Lipid Production Process Research Articles

Kinetics of continuous cultivation of the oleaginous yeast Rhodosporidium toruloides

Microbial lipids are potential alternative feedstock for biofuel and oleochemical industries. The oleaginous yeast Rhodosporidium toruloides AS 2.1389 is an excellent lipid producer. To attain parameters for the understanding of the lipid production process, we performed continuous cultivation experiments under either carbon or nitrogen limitation. The maintenance coefficient and maximum cell mass yield for this yeast were determined as 5.7mg glucose/g cell/h and 0.42g cell/g glucose, respectively, under carbon limitation. Under nitrogen limitation, the highest lipid yield of 0.19g/g was observed at the dilution rate of 0.02h−1 while the highest specific lipid formation rate of 0.058g/g cell/h at the dilution rate of 0.08h−1. A kinetic model of lipid formation under steady state conditions was developed, parameters estimated, and optimal continuous cultivation conditions forecasted. These data should be very helpful to develop and design more efficient bioprocesses for microbial lipid production.

Optimization of outdoor cultivation in flat panel airlift reactors for lipid production by Chlorella vulgaris

Microalgae are discussed as a potential renewable feedstock for biofuel production. The production of highly concentrated algae biomass with a high fatty acid content, accompanied by high productivity with the use of natural sunlight is therefore of great interest. In the current study an outdoor pilot plant with five 30 L Flat Panel Airlift reactors (FPA) installed southwards were operated in 2011 in Stuttgart, Germany. The patented FPA reactor works on the basis of an airlift loop reactor and offers efficient intermixing for homogeneous light distribution. A lipid production process with the microalgae Chlorella vulgaris (SAG 211-12), under nitrogen and phosphorous deprivation, was established and evaluated in regard to the fatty acid content, fatty acid productivity and light yield. In the first set of experiments limitations caused by restricted CO₂ availability were excluded by enriching the media with NaOH. The higher alkalinity allows a higher CO₂ content of supplied air and leads to doubling of fatty acid productivity. The second set of experiments focused on how the ratio of light intensity to biomass concentration in the reactor impacts fatty acid content, productivity and light yield. The specific light availability was specified as mol photons on the reactor surface per gram biomass in the reactor. This is the first publication based on experimental data showing the quantitative correlation between specific light availability, fatty acid content and biomass light yield for a lipid production process under nutrient deprivation and outdoor conditions. High specific light availability leads to high fatty acid contents. Lower specific light availability increases fatty acid productivity and biomass light yield. An average fatty acid productivity of 0.39 g L⁻¹ day⁻¹ for a 12 days batch process with a final fatty acid content of 44.6% [w/w] was achieved. Light yield of 0.4 g mol photons⁻¹ was obtained for the first 6 days of cultivation.

Efficient conversion of biomass into lipids by using the simultaneous saccharification and enhanced lipid production process

BackgroundMicrobial lipid production by using lignocellulosic biomass as the feedstock holds a great promise for biodiesel production and biorefinery. This usually involves hydrolysis of biomass into sugar-rich hydrolysates, which are then used by oleaginous microorganisms as the carbon and energy sources to produce lipids. However, the costs of microbial lipids remain prohibitively high for commercialization. More efficient and integrated processes are pivotal for better techno-economics of microbial lipid technology.ResultsHere we describe the simultaneous saccharification and enhanced lipid production (SSELP) process that is highly advantageous in terms of converting cellulosic materials into lipids, as it integrates cellulose biomass hydrolysis and lipid biosynthesis. Specifically, Cryptococcus curvatus cells prepared in a nutrient-rich medium were inoculated at high dosage for lipid production in biomass suspension in the presence of hydrolytic enzymes without auxiliary nutrients. When cellulose was loaded at 32.3 g/L, cellulose conversion, cell mass, lipid content and lipid coefficient reached 98.5%, 12.4 g/L, 59.9% and 204 mg/g, respectively. Lipid yields of the SSELP process were higher than those obtained by using the conventional process where cellulose was hydrolyzed separately. When ionic liquid pretreated corn stover was used, both cellulose and hemicellulose were consumed simultaneously. No xylose was accumulated over time, indicating that glucose effect was circumvented. The lipid yield reached 112 mg/g regenerated corn stover. This process could be performed without sterilization because of the absence of auxiliary nutrients for bacterial contamination.ConclusionsThe SSELP process facilitates direct conversion of both cellulose and hemicellulose of lignocellulosic materials into microbial lipids. It greatly reduces time and capital costs while improves lipid coefficient. Optimization of the SSELP process at different levels should further improve the efficiency of microbial lipid technology, which in turn, promote the biotechnological production of fatty acid-derived products from lignocellulosic biomass.

Differential roles of pyruvate decarboxylase in aerial and embedded mycelia of the ascomycete Gibberella zeae

The pyruvate-acetaldehyde-acetate (PAA) pathway has diverse roles in eukaryotes. Our previous study on acetyl-coenzyme A synthetase 1 (ACS1) in Gibberella zeae suggested that the PAA pathway is important for lipid production, which is required for perithecia maturation. In this study, we deleted all three pyruvate decarboxylase (PDC) genes, which encode enzymes that function upstream of ACS1 in the PAA pathway. Results suggest PDC1 is required for lipid accumulation in the aerial mycelia, and deletion of PDC1 resulted in highly wettable mycelia. However, the total amount of lipids in the PDC1 deletion mutants was similar to that of the wild-type strain, likely due to compensatory lipid production processes in the embedded mycelia. PDC1 was expressed both in the aerial and embedded mycelia, whereas ACS1 was observed only in the aerial mycelia in a PDC1-dependent manner. PDC1 is also involved in vegetative growth of embedded mycelia in G.zeae, possibly through initiating the ethanol fermentation pathway. Thus, PDC1 may function as a key metabolic enzyme crucial for lipid production in the aerial mycelia, but play a different role in the embedded mycelia, where it might be involved in energy generation by ethanol fermentation.

