Cultivation Of Filamentous Fungi Research Articles

Light Signaling Regulates Aspergillus niger Biofilm Formation by Affecting Melanin and Extracellular Polysaccharide Biosynthesis.

ABSTRACTLight is an important signal source in nature, which regulates the physiological cycle, morphogenetic pathways, and secondary metabolites of fungi. As an external pressure on Aspergillus niger, light signaling transmits stress signals into the cell via the mitogen-activated protein kinase (MAPK) signaling pathway. Studying the effect of light on the biofilm of A. niger will provide a theoretical basis for light in the cultivation of filamentous fungi and industrial applications. Here, the characterization of A. niger biofilm under different light intensities confirmed the effects of light signaling. Our results indicated that A. niger intensely accumulated protective mycelial melanin under light illumination. We also discovered that the RlmA transcription factor in the MAPK signaling pathway is activated by light signaling to promote the synthesis of melanin, chitin, and other exopolysaccharides. However, the importance of melanin to A. niger biofilm is rarely reported; therefore, we knocked out key genes of the melanin biosynthetic pathway—Abr1 and Ayg1. Changes in hydrophobicity and electrostatic forces resulted in the decrease of biofilm caused by the decrease of melanin in mutants.

Total Phenolic and Flavonoid Content, Antioxidant Activity of Talaromyces funiculosus Strains

Microscopic fungi synthesize a wide range of phenolic compounds that play an important role in cell wall formation, as well as perform signaling and protective functions. One of the important functions of phenolic compounds, primarily polyphenols, is antioxidant activity. The most active polyphenols are flavonoids, their synthesis in microscopic fungi has not been studied enough. The aim of the study was to determine the total phenols and flavonoids content and antioxidant activity of Talaromyces funiculosus strains isolated from different econiches. Methods. 20 strains of the microscopic fungus T. funiculosus – 10 endophytes and 10 saprotrophs were used in the experiments. Cultivation of filamentous fungi was performed under submerged conditions with shaking at 210–230 rpm in Erlenmeyer flasks containing 200 ml of Czapek liquid medium at a temperature 26±2°C for 6 days. The biomass of the strains was filtered, dried at 70°C and extracted with 95% methanol (1:50 w/v). The total phenols content was determined using Folin-Ciocalteu reagent by the method of Arora, Chandra (2010) with some modifications and expressed as mg of gallic acid equivalents (GAE) per g of dry weight mycelium. Flavonoids in each extract were determined spectrophotometrically with AlCl3 and expressed as mg of quercetin equivalents (QE) per g of dry weight mycelium. Antioxidant activity of methanolic extracts was measured as radical scavenging activity by the percentage inhibition of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. Results. The total phenolic content of methanolic extracts of T. funiculosus strains ranged from 0.12±0.007 to 1.97±0.150 mg GAE/g. The highest level of phenolic compounds was found for saprotroph UCM F-16790 (1.97±0.150) and endophyte UCM F-16795 (1.71±0.137 mg GAE/g). The flavonoid content in methanolic extracts of the studied strains varied in the range of 0.02±0.001–0.39±0.041 mg QE/g. All studied strains of T. funiculosus showed antioxidant activity against DPPH radical, which was 12.19±2.34–60.37±7.24% inhibition. Statistical analysis revealed a strong positive correlation between the total phenols and flavonoids content (Pearson’s correlation coefficient r=0.92); between antioxidant activity, phenols (r=0.82) and flavonoids (r=0.81) content in methanol extracts of T. funiculosus strains. The total content of phenols and flavonoids and antioxidant activity did not depend on the trophic group of the studied strains. The moderate correlation between flavonoids content and antioxidant activity of methanolic extracts of endophytic T. funiculosus strains (r=0.46) may be related to their ability to synthesize a wider range of polyphenolic compounds with antioxidant activity. Conclusions. The studied T. funiculosus strains have the ability to synthesize phenolic compounds and flavonoids in different concentrations. Correlation analysis revealed strong correlations between total phenolic and flavonoid contents and antioxidant activity. The reason for phenomenon of moderate correlation (r=0.46) between flavonoid content and antioxidant activity in endophytic strains may be the synthesis of other compounds by individual strains. The determination of individual components synthesized by the most active strains is of scientific interest for further study. T. funiculosus UСM F-16795 and UСM F-16790 strains had a quite high level of phenols and flavonoids. Obtained data may be promising for the search for phenolic compounds with antioxidant properties to increase plant resistance to negative impacts of environmental stress factors.

