to microbial lipid accumulation. Herein, we developed a novel detoxification strategy of rice straw hydrolysate using immobilized laccase on magnetic Fe3O4 nanoparticles for improving lipid production of Rhodotorula glutinis. Compared with free laccase, the immobilized laccase on magnetic nanoparticles showed better stability, which still retained 76% of original activity at 70 °C and 56% at pH 2 for 6 h. This immobilized laccase was reused to remove inhibitors in acid-pretreated rice straw hydrolysate through recycling with external magnetic field. The results showed that most of phenols, parts of furans, and formic acids could be removed by immobilized laccase after the first batch. Notably, the immobilized laccase exhibited good reusability in repeated batch detoxification. 78.2% phenols, 43.8% furfural, 30.4% HMF, and 16.5% formic acid in the hydrolysate were removed after the fourth batch. Furthermore, these detoxified rice straw hydrolysates, as substrates, were applied to the lipid production of Rhodotorula glutinis. The lipid yield in detoxified hydrolysate was significantly higher than that in undetoxified hydrolysate. These findings suggest that the immobilized laccase on magnetic nanoparticles has a potential to detoxify lignocellusic hydrolysate for improving microbial lipid production.

Marine Protists and Rhodotorula Yeast as Bio-Convertors of Marine Waste into Nutrient-Rich Deposits for Mangrove Ecosystems.

This paper represents a comprehensive study of two new thraustochytrids and a marine Rhodotorula red yeast isolated from Australian coastal waters for their abilities to be a potential renewable feedstock for the nutraceutical, food, fishery and bioenergy industries. Mixotrophic growth of these species was assessed in the presence of different carbon sources: glycerol, glucose, fructose, galactose, xylose, and sucrose, starch, cellulose, malt extract, and potato peels. Up to 14g DW/L (4.6gDW/L-day and 2.8gDW/L-day) of biomass were produced by Aurantiochytrium and Thraustochytrium species, respectively. Thraustochytrids biomass contained up to 33% DW of lipids, rich in omega-3 polyunsaturated docosahexaenoic acid (C22:6, 124mg/g DW); up to 10.2mg/gDW of squalene and up to 61µg/gDW of total carotenoids, composed of astaxanthin, canthaxanthin, echinenone, and ß-carotene. Along with the accumulation of these added-value chemicals in biomass, thraustochytrid representatives showed the ability to secrete extracellular polysaccharide matrixes containing lipids and proteins. Rhodotorula sp lipids (26% DW) were enriched in palmitic acid (C16:0, 18mg/gDW) and oleic acid (C18:1, 41mg/gDW). Carotenoids (87µg/gDW) were mainly represented by ß-carotene (up to 54µg/gDW). Efficient growth on organic and inorganic sources of carbon and nitrogen from natural and anthropogenic wastewater pollutants along with intracellular and extracellular production of valuable nutrients makes the production of valuable chemicals from isolated species economical and sustainable.

Microbial lipid production by oleaginous yeasts grown on Scenedesmus obtusiusculus microalgae biomass hydrolysate.

Due to increasing oil prices and climate change concerns, biofuels have become increasingly important as potential alternative energy sources. However, the use of arable lands and valuable resources for the production of biofuel feedstock compromises food security and negatively affect the environment. Single cell oils (SCOs), accumulated by oleaginous yeasts, show great promise for efficient production of biofuels. However, the high production costs attributed to feedstocks or raw materials present a major limiting factor. The fermentative conversion of abundant, low-value biomass into microbial oil would alleviate this limitation. Here, we explore the feasibility of utilizing microalgae-based cell residues as feedstock for yeast oil production. We developed an efficient, single-step enzymatic hydrolysis to generate Scenedesmus obtusiusculus hydrolysate (SH) without thermo-chemical pretreatment. With this eco-friendly process, glucose conversion efficiencies reached 90-100%. Cutaneotrichosporon oleaginosus, Cryptococcus curvatus and Rhodosporidium toruloides were cultivated on SH as sole nutrients source. Only C. oleaginosus was able to accumulate intracellular lipids, with a 35% (g lipid/g DCW) content and a yield of 3.6 g/L. Our results demonstrate the potential valorization of algal biomass into desired end-products such as biofuels.

Reutilization of residual glycerin for the produce ß-carotene by Rhodotorula minuta.

