Carbon Source In Medium Research Articles

Screening and Identification of Cellulase Producing Yeast from Rongkho Forest, Ubon Ratchathani University

In this present study, a total of 82 yeast strains were obtained from 111 samples collected from various sources such as soil, tree barks and insect frass from RongKho forest, Ubon Ratchathani University. They were screened and identified for cellulase production. Based on their capabilities of degrading carboxymethyl cellulose as a sole carbon source in solid medium, 45 strains performed cellulose hydrolysis which was verified by the colorless halo in the solid medium. Among them, 3 strains namely UBU-SK6, UBU-JK8 and UBU-JK9/1 showed higher levels of enzyme activity. After the step of solid medium selection, yeast cells were grown in liquid medium containing 1% yeast extract and different concentration of carboxymethyl cellulose (0.5-1.5%) under shaking with 150 rpm at 30℃ and for 168 h. The enzyme activity of these 3 strains exhibited 0.224, 0.238 and 0.26 UmL-1, respectively. Afterwards, these wild yeast strains were molecularly identified by sequencing the ITS1-5.8S-ITS2 and D1/D2 domains of the subunit (26S) ribosomal DNA. The identification of UBU-SK6, UBU-JK8 and UBU-JK9/1 showed 98-100% similarity as Candida sp. 05-7-186T, Candida easanensis and Candida sp. ST-390 respectively.

Optimization of nitrogen source for Bifidobacterium bifidum using response surface methodology

Abstract In order to improve the viable counts of Bifidobacterium bifidum BB01 in the liquid medium, the Central Composite Design (CCD) was used to optimize the nitrogen source in the medium of B. bifidum BB01. The results showed that the nitrogen source composition of B. bifidum BB01 was: peptone 0.9%, yeast extracts 0.3%, beef paste 0.7%. Under the optimal conditions, the viable counts of B. bifidum BB01 reached (2.49±0.06)×109CFU/mL after cultured at 18h, which was 42.97% higher than MRS (lactose), and 12.85% higher than the optimized MRS medium (carbon source and prebiotics were optimized). Therefore, the CCD used in this study is workable for promoting the growth of B. bifidum BB01.

Biodegradation of the Organophosphate Trichlorfon and Its Major Degradation Products by a Novel Aspergillus sydowii PAF-2.

Trichlorfon (TCF) is an important organophosphate pesticide in agriculture. However, limited information is known about the biodegradation behaviors and kinetics of this pesticide. In this study, a newly isolated fungus (PA F-2) from pesticide-polluted soils was identified as Aspergillus sydowii on the basis of the sequencing of internal transcribed spacer rDNA. This fungus degraded TCF as sole carbon, sole phosphorus, and sole carbon-phosphorus sources in a mineral salt medium (MSM). Optimal TCF degradation conditions were determined through response surface methodology, and results also revealed that 75.31% of 100 mg/L TCF was metabolized within 7 days. The degradation of TCF was accelerated, and the mycelial dry weight of PA F-2 was remarkably increased in MSM supplemented with exogenous sucrose and yeast extract. Five TCF metabolic products were identified through gas chromatography-mass spectrometry. TCF could be initially hydrolyzed to dichlorvos and then be degraded through the cleavage of the P-C bond to produce dimethyl hydrogen phosphate and chloral hydrate. These two compounds were subsequently deoxidized to produce dimethyl phosphite and trichloroethanal. These results demonstrate the biodegradation pathways of TCF and promote the potential use of PA F-2 to bioremediate TCF-contaminated environments.

