Enhanced protein and amino acids of corn-ethanol co-product by Mucor indicus and Rhizopus oryzae.

Upcycle of co-products from corn-ethanol plant into protein-rich animal feed with balanced key amino acids via solid-state fermentation is a promising approach to economically support both biofuel and animal feed industries. However, there are multiple types of solid-state fermentation microorganisms and growth conditions that have not been tested. In this study, Mucor indicus and Rhizopus oryzae were used to ferment corn-based wet distiller's grains with solubles (WDGS). The effects of fermentation conditions (temperature, agitation, and moisture) and supplementations (extraneous carbon and nitrogen sources) were evaluated on protein production and amino acids profiles before and after fermentation. The study established best fermentation conditions (23 °C, static incubation for 4 days at 70% initial moisture content) to improve protein content for both R. oryzae and M. indicus. Moreover, urea supplied to R. oryzae and M. indicus improved protein concentration by 35 and 38%, and total amino acids content by 28 and 18%, respectively. The amount of 693.1 and 451.8 mg of additional total amino acids including 262.8 and 227.7 mg of key amino acids (lysine, methionine, tryptophan, and arginine) was synthesized by R. oryzae and M. indicus, respectively, per supply of 536 mg urea in 25 g of WDGS. This study demonstrated the feasibility of urea as a low-cost nitrogen source for amino acid biosynthesis in fungal fermentation of WDGS, which could contribute to the increasing demand for high-value monogastric animal feed.

Activity of selected essential oils on spoiling fungi cultured from Marzolino cheese.

Microscopic fungi can be present on a variety of foodstuff, including cheese. They can be responsible for fungal spoilage, causing sensory changes making food unacceptable for human consumption, and posing severe health concerns. Furthermore, some of these organisms are able to resist antimicrobial preservatives provided for by law. Antifungal activity of 15 chemically defined EOs, alone and in mixture, were checked by a microdilution test against isolates of Penicillium funiculosum and Mucor racemosus cultured from rinds of Marzolino, a typical Italian fresh pecorino cheese. Origanum vulgare yielded the lowest MIC values, followed by Salvia sclarea, Ocimum basilicum and Cymbopogon citratus, while Citrus paradisi and Citrus limon were not active. All mixtures showed antifungal activity at lower concentration with respect to MIC values of each EO component, when not in combination. This study is the first to describe the setting up of EOs mixtures to limit spoiling moulds.

Pretreatment of different food rest materials for bioconversion into fungal lipid-rich biomass.

Food rest materials have the potential to be used as media components in various types of fermentations. Oleaginous filamentous fungi can utilize those components and generate a high-value lipid-rich biomass, which could be further used for animal and human use. One of the main limitations in this process is the pretreatment of food rest materials, needed to provide homogenization, sterilization and solubilization. In this study, two pretreatment processes-steam explosion and enzymatic hydrolysis-were evaluated for potato and animal protein-rich food rest materials. The pretreated food rest materials were used for the production of fungal lipid-rich biomass in submerged fermentation by the oleaginous fungus Mucor circinelloides. Cultivation media based on malt extract broth and glucose were used as controls of growth and lipid production, respectively. It was observed that media based on food rest materials can support growth and lipid production in M. circinelloides to a similar extent as the control media. More specifically, the use of potato hydrolysate combined with chicken auto-hydrolysate resulted in a higher fungal total biomass weight than using malt extract broth. When the same C/N ratio was used for glucose and rest materials-based media, similar lipid content was obtained or even higher using the latter media.

Morphological evolution of various fungal species in the presence and absence of aluminum oxide microparticles: Comparative and quantitative insights into microparticle-enhanced cultivation (MPEC).

