Effect of cyanocobalamin and p-toluic acid on the fatty acid composition of Schizochytrium limacinum (Thraustochytriaceae, Labyrinthulomycota)

Changes in the fatty acid composition of docosahexaenoic acid (DHA)-producing Schizochytrium limacinum SR21 were investigated. The addition of cyanocobalamin, which is an active component of vitamin B₁₂, decreased the content of odd-chain fatty acids such as pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0). Cyanocobalamin may upregulate the cobalamin-dependent methylmalonyl-CoA mutase, which converts propionic acid to succinic acid, thereby decreasing the content of odd-chain fatty acids. The addition of p-toluic acid resulted in a decrease in docosapentaenoic acid (DPA, 22:5n-6) content and an increase in eicosapentaenoic acid (EPA, 20:5n-3) content in a dose-dependent manner. Two additional peaks of fatty acids, characterized as Δ4,7,10,14-eicosatetraenoic acid (20:4n-7) and Δ4,7,10,14-docosatetraenoic acid (22:4n-9), were detected.     

Improvement of ferulic acid bioconversion into vanillin by use of high-density cultures of Pycnoporus cinnabarinus

High-density cultures of Pycnoporus cinnabarinus were tested with a view to optimisation of ferulic acid bioconversion into vanillin. The dry weight was increased fourfold by using glucose, fructose or a mixture of glucose and phospholipids as carbon source instead of maltose, the carbon source previously used. 5 mmol l⁻¹ vanillin, i.e. 760 mg l⁻¹, was produced over 15 days with glucose-phospholipid medium. In contrast, formation of vanillin was lower using glucose or fructose compared to the maltose control. A bioreactor (2 l) with a glucose-phospholipid medium gave a molar yield of vanillin of 61% (4 mmol l⁻¹). An alternative strategy was to grow the fungus on a glucose or fructose medium for 3 days, then switch to maltose during the bioconversion phase: this method allowed 3.3 mmol l⁻¹ vanillin to be obtained in 10 days. Many by-products such as methoxyhydroquinone and vanillyl alcohol were also produced. Received: 19 February 1999 / Received revision: 4 June 1999 / Accepted: 4 June 1999     

Propionic acid fermentation from glycerol: comparison with conventional substrates

Instead of the conventional carbon sources used for propionic acid biosynthesis, the utilization of glycerol is considered here, since the metabolic pathway involved in the conversion of glycerol to propionic acid is redox-neutral and energetic. Three strains, Propionibacterium acidipropionici, Propionibacterium acnes and Clostridium propionicum were tested for their ability to convert glycerol to propionic acid during batch fermentation with initially 20 g/l glycerol. P. acidipropionici showed higher efficiency in terms of fermentation time and conversion yield than did the other strains. The fermentation profile of this bacterium consisted in propionic acid as the major product (0.844 mol/mol), and in minimal by-products: succinic (0.055 mol/mol), acetic (0.023 mol/mol) and formic (0.020 mol/mol) acids and n-propanol (0.036 mol/mol). The overall propionic acid productivity was 0.18 g l⁻¹h⁻¹. A comparative study with glucose and lactic acid as carbon sources showed both less diversity in end-product composition and a 17% and 13% lower propionic acid conversion yield respectively than with glycerol. Increasing the initial glycerol concentration resulted in an enhanced productivity up to 0.36 g l⁻¹h⁻¹ and in a maximal propionic acid concentration of 42 g/l, while a slight decrease of the conversion yield was noticed. Such a propionic acid production rate was similar or higher than the values obtained with lactic acid (0.35 g l⁻¹h⁻¹) or glucose (0.28 g l⁻¹h⁻¹). These results demonstrated that glycerol is a carbon source of interest for propionic acid production. Received: 15 July 1996 / Received revision: 11 November 1996 / Accepted: 11 November 1996     

Large-scale expression,purification, and glucose uptake activity of recombinant human FGF21 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

