Semi-pilot scale production of biodiesel from waste frying oil by genetically improved fungal lipases.

This paper addresses the issue of combining the usage of waste frying oil (WFO), as a feedstock, and a lipase produced in solid-state fermentation (SSF), as a biocatalyst, for semi-pilot scale production of biodiesel as fatty acid methyl esters (FAME). Two fungal mutants namely; Rhizopus stolonifer 1aNRC11 mutant F (1F) and Aspergillus tamarii NDA03a mutant G (3G) were used as a cocatalyst. The two mutants were cultivated separately by SSF in a tray bioreactor. The dried fermented solid of 1F and 3G mutants were used in a ratio of 3:1, respectively, for WFO transesterification. Optimization of several semi-pilot process stages including SSF and WFO transesterification reaction conditions resulted in 92.3% conversion of WFO to FAME. This FAME yield was obtained after 48 h using 10% cocatalyst (w/w of WFO), 10% water (w/w of WFO) and 3:1 methanol/ WFO molar ratio at 30 °C and 250 rpm. A preliminary economic evaluation of produced biodiesel price (190 $/Ton) is less than half the price of petroleum diesel in Egypt (401$/Ton) and is about 40.3% the price of biodiesel produced using a pure enzyme, which is a promising result. This strategy makes the biodiesel synthesis process greener, economical and sustainable.

Iron restriction inside macrophages regulates pulmonary host defense against Rhizopus species.

Mucormycosis is a life-threatening respiratory fungal infection predominantly caused by Rhizopus species. Mucormycosis has incompletely understood pathogenesis, particularly how abnormalities in iron metabolism compromise immune responses. Here we show how, as opposed to other filamentous fungi, Rhizopus spp. establish intracellular persistence inside alveolar macrophages (AMs). Mechanistically, lack of intracellular swelling of Rhizopus conidia results in surface retention of melanin, which induces phagosome maturation arrest through inhibition of LC3-associated phagocytosis. Intracellular inhibition of Rhizopus is an important effector mechanism, as infection of immunocompetent mice with swollen conidia, which evade phagocytosis, results in acute lethality. Concordantly, AM depletion markedly increases susceptibility to mucormycosis. Host and pathogen transcriptomics, iron supplementation studies, and genetic manipulation of iron assimilation of fungal pathways demonstrate that iron restriction inside macrophages regulates immunity against Rhizopus. Our findings shed light on the pathogenetic mechanisms of mucormycosis and reveal the role of macrophage-mediated nutritional immunity against filamentous fungi.

Polyvinyl acetate processing wastewater treatment using combined Fenton's reagent and fungal consortium: Application of central composite design for conditions optimization.

The Fenton reaction as an oxidative degradation process was used for industrial chemical wastewater (ICW) pretreatment. The biodegradation of pretreated ICW was performed, in aqueous environment under aerobic condition, by a defined fungal consortium. The central composite design (CCD) was used to study the effect of nitrogen and phosphorus addition and the concentration of the pollution on the removal of polyvinyl alcohol (PVA) and organic compounds. The interaction between parameters was modeled using the response surface methodology (RSM). Results of optimization showed COD, PVA and color removal yields of 97.8%, 98.5% and 99.75%, respectively with a supplementof 1.4 gL-1 of (NH4)2SO4, 1.2 gL-1 of KH2PO4 and 75% of concentrated ICW. Enzymatic analysis proved that laccase and lignin peroxidase were involved in the biodegradation with 45 UIL-1 and 450 UIL-1, respectively. Furthermore, the analysis of metabolic products using Fourier transforms infrared spectroscopy (FTIR) and nuclear magnetic resonance (1HNMR) showed clearly the mineralization of organic compounds and the formation of formic acid and ethanol. Therefore, the effective treatment of ICW was achieved by developing an integrated chemical and biological process which met the requirement for a safety effluent respectful for environment without risks for public health.

Effects of solid-state fermentation with two filamentous fungi on the total phenolic contents, flavonoids, antioxidant activities and lipid fractions of plum fruit (Prunus domestica L.) by-products.