The application of microparticle-enhanced cultivation (MPEC) is an attractive method to control mycelial morphology, and thus enhance the production of metabolites and enzymes in the submerged cultivations of filamentous fungi. Unfortunately, most literature data deals with the spore-agglomerating species like aspergilli. Therefore, the detailed quantitative study of the morphological evolution of four different fungal species (Aspergillus terreus, Penicillium rubens, Chaetomium globosum, and Mucor racemosus) based on the digital analysis of microscopic images was presented in this paper. In accordance with the current knowledge, these species exhibit different mechanisms of agglomerates formation. The standard submerged shake flask cultivations (as a reference) and MPEC involving 10 µm aluminum oxide microparticles (6 g·L-1 ) were performed. The morphological parameters, including mean projected area, elongation, roughness, and morphology number were determined for the mycelial objects within the first 24 hr of growth. It occurred that heretofore observed and widely discussed effect of microparticles on fungi, namely the decrease in pellet size, was not observed for the species whose pellet formation mechanism is different from spore agglomeration. In the MPEC, C. globosum developed core-shell pellets, and M. racemosus, a nonagglomerative species, formed the relatively larger, compared to standard cultures, pellets with distinct cores.

Activity of Salvia dolomitica and Salvia somalensis Essential Oils against Bacteria, Molds and Yeasts.

Essential oils (EOs) from Salvia dolomitica and Salvia somalensis, widely employed in the cosmetic and perfume industry, were analyzed for composition and tested against bacterial and fungal pathogens isolated from clinical and environmental specimens. The analyses were carried out against Staphylococcus aureus, Staphylococcus pseudointermedius, Pseudomonas aeruginosa, Escherichia coli, Streptococcus canis, Streptococcus pyogenes, Klebsiella pneumoniae, Proteus mirabilis, Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, Aspergillus niger, Aspergillus flavus, Candida albicans, Candida krusei, Mucor sp. and Trichothecium roseum. Both EOs showed similar percentages of total monoterpenes and sesquiterpene hydrocarbons. The main constituents were 1,8-cineole and ß-caryophyllene in S. dolomitica and bornyl acetate and camphor in S. somalensis. The selected EOs have no relevant antifungal or antibacterial activities if compared to conventional drugs.

Sageretia thea (Osbeck.) modulated biosynthesis of NiO nanoparticles and their in vitro pharmacognostic, antioxidant and cytotoxic potential.

NiO nanoparticles are biosynthesized using Sageretia thea (Osbeck.) aqueous leave extracts and their biological activities are reported. Nanoparticles (∼18 nm) were characterized through XRD, ATR-FTIR, EDS, SAED, HR-SEM/TEM and Raman spectroscopy. Antibacterial activity was investigated against six pathogenic bacterial strains (gram positive and gram negative) and their corresponding minimum inhibitory concentrations (MICs) were calculated. UV-exposed nanoparticles were investigated to have reduced MICs relative to the NiO nanoparticles have not been exposed to UV. Moderate linear fungal growth inhibition was observed while Mucor racemosus (percentage inhibition 64% ± 2.30) was found to be most susceptible. Cytotoxicity was confirmed using brine shrimps lethality assay (IC50 42.60 µg/ml). MTT cytotoxicity was performed against Leishmania tropica-KWH23 promastigotes and amastigotes revealed significant percentage inhibition across the applied concentrations. IC50 values were calculated as 24.13 µg/ml and 26.74 µg/ml for the promastigote and amastigote cultures of Leishmania tropica. NiO nanoparticles were found. Moderate, antioxidant potential was concluded through assays like DPPH, TAP and TAC. Furthermore, protein kinase inhibition and alpha amylase inhibition is also reported.

Screening of Phosphorus-Accumulating Fungi and Their Potential for Phosphorus Removal from Waste Streams.

