Oleaginous Yeast Trichosporon Cutaneum Research Articles

Simultaneous enhancement of lignin-derived inhibitor tolerance and lipid accumulation of oleaginous yeast Trichosporon cutaneum by adaptive evolution

Inhibitory compounds should be removed from the pretreated lignocellulose feedstock before fermentative production of microbial lipid by oleaginous yeast. Biodetoxification effectively degrades furan aldehydes and weak organic acids, but is less efficient against complex and various lignin-derived phenolic aldehydes. Residual phenolics in lignocellulose hydrolysates require a tolerant fermentation strain. This study adopted the long-term adaptive evolution combined with centrifugal enrichment in corn stover hydrolysate to simultaneously improve the phenolic inhibitors tolerance and lipid synthesis of Trichosporon cutaneum. The obtained mutant T. cutaneum MS28 showed significant improved performances of phenolic aldehydes tolerance and lipid accumulation. The final cellulosic lipid production of T. cutaneum MS28 utilizing corn stover reached 33.8 ± 0.1 g/L, approximately 6-fold higher than that of the starting strain. Transcriptional analysis suggested that T. cutaneum MS28 had more active lignocellulose-derived sugars metabolism and lipid synthesis shunts. The intracellular contents of lipid synthesis precursors including NADPH and acetyl-CoA increased 1.7–3.0 folds. This adaptive evolution in lignocellulose hydrolysate combined with centrifugal enrichment is an efficient tool to targeted enhancement of inhibitors tolerance and cellulosic lipid production of oleaginous yeast.

Coproduction of single cell protein and lipid from lignocellulose derived carbohydrates and inorganic ammonia salt with soluble ammonia recycling

Co-production of single cell protein (SCP) and lipid from lignocellulose-derived carbohydrates and inorganic ammonia offers a promising alternative for poultry or aquaculture feeds. An engineered oleaginous yeast Trichosporon cutaneum MP11 showed great potential for producing SCP and lipid from wheat straw and ammonia sulfate with minimum nutrient input. Trichosporon cutaneum MP11 showed stronger SCP and lipid fermentability using dry acid pretreated and biodetoxified wheat straw than using pure sugars. The residual ammonium sulfate in fermentation broth was recycled up to five times, resulting in ∼70% of nitrogen fixation into SCP. The overall yield of SCP and lipid from lignocellulose-derived sugars was 0.15 g/g and 0.11 g/g, respectively. This translates to the production of one ton of SCP (0.56 ton) and lipid (0.44 ton) from 6.6 tons of wheat straw, or one ton of SCP and lipid containing yeast cells (dry) from 4.8 tons of wheat straw.

Cellulase mediated stress triggers the mutations of oleaginous yeast Trichosporon cutaneum with super-large spindle morphology and high lipid accumulation.

Accumulation of intracellular lipid bodies in oleaginous yeast cells is highly restricted by their natural intracellular space. Here we show a cellulase mediated adaptive evolution with ultra-centrifugation fractionation of oleaginous yeast Trichosporon cutaneum to obtain the favorable cell structure for lipid accumulation. Cellulase was added to the wheat straw hydrolysate during long-term adaptive evolution for disruption of cell wall integrity of T. cutaneum cells. The cellulase, together with ultracentrifugation force, triggered multiple mutations and transcriptional expression changes of the functional genes associated with cell wall integrity and lipid synthesis metabolism. The fractionated mutant T. cutaneum YY52 demonstrated the heavily weakened cell wall and high lipid accumulation by the super-large expanded spindle cells (two orders of magnitude greater than the parental). A record-high lipid production by T. cutaneum YY52 was achieved (55.4±0.5gL-1 from wheat straw and 58.4±0.1gL-1 from corn stover). This study not only obtained an oleaginous yeast strain with industrial application potential for lipid production but also provided a new method for generation of mutant cells with high intracellular metabolite accumulation.

Microbial Lipid Production from High Concentration of Volatile Fatty Acids via Trichosporon cutaneum for Biodiesel Preparation.

