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Trichosporon oleaginosus is an unconventional oleaginous yeast distinguished by its remarkable capacity to accumulate lipids in excess of 70% of its dry weight, particularly when cultivated in nitrogen-restricted conditions with ample carbon sources. A pivotal question that arises pertains to the nutrient dynamics in the culture medium, which give rise to both the excessive lipid content and corresponding lipid concentration. While previous research has predominantly focused on evaluating the impact of the initial carbon-to-nitrogen (C/N) ratio on lipid production, the precise critical thresholds of glucose and ammonium sulfate ((NH4)2SO4) at which growth and intracellular lipid production are either stimulated or impeded remain inadequately defined. This study employs an experimental design and response surface methodology to investigate the complex mechanism of lipid accumulation and its interaction with cellular growth. Application of the aforementioned methodologies resulted in the production of 10.6 g/L of microbial oil in batch cultures under conditions that correspond to a C/N ratio of 76. However, the primary objective is to generate knowledge to facilitate the development of efficient fed-batch cultivation strategies that optimize lipid production exclusively employing inorganic nitrogen sources by finely adjusting carbon and nitrogen levels. The intricate interaction between these levels is comprehensively addressed in the present study, while it is additionally revealed that as glucose levels rise within a non-inhibitory range, lipid-free biomass production decreases while lipid accumulation simultaneously increases. These findings set the stage for further exploration and the potential development of two-stage cultivation approaches, aiming to fully decouple growth and lipid production. This advancement holds the promise of bringing microbial oil production closer to commercial viability. [ABSTRACT FROM AUTHOR]

Trichosporon oleaginosus is an unconventional oleaginous yeast distinguished by its remarkable capacity to accumulate lipids in excess of 70% of its dry weight, particularly when cultivated in nitrogen-restricted conditions with ample carbon sources. A pivotal question that arises pertains to the nutrient dynamics in the culture medium, which give rise to both the excessive lipid content and corresponding lipid concentration. While previous research has predominantly focused on evaluating the impact of the initial carbon-to-nitrogen (C/N) ratio on lipid production, the precise critical thresholds of glucose and ammonium sulfate ((NH4)2SO4) at which growth and intracellular lipid production are either stimulated or impeded remain inadequately defined. This study employs an experimental design and response surface methodology to investigate the complex mechanism of lipid accumulation and its interaction with cellular growth. Application of the aforementioned methodologies resulted in the production of 10.6 g/L of microbial oil in batch cultures under conditions that correspond to a C/N ratio of 76. However, the primary objective is to generate knowledge to facilitate the development of efficient fed-batch cultivation strategies that optimize lipid production exclusively employing inorganic nitrogen sources by finely adjusting carbon and nitrogen levels. The intricate interaction between these levels is comprehensively addressed in the present study, while it is additionally revealed that as glucose levels rise within a non-inhibitory range, lipid-free biomass production decreases while lipid accumulation simultaneously increases. These findings set the stage for further exploration and the potential development of two-stage cultivation approaches, aiming to fully decouple growth and lipid production. This advancement holds the promise of bringing microbial oil production closer to commercial viability.

Abstract Background Cryptococcal meningitis is mainly caused by Cryptococcus neoformans/C. gattii complex. We compared the clinical, biological, and antifungal susceptibility profiles of isolates from HIV-Infected Patients (HIVIP) with C. neoformans (Cn) versus C. curvatus/C. laurentii (Cc/Cl) meningitis. Methods Comparative analytical study were conducted. Apart from patients’ clinical data, the following analysis were performed and the results were compared in both groups: biochemical examination, cryptococcal antigen test, India ink staining, and culture on Cerebral Spinal Fluid (CSF), strains identification by mass spectrometry, ITS sequencing, PCR serotyping and antifungal susceptibility. The main outcome variable was the “species of Cryptococcus identified”, which was compared to other variables of the same type using the Pearson Chi-square test or the Fisher exact test. Results A total of 23 (79.3%) Cn meningitis cases versus 6 (20.7%) Cc/Cl meningitis were retained. Cn meningitis was more frequently associated with headache (100% vs 50%, p = 0.005) than Cc/Cl meningitis and meningeal signs were more frequent in Cn infected patients. Biologically, hypoglycorrhachia and low CD4 count were more observed in Cn group (90% vs 20% of patients, p = 0.01; 45.6 vs 129.8 cells/µL, p = 0.02, respectively). A higher proportion of Cn strains (91.3%) showed a low Minimum Inhibitory Concentration (MIC) (