Evolutions of phenolic contents and antioxidant activities during solid-state fermentation (SSF) of plum pomaces (from the juice industry) and brandy distillery wastes with Aspergillus niger and Rhizopus oligosporus were investigated. The effect of fermentation time on the oil content and major lipid classes in the plum kernels was also studied. Results showed that total phenolic (TP) amounts increased by over 30% for SSF with Rhizopus oligosporus and by >21% for SSF with A. niger. The total flavonoid contents presented similar tendencies to those of the TPs. The free radical scavenging activities of methanolic extracts were also significantly enhanced. The HPLC-MS analysis showed that quercetin-3-glucoside was the major phenolic compound in both fermented plum by-products. The results also demonstrated that SSF not only helped to achieve higher lipid recovery from plum kernels, but also resulted in oils with better quality attributes (high sterol ester and n-3 PUFA-rich polar lipid contents).

Effect on tomato plant and fruit of the application of biopolymer-oregano essential oil coatings.

BACKGROUND: Oregano essential oil (EO) was incorporated into film-forming dispersions (FFDs) based on biopolymers (chitosan and/or methylcellulose) at two different concentrations. The effect of the application of the FFDs was evaluated on tomato plants (cultivar Micro-Tom) at three different stages of development, and on pre-harvest and postharvest applications on tomato fruit. RESULTS: The application of the FFDs at '3 Leaves' stage caused phytotoxic problems, which were lethal when the EO was applied without biopolymers. Even though plant growth and development were delayed, the total biomass and the crop yield were not affected by biopolymer-EO treatments. When the FFDs were applied in the 'Fruit' stage the pre-harvest application of FFDs had no negative effects. All FFDs containing EO significantly reduced the respiration rate of tomato fruit and diminished weight loss during storage. Moreover, biopolymer-EO FFDs led to a decrease in the fungal decay of tomato fruit inoculated with Rhizopus stolonifer spores, as compared with non-treated tomato fruit and those coated with FFDs without EO. CONCLUSION: The application of biopolymer-oregano essential oil coatings has been proven to be an effective treatment to control R. stolonifer in tomato fruit. © 2016 Society of Chemical Industry.

Compositional changes in (iso)flavonoids and estrogenic activity of three edible Lupinus species by germination and Rhizopus-elicitation.

The effects of germination and elicitation on (iso)flavonoid composition of extracts from three edible lupine species (Lupinus luteus, Lupinus albus, Lupinus angustifolius) were determined by RP-UHPLC-MS(n). The total (iso)flavonoid content of lupine increased over 10-fold upon germination, with the total content and composition of isoflavonoids more affected than those of flavonoids. Glycosylated isoflavones were the most predominant compounds found in lupine seedlings. Lesser amounts of isoflavone aglycones, including prenylated ones, were also accumulated. Elicitation with Rhizopus oryzae, in addition to germination, raised the content of isoflavonoids further: the total content of 2'-hydroxygenistein derivatives was increased considerably, without increasing that of genistein derivatives. Elicitation by fungus triggered prenylation of isoflavonoids, especially of the 2'-hydroxygenistein derivatives. The preferred positions of prenylation differed among the three lupine species. The change in isoflavone composition increased the agonistic activity of the extracts towards the human estrogen receptors, whereas no antagonistic activity was observed.

Enhancement and modeling of microparticle-added Rhizopus oryzae lactic acid production.

Lactic acid has a wide industrial application area and can be produced by fungal strains. However, excessive bulk growth form of fungi during the fermentations is a major problem, which limits the fermentation performance. Microparticles are excellent tools to prevent bulk fungal growth and provide homogenized fermentation broth to increase uniformity and the prediction performance of the models. Therefore, in this study, addition of aluminum oxide and talcum microparticles into fermentations was evaluated to enhance the production of lactic acid by Rhizopus oryzae. The results showed that the bulk fungal growth was prevented and the lactic acid concentration increased from 6.02 to 13.88 and 24.01 g/L, when 15 g/L of aluminum oxide or 10 g/L of talcum was used, respectively, in the shake-flask fermentations. Additionally, substrate concentration, pH, and agitation were optimized in the bioreactors using response surface methodology, and optimum values were determined as 126 g/L of glucose, 6.22 pH, and 387 rpm, respectively. Under these conditions, lactic acid production further increased to 75.1 ± 1.5 g/L with 10 g/L of talcum addition. Also, lactic acid production and glucose consumption in the batch fermentation were successfully modeled with modified Gompertz model and modified logistic model. RMSE and MAE values for lactic acid production were calculated as 2.279 and 1.498 for the modified Gompertz model; 3.6 and 4.056 for the modified logistic model. Additionally, modified logistic model predicted glucose consumption with -2.088 MAE and 2.868 RMSE, whereas these values were calculated as 2.035 and 3.946 for the modified Gompertz model.

