Oleaginous Yeast Cryptococcus Curvatus Research Articles

Lipid production by eukaryotic microorganisms isolated from palm oil mill effluent

Microbial oil production combined with wastewater management is one option for a more sustainable future. Micrographs of microbial cultures enriched from palm oil mill effluent (POME) showed lipid inclusion in the eukaryotic cells, indicating the cells can accumulate lipids. However, enriching the culture did not increase the total lipids. Therefore, eukaryotic microorganisms were isolated from POME to investigate whether these microorganisms are potential lipid producers. Four strains were isolated, and their lipid synthesis capabilities were compared with known oleaginous yeasts in a synthetic oil-free medium. Two strains (identified as Galactomyces geotrichum and Graphium penicillioides) had the potential to accumulate lipid accumulation based on the increase in triacylglycerol content. G. penicillioides was the most promising strain for lipid production as this strain accumulated more lipids than the well-known oleaginous yeast Cryptococcus curvatus (29.1±3.0wt% vs. 20.2±2.9wt%). To our knowledge, oil synthesis and accumulation by G. penicillioides have not previously been reported.

Oleaginous yeast Cryptococcus curvatus exhibits interplay between biosynthesis of intracellular sugars and lipids

AbstractPatterns of the biosynthesis of major metabolites of the oleaginous yeast Cryptococcus curvatus NRRL Y‐1511 were investigated during cultivation on sugar‐based media. When lactose or sucrose was employed as substrate under nitrogen‐limited conditions, the yeast strain accumulated high quantities of intra‐cellular total sugars (ITS) at the beginning of fermentation (up to 68% w/w), with ITS values progressively decreasing to 20%, w/w, at the end of the fermentation. Decrease in ITS content and consumption of extra‐cellular lactose led to a subsequent rise in lipid accumulation, reaching 29.8% in dry cell weight at 80 g/L of initial lactose concentration. Lactose was a more favorable substrate for lipid production than sucrose. In nitrogen‐excess conditions, ITS were produced in significant quantities despite the continuous presence of nitrogen in the medium. Growth on lactose was not followed by secretion of extra‐cellular β‐galactosidase. High quantities of extra‐cellular invertase were observed during growth on sucrose. The composition of ITS was highly influenced by the sugar used as substrate. Cellular lipids contained mainly palmitic and to lesser extent linoleic and stearic acids. This is the first report in the literature that demonstrates the interplay between the biosynthesis of intra‐cellular total sugars and lipid synthesis for oleaginous yeast strains.Practical applications: Cryptococcus curvatus represents a promising candidate for the successful production of microbial lipids and polysaccharides during growth on several types of low‐cost sugars utilized as substrates. Thus, C. curvatus could be used to produce a variety of metabolites useful for industrial biotechnology.Interplay between the biosynthesis of intra‐cellular total sugars produced per unit of dry weight (YITS/X %, w/w) and intra‐cellular lipids produced per unit of dry weight (YL/X %, w/w) of Cryptococcus curvatus, during growth on lactose, under nitrogen‐limited conditions.

Bioconversion of volatile fatty acids from macroalgae fermentation into microbial lipids by oleaginous yeast

Marine biomass is a potential carbon resource for the production of bio-based energy and chemicals. Microbial lipids are competitive in producing biodiesel. In this study, Laminaria japonica (Saccharina japonica) was chosen as a model marine biomass producer due to its fast rate and well-established cultivation technology. A mixed culture system was established by inoculating it with sludge from an anaerobic digester, and then operated in a continuous mode for over 1000days by feeding it a non-sterile suspension of dried L. japonica in tap water without additional nutrients. The average yield and productivity of total organic acids during the stationary phase were as high as 0.47g/g COD and 2.45g/L/day at pH 7 with 84.08% of acetate in total organic acids. The volatile fatty acids (VFAs) from the continuously-stirred tank reactor (CSTR) were used by oleaginous yeast (Cryptococcus curvatus) to produce lipids in a repeated batch system. The highest lipid content was 61%. The composition of the fatty acids was quite similar to that of vegetable oils. This system has potential application to biodiesel production.