Microbial 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 & Sons, Ltd [ABSTRACT FROM AUTHOR]

Abstract Coal fly ash originated from coal combustion has high concentrations of metals. If suitable leaching techniques are identified, then coal fly ash could serve as a useful source of valuable minerals including rare earth elements (REEs). In this study, three microbial strains, Candida bombicola, Phanerochaete chrysosporium and Cryptococcus curvatus were tested on their performance of leaching trace elements and REEs from fly ash. Through comparing mineral loss and leaching efficiencies resulting from indirect leaching or use of the culture supernatant, C. bombicola was identified to be the best leading to the highest mineral loss and extracting efficiencies of trace elements and REEs among the three strains. The highest mineral loss observed from using the supernatant of this yeast strain was 59.7%. Among all trace elements, As and Mo had the highest leaching efficiency of 80.9% and 79.5%, respectively. The same leaching test led to 67.7% of Yb and 64.6% of Er dissolved from the ash. This study, thus, demonstrated that bioleaching is feasible for leaching metals out of fly ash. The C. bombicola strain deserves further investigation due to its robust actions on metal leaching.

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. [ABSTRACT FROM AUTHOR]

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 0.10 g g−1. Furthermore, cellular lipid contents were higher than 20% when most AA were used individually. High lipid coefficients of over 0.23 g g−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. [ABSTRACT FROM AUTHOR]

Abstract Cutaneotrichosporon oleaginosus ATCC 20509, previously known as Trichosporon oleaginosus, Cryptococcus curvatus, Apiotrichum curvatum or Candida curvata D is an oleaginous yeast with several favorable qualities: it is fast growing, accumulates high amounts of lipid and has a very broad substrate spectrum. Its resistance to hydrolysis byproducts and genetic accessibility make it a promising cell factory for custom tailored microbial oils. However, literature about this organism is of varying degree of quality. Moreover, due to numerous changes of the species name, reports are highly scattered and poorly cited. This led to a poor integration of the findings into a unified body of knowledge. Particularly, errors in strain name usage and consequently citation are found even in most recent literature. To simplify future work, this review provides an overview of published studies and main findings regarding the metabolic capacities of C. oleaginosus.

Poplar wood hemicellulose prehydrolysate was used for microbial oil production using an oleaginous microorganism Cryptococcus curvatus. Initially, the effect of substrate concentration and nitrogen content was investigated on synthetic media. Then poplar wood prehydrolysate without detoxification was used as substrate in the fermentation. The result showed that this strain is capable of consuming both hexose and pentose sugars, a challenge in fermentation of hemicellulosic streams. It was able to accumulate 36.98% of lipid and the fermentation resulted in 13.78 g/L of biomass and 5.13 g/L of lipid under optimum conditions after 164 h of fermentation. The lipid product obtained was characterized in terms of their fatty acid profiles. Overall, this study shows that it is possible to produce microbial oil from a sustainable renewable feedstock like poplar wood hemicellulose prehydrolysate. This robust strain used has the ability to grow on industrially produced hemicellulose which can help in the development of an integrated biorefinery, where all the three components of lignocellulosic biomass are utilized. [ABSTRACT FROM AUTHOR]

Coal fly ash originated from coal combustion has high concentrations of metals. If suitable leaching techniques are identified, then coal fly ash could serve as a useful source of valuable minerals including rare earth elements (REEs). In this study, three microbial strains, Candida bombicola, Phanerochaete chrysosporium and Cryptococcus curvatus were tested on their performance of leaching trace elements and REEs from fly ash. Through comparing mineral loss and leaching efficiencies resulting from indirect leaching or use of the culture supernatant, C. bombicola was identified to be the best leading to the highest mineral loss and extracting efficiencies of trace elements and REEs among the three strains. The highest mineral loss observed from using the supernatant of this yeast strain was 59.7%. Among all trace elements, As and Mo had the highest leaching efficiency of 80.9% and 79.5%, respectively. The same leaching test led to 67.7% of Yb and 64.6% of Er dissolved from the ash. This study, thus, demonstrated that bioleaching is feasible for leaching metals out of fly ash. The C. bombicola strain deserves further investigation due to its robust actions on metal leaching. [ABSTRACT FROM AUTHOR]

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.