As a novel important regulator of glucose and lipid metabolism homeostasis, human fibroblast growth factor 21 (hFGF21) has become a potential drug candidate for the treatment of metabolic diseases including obesity, and type 2 diabetes, as well as non-alcoholic fatty liver disease. To improve the production of recombinant hFGF21 to meet the increasing demand in clinical applications, an artificial gene encoding its mature peptide sequence was constructed, cloned into vector pET-3c and then expressed in Escherichia coli Origami B (DE3). Under optimal conditions in a 50-L fermentor, the average bacterial yield and the soluble expression level of recombinant hFGF21 of six batches attained 1750 ± 185 g and 32 ± 1.5%, respectively. The target protein was purified by the combination of nickel-nitrilotriacetic acid affinity chromatography and Sephadex S-100 resin. 5% (w/v) trehalose solution was able to prevent rhFGF21 from degradation effectively. The purity of rhFGF21 was higher than 97%, and the yield was 213 ± 17 mg/L. The preliminary biochemical characterization of rhFGF21 was confirmed using Western blot and peptide map finger analysis. Based on the glucose oxidase–peroxidase assay, the EC50 of glucose uptake activity of the purified rhFGF21 was 22.1 nM.     

Limonene bioconversion to high concentrations of a single and stable product, perillic acid, by a solvent-resistant Pseudomonas putida strain

The white-rot basidiomycete Phanerochaete chrysosporium BKM-F-1767 was tested for its capacity to degrade dehydroabietic acid (DHA). In anaerobic treatment, this molecule is the most recalcitrant member of the resin acid group, which is known to cause operational problems to anaerobic reactors treating pulp and paper industry wastewaters. In this study the effect of DHA on different parameters, such as growth, ligninolytic enzyme activity, extracellular protein production as well as both glycerol and ammonium consumption by the fungus, was determined. Although the above parameters were affected by the addition of DHA, the results show that the fungus could still produce significant titres of ligninolytic enzymes. The fungus removed 47% of the DHA initially present in the static culture, after 10 days of incubation. Anaerobic toxicity assays showed that the treatment of DHA with P. chrysosporium reduced the methanogenesis and acetogenesis inhibition caused by DHA and allowed improved methane production by the anaerobic bacteria. Received: 10 June 1997 / Received revision: 6 January 1998 / Accepted: 24 January 1998     

Analysis of the exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 grown in continuous culture on glucose and fructose

The exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 grown in defined medium were investigated. At equal cell densities, the strain produced 95 mg l⁻¹ exopolysaccharides with glucose and 30 mg l⁻¹ with fructose as the carbohydrate source. High-performance size-exclusion chromatography of the exopolysaccharides produced on glucose showed the presence of two fractions with relative molecular masses (M _r) of 1.7 × 10⁶ and 4 × 10⁴ in almost equal amounts. The exopolysaccharides produced on fructose contained mainly a fraction of low M _r of 4 × 10⁴. The high-M _r fraction of the purified exopolysaccharides produced on glucose appeared to have a sugar composition of galactose, glucose and rhamnose in the molar ratio of 5:1:1, whereas the low-M _r weight fraction contained galactose, glucose and rhamnose in the molar ratio of approximately 11:1:0.4. The purified exopolysaccharide fractions produced on fructose showed comparable ratios. The high-molecular-mass fractions contained terminally linked galactose, 1,2,3-linked galactose, 1,3,4-linked galactose, 1,3-linked glucose and terminally linked rhamnose. The low-molecular-mass fractions contained mainly 1,3-linked galactose and 1,6-linked galactose and lower amounts of other sugar linkages. The production of the high-M _r fractions appeared to be dependent on the carbohydrate source, whereas the low-M _r fractions were produced more continuously. Received: 30 April 1997 / Received revision: 11 June 1997 / Accepted: 14 June 1997     

Simultaneous consumption of glucose and fructose from sugar mixtures during batch growth of Corynebacterium glutamicum

Growth of Corynebacterium glutamicum on mixtures of glucose and fructose leads to simultaneous consumption of both sugars in which the uptake of each sugar is directly related to the expression of the corresponding sugar uptake mechanism. The overall rate of sugar uptake was higher on sugar mixtures than on either glucose or fructose alone and was similar to that observed during sucrose metabolism. The results suggest that sugar uptake limits metabolic rates though, in the case of fructose, overflow metabolism of both lactate and dihydroxyacetone was observed. Such products could reflect a higher flux through glycolysis rather than the pentose pathway during catabolism of fructose. Received: 24 October 1996 / Received revision: 10 January 1997 / Accepted: 10 January 1997     