Isolation of Rhodococcus sp. CMGCZ Capable to Degrade High Concentration of Fluoranthene

A bacterial strain CMGCZ was isolated from an abandoned oil field soil sample and identified as Rhodococcus sp. by 16S rRNA sequencing. Rhodococcus sp. CMGCZ was investigated for the degradation of model polycyclic aromatic hydrocarbons (PAHs) and Iranian light crude oil (ILCO) as a sole carbon source in minimal medium. Biodegradation enhancement was attained by supplementing the minimal medium with yeast extract (YE). Rhodococcus sp. CMGCZ was capable to degrade 13.2 % naphthalene (Nap), 13.1 % phenanthrene (Phe), and 99.3 % fluoranthene (Fla) in 1 week and 11 % aliphatic fraction of ILCO in 2 weeks as a sole carbon and energy source. Effect of YE supplementation on degradation potential of Rhodococcus sp. CMGCZ depended upon the added hydrocarbon in the medium. YE completely inhibited Nap degradation, slightly enhanced degradation of Phe (14.8 %) and ILCO aliphatics (13.2 %), and promoted a more rapid degradation of Fla (100 %). YE addition promoted rapid degradation of Fla and eliminated delay of 24 h in Fla degradation that was observed in minimal medium. Rhodococcus sp. CMGCZ was capable to degrade high concentrations of Fla (1000 mg l−1). Rieske [Fe2-S2] center was amplified in Rhodococcus sp. CMGCZ that exhibited homology with Rieske [Fe2-S2] domain protein of Mycobacterium species and pahAC gene of uncultured bacterium clones.

Micro-organism isolation and process optimization for lipase production

A highly lipase producing Bacillus sp. was isolated from soil under optimized culture conditions such as medium pH, temperature, incubation period, carbon sources, nitrogen sources, lipid sources and various surfactants at different concentrations. The medium pH of 7.0 and temperature of 40 °C were optimum for maximizing lipase production. The maximal yield of lipase production by Bacillus sp. was obtained after incubation periods ranging between 3 and 4 days. Casein produced maximum lipase (6.5±0.015) U/ml) as compared to others nitrogen sources and the medium containing starch was more suitable for maximum lipase (15.60±0.20) U/ml) production than other carbon sources. The gingily oil was found to be most suitable for maximizing the lipase production (20.52±0.20) U/ml). The studies on the influence of surfactants on lipase production revealed that maximum lipase production was induced by tween-20 ((27.10±0.01) U/ml).

Biodegradation of Unsymmetrical Dimethylhydrazine in Solution and Soil by Bacteria Isolated from Activated Sludge

The biodegradation effect and pathway of unsymmetrical dimethylhydrazine (UDMH), which is a major rocket propellant with highly toxic properties, with two strains isolated from the acclimated activated sludge were investigated in solution and in soil. The results demonstrated that Stenotrophomonas sp. M12 (M12) was able to degrade UDMH of 50 mg·L−1 as the sole carbon source in aqueous mineral salt medium (MSM), but could not degrade UDMH in soil. Comamonas sp. P4 (P4) barely degraded UDMH of 50 mg·L−1 as the sole carbon source in aqueous MSM, but the degrading capacity of P4 could be improved by the addition of an extra carbon source. Meanwhile, P4 was able to degrade UDMH of 100–600 mg·kg−1 in the soil. The degradation of UDMH in the soil was influenced by organic matter, autochthonous microorganisms, and metal ions. UDMH could inhibit metabolism of M12 and P4, and the inhibition influence was more severe in aqueous MSM than in soil. Oxygen content was important for M12 biodegrading UDMH, and co-metabolism helped P4 to self-detoxify and self-recover. The main intermediates of UDMH were identified by Gas Chromatography-Mass Spectrometer (GC/MS) qualitative analysis, and the concentrations of UDMH and its important transformation products were determined in solution and soil. According to the determination results, the synchronous degradation theory was proposed, and the degradation pathway was discussed.