Co-utilization of glucose, xylose and cellobiose by the oleaginous yeast Cryptococcus curvatus

Simultaneous utilization of mixed sugars is one of the major challenges for biofuel production utilizing lignocellulosic biomass as feedstock. Our previous work proved that the oleaginous yeast Cryptococcus curvatus could efficiently produce lipids, the precursors of hydrocarbons with high energy density, from lignocellulosic hydrolysates. However, the strain's capability of simultaneously utilizing primary sugars was still unknown. Thus, this work comprehensively explored the co-utilization of glucose, xylose and cellobiose by C. curvatus. The results indicated that the consumption of both xylose and cellobiose was repressed by glucose, while xylose and cellobiose could be simultaneously consumed at similar rates. The total sugar consumption rates remained constant at about 0.6 g L−1 h−1 regardless of the sugar composition in the mixtures, and the cell biomass and lipid production by C. curvatus cultured on the different sugar mixtures were similar. Moreover, compared with glucose and xylose, cellobiose could lead to higher dry cell weights and lipid yields, suggesting an excellent carbon source for lipid production. Based on these findings, this study demonstrated an effective approach for alleviating glucose repression for microbial lipid production by C. curvatus through xylose/cellobiose co-utilization which would greatly contribute to a more efficient and economical cellulosic biofuel production.

Lipid production from corn stover by the oleaginous yeast Cryptococcus curvatus

BackgroundMicrobial lipids produced from lignocellulosic biomass hold great promise for the biodiesel industry. These lipids usually consist of three major processes: pretreatment, enzymatic hydrolysis and lipid production. However, the conventional strategy of using biomass hydrolysates as the feedstock for lipid production suffers from low lipid coefficient and prohibitively high costs. More cost-effective and integrated processes are required to advance lignocellulosic biomass-based microbial lipid technology.ResultsThree different strategies were tested using the oleaginous yeast Cryptococcus curvatus ATCC 20509 as a lipid producer and alkaline-pretreated corn stover as a model material. It was found that the separate hydrolysis and enhanced lipid production process required more cellulolytic enzymes yet afforded a low lipid coefficient of 115.6 mg/g pretreated corn stover. When biomass hydrolysis and lipid production were integrated, the amounts of cellulase and xylanase were reduced and no β-glucosidase was required. The simultaneous saccharification and lipid production process gave a lipid coefficient of 129.4 mg/g pretreated corn stover. A higher lipid coefficient of 159.4 mg/g pretreated corn stover was obtained using the simultaneous saccharification and enhanced lipid production (SSELP) process. Furthermore, cellulolytic enzymes were found recoverable and reusable upon recycling the spent supernatants of the SSELP process, which could reduce enzyme consumption and wastewater discharge.ConclusionsThe SSELP process was superior to other processes in terms of converting alkaline-pretreated corn stover into lipids by C. curvatus, as it required less cellulolytic enzymes and had a higher lipid coefficient. Moreover, the process facilitated easy enzyme recycling that should lead to further reduction of enzyme consumption. These results provide valuable information for cost-effective lipid production from lignocelluloses, which should be particularly important in achieving a sustainable production of biodiesel.

Cultivation of oleaginous yeast using aqueous fractions derived from hydrothermal pretreatments of biomass

This study addresses some of the current challenges in producing biofuels from yeast oils. Specifically, it valorizes byproduct waste streams of biomass processing technologies by integrating them as alternative carbon or nutrient sources in oleaginous yeast cultivation. Crude glycerol recovered from the thermal hydrolysis of various fats and oils was successfully used in culturing of the oleaginous yeast Cryptococcus curvatus, with growth and lipid accumulation occurring at levels identical to those achieved when commercially purchased glycerol was used. The aqueous byproduct stream from the hydrothermal processing of C. curvatus can also be recycled as a growth substrate for subsequent C. curvatus cultures. The addition of this stream promoted higher biomass production without affecting lipid accumulation and only moderately changing the fatty acid profile. Use of these recycling strategies reduces costs and environmental impact of current microbial biofuels production by providing accessible, non-expensive carbon sources and nutrients for oleaginous yeast cultivation.