In vitro Antifungal Activity and Mechanism of Action of Tea Polyphenols and Tea Saponin against Rhizopus stolonifer.

The in vitro antifungal activities and mechanism of action of tea polyphenols (TP), tea saponin (TS) and their combination were evaluated against Rhizopus stolonifer. The results showed that both TP and TS inhibited the mycelial growth in a dose-dependent manner, and their combination at the ratio of 7:3 exhibited synergistic antifungal interaction. We also observed that the treatment of TP or TS significantly induced the production of H2O2 and resulted in membrane lipid peroxidation, thus leading to an increase in cell membrane permeability and the leakage of K(+), soluble protein and soluble sugar. Moreover, combining them for treatment increased the induction of H2O2 production and oxidative damage. Scanning electron microscopic observations also showed the damage to the hyphal cell structure. It was concluded that TP, TS and their combination inhibit the growth of R. stolonifer through the induction of H2O2 production, leading to cell membrane oxidative damage and intracellular constituent leakage. These findings suggest that TP and TS can potentially be used as an alternative to control postharvest fruit diseases caused by R. stolonifer.

Effects of 7-hydroxycalamenene isolated from Croton cajucara essential oil on growth, lipid content and ultrastructural aspects of Rhizopus oryzae.

The leaves and bark of Croton cajucara, a shrub from the Amazon region, have been used in folk medicine to treat diabetes, malaria, and gastrointestinal and liver disorders. The essential oil from the leaves, rich in linalool, presented antileishmanial and antimicrobial activities. A chemotype of this species was found with an essential oil rich in 7-hydroxycalamenene. During our studies of the C. cajucara essential oil, we isolated 7-hydroxycalamenene at > 98 % purity. The minimum inhibitory concentration of 7-hydroxycalamenene against Absidia cylindrospora, Cunninghamella elegans, Mucor circinelloides, Mucor circinelloides f. circinelloides, Mucor mucedo, Mucor plumbeus, Mucor ramosissimus, Rhizopus microsporus, Rhizopus oryzae, and Syncephalastrum racemosum ranged from 19.53 to 2500 µg/mL. The reference drug used, amphotericin B, presented a minimum inhibitory concentration ranging from 0.085 µg/mL to 43.87 µg/mL. 7-Hydroxycalamenene also altered spore differentiation and total lipid content. Ultrastructural analysis by transmission electron microscopy showed significant alterations in the cellular structure of R. oryzae.

Preparation and application of a novel bioflocculant by two strains of Rhizopus sp. using potato starch wastewater as nutrilite.

A complex bioflocculant MBF917 was prepared by Rhizopus sp. M9 and M17 using potato starch wastewater (PSW) as nutrilite, and its flocculation characteristics of treating PSW were studied. Culture conditions of the two strains were optimized, and flocculating conditions of the bioflocculant for treating PSW were also investigated. The optimal and economical culture conditions were determined as COD of about 1600 mg/L, 0.3 g/L urea and 0.04 g/L potassium dihydrogen phosphate, with no need of adding carbon sources or adjusting pH. When the bioflocculant was used to flocculate PSW, the optimal dosage was 0.1 mL/L with addition of 5 mL/L 10% CaCl2 as coagulant aid, and there was no need to adjust pH. After flocculation, COD and turbidity removal rates of the PSW could reach 54.09% and 92.11%, respectively, and 1.1g/L proteic substance was recycled from the PSW as a byproduct that could be used for animal feed.

In vitro antifungal activity of three geophytic plant extracts against three post-harvest pathogenic fungi.

