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Xylooligosaccharides (XOS) are non-digestible and fermentable oligomers that stand out for their efficient production by enzymatic hydrolysis and beneficial effects on human health. This study aimed to investigate the influence of the main reaction parameters of the beechwood xylan hydrolysis using crude xylanase from Aureobasidium pullulans CCT 1261, thus achieving the maximum XOS production. The effects of temperature (40 to 50 °C), reaction time (12 to 48 h), type of agitation, substrate concentration (1 to 6%, w/v), xylanase loading (100 to 300 U/g xylan), and pH (4.0 to 6.0) on the XOS production were fully evaluated. The most suitable conditions for XOS production included orbital shaking of 180 rpm, 40 °C, and 24 h of reaction. High contents of total XOS (10.1 mg/mL) and XOS with degree of polymerization (DP) of 2-3 (9.7 mg/mL), besides to a high percentage of XOS (99.1%), were obtained at 6% (w/v) of beechwood xylan, xylanase loading of 260 U/g xylan, and pH 6.0. The establishment of the best hydrolysis conditions allowed increasing both the content of total XOS 1.5-fold and the percentage of XOS by 9.4%, when compared to the initial production (6.7 mg/mL and 89.7%, respectively). Thus, this study established an efficient enzymatic hydrolysis process that results in a hydrolysate containing XOS with potential prebiotic character (i.e., rich in XOS with DP 2-3) and low xylose amounts.

To improve β-1,3-1,6-D-glucan (β-glucan) production by Aureobasidium pullulans, an Agrobacterium tumefaciens-mediated transformation method was developed to screen a mutant A. pullulans CGMCC 19650. Based on thermal asymmetric-interlaced PCR detection, DNA sequencing, BLAST analysis, and quantitative real-time PCR assay, the T-DNA was identified to be inserted in the coding region of mal31 gene, which encodes a sugar transporter involved in pullulan biosynthesis in the mutant. The maximal biomass and β-glucan production under batch fermentation were significantly increased by 47.6% and 78.6%, respectively, while pullulan production was decreased by 41.7% in the mutant, as compared to the parental strain A. pullulans CCTCC M 2012259. Analysis of the physiological mechanism of these changes revealed that mal31 gene disruption increased the transcriptional levels of pgm2, ugp, fks1, and kre6 genes; increased the amounts of key enzymes associated with UDPG and β-glucan biosynthesis; and improved intracellular UDPG contents and energy supply, all of which favored β-glucan production. However, the T-DNA insertion decreased the transcriptional levels of ags2 genes, and reduced the biosynthetic capability to form pullulan, resulting in the decrease in pullulan production. This study not only provides an effective approach for improved β-glucan production by A. pullulans, but also presents an accurate and useful gene for metabolic engineering of the producer for efficient polysaccharide production. KEY POINTS: • A mutant A. pullulans CGMCC 19650 was screened by using the ATMT method. • The mal31 gene encoding a sugar transporter was disrupted in the mutant. • β-Glucan produced by the mutant was significantly improved.

Cv. Arbequina fruits were inoculated during one day with suspensions of Aureobasidium pullulans, Epicoccum nigrum or both species (104 spores/g olives), isolated from olive leaves, aiming to increase the phenolic content of the olive oils. Oils were extracted from non-inoculated (control) and inoculated olives, being evaluated for their oxidative stability, and phenolic composition. Compared to the control oils, those extracted from inoculated olives showed higher oxidative stability (+1629%), phenolic acids contents (up to+37%), phenolic alcohols (up to+101%), flavonoids (up to+154%) and secoiridoid derivatives (up to + 12%). These positive effects could be attributed to the hydrolytic activity of enzymes secreted by the studied fungi on the fruit cellular membranes and extracted phenolics, increasing the extractability and, consequently, altering the phenolic concentration. Considering the two fungi species, E. nigrum inoculation had a more significant positive impact on the contents of tyrosol, oleuropein, and ligstroside derivatives, showing an inhibition effect regarding the oleacein formation. Contrary, A. pullulans inoculation mainly resulted into an accentuated increase of luteolin and oleacein contents (3 and 12-times higher compared with control oils, respectively). Overall, no enhanced positive synergetic effect could be attributed to the inoculation with a suspension containing both fungi. The significant changes observed on the phenolic contents, due to the fungal inoculation, allowed the successful discrimination of the four types of extracted oils using an electronic tongue. This device acts as an artificial taste device allowing anticipating an impact at the oils basic taste sensations due to the fungi inoculation., The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for fnancial support by national funds FCT/MCTES (PIDDAC) to CIMO (UIDB/00690/2020 and UIDP/00690/2020), CEB (UIDB/04469/2020), REQUIMTE-LAQV (UIDB/50006/2020) units and SusTEC (LA/P/0007/2020); to Project “GreenHealth—Digital strategies in biological assets to improve well being and promote green health” (Norte-01–0145-FEDER-000042) and to Project AgriFood XXI (NORTE-01–0145-FEDER-000041), funded by the European Regional Development Fund under the scope of Norte2020 ‐ Programa Operacional Regional do Norte. Ítala M.G. Marx acknowledges the Ph.D. grant (SFRH/BD/137283/2018) pro vided by FCT. Nuno Rodrigues thanks to National funding by FCT-Foundation for Science and Technology, P.I., through the institutional scientifc employment program-contract, info:eu-repo/semantics/publishedVersion

