Oleaginous Yeast Cryptococcus Curvatus Research Articles

Biorefinery of vineyard winter prunings for production of microbial lipids by the oleaginous yeast Cryptococcus curvatus

Spent biomass from agricultural and forestry industries are substantial low-cost carbon source for reducing the input of microbial lipid production. Herein, the components of the vineyard winter prunings (VWPs) from 40 grape cultivars were analyzed. The VWPs contained (w/w) cellulose ranged from 24.8% to 32.4%, hemicellulose 9.6% to 13.8%, lignin 23.7% to 32.4%. The VWPs from Cabernet Sauvignon was processed with the alkali-methanol pretreatment, and 95.8% of the sugars was released from the regenerated VWPs after enzymatic hydrolysis. The hydrolysates from the regenerated VWPs was suitable for lipid production without further treatment as a lipid content of 59% could be achieved with Cryptococcus curvatus. The regenerated VWPs was also used for lipid production via simultaneous saccharification and fermentation (SSF), which led to a lipid yield of 0.088 g/g raw VWPs, 0.126 g/g regenerated VWPs and 0.185 g/g from the reducing sugars. This work demonstrated that the VWPs can be explored for co-production of microbial lipids.

Evaluation and Identification of Key Economic Bottlenecks for Cost-Effective Microbial Oil Production from Fruit and Vegetable Residues

Intensive horticultural systems for the production of vegetables in greenhouses represent one of the main industries generating organic waste, as those that do not meet the quality standards for the fresh market or the processing industry are discarded. This highlights the importance of using these residues as raw material for other applications, such as bioenergy and bioproducts production, within the framework of a bio-based economy that maximizes the utilization of biomass resources in a sustainable manner. In this work, the microbial oil production from discarded pepper using the oleaginous yeast Cryptococcus curvatus was evaluated. Overall, a total lipid accumulation of 16.8 g/L was achieved with a fatty acid profile suitable to produce biodiesel. The lipid yield obtained was 0.12 g/g sugars. In addition, experimental results were used to assess the techno-economic feasibility of a proposed microbial oil plant using the software Aspen Plus. This plant yields approximately 96 kg of microbial oils/ton dry discarded pepper, with an estimated Minimum Selling Price of 7 €·kg−1. These figures point out the necessity of increasing the yield of microbial oil production and considering the utilization of possible by-products, such as mannitol and cell debris, to improve the economic performance of the process.

Conversion of Arthrospira platensis Biomass into Microbial Lipids by the Oleaginous Yeast Cryptococcus curvatus

Conversion of <i>Arthrospira platensis</i> Biomass into Microbial Lipids by the Oleaginous Yeast <i>Cryptococcus curvatus</i>

Co‐utilization of amino acid‐rich wastes and glycerol for microbial lipid production

AbstractMicrobial lipids are considered to be potential raw materials for biodiesel production. However, the costs of such a process remain high due to the use of expensive feedstocks for microbial lipid production. The meat industry and biodiesel refineries generate amino acid (AA) wastes and crude glycerol, respectively, which could be explored as relatively cheap feedstocks for oleaginous yeasts. Unfortunately, AA wastes have a high nitrogen content and crude glycerol contains several impurities, discouraging microbial lipid production. Here, glycerol was co‐used with AA, or with AA blends with compositional profiles matching those of different meats, so that the media had favorable carbon‐to‐nitrogen (C/N) ratios to promote AA conversion into lipids using the oleaginous yeast Cryptococcus curvatus ATCC 20509 under two‐stage culture conditions. Co‐utilization of glycerol and AA resulted in improved lipid production. The highest lipid yield of 20.9 g 100 g–1 was achieved with lysine and glycerol, where the lipid yield was 13.9 g 100 g–1, using an AA blend of similar composition to sheep viscera and glycerol. The lipid products comprised over 93% of 16 and 18 carbon‐containing fatty acids, showing high potential for biodiesel production. The co‐use of glycerol with AA improved AA conversion into lipids while decreasing the inhibitory effects at relatively high AA concentrations. The utilization of crude glycerol and AA‐rich wastes from the meat industry could help both the development of biorefineries and waste minimization. © 2021 Society of Chemical Industry and John Wiley &amp; Sons, Ltd

Automated Pressurized Liquid Extraction of Microbial Lipids from Oleaginous Yeasts

