Thamnidium Elegans Research Articles

Polyunsaturated Fatty Acids from Thamnidium elegans and Mortierella alpina Suppress Prostate Cancer Cell Proliferation and Migration

Thamnidium elegans and Mortierella alpina are two oleaginous fungi that belong to Mucoromycota that synthesize polyunsaturated fatty acids, which are credited with multiple health benefits and possible anticancer properties. These fungi were cultivated on culture media, with glucose or glycerol as a carbon source. After extracting the lipids, we transformed them into fatty acid lithium salts (FALSs), which are water-soluble and absorbable mammalian cells, including DU-145 and PC-3 cancer cells. The two cell lines, both long-established prostate cancer models, were treated with FALSs and indicated increased susceptibility to the lipid derivatives. The viability and proliferation rates were significantly reduced, as well as their migratory capabilities, which were significantly impaired compared to olive oil-derived FALS, which was used as a control substance. We conclude that the FALS derivatives of microbial lipids from these organisms exhibit anticancer effects, by suppressing the proliferation and migration of human prostate cancer cell lines.

Microbial Oil Production from Alkali Pre-Treated Giant Reed (Arundo donax L.) by Selected Fungi

This study aimed to evaluate the microbial oil production by three selected strains, Mortierella isabellina, Cunninghamella echinulata, and Thamnidium elegans, after fermentation of an enzymatic hydrolysate from alkali pre-treated giant reed biomass, in comparison to a semi-synthetic medium, at three increasing nitrogen supplementation levels (0.14, 0.25, and 0.47 g/L). M. isabellina showed the fastest sugar consumption, the highest final cell and oil concentrations (10.9 and 5.6 g/L, respectively), as well as the highest cellular oil content, oil yield/g sugar consumed, and oil productivity (63.4%, 0.19 g/g, and 1 g/L/day, respectively) in the giant reed hydrolysate. The oil yield tended to decrease with an increasing nitrogen content in the cultures. Oleic acid was the most copious fatty acid in the oil for all the fungi. On the contrary, T. elegans exhibited the poorest performances. In particular, M. isabellina showed, respectively, the highest and lowest values of oleic and γ-linolenic acid (52.2 and 3.1%, on average). In comparison, C. echinulata and T. elegans showed much higher γ-linolenic acid content (15.3 and 21.6%, on average). Notably, the C. echinulata cultures showed by far the highest γ-linolenic acid concentration in both substrates (345 and 595 g/L in the giant reed hydrolysate and in the synthetic medium, respectively). Finally, the estimated biodiesel properties of all the oils fell within the limits of the U.S. standards, while the oil of M. isabellina only respected the tighter limits fixed by the E.U. regulations.

A Green Biocomposite Produced by Passive Cell Immobilization onto Waste Biomass Support for Biodecolorization of Reactive Dye Contamination

AbstractThe waste biomass of grape pomace was utilized for the immobilization of model fungal cells (Thamnidium elegans) providing a green, innovative, and practical biocomposite for efficient biodecolorization of a model reactive dye (Reactive Yellow 2). Appreciable decolorization capacity at optimized conditions was observed as qmax=151.0 mg g‐1 with the suggested biocomposite (TECWGP). During the batch process, the biocomposite exhibited the highest dye removal of 98.57 % in the RY2‐contaminated solutions. The biosorptive decolorization process was the best predicted by the Langmuir isotherm and the pseudo‐second‐order kinetic models. The possible interactions between RY2 and TECWGP were also examined. Synthetic wastewater treatment experiments indicated that TECWGP has a good decolorization potential. Overall, the findings indicated that TECWGP could be a good alternative to synthetic materials for the treatment of reactive dye‐contaminated aqueous phase. Furthermore, the utilization of agro‐industrial waste to support fungal cells contributes to the growth of a solid circular bio‐based economy.