This study aimed to evaluate the production of carotenoid pigments by Rhodotorula spp. in submerged fermentation, using residual glycerin from biodiesel production as a carbon source. Chromatographic analysis by HPLC showed that the residual glycerin used as substrate was 57.88% composed of glycerol. The best growth conditions were found in the fermentation medium composed of residual glycerin at a concentration of 30 g/L and pH 9. From all the Rhodotorula strains tested, R. minuta URM6693 was selected because of their performance and adaptation in all culture media assayed. The maximum volumetric production of carotenoids was found at 48 h (equivalent to 17.20 mg/L, for the R. minuta). The production of ß-carotene since the first 24 h of fermentation reach a final concentration of 1.021 mg/L. The yeast Rhodotorula minuta proved its capability to efficiently convert the substrate (mainly at the concentration of 50 g/L), obtaining products of biotechnological interest.

Fatty acids profiles and estimation of the biodiesel quality parameters from Rhodotorula spp. from Antarctica.

OBJECTIVE: Oleaginous yeasts are a renewable and alternative source of oil for third-generation biodiesel. This work aimed to evaluate the effects of glucose concentration (30-100 g L-1) on growth, lipid synthesis, and fatty acids (FA) profile of three Rhodotorula spp. (R. glacialis R15, R. glutinis R4, and R. glutinis R48) isolated from Antarctica, and estimate the key quality parameters of the biodiesel produced by yeasts to confirm their potential as feedstocks for third-generation biodiesel synthesis. RESULTS: Yeasts accumulated 50-69.5% of lipids (w/w) under nitrogen-limitation and glucose-excess (C/N = 40-133). Glucose concentration increase influenced positively lipid accumulation (69.5% w/w) and FA profile of R. glacialis R15. Lipid accumulation (53% on average) of R. glutinis strains was not significantly affected by glucose concentration; content of saturated (~ 30%) and polyunsaturated FA (~ 29-30%) was slightly influenced. FA profiles of lipids synthesized by R15, R4, and R48 are similar to vegetable oils used in biodiesel industry with C16 and C18 FA (95-99%) as the major components, and contain mainly oleic (C18:1), palmitic (C16:0), and linoleic (C18:2) acids, which are suitable for biodiesel synthesis. Estimated fuel properties for biodiesel produced by R15, R4, and R48 satisfied all the criteria established by ASTM D6751 and EN 14214 with good cetane number, iodine value, and oxidation stability. An improvement in biodiesel quality of R15 was observed with the glucose increase. The best global properties of biodiesel from R4 were obtained with 30 g L-1 of glucose. CONCLUSIONS: Rhodotorula spp. from Antarctica are promising candidates for third-generation biodiesel synthesis.

Evaluation of seven chemical pesticides by mixed microbial culture (PCS-1): Degradation ability, microbial community, and Medicago sativa phytotoxicity.

Environmental problems caused by the large-scale use of chemical pesticides are becoming more and more serious, and the removal of chemical pesticides from the ecological environment by microbial degradation has attracted wide attention. In this study, using enrichment screening with seven chemical pesticides as the sole carbon source, a mixed microbial culture (PCS-1) was obtained from the continuous cropping of strawberry fields. The microbial community composition, degradation ability, and detoxification effect of PCS-1 was determined for the seven pesticides. Inoculation with PCS-1 showed significant degradation of and tolerance to the seven pesticides. Microbial community composition analysis indicated that Pseudomonas, Enterobacter, Aspergillus, and Rhodotorula were the dominant genera for the degradation of the seven pesticides by PCS-1. The concentration of the seven pesticides was 10 mg L-1 in hydroponic and soil culture experiments. The fresh weight, plant height, and root length of PCS-1-inoculated alfalfa (Medicago sativa) significantly increased compared with those of non-PCS-1-inoculated M. sativa. PCS-1 not only effectively degraded the residual content of the seven pesticides in water and soil but also reduced the pesticide residues in the roots, stems, and leaves of M. sativa. This study shows that PCS-1 may be important in environmental remediation involving the seven pesticides.

A Tripartite Interaction among the Basidiomycete Rhodotorula mucilaginosa, N2-Fixing Endobacteria, and Rice Improves Plant Nitrogen Nutrition.