The lipids produced by oleaginous microbes are considered sustainable resources for biofuels. To facilitate controlled lipid production and lipid analysis, more efficient lipid extraction methods are required. This study describes the automated pressurized liquid extraction (APLE) method for lipid extraction from dried cells of the oleaginous yeast species Rhodosporidium toruloides and Cryptococcus curvatus. Cells were mixed with diatomite in a mortar, added to the sample chamber, and treated with a mixture of chloroform and methanol at 105°C. More than 95% lipids were extracted. Analysis by using high-performance thin-layer chromatography showed that the neutral lipid contents in the obtained samples by APLE method were similar to those by the ball milling-assisted extraction method. The lipids had an essentially identical fatty acid composition compared with lipids extracted with the acid-heating extraction (AHE) method. This demonstrated that lipids can be efficiently extracted from oleaginous yeasts in less time and without harsh pretreatment procedures.

Utilization of Microbial Oil from Poplar Wood Hemicellulose Prehydrolysate for the Production of Polyol Using Chemo-enzymatic Epoxidation

The aim of this work was to demonstrate the production of polyol from microbial oil using wood-based hemicellulose stream through a greener enzymatic process. Yeast biomass concentration of 16.54 ± 0.65 g/L and lipid concentration of 6.97 ± 0.58 g/L were obtained by batch fermentation of hemicellulose prehydrolysate of poplar wood using an oleaginous yeast Cryptococcus curvatus. The produced microbial oil was successfully converted to epoxidized oil catalyzed by lipase with 84.55 ± 1.80% conversion. The epoxidation followed by ring opening reaction to produce polyol with hydroxyl value of 299.53 ± 1.24 mg KOH/g. This showed the possible use of the hemicellulose stream of lignocellulosic biomass to microbial oil and its subsequent conversion to polyol, a precursor to polyurethane and a number of value-added products. This bio-based polyol could be used to substitute the conventional polyols.

Utilization of Amino Acid-Rich Wastes for Microbial Lipid Production.

To produce microbial lipids for biofuel production, carbohydrates and related compounds from biomass have been routinely utilized, yet amino acids (AA) from protein-rich wastes have been overlooked so far. We use the oleaginous yeast Cryptococcus curvatus ATCC 20509 as a lipid producer and evaluate the capacity for lipid production on proteinogenic AA individually or in designated blends under two-staged culture conditions. It was found that cellular lipid contents reached 48.8%, 44.5% and 29.0% when yeast cells were cultivated in media-contained AA blends with compositional profiles similar to those of sheep viscera, meat industry by-products and fish muscle, respectively, and that lipid coefficients were more than 0.10gg-1. Furthermore, cellular lipid contents were higher than 20% when most AA were used individually. High lipid coefficients of over 0.23gg-1 were observed when Pro, Trp or Leu were used as a substrate. Results also indicated that higher initial media pH or reduced phosphate concentration was beneficial for lipid production on AA. This work demonstrated the potential to use AA and related wastes as substrates for microbial lipid production by the yeast C. curvatus, which fit well with the protein-based biorefinery concept. Further efforts should be devoted to recognizing the metabolic features, identifying more robust lipid producer and optimizing lipid production processes.

Using non-ionic surfactant as an accelerator to increase extracellular lipid production by oleaginous yeast Cryptococcus curvatus MUCL 29819

The aim of this work was to study the effects of non-ionic surfactant on the accumulation of total microbial lipids and extracellular lipid by Cryptococcus curvatus MUCL 29819 with acetic acid as carbon source. Compared with Brij 58 and Triton X-100, Brij 58 most increased the total lipids, with a yield up to 2.84 g/L (extracellular lipid up to 47%). Brij 58 also increased the metabolic flow of acetic acid to lipid accumulation (maximum conversion of 0.54 g/g at 1.0 g/L Brij 58) and limited its conversion to non-lipid biomass (minimum conversion 0.12 g/g at 0.5 g/L Brij 58). The improvement in the proportion of extracellular lipid by tea saponin and Brij 58 was due to changes in cell membrane permeability and improvement of cell membrane fluidity. Triton X-100, having weaker surface activity, promoted release of extracellular lipid and also increased the proportion of polyunsaturated fatty acid (C22:6, docosahexaenoic acid).

Integrated Process for the Enzymatic Production of Fatty Acid Sugar Esters Completely Based on Lignocellulosic Substrates.