Cucumber mosaic virus (CMV), the most prevalent virus in chili pepper (Capsicum annuum L.), negatively affects chili pepper production in South Korea. In this study, foliar spraying with β-glucans obtained from the mycelial walls of the yeast-like fungus Aureobasidium pullulans inhibited CMV infection of chili pepper if applied before virus inoculation. At three concentrations, β-glucans from A. pullulans significantly ameliorated CMV symptoms in treated chili pepper; the effect was greater in plants treated with 0.01% β-glucans than 0.005% or 0.001% β-glucans. Double antibody sandwich enzyme-linked immunosorbent assay showed that these β-glucans treatments resulted in 1.7- to 10-fold reductions in CMV accumulation in the treated chili pepper. The glucans did not act directly on the virus and did not interfere with virus disassembly or replication. Foliar spraying with 0.01% β-glucans from A. pullulans at 24 hr intervals for 3 days significantly increased plant height, the total number of fruit, and the fresh weight of chili pepper fruit. However, the stem diameter of chili pepper treated with β-glucans did not increase significantly. These results indicate that foliar spraying with β-glucans from A. pullulans acts an antiviral agent against CMV infection and stimulates chili pepper growth.

Seed sprouts are nutrient-rich components, and their use in human and animal diets have increased in recent times. Microbial fermentation of seed sprouts not only improves the nutrient digestibility but also elevates the nutritional characteristics by lowering the level of antinutritional factors (ANFs). This study evaluates the effect of sprouting followed by fungal fermentation on the nutritional characteristics of canola seed sprouts. Canola seeds were soaked overnight and sprouted over a 6-day period. Dried and ground seed sprouts were subjected to submerged fermentation for 5 days using three fungal strains (Aureobasidium pullulans, Trichoderma reesei, Neurospora crassa). Results showed an improvement in proteins and reduction in ANFs of sprouts when compared to seeds. Fermentation with T. reesei resulted in higher protein titers, while N. crassa fermented sprouts exhibited slight reduction in crude fiber. Sprouting prior to fermentation helped in enhancing the protein solubility of canola seed and reducing the overall fermentation duration. Based on these findings it could be concluded that sprouting prior to fermentation can potentially lead to the production of nutritionally enriched canola based high protein ingredient. However, further research needs to be conducted to develop a mechanism for minimizing the oil loss during seed sprouting process.

Extracellular rhodanese obtained from Aureobasidium pullulans was employed in both free and immobilized forms for the biodegradation of cyanide present in cassava processing mill effluent (CPME). Crosslinking with glutaraldehyde (at an optimum concentration of 5% v/v) before entrapment in alginate beads resulted in the highest immobilization yield of 94.5% and reduced enzyme leakage of 1.8%. Rhodanese immobilized by cross-linking before entrapment (cbe) retained about 46% of its initial activity after eight cycles of catalysis compared to the entrapment in alginate alone (eaa) which lost more than 79% after the fifth catalytic cycle. A cross-examination of thermodynamic ( Δ G d * , Δ S d * , Δ H d * ) kinetic ( k d , t 1 / 2 , D and z - values ) parameters at 30-70 °C showed that cbe displayed a higher resistance to thermal inactivation when compared to the free enzyme (fe) and (eaa). The efficiency of cyanide biodegradation from the CPME by the fe, eaa and cbe were 55, 62, and 74% respectively after 6 h. Biodegradation of cyanide using the cbe was monitored using FTIR spectroscopy. Rhodanese immobilized via cbe had a higher resistance to thermal denaturation over other enzyme forms. Hence, this makes cbe adaptable for large-scale detoxification of cyanide from CPME.