Nitrogen (N) limits crop yield, and improvement of N nutrition remains a key goal for crop research; one approach to improve N nutrition is identifying plant-interacting, N2-fixing microbes. Rhodotorula mucilaginosa JGTA-S1 is a basidiomycetous yeast endophyte of narrowleaf cattail (Typha angustifolia). JGTA-S1 could not convert nitrate or nitrite to ammonium but harbors diazotrophic (N2-fixing) endobacteria (Pseudomonas stutzeri) that allow JGTA-S1 to fix N2 and grow in a N-free environment; moreover, P. stutzeri dinitrogen reductase was transcribed in JGTA-S1 even under adequate N. Endobacteria-deficient JGTA-S1 had reduced fitness, which was restored by reintroducing P. stutzeri JGTA-S1 colonizes rice (Oryza sativa), significantly improving its growth, N content, and relative N-use efficiency. Endofungal P. stutzeri plays a significant role in increasing the biomass and ammonium content of rice treated with JGTA-S1; also, JGTA-S1 has better N2-fixing ability than free-living P. stutzeri and provides fixed N to the plant. Genes involved in N metabolism, N transporters, and NODULE INCEPTION-like transcription factors were upregulated in rice roots within 24 h of JGTA-S1 treatment. In association with rice, JGTA-S1 has a filamentous phase and P. stutzeri only penetrated filamentous JGTA-S1. Together, these results demonstrate an interkingdom interaction that improves rice N nutrition.

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.

Allium sativum Extract Chemical Composition, Antioxidant Activity and Antifungal Effect against Meyerozyma guilliermondii and Rhodotorula mucilaginosa Causing Onychomycosis.

Onychomycosis is a major health problem due to its chronicity and resistance to therapy. Because some cases associate paronychia, any therapy must target the fungus and the inflammation. Medicinal plants represent an alternative for onychomycosis control. In the present work the antifungal and antioxidant activities of Alium sativum extract against Meyerozyma guilliermondii (Wick.) Kurtzman & M. Suzuki and Rhodotorula mucilaginosa (A. Jörg.) F.C. Harrison, isolated for the first time from a toenail onychomycosis case, were investigated. The fungal species were confirmed by DNA molecular analysis. A. sativum minimum inhibitory concentration (MIC) and ultrastructural effects were examined. At the MIC concentration (120 mg/mL) the micrographs indicated severe structural alterations with cell death. The antioxidant properties of the A. sativum extract were evaluated is a rat turpentine oil induced inflammation, and compared to an anti-inflammatory drug, diclofenac, and the main compound from the extract, allicin. A. sativum reduced serum total oxidative status, malondialdehyde and nitric oxide production, and increased total thiols. The effects were comparable to those of allicin and diclofenac. In conclusion, the garlic extract had antifungal effects against M. guilliermondii and R. mucilaginosa, and antioxidant effect in turpentine-induced inflammation. Together, the antifungal and antioxidant activities support that A. sativum is a potential alternative treatment in onychomycosis.

Effect of exogenous stress factors on the biosynthesis of carotenoids and lipids by Rhodotorula yeast strains in media containing agro-industrial waste.

In this study, we aimed to determine the effect of exogenous stress factors (sodium chloride as osmotic stressor, hydrogen peroxide as an inducer of oxidative stress, white light irradiation, and low temperature) on the biosynthesis of carotenoids and lipids by red yeast (Rhodotorula glutinis, R. mucilaginosa, and R. gracilis) during cultivation in media containing potato wastewater and glycerol. According to our results, the yeast were able to grow and biosynthesize lipids and carotenoids in the presence of the applied stress factors. Low temperature caused an increase in the biosynthesis of intracellular lipids and carotenoids. R. gracilis synthesized lipids (21.1 g/100 gd.w.) and carotenoids (360.4 µg/gd.w.) in greater quantities than that of other strains. Under these conditions, there was also an increase in the content of unsaturated fatty acids, especially linoleic and linolenic acids. The highest percentage of polyunsaturated fatty acid (PUFA) (30.4%) was synthesized by the R. gracilis yeast after cultivation at 20°C. Their quantity was 2.5-fold greater than that of the biomass grown in control conditions. The contribution of individual carotenoid fractions depended both on the yeast strain and the culture conditions. Induction of osmotic stress and low temperature intensified the biosynthesis of ß-carotene (up to 73.9% of the total carotenoid content). In oxidative stress conditions, yeast synthesized torulene (up to 82.2%) more efficiently than under other conditions, whereas white light irradiation increased the production of torularhodin (up to 20.0%).

Simultaneous Production of Lipids and Carotenoids by the Red Yeast Rhodotorula from Waste Glycerol Fraction and Potato Wastewater.