Mitochondrial pyruvate dehydrogenase (PDH) is important in the production of lipids in oleaginous yeast, but other yeast may bypass the mitochondria (PDH bypass), converting pyruvate in the cytosol to acetaldehyde, then acetate and acetyl CoA which is further converted to lipids. Using a metabolic model based on the oleaginous yeast Yarrowia lipolytica, we found that introduction of this bypass to an oleaginous yeast should result in enhanced yield of triacylglycerol (TAG) on substrate. Trichosporon oleaginosus (formerly Cryptococcus curvatus) is an oleaginous yeast which can produce TAGs from both glucose and xylose. Based on the sequenced genome, it lacks at least one of the enzymes needed to complete the PDH bypass, acetaldehyde dehydrogenase (ALD), and may also be deficient in pyruvate decarboxylase and acetyl-CoA synthetase under production conditions. We introduced these genes to T. oleaginosus in various combinations and demonstrated that the yield of TAG on both glucose and xylose was improved, particularly at high C/N ratio. Expression of a phospholipid:diacyltransferase encoding gene in conjunction with the PDH bypass further enhanced lipid production. The yield of TAG on xylose (0.27 g/g) in the engineered strain approached the theoretical maximum yield of 0.289 g/g. Interestingly, TAG production was also enhanced compared to the control in some strains which were given only part of the bypass pathway, suggesting that these genes may contribute to alternative routes to cytoplasmic acetyl CoA. The metabolic model indicated that the improved yield of TAG on substrate in the PDH bypass was dependent on the production of NADPH by ALD. NADPH for lipid synthesis is otherwise primarily supplied by the pentose phosphate pathway (PPP). This would contribute to the greater improvement of TAG production from xylose compared to that observed from glucose when the PDH bypass was introduced, since xylose enters metabolism through the non-oxidative part of the PPP. Yield of TAG from xylose in the engineered strains (0.21–0.27 g/g) was comparable to that obtained from glucose and the highest so far reported for lipid or TAG production from xylose.

N -acetylglucosamine (GlcNAc), the monomeric constituent of chitin, is rarely studied for lipid production by oleaginous species. This study demonstrated that Cryptococcus curvatus had a great capacity to convert GlcNAc into lipid with high yield using a two-stage production process. Optimal inoculum age and inoculation size strongly improved the two-stage lipid production efficiency. More interestingly, this process rendered superior lipid production under non-sterile condition. The acetate liberated from GlcNAc was consumed timely, while the NH 4 + released was rarely assimilated. Lipid titre, lipid content and lipid yield reached 9.9 g/L, 56.9% and 0.23 g/g, respectively, which were significantly higher than those from the conventional process where cell growth and lipid accumulation were coupled. The resulting lipid samples had similar fatty acid compositional profiles to those of vegetable oil, suggesting their potential for biodiesel production. These findings strongly supported the two-stage process as an attractive strategy for better techno-economics of the chitin-to-biodiesel routes. [ABSTRACT FROM AUTHOR]

Research and development on hydrothermal liquefaction (HTL) of wet microbial biomass have been on a dramatic rise. Although microalgae have been the main feedstocks, investigations of HTL of yeast species were few, not to mention yeast biomass cultivated on cellulosic hydrolysates. In this study, six catalysts were tested regarding their effects on yields of biocrude and biochar from partially defatted Cryptococcus curvatus . Among the six, K 2 CO 3 at 350 and 300 °C and KOH at 350 °C led to the highest yield of biocrude, 68.9%, 63.9% and 67.0%, respectively. These biocrudes had low content of sulfur and nitrogen but high HHVs in the range of 36.9 and 39.0 MJ/kg. The biocrudes from the top three running conditions were dominated by fatty acids and fatty acid esters based on GC/MS identification. The corresponding aqueous phase samples contained high concentrations of fatty acids among all that were identifiable. The successful HTL of the partially defatted yeast cell residues promises a platform where lignocellulosic sugars can be converted to biodiesel from yeast cell lipids and biocrude from the remaining yeast cells. [ABSTRACT FROM AUTHOR]