BIOCONVERSION OF VARIOUS AGRO-INDUSTRIAL BYPRODUCTS INTO BIOETHANOL BY TWO LOCAL YEAST STRAINS USING STATIC CULTURE TECHNIQUE

Some agro-industrial byproducts [sugar cane molasses (SM), corncobs waste (CW), sugar cane bagasse (SB), sawdust (SD), sugar beet pulp (SBP) and fruit juice waste (FJW)] were used as substrates for bioethanol production by Clavispora lusitaniae Gr45 and Saccharomyces cerevisiae B1 after 3 days incubation at 30°C in static batch culture. Different treatments of these materials as a sole carbon source were applied with or without nitrogen sources (yeast extract or ammonium sulfate). The highest bioethanol concentration when SM is used (13.05 gl-1) was obtained by Sacch. cerevisiae at the treatments: [yeast fermentation medium (YFM) -glucose + 4 % SM], (yeast extract + 6 % SM) and (10% SM), which represent about 10.6 % increase comparing to YFM medium (control). The addition of acid CW hydrolyzate as the sole carbon source to YFM medium was the best of the CW treatments by Cl. lusitaniae Gr45 which recorded the highest bioethanol concentration, productivity, yield and conversion coefficient being, 13.32 gl-1, 0.18 gl-1h-1, 26.64 % and 26.65 %; respectively. These figures were increased to 15.8 gl-1, 0.22 gl-1h-1, 31.6 % and 35.9 %, respectively, on YFM medium containing acid SB hydrolyzate as the sole carbon source. All acid SD hydrolyzate treatments increased bioethanol production by Cl. lusitaniae Gr45 and decreased it by Sacch. cerevisiae B1. The highest bioethanol production obtained on SD was on SD hydrolyzate containing yeast extract followed by SD hydrolyzate only and SD hydrolyzate containing ammonium sulfate being 16.5, 13.76 and 13.54 gl-1; respectively. Using SBP as sole carbon source added to YFM medium decreased significantly the growth and bioethanol production by both strains. Generally, FJW was the best carbon source in YFM medium which increased bioethanol production to about 68 % and 38 % by Sacch. cerevisiae B1 and Cl. lusitaniae Gr45; respectively using static culture technique.

Reduction of aldehydes and terpenes within pine wood by microbial activity

Abstract Aldehydes and terpenes are the most frequently emitted volatile organic compounds (VOCs) in the wood-processing industry. These emissions are classified as injurious to health and hazardous to the environment. To address this problem, the growth conditions have been identified under which bacteria extensively degrade both aldehydes and particularly terpenes in pine wood particles as a raw material for the production of particle boards. In this study, the focus was on pentanal and hexanal (aldehydes) as well as α- and β-pinene (terpenes). Bacterial strains were selected for their ability to metabolize α-pinene as single carbon source in liquid culture medium. α-Pinene degradation was then determined by gas chromatography (GC) analysis. Strains belonging to the genus Pseudomonas showed the best results (98% degradation after 72 h). Comparably good results were also achieved with the thermophilic strain Bacillus pallidus (90% degradation) at 55°C. Furthermore, an adapted mixed culture of Pseudomonas species was inoculated onto wood particles and incubated at room temperature for 3 days. Solid-phase microextraction (SPME) measurements of emitted volatiles and subsequent gas chromatography-mass spectrometry (GC-MS) analysis indicated a complete removal (100%) of aldehydes and, even more importantly, α- and β-pinene. Pre-treatment of unsterile pine wood particles with Pseudomonas species may have potential for industrial application.

The usefulness of apricot gum as an organic additive in grapevine tissue culture media

The growth and morphogenesis of cultured plant tissues can be improved by small amounts of some organic elements. In addition to being a natural source of carbon, organic additives may contain natural vitamins, phenols, fiber, hormones and also proteins. Hence, the physiological effects of apricot gum on the regeneration capacity and growth rate of three different plant species i.e. carrot (as a model plant), stevia (as an herbaceous plant), and grapevine (as a woody plant) were examined. The proliferated callus cultures of carrot and in vitro-derived microcuttings of stevia and grapevine were inoculated on their respective standardized proliferation media supplemented with 2.0-6.0 g/l apricot gum. The growth parameters of treated samples were measured and compared to gum-free medium. Earlier callus initiations with greater fresh weight, volume, as well as improved pigmentation were recorded in media fortified with apricot gum. The usefulness of gum application was also obvious in both stevia and grapevine with respect to better shoot multiplication and rooting parameters. Due to positive effects of apricot gum, longer vines with a higher number of lateral shoots, internodes and leaf area were achieved. Overall, the gum at the rate of 4.0 g/L was found to be a logical concentration with respect to encouraging response in all three species. Owing to promising results evolved in the present research, the application of gum in commercial tissue culture protocols is highly recommended. However, further studies are needed to exploit plant derived gums as an alternative carbon source in plant tissue culture media.