Direct bioconversion of high concentration of volatile fatty acids (VFAs) into microbial lipid is challenging due to the aggravated cytotoxicity of VFAs at high loadings. Herein, a robust oleaginous yeast Trichosporon cutaneum was screened for lipogenesis from high concentration of VFAs using a regular batch culture. Biomass and lipid content of 8.9g/L and 49.1%, respectively, were attained from 50g/L acetic acid with 90.9% of which assimilated within 10days. The blend of VFAs (50g/L), with mass ratio of acetic, propionic, and butyric acids of 6:3:1, was found superior to acetic acid for lipogenesis. Biomass and lipid titer increased by 16.9% and 18.2%, respectively, with the three VFAs completely consumed within 8days. Butyric acid was assimilated simultaneously with acetic acid at the beginning of the culture. Heptadecanoic acid (C17:0) and heptadecenoic acid (C17:1) were produced when propionic acid co-existed with acetic and butyric acids. The estimation of biodiesel properties indicated that lipid prepared from VFA blend showed superiority to acetic acid for high-quality biodiesel production. This study strongly supported that T. cutaneum permitted high concentration of VFA mixture for lipid production.

Microbial lipid fermentation of Trichosporon cutaneum in high saline water

Fermentative production of microbial lipid requires high fresh water input. The utilization of high saline seawater or industrial wastewater is an important alternative to reduce the freshwater consumption. This study revealed that oleaginous yeast Trichosporon cutaneum was tolerant to a high salinity up to 130 g/L of NaCl after long-term adaptive evolution. Lipid fermentation of T. cutaneum in seawater achieved the lipid production of 31.7 g/L with approximately 36% greater than that in freshwater. The saline water containing phenol was also tested for lipid fermentation and 23.6 g/L of lipid was produced simultaneously with the complete biodegradation of phenol. An interesting phenomenon was also observed that the yeast cells spontaneously segregated onto the upper surface of the saline water. This study extended the lipid fermentation options with practical application potentials.

Efficient bioconversion from acid hydrolysate of waste oleaginous yeast biomass after microbial oil extraction to bacterial cellulose by Komagataeibacter xylinus

ABSTRACTBiomass acid hydrolysate of oleaginous yeast Trichosporon cutaneum after microbial oil extraction was applied as substrate for bacterial cellulose (BC) production by Komagataeibacter xylinus (also named as Gluconacetobacter xylinus previously) for the first time. BC was synthesized in static culture for 10 days, and the maximum BC yield (2.9 g/L) was got at the 4th day of fermentation. Most carbon sources in the substrate (glucose, mannose, formic acid, acetic acid) can be utilized by K. xylinus. The highest chemical oxygen demand (COD) removal (40.7 ± 3.0%) was obtained at the 6th day of fermentation, and then the COD increased possibly due to the degradation of BC. The highest BC yield on COD consumption was 38.7 ± 4.0% (w/w), suggesting that this is one efficient bioconversion for BC production. The BC structure was affected little by the substrate by comparison with that generated in classical HS medium using field-emission scanning electron microscope (FE-SEM), Fourier transform infrared, and X-ray diffraction. Overall, this technology can both solve the issue of waste oleaginous yeast biomass and produce valuable biopolymer (BC).

Lumping kinetics of ABE fermentation wastewater treatment by oleaginous yeast Trichosporon cutaneum

ABSTRACTLumping kinetics models were built for the biological treatment of acetone–butanol–ethanol (ABE) fermentation wastewater by oleaginous yeast Trichosporon cutaneum with different fermentation temperatures. Compared with high temperature (33°C, 306 K) and low temperature (23°C, 296 K), medium temperature (28°C, 301 K) was beneficial for the cell growth and chemical oxygen demand (COD) degradation during the early stage of fermentation but the final yeast biomass and COD removal were influenced little. By lumping method, the materials in the bioconversion network were divided into five lumps (COD, lipid, polysaccharide, other intracellular products, other extracellular products), and the nine rate constants (k1–k9) for the models can well explain the bioconversion laws. The Gibbs free energy (G) for this bioconversion was positive, showing that it cannot happen spontaneous, but the existence of yeast can after the chemical equilibrium and make the bioconversion to be possible. Overall, the possibility of using lumping kinetics for elucidating the laws of materials conversion in the biological treatment of ABE fermentation wastewater by T. cutaneum has been initially proved and this method has great potential for further application.