AbstractThe objective of this study was to determine the possibility of simultaneous biosynthesis of lipids and carotenoids by the Rhodotorula yeast strains in media with waste glycerol and deproteinized potato wastewater and to determine the level of pollution reduction by media. On the basis of results obtained during the yeast microcultures in the Bioscreen C system, it was found that potato wastewater and glycerol can be used as components of media for Rhodotorula glutinis, Rhodotorula mucilaginosa, and Rhodotorula gracilis yeast strains. The amount of glycerol added to media higher than 10% significantly decreased the growth rate of yeast. The results of yeast culture in the laboratory shaker flasks showed a possibility of simultaneous production of lipids and carotenoids by R. glutinis, R. mucilaginosa, and R. gracilis yeast strains during cultivation in media containing only waste glycerol and deproteinized potato wastewater. A higher intracellular lipid content (approximately 15 g/100 gd.w.) was obtained for R. mucilaginosa and R. gracilis yeast biomass after cultivation in experimental media with waste glycerol and potato wastewater. In conclusion, the yeast grown in media with potato wastewater supplemented with 3% or 5% glycerol synthesized carotenoids, and their content in biomass did not exceed 230 µg/gd.w.

Simultaneous mitigation of aluminum, salinity and drought stress in Lactuca sativa growth via formulated plant growth promoting Rhodotorula mucilaginosa CAM4.

In the present study, a potent Aluminum (Al) resistant yeast strain CAM4 was isolated from rhizosphere soil of Rubus geoides, grown in acidic Andisols and identified as Rhodotorula mucilaginosa by 18S rRNA gene sequence analysis. The strain CAM4 was selected in terms of abiotic stress tolerance to Al, salinity and drought with multiple plant growth promoting (PGP) traits. Besides, strain CAM4 also exhibited Al removal efficiency (80-88%) from the culture medium even under combined stresses of salinity and drought. The sawdust-based formulation of strain CAM4 (sawdust-molasses 5%-PEG 1%-strain CAM4) showed higher cell viability of up to 24 weeks (8.54 log CFU g-1). Inoculation of formulated strain CAM4 significantly enhanced the various morphological and biochemical characters of Lactuca sativa grown under abiotic stress conditions. The formulated strain CAM4 also reduced the accumulation of Al in L. sativa as well that conferring Al tolerance to the plants. The study concludes that strain CAM4 could be used as a biofertilizer for healthy and safe crop production in soils, with Al toxicity as well as combined salt and drought stresses.

Biochemical changes of polysaccharides and proteins within EPS under Pb(II) stress in Rhodotorula mucilaginosa.

Microorganisms have been widely applied to heavy metal adsorption due to their strong secretion of extracellular polymeric substances (EPS). This study explored the responses of Rhodotorula mucilaginosa (R1, a red yeast with substantial EPS supply) under Pb stress. The maximum sorption of Pb cations by R1 was ~650 mg/L. In particular, despite the declined microbial biomass, the total Pb sorption after incubation was actually elevated in the solution with high Pb concentration. At 0-1000 mg/L Pb(NO3)2 level, the longitudinal sizes of the yeast capsules increased from 2.04 to 2.90 µm. At 1500 mg/L, however, the survived yeast started to lose the membrane integrity of the cells. Meanwhile, the percentages of organic carbon contents of EPS decreased from 40% to 33% when the Pb(NO3)2 concentration raised to 2500 mg/L, confirming the incorporation of Pb2+ cations into the fungal EPS during the sorption. For the survived R1 cells, function of polysaccharides to resist Pb toxicity only worked at extremely high Pb(NO3)2 levels (>= 1500 mg/L). In contrast, proteins showed continuously enhanced ability to resist Pb toxicity, consistent with their increasing content (per cell) in the EPS. Moreover, ATR-IR spectra showed that the intensity of amide II peak at 1540 cm-1 was significantly increased, indicating elevated glutathione (GSH) in EPS. This suggested that GSH could be the critical Pb-binding component in EPS proteins. This study hence elucidated roles of polysaccharides and proteins in EPS under the toxicity caused by heavy metals.

Multi-omics metabolism analysis on irradiation-induced oxidative stress to Rhodotorula glutinis.

Oxidative stress is induced in many organisms by various natural abiotic factors including irradiation. It has been demonstrated that it significantly improves growth rate and lipid production of Rhodotorula glutinis. However, the specific mechanism of how irradiation influences the metabolism of R. glutinis remains still unavailable. To investigate and better understand the mechanisms involved in irradiation-induced stress resistance in R. glutinis, a multi-omics metabolism analysis was implemented. The results confirmed that irradiation indeed not only improved cell biomass but also accelerated the production of carotenoids and lipids, especially neutral lipid. Compared with the control, metabolome profiling in the group exposed to irradiation exhibited an obvious difference in the activation of the tricarboxylic acid cycle and triglyceride (TAG) production. The results of proteome analysis (data are available via ProteomeXchange with identifier PXD009678) showed that 423 proteins were changed significantly, and proteins associated with protein folding and transport, the Hsp40 and Sec12, were obviously upregulated, indicating that cells responded to irradiation by accelerating the protein folding and transport of correctly folded proteins as well as enhanced the degradation of misfolded proteins. A significant upregulation of the carotenoid biosynthetic pathway was observed which revealed that increased carotenoid content is a cellular defense mechanism against oxidative stress generated by irradiation. Therefore, the results of comprehensive omics analysis provide intensive insights on the response mechanism of R. glutinis to irradiation-induced oxidative stress which could be helpful for using irradiation as an effective strategy to enhance the joint production of the neutral lipid and carotene.