β-poly(L-malic acid) (PMLA) has attracted industrial interest for its potential applications in medicine and other industries. For a sustainable PMLA production, it requires replacing/reducing the CaCO3 usage, since the residual CaCO3 impeded the cells’ utilization, and a large amount of commercially useless gypsum was accumulated. In this study, it was found that more glucose was converted into CO2 using soluble alkalis compared with CaCO3 usage. Moreover, since the high ion strength and respiration effect of soluble alkalis also inhibited PMLA production, they could not effectively replace CaCO3. Furthermore, comparing the fermentations with different neutralizers (soluble alkali vs. CaCO3), it was found that the differential genes are mainly involved in the pathway of starch and sucrose metabolism, pentose and glucuronate interconversions, histidine metabolism, ascorbate and aldarate metabolism, and phagosome. In detail, in the case with CaCO3, 562 genes were downregulated and 262 genes were upregulated, and especially, those genes involved in energy production and conversion were downregulated by 26.7%. Therefore, the irreplaceability of CaCO3 was caused by its effect on the PMLA metabolic pathway rather than its usage as neutralizer. Finally, a combined pH shift control strategy with CaCO3 addition was developed. After the fermentation, 64.8 g/L PMLA and 38.9 g/L biomass were obtained with undetectable CaCO3 and less CO2 emission. • The effect of CaCO3 on PMLA metabolic pathway resulted in its irreplaceability. • A pH shift control strategy with CaCO 3 addition was developed. • Undetectable CaCO 3 and less CO 2 emission were detected with the new strategy.

Aureobasidium pullulans LB83 was evaluated for cellulase production under submerged fermentation conditions. Different process variables such as carbon sources (corn cob, sugarcane bagasse, and sug...

Cellulose is the most abundant renewable carbon source found in nature and is of great interest to various industries, including the food industry. Generally, microorganisms can be used to convert ...

Epiphytic yeasts were isolated from different cultivars of apples and lemons and identified by a combination of PCR-RFLP of 5.8S rRNA region and sequencing of D1/D2 domain of the 26S rRNA gene. Among 69 isolates, Aureobasidium pullulans GE17 and Meyerozyma guilliermondii KL3 strains showed the greatest antagonistic activity against two significant apple and lemon postharvest pathogens, Penicillium expansum DSM62841 (blue mold) and Penicillium digitatum DSM2750 (green mold), after preliminary screening. Yeasts were applied as single and mixed cultures with two different cell concentrations of 106 and 108 cells/ml in the present study. It was determined that antagonistic activity of two yeast strains studied emerged with a combination of several mechanisms of action including competition for space and nutrients, production of volatile organic compounds (VOCs), secretion of extracellular lytic enzymes and inhibition of fungal spore germination. The highest inhibition of mycelial growth on P. expansum DSM62841 and P. digitatum DSM2750 (83.4% and 74.7%, respectively) was achieved by utilization of single culture of A. pullulans GE17. Otherwise, the application of mixed culture at the ratio of 108 cells/ml inhibited spore germination of both pathogens from 86% to 95%. Results of this study suggest that an increase in yeast cell concentrations positively affected their biocontrol activity against blue and green molds. According to the results, employing single culture of M. guilliermondii KL3 did not exhibit effective antagonistic activity against blue and green molds. However, utilization of A. pullulans GE17 alone and mixed culture showed succesfull controlling against both P. expansum DSM62841 and P. digitatum DSM2750.