Converting Chemical Oxygen Demand (COD) of Cellulosic Ethanol Fermentation Wastewater into Microbial Lipid by Oleaginous Yeast Trichosporon cutaneum.

Cellulosic ethanol fermentation wastewater is the stillage stream of distillation column of cellulosic ethanol fermentation broth with high chemical oxygen demand (COD). The COD is required to reduce before the wastewater is released or recycled. Without any pretreatment nor external nutrients, the cellulosic ethanol fermentation wastewater bioconversion by Trichosporon cutaneum ACCC 20271 was carried out for the first time. The major components of the wastewater including glucose, xylose, acetic acid, ethanol, and partial of phenolic compounds could be utilized by T. cutaneum ACCC 20271. In a 3-L bioreactor, 2.16g/L of microbial lipid accumulated with 55.05% of COD reduced after a 5-day culture of T. cutaneum ACCC 20271 in the wastewater. The fatty acid composition of the derived microbial lipid was similar with vegetable oil, in which it could be used as biodiesel production feedstock. This study will both solve the environmental problem and offer low-cost lipid feedstock for biodiesel production.

Use of elephant grass (Pennisetum purpureum) acid hydrolysate for microbial oil production by Trichosporon cutaneum

ABSTRACTElephant grass (Pennisetum purpureum) dilute acid hydrolysate contains 34.6 g/L total sugars. The potential of lipid production by oleaginous yeast Trichosporon cutaneum grown on elephant grass acid hydrolysate was investigated for the first time. During the fermentation process on the elephant grass acid hydrolysate, glucose, xylose, and arabinose could be well utilized as carbon sources by T. cutaneum. Interestingly, xylose was almost no use before glucose was consumed completely. This illustrated that simultaneous saccharification of xylose and glucose by T. cutaneum did not occur on elephant grass acid hydrolysate. The highest biomass, lipid content, lipid yield, and lipid coefficient of T. cutaneum were measured after the sixth day of fermentation and were 22.76 g/L, 24.0%, 5.46 g/L, and 16.1%, respectively. Therefore, elephant grass is a promising raw material for microbial oil production by T. cutaneum.

Inhibitor degradation and lipid accumulation potentials of oleaginous yeast Trichosporon cutaneum using lignocellulose feedstock

Oleaginous yeast Trichosporon cutaneum is robust to high levels of lignocellulose derived inhibitor compounds with considerable lipid accumulation capacity. The potential of lipid accumulation of T. cutaneum ACCC 20271 was investigated using corn stover hydrolysates with varying sugar and inhibitor concentrations. Biodiesel was synthesized using the extracted lipid and the product satisfied the ASTM standards. Among the typical inhibitors, T. cutaneum ACCC 20271 is relatively sensitive to furfural and 4-hydroxybenzaldehyde, but strongly tolerant to high titers of formic acid, acetic acid, levulinic acid, HMF, vanillin, and syringaldehyde. It is capable of complete degradation of formic acid, acetic acid, vanillin and 4-hydroxybenzaldehyde. Finally, the inhibitor degradation pathways of T. cutaneum ACCC 20271 were constructed based on the newly sequenced whole genome information and the experimental results. The study provided the first insight to the inhibitor degradation of T. cutaneum and demonstrated the potentials of lipid production from lignocellulose.

Genome sequence of Trichosporon cutaneum ACCC 20271: An oleaginous yeast with excellent lignocellulose derived inhibitor tolerance

Oleaginous yeast Trichosporon cutaneum demonstrated excellent lipid accumulation performance and inhibitor tolerance derived from lignocellulose pretreatment. Here we firstly report a 30.45Mb assembly genome of T. cutaneum ACCC 20271 for understanding the microbial lipid biosynthesis and mechanism of inhibitor tolerance and degradation.