Lignocellulose can be converted sustainably to fuels, power and value-added chemicals like fatty acid esters. This study presents a concept for the first eco-friendly enzymatic synthesis of economically important fatty acid sugar esters based on lignocellulosic biomass. To achieve this, beech wood cellulose fiber hydrolysate was applied in three manners: as sugar component, as part of the deep eutectic solvent (DES) reaction system and as carbon source for the microbial production of the fatty acid component. These fatty acids were gained from single cell oil produced by the oleaginous yeast Cryptococcus curvatus cultivated with cellulose fiber hydrolysate as carbon source. Afterwards, an immobilized Candida antarctica lipase B was used as the biocatalyst in DES to esterify sugars with fatty acids. Properties of the DES were determined and synthesized sugar mono- and di-esters were identified and characterized using TLC, MS, and NMR. Using this approach, sugar esters were successfully synthesized which are 100% based on lignocellulosic biomass.

Enhanced production of microbial lipids from waste office paper by the oleaginous yeast Cryptococcus curvatus

Waste paper has a potential to serve as renewable feedstock for the biorefineries of fuels, chemicals and materials due to rich in cellulose and its abundance at low cost. In the present study, pretreated waste office paper (WOP) was enzymatically hydrolysed and used for lipid production by Cryptococcus curvatus. The results suggested that the WOP hydrolysate supplemented with ammonium sulphate (2 g/L) and yeast extract (0.5 g/L) as nitrogen source at a C/N ratio of 80 were the most suitable for high yield of lipids. The biomass, lipid yield, lipid content and lipid coefficient achieved from batch cultivation of C. curvatus using untreated and pretreated WOP hydrolysates were 6.32 and 15.20 g/L, 1.39 and 5.75 g/L, 22 and 37.8%, and 99.9 and 234.6 mg/g sugar with the productivity of 0.02 and 0.08 g/L/h, respectively. The fatty acid profile of the lipids indicated that the oleic acid was the major fatty acid followed by palmitic acid, stearic acid and linoleic acid which is quite similar to plant/vegetable oils. Thus, the results suggested that the waste office paper could be an alternative feedstock for production of microbial lipids for biodiesel.

Enhancement of extracellular lipid production by oleaginous yeast through preculture and sequencing batch culture strategy with acetic acid

Oleaginous yeast Cryptococcus curvatus MUCL 29819, an acid-tolerant lipid producer, was tested to spill lipids extracellularly using different concentrations of acetic acid as carbon source. Extracellular lipids were released when the yeast was cultured with acetic acid exceeding 20g/L. The highest production of lipid (5.01g/L) was obtained when the yeast was cultured with 40g/L acetic acid. When the yeast was cultivated with moderate concentration (20g/L) of acetic acid, lipid production was further increased by 49.6% through preculture with 40g/L acetic acid as stimulant. When applying high concentration (40g/L) of acetic acid as carbon source in sequencing batch cultivation, extracellular lipids accounted up to 50.5% in the last cycle and the extracellular lipids reached 5.43g/L through the whole process. This study provides an effective strategy to enhance extracellular lipid production and facilitate the recovery of microbial lipids.

Feasibility of Lipid Production from Waste Paper by the Oleaginous Yeast Cryptococcus curvatus

Waste paper was studied as a potential source for lipid production using the oleaginous yeast Cryptococcus curvatus for the first time. Three common types of waste paper, office paper, newspaper, and cardboard, were directly hydrolyzed by an enzyme cocktail to generate sugar-rich and nitrogen-limited hydrolysates. When these hydrolysates were used without any auxiliary nutrients by C. curvatus, the lipid content and lipid yield were higher than 50% and 200 mg/g, respectively. The nitrogen-rich enzyme cocktail exerted no negative effects on lipid production. Moreover, the integrated processes of enzymatic hydrolysis and lipid fermentation achieved comparable lipid yield to the separate hydrolysis and lipid production process. The resulting lipid samples had similar fatty acid compositional profiles to those of vegetable oils, which suggested their potential for biodiesel production. These findings strongly supported waste paper as appealing substrates for lipid production via oleaginous yeast, which provided cost-effective waste paper-to-lipids routes for sustainable biodiesel production.