Pullulan has many useful characteristics but, its high cost limits its potential applications. In the present work, kitchen waste (KW), which otherwise has zero commercial value, was evaluated for the economical production of pullulan. Before fermentation, the KW was hydrolyzed into free sugars using an in-house produced cocktail of enzymes. During hydrolysis, 46 ± 3.5 g/l and 31 ± 2.2 g/l of total reducing sugars and glucose were released, respectively. Hydrolyzed kitchen waste was then used as substrate for fermentation by Aureobasidium pullulans MTCC 2013 yielding 20.46 ± 2.01 g/l pullulan. Further, effect of different nitrogen sources was evaluated and yeast extract (3%) was found to be the best, yielding (24.77 ± 1.06 g/l) exopolysaccharide (EPS). The pullulan produced from KW was characterized in terms of organoleptic properties, physical strength, Fourier-transform infrared spectroscopy (FTIR), and H nuclear magnetic resonance (H NMR) analysis. The results corroborated well with commercial pullulan. The biodegradable nature and water solubility of the film developed from pullulan was also confirmed. To the best of our knowledge, this is the first report on the validation of the biodegradability of in-house produced pullulan. Thus, kitchen waste appears to be a promising option for economical pullulan production. Additionally, the method may also prove to be helpful for managing the increasing load of municipal solid waste in an eco-friendly and scientific way.

The ability of Aureobasidium pullulans NCYC 4012 to produce gluconic acid (GA) was firstly studied in flask batch cultures and it was found that GA productivity was not affected by initial glucose concentration, but highest substrate uptake rate was observed at glucose concentration of 80gL-1. The effect of oxygen mass transfer, varied by changing agitation and aeration rates or total air pressure, was evaluated in batch cultures of A. pullulans in stirred tank bioreactors. Higher productivities were attained in conditions of high oxygen mass transfer and it was proven that the use of increased total pressure of 4bar is an effective means of oxygen supply to A. pullulans cultures. At increased pressure, cells induced the antioxidant intracellular enzyme superoxide dismutase to cope with oxidative stress due to high oxygenation conditions. On the other hand, extracellular glucose oxidase activity was reduced by highly oxygenated conditions, suggesting opposite effects of oxygen on this enzyme secretion and GA production., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and the Doctoral grant (SFRH/BD/129475/2017)., info:eu-repo/semantics/publishedVersion

Fructooligosaccharides (FOS) can be used as feed prebiotics, but are limited by high production costs. In this study, low-cost sugarcane molasses was used to produce whole-cell biocatalysts containing transfructosylating enzymes by Aureobasidium pullulans FRR 5284, followed by FOS production from molasses using the whole-cells of A. pullulans. A. pullulans in molasses-based medium produced cells and broth with a total transfructosylating activity of 123.6 U/mL compared to 61.0 and 85.8 U/mL in synthetic molasses-based and sucrose-based media, respectively. It was found that inclusion of glucose in sucrose medium reduced both transfructosylating and hydrolytic activities of the produced cells and broth. With the use of pure glucose medium, cells and broth had very low levels of transfructosylating activities and hydrolytic activities were not detected. These results indicated that A. pullulans FRR 5284 produced both constitutive and inducible enzymes in sucrose-rich media, such as molasses while it only produced constitutive enzymes in the glucose media. Furthermore, treatment of FOS solutions generated from sucrose-rich solutions using an invertase-deficient Saccharomyces yeast converted glucose to ethanol and acetic acid and improved FOS content in total sugars by 20–30%. Treated FOS derived from molasses improved the in vitro growth of nine probiotic strains by 9–63% compared to a commercial FOS in 12 h incubation. This study demonstrated the potential of using molasses to produce FOS for feed application.

A low molecular weight intracellular and extracellular fructosyltransferase was purified from the wild (Wt) and improved mutant-type (Mt) Aureobasidium pullulans and its characteristics and thermod...

The production of cell-wall degrading enzymes (CWDE) such as cellulase and pectinase by Aureobasidium pullulans NAC8 through induction using orange peels was investigated for the potential ...

The fungi Aureobasidium pullulans, Mortierella humilis, Trichoderma harzianum and Phoma glomerata were used to investigate the formation of selenium- and tellurium-containing nanoparticles during growth on selenium- and tellurium-containing media. Most organisms were able to grow on both selenium- and tellurium-containing media at concentrations of 1 mM resulting in extensive precipitation of elemental selenium and tellurium on fungal surfaces as observed by the red and black colour changes. Red or black deposits were confirmed as elemental selenium and tellurium, respectively. Selenium oxide and tellurium oxide were also found after growth of Trichoderma harzianum with 1 mM selenite and tellurite as well as the formation of elemental selenium and tellurium. The hyphal matrix provided nucleation sites for metalloid deposition with extracellular protein and extracellular polymeric substances localizing the resultant Se or Te nanoparticles. These findings are relevant to remedial treatments for selenium and tellurium and to novel approaches for selenium and tellurium biorecovery.

