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Celldisruptionis animportantstepused tosubstantially increase lipidrecoverypriorto extraction.Thisis 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%forM.isabellinaand 12.8%forC.sorokiniana, followed byaciddigestion. The highestlipid 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.

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.

Oleaginous yeasts R. glutinis and C. curvatus were grown in batch culture on high strength distillery wastewaters from the Tequila production process (Tequila's vinasses). Both strains were able to grow either on decolorized charcoal or on naturally colored regular vinasses, with no extra nutrients addition. After 144 h of culture, lipids were accumulated up to 25.2 ± 1.98 and 27.02 ± 2.36% of cell dry weight, in C. curvatus and R. glutinis, respectively. At the same time, the chemical oxygen demand of vinasses (23,125 ± 5,557) was removed by C. curvatus and R. glutinis up to 78.98 ± 1.38 and 84.44 ± 3.52%, respectively. The fatty acid profile of the lipids accumulated by both strains was composed by myristic, palmitic, stearic, oleic, and linoleic. Linolenic acid was also found but only in the intracellular lipids of R. glutinis. This is the first report of the utilization of Tequila's vinasses for the production of microbial oil by oleaginous yeast. This process could become a strategy to transform this highly contaminant wastewater into value added lipids. © 2011 American Institute of Chemical Engineers Environ Prog, 32: 69–74, 2013.

In the first part of this review, the biochemistry of lipid accumulation in the oleaginous microorganisms is depicted. Lipid biosynthesis form sugars and related substrates is a secondary anabolic activity, conducted after essential nutrient (usually nitrogen) depletion in the medium. Due to this exhaustion, the carbon flow is directed towards the accumulation of intracellular citric acid that is used as acetyl-CoA donor in the cytoplasm. Acetyl-CoA generates cellular fatty acids and subsequently triacylglycerols. Lipid accumulation from hydrophobic substrates is a growth associated process, being independent from nitrogen exhaustion in the medium. Medium fatty acids are incorporated with various incorporation rates and are either dissimilated for growth needs or become “substrate” for intracellular biotransformations. “New” fatty acid profiles (in both extra- and intracellular lipids) that did not previously exist in the medium are likely to be produced. Oleaginous microorganisms consume their own storage lipids when their metabolic abilities cannot be saturated by the extracellular carbon source. Reserve lipid breakdown is independent from the type of the carbon source used for lipid accumulation. In most cases it is accompanied by lipid-free biomass production. Lipid mobilization is a specific process, as preferential degradation of the neutral lipid fractions is observed.

The process of lipid accumulation in the oleaginous yeasts cultivated in various fermentation configurations when either sugars and related compounds or hydrophobic substances are used as substrates is presented and kinetic models describing both de novo and ex novo lipid accumulation are analyzed. Technological aspects related with single cell oil (SCO) produced by oleaginous yeasts are depicted. The influence of culture parameters upon lipid production process is presented. Lipid production has been studied in batch, fed-batch, and continuous cultivation systems using yeasts belonging to the species Lipomyces starkeyi, Rhodosporidium toruloides, Apiotrichum curvatum, Candida curvata, Cryptococcus curvatus, Trichosporon fermentans, and Yarrowia lipolytica. The potentiality of yeasts to produce SCO as starting material of 2nd generation biodiesel is indicated and discussed. Of significant importance is also the utilization of yeast lipids as substitutes of high added value exotic fats (e.g., cocoa butter). Lipid produced by the various yeasts presents, in general, similar composition with that of common vegetable oils being composed of unsaturated fatty acids, whereas cocoa butter is principally composed of saturated fatty acids, consequently the various strategies that are followed in order to increase the cellular saturated fatty acid content of the yeast lipid are presented and comprehensively discussed.