While bacteria have been primarily studied for phosphorus (P) removal in wastewater treatment, fungi and their ability to accumulate intracellular polyphosphate are less investigated. P-accumulating fungal strains were screened from soybean plants and surrounding soil by flask cultivation with potato dextrose broth and KH2PO4 in this study. Mucor circinelloides was selected for its high efficiency in P removal efficiency and high cellular P content. Neisser staining and growth-curve analysis confirmed that M. circinelloides stored polyphosphate intracellularly by luxury phosphate uptake. The effect of culture medium compositions on P removal efficiency and cellular P content was also investigated. Monosaccharides (such as glucose and fructose) and organic nitrogen (N, such as urea, and peptone) promoted fungi growth and P accumulation. M. circinelloides also preferred organic phosphates. When glucose, urea, and phytic acid sodium salt were used as the carbon, N, and P source, respectively, the maximum utilization efficiency was 40.1% for P and 7.08% for cellular P content. In addition, the potential of M. circinelloides for P removal from waste streams was investigated. Compared with the non-inoculated control culture, inoculation with M. circinelloides improved the soluble P removal in treating wastewater centrate, screened manure, and digested manure.

Effects of Plant Growth Hormones on Mucor indicus Growth and Chitosan and Ethanol Production.

The objective of this study was to investigate the effects of indole-3-acetic acid (IAA) and kinetin (KIN) on Mucor indicus growth, cell wall composition, and ethanol production. A semi-synthetic medium, supplemented with 0-5 mg/L hormones, was used for the cultivations (at 32 °C for 48 h). By addition of 1 mg/L of each hormone, the biomass and ethanol yields were increased and decreased, respectively. At higher levels, however, an inverse trend was observed. The glucosamine fraction of the cell wall, as a representative for chitosan, followed similar but sharper changes, compared to the biomass. The highest level was 221% higher than that obtained without hormones. The sum of glucosamine and N-acetyl glucosamine (chitin and chitosan) was noticeably enhanced in the presence of the hormones. Increase of chitosan was accompanied by a decrease in the phosphate content, with the lowest phosphate (0.01 g/g cell wall) being obtained when the chitosan was at the maximum (0.45 g/g cell wall). In conclusion, IAA and KIN significantly enhanced the M. indicus growth and chitosan production, while at the same time decreasing the ethanol yield to some extent. This study shows that plant growth hormones have a high potential for the improvement of fungal chitosan production by M. indicus.

Utilisation of potato processing wastewater for microbial lipids and γ-linolenic acid production by oleaginous fungi.

BACKGROUND: Microbial lipids are considered as the starting material for production of second-generation biofuels and their polyunsaturated fatty acids are rich sources of neutraceuticals. Exploring cheap feedstock for producing microbial lipids is necessary. The present study examined the potential of microbial lipids and γ-linolenic acid (GLA) production by two oleaginous fungi, Aspergillus flavus I16-3 and Mucor rouxii, with potato processing wastewater as a low-cost or no-cost nutrient source. RESULTS: Biochemistry and physiology of two oleaginous fungi, A. flavus I16-3 and M. rouxii, on lipid accumulation showed the two fungi grew well and efficiently utilised the starch in wastewater. On average (P < 0.05), 2.8 and 3.6 g L(-1) of lipids were produced by A. flavus I16-3 and M. rouxii, respectively, with maximum GLA yields of 60 and 100 mg L(-1) . Addition of nutrients to raw wastewater significantly improved (P < 0.05) the lipid and GLA yields; 3.5 and 4.2 g L(-1) of lipids, and 100 and 140 mg L(-1) of GLA were produced by A. flavus I16-3 and M. rouxii, respectively. In addition, the wastewater was efficiently treated, with soluble chemical oxygen demand, total soluble nitrogen and total soluble phosphorus removals up to 60% and 90%, 100% and 98%, and 92% and 81% by A. flavus I16-3 and M. rouxii, respectively. CONCLUSION: This study demonstrated an alternative approach to valorise potato processing wastewater to produce microbial lipids and GLA (nutraceuticals).

Simultaneous enrichment of cereals with polyunsaturated fatty acids and pigments by fungal solid state fermentations.