Since cold atmospheric pressure plasma (CAP) has not only bactericidal activity but also fungicidal activity without toxic residues and thermal damage, it is considered as an alternative method for sterilization of fungi on the surfaces of perishable foodstuffs and human bodies. Aureobasidium pullulans is a ubiquitous yeast-like fungus and called black yeast because it produces melanin, a dark biological pigment. It is well known that various melanized fungi show hyper-resistance to extreme stress conditions including high levels of radioactivity. Curiously, however, there is very little information about the fungicidal effects of CAP on melanized fungi. Therefore, we herein investigated the effects of CAP on A. pullulans, using cold atmospheric argon plasma (Ar plasma). We found that ammonium sulfate repressed the synthesis of melanin in A. pullulans as well as Aureobasidium melanogenum. Although the non-melanized A. pullulans cells were efficiently killed by the exposure of Ar plasma, the melanized cells showed the significant resistance to Ar plasma as well as to hydrogen peroxide and thermal stress. In order to improve the fungicidal efficacy of Ar plasma, we examined the combination of Ar plasma and Fenton reaction. We realized that FeCl2 and FeSO4 significantly improved the sterilization efficacy of Ar plasma on the melanized A. pullulans.

Liamocins are biosurfactants produced by the fungus Aureobasidium pullulans. A. pullulans belongs to the black yeasts and is known for its ability to produce pullulan and melanin. However, the production of liamocins has not been investigated intensively. Initially, HPLC methods for the quantification of liamocin and the identification of liamocin congeners were established. Eleven congeners could be detected, differing in the polyol head groups arabitol or mannitol. In addition, headless molecules, so-called exophilins, were also identified. The HPLC method reported here allows quick and reliable quantification of all identified congeners, an often-overlooked prerequisite for the investigation of valuable product formation. Liamocin synthesis was optimized during cultivation in lab-scale fermenters. While the pH can be kept constant, the best strategy for liamocin synthesis consists of a growth phase at neutral pH and a subsequent production phase induced by a manual pH shift to pH 3.5. Finally, combining increased nitrogen availability with a pulsed fed-batch fermentation, cell growth, and liamocin titers could be enhanced. Here, the maximal titers of above 10 g/L that were reached are the highest reported to date for liamocin synthesis using A. pullulans in lab-scale fermenters.

β-poly(L-malic acid) (PMLA) is a highly water-soluble biopolymer used in medicine and other industries. PMLA is usually biosynthesized by Aureobasidium pullulans, but in low concentrations, which has restricted its further large-scale production. Here effects of surfactants on PMLA production were investigated. Of various surfactants examined, the addition of Tween 80 at the start of cell growth increased mycelial growth, PMLA production, PMLA productivity and the ratio of PMLA to cell biomass concentration (Yp/x) of A. pullulans. Transmission electron microscopy and flow cytometry showed that cell membrane permeability was increased, improving the secretion of PMLA and uptake of nutrients and oxygen. Proteomic analysis showed that key pathways of PMLA synthesis, along with cellular energy metabolism, were enhanced. Moreover, glutathione (GSH) synthesis and anti-oxidation capacity were also improved. These results indicated that the addition of Tween 80 is an efficient approach for improving the production of PMLA.

Ergothioneine (EGT) is a sulfur-containing, anti-oxidative amino acid derived from histidine. EGT is synthesized in bacteria and fungi but not in animals and plants, and is now recognized as important for human health. Its cost-effective fermentative production has not been elucidated due to the lack of information for productive microorganisms. In this study, we doubled the gene copy for EGT synthesis and deleted the histidine ammonia-lyase gene in a potent EGT-producing methylotrophic bacterium Methylobacterium aquaticum strain 22A, and optimized its culture conditions, resulting in increased EGT production of 7.0 mg EGT/g dry cell weight and 100 μg EGT/5 mL/7 days. In addition, through screening we found EGT-producing eukaryotic strains of Aureobasidium pullulans and Rhodotorula mucilaginosa, which can produce 1.0 and 3.2 mg EGT/g dry cell weight, 70 and 120 μg EGT/5 mL/7 days, respectively. This study proposes practical uses of potent EGT-producing recombinant Methylobacterium species and non-recombinant yeast and fungal strains.