Bioprospecting and evolving alternative xylose and arabinose pathway enzymes for use in Saccharomyces cerevisiae.

Bioprospecting is an effective way to find novel enzymes from strains with desirable phenotypes. Such bioprospecting has enabled organisms such as Saccharomyces cerevisiae to utilize nonnative pentose sugars. Yet, the efficiency of this pentose catabolism (especially for the case of arabinose) remains suboptimal. Thus, further pathway optimization or identification of novel, optimal pathways is needed. Previously, we identified a novel set of xylan catabolic pathway enzymes from a superior pentose-utilizing strain of Ustilago bevomyces. These enzymes were used to successfully engineer a xylan-utilizing S. cerevisiae through a blended approach of bioprospecting and evolutionary engineering. Here, we expanded this approach to xylose and arabinose catabolic pathway engineering and demonstrated that bioprospected xylose and arabinose catabolic pathways from U. bevomyces offer alternative choices for enabling efficient pentose catabolism in S. cerevisiae. By introducing a novel set of xylose catabolic genes from U. bevomyces, growth rates were improved up to 85 % over a set of traditional Scheffersomyces stipitis pathway genes. In addition, we suggested an alternative arabinose catabolic pathway which, after directed evolution and pathway engineering, enabled S. cerevisiae to grow on arabinose as a sole carbon source in minimal medium with growth rates upwards of 0.05 h(-1). This pathway represents the most efficient growth of yeast on pure arabinose minimal medium. These pathways provide great starting points for further strain development and demonstrate the utility of bioprospecting from U. bevomyces.

Bioethanol production from taro waste using thermo-tolerant yeast Kluyveromyces marxianus K21

In the present study, evaluation and optimization of taro waste (TW), which was mainly composed of taro peels that contain many starch residues, as the main carbon source in medium were studied. The flask studies showed the optimal medium was using 170g/L of TW which is about 100g/L of glucose and 9g/L of CGM as alternative nitrogen source. Simultaneous saccharification and fermentation (SSF) exhibited higher bioethanol productivity toward separation hydrolysis and fermentation (SHF). The optimal condition of SSF was 5% of Kluyveromyces marxianus K21 inoculum at 40°C resulting in the maximum ethanol concentration (48.98g/L) and productivity (2.23g/L/h) after 22h of cultivation. The scaling up experiment in a 5L bioreactor demonstrated that K21 can still maintain its capability. After 20h of cultivation, 43.78g/L of ethanol (2.19g/L/h of productivity) was achieved corresponding to a 94.2% theoretical ethanol yield.

Engineering of Corynebacterium glutamicum for growth and succinate production from levoglucosan, a pyrolytic sugar substrate.

Thermochemical processing provides continuous production of bio-oils from lignocellulosic biomass. Levoglucosan, a pyrolytic sugar substrate C6H10O5 in a bio-oil, has been used for ethanol production using engineered Escherichia coli. Here we provide the first example for succinate production from levoglucosan with Corynebacterium glutamicum, a well-known industrial amino acid producer. Heterologous expression of a gene encoding a sugar kinase from Lipomyces starkeyi, Gibberella zeae or Pseudomonas aeruginosa was employed for levoglucosan conversion in C. glutamicum because the wild type was unable to utilize levoglucosan as sole carbon source. As result, expression of a levoglucosan kinase (LGK) of L. starkeyi only enabled growth with levoglucosan as sole carbon source in CgXII minimal medium by catalyzing conversion of levoglucosan to glucose-6-phosphate. Subsequently, the lgk gene was expressed in an aerobic succinate producer of C. glutamicum, strain BL-1. The recombinant strain showed a higher succinate yield (0.25 g g(-1)) from 2% (w/v) levoglucosan than the reference strain BL-1 from 2% (w/v) glucose (0.19 g g(-1)), confirming that levoglucosan is an attractive carbon substrate for C. glutamicum producer strains. In summary, we demonstrated that a pyrolytic sugar could be a potential carbon source for microbial cell factories.