Bioconversion of mixed volatile fatty acids into microbial lipids by Cryptococcus curvatus ATCC 20509

The oleaginous yeast Cryptococcus curvatus ATCC 20509 can use 5–40g/L of acetic, propionic, or butyric acid as sole carbon source to produce lipids. High concentrations (30g/L) of mixed volatile fatty acids (VFAs) were used to cultivate C. curvatus to explore the effects of different ratios of mixed VFAs on lipid production and composition. When mixed VFAs (VFA ratio was 15:5:10) were used as carbon sources, the highest cell mass and lipid concentration were 8.68g/L and 4.93g/L, respectively, which were significantly higher than those when 30g/L of acetic acid was used as sole carbon source. The highest content and yield of odd-numbered fatty acids were 45.1% (VFA ratio was 0:15:15) and 1.62g/L (VFA ratio was 5:15:10), respectively. These results indicate that adjusting the composition ratios of mixed VFAs effectively improves microbial lipid synthesis and the yield of odd-numbered fatty acids.

Efficient bioconversion of high-content volatile fatty acids into microbial lipids by Cryptococcus curvatus ATCC 20509

The lower utilization ratios of high-content VFAs resulted in less lipid yield by the oleaginous yeast Cryptococcus curvatus ATCC 20509. In this work, increasing the inoculation concentration to OD600=5.1 improved the acetic acid utilization ratio (99.8%) and lipids production (4.63g/L) in mediums with 30g/L of acetic acid. For the higher acetic acid concentration (40g/L), increasing the nitrogen to carbon ratio (0.033) and raising the initial pH (pH=8) was superior over improvement in the inoculate, with the lipid production increased from 1.08g/L to 6.49g/L. Subsequently, mixed VFAs at concentrations of 30g/L and 40g/L were used as the carbon source to simulate waste-derived VFAs. High lipid production (4.82 and 7.45g/L, respectively) was correspondingly achieved with similar high lipid yield (0.187g/g). This study provides an effective strategy to enhance the bioconversion of high-content VFAs into microbial lipids.

Proof of concept for biorefinery approach aiming at two bioenergy production compartments, hydrogen and biodiesel, coupled by an external membrane

ABSTRACTA biorefinery is a facility that integrates biomass conversion processes and equipment to produce fuels, power and chemicals from biomass. This concept is applied here to hydrogen production by anaerobic fermentation. Its biological production is increasingly perceived as a potential pathway for developing renewable sources of energy, due to hydrogen's high energy content (142 kJ g−1) and the absence of harmful emissions during utilization in a hydrogen fuel cell. The main secondary metabolites produced during dark fermentation (volatile fatty acids, VFAs) are building blocks and can also serve as an alternate carbon source for lipid production from oleaginous yeast strains for biodiesel production.The concept proposed here is the coupling of two main compartments. In the first one, lignocellulosic biomass is transformed by dark fermentation into hydrogen and VFAs. The medium used in the first compartment is transferred via an immersed membrane bioreactor to the second compartment where the production of single-cell oil with oleaginous yeast occurs using VFAs as carbon source. This study aims at presenting the feasibility of this bioprocess, through hydrogen production from glucose followed by lipid production by the oleaginous yeast Cryptococcus curvatus grown on the VFA-enriched supernatant resulting from the dark fermentation step.

Microbial conversion of mixed volatile fatty acids into microbial lipids by sequencing batch culture strategy

Four mixed volatile fatty acids (VFAs) were used as sole carbon source to culture oleaginous yeast Cryptococcus curvatus by sequencing batch culture strategy. The highest lipid content (42.7%) and concentration (1.77g/L) were achieved when the ratio of VFAs (acetic, propionic, and butyric acids) was 6:3:1. The oleaginous yeast favored to use VFAs for lipid biosynthesis rather than cell proliferation. With regard to the utilization ratio of VFAs, acetic acid reached over 99%, whereas propionic acid was barely 35%. The produced lipids contained nearly 45% of monounsaturated fatty acids, which can be the ideal raw materials for biodiesel production. Additionally, the produced odd-numbered fatty acid content reached 23.6% when the propionate acid content of VFAs was 50%. Further analysis showed that increasing the ratio of acetic acid was most beneficial to cell mass and lipid production, whereas propionic acid and butyric acid were more conducive to lipid and cell mass synthesis, respectively.