More than 30,000 tons of menthol are produced every year as a flavor and fragrance compound or as a medical component. So far, only extraction from plant material and chemical synthesis are possible. An alternative approach for menthol production could be a biotechnological-chemical process with ideally only two conversion steps, starting from (+)-limonene, which is a side product of the citrus processing industry. The first step requires a limonene-3-hydroxylase (L3H) activity that specifically catalyzes hydroxylation of limonene at carbon atom 3. Several protein engineering strategies have already attempted to create limonene-3-hydroxylases from bacterial cytochrome P450 monooxygenases (CYPs, or P450s), which can be efficiently expressed in bacterial hosts. However, their regiospecificity is rather low compared to that of the highly selective L3H enzymes from the biosynthetic pathway for menthol in Mentha species. The only naturally occurring limonene-3-hydroxylase activity identified in microorganisms so far was reported for a strain of the black yeast-like fungus Hormonema sp. in South Africa. We have discovered additional fungi that can catalyze the intended reaction and identified potential CYP-encoding genes within the genome sequence of one of the strains. Using heterologous gene expression and biotransformation experiments in yeasts, we were able to identify limonene-3-hydroxylases from Aureobasidium pullulans and Hormonema carpetanum. Further characterization of the A. pullulans enzyme demonstrated its high stereospecificity and regioselectivity, its potential for limonene-based menthol production, and its additional ability to convert α- and β-pinene to verbenol and pinocarveol, respectively. IMPORTANCE (−)-Menthol is an important flavor and fragrance compound and furthermore has medicinal uses. To realize a two-step synthesis starting from renewable (+)-limonene, a regioselective limonene-3-hydroxylase enzyme is necessary. We identified enzymes from two different fungi which catalyze this hydroxylation reaction and represent an important module for the development of a biotechnological process for (−)-menthol production from renewable (+)-limonene.

Fructooligosaccharides (FOS) are a type of important prebiotics and produced by transfructosylating enzymes. In this study, sugarcane molasses was used as the substrate for production of transfructosylating enzymes by Aureobasidium pullulans FRR 5284. NaNO3 was a superior nitrogen source to yeast extract for production of transfructosylating enzymes by A. pullulans FRR 5284 and decreasing the ratio of NaNO3 to yeast extract nitrogen from 1:0 to 1:1 resulted in the reduction of the total transfructosylating activity from 109.8 U/mL to 82.5 U/mL. The addition of only 4.4 g/L NaNO3 into molasses-based medium containing 100 g/L mono- and di-saccharides resulted in total transfructosylating activity of 123.8 U/mL. Scale-up of the A. pullulans FRR 5284 transfructosylating enzyme production process from shake flasks to 1 L bioreactors improved the enzyme activity and productivity to 171.7 U/mL and 3.58 U/mL/h, 39% and 108% higher than those achieved from shake flasks, respectively. Sucrose (500 g/L) was used as a substrate for extracellular, intracellular, and total A. pullulans FRR 5284 transfructosylating enzymes, with a maximum yield of 61%. Intracellular, extracellular, and total A. pullulans FRR 5284 transfructosylating enzymes from different production systems resulted in different FOS profiles, indicating that FOS profiles can be controlled by adjusting intracellular and extracellular enzyme ratios and, hence prebiotic activity.

Abstract Fungi cause diverse, serious socio-economic problems, including biodeterioration of valuable products and materials that spawns a biocides industry worth ~$11 billion globally. To help combat environmental fungi that commonly colonise material products, this study tested the hypothesis that combination of an approved fungicide with diverse agents approved by the FDA (Food and Drug Administration) could reveal potent combinatorial activities with promise for fungicidal applications. The strategy to use approved compounds lowers potential development risks for any effective combinations. A high-throughput assay of 1280 FDA-approved compounds was conducted to find those that potentiate the effect of iodopropynyl-butyl-carbamate (IPBC) on the growth of Trichoderma virens; IPBC is one of the two most widely used Biocidal Products Regulations–approved fungicides. From this library, 34 compounds in combination with IPBC strongly inhibited fungal growth. Low-cost compounds that gave the most effective growth inhibition were tested against other environmental fungi that are standard biomarkers for resistance of synthetic materials to fungal colonisation. Trifluoperazine (TFZ) in combination with IPBC enhanced growth inhibition of three of the five test fungi. The antifungal hexetidine (HEX) potentiated IPBC action against two of the test organisms. Testable hypotheses on the mechanisms of these combinatorial actions are discussed. Neither IPBC + TFZ nor IPBC + HEX exhibited a combinatorial effect against mammalian cells. These combinations retained strong fungal growth inhibition properties after incorporation to a polymer matrix (alginate) with potential for fungicide delivery. The study reveals the potential of such approved compounds for novel combinatorial applications in the control of fungal environmental opportunists. Key points • Search with an approved fungicide to find new fungicidal synergies in drug libraries. • New combinations inhibit growth of key environmental fungi on different matrices. • The approach enables a more rapid response to demand for new biocides.