Metabolomics Analysis on Antifungal Activities Produced by Penicillium oxalicum T3.3 Grown on Different Types of Carbon Sources

Anthracnose is a disease caused by a fungal Colletotrichum gleosporioides. It is a common disease to dragon fruit plant and has caused a great loss to this industry. This pathogenic fungus can cause damage to most parts of plants including roots, stems, leaves, flowers and fruits. One of the potential microorganisms that able to produce antimicrobial activity against this plant pathogen is the endophytic fungus. However, research carried out on metabolomics of cellular metabolites produced by endophytic Penicillium oxalicum against C. gleosporioides is very limited. Thus, this study was conducted with the aims to evaluate the antifungal activities produced by Penicillium oxalicum T3.3 against Colletotrichum gloeosporiodes by using different types of carbon sources in fermentation medium and to identify the possible compounds produced by this strain through metabolomics approach. Preliminary study on locally isolated; P. oxalicum T3.3 had shown a promising antagonism through antifungal secondary metabolites which suppressed the growth of C. gloeosporioides. Disc diffusion test was conducted in order to determine the inhibition zone on the growth of C. gloeosproides. Among the six carbon sources tested, glucose exhibited the highest percent inhibition of radial growth (PIRG) of 75% and producing inhibition zone ranges from 4 to 7 mm. The minimum inhibition concentration (MIC) and minimum fungicidal concentration (MFC) of the glucose crude extract was the highest with 78 μg/mL and 2500 μg/mL, respectively. Meanwhile, starch and xylitol crude extracts gave the lowest MIC value of 1250 μg/mL and no MFC can be recorded. The compounds were extracted with ethyl acetate and 1H NMR analysis was carried out to identify candidate compounds that had been produced. Metabolomics studies were conducted by analyzing the spectrum using SIMCA P+, Principal Component Analysis (PCA) and Partial Least Square (PLS) multivariate statistical method. The conducted metabolomics study showed that sugar crude extracts (glucose, sucrose and maltose) tend to produce more threonine, 2-heptanone, lactate, valine, butyrate, O-phosphoserine. Meanwhile complex sugar substrates (xylitol, starch and Undaria pinnatifida) produced more acetic acid, methionine and leucine. From this analysis, different metabolites produced by P. oxalicum T3.3 were clustered according to the carbon sources used. This research demonstrates the potential of using a combination of 1H NMR spectroscopy and multivariate data analyses in differentiating the effect of carbon sources used based on the identification of possible metabolites contributing to their differences.

Biotransformation of tributyltin chloride to less toxic dibutyltin dichloride and monobutyltin trichloride by Klebsiella pneumoniae strain SD9

A tributyltin chloride (TBTCl) resistant bacterial strain was isolated from the Zuari estuary, in Goa, India, and identified as Klebsiella pneumoniae based on biochemical characteristics and 16S rRNA sequence analysis, and designated as strain SD9. It could utilize TBTCl as a sole carbon source in mineral salt medium and tolerated up to 2.5 mM TBTCl with maximum growth at 2 mM. Nuclear magnetic resonance spectroscopic analysis of column purified TBTCl degradation products clearly demonstrated the presence of dibutyltin dichloride (DBTCl2) and monobutyltin trichloride (MBTCl3). Mass spectrometry further confirmed degradation of toxic TBTCl into its less toxic derivatives, viz., DBTCl2 and MBTCl3. This strain also showed enhanced siderophore production in the presence of TBTCl, which was demonstrated by chrome azurol S agar assay as an increase in diameter of the orange halo around the bacterial colony in the presence of 2 mM TBTCl; this seems to be a mechanism to counteract TBTCl toxicity. Furthermore, scanning electron microscopy revealed significant morphological alterations as shrinkage in cell size along with roughness of cell surface when bacterial cells were exposed to 2 mM TBTCl. These interesting characteristics of this estuarine bacterium make it a potential tool for bioremediation of TBTCl-contaminated sites since it possesses biotransformation capability to convert TBTCl into DBTCl2 and MBTCl3.