BACKGROUND Refining of renewable resources for the production of various end-products should be applied in order to develop sustainable processes and ensure the transition to the bio-economy era. With this principle in mind, a novel integrated biorefinery has been developed based on cheese whey and wine lees valorization. RESULTS Polyphenols, tartrate salts and ethanol were extracted from wine lees, while the remaining solids enriched in yeast cells were converted into nutrient-rich fermentation supplements. The composition of phenolics varied between solid and liquid fractions of wine lees. Protein concentrate was separated from cheese whey via ultrafiltration, while the concentrated lactose-rich permeate stream was supplemented with wine lees derived hydrolysates to form fermentation media for microbial oil production by Cryptococcus curvatus and Mortierella ramanniana. Functional oil containing 4.5% (w/w) of the omega-6 fatty acid γ-linolenic acid was produced in shake flask cultures by Mortierella ramanniana with total dry weight of 25.8 g L−1 and 30.6% (w/w) lipid content. Fed-batch bioreactor cultures with Cryptococcus curvatus using only crude resources led to one of the highest lipid concentrations (33.1 g L−1) reported in literature-cited publications using cheese whey. CONCLUSION This is the first study proposing the integrated refining of cheese whey and wine lees for the production of both commodity and speciality products, namely whey protein concentrate, antioxidants, ethanol, tartrate salts and microbial oil. © 2017 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Tailoring lipids from oleaginous yeasts to contain specific types of fatty acid is of considerable interest to food, fuel, and pharmaceutical industries. In this study, the essential oil obtained from Citrus sinesus L. has been used to alter the fatty acid composition of two common oleaginous yeasts, Rhodosporidium toruloides and Cryptococcus curvatus. With increasing levels of essential oil in the medium, the metabolic flux of the fatty acid biosynthesis pathway shifted towards saturated fatty acid production. Essential oil reduced the activities of elongase and ∆9 desaturase. This made the lipid obtained from both these yeasts rich in saturated fatty acids. At certain specific concentrations of the essential oil in the medium, the lipid obtained from R. toruloides and C. curvatus cultures was similar to mahuwa butter and palm oil, respectively. Limonene is the major constituents of orange essential oil. Its effect on one of the oleaginous yeasts, R. toruloides, was also studied separately. Effects similar to orange essential oil were obtained with limonene. Thus, we can conclude that limonene in orange essential oil brings about compositional change of microbial lipid produced in this organism. [ABSTRACT FROM AUTHOR]

Cutaneotrichosporon oleaginosus ATCC 20509, previously known as Trichosporon oleaginosus, Cryptococcus curvatus, Apiotrichum curvatum or Candida curvata D is an oleaginous yeast with several favorable qualities: it is fast growing, accumulates high amounts of lipid and has a very broad substrate spectrum. Its resistance to hydrolysis byproducts and genetic accessibility make it a promising cell factory for custom tailored microbial oils. However, literature about this organism is of varying degree of quality. Moreover, due to numerous changes of the species name, reports are highly scattered and poorly cited. This led to a poor integration of the findings into a unified body of knowledge. Particularly, errors in strain name usage and consequently citation are found even in most recent literature. To simplify future work, this review provides an overview of published studies and main findings regarding the metabolic capacities of C. oleaginosus. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In a world of finite resources, the transition to a sustainable post-fossil resources economy is essential. Within this concept, spent coffee grounds (SCGs) could be used for the production of bio-based chemicals, biopolymers and value-added products. Lipids and phenolics were initially extracted from SCGs. Alkaline pretreatment of the remaining SCGs solids has been optimised in this study via Central Composite Design to maximise lignin removal and overall sugar to polysaccharide conversion yield. The optimal SCGs pretreatment conditions were 0.06% (w/v) NaOH and 99.5 °C resulting in 35.7% lignin removal at 60 min pretreatment duration with low glucan and hemicellulose losses (ca. 2.5%). Enzymatic hydrolysis of the remaining SCGs alkaline pretreated solids resulted in 79.4% glucan, 74.5% mannan and 54.8% hemicellulose hydrolysis yield. The SCGs hydrolysate was used as fermentation feedstock in bioreactor cultures of Cryptococcus curvatus resulting in 36.1 g/L microbial oil with 0.25 g/g yield and 0.41 g/(L∙h) productivity. The total dry weight reached 62.7 g/L corresponding to 57.5% (w/w) intracellular lipid content. The microbial oil was rich in oleic acid (55.6%) followed by palmitic acid (18.9%) and stearic acid (14.0%). The proposed process led to the production of 7.81 g microbial oil per 100 g antioxidant- and oil-free SCGs. • Antioxidant- and oil-free SCGs were efficiently used for microbial oil production. • Optimal SCGs pretreatment conditions were 0.06% (w/v) NaOH and 99.5 °C. • Lignin removal of 35.7% with low glucan and hemicellulose losses was achieved. • Fermentation of SCGs hydrolysate led to 57.5% (w/w) intracellular lipid content. • 7.81 g microbial oil obtained per 100 g antioxidant and oil-free SCGs. [ABSTRACT FROM AUTHOR]