Plant extracts appear to be one of the most effective alternative methods of plant diseases control which are less harmful to human beings and environment. In vitro antifungal activity of methanolic extracts of three promising wild geophytic plants against three post-harvest pathogenic fungi using radial growth technique was conducted. These extracts included the shoot system (S) and underground parts (R) of Asparagus stipularis, Cyperus capitatus and Stipagrostis lanata. The tested fungi were Alternaria solani, Aspergillus niger and Rhizopus stolonifer. The results exhibited that, all plant extracts had antifungal activity against the tested fungi. The antifungal activity greatly varied depending on plant parts and/or plant species. R. stolonifer was the most susceptible fungus to the tested plant extracts followed by A. niger and then A. solani. On the other hand, the most effective plant extracts against tested fungi were S. lanata (S) and A. stipularis (R). The most effective plant extracts against R. stolonifer were S. lanata (R) and C. capitatus (S). While, the extracts of A. stipularis (R) and S. lanata (S) were the most effective against A. niger. The extracts of C. capitatus (S) and S. lanata (S) exhibited the highest antifungal activity against A. solani. The results demonstrated that, the methanolic extracts of A. stipularis, C. capitatus and S. lanata had potential antifungal activity against A. solani, A. niger and R. stolonifer.

Efficacy of the application of a coating composed of chitosan and Origanum vulgare L. essential oil to control Rhizopus stolonifer and Aspergillus niger in grapes (Vitis labrusca L.).

This study evaluated the efficacy of the combined application of chitosan (CHI) and Origanum vulgare L. essential oil (OV) in the inhibition of Rhizopus stolonifer URM 3728 and Aspergillus niger URM 5842 on laboratory media and on grapes (Vitis labrusca L.) and its influence on the physical, physicochemical and sensory characteristics of the fruits during storage (25 °C, 12 days and 12 °C, 24 days). The application of mixtures of different CHI and OV concentrations (Minimum Inhibitory Concentration - MIC, 1/2 MIC and 1/4 MIC) inhibited the mycelial growth of the test fungi. The application of CHI and OV at sub-inhibitory concentrations (CHI 1/2 MIC + OV 1/4 MIC; CHI 1/2 MIC + OV 1/2 MIC) inhibited spore germination and caused morphological changes in fungal spores and mycelia, in addition to inhibiting the growth of the assayed fungi strains in artificially infected grapes as well as the autochthonous mycoflora of grapes stored at both room and cold temperature. In general, the application of a coating composed of CHI and OV at sub-inhibitory concentrations preserved the quality of grapes as measured by their physical and physicochemical attributes, while some of their sensory attributes improved throughout the assessed storage time. These results demonstrate the potential of the combination of CHI and OV at sub-inhibitory concentrations to control post-harvest pathogenic fungi in fruits, in particular, R. stolonifer and A. niger in grapes.

Antifungal effect of Mexican oregano (Lippia berlandieri Schauer) essential oil on a wheat flour-based medium.

The antimicrobial activity of oregano has been attributed mainly to the presence of volatile compounds found in its essential oil (EO), mainly carvacrol and thymol. The search for antimicrobial activity of oregano EO with different concentrations of thymol and carvacrol, can lead to products with a wider range of applications. The aim of this work was to describe the in vitro antifungal effect of Mexican oregano (Lippia berlandieri Schauer) EO fractions on the growth of Aspergillus, Penicillium, and Rhizopus sp. The Mexican oregano EO fractions studied had different concentrations of carvacrol, which decreased from fraction 1 to 5 (81% to 23%), while thymol content increased from 3% to 64%. Fungal inhibition was evaluated on a wheat flour-based medium with EO fractions concentrations ranging from 50 to 200 mg/kg. Radial growth curves were fitted using the modified Gompertz model (R(2)(adj) = 0.989 ± 0.01). No significant differences (P > 0.05) were found with the different composition of the Mexican oregano EO fractions; nevertheless, fraction concentration presented significant (P < 0.05) mold inhibition as concentration increased. Rhizopus sp. (Rh18) showed a linear reduction on specific growth rate, on the maximum mold growth at the stationary phase, and an increase in the lag time as the concentration of the oregano EO increased; mold growth inhibition were achieved at 150 mg/kg in fractions 1 to 4, and at 100 mg/kg for fraction 5. Aspergillus sp. (As6) and Penicillium sp. (Pe36) were inhibited at 150 and 200 mg/kg, respectively. Results obtained suggest that Mexican oregano EO (Lippia berlandieri Schauer) compounds could be used as antimicrobial agents to prevent fungal growth in bakery products.

Antifungal activity of Thymus vulgaris L. essential oil and its constituent phytochemicals against Rhizopus oryzae: interaction with ergosterol.