Docosapentaenoic acid (C22:5, ω-3) production by Pythium acanthicum

The physiological roles of omega-3 fatty acids, eicosapentaenoic acid and docosahexaenoic acid have been investigated in detail and microbial strains producing these polyunsaturated fatty acids have been characterised. It has recently been suggested that docosapentaenoic acid may have an important role, especially in infant nutrition, and that its positive health effects have been overlooked. Docosapentaenoic acid (C22:5, ω-3) production by a strain of Pythium acanthicum ATCC 18660 was thus investigated. Optimum conditions for growth of P. acanthicum ATCC 18660 and docosapentaenoic acid production were: pH 6.0, temperature 20°C and incubation time, 10 days. Among different saccharides and complex nitrogen sources tested, glucose and sodium glutamate were preferred carbon and nitrogen sources, respectively. Maximum biomass content (10.4 g L⁻¹) and docosapentaenoic acid yield (49.9 mg L⁻¹) were obtained in 10 days. An increase in docosapentaenoic acid volumetric yields to 108–110 mg L⁻¹ was obtained when linseed oil was used to supplement glucose or soy flour-containing medium. Batch feeding of additional glucose or linseed oil further enhanced the docosapentaenoic acid volumetric yield to 132 mg L⁻¹ and 125 mg L⁻¹, respectively, in 14 days. The specific production of docosapentaenoic acid in preliminary experiments ranged from 1.0–5.0 mg g⁻¹ biomass. As conditions were optimised, docosapentaenoic acid specific production titers were generally in the range of 4.0–5.5 mg g⁻¹ and increases in docosapentaenoic acid volumetric production could be attributed to increased biomass production. The limited improvement obtained by modifying culture conditions indicates that increasing volumetric yields of docosapentaenoic acid by modifying culture conditions appears to represent a significant barrier to commercialisation of such a process and suggests a more fundamental manipulation of metabolism and physiology is required. Received 06 November 1997/ Accepted in revised form 10 January 1998     

Relationship between cadmium sensitivity and degree of plasma membrane fatty acid unsaturation in Saccharomyces cerevisiae

The sensitivity of Saccharomyces cerevisiae to the redox-active metal copper has recently been found to be influenced by cellular fatty acid composition. This study sought to investigate whether fatty acid composition affected plasma membrane permeabilisation and whole-cell toxicity induced by the redox-inactive metal cadmium. S. cerevisiae NCYC 1383 was enriched with the polyunsaturated fatty acids linoleate (18:2) and linolenate (18:3) by growth in 18:2- or 18:3-supplemented medium. Incorporation of the exogenous fatty acids resulted in them comprising more than 65% of the total fatty acids in plasma membrane lipids. Inhibition of cell division in the presence of Cd(NO₃)₂ was accentuated by growth in the presence of a polyunsaturated fatty acid. Furthermore, susceptibility to Cd²⁺-induced plasma membrane permeabilisation increased with the degree of fatty acid unsaturation. Thus, during exposure to Cd²⁺, K⁺ efflux from 18:2- and 18:3-enriched cells was up to 2.5-fold or 3-fold greater, respectively than that from unsupplemented cells. In addition, reductions in cell viability during exposure to Cd²⁺ were most marked in polyunsaturated-fatty-acid-supplemented cells. At certain times, unsupplemented Cd²⁺-exposed cells displayed up to 7-fold greater viability than supplemented Cd²⁺-exposed cells. The study demonstrates that the toxicity of the redox-inactive metal Cd²⁺ towards S. cerevisiae becomes markedly amplified with increased cellular and plasma membrane fatty acid unsaturation. Received: 14 March 1997 / Received revision: 4 June 1997 / Accepted: 7 June 1997     

Docosahexaenoic Acid Production and Lipid-Body Formation in Schizochytrium limacinum SR21