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 18.0 g/100 g and 0.13 g/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. [ABSTRACT FROM AUTHOR]

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 26.7 g/L containing 48–53% of lipid content. A maximal lipid productivity of 0.54 g L −1 h −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. [ABSTRACT FROM AUTHOR]

Disseminated cryptococcosis is rare but can often become severe with a poor outcome. Given recent reports that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyser is useful for Cryptococcus species identification, it was applied retrospectively to past cases of disseminated cryptococcosis at our hospital over the past 10 years, and their clinical courses were reviewed. For each case, the retained Cryptococcus spp. were used for identification using both MALDI-TOF MS and genetic sequencing, as well as for drug susceptibility testing. A total of eight cases were found. Cryptococcus spp. were found in cerebrospinal fluid in 3 cases and blood in 5 cases; anti-HIV antibody was either negative or untested. MALDI-TOF MS identified Cryptococcus neoformans as the pathogen in all 8 cases, but genetic testing identified one of these as Cryptococcus curvatus. The outcome was death within 30 days in 5 of the total 8 cases and in 2 of the 3 cases in which C. neoformans was detected in the cerebrospinal fluid, despite regimens and dosages that followed IDSA Guidelines in all 3 cases. Drug susceptibility testing showed no drug resistance that would have affected the therapy. In conclusion, the outcomes were very poor in these drug-susceptible cases, despite treatment in full accordance with standard guidelines. This study confirmed the need to develop newer therapies as well as the high capability of MALDI-TOF MS for the identification of C. neoformans. Genetic testing, however, may be necessary if non-neoformans Cryptococcus is suspected. [ABSTRACT FROM AUTHOR]

Background: Microbial lipids can represent a valuable alternative feedstock for biodiesel production in the context of a viable bio-based economy. This production can be driven by cultivating some oleaginous microorganisms on crude-glycerol, a 10 % (w/w) by-product produced during the transesterification process from oils into biodiesel. Despite attractive, the perspective is still economically unsustainable, mainly because impurities in crude glycerol can negatively affect microbial performances. In this view, the selection of the best cell factory, together with the development of a robust and effective production process are primary requirements. Results: The present work compared crude versus pure glycerol as carbon sources for lipid production by three different oleaginous yeasts: Rhodosporidium toruloides (DSM 4444), Lipomyces starkeyi (DSM 70295) and Cryptococcus curvatus (DSM 70022). An efficient yet simple feeding strategy for avoiding the lag phase caused by growth on crude glycerol was developed, leading to high biomass and lipid production for all the tested yeasts. Flow-cytometry and fourier transform infrared (FTIR) microspectroscopy, supported by principal component analysis (PCA), were used as non-invasive and quick techniques to monitor, compare and analyze the lipid production over time. Gas chromatography (GC) analysis completed the quali-quantitative description. Under these operative conditions, the highest lipid content (up to 60.9 % wt/wt) was measured in R. toruloides, while L. starkeyi showed the fastest glycerol consumption rate (1.05 g L-1 h-1). Being productivity the most industrially relevant feature to be pursued, under the presented optimized conditions R. toruloides showed the best lipid productivity (0.13 and 0.15 g L-1 h-1 on pure and crude glycerol, respectively). Conclusions: Here we demonstrated that the development of an efficient feeding strategy is sufficient in preventing the inhibitory effect of crude glycerol, and robust enough to ensure high lipid accumulation by three different oleaginous yeasts. Single cell and in situ analyses allowed depicting and comparing the transition between growth and lipid accumulation occurring differently for the three different yeasts. These data provide novel information that can be exploited for screening the best cell factory, moving towards a sustainable microbial biodiesel production. [ABSTRACT FROM AUTHOR]