Production of biodiesel from carbon sources of macroalgae, Laminaria japonica

As aquatic biomass which is called “the third generation biomass”, Laminaria japonica (also known as Saccharina japonica) consists of mannitol and alginate which are the main polysaccharides of algal carbohydrates. In this study, oleaginous yeast (Cryptococcus curvatus) was used to produce lipid from carbon sources derived from Laminaria japonica. Volatile fatty acids (VFAs) were produced by fermentation of alginate extracted from L. japonica. Thereafter, mannitol was mixed with VFAs to culture the oleaginous yeast. The highest lipid content was 48.30%. The composition of the fatty acids was similar to vegetable oils. This is the first confirmation of the feasibility of using macroalgae as a carbon source for biodiesel production.

Combination of algae and yeast fermentation for an integrated process to produce single cell oils.

Economic and ecological reasons cause the industry to develop new innovative bio-based processes for the production of oil as renewable feedstock. Petroleum resources are expected to be depleted in the near future. Plant oils as sole substituent are highly criticized because of the competitive utilization of the agricultural area for food and energy feedstock production. Microbial lipids of oleaginous microorganisms are therefore a suitable alternative. To decrease production costs of microbial lipids and gain spatial independence from industrial sites of CO2 emission, a combination of heterotrophic and phototrophic cultivation with integrated CO2 recycling was investigated in this study. A feasibility study on a semi-pilot scale was conducted and showed that the cultivation of the oleaginous yeast Cryptococcus curvatus on a 1.2-L scale was sufficient to supply a culture of the oleaginous microalgae Phaeodactylum tricornutum in a 21-L bubble column reactor with CO2 while single cell oils were produced in both processes due to a nutrient limitation.

Efficient conversion from cheese whey to lipid using Cryptococcus curvatus

Cheese whey, which is a byproduct of the cheese-making process, was pretreated by means of hydrodynamic cavitation (HC) under an alkaline condition and then was converted to lipid using our newly isolated oleaginous yeast Cryptococcus curvatus. The growth rate (7.2g/L/day) and lipid content (65%) were nearly double those obtained using commercial types of sugars such as glucose, galactose, and lactose. The lipid productivity level (4.68g/L/day) was the highest ever reported. HC-pretreated cheese whey under alkaline condition can serve as an excellent complete nutrient source and/or as a stimulant of lipid production.

Conversion of biomass-derived oligosaccharides into lipids

BackgroundOligocelluloses and oligoxyloses are partially hydrolyzed products from lignocellulosic biomass hydrolysis. Biomass hydrolysates usually contain monosaccharides as well as various amounts of oligosaccharides. To utilize biomass hydrolysates more efficiently, it is important to identify microorganisms capable of converting biomass-derived oligosaccharides into biofuels or biochemicals.ResultsWe have demonstrated that the oleaginous yeast Cryptococcus curvatus can utilize either oligocelluloses or oligoxyloses as sole carbon sources for microbial lipid production. When oligocelluloses were used, lipid content and lipid coefficient were 35.9% and 0.20 g/g consumed sugar, respectively. When oligoxyloses were used, lipid coefficient was 0.17 g/g consumed sugar. Ion chromatography analysis showed oligocelluloses with a degree of polymerization from 2 to 9 were assimilated. Our data suggested that these oligosaccharides were transported into cells and then hydrolyzed by cytoplasmic enzymes. Further analysis indicated that these enzymes were inducible by oligocelluloses. Lipid production on cellulose by C. curvatus using the simultaneous saccharification and lipid production process in the absence of cellobiase achieved essentially identical results to that in the presence of cellobiase, suggesting that oligocelluloses generated in situ were utilized with high efficiency. This study has provided inspiring information for oligosaccharides utilization, which should facilitate biorefinery based on lignocellulosic biomass.ConclusionsC. curvatus can directly utilize biomass-derived oligosaccharides. Oligocelluloses are transported into the cells and then hydrolyzed by cytoplasmic enzymes. A simultaneous saccharification and lipid production process can be conducted without oligocelluloses accumulation in the absence of cellobiase by C. curvatus, which could reduce the enzyme costs.