Characterization of Inulin Hydrolyzing Enzyme(s) in Oleaginous Yeast Trichosporon cutaneum in Consolidated Bioprocessing of Microbial Lipid Fermentation.

Oleaginous yeast Trichosporon cutaneum CGMCC 2.1374 was found to utilize inulin directly for microbial lipid fermentation without a hydrolysis step. The potential inulinase-like enzyme(s) in T. cutaneum CGMCC 2.1374 were characterized and compared with other inulinase enzymes produced by varied yeast strains. The consolidated bioprocessing (CBP) for lipid accumulated using inulin was optimized with 4.79 g/L of lipid produced from 50 g/L inulin with the lipid content of 33.6% in dry cells. The molecular weight of the enzyme was measured which was close to invertase in Saccharomyces cerevisiae. The study provided information for inulin hydrolyzing enzyme(s) in oleaginous yeasts, as well as a preliminary CBP process for lipid production from inulin feedstock.

Acetone-Butanol-Ethanol (ABE) Fermentation Wastewater Treatment by Oleaginous Yeast Trichosporon cutaneum.

In the present study, acetone-butanol-ethanol (ABE) fermentation wastewater with high chemical oxygen demand (COD) value (about 18,000 mg/L) was biologically treated by oleaginous yeast Trichosporon cutaneum without any pretreatment. During fermentation, most COD degradation was finished within 48 h and finally, a maximum COD degradation of 68% was obtained. The highest biomass and lipid content was 4.9 g/L and 14.7%, respectively. Various materials including sugars (glucose and xylose), organic acids (acetic acid and butyric acid), and alcohol compounds (ethanol and butanol) could be utilized as carbon sources by T. cutaneum simultaneously; thus, it has a broad carbon source spectrum and is a potential microorganism for biological treatment for various wastewaters. Overall, the lipid composition of microbial oils produced by this bioconversion is similar to that of vegetable oils, and thus, it could be used for biodiesel production.

Lipid fermentation of corncob residues hydrolysate by oleaginous yeast Trichosporon cutaneum

Corncob residues (CCR) are cellulose residues of corncob after xylan (hemicellulose) is extracted for production of xylitol. Here, an oleaginous yeast Trichosporon cutaneum ACCC 20271 was screened for lipid fermentation using CCR hydrolysate. The initial carbon-to-nitrogen molar ratio (C/N ratio) and the initial sugar concentration of the CCR hydrolysate were investigated in the lipid fermentation of T. cutaneum ACCC 20271. A C/N ratio gradient was generated by changing the corn steep liquor (CSL) addition and an optimal C/N ratio of 49.3 was obtained. The different initial sugar concentration was obtained by changing the cellulase amount and the lipid titer was enhanced by the increased sugar concentration. To our knowledge, this is the first report on using CCR as the feedstock for lipid fermentation. The lipid titer of 12.3g/L and dry cell weight (DCW) of 38.4 g/L were the highest values among the studies using lignocellulose for lipid production.

Evaluating the effect of medium composition and fermentation condition on the microbial oil production by Trichosporon cutaneum on corncob acid hydrolysate

The effect of medium composition and cultural condition on the growth and lipid accumulation of oleaginous yeast Trichosporon cutaneum on corncob acid hydrolysate was systematically investigated. Glucose, xylose, and cellobiose were shown to be promising sugar for lipid production by T. cutaneum. Adding other nitrogen sources into the hydrolysate was not beneficial for the lipid production possibly due to the existence of other nitrogen sources in it. Interestingly, adding MgSO4·7H2O, CuSO4·5H2O, MnSO4·H2O, and KCl (optimal concentration were 0.3, 3.0×10(-3), 3.0×10(-3), and 0.4 g/L, respectively) could stimulate the lipid production by T. cutaneum. Additionally, inoculum concentration, temperature, and initial pH (optimal value were 5%, 28 °C, and 6.0, respectively) showed influence on the lipid production of T. cutaneum. Under the optimum conditions, the biomass (22.9 g/L) had a weak increase (3.6%), while the lipid content (45.4%) and lipid coefficient (22.9%) increased obviously (about 26.5% and 31.6%) compared with the initial conditions.