The process of lignocellulosic biomass routinely produces a stream that contains sugars plus various amounts of acetic acid. As acetate is known to inhibit the culture of microorganisms including oleaginous yeasts, little attention has been paid to explore lipid production on mixtures of acetate and sugars. Here we demonstrated that the yeast Cryptococcus curvatus can effectively co-ferment acetate and sugars for lipid production. When mixtures of acetate and glucose were applied, C. curvatus consumed both substrates simultaneously. Similar phenomena were also observed for acetate and xylose mixtures, as well as acetate-rich corn stover hydrolysates. More interestingly, the replacement of sugar with equal amount of acetate as carbon source afforded higher lipid titre and lipid content. The lipid products had fatty acid compositional profiles similar to those of cocoa butter, suggesting their potential for high value-added fats and biodiesel production. This co-fermentation strategy should facilitate lipid production technology from lignocelluloses. [ABSTRACT FROM AUTHOR]

Background: Acetic 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. Results: Ten 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. Conclusions: Acetate 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. [ABSTRACT FROM AUTHOR]

The economic feasibility and environmental impact is investigated for the conversion of agricultural waste, delactosed whey permeate, through yeast fermentation to a renewable diesel via hydrothermal liquefaction. Process feasibility was demonstrated at laboratory-scale with data leveraged to validate systems models used to perform industrial-scale economic and environmental impact analyses. Results show a minimum fuel selling price of $4.78 per gallon of renewable diesel, a net energy ratio of 0.81, and greenhouse gas emissions of 30.0 g-CO 2 -eq MJ −1 . High production costs and greenhouse gas emissions can be attributed to operational temperatures and durations of both fermentation and hydrothermal liquefaction. However, high lipid yields of the yeast counter these operational demands, resulting in a favorable net energy ratio. Results are presented on the optimization of the process based on economy of scale and a sensitivity analysis highlights improvements in conversion efficiency, yeast biomass productivity and hydrotreating efficiency can dramatically improve commercial feasibility. [ABSTRACT FROM AUTHOR]

Microbial lipids produced by oleaginous yeasts serve as promising alternatives to traditional oils and fats for the production of biodiesel and oleochemicals. To improve its techno-economics, it is pivotal to use wastes and produce high quality lipids of special fatty acid composition. In the present study, four oleaginous yeasts were tested to use free fatty acids for lipid production under non-growth conditions. Microbial lipids of exceptionally high fatty acid relative contents, e.g. those contained over 70% myristic acid or 80% oleic acid, were produced that may be otherwise inaccessible by growing cells on various carbon sources. It was found that Cryptococcus curvatus is a robust strain that can efficiently use oleic acid as well as even-numbered saturated fatty acids with carbon atoms ranging from 10 to 20. Our results provided new opportunity for the production of functional lipids and for the exploitation of organic wastes rich in free fatty acids. [ABSTRACT FROM AUTHOR]

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 80 g/L biomass within 60 h with a maximal growth rate of 0.28 h −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. [ABSTRACT FROM AUTHOR]

Cell disruption is an important step used to substantially increase lipid recovery prior to extraction. This is the first study exploring the effects of cell disruption methods on lipid recovery from different oleaginous microorganisms. Specifically, we examined the effects of cell disruption methods, including autoclaving, bead-beating, microwaving, sonication, acid digestion, and Soxhlet extraction on the yeast Cryptococcus curvatus, the fungus Mortierella isabellina and the microalga Chlorella sorokiniana. Although lipid recovery by cell disruption methods varied with microbial species, autoclaving and microwaving resulted in the lowest lipid contents for all tested strains. The Soxhlet method achieved the highest lipid content of 63.4% for M. isabellina and 12.8% for C. sorokiniana, followed by acid digestion. The highest lipid content of C. curvatus was achieved at 47.3% through acid digestion, compared to sonication, bead-beating and Soxhlet methods. This study suggests that acid digestion may be the most simple and effective method for lipid recovery from the tested species in terms of time and reaction temperature. Moreover, fatty acid profile analysis revealed that lipids extracted by acid digestion were similar to those from untreated microbial cells, indicating a favorable feedstock for use in biodiesel production. Practical applications: An effective cell disruption method is critical for not only enhancing the lipid recovery in a large scale but also reporting the accurate values of lipid contents of microorganisms in a lab scale. Different cell disruption methods have different effects on lipid extraction from different microorganisms. In this work, for the first time, we performed comparative investigations among yeast, fungus, and microalga to reveal the lipid recovery yields by cell disruption methods for microbial lipid extraction from different oleaginous microorganisms and identified their most effective methods for analytical lipid determination. An effective cell disruption method is critical for enhancing lipid recovery and reporting the accurate lipid contents of microorganisms. In this study, the authors performed comparative investigations among yeast, fungus, and microalga to determine the lipid recovery yields from these oleaginous micro organisms using various cell disruption methods. They identified the most effective cell disruption methods for analytical lipid determination in each microorganism. [ABSTRACT FROM AUTHOR]