Conversion of fish processing wastewater into fish feed ingredients through submerged cultivation of Aspergillus oryzae

Fish processing towards production of fillet gives rise to wastewater streams that are ultimately directed to biogas production and/or wastewater treatment. However, these wastewater streams are rich in minerals, fat, and proteins that can be converted to protein-rich feed ingredients through submerged cultivation of edible filamentous fungi. In this study, the origin of wastewater stream, initial pH, cultivation time, and extent of washing during sieving, were found to influence the amount of recovered material from the wastewater streams and its protein content, following cultivation with Aspergillus oryzae. Through cultivation of the filamentous fungus in sludge, 330 kg of material per ton of COD were recovered by sieving, corresponding to 121 kg protein per ton of COD, while through its cultivation in salt brine, 210 kg of material were recovered per ton of COD, corresponding to 128 kg protein per ton of COD. Removal ranges of 12–43%, 39–92%, and 32–66% for COD, total solids, and nitrogen, respectively, were obtained after A. oryzae growth and harvesting in the wastewater streams. Therefore, the present study shows the versatility that the integration of fungal cultivation provides to fish processing industries, and should be complemented by economic, environmental, and feeding studies, in order to reveal the most promising valorization strategy.Graphic abstract

Microscopic fungi of lithobiont communities of Argentine Islands Region: Data from the 22nd Ukrainian Antarctic Expedition

The identification of the diversity of microscopic fungi of lithobiont communities of the Argentine Islands in specimens collected during the 22nd Ukrainian Antarctic Expedition was the purpose of this work. Samples of rock, soil, mosses and lichens of rock micro-habitats of “Crustose lichen sub-formation and fruticose lichen and moss cushion sub-formation” were used in the work. These samples were used for extracting and cultivation of filamentous fungi on dense nutrient media. Determination of physiological and biochemical characteristics and identification of yeast-like fungi were performed using a microbiological analyser ‘Vitek-2’ (‘Bio Merieux’, France). Cultivation of microorganisms was carried out at temperatures from +2 to +37 °C. In results cultures of microscopic fungi of Zygomycota (Mucor circinelloides), Ascomycota (species of the genera cf. Tlielebolus, Talaromyces), representatives of the Anamorphic fungi group (Geomyces pannorum, species of the genera Alternaria, Acremonium, Aspergillus, Penicillium, and Cladosporium) were isolated from Antarctic samples. Microscopic fungi Penicillium spp. were dominated after the frequency in the studied samples (54.5%). Rhodotorula rubra and Candida sp. among isolated yeast fungi, and dark pigmented fungi represented by Aureobasidium pulhdans and Exophiala spp. were identified. The biological properties of a number of isolated fungi (the potential ability to synthesise important biologically active substances: melanins, carotenoids, lipids) are characterised. Mycobiota of rock communities of Argentine Islands is rich on filamentous and yeast fungi similarly to other regions of Antarctica. A number of fungi investigated are potentially able to synthesise biologically active substances. The dark pigmented species of the genera Cladosporium, Exophiala, Aureobasidium pulhdans, capable of melanin synthesis; ‘red’ yeast Rhodotorula rubra (carotenoid producers and resistant to toxic metals); Mucor circinelloides and Geomyces pannorum, lipid producers, are among these fungi. Yeast-like fungi assimilated a wide range of carbohydrates, which will allow them to be further used for cultivation in laboratory and process conditions. The collection of technologically promising strains of microorganisms, part of the Culture Collection of Fungi at Taras Shevchenko National University of Kyiv (Ukraine), is updated with isolated species (strains) of filamentous fungi and yeast – potential producers of biologically active substances, obtained within this study.

Anti-oxidant, Histopathology and LD50 Evaluation of Ethanol Stem Bark Extract of Enantia chlorantha in Rodents

In this study, it was shown that how rheology and fluid dynamics effected the behavior of cell growth and L-Lactic acid productivity during cultivation of filamentous fungi (Aspergillus oryzae) in a stirred-tank. Submerged culture of A. oryzae was a shear-thinning fluid with complex rheology and morphology. It is a shear-sensitive fluid. The scope of this study, was enhancing the gas-liquid flow of fermentation fluid to improve the liquid-gas mass transfer, L-lactic acid production and stagnant zone reduction at low energy consumption by effective shear rate generation using multi-large blade agitator with global axial pattern of mixing. Due to this reason, the advantage of Fullzone® (FZ), and impeller was investigated. Experimental results when using multi-large and multi-radial impeller, showed that the same volumetric power consumption of the dissolved oxygen concentration, Re, macro and micro-morphology size, biomass and L-lactic acid production when using multi large impeller was higher and more stable than that of the multi radial impeller. Most of above positive properties were due to the reduction in viscosity and apparent consistence index of fermented fluid.

Carbazole hydroxylation by the filamentous fungi of the Cunninghamella species.

Nitrogen heterocyclic compounds, especially carbazole, quinolone, and pyridine are common types of environmental pollutants. Carbazole has a toxic influence on living organisms, and the knowledge of its persistence and bioconversion in ecosystems is still not complete. There is an increasing interest in detoxification of hazardous xenobiotics by microorganisms. In this study, the ability of three filamentous fungi of the Cunninghamella species to eliminate carbazole was evaluated. The Cunninghamella elegans IM 1785/21Gp and Cunninghamella echinulata IM 2611 strains efficiently removed carbazole. The IM 1785/21Gp and IM 2611 strains converted 93 and 82 % of the initial concentration of the xenobiotic (200 mg L−1) after 120 h incubation. 2-Hydroxycarbazole was for the first time identified as a carbazole metabolite formed by the filamentous fungi of the Cunninghamella species. There was no increase in the toxicity of the postculture extracts toward Artemia franciscana. Moreover, we showed an influence of carbazole on the phospholipid composition of the cells of the tested filamentous fungi, which indicated its harmful effect on the fungal cell membrane. The most significant modification of phospholipid levels after the cultivation of filamentous fungi with the addition of carbazole was showed for IM 1785/21Gp strain.