Effect of lignocellulose degradation products on microbial biomass and lipid production by the oleaginous yeast Cryptococcus curvatus

This work investigated effects of lignocellulose degradation products on cell biomass and lipid production by Cryptococcus curvatus. Furfural was found to have the strongest inhibitory effect. For the three phenolic compounds tested, vanillin was the most toxic, while PHB and syringaldehyde showed comparable inhibitions in the concentration range of 0–1.0g/L. Generally little significant differences on the relative cell biomass and lipid contents at the same concentrations of tested compounds were observed between glucose and xylose as a sole carbon source. At 1.0g/L of furfural, the cell biomass and lipid content decreased by 78.4% and 61.0% for glucose as well as 72.0% and 59.3% for xylose, respectively. C. curvatus ceased to grow at concentrations of PHB over 1.0g/L or vanillin over 1.5g/L. The strain could survive in the presence of syringaldehyde up to 2.0g/L for glucose or 1.5g/L for xylose. The compounds’ negative impact was reduced by an increase in inoculum size and a 10% (v/v) seed was detected to be optimal for cell biomass and lipid production. The results demonstrated C. curvatus could effectively utilize most of the dominant monosaccharides and cellobiose existing in lignocellulosic biomass hydrolysate in the presence of toxic compounds.

Cultivation and lipid production of yeast Cryptococcus curvatus using pretreated waste active sludge supernatant

Oleaginous yeast Cryptococcus curvatus has become some of the most promising feedstock for biodiesel production due to their high production efficiency. However, high cost of cultivation, especially substrate cost, hinders rapid commercialization of yeast-based biodiesel. In this study, waste activated sludge (WAS), which is rich in nutrients and organic matters, was examined as an economic substitute for organic substances. To be efficiently bioavailable, WAS must be pretreated. Hydrodynamic cavitation reaction time and pH were identified as experimental factors, whereas growth rate was selected as response parameter. The experimental factors were optimized employing Response Surface Methodology (RSM). Growth rate of 1.13/h was optimized at reaction time 12.5min and pH 8.65. When sludge pretreated under these optimal conditions was used as a substitute to yeast extract, 9.84g/L of biomass was obtained in a day and lipid was accumulated up to 23% of dry weight.

High-cell-density cultivation of oleaginous yeast Cryptococcus curvatus for biodiesel production using organic waste from the brewery industry

Waste spent yeast from brewery industry was used as a sole growth substrate to grow an oleaginous yeast Cryptococcus curvatus for the purpose of biodiesel production. Approximately 7g/l/d of biomass productivity was obtained using only spent yeast (30g/l) without additional nutrients and pretreatment of any kind. To make best use of available nutrients in the spent yeast, stepwise cultivation was carried out in a batch culture mode and the highest biomass and lipid content, which were 50.4g/l and 37.7%, respectively, were obtained at 35:1 of C/N ratio. Lipid from C. curvatus was found to be a quality-sufficient source of oil as a transportation fuel in terms of cetane, iodine values, and oxidation stability, although the values of cold filter plugging point were less desirable. Economic evaluation revealed that the use of the spent yeast could significantly reduce the unit cost of yeast-based biodiesel production.

Oleaginous yeast Cryptococcus curvatus for biofuel production: Ammonia’s effect

Abstract Culturing oleaginous yeast with organic residues can provide cheap feedstock for biodiesel production, but the high content of nitrogen in some feedstocks (such as food residues) can lead to inhibition to cell growth and lipid synthesis, and this may be caused by ammonia. Thus, the inhibitory effect of ammonia on the growth of yeast Cryptococcus curvatus and its possible mechanism were evaluated in this study. The biomass production, lipid contents, affected metabolic pathways, and activities of the related pathway enzymes were investigated. The results showed that the yeast biomass production on acetate decreased from 5.9 kg m −3 to 2.0 kg m −3 when the initial ammonia concentration (in nitrogen equivalent terms) increased from 131 g m −3 to 3140 g m −3 . Ammonia significantly inhibited the yeast growth with volatile fatty acids (VFA) as carbon sources, but not in the culture with pyruvate generating substances (PGS) as carbon sources. However, the fatty acid synthesis was suppressed by ammonia in cultures with either VFA or PGS. The study on mechanism of ammonia inhibition suggested it might include an inhibition on the acyl-CoA synthetase activity, since a 61% decrease in the enzyme activity was found when 3140 g m −3 of ammonia (in nitrogen equivalent terms) was added. This study firstly reported the effect of ammonia with high concentrations to oleaginous yeast growth and lipid accumulation, and provided preliminary evidences on its possible mechanism of acyl-CoA synthetase inhibition.