Xylan catabolism is improved by blending bioprospecting and metabolic pathway engineering in Saccharomyces cerevisiae.

Complete utilization of all available carbon sources in lignocellulosic biomass still remains a challenge in engineering Saccharomyces cerevisiae. Even with efficient heterologous xylose catabolic pathways, S. cerevisiae is unable to utilize xylose in lignocellulosic biomass unless xylan is depolymerized to xylose. Here we demonstrate that a blended bioprospecting approach along with pathway engineering and evolutionary engineering can be used to improve xylan catabolism in S. cerevisiae. Specifically, we perform whole genome sequencing-based bioprospecting of a strain with remarkable pentose catabolic potential that we isolated and named Ustilago bevomyces. The heterologous expression of xylan catabolic genes enabled S. cerevisiae to grow on xylan as a single carbon source in minimal medium. A combination of bioprospecting and metabolic pathway evolution demonstrated that the xylan catabolic pathway could be further improved. Ultimately, engineering efforts were able to achieve xylan conversion into ethanol of up to 0.22 g/L on minimal medium compositions with xylan. This pathway provides a novel starting point for improving lignocellulosic conversion by yeast.

Production of xylanase, α-l-arabinofuranosidase, β-xylosidase, and β-glucosidase by Aspergillus awamori using the liquid stream from hot-compressed water treatment of sugarcane bagasse

Aiming to decrease the cost of enzymes used to hydrolyze biomass, the liquid stream from the hot-compressed water (HCW) treatment of sugarcane bagasse was used in the growth medium of Aspergillus awamori 2B.361 U2/1. Statistical experimental design was used to determine the HCW1 temperature and residence time for maximum hemicellulose-derived sugar recovery to the liquid stream. The HCW reactor was fed with 10 % (w/v) bagasse, and the treatment was performed under agitation using an initial pressure of 20 bar (N2) at 170 °C for 40 min. The HCW liquid stream (23.70 ± 0.30 g/L xylan) was diluted and used as the culture medium carbon source for fungal enzyme production. In growth medium containing 5 g/L xylan at initial pH 5.0, the fungus produced 46 U/mL of xylanase (EC 3.2.1.8), 3.3 U/mL of β-glucosidase (EC 3.2.1.21), 0.24 U/mL of β-xylosidase (EC 3.2.1.37), and 0.6 U/mL of α-l-arabinofuranosidase (EC 3.2.1.55) in the supernatant. The use of a residual hemicellulosic stream would decrease the cost of enzyme production and by extension favors the economics of ethanol production from lignocellulosic biomass.

Evaluation of Synthetic and Semi- synthetic Culture Media for Endo-1,4-β- Glucanases Secretion by Trichoderma koningiopsis

The actual demand of energy and the environmental concerns together with the reduced fossil fuel reserves have played an important role to convert the second generation bioethanol production into an attractive research area. To convert lignocellulosic biomass to bioethanol the cellulosic components must be hydrolyzed to fermentable sugars. Trichoderma fungi secrete large amounts of enzymes of industrial interest such as cellulases, able to degrade holocellulose in the saccharification of lignocellulosic biomass. In this work we evaluated endo-1.4-β-glucanases enzymatic secretion of Trichoderma koningiopsis from Misiones province, in synthetic medium, with carboxymethylcellulose as carbon source; and semi-synthetic medium, with pine sawdust as carbon source. Higher values of endo-1.4-β-glucanases were reached when the semi-synthetic medium was used. It could be concluded that pine sawdust seems to be a good candidate for utilization as carbon source in culture media aiming to obtain good enzyme secretion, being also an economic and easily available substrate.