The coproduction of polymalic acid (PMA) and liamocins, two important metabolites secreted by Aureobasidium pullulans, from two waste by-products from the xylitol and gluconate industries was investigated in shake flasks and fermentors, confirming that waste xylose mother liquor (WXML) could be utilized as an economical feedstock without any pretreatment. Gluconate could strengthen carbon flux and NADPH supply for the synergetic biosynthesis of PMA and liamocins. High PMA and liamocin titers of 82.9 ± 2.1 and 28.3 ± 2.7 g/L, respectively, were obtained from the coupled WXML and waste gluconate mother liquor (WGML) in batch fermentation, with yields of 0.84 and 0.25 g/g, respectively. These results are comparable to those obtained from renewable feedstocks. Economic assessment of the process revealed that PMA and liamocins could be coproduced from two by-products at costs of $1.48/kg or $0.67/kg (with liamocins credit), offering an economic and sustainable process for the application of waste by-products.

Polymalic acid (PMA), a homopolymer of L-malic acid (MA) generated from a yeast-like fungus Aureobasidium pullulans, has unique properties and many applications in food, biomedical, and environmental fields. Acid hydrolysis of PMA, releasing the monomer MA, has become a novel process for the production of bio-based MA, which currently is produced by chemical synthesis using petroleum-derived feedstocks. Recently, current researches attempted to develop economically competitive process for PMA and MA production from renewable biomass feedstocks. Compared to lignocellulosic biomass, PMA and MA production from low-value food processing wastes or by-products, generated from corn, sugarcane, or soybean refinery industries, showed more economical and sustainable for developing a MA derivatives platform from biomass biorefinery to chemical conversion. In the review, we compared the process feasibility for PMA fermentation with lignocellulosic biomass and food process wastes. Some useful strategies for metabolic engineering are summarized. Its changeable applicability and future prospects in food and biomedical fields are also discussed.

The effects of several surfactants on the biosynthesis of β-1,3-D-glucan (β-glucan) and pullulan by Aureobasidium pullulans CCTCC M 2012259 were investigated, and Triton X-100 was found to decrease biomass formation but increase β-glucan and pullulan production. The addition of 5 g/L Triton X-100 to the fermentation medium and bioconversion broth significantly increased β-glucan production by 76.6% and 69.9%, respectively, when compared to the control without surfactant addition. To reveal the physiological mechanism underlying the effect of Triton X-100 on polysaccharides production, the cell morphology and viability, membrane permeability, key enzyme activities, and intracellular levels of UDPG, NADH, and ATP were determined. The results indicated that Triton X-100 increased the activities of key enzymes involved in β-glucan and pullulan biosynthesis, improved intracellular UDPG and energy supply, and accelerated the transportation rate of precursors across the cell membrane, all of which contributed to the enhanced production of β-glucan and pullulan. Moreover, a two-stage culture strategy with combined processes of batch fermentation and bioconversion was applied, and co-production of β-glucan and pullulan in the presence of 5 g/L Triton X-100 additions was further improved. The present study not only provides insights into the effect of surfactant on β-glucan and pullulan production but also presents a feasible approach for efficient production of analogue exopolysaccharides. KEY POINTS: • Triton X-100 increased β-glucan and pullulan production under either batch fermentation or bioconversion. • Triton X-100 increased the permeability of cell membrane and accelerated the transportation rate of precursors across cell membrane. • Activities of key enzymes involved in β-glucan and pullulan biosynthesis were increased in the presence of Triton X-100. • Intracellular UDPG levels and energy supply were improved by Triton X-100 addition.