A hybrid biocomposite of Thamnidium elegans/olive pomace/chitosan for efficient bioremoval of toxic copper

Immobilized biomaterials have recently attracted researchers' attention in the field of environmental biotechnology due to their effective biosorption performances. In this respect, a novel hybrid biocomposite based on Thamnidium elegans cells, olive pomace, and chitosan (TE-OP@C) was produced and tested for the first time to remove a target pollutant. It was successfully employed to eliminate toxic Cu(II) ions. Uptake efficiency of the biocomposite was significantly greater than that of T. elegans and T. elegans-olive pomace, despite the much lesser amount of biocomposite used. Freundlich model best fitted the equilibrium data, and the pseudo-second-order kinetic model followed Cu(II) uptake. The maximum removal efficiencies in batch and continuous systems were determined to be 96 % and 98 %, respectively. After eight cycles, the biosorption and recovery efficiencies of TE-OP@C were higher than 90 %. Biocomposite was able to remove approximately 90 % and 88 % of Cu(II) from real wastewater in batch and continuous systems, respectively. FTIR analysis, zeta potential measurements, EDX, and SEM findings confirmed the Cu(II) uptake. XRD and BET analysis were also performed for biocomposite characterization. Breakthrough and exhausted points were determined as 80 and 150 min, respectively. The findings potentially lead to a new perspective on the treatment of copper contamination.

Latent Functions and Applications of Cytochrome P450 Monooxygenases from Thamnidium elegans: A Novel Biocatalyst for 14α-Hydroxylation of Testosterone.

Cytochrome P450 monooxygenases (P450s) are ubiquitous enzymes with high availability and diversity in nature. Fungi provide a diverse and complex array of P450s, and these enzymes play essential roles in various secondary metabolic processes. Besides the physiological impacts of P450s on fungal life, their versatile functions are attractive for use in advanced applications of the biotechnology sector. Herein, we report gene identification and functional characterization of P450s from the zygomycetous fungus Thamnidium elegans (TeCYPs). We identified 48 TeCYP genes, including two putative pseudogenes, from the whole-genome sequence of T. elegans. Furthermore, we constructed a functional library of TeCYPs and heterologously expressed 46 TeCYPs in Saccharomyces cerevisiae. Recombinants of S. cerevisiae were then used as whole-cell biocatalysts for bioconversion of various compounds. Catalytic potentials of various TeCYPs were demonstrated through a functionomic survey to convert a series of compounds, including steroidal substrates. Notably, CYP5312A4 was found to be highly active against testosterone. Based on nuclear magnetic resonance analysis, enzymatic conversion of testosterone to 14α-hydroxytestosterone by CYP5312A4 was demonstrated. This is the first report to identify a novel fungal P450 that catalyzes the 14α-hydroxylation of testosterone. In addition, we explored the latent potentials of TeCYPs using various substrates. This study provides a platform to further study the potential use of TeCYPs as catalysts in pharmaceutical and agricultural industries and biotechnology.

Microbial Transformation of (–)-α-Bisabolol Towards Bioactive Metabolites

Bisabolol is one of the bioactive constituents of chamomile. It was aimed to biotransform (–)-α-bisabolol by different fungi for the production of new bioactive metabolites, which was converted to α-bisabolol oxide A and B by Thamnidium elegans. Additionally, the biotransformation by Penicillium neocrassum yielded a new metabolite, which was characterized as 2-(5-methyl-5-(6-methyl-7-oxabicyclo[4.1.0]heptan-3-yl)tetrahydrofuran-2-yl)propan-2-ol = bisafuranol. The substrate and its metabolite mixtures were tested using antioxidant DPPH• scavenging assay. Antimicrobial activity was evaluated by an in vitro microdilution assay against a panel of human pathogenic bacteria and yeasts resulting in the lowest MIC and MFC values of 150 μg/mL. (–)-α-Bisabolol was effective against Propionibacterium acnes and Staphylococcus epidermidis (75, 37.5 μg/mL, respectively). The antioxidant activity of the metabolites was found to be more effective in scavenging free radicals.

Metabolic Potential, Ecology and Presence of Associated Bacteria Is Reflected in Genomic Diversity of Mucoromycotina.