Schizochytrium limacinum SR21, a thraustochytrid (Labyrinturomycota), is a heterotrophic marine microorganism. SR21 has attracted recent attention because of the production of docosahexaenoic acid (DHA). We obtained highly concentrated SR21 zoospores and successfully observed synchronous growth. We investigated changes of lipid content and fatty acid composition during the growth. The morphological features of the lipid bodies were also described via fluorescent and electron microscopy. The cells developed quickly after zoospore settlement. Lipid bodies developed in accordance with an increase in lipid content during the 8-h synchronous growth. The total lipid was composed mainly of triacylglycerol, sterol esters, and phosphatidylcholine. The proportion of neutral lipids (triacylglycerol and sterol esters) in the total lipid was fairly constant during growth. The fatty acid composition of neutral lipids, primary components of the lipid body, and phospholipids, primary components of the cell membranes, was nearly unchanged during the synchronous growth. However, the DHA content of the phospholipids decreased drastically after a 10-day culture. Electron micrographs prepared using a high-pressure freeze substitution technique revealed a fine structure of light- and dark-staining bands inside the lipid bodies in many stages of the cells.     

Study of a two-stage growth of DHA-producing marine algae <Emphasis Type="Italic">Schizochytrium limacinum</Emphasis> SR21 with shifting dissolved oxygen level

The culture protocol of Schizochytrium limacinum SR 21, a known docosahexaenoic acid (DHA) producing marine algae was modified in this study to better fit fermentation parameters, particularly control of dissolved oxygen (DO) to the known reproductive and growth biology of the microorganism. The cultures controlled at 50% DO saturation produced a cell density of 181 million cells/ml, whereas cultures with 10% DO produced only 98.4 million cells/ml. A fixed-agitation rate of 150 rpm resulted in an even lower density of 22.5 million cells/ml. Fifty percent DO saturation level led to a decreased pH, as well as a negative correlation with lipid accumulation, while low oxygen concentration was obligatory for lipid accumulation. This study indicated that high DO was preferred for the cells’ reproduction via release of zoospores. Thus, the culture of S. limacinum SR21 should be best divided into two stages: (1) a cell-number-increasing stage in which cell reproduction and cell number increase with little increase in the size and weight of each cell; and (2) a cell-size-increasing stage in which cells stop reproduction but cell size enlarges due to lipids accumulation. With such a protocol, the production of algae biomass and DHA was improved to levels of 37.9 g/L and 6.56 g/L, respectively. The two-stage culture process could be potentially used not only for omega-3 PUFA production, but also in other single cell oil (SCO)-producing processes, including biodiesel production from algae.     

Propionic acid fermentation of glycerol and glucose by Propionibacterium acidipropionici and Propionibacterium freudenreichii ssp.shermanii

A comparative study was carried out in anaerobic batch cultures on 20 g/l of either glycerol or glucose using two propionibacteria strains, Propionibacterium acidipropionici and Propionibacterium freudenreichii ssp. shermanii. In all cases, fermentation end-products were the same and consisted of propionic acid as the major product, acetic acid as the main by-product and two minor metabolites, n-propanol and succinic acid. Evidence was provided that greater production of propionic acid by propionibacteria was obtained with glycerol as carbon and energy sources. P. acidipropionici showed higher efficiency in glycerol conversion to propionic acid with a faster substrate consumption (0.64 g l⁻¹ h⁻¹) and a higher propionic acid production (0.42 g l⁻¹ h⁻¹ and 0.79 mol/mol). The almost exclusive production of propionic acid from glycerol by this bacterium suggested an homopropionic tendency of this fermentation. Acetic acid final concentration was two times lower on glycerol (2 g/l) than on glucose (4 g/l) for both micro-organisms. P. freudenreichii ssp. shermanii exhibited a glycerol fermentation pattern typical of non-associated glycerol-consumption-product formation. This could indicate a particular metabolism for P. freudenreichii ssp. shermanii oriented towards the production of other specific components. These results tend to show that glycerol could be an excellent alternative to conventional carbon sources such as carbohydrates for propionic acid production. Received: 21 May 1999 / Accepted: 1 November 1999     

Regulation of lysine ɛ-aminotransferase by carbon source and lack of control by phosphate in Streptomyces clavuligerus