Modeling growth, lipid accumulation and lipid turnover in submerged batch cultures of Umbelopsis isabellina

The production of lipids by oleaginous yeast and fungi becomes more important because these lipids can be used for biodiesel production. To understand the process of lipid production better, we developed a model for growth, lipid production and lipid turnover in submerged batch fermentation. This model describes three subsequent phases: exponential growth when both a C-source and an N-source are available, carbohydrate and lipid production when the N-source is exhausted and turnover of accumulated lipids when the C-source is exhausted. The model was validated with submerged batch cultures of the fungus Umbelopsis isabellina (formerly known as Mortierella isabellina) with two different initial C/N-ratios. Comparison with chemostat cultures with the same strain showed a significant difference in lipid production: in batch cultures, the initial specific lipid production rate was almost four times higher than in chemostat cultures but it decreased exponentially in time, while the maximum specific lipid production rate in chemostat cultures was independent of residence time. This indicates that different mechanisms for lipid production are active in batch and chemostat cultures. The model could also describe data for submerged batch cultures from literature well.

Lipids of oleaginous yeasts. Part II: Technology and potential applications

AbstractThe 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.

The proteome analysis of oleaginous yeast Lipomyces starkeyi

Oleaginous yeast Lipomyces starkeyi, a species in the Saccharomycetales order, has the capability to accumulate over 70% of its cell biomass as lipid under defined culture conditions. In this study, analysis of L. starkeyi AS 2.1560 proteome samples from different culture stages during a typical lipid production process was performed using an online multidimensional μRPLC/MS/MS method. Data searching against the proteome database of the yeast Saccharomyces cerevisiae led to the identification of 289 protein hits. Further comparative and semi-quantitative analysis under more stringent criteria revealed 81 proteins with significant expression-level changes. Among them, 52 proteins were upregulated and 29 proteins were downregulated. Gene ontology annotation indicated that global responses occurred when cells were exposed to the nitrogen deficiency environment for lipid production. Protein hits were annotated and largely concerned metabolic processes for alternative nitrogen sources usage or lipid accumulation. Many of the downregulated proteins were related to glycolysis, whereas the majority of the upregulated proteins were involved in proteolysis and peptidolysis, carbohydrate metabolism and lipid metabolism. Insights were provided in terms of cellular responses to nutrient availability as well as the basic biochemistry of lipid accumulation. This work presented potentially valuable information for understanding the biochemical events related to microbial oleaginity and rational engineering of oleaginous yeasts.

Comparative proteomic analysis of Rhodosporidium toruloides during lipid accumulation

Intracellular lipid accumulation is a common biological process for some eukaryotic microorganisms under specific growth conditions, yet study on this phenomenon at an '-omics' level remains rare. In this study we induced lipid accumulation by the oleaginous yeast Rhodosporidium toruloides by transferring cells into a nitrogen-limited medium and performed a comparative and semi-quantitative proteomic analysis of cell samples obtained thereafter by a 2D-LC-MS/MS approach. A total of 184 proteins were identified, based on the database of yeast Saccharomyces cerevisiae. Semi-quantitative analysis suggested that 46 proteins were notably changed during the lipid production process. Among them, seven, three and four proteins were significantly upregulated only at the late stage, the early stage and both stages, respectively. There were 26 proteins drastically downregulated at both stages. The majority of the downregulated proteins are related to protein metabolism and carbohydrate metabolism, whereas the upregulated proteins are mainly involved in alternative nitrogen sources metabolism and lipid biosynthesis. Our data indicated that a nitrogen deficiency environment had a key impact on cellular metabolism that likely stimulated the lipid accumulation process by R. toruloides. This work provids valuable information for further exploration of the molecular mechanism of cellular lipid metabolism and should be of great interest in oleaginous microorganisms engineering.

Extracellular matrix stimulates production and breakdown of inositol phospholipids.

Adhesion of rat glomerular epithelial cells (GEC) to collagen stimulates production of D-myo-inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol. This process is mediated via beta 1-integrins, and it modulates GEC proliferation. In this study, we address the changes in inositol-lipid turnover induced by GEC adhesion to extracellular matrix (ECM). The masses of both phosphatidylinositol 4,5-bisphosphate (PIP2) and IP3, as well as [3H]inositol phosphates, were increased in GEC adherent to collagen, compared with plastic substratum. Phosphatidylinositol-4-phosphate (PIP) 5-kinase activity was predominantly membrane associated and was enhanced in GEC on collagen. Phospholipase C (PLC) activity and PLC-gamma 1 protein were increased in membrane fractions of GEC adherent to collagen, compared with plastic. Stable overexpression of PLC-gamma 1 in GEC amplified the effect of ECM on the production of [3H]inositol phosphates. In addition, the PLC-gamma 1 that was membrane associated in collagen-adherent GEC was tyrosine phosphorylated. Thus production of IP3 in GEC adherent to ECM is associated with increased production of PIP2. Moreover, adhesion to ECM increases tyrosine phosphorylation and membrane association of PLC-gamma 1, which may facilitate PIP2 hydrolysis by increasing the catalytic activity of PLC-gamma 1 and the proximity of PLC-gamma 1 and its substrate. Understanding the process of ECM-induced inositol lipid production and breakdown in GEC may provide insights into the regulation of GEC proliferation and differentiated functions in normal conditions and during glomerular injury.