Mucoromycotina are often considered mainly in pathogenic context but their biology remains understudied. We describe the genomes of six Mucoromycotina fungi representing distant saprotrophic lineages within the subphylum (i.e., Umbelopsidales and Mucorales). We selected two Umbelopsis isolates from soil (i.e., U. isabellina, U. vinacea), two soil-derived Mucor isolates (i.e., M. circinatus, M. plumbeus), and two Mucorales representatives with extended proteolytic activity (i.e., Thamnidium elegans and Mucor saturninus). We complement computational genome annotation with experimental characteristics of their digestive capabilities, cell wall carbohydrate composition, and extensive total lipid profiles. These traits inferred from genome composition, e.g., in terms of identified encoded enzymes, are in accordance with experimental results. Finally, we link the presence of associated bacteria with observed characteristics. Thamnidium elegans genome harbors an additional, complete genome of an associated bacterium classified to Paenibacillus sp. This fungus displays multiple altered traits compared to the remaining isolates, regardless of their evolutionary distance. For instance, it has expanded carbon assimilation capabilities, e.g., efficiently degrades carboxylic acids, and has a higher diacylglycerol:triacylglycerol ratio and skewed phospholipid composition which suggests a more rigid cellular membrane. The bacterium can complement the host enzymatic capabilities, alter the fungal metabolism, cell membrane composition but does not change the composition of the cell wall of the fungus. Comparison of early-diverging Umbelopsidales with evolutionary younger Mucorales points at several subtle differences particularly in their carbon source preferences and encoded carbohydrate repertoire. Nevertheless, all tested Mucoromycotina share features including the ability to produce 18:3 gamma-linoleic acid, use TAG as the storage lipid and have fucose as a cell wall component.

Structural and Functional Characterization of Three Novel Fungal Amylases with Enhanced Stability and pH Tolerance.

Amylases are probably the best studied glycoside hydrolases and have a huge biotechnological value for industrial processes on starch. Multiple amylases from fungi and microbes are currently in use. Whereas bacterial amylases are well suited for many industrial processes due to their high stability, fungal amylases are recognized as safe and are preferred in the food industry, although they lack the pH tolerance and stability of their bacterial counterparts. Here, we describe three amylases, two of which have a broad pH spectrum extending to pH 8 and higher stability well suited for a broad set of industrial applications. These enzymes have the characteristic GH13 α-amylase fold with a central (β/α)8-domain, an insertion domain with the canonical calcium binding site and a C-terminal β-sandwich domain. The active site was identified based on the binding of the inhibitor acarbose in form of a transglycosylation product, in the amylases from Thamnidium elegans and Cordyceps farinosa. The three amylases have shortened loops flanking the nonreducing end of the substrate binding cleft, creating a more open crevice. Moreover, a potential novel binding site in the C-terminal domain of the Cordyceps enzyme was identified, which might be part of a starch interaction site. In addition, Cordyceps farinosa amylase presented a successful example of using the microseed matrix screening technique to significantly speed-up crystallization.

Corrigendum to “The feasibility of Thamnidium elegans cells for color removal from real wastewater” [Process Saf. Environ. Prot. 105 (2017) 316–325

Corrigendum to “The feasibility of Thamnidium elegans cells for color removal from real wastewater” [Process Saf. Environ. Prot. 105 (2017) 316–325

Anionically reinforced hydrogel network entrapped fungal cells for retention of cadmium in the contaminated aquatic media

A novel biomass/polymer composite was fabricated by embedding Thamnidium elegans cells in acrylic network of p(3-Methoxyprophyl)acrylamide p(MPA) enriched with 2-Akrylamido-2-methyl-1-propane sulfonic acid (AMPS). Cd(II) retention potential of hydrogel (p(MPA-co-AMPS)) increased by 20.66% times after this enrichment. The gel matrix could be effectively entrapped the biomass and resulting sorbent applied to remove Cd(II) from water in batch and continuous modes. The main physico-chemical parameters are discussed in addition to characterization, regeneration and application studies of the suggested sorbent. Equilibrium occurred within 30 min and Langmuir model predicted the equilibrium data. Kinetics of Cd(II) removal onto immobilized biomass is modeled using the pseudo-second-order rate equation. Maximum monolayer sorption capacity was estimated to be 123.76 mg g−1 at 25 °C. Designed composite was successfully applied for the removal of Cd(II) from industrial wastewater. EDTA and HNO3 can be efficiently used for Cd(II) recovery and composite sorbent recycled for at least 12 cycles with nearly stable sorption performance.