BACKGROUND: The byproducts from shrimp processing are heads and shells which contain a wealth of carbon and could be converted into oils via oleaginous microorganisms. The objective of this investigation was to determine the feasibility of using oleaginous microorganisms to convert N-acetylglucosamine (GlcNAc), the major carbohydrate of the hydrolysate of shrimp processing waste, to triacylglycerols as a biodiesel feedstock. RESULTS: Screening experiments were conducted among Rhodotorula glutinis, Rhodococcus opacus and Cryptococcus curvatus using GlcNAc as sole carbon and energy source at 30 °C. All three microorganisms were found to grow well on GlcNAc, but the lipid contents in the cells were consistently low (lower than 5%) in the growth phase. However, lipid accumulation by C. curvatus was greatly enhanced upon entering the death phase in the absence of GlcNAc and the lipid content increased to 28.4% at 167.7 h. This indicated that C. curvatus was the optimal tested microorganism for the production of microbial oils from GlcNAc. Phosphate was further evaluated on the growth and lipid production by C. curvatus from GlcNAc. Results indicated that the yields of both biomass during growth phase and lipids at death phase increased with the increase of the ratio of C to P. But the fatty acid profiles of the accumulated lipids did not change significantly. CONCLUSION: Results indicated that shrimp processing waste could be utilized to produce oils as a biodiesel feedstock. The results could be applied to maximize production of oils from shrimp processing waste. Copyright © 2011 Society of Chemical Industry

Summary We review the prospects of using yeasts and microalgae as sources of cheap oils that could be used for biodiesel. We conclude that yeast oils, the cheapest of the oils producible by heterotrophic microorganisms, are too expensive to be viable alternatives to the major commodity plant oils. Algal oils are similarly unlikely to be economic; the cheapest form of cultivation is in open ponds which then requires a robust, fast-growing alga that can withstand adventitious predatory protozoa or contaminating bacteria and, at the same time, attain an oil content of at least 40% of the biomass. No such alga has yet been identified. However, we note that if the prices of the major plant oils and crude oil continue to rise in the future, as they have done over the past 12 months, then algal lipids might just become a realistic alternative within the next 10 to 15 years. Better prospects would, however, be to focus on algae as sources of polyunsaturated fatty acids.

The oleaginous yeast Cryptococcus curvatus is of industrial interest because it can accumulate triacylglycerols up to 60% of the cell dry weight. We are aiming at genetic modification of fatty acid biosynthesis for the production of tailor-made triacylglycerols in C. curvatus. As a first step in the development of a transformation and expression system a gene encoding the delta-9 fatty acid desaturase of C. curvatus (CBS 570) was cloned. The 1470 bp gene encodes a protein of 493 amino acids with a calculated molecular mass of 55 kDa. The gene shows strong similarity to previous cloned delta-9 desaturase genes from rat and Saccharomyces cerevisiae, 62 and 72%, respectively. Expression of the delta-9 desaturase gene was studied. Supplementation of the growth medium with oleic acid (C18:1(c9)) showed a strong repression (90%) on the mRNA level, while supplementation with petroselinic acid (C18:1(c6)) had no effect on the amount of mRNA.

The oleaginous yeast, Candida curvata D, was grown in both batch and continuous culture on 5 different carbon sources to compare the efficiency of fat production from the various substrates. Maximum lipid accumulation occurred in batch culture with xylose as the carbon source on nitrogen-limited medium reaching a level of 49% (w/w) of the biomass, but this was reduced to 37% at the optimum dilution rate (D = 0.05/hr) in a chemostat. Both the highest biomass and lipid yields were attained in continuous culture with lactose as the sole carbon source at a dilution rate of D = 0.04/hr, giving an efficiency of substrate conversion of 60 g of biomass and 18.6 g lipid per 100 g lactose utilized. The relative proportions of the major fatty acids (16:0, 18:0, 18:1, 18:2) in the lipid were found to vary considerably in batch culture and in continuous culture under carbon-limited conditions. However, on nitrogen-limited media in the chemostat, the fatty acid composition remained relatively constant over the whole range of dilution rates employed. Lipid from xylose-grown cells contained the greatest percentage of stearic acid (18:0) 15% and the lowest linoleic acid (18:2) 4%, whereas lipid from ethanol-grown cells contained elevated levels of oleic acid (18:1) 51% and decreased palmitic acid (16:0) 25%.