Fungal Biotransformation Research Articles

Rendered-Protein Hydrolysates as a Low-Cost Nitrogen Source for the Fungal Biotransformation of 5-Hydroxymethylfurfural

5-hydroxymethylfurfural (HMF) is a platform chemical that can be converted into a wide range of high-value derivatives. Industrially, HMF-based derivatives are synthesized via chemical catalysis. However, biocatalytic transformation has emerged as an attractive alternative. Significant advances have been made in the last years using isolated enzymes and whole-cell biocatalysts in HMF biotransformation. Nonetheless, one of the major bottlenecks is the cost of the process, mainly due to the microorganism growth substrate. In this work, biotransformation studies to transform HMF into 2,5-di(hydroxymethyl)furan (DHMF) were carried out with the fungus Fusarium striatum using low-cost protein hydrolysates. The protein hydrolysates were obtained from fines, an unexploited material produced during the rendering process of meat industry waste residues. Given the high content in the protein of fines, of around 46%, protein hydrolysis was optimized using two commercially available proteases, Alcalase 2.4 L and Neutrase 0.8 L. The maximum degree of hydrolysis (DH) achieved with Alcalase 2.4 L was 21.4% under optimal conditions of 5% E/S ratio, pH 8, 55 °C, and 24 h. On the other hand, Neutrase 0.8 L exhibited lower efficiency, and therefore, lower protein recovery. After optimization of the Neutrase 0.8 L process using the response surface methodology (RSM), the maximum DH achieved was 7.2% with the variables set at 15% E/S ratio, initial pH 8, 40 °C, and 10.5 h. Using these hydrolysates as a nitrogen source allowed higher sporulation of the fungus and, therefore, the use of a lower volume of inoculum (three-fold), obtaining a DHMF yield > 90%, 50% higher than the yield obtained when using commercial peptones. The presented process allows the transformation of animal co- and by-products into low-cost nitrogen sources, which greatly impacts the industrial feasibility of HMF biotransformation.

Sustainable environmental remediation via biomimetic multifunctional lignocellulosic nano-framework

Chemical pollution threatens human health and ecosystem sustainability. Persistent organic pollutants (POPs) like per- and polyfluoroalkyl substances (PFAS) are expensive to clean up once emitted. Innovative and synergistic strategies are urgently needed, yet process integration and cost-effectiveness remain challenging. An in-situ PFAS remediation system is developed to employ a plant-derived biomimetic nano-framework to achieve highly efficient adsorption and subsequent fungal biotransformation synergistically. The multiple component framework is presented as Renewable Artificial Plant for In-situ Microbial Environmental Remediation (RAPIMER). RAPIMER exhibits high adsorption capacity for the PFAS compounds and diverse adsorption capability toward co-contaminants. Subsequently, RAPIMER provides the substrates and contaminants for in situ bioremediation via fungus Irpex lacteus and promotes PFAS detoxification. RAPIMER arises from cheap lignocellulosic sources, enabling a broader impact on sustainability and a means for low-cost pollutant remediation.

Fungal biotransformation of carvone and camphor by Aspergillus flavus and investigation of cytotoxic activities of naturally obtained essential oils

In this study, the biotransformation of carvone and camphor by Aspergillus flavus and the products were investigated. The biotransformation reaction of carvone by A. flavus resulted in the production of neodihydrocarveol, dihydrocarvone, 2-cyclohexene-1-one,2-methyl-5-(1-methylethenyl), limonene-1,2-diol, trans-p-mentha-1(7),8-dien-2-ol, p-menth-8(10)-ene-2,9-diol, and the biotransformation reaction of camphor resulted in the production of 2 -campholenic acid, 2-cyclohexene-1-one,2-hydroxy-4,4,6,6-tetramethyl, α-campholene aldehyde. The naturally produced essential oils by biotransformation of carvone and camphor were observed to be cytotoxic to breast cancer cells while no significant inhibition was seen in the healthy cell line. Additionally, biotransformation products had the highest inhibition (74%) against aflatoxin B1. The bioactivities of biotransformation products are promising, and they can be further investigated for their therapeutic potential as active agents.

Structural properties and evaluation of the antiproliferative activity of limonene-1,2-diol obtained by the fungal biotransformation of R-(+)- and S-(-)-limonene.

Limonene-1,2-diol is a limonene oxygenated metabolite that possesses eight different stereoisomers, which could result in different biological properties. Nonetheless, the relation between its spatial configuration and biological function is still little explored. The present study aimed to perform the stereoisomers identification using nuclear magnetic resonance (NMR) investigation of the limonene-1,2-diol produced via R-(+)- and S-(-)-limonene biotransformation by Colletotrichum nymphaeae and S-(-)-limonene biotransformation by Fusarium oxysporum 152B. Besides, in vitro antiproliferative activity was evaluated against human tumor and nontumor cell lines. The NMR analysis showed that R-(+)-limonene biotransformation afforded exclusively (+)-(1S,2S,4R-limonene-1,2-diol), whereas S-(-)-limonene biotransformation afforded exclusively (-)-(1R,2R,4S-limonene-1,2-diol) independent on the fungi used. Despite no significant cytostatic effects, a possible influence of stereogenic center on the antiproliferative activity of these limonene biotransformation products was evidenced. Moreover, the lack of in vitro antiproliferative effect of limonene-1,2-diol against nontumor cells suggested a safe dose range for further in vivo evaluations, including food applications.

Fungal biotransformation of limonene and pinene for aroma production

Fungal biotransformation of limonene and pinene for aroma production

White Rot Fungi Produce Novel Tire Wear Compound Metabolites and Reveal Underappreciated Amino Acid Conjugation Pathways.

There is increasing concern about tire wear compounds (TWCs) in surface water and stormwater as evidence grows on their toxicity and widespread detection in the environment. Because TWCs are prevalent in stormwater, there is a need to understand fate and treatment options including biotransformation in