The feasibility of Thamnidium elegans cells for color removal from real wastewater

Abstract Biosorptive treatment of contaminated solutions with different biomaterials has been extensively studied in recent years. However, application of the suggested biosorbents in industrial scale has been very limited so far. Real wastewater conditions play an important role in the commercial success of the biosorption process. This study describes the potential use of Thamnidium elegans (T. elegans) cells for the biosorptive treatment of real industrial wastewater. Biosorption mechanism was characterized by zeta potential, Brunauer, Emmett and Teller (BET), Infrared (IR) spectrometry, Atomic force microscopy (AFM) and elemental analysis. A quadratic model was built for the batch process and optimum values of variables were recorded as pH 2.0, biosorbent amount: 0.06 g and contact time: 39.3 min with the removal yield of 99.42%. Isotherm studies indicated that the process follows the Langmuir model. Relatively fast decolorization (40 min) process was well described by the pseudo-second-order kinetics. Conversely, the biosorbent exhibited high biosorption efficiency (97.55%) in continuous mode. In addition to high batch biosorption capacity (288.08 mg g−1) of the biosorbent, another impressive aspect of this study, even after ten regeneration cycles, is high biosorption yield (95%). Consequently, T. elegans cells showed great potential for the treatment of colored real wastewaters.

Biosynthesis of Essential Polyunsaturated Fatty Acids in Wheat Triggered by Expression of Artificial Gene.

The artificial gene D6D encoding the enzyme ∆6desaturase was designed and synthesized using the sequence of the same gene from the fungus Thamnidium elegans. The original start codon was replaced by the signal sequence derived from the wheat gene for high-molecular-weight glutenin subunit and the codon usage was completely changed for optimal expression in wheat. Synthesized artificial D6D gene was delivered into plants of the spring wheat line CY-45 and the gene itself, as well as transcribed D6D mRNA were confirmed in plants of T0 and T1 generations. The desired product of the wheat genetic modification by artificial D6D gene was the γ-linolenic acid. Its presence was confirmed in mature grains of transgenic wheat plants in the amount 0.04%–0.32% (v/v) of the total amount of fatty acids. Both newly synthesized γ-linolenic acid and stearidonic acid have been detected also in leaves, stems, roots, awns, paleas, rachillas, and immature grains of the T1 generation as well as in immature and mature grains of the T2 generation. Contents of γ-linolenic acid and stearidonic acid varied in range 0%–1.40% (v/v) and 0%–1.53% (v/v) from the total amount of fatty acids, respectively. This approach has opened the pathway of desaturation of fatty acids and production of essential polyunsaturated fatty acids in wheat.

Production of polyunsaturated single cell oils possessing antimicrobial and anticancer properties

Natural bioactive agents, with antimicrobial and/or anticancer activity, such as polyunsaturated fatty acids (PUFAs), are at the forefront of biotechnological research due to the high interest in these compounds in pharmacy. This paper reports production of polyunsaturated lipids by the fungus Thamnidium elegans cultivated on raw glycerol, and the microalga Nannochloropsis salina cultivated under autotrophic conditions. Fungal lipids, containing gamma linolenic acid, consisted of 82 % neutral lipids. Algal lipids, containing eicosapentaenoic acid (EPA), consisted of 52.7 % glycolipids plus sphingolipids, 28.5 % neutral lipids and 18.8 % phospholipids. EPA was located mainly in polar lipids. Fatty acid potassium salts (FAPS) produced from the above lipids effectively inhibited the growth of many Gram negative bacteria [such as Serratia sp., Neisseria gonorrhoeae, Shigella sonnei, Proteus mirabilis, P. vulgaris, Escherichia coli ATCC BAA1001 Enterobacter cloacae, Klebsiella pneumoniae and Salmonella typhimurium but not E. coli ATCC 11229 and NCTC 12241 (ATCC 25922) and Moraxella catarrhalis], Gram positive bacteria (i.e. Micrococcus luteus, Staphylococcus aureus ATCC 25923 and ATCC 43330, S. epidermidis, S. saprophyticus, Enterococcus faecalis but not S. aureus ATCC 29213 and Streptococcus agalactiae) and Candida albicans and provoked a marked lethality on an MCF-7 cancer cell line, even at low concentrations. It was concluded that FAPS derived from polyunsaturated microbial lipids could be the basis for the development of new therapeutic agents.