Four Mucor strains were tested for their ability to grow on four cereal substrates and enriched them with gamma-linolenic acid (GLA) and ß-carotene. M. circinelloides CCF-2617 as the best producer accumulated of both GLA and ß-carotene in high amounts during utilization of rye bran/spent malt grains (3:1). The first growth phase was characterized by rapid GLA biosynthesis, while distinct ß-carotene formation was found in the stationary fungal growth. Therefore various cultivation conditions were tested in order to optimize the yield of either GLA or ß-carotene. The fungus grown on cereal substrate supplemented with glucose produced maximal 8.5 mg ß-carotene and 12.1 g GLA in 1 kg fermented substrate, respectively. On the other hand, the highest amount of GLA in the fermented substrate (24.2 g/kg) was achieved when 30% of sunflower oil was employed to the substrate. Interestingly, ß-carotene biosynthesis was completely inhibited when either whey or linseed oil were added to the substrate.

The effects of palm oil breakdown products on lipid turnover and morphology of fungi.

The oleaginous fungi Cryptococcus curvatus and Mucor circinelloides were used to determine the effect of palm oil breakdown products, measured as polymerized triglycerides (PTGs), on lipid turnover and on fungal growth and morphology. In M. circinelloides, we found after 7 days of growth, a decrease in biomass and in lipid utilization and accumulation at increased PTG levels, both at low and neutral pH. In C. curvatus, there was also a decrease in lipid utilization and biomass production at increased PTG levels, at both low and neutral pH. However, an increase in oil accumulation was observed at low pH while it remained similar at neutral pH for all PTG levels tested. Hairy and warty protuberances on the cell surface were observed when C. curvatus was grown on oils with 15% and 45% PTGs, respectively. Using nano scanning Auger microscopy, we found no evidence to suggest a difference in elemental composition of the surfaces of the warty protuberances compared with the rest of the cell wall surface. We conclude that the warty protuberances are outgrowths of cell walls and that the changes observed in lipid turnover in both fungi are due to the presence of palm oil breakdown products.

Gamma-linolenic acid production of Mucor rouxii by solid-state fermentation using agricultural by-products.

AIMS: This study aims to maximize the yield of gamma-linolenic acid by a filamentous fungus, Mucor rouxii, using low cost production by solid-state fermentation. METHODS AND RESULTS: We optimized substrate types and culture conditions including inoculum size and temperature. The optimal growth of M. rouxii was found in the cultures inoculated with 5 x 10(5) spores g(-1) substrate. The fungal cells grew well on rice bran and soy bean meal, whereas a lower biomass was found in the cultures grown on polished rice, broken rice and spent malt grain. The GLA content was highly accumulated in rice bran ferment and its maximal content of about 6 g kg(-1) fermented mass was observed in the 5th-day culture grown at 30 degrees C. However, the GLA content in the rice bran ferment was not enhanced by low temperature. CONCLUSIONS: The GLA production of M. rouxii could be enhanced by optimizing the agricultural by-product substrates and culture condition. SIGNIFICANCE AND IMPACT OF THE STUDY: Low cost GLA production process was achieved, and fermented product containing GLA can be incorporated into feed additives without further oil extraction to alternate the expensive plant oils.

Significance of fatty acid supplementation on profiles of cell growth, fatty acid, and gene expression of three desaturases in Mucor rouxii.

Molecular and biochemical studies investigating the biosynthesis of gamma-linolenic acid (GLA), a nutritionally important polyunsaturated fatty acid, were implemented in Mucor rouxii by supplementation with exogenous fatty acids. Compared to the fatty acid-free cultures, addition of C18:2Delta(9,12) to the cultures showed up to threefold decrease in the GLA content from the beginning of the culture, whereas a significantly increased GLA amount (P

Characterization of an extracellular endopolygalacturonase from the saprobe Mucor ramosissimus Samutsevitsch and its action as trigger of defensive response in tropical plants.

In recent years, interest in the ability of non-pathogenic microorganisms to induce resistance in plants has grown, particularly with respect to their use as environmentally safe controllers of plant disease. In this study, we investigated the capacity of Mucor ramosissimus Samutsevitsch to release pectinases able to degrade cell walls of Palicourea marcgravii St. Hil., a tropical forest native Rubiaceae on which the spores of this saprobic fungus have been found. The fungus was grown in liquid culture medium containing pectin as the sole carbon source and filtrates were analyzed for pectinase activity. An endopolygalacturonase was partially purified by ion exchange chromatography, gel filtration, and preparative isoelectrofocusing, and characterized. This enzyme was more active upon pectic substrates with a low degree of methyl esterification. The products of hydrolysis of different pectic substrates (including pectin from P. marcgravii) by the action of this endopolygalacturonase elicited to different extents the phytoalexin production in soybean cotyledons. Also, the enzyme itself and the products of its action on the pectic fraction of P. marcgravii elicited the production of defensive compounds in the leaves of the plant. These results suggest that, besides the role in recycling organic matter, saprobes may also play an important role in the induction of defensive mechanisms in wild plants by enhancing their non-specific resistance against pathogens. Furthermore, they set the stage for future studies on the role of saprobic fungi in inducing resistance of host plants to pathogens.

Detection of NAD+-dependent alcohol dehydrogenase activities in YR-1 strain of Mucor circinelloides, a potential bioremediator of petroleum contaminated soils.

Different soluble NAD+-dependent alcohol dehydrogenase (ADH) isozymes were detected in cell-free homogenates from aerobically grown mycelia of YR-1 strain of Mucor circinelloides isolated from petroleum- contaminated soil samples. Depending on the carbon source present in the growth media, multiple NAD+-dependent ADHs were detected when hexadecane or decane was used as the sole carbon source in the culture media. ADH activities from aerobically or anaerobically grown mycelium or yeast cells, respectively, were detected when growth medium with glucose added was the sole carbon source; the enzyme activity exhibited optimum pH for the oxidation of different alcohols (methanol, ethanol, and hexadecanol) similar to that of the corresponding aldehyde (approximately 7.0). Zymogram analysis conducted with partially purified fractions of extracts from aerobic mycelium or anaerobic yeast cells of the YR-1 strain grown in glucose as the sole carbon source indicated the presence of a single NAD+-dependent ADH enzyme in each case, and the activity level was higher in the yeast cells. ADH enzyme from mycelium grown in different carbon sources showed high activity using ethanol as substrate, although higher activity was displayed when the cells were grown in hexadecane as the sole carbon source. Zymogram analysis with these extracts showed that this particular strain of M. circinelloides has four different isozymes with ADH activity and, interestingly, one of them, ADH4, was identified also as phenanthrene-diol- dehydrogenase, an enzyme that possibly participates in the aromatic hydrocarbon biodegradation pathway.

Substrate specificity and preference of Delta6-desaturase of Mucor rouxii.

The Delta(6)-fatty acid desaturase is a key enzyme in the synthesis of an important fatty acid, gamma-linolenic acid. We have characterized, by heterologous expression in Saccharomyces cerevisiae, substrate specificity and preference of Delta(6)-desaturase of Mucor rouxii. Fatty acid supplementation was carried out based on the predicted enzyme topology, fatty acid phenotype and the corresponding metabolic pathway in M. rouxii. The enzyme has a broad substrate specificity as based on C15-C18. The result also supported classification of the M. rouxii Delta(6)-desaturase into a front-end desaturase. Interestingly, a relatively rare activity based on odd acyl chains and not described previously in other eukaryotic Delta(6)-desaturases was also observed.

Evaluation of aqueous Moringa seed extract as a seed treatment biofungicide for groundnuts.

In a search for alternatives to currently used fungicides, the potential of aqueous Moringa seed extract (AMSE) as a seed treatment was evaluated. Seeds of groundnut, Arachis hypogea L cv Dakar, were soaked in AMSE at concentrations of 1, 5, 10, 15 and 20 g litre(-1) for 24 h. Comparison was made with Apron Plus (metalaxyl+carboxin+furathiocarb), until recently a recommended seed-treatment chemical, and distilled water, which was the medium for extraction of Moringa seeds. The results showed that AMSE has potential for use as a biofungicide on groundnut seeds, since all the concentrations used except 1 g litre(-1) brought about significant reduction in the incidence of fungi on the seeds, such reduction increasing as the dosage of AMSE increased. There were no significant differences in control between the highest concentration of AMSE (20 g litre(-1)) and Apron Plus at the manufacturer's recommended level. Water also produced slight reductions in the incidence of fungi, although this was not significant at P = 0.05. The sensitivity to AMSE of the fungi tested varied, Mucor sp being the most sensitive and Aspergillus niger the least, with Rhizopus stolonifer and Aspergillus flavus intermediate.

Development and application of an assay for uranyl complexation by fungal metabolites, including siderophores.

An assay to detect UO(2)(2+) complexation was developed based on the chrome azurol S (CAS) assay for siderophores (B. Schwyn and J. B. Neilands, Anal. Biochem. 160:47-56, 1987) and was used to investigate the ability of fungal metabolites to complex actinides. In this assay the discoloration of two dyed agars (one containing a CAS-Fe(3+) dye and the other containing a CAS-UO(2)(2+) dye) caused by ligands was quantified. The assay was tested by using the siderophore desferrioxamine B (DFO), and the results showed that there was a regular, reproducible relationship between discoloration and the amount of siderophore added. The ratio of the discoloration on the CAS-UO(2)(2+) agar to the discoloration on the CAS-Fe(3+) agar was independent of the amount of siderophore added. A total of 113 fungi and yeasts were isolated from three soil samples taken from the Peak District National Park. The fungi were screened for the production of UO(2)(2+) chelators by using the CAS-based assay and were also tested specifically for hydroxamate siderophore production by using the hydroxamate siderophore auxotroph Aureobacterium flavescens JG-9. This organism is highly sensitive to the presence of hydroxamate siderophores. However, the CAS-based assay was found to be less sensitive than the A. flavescens JG-9 assay. No significant difference between the results for each site for the two tests was found. Three isolates were selected for further study and were identified as two Pencillium species and a Mucor species. Our results show that the new assay can be effectively used to screen fungi for the production of UO(2)(2+) chelating ligands. We suggest that hydroxamate siderophores can be produced by mucoraceous fungi.

Morphology and physiology of the dimorphic fungus Mucor circinelloides (syn. M. racemosus) during anaerobic growth.

The dimorphic Mucor circinelloides requires an anaerobic atmosphere and the presence of 30% CO2 to grow as a multipolar budding yeast, otherwise hyphal growth predominates. Establishing other means to control the morphology would be a distinct advantage in the development of a fermentation process for this organism for the production of heterologous proteins. Thus, conditions suppressing polarised growth while at the same time abolishing the CO2 requirement were investigated in submerged cultivations. It was found that supplementing cultures with mixtures of ergosterol and Tween 80 resulted in yeast-like growth under 100% N2. Their impact on growth and morphological development was assessed at a range of concentrations. Maximum biomass levels and the specific growth rate decreased at elevated levels of ergosterol and Tween 80. Possible effects of carbon dioxide and the added fatty acid/sterol mixture on supporting yeast growth by influencing the fluidity of the plasma membrane or affecting polarised growth are discussed.

[Synthesis of biologically active lipids by fungus Mucor lusitanicus 306D grown on media with various composition]. / Sintez biologicheski aktivnykh lipidov gribom Mucor lusitanicus 306D na sredakh raznogo sostava.

Growth and lipogenesis of fungus Mucor lusitanicus 306 D producing gamma-linolenic acid was studied under various regimes of nitrogen and carbon nutrition. Media containing food industry wastes such as maize extract, molasses, and protein hydrolysate were used. Content of gamma-linolenic acid was higher when using carbohydrates such as glucose and molasses as carbon sources and urea as a nitrogen source. At high glucose concentration (100 g/l), fed batch cultivation provided high content of gamma-linolenic acid in lipids (1 g/l). After extraction of lipids, fungus biomass contained 42% proteins with all essential amino acids. Defatted biomass was shown to be effectively assimilated by minks.