Abstract Aureobasidium pullulans is a ubiquitous and widely distributed fungus in the environment, and exhibits substantial tolerance against toxic metals. However, the interactions between metals and metalloids with the copious extracellular polymeric substances (EPS) produced by A. pullulans and possible relationships to tolerance are not well understood. In this study, it was found that mercury (Hg) and selenium (Se), as selenite, not only significantly inhibited growth of A. pullulans but also affected the composition of produced EPS. Lead (Pb) showed little influence on EPS yield or composition. The interactions of EPS from A. pullulans with the tested metals and metalloids depended on the specific element and their concentration. Fluorescence intensity measurements of the EPS showed that the presence of metal(loid)s stimulated the production of extracellular tryptophan-like and aromatic protein-like substances. Examination of fluorescence quenching and calculation of binding constants revealed that the fluorescence quenching process for Hg; arsenic (As), as arsenite; and Pb to EPS were mainly governed by static quenching which resulted in the formation of a stable non-fluorescent complexes between the EPS and metal(loid)s. Se showed no significant interaction with the EPS according to fluorescence quenching. These results provide further understanding of the interactions between metals and metalloids and EPS produced by fungi and their contribution to metal(loid) tolerance. Key points • Metal(loid)s enhanced production of tryptophan- and aromatic protein-like substances. • Non-fluorescent complexes formed between the EPS and tested metal(loid)s. • EPS complexation and binding of metal(loid)s was dependent on the tested element. • Metal(loid)-induced changes in EPS composition contributed to metal(loid) tolerance.

Yeasts such as Aureobasidium pullulans are unicellular fungi that occur in all environments and play important roles in biotechnology, medicine, food and beverage production, research, and agriculture. In the latter, yeasts are explored as biocontrol agents for the control of plant pathogenic fungi (e.g., Botrytis cinerea, Fusarium sp.); mainly on flowers and fruits. Eventually, such yeasts must be evaluated under field conditions, but such trials require a lot of time and resources and are often difficult to control. Experimental systems of intermediate complexity, between in vitro Petri dish assays and field trials, are thus required. For pre- and post-harvest applications, competition assays on fruits are reproducible, economical and thus widely used. Here, we present a general protocol for competition assays with fruits that can be adapted depending on the biocontrol yeast, plant pathogen, type of assay or fruit to be studied.

Microbial oils can be used for biodiesel production and fumaric acid (FA) is widely used in the food and chemical industries. In this study, the production of lipids and FA by Aureobasidium pullulans var. aubasidani DH177 was investigated. A high initial carbon/nitrogen ratio in the medium promoted the accumulation of lipids and FA. When the medium contained 12.0% glucose and 0.2% NH4NO3, the yeast strain DH177 accumulated 64.7% (w/w) oil in its cells, 22.4 g/l cell biomass and 32.3 g/l FA in a 5-L batch fermentation. The maximum yields of oil and FA were 0.12 g/g and 0.27 g/g of consumed sugar, respectively. The compositions of the produced fatty acids were C14:0 (0.6%), C16:0 (24.9%), C16:1 (4.4%), C18:0 (2.1%), C18:1 (57.6%), and C18:2 (10.2%). Biodiesel obtained from the extracted oil burned well. This study provides the pioneering utilization of the yeast strain DH177 for the integrated production of oil and FA.

Polymalic acid (PMA) is a linear anionic polyester composed of l-malic acid monomers, which have potential applications as drug carriers, surgical suture, and biodegradable plastics. In this study, a novel strain of Aureobasidium pullulans var. melanogenum GXZ-6 was isolated and identified according to the morphological observation and deoxyribonucleic acid internal-transcribed spacer sequence analysis, and the product of PMA was characterized by FT-IR, 13C-NMR, and 1H-NMR spectra. The PMA titer of GXZ-6 reached 62.56 ± 1.18 g L−1 with productivity of 0.35 g L−1 h−1 using optimized medium with addition of metabolic intermediates (citrate and malate) and inhibitor (malonate) by batch fermentation in a 10-L fermentor. Besides that the malate for PMA synthesis in GXZ-6 might mainly come from the glyoxylate cycle, based on results, citrate, malate, malonate, and maleate increased while succinate and fumarate inhibited the production of PMA, which was different from that of other A. pullulans. This study provided a potential strain and a simple metabolic control strategy for high-titer production of PMA and shared novel information on the biosynthesis pathway of PMA in A. pullulans.