Carbon Sources for Yeast Growth as a Precondition of Hydrogen Peroxide Induced Hormetic Phenotype.

Hormesis is a phenomenon of particular interest in biology, medicine, pharmacology, and toxicology. In this study, we investigated the relationship between H2O2-induced hormetic response in S. cerevisiae and carbon sources in yeast growth medium. In general, our data indicate that (i) hydrogen peroxide induces hormesis in a concentration-dependent manner; (ii) the effect of hydrogen peroxide on yeast reproductive ability depends on the type of carbon substrate in growth medium; and (iii) metabolic and growth rates as well as catalase activity play an important role in H2O2-induced hormetic response in yeast.

Enhanced Bioactive Exopolysaccharide Production by Mossy Maze Polypore, Cerrena unicolor (Higher Basidiomycetes) in Submerged Culture Conditions.

In this study, the culture requirements of the Cerrena unicolor OBCC 5005 strain were determined to optimize bioactive exopolysaccharide production in submerged culture. The effects of initial medium pH, carbon and nitrogen sources, inoculum age and amount, and mineral source on exopolysaccharide and mycelial biomass production by the C. unicolor OBCC 5005 strain were studied using a one-factor-at-a-time method. The highest exopolysaccharide production was obtained when culture parameters were used as initial medium pH: 5.5, 5% sucrose, 5% mycological peptone, and 5% of 4-day inoculants in the presence of 5 mM Fe2+. Optimized culture conditions at a flask scale were applied to a 3-L stirred tank reactor. As a result, 7.92 g/L and 7.34 g/L maximum exopolysaccharide production in optimized conditions at flask and stirred-tank reactor scales were achieved, respectively. The present study is the first to prove that C. unicolor can yield high bioactive exopolysaccharide production at flask and stirred-tank reactor scales.

Triacylglycerol Storage in Lipid Droplets in Procyclic Trypanosoma brucei.

Carbon storage is likely to enable adaptation of trypanosomes to nutritional challenges or bottlenecks during their stage development and migration in the tsetse. Lipid droplets are candidates for this function. This report shows that feeding of T. brucei with oleate results in a 4–5 fold increase in the number of lipid droplets, as quantified by confocal fluorescence microscopy and by flow cytometry of BODIPY 493/503-stained cells. The triacylglycerol (TAG) content also increased 4–5 fold, and labeled oleate is incorporated into TAG. Fatty acid carbon can thus be stored as TAG in lipid droplets under physiological growth conditions in procyclic T. brucei. β-oxidation has been suggested as a possible catabolic pathway for lipids in T. brucei. A single candidate gene, TFEα1 with coding capacity for a subunit of the trifunctional enzyme complex was identified. TFEα1 is expressed in procyclic T. brucei and present in glycosomal proteomes, Unexpectedly, a TFEα1 gene knock-out mutant still expressed wild-type levels of previously reported NADP-dependent 3-hydroxyacyl-CoA dehydrogenase activity, and therefore, another gene encodes this enzymatic activity. Homozygous Δtfeα1/Δtfeα1 null mutant cells show a normal growth rate and an unchanged glycosomal proteome in procyclic T. brucei. The decay kinetics of accumulated lipid droplets upon oleate withdrawal can be fully accounted for by the dilution effect of cell division in wild-type and Δtfeα1/Δtfeα1 cells. The absence of net catabolism of stored TAG in procyclic T. brucei, even under strictly glucose-free conditions, does not formally exclude a flux through TAG, in which biosynthesis equals catabolism. Also, the possibility remains that TAG catabolism is completely repressed by other carbon sources in culture media or developmentally activated in post-procyclic stages in the tsetse.