Production of Oils from Acetic Acid by the Oleaginous Yeast Cryptococcus curvatus

The feasibility of the conversion of acetic acid, a metabolite commonly obtained during anaerobic fermentation processes, into oils using the yeast Cryptococcus curvatus was reported. This microorganism exhibited very slow growth rates on acetate as carbon source, which led to design a two-stage cultivation process. The first consisted of cell growth on glucose as carbon source until its complete exhaustion. The second step involved the use of acetate as carbon source under nitrogen limitation in order to induce lipid accumulation. A typical experiment performed in a bioreactor involved a preliminary yeast growth with a glucose initial concentration of 15g/L glucose. Further additions of acetate and nitrogen source allowed a final lipid accumulation up to 50% (w/w). These promising results demonstrated the suitability of the technique proposed.

Process for biodiesel production from Cryptococcus curvatus

The objective of the current report is process optimization for economical production of lipids by the well known oleaginous yeast Cryptococcus curvatus and conversion of the lipids to biodiesel. A high cell density fed-batch cultivation on low cost substrate viz. crude glycerol resulted in a dry biomass and oil yield of up to 69g/L and 48% (w/w), respectively. The process was scaled up easily to 26L. The oil extraction process was also optimized using environmentally safe solvents. The oil profile indicated a high oleic acid content followed by palmitic acid, stearic acid and linoleic acid. The oil was trans-esterified to biodiesel and thoroughly characterized. This is the first end to end report on production of biodiesel from the C. curvatus oil.

Oleaginous yeast Cryptococcus curvatus culture with dark fermentation hydrogen production effluent as feedstock for microbial lipid production

Volatile fatty acids (VFA) from dark fermentation hydrogen production were tested as carbon sources for the culture of oleaginous yeast Cryptococcus curvatus, which is a promising feedstock for biofuel production. The optimal acetate concentration and pH were investigated when potassium acetate was used as the sole carbon source. Comparisons were then made when hydrogen production effluent (HPE) from synthetic wastewater was tested as feedstock. A pH-stat culture fed with acetic acid ultimately produced 168 g/L biomass, with a lipid content of 75.0%. No inhibitor to yeast growth was produced in the hydrogen production process. However, inhibition occurred in culture with HPE from food waste (FW), indicating that inhibitors may be present in the original raw food waste. This inhibition could be avoided by a process that uses glucose as the initial carbon source and then is continuously fed with FW-HPE. The biomass productivity in this continuous culture process reached 0.34 g/L/h, but the lipid content was only 13.5%. These results suggest that FW-HPE alone is not an optimal feedstock, but HPE derived from nitrogen-deficient waste streams could be good feedstocks. This study provides preliminary evidence for the feasibility of using organic waste for the co-production of hydrogen and lipid.

Microbial lipid production by the oleaginous yeast Cryptococcus curvatus O3 grown in fed-batch culture

