Biodiesel production from oleaginous microorganisms

Fermentation of lignocellulosic hydrolysates for ethanol production.

The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms

Production of bioethanol from forest and agricultural products requires a fermenting organism that converts all types of sugars in the raw material to ethanol in high yield and with a high rate. This review summarizes recent research aiming at developing industrial strains of Saccharomyces cerevisiae with the ability to ferment all lignocellulose-derived sugars. The properties required from the industrial yeast strains are discussed in relation to four benchmarks: (1) process water economy, (2) inhibitor tolerance, (3) ethanol yield, and (4) specific ethanol productivity. Of particular importance is the tolerance of the fermenting organism to fermentation inhibitors formed during fractionation/pretreatment and hydrolysis of the raw material, which necessitates the use of robust industrial strain background. While numerous metabolic engineering strategies have been developed in laboratory yeast strains, only a few approaches have been realized in industrial strains. The fermentation performance of the existing industrial pentose-fermenting S. cerevisiae strains in lignocellulose hydrolysate is reviewed. Ethanol yields of more than 0.4 g ethanol/g sugar have been achieved with several xylose-fermenting industrial strains such as TMB 3400, TMB 3006, and 424A(LNF-ST), carrying the heterologous xylose utilization pathway consisting of xylose reductase and xylitol dehydrogenase, which demonstrates the potential of pentose fermentation in improving lignocellulosic ethanol production.

Towards industrial pentose-fermenting yeast strains

A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae

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 nitrogenlimited 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%.

A comparison of the oleaginous yeast,Candida curvata, grown on different carbon sources in continuous and batch culture

The effect of various nitrogen sources on lipid accumulation by 17 species and strains of yeast was examined. Organic nitrogen sources resulted in considerably increased lipid contents only in strains of Rhodosporidium toruloides. Lipid accumulation in Rs. toruloides CBS 14 increased from 18% (w/w), with NH4Cl as nitrogen source, to above 50% (w/w) when glutamate, urea or arginine was used. Stimulation of lipid production by glutamate was not observed when the yeast was grown in continuous culture with nitrogen-limiting medium. The increase in lipid content of glutamate-grown cells in batch culture was accompanied by a marked increase in the intracellular citrate concentration and its excretion from the cells. The pattern of citrate accumulation in glutamate-grown cells was mirrored by the accumulation of other metabolites, especially 2-oxoglutarate and NH+ 4 ions, which were produced as a result of the increased catabolism of glutamate. It is proposed that the products of glutamate metabolism in Rs. toruloides play a major role in regulating the flux of carbon to precursors of lipid biosynthesis, such as citrate.

Effect of Nitrogen Source on Lipid Accumulation in Oleaginous Yeasts

Citrate efflux from mitochondria of 10 different yeasts was investigated.



1
Isolated mitochondria of all the yeasts show the possession of a carrier system for dicarboxylic and tricarboxylic anions, phosphate and pyruvate.

2
The tricarboxylate carrier is also specific for l-malate in all 10 yeasts.

3
In citrate-loaded mitochondria, citrate efflux and l-malate uptake are directly proportional to the concentration of extramitochondrial l-malate until the translocator becomes saturated (> 8 mM l-malate in Candida curvata D).

4
Rates of citrate efflux in the presence of l-malate are linear for a period of 2 min and are approximately 2.5-times greater in oleaginous than in non-oleaginous yeasts.

5
The malate-citrate translocator has a significantly lower Km for l-malate in oleaginous yeasts (∼ 4.0 mM) than in non-oleaginous yeasts (∼ 8.4 mM).

6
Both pyruvate and phosphate appear to stimulate the malate-citrate exchange in mitochondria of C. curvata D.

7
Oleginous yeast mitochondria contain 3—4-times higher intramitochondrial citrate levels than non-oleaginous yeast. In all these yeasts the enzymes for synthesis and metabolism of citrate were exclusively mitochondrial, the only exception being the possession of a cytosolic ATP; citrate lyase by oleaginous yeasts.

8
In metabolically active mitochondria, once the malate-citrate carrie is saturated with l-malate, efflux of citrate is directly proportional to the concentration of extramitochondrial pyruvate (as acetyl donor).

9
The rates of efflux of newly synthesised citrate from metabolically active mitochondria, in the presence of saturating l-malate and pyruvate, are 6–8-times greater in oleaginous yeasts and the citric-acid-producing yeast.

10
The inhibition of aconitase by fluorocitrate produced a ninefold increase in citrate efflux from non-oleaginous yeast mitochondria but only a slight increase in oleaginous yeasts.

11
It is concluded that in fully functional yeast mitochondria, citrate efflux is limited by the amount of intra-mitochondrial citrate made available to the citrate translocator for exchange.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1983.tb07136.x

A Comparative Study of Citrate Efflux from Mitochondria of Oleaginous and Non-oleaginous Yeasts

Activity of a pentulose (xylulose 5-phosphate) phosphoketolase was detected in 20 out of 25 yeasts examined. No significant activity was detected in any yeast grown with glucose, and the enzyme was induced by up to 70-fold when the yeasts were grown on xylose as sole carbon source. Biomass yields from xylose were greater than, or approximately equal to, those from glucose in 15 of the 19 yeasts which possessed phosphoketolase activity. The molar yield of C2 units from xylose, by metabolism via the pentose phosphate pathway, can be calculated to be insufficient to account for the high yields of biomass and ethanol obtained from xylose. We have shown that the presence of a phosphoketolase system can account for such yields by producing 2 mol C2 from 1 mol C5. This pathway must therefore be regarded as a major route of pentose dissimilation in such yeasts.

Induction of xylulose-5-phosphate phosphoketolase in a variety of yeasts grown ond-xylose: the key to efficient xylose metabolism

Rhodosporidium toruloides CBS 14 was grown in batch culture with urea as principal nitrogen source. The lipid content increased from 18% (w/w) with NH+
4-grown cells to 52% (w/w) after 90 h growth. Urea was rapidly taken up and catabolized to release intracellular NH+
4, which accumulated between 10 and 30 h growth. The increase in pool NH+
4 content was mirrored by an increase in citric acid accumulation and excretion from the cells. The production of intracellular NH+
4, sufficient to permit lipid accumulation, could be attributed to the increase in activity of urease over this period. Similarly, other catabolic enzymes, such as arginase, threonine dehydratase and NAD+: glutamate dehydrogenase, were also induced (or derepressed) when the respective amino acids were used as medium nitrogen source. Growth with mixed organic and inorganic nitrogen compounds considerably decreased the lipid content and was accompanied by a reduction in activity of the various catabolic enzymes concerned. The significance of nitrogen catabolism during lipid accumulation in this yeast is discussed.

/pdf/influence-of-nitrogen-metabolism-on-lipid-accumulation-by-3dajf9bajj.pdf

Christopher T. Evans

Papers

A comparison of the oleaginous yeast,Candida curvata, grown on different carbon sources in continuous and batch culture

Effect of Nitrogen Source on Lipid Accumulation in Oleaginous Yeasts

A Comparative Study of Citrate Efflux from Mitochondria of Oleaginous and Non-oleaginous Yeasts

Induction of xylulose-5-phosphate phosphoketolase in a variety of yeasts grown ond-xylose: the key to efficient xylose metabolism

Influence of Nitrogen Metabolism on Lipid Accumulation by Rhodosporidium toruloides CBS 14