Co-utilization of corn stover hydrolysates and biodiesel-derived glycerol by Cryptococcus curvatus for lipid production

In the present study, synergistic effects were observed when glycerol was co-fermented with glucose and xylose for lipid production by the oleaginous yeast Cryptococcus curvatus. Glycerol was assimilated simultaneously with sugars at the beginning of the culture without adaption time. Furthermore, better lipid production results, i.e., lipid yield and lipid productivity of 18.0g/100g and 0.13g/L/h, respectively, were achieved when cells were cultured in blends of corn stover hydrolysates and biodiesel-derived glycerol than those in the hydrolysates alone. The lipid samples had fatty acid compositional profiles similar to those of vegetable oils, suggesting their potential for biodiesel production. This co-utilization strategy provides an extremely simple solution to advance lipid production from both lignocelluloses and biodiesel-derived glycerol in one step.

Validation of a predictive model for fed-batch and continuous lipids production processes from acetic acid using the oleaginous yeast Cryptococcus curvatus

This study focuses on the use of acetic acid as carbon source for cell mass and lipid production using an oleaginous yeast, Cryptococcus curvatus. To improve this cell mass and lipid production on acetic acid, a numerical model was built to predict the yeast behavior. The model was first calibrated and validated on a fed-batch culture of the oleaginous yeast grown on acetic acid. After the validation step, this model was used as a predictive tool to search for optimal dilution rate and C/N ratios for continuous culture. A continuous culture was then launched using these culture parameters, revealing that the experimental data fitted the model predictions. At steady-state, cell mass concentration was stabilized at 26.7g/L containing 48–53% of lipid content. A maximal lipid productivity of 0.54gL−1h−1 was obtained, which was superior to those reported in literature. This model could be qualified as a mechanistic one since it allows understanding reactions underlying the lipid production process. The experimental data fitted the model predictions, which confirmed that the use of a model is a powerful and predictive tool which could help the operator to process parameters to improve the culture.

Efficient conversion of acetate into lipids by the oleaginous yeast Cryptococcus curvatus.

BackgroundAcetic acid is routinely generated during lignocelluloses degradation, syngas fermentation, dark hydrogen fermentation and other anaerobic bioprocesses. Acetate stream is commonly regarded as a by-product and detrimental to microbial cell growth. Conversion of acetate into lipids by oleaginous yeasts may be a good choice to turn the by-product into treasure.ResultsTen well-known oleaginous yeasts were evaluated for lipid production on acetate under flask culture conditions. It was found that all of those yeasts could use acetate for microbial lipid production. In particular, Cryptococcus curvatus accumulated lipids up to 73.4 % of its dry cell mass weight. When the culture was held in a 3-L stirred-tank bioreactor, cell mass, lipid content, lipid yield and acetate consumption rate were 8.1 g/L, 49.9 %, 0.15 g/g and 0.64 g/L/h, respectively. The fatty acid compositional profiles of the acetate-derived lipids were similar to those of vegetable oil, suggesting their potential for biodiesel production. Continuous cultivation of C. curvatus was conducted under nitrogen-rich condition at a dilution rate of 0.04 h−1, the maximal lipid content and lipid yield were 56.7 % and 0.18 g/g, respectively. The specific lipid formation rate, lipid content and lipid yield were all higher under nitrogen-rich conditions than those obtained under nitrogen-limited conditions at the same dilution rates. Effective lipid production by C. curvatus was observed on corn stover hydrolysates containing 15.9 g/L acetate.ConclusionsAcetate is an effective carbon source for microbial lipid production by oleaginous yeasts. Continuous cultivation of C. curvatus on acetate was promising for lipid production under both nitrogen-rich and nitrogen-limited conditions. These results provide valuable information for developing and designing more efficient acetate-into-lipids bioprocess.

Improvement and modeling of culture parameters to enhance biomass and lipid production by the oleaginous yeast Cryptococcus curvatus grown on acetate

The improvement of culture parameters for lipid production from acetate as carbon source was investigated using the oleaginous yeast Cryptococcus curvatus. A new pH regulation system dispensing acetate was developed for fed-batch culture and allowed obtaining nearly 80g/L biomass within 60h with a maximal growth rate of 0.28h−1. A biological model was developed from experimental data. The influence of three C/N ratios of 300, 500 and 900 were tested during a multi-phases process on lipid accumulation. The C/N ratio of 300 was reported to be the most suitable for lipid storage. No significant increase of lipids content was obtained with higher value. A maximal content of 60% DCW of lipid was obtained. The determination of fatty acids profiles of the microbial oils has confirmed that the valorization of acetate by microbial oils production was a promising perspective.