Carotenoid production in sugarcane juice and synthetic media supplemented with nutrients by Rhodotorula rubra l02

ABSTRACT In order for the use of biological carotenoids to become feasible, it is necessary to have adequate low cost sources and improved methods of cultivation. The aim of this study was to evaluate the effect of supplementation with nitrogen, phosphorus, zinc, and magnesium, on the biomass and carotenoid volumetric production by yeast Rhodotorula rubra L02 using a complex medium (sugarcane juice) and synthetic media (sucrose and maltose) as substrates. The experimental design used for each substrate was randomized in blocks with 16 treatments and 3 repetitions. The treatments were compound for 15 different combinations of nutrients (N; Mg; Zn; P, N + Mg; N + Zn; N + P; Mg + Zn; Mg + P; Zn + P; N + P + Zn; N + P + Mg; N + Zn + Mg; P + Zn + Mg; N + Zn + Mg + P) alone and combined, and a control. The results were submitted to analysis of variance and Tukey test at 5% significance level. Among the treatments evaluated, the highest production of dry biomass, with both maltose and sucrose, was observed for Mg (1.60 g/L and 1.94 g/L, respectively). Additionally, another treatment that stood out in terms of biomass production was the control treatment with maltose (1.54 g/L). After the incubation time, killer activity was not observed since there was no formation of inhibition halo around the L02 yeast.

Valorization of Brewers' Spent Grain for the Production of Lipids by Oleaginous Yeast.

Brewers' spent grain (BSG) accounts for 85% of the total amount of by-products generated by the brewing industries. BSG is a lignocellulosic biomass that is rich in proteins, lipids, minerals, and vitamins. In the present study, BSG was subjected to pretreatment by two different methods (microwave assisted alkaline pretreatment and organosolv) and was evaluated for the liberation of glucose and xylose during enzymatic saccharification trials. The highest amount of glucose (46.45 ± 1.43 g/L) and xylose (25.15 ± 1.36 g/L) were observed after enzymatic saccharification of the organosolv pretreated BSG. The glucose and xylose yield for the microwave assisted alkaline pretreated BSG were 34.86 ± 1.27 g/L and 16.54 ± 2.1 g/L, respectively. The hydrolysates from the organosolv pretreated BSG were used as substrate for the cultivation of the oleaginous yeast Rhodosporidium toruloides, aiming to produce microbial lipids. The yeast synthesized as high as 18.44 ± 0.96 g/L of cell dry weight and 10.41 ± 0.34 g/L lipids (lipid content of 56.45 ± 0.76%) when cultivated on BSG hydrolysate with a C/N ratio of 500. The cell dry weight, total lipid concentration and lipid content were higher compared to the results obtained when grown on synthetic media containing glucose, xylose or mixture of glucose and xylose. To the best of our knowledge, this is the first report using hydrolysates of organosolv pretreated BSG for the growth and lipid production of oleaginous yeast in literature. The lipid profile of this oleaginous yeast showed similar fatty acid contents to vegetable oils, which can result in good biodiesel properties of the produced biodiesel.

Engineering the oleaginous red yeast Rhodotorula glutinis for simultaneous ß-carotene and cellulase production.

Rhodotorula glutinis, an oleaginous red yeast, intrinsically produces several bio-products (i.e., lipids, carotenoids and enzymes) and is regarded as a potential host for biorefinery. In view of the limited available genetic engineering tools for this yeast, we have developed a useful genetic transformation method and transformed the ß-carotene biosynthesis genes (crtI, crtE, crtYB and tHMG1) and cellulase genes (CBHI, CBHII, EgI, EgIII, EglA and BGS) into R. glutinis genome. The transformant P4-10-9-63Y-14B produced significantly higher ß-carotene (27.13 ± 0.66 mg/g) than the wild type and also exhibited cellulase activity. Furthermore, the lipid production and salt tolerance ability of the transformants were unaffected. This is the first study to engineer the R. glutinis for simultaneous ß-carotene and cellulase production. As R. glutinis can grow in sea water and can be engineered to utilize the cheaper substrates (i.e. biomass) for the production of biofuels or valuable compounds, it is a promising host for biorefinery.