This study investigates the capability of the oleaginous yeast Cryptococcus curvatus O3 to synthesize microbial lipids using glucose as its sole carbon source. Both glucose concentration and varying nitrogen sources have a significant effect on cell growth and microbial lipid accumulation in batch and fed-batch cultures. When cultivated in a shaking flask at 30 °C with glucose as sole carbon source, the cellular biomass and lipid content reached 51.8 kg m −3 and 651 g kg −1 , respectively. The fed-batch culture in a 30 × 10 −3 m 3 stirred-tank fermentor run for 185 h produced a cellular biomass, lipid content, and lipid productivity rate of up to 104.1 kg m −3 , 827 g kg −1 , and 0.47 kg m −3 h −1 , respectively. These data indicate that C . curvatus O3 can be used as an ideal oleaginous yeast for microbial lipid production. Gas chromatography analysis of the synthesized microbial lipids revealed that the major constituents are long-chain fatty acids, such as palmitic acid, stearic acid, oleic acid, and linoleic acid. The results suggest that the microbial lipids produced by C. curvatus O3 can be used to produce biodiesel. ► In this manuscript, we use the fed-batch ferementation method to improve the lipid production. ► In a 30 × 10 −3 m 3 stirred-tank fermentor run for 185 h produced a cellular biomass, lipid content, and lipid productivity rate of up to 104.1 kg m −3 , 827 g kg −1 , and 0.47 kg m −3 h −1 , respectively. ► These data indicate that C. curvatus O3 can be used as an ideal oleaginous yeast for microbial lipid production.

Oil production by oleaginous yeasts using the hydrolysate from pretreatment of wheat straw with dilute sulfuric acid

This paper explores the use of the hydrolysate from the dilute sulfuric acid pretreatment of wheat straw for microbial oil production. The resulting hydrolysate was composed of pentoses (24.3 g/L) and hexoses (4.9 g/L), along with some other degradation products, such as acetic acid, furfural, and hydroxymethylfurfural (HMF). Five oleaginous yeast strains, Cryptococcus curvatus, Rhodotorula glutinis, Rhodosporidium toruloides, Lipomyces starkeyi, and Yarrowia lipolytica, were evaluated by using this hydrolysate as substrates. The results showed that all of these strains could use the detoxified hydrolysate to produce lipids while except R. toruloides non-detoxified hydrolysate could also be used for the growth of all of the selective yeast strains. C. curvatus showed the highest lipid concentrations in medium on both the detoxified (4.2 g/L) and non-detoxified (5.8 g/L) hydrolysates. And the inhibitory effect studies on C. curvatus indicated HMF had insignificant impacts at a concentration of up to 3 g/L while furfural inhibited cell growth and lipid content by 72.0% and 62.0% at 1 g/L, respectively. Our work demonstrates that lipid production is a promising alternative to utilize hemicellulosic sugars obtained during pretreatment of lignocellulosic materials.

Production of lipid from N‐acetylglucosamine by Cryptococcus curvatus

AbstractN‐Acetylglucosamine (GlcNAc), the monomeric constituent of chitin, is rarely used as a carbon source for fermentation technology. In this study, we demonstrate that the oleaginous yeast Cryptococcus curvatus ATCC 20509 can produce intracellular lipid during the cultivation process and total lipid content can reach 54% on a GlcNAc‐based medium. Culture of C. curvatus under various conditions indicated that lipid accumulation also occurred at a relatively broad range of temperatures as well as relatively high initial GlcNAc concentrations. Fatty acid analysis indicated that the product was rich in palmitic acid, stearic acid, and oleic acid, closely resembling the composition of palm oil. More importantly, the lipid sample produced at 22 °C had a total saturated fatty acid content of 54.2 wt%, suggesting that it may be explored as cocoa‐butter equivalent. Our data suggested that GlcNAc could be used as a feedstock for industrial biotechnology and that C. curvatus ATCC 20509 is a strain capable of accumulating high intracellular lipid using this nitrogen‐rich renewable material.Practical applications: Microbial lipid is a versatile material, especially for biodiesel production. Stable and abundant renewable raw substrates remain to be explored for large‐scale production of microbial lipid. The present work reports lipid production using N‐acetylglucosamine (GlcNAc) by the oleaginous yeast Cryptococcus curvatus ATCC 20509 to yield up to 54% intracellular lipid content. More significantly, the lipid sample produced at 22 °C had a total saturated fatty acid content of 54.2 wt%, suggesting that it may be explored as cocoa‐butter equivalent. Our technology provides the opportunity to effectively convert GlcNAc, available from one of the most abundant renewable materials chitin, into lipid. This procedure should prove valuable in terms of renewable energy production as well as environmental pollution control.