Transgenic barley producing essential polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFAs) affect diverse physiological processes and human health. Most cereals are poor in n-3 and n-6 PUFAs. Using biolistics, barley (Hordeum vulgare L. cv. Golden Promise) was transformed with an artificial gene encoding Δ6-desaturase (D6D) under an endosperm-specific promoter. This artificial gene was designed from the sequence of D6D of the filamentous fungus Thamnidium elegans, but codon usage was optimised for cereals. A signal sequence from the gene encoding for high molecular mass glutenin Dx5 was added to a destinate mature protein. Successful transformation was confirmed in T0 plants at the genomic level and in T1 seeds at the transcriptomic and metabolomic levels. Transformed plants produced up to 0.141 % of γ-linolenic acid (GLA) and 0.294 % of stearidonic acid (SDA) of the total amount of fatty acids in their grains. Although the content of these fatty acids was relatively low, the current study provides the first evidence that transgenic barley can be a source of GLA/SDA.

The olive mill wastewater as substrate for single cell oil production by Zygomycetes

The conversion of olive mill wastewater (OMW) into high added value lipids containing polyunsaturated fatty acids (PUFA), in parallel with a significant phenolic removal by selected strains of Zygomycetes, is reported here for the first time. The growth of Mortierella isabellina, Mortierella ramanniana, Cunninghamella echinulata, Mucor sp., Thamnidium elegans and Zygorhynchus moelleri on solidified media was not significantly affected by the presence of OMW used in the growth medium up to 50% (v/v). Kinetic parameter values and conversion yields, estimated using a mathematical model which was fitted on the experimental data originated from submerged cultures, shows the ability of some Zygomycetes (i.e. T. elegans and Z. moelleri) to grow on OMW and accumulate storage material, i.e. lipids rich in PUFA, and these findings open new perspectives in OMW management and valorization. In liquid media containing OMW as sole carbon source, T. elegans and Z. moelleri produced 4.4 and 3.5g/L cell mass in surface (SC) and submerged (SMC) cultures, respectively, containing around 60% (w/w) of lipids. Oleic and palmitic acids were the predominant fatty acids. Gamma-linolenic acid was found in high percentages (up to 17.7%, w/w) in the lipid of Z. moelleri, in SMC with OMW as sole carbon source, while PUFA biosynthesis was not favored in SC.

Utilization of Thamnidium elegans fungal culture in environmental cleanup: A reactive dye biosorption study

In this study, the potential of biomaterial obtained from filamentous fungi, Thamnidium elegans was investigated for the batch and dynamic flow mode biosorption of Reactive Red 198 (RR198). The effects of pH, biosorbent amount, contact time, initial dye concentration and ionic strength were evaluated in addition to the investigation of its biosorption performance in the presence of other dyes in the biosorption medium. High biosorption yields were recorded by using small amount of biosorbent in a relatively short time. The biosorption process followed by the pseudo-second-order kinetic and the Langmuir isotherm models. Maximum monolayer dye biosorption capacity of biosorbent was found as 234.24mgg−1 in batch conditions. Biosorption–desorption studies indicated that T. elegans has relatively good regeneration ability. Also the suggested biomaterial exhibited high biosorption yield in competitive and real wastewater conditions. The dye removal mechanism was evaluated by IR, SEM and zeta potential analysis. Overall, batch and dynamic-flow mode studies indicated that this environmentally friendly biosorbent may be an alternative for the removal of reactive dyes from contaminated media.

Effective decolorization potential of Thamnidium elegans: Biosorption optimization, modelling, characterization and application studies

The biosorption of MV onto untried biomass prepared from Thamnidium elegans culture was investigated in batch and continuous systems. The effects of experimental parameters on the biosorption process (initial pH, biosorbent amount, time, flow rate, initial dye concentration and competing ions) were examined. Biosorption process was the best described by the pseudo-second order kinetic and Langmuir isotherm models. The high biosorption yield was recorded with lower amount of biosorbent in a short equilibrium time. Maximum biosorption capacity of biosorbent was determined as 579.37mgg−1 in batch mode. Biosorption–desorption studies indicated that T. elegans has a relatively good regeneration and recovery potential. The possible dye-biosorbent interactions were investigated by zeta potential measurements and IR analysis. Findings indicated that the biosorbent prepared from T. elegans may be an effective alternative for the removal of MV from aqueous media with the high biosorption capacity and satisfactory recovery potential.