Analysis of metabolic fluxes for better understanding of mechanisms related to lipid accumulation in oleaginous yeast Trichosporon cutaneum

Microbial fermentation for producing biodiesel from lignocellulosic hydrolysates is receiving increasing attention and attempts have been made to screen an oleaginous Trichosporon sp. with high lipid content and a strong tolerance to lignocellulose hydrolysates. In order to better understand mechanisms related to its lipid accumulation, metabolic flux analysis was performed under 5gL−1 ammonium sulfate (high nitrogen) and/or 0.4gL−1 ammonium sulfate (low nitrogen) conditions. Cell growth phase and lipid accumulation phase were shown for cells grown under low nitrogen condition. Results of flux distribution demonstrated that NADPH provided by cytosolic malic enzyme and the acetyl-CoA from cytoplasmic citrate by the ATP: citrate lyase were the two primary sources for excess lipid accumulation. Flux data also supported the fact that the citrate pyruvate cycle plays an essential role in the lipid accumulation. The flux information obtained could also motivate new design strategies for oleaginous yeasts for enhanced biodiesel production.

Repeated batch fermentation with water recycling and cell separation for microbial lipid production

Large waste water disposal was the major problem in microbial lipid fermentation because of low yield of lipid. In this study, the repeated batch fermentation was investigated for reducing waste water generated in the lipid fermentation of an oleaginous yeast Trichosporon cutaneum CX1 strain. The waste fermentation broth was recycled in the next batch operation after the cells were separated using two different methods, centrifugation and flocculation. Two different sugar substrates, glucose and inulin, were applied to the proposed operation. The result showed that at least 70% of the waste water was reduced, while lipid production maintained satisfactory in the initial four cycles. Furthermore, it is suggested that T. cutaneum CX1 cells might produce certain naturally occurring inulin hydrolyzing enzyme(s) for obtaining fructose and glucose from inulin directly. Our method provided a practical option for reducing the waste water generated from microbial lipid fermentation.

Simultaneous saccharification and microbial lipid fermentation of corn stover by oleaginous yeast Trichosporon cutaneum

Simultaneous saccharification and fermentation (SSF) is the most commonly practiced operation in lignocellulose bioconversion to avoid the sugar product inhibition to cellulase enzymes. In this study, for the first time SSF was tested on microbial lipid fermentation using the diluted acid pretreated and biodetoxified corn stover. The results show that SSF was effective than the separate hydrolysis and fermentation (SHF) on lipid accumulation of Trichosporon cutaneum CX1 cells in both the small scale (5L) and the enlarged scale (50L) bioreactors. The solutions for the oxygen transfer and the lipid extraction in SSF practically worked well. The process parameters were optimized and the lipid yield obtained were 3.03g/L in the 5L, and 3.23g/L in the 50L, respectively. The result also shows that the cellulase enzyme could be partially recycled in the SSF. The study provided a practical and efficient way for microbial lipid production from lignocellulose material.

Purification and properties of two lactose hydrolases from Trichosporon cutaneum

Two enzymes that hydrolysed lactose were purified essentially to homogeneity from cell extracts of the oleaginous yeast Trichosporon cutaneum. One enzyme of Mr 120,000 had properties typical of a beta-galactosidase (EC 3.2.1.23). It hydrolysed lactose, lactulose and nitrophenyl-beta-D-galactosides. The enzyme required K+ or Rb+ for activity, and other monovalent cations tested were not effective. Enzyme activity was abolished by EDTA and stimulated by Mg2+, Mn2+ and Ca2+. The beta-galactosidase was induced by lactose, galactose, lactulose and lactobionic acid. The other enzyme, a beta-glycosidase (EC 3.2.1.21) of Mr 52,000 showed no ionic requirements and it hydrolysed lactose, nitrophenyl-beta-D-galactosides, 4-nitrophenyl-beta-D-glucoside, cellobiose, laminaribiose, laminaritriose and sophorose, but not gentiobiose, 4-nitrophenyl-beta-D-mannoside or sucrose. This enzyme was induced by lactose, galactose and lactulose, and also by cellobiose.