Patterns 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. [ABSTRACT FROM AUTHOR]

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 1000 days 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 0.47 g/g COD and 2.45 g/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. [ABSTRACT FROM AUTHOR]

This research explores the application of glucose and xylose from corn stover pretreated with nitric acid (HNO 3 ) for the co-production of bioethanol and biodiesel. Response surface methodology was employed to optimize HNO 3 pretreatment condition including HNO 3 concentration (0.2–1.0%), temperature (140–160 °C), and reaction time (1–10 min). The optimal reaction condition was 151.9 °C, 0.68% HNO 3 and 2.5 min, which resulted in the highest xylose yield of 77.8% and glucan content of 57.1%. Quasi-simultaneous saccharification and fermentation (Q-SSF) of pretreated corn stover with Saccharomyces cerevisiae gave an ethanol concentration of 22.4 g/L, corresponding to 69.1% theoretical ethanol yield based on initial cellulose weight. In addition, Cryptococcus curvatus in hydrolysate medium showed the lipid yield (final cell weight × lipid content) of 1.04 g/L, which was higher than those in defined medium using pure xylose. Our work demonstrates that the simultaneous production of ethanol and lipid can be one potentially promising option for lignocellulose-derived fuel production. [ABSTRACT FROM AUTHOR]

Cryptococcus curvatus , an oleaginous yeast strain was observed to grow on sweet sorghum syrup derived from sorghum juice. In a batch cultivation mode, within the tested range of total sugar concentrations from 11.7 to 34.0 g/L, no substrate inhibition to cell growth was detected. C . curvatus utilized glucose, fructose, and sucrose sequentially. In a fed-batch process, however, this yeast consumed the three sugars more rapidly and simultaneously. Within three days, a biomass density of 23.6 g/L was obtained. The overall lipid productivity was 4.0 g/L-day. Through use of microwave-assisted lipid extraction, 78.1% of available lipid was extracted out of C. curvatus cells in 4 min using methanol as the only solvent. This study demonstrated that fermentation of C . curvatus using sorghum syrup will result in high lipid yield. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background Microbial 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. Results Three 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. Conclusions The 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. [ABSTRACT FROM AUTHOR]

Microbial oils hold great potential as a suitable feedstock for the renewable production of biofuels. Specifically, the use of oleaginous yeasts offers several advantages related to cultivation and quality of lipid products. However, one of the major bottlenecks for large-scale production of yeast oils is found in the lipid extraction process. This work investigated the hydrothermal treatment of oleaginous yeast for hydrolysis and lipid extraction resulting in fatty acids used for biofuel production. The oleaginous yeast, Cryptococcus curvatus , was grown in 5 L bioreactors and the biomass slurry with 53 ± 4% lipid content (dry weight basis) was treated at 280 °C for 1 h with an initial pressure of 500 psi in batch stainless steel reactors. The hydrolysis product was separated and each of the resulting streams was further characterized. The hexane soluble fraction contained fatty acids from the hydrolysis of yeast triacylglycerides, and was low in nitrogen and minerals and could be directly integrated as feedstock into pyrolysis processing to produce biofuels. The proposed hydrothermal treatment addresses some current technological bottlenecks associated with traditional methodologies such as dewatering, oil extraction and co-product utilization. It also enhances the feasibility of using microbial biomass for production of renewable fuels and chemicals. [ABSTRACT FROM AUTHOR]