High cell density co-culture for production of recombinant hydrolases

Sugarcane bagasse is a residue with great potential as a feedstock for second-generation ethanol production. One of the approaches studied for making use of this material is the utilization of enzymes to hydrolyze the cell wall carbohydrates and generate fermentable sugars. These enzymes can be produced by cultivation of filamentous fungi or bacteria; however, the high production cost still represents a bottleneck to second-generation ethanol production. Expression of recombinant hydrolases through a co-culture strategy could be an interesting alternative for producing a recombinant cocktail at high levels of productivity that is tailor-made for each material to be hydrolyzed. In this study we evaluate the production of hydrolases by co-culturing two recombinant Escherichia coli, each expressing a specific hydrolase, β-1,3-1,4-glucanase or β-1,4-xylanase, both isolated from Bacillus subtilis. The cultures were conducted in bioreactors in batch and fed-batch mode in order to reach high cell densities. Co-culture in batch cultivation reached a dry cell weight of 10.4g/L and volumetric activities of 31.96U/mL and 11.89U/mL for xylanase and endoglucanase, respectively. Fed-batch cultivation reached a dry cell weight of 60g/L and the volumetric activities of xylanase and endoglucanase were respectively up to 5 and 1.3 times higher than those in batch mode. A competition assay indicates that no clone predominates over the other during cultivation. These results suggest that co-culture is a potential technique for producing recombinant hydrolase cocktails at lower cost than those associated with the production of a single culture.

Produção de complexos enzimáticos por A. niger a partir de soja por fermentação em estado sólido

Soybean has a wide availability of biochemical compounds as proteins, polysaccharides, lipids, carbohydrates, minerals, vitamins, fibers, lecithin, among others, making interesting its employment as substrate for solid state fermentation. Although the process of SSF is not extensively used industrially like the submerged fermentation (SmF), it can be a viable alternative, since presents higher results of productivity, especially at the cultivation of filamentous fungi and enzymes production. In this way, this study compared the production of different types of enzymes through solid-state fermentation, using different types of soybean cultures (organic, transgenic and conventional) with the fungus A. niger. We accomplished the characterization of the substrates for the production of amylase, protease and lipase, evaluation of the growth curves of the microorganism and the optimal conditions of enzymes production using experimental design. The highest enzyme activities in the study of fermentation parameters were observed for the protease, using conventional soybeans with initial moisture of 50%, 144h of fermentation, initial inoculum concentration of 4.10 6 spores g -1 and particle size of 0.6 mm. The study of process optimization indicated that the best results of enzyme activity for the protease were obtained at pH 3.0 and particle size of soybean fragmentation of 0.6 mm.

Estudo do crescimento de A. casiellus em farelo de soja convencional para produção de enzimas

Soybean meal is one of the major wastes from the Brazilian agribusiness, in view of the large domestic production of grain. This byproduct has characteristics that make it interesting to be used as substrate in the process of solid-state fermentation (SSF). Although it is not much used industrially as the submerged fermentation (SmF), it may be a viable alternative, as it has shown superior results of productivity, specially in the cultivation of filamentous fungi and the production of enzymes. The present study aimed the production of the enzymes protease, amylase and invertase by SSF using conventional soybean meal and the fungus A. casiellus. The pH and nutrient sources were evaluated and, finally, an optimization of the parameters of the SSF through experimental design and construction of growth curves of the fungus were carried out. The optimization of the invertase production process reached the best values for specific enzymatic activity (AEesp) at neutral pH (6.7) and 3% additional source of nitrogen to the medium, with initial moisture of 50% and temperature of 28oC. The best result for the protease activity was obtained in the evaluation of medium nutrient sources, with the use of 2% of carbon and nitrogen sources and 2% of mineral sources. Already, amylase did not show significant results for the variables used. Key words: SSF, soybean meal, A. casiellus.

The influence of geometry on hydrodynamic and mass transfer characteristics in an external airlift reactor for the cultivation of filamentous fungi

The influences of geometric configuration, mycelial broth rheology and superficial gas velocity (Usg) were investigated with respect to the following hydrodynamic parameters: gas holdup (e), oxygen transfer coefficient (KLa) and mixing time (tm). Increases in Usg and height of gas separator (Ht) caused an increase in e and KLa, and a decrease in tm. Consequently, a diameter ratio (Dd/Dr) of 0.71 and Ht 0.20 m were found to be the best geometry and operation parameters to achieve high aeration and mixing efficiency for the high viscous broth system in the cultivation of filamentous fungi. An external airlift reactor (EALR) was developed and designed for the cultivation of filamentous fungi. The EALR with two spargers excels in reliability and high aeration and mass transfer coefficiency, resulting in a fast mycelial growth and high biomass productivity in the cultivation of the fungus Rhizopus oryzae.