Cephalosporin production by Streptomyces clavuligerus is known to be negatively regulated by carbon sources, e.g., glycerol and starch, and by phosphate at high concentrations. Formation of lysine ɛ-aminotransferase (LAT) activity, the first enzyme of the biosynthetic pathway, was affected by a high concentration of carbon source. Whereas 3% starch more than doubled LAT activity production as compared to 1% starch, 3% glycerol repressed LAT activity formation by 20%–30%. LAT activity production was not affected by 100 mM K₂HPO₄. Our results thus show that the negative effects of 2% glycerol and 3% starch and 100 mM phosphate on cephalosporin production are not due to an effect on production of LAT activity. However, repression of LAT activity by 3% glycerol would be expected to play a negative role in antibiotic production. Received: 13 June 1997 / Received revision: 20 August 1997 / Accepted: 25 August 1997     

Hydrolysis of sucrose within isolated vacuoles from Solanum tuberosum L. tubers

The soluble acid invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) from potato (Solanum tuberosum L. cv. Kennebec) tubers was located in the vacuoles. Although the functionality of this invertase in the vacuoles has been assumed, the activity of the enzyme has never been shown within isolated vacuoles. Vacuoles were prepared by gentle osmotic shock from free protoplasts obtained by enzymic digestion of tuber tissues. The mean volume of these vacuoles, (0.26 ± 0.05) × 10⁻² μl, was estimated by optical microscopy. Sucrose, glucose and fructose concentrations were calculated to be 100 mM, 20 mM and 40 mM, respectively, in the vacuoles. Sucrose hydrolysis and the increase in glucose and fructose concentrations within the vacuoles were measured during vacuolar incubations. An almost identical pattern of sucrose hydrolysis by invertase was found by an in-vitro assay reproducing the vacuolar conditions. In view of the determinations of internal vacuolar pH (5.2), the possibility of spontaneous hydrolysis of sucrose was disregarded. Vacuoles were shown to be free from proteinaceous inhibitors, confirming the extravacuolar location of these inhibitors. The vacuolar hydrolytic pattern of sucrose confirms the regulatory role of the reaction products previously proposed for in-vitro assays. Received: 21 July 1997 / Accepted: 31 August 1997     

Docosahexaenoic acid (DHA) production by Thraustochytrium sp. ATCC 20892

Thraustochytrium sp. ATCC 20892 produced high yields of docosahexaenoic acid (DHA), more than four other strains of Thraustochytrium and Schizochytrium tested, but insignificant amounts of other polyunsaturated fatty acids. Glucose and sodium glutamate were the preferred carbon and nitrogen sources, respectively, and the optimum conditions for growth and DHA production were pH 7.0 at 25°C with 40 g glucose 1^-1 for 4 days. Temperature profiling under these optimum conditions further enhanced the yield and volumetric productivity of DHA.     

Eicosapentaenoic and docosahexaenoic acids production by and okara-utilizing potential of thraustochytrids

Nine thraustochytrid strains isolated from subtropical mangroves were screened for their eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) production potential in a glucose yeast extract medium. Their ability to utilize okara (soymilk residue) for growth and EPA and DHA production was also evaluated. EPA yield was low in most strains, while DHA level was high on glucose yeast extract medium, producing 28.1–41.1% of total fatty acids, for all strains, with the exception of Ulkenia sp. KF13. The DHA yield of Schizochytrium mangrovei strains ranged from 747.7 to 2778.9 mg/l after 52 h of fermentation at 25°C. All strains utilized okara as a substrate for growth, but DHA yield was lower when compared with fermentation in a glucose yeast extract medium. Journal of Industrial Microbiology & Biotechnology (2001) 27, 199–202. Received 11 December 2000/ Accepted in revised form 29 June 2001     

Optimal conditions for bioconversion of ferulic acid into vanillic acid by Pseudomonas fluorescens BF13 cells