Mucormycoses are emerging infections that have high rates of morbidity and mortality. They show high resistance to antifungal agents, and there is a limited therapeutic arsenal currently available, therefore, there is a great need to give priority to testing therapeutic agents for the treatment of mucormycosis. Along this line, the use of essential oils and phytoconstituents has been emphasized as a new therapeutic approach. The objective of this work was to investigate the antifungal activity of the essential oil (EO) of Thymus vulgaris, and its constituents thymol and p-cymene against Rhizopus oryzae, through microbiological screening, determination of minimal inhibitory concentration (MICs) and minimal fungicidal concentration (MFCs), effects on mycelial growth and germination of sporangiospores and interaction with ergosterol. The MIC of EO and thymol varied 128-512 µg/mL, but the MFC of EO and thymol varied 512-1024 µg/mL and 128-1024 µg/mL, respectively. The results also showed that EO and thymol significantly inhibited mycelial development and germination of sporangiospores. Investigation of the mechanism of antifungal action showed that EO and thymol interact with ergosterol. These data indicate that EO of T. vulgaris and thymol possess strong antifungal activity, which can be related to their interaction with ergosterol, supporting the possible use of these products in the treatment of mucormycosis.

Green processing of tropical banagrass into biofuel and biobased products: an innovative biorefinery approach.

Banagrass (Pennisetum purpureum) approximately 4 months old was hand-harvested and shredded. Half of the sample was dewatered using a screw-press, whereby the extracted juice was used for cultivating an edible fungus, Rhizopus microsporus, for aquaculture feed supplementation. The remaining biomass was divided into four separate streams: (1) wet, juiced; (2) dry, juiced; (3) wet, unjuiced; and (4) dry, unjuiced. Each stream was pretreated with dilute sulfuric acid and compared on the basis of sugar release at varying acid concentrations, temperatures, and residence times. Wet, juiced banagrass released the most soluble sugars (theoretical xylose and ∼85% glucose). Ultrasonication (20 kHz) was applied to further increase monomeric sugar release but demonstrated little improvement on total sugar yields. Fungal biomass generated from banagrass juice exhibited potential as a fungal-protein production medium producing 1.16±0.34 g biomass increase/g initial biomass.

A combination of heat treatment and Pichia guilliermondii prevents cherry tomato spoilage by fungi.

This study investigated the effectiveness of heat treatment and Pichia guilliermondii, either alone or in combination, to combat postharvest fungal spoilage in cherry tomato fruit. In vitro experiments demonstrated that heat treatment at 38 degrees C significantly inhibited mycelial growth of three different pathogens (Botrytis cinerea, Alternaria alternata and Rhizopus stolonifer Ehrenb). In vivo experiments unveiled that either heat treatment or P. guilliermondii reduced decay caused by these pathogens. Furthermore, a combination of heat treatment followed by the application of P. guilliermondii (H+P) provided the best efficacy in prevention of cherry tomato from fungal spoilage. Following, H+P treatment, electronic nose detected a reduction of volatility in cherry tomato fruit odor, an indicator of preserving fruit's freshness. Scanning electron microscopy unveiled that heat treatment at 38 degrees C for 24h inhibited hyphae growth and spore germination of R. stolonifer Ehrenb while P. guilliermondii multiplied rapidly on fruit wounds, and its cells had a strong capability of adhesion to the hyphae of R. stolonifer Ehrenb. However, heat treatment also seriously injured P. guilliermondii, therefore P. guilliermondii should be applied after heat treatment. A combination of heat treatment and P. guilliermondii is one of the most effective techniques at controlling postharvest fungal spoilage in cherry tomato fruit.

Chemical constituents of Nauclea latifolia and their anti-GST and anti-fungal activities.

The crude ethanolic extract of Nauclea latifolia yielded five known compounds, strictosamide (1), naucleamides A (2), naucleamide F (3), quinovic acid-3-O-beta-rhamnosylpyranoside (4), and quinovic acid 3-O-beta-fucosylpyranoside (5). Microbial reactions, using a whole cell culture of Rhizopus circinans on compound (1), yielded three analogues, 10-hydroxystrictosamide (6), 10-beta-glucosyloxyvincoside lactam (7) and 16,17-dihydro-10-beta-glucosyloxyvincoside lactam (8). Compounds 1-8 were identified with the aid of extensive NMR spectral studies. Compound 8 was found to be a new metabolite. Compounds 1-8 exhibited different levels of anti-GST and anti-fungal activities.