Evaluating glucose and xylose as cosubstrates for lipid accumulation andγ-linolenic acid biosynthesis ofThamnidium elegans

To study the biotechnological production of lipids containing rich amounts of the medically and nutritionally important γ-linolenic acid (GLA), during cultivation of the Zygomycetes Thamnidium elegans, on mixtures of glucose and xylose, abundant sugars of lignocellulosic biomass. Glucose and xylose were utilized as carbon sources, solely or in mixtures, under nitrogen-limited conditions, in batch-flask or bioreactor cultures. On glucose, T. elegans produced 31.9 g l(-1) of biomass containing 15.0 g l(-1) lipid with significantly high GLA content (1014 mg l(-1)). Xylose was proved to be an adequate substrate for growth and lipid production. Additionally, xylitol secretion occurred when xylose was utilized as carbon source, solely or in mixtures with glucose. Batch-bioreactor trials on glucose yielded satisfactory lipid production, with rapid substrate consumption rates. Analysis of intracellular lipids showed that the highest GLA content was observed in early stationary growth phase, while the phospholipid fraction was the most unsaturated fraction of T. elegans. Thamnidium elegans represents a promising fungus for the successful valorization of sugar-based lignocellulosic residues into microbial lipids of high nutritional and pharmaceutical interest. Xylitol production and cultivation in bioreactor trials is reported for the first time for T. elegans, while cultivation on xylose-based media resulted in high GLA production by this fungus.

Modulation of phenolic content and antioxidant activity of maize by solid state fermentation with Thamnidium elegans CCF 1456

Thamnidium elegans CCF 1456, a filamentous fungus, was used to enhance the total phenolic content and radical scavenging activity of maize via solid-state fermentation. Thamnidium fermented maize (TFM) and unfermented maize (UFM) grains were extracted with 65% ethanol and dimethylsulfoxide (DMSO). Total phenolic content (TPC), and radical scavenging capacity — determined with 1,1-diphenyl-2-picrylhydrazyl (DPPH) and radical cations of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS·+) found for TFM — were significantly (P < 0.05) higher on the 5th day of incubation than that of UFM extracts. A linear correlation was observed among TPC, DPPH and ABTS scavenging activities, and also among TPC and various carbohydrate-cleaving enzymes (α-amylase, β-glucosidase and xylanase), suggesting that this? strategy may help to enrich? TFM with improved phytochemical properties and antioxidant activities.

Prefermented cereals containing fungal gamma-linolenic acid and their effect on rumen metabolism in vitro

The application of Thamnidium elegans fungal strain CCF 1456 (TE) for effective utilization of various agroindustrial materials creates new perspectives for animal cereal diets enriched with microbial &amp;gamma;-linolenic acid (GLA). Diets consisting of lucerne hay (LH) plus prefermented cereals (wheat bran/spent malt grains, WB+TE or WB+TE enriched with sunflower oil, WB+SO+TE in the first experiment and ground maize grains, GC+TE in the second experiment) were used in the artificial rumen. We examined their effect on the rumen fermentation pattern and lipid metabolism. The diet affected the results of degradability of dry matter, organic matter, neutral detergent fibre and acid detergent fibre of LH+WB diets (P &amp;lt; 0.05 and P &amp;lt; 0.01). The GLA daily output of prefermented diet substrates LH+WB+TE and LH+WB+SO+TE, or LH+GC+TE was higher compared to the non-prefermented LH+WB or LH+GC, respectively (P &amp;lt; 0.05 and P &amp;lt; 0.01). Daily outputs of trans11 oleic (TVA) of the LH+GC+TE diet were higher versus the non-prefermented LH+GC (P &amp;lt; 0.01). The biohydrogenation of fatty acids (C18:1 cis9 oleic, C18:2 linoleic, C18:3n-3 alpha-linolenic, C18:3n-6 GLA and total FA) of prefermented cereal diets was not influenced. Cereal diets containing microbial GLA might positively enhance GLA daily outputs in the RUSITEC effluent, but they are not effective enough to decrease the biohydrogenation of unsaturated fatty acids.