Pseudomonas fluorescens BF13 is especially capable of promoting the formation of vanillic acid during ferulic acid degradation. We studied the possibility of enhancing the formation of this intermediary metabolite by using suspensions of cells at high density. The bioconversion of ferulic into vanillic acid was affected by several parameters, such as the concentration of the biomass, the amount of ferulic acid that was treated, the carbon source on which the biomass was grown. The optimal yield of vanillic acid was obtained with 6 mg/ml cells pre-grown on p-coumaric acid and 2 mg/ml ferulic acid. Under these conditions the bioconversion rate was 95% in 5 h. Therefore BF13 strain represents a valid biocatalyst for the preparative synthesis of vanillic acid. Received: 1 July 1997 / Received revision: 28 October 1997 / Accepted: 16 November 1997     

Exploring potential use of Australian thraustochytrids for the bioconversion of glycerol to omega-3 and carotenoids production

Marine microbes have the potential for accumulating large quantities of lipids and are therefore suitable candidate as feedstock in unsaturated fatty acid production. The efficient utilisation of glycerol as an alternative carbon source to glucose was demonstrated in the fermentation of newly isolated thraustochytrid strains from the Queenscliff, Victoria, Australia. The isolates exhibited the presence of omega-3 and omega-6 polyunsaturated fatty acids, with the major fatty acids for all isolates being (as percent total fatty acid), palmitic acid (25.1–40.78%), stearic acid (4.24–13.2%), eicosapentaenoic acid EPA (2.31–8.5%) and docosapentaenoic acid (7.24–10.9%). Glycerol as a carbon source gave promising biomass growth with significant lipid and DHA productivity. An approximate three-fold increase in carotenoid content in all isolates was achieved when glycerol was used as a carbon source in the production medium.     

Influence of acetic acid on the growth of Escherichia coli K12 during high-cell-density cultivation in a dialysis reactor

High-cell-density cultivations of Escherichia coli K12 in a dialysis reactor with controlled levels of dissolved oxygen were carried out with different carbon sources: glucose and glycerol. Extremely high cell concentrations of 190 g/l and 180 g/l dry cell weight were obtained in glucose medium and in glycerol medium respectively. Different behaviour was observed in the formation of acetic acid in these cultivations. In glucose medium, acetic acid was formed during the earlier phase of cultivation. However, in glycerol medium, acetic acid formation started later and was particularly rapid at the end of the cultivation. In order to estimate the influence of acetic acid during these high-cell-density cultivations, the inhibitory effect of acetic acid on cell growth was investigated under different culture conditions. It was found that the inhibition of cell growth by acetic acid in the fermentor was much less than that in a shaker culture. On the basis of the results obtained in these investigations of the inhibitory effect of acetic acid, and the mathematical predictions of cell growth in a dialysis reactor, the influence of acetic acid on high-cell-density cultivation is discussed. Received: 20 May 1997 / Received revision: 12 August 1997 / Accepted: 25 August 1997     

Thraustochytrid Marine Protists: Production of PUFAs and Other Emerging Technologies

Thraustochytrids, the heterotrophic, marine, straminipilan protists, are now established candidates for commercial production of the omega-3 polyunsaturated fatty acid (ω-3 PUFA), docosahexaenoic acid (DHA), that is important in human health and aquaculture. Extensive screening of cultures from a variety of habitats has yielded strains that produce at least 50% of their biomass as lipids, and DHA comprising at least 25% of the total fatty acids, with a yield of at least 5 g L⁻¹. Most of the lipids occur as triacylglycerols and a lesser amount as phospholipids. Numerous studies have been carried out on salinity, pH, temperature, and media optimization for DHA production. Commercial production is based on a fed batch method, using high C/N ratio that favors lipid accumulation. Schizochytrium DHA is now commercially available as nutritional supplements for adults and as feeds to enhance DHA levels in larvae of aquaculture animals. Thraustochytrids are emerging as a potential source of other PUFAs such as arachidonic acid and oils with a suite of PUFA profiles that can have specific uses. They are potential sources of asataxanthin and carotenoid pigments, as well as other lipids. Genes of the conventional fatty acid synthesis and the polyketide-like PUFA synthesis pathways of thraustochytrids are attracting attention for production of recombinant PUFA-containing plant oils. Future studies on the basic biology of these organisms, including biodiversity, environmental adaptations, and genome research are likely to point out directions for biotechnology explorations. Potential areas include enzymes, polysaccharides, and secondary metabolites.