Lipid formation and gama-linolenic acid production by Mucor circinelloides and Rhizopus sp., grown on vegetable oil / Formação de lipídeos e produção de ácido gama-linolênico por Mucor circinelloides e Rhizopus sp., em óleos vegetais

The fungi strains were tested in Bioscreen automated system to select the best nutritional source. Following, shaking submserse cultures were studied in media containing sole carbon or nitrogen source. The growth of these strains improved in media containing vegetable oil, with high concentration of lipids. The high concentration of γ-linolenic acid was obtained with M. circinelloides in culture containing sesame oil.

Linhagens de fungos foram testadas em sistema automatizado Bioscreen para selecionar melhor fonte nutricional. Em seguida, foram estudadas culturas submersas em meios contendo uma única fonte de carbono e de nitrogênio. As linhagens contendo alta concentração de lipídeos tiveram melhor crescimento em meio contendo óleos de gergelim ou de dendê. Maior concentração de ácido γ-linolênico foi obtida com M. circinelloides nas culturas em óleo de gergelim.

Converting corn wet-milling effluent into high-value fungal biomass in a biofilm reactor.

Rhizopus microsporus was grown in an attached growth system using corn wet-milling effluent as a growth medium. This strain was chosen due to its ability to effectively degrade organic matter in corn wet-milling effluent and for its properties to produce significant levels of protein, chitin and chitosan. Fungal growth and organic removal efficiency were examined under both aseptic and non-aseptic conditions with and without nutrient supplementation. Plastic composite support (PCS) tubes, composed of 50% (w/w) polypropylene (PP) and 50% (w/w) agricultural products were used as support media. Significantly higher organic removal measured as chemical oxygen demand (COD) and biomass yield were observed in the bioreactor with PCS tubes than in two control bioreactors; that is with PP tubes alone and suspended growth (without support media). This confirmed that the PCS support medium with agricultural components enhanced fungal growth and organic removal. The results showed that supplementation of nutrients (e.g., mineral salts) under aseptic conditions enhanced the COD removal from 50% to 55% and observed biomass yield from 0.11 to 0.16 g (dry-weight)/g COD(removed) (i.e., from 0.10 to 0.14 g volatile solids (VS)/g COD(removed) approximately). Non-aseptic operation without nutrient supplementation resulted in an observed biomass yield of 0.32 g volatile suspended solids (VSS)/g COD(removed) with no significant improvement in COD removal ( approximately 53%); whereas with nutrient supplementation, the observed biomass yield increased to 0.56 g VSS/g COD(removed) and COD removal improved to 85%. The fungal system was able to degrade the organic matter with concomitant production of high-value fungal biomass. This is the first study that examined the conversion of corn milling waste stream into high value fungal protein.

Co-production of lactic acid and chitin using a pelletized filamentous fungus Rhizopus oryzae cultured on cull potatoes and glucose.

AIMS: This paper developed a novel process for lactic acid and chitin co-production of the pelletized Rhzious oryzae NRRL 395 fermentation using underutilized cull potatoes and glucose as nutrient source. METHODS AND RESULTS: Whole potato hydrolysate medium was first used to produce the highest pelletized biomass yield accompanying the highest chitin content in biomass. An enhanced lactic acid production then followed up using batch, repeated batch and fed batch culture with glucose as carbon source and mixture of ammonia and sodium hydroxide as neutralizer. The lactic acid productivity peaked at 2.8 and 3 g l(-1 )h(-1) in repeated batch culture and batch culture, respectively. The fed batch culture had the highest lactate concentration of 140 g l(-1). CONCLUSIONS: Separation of the biomass cultivation and the lactic acid production is able to not only improve lactic acid production, but also enhance the chitin content. Cull potato hydrolysate used as a nutrient source for biomass cultivation can significantly increase both biomass yield and chitin content. SIGNIFICANCE AND IMPACT OF THE STUDY: The three-step process using pelletized R. oryzae fermentation innovatively integrates utilization of agricultural residues into the process of co-producing lactic acid and chitin, so as to improve the efficiency, revenues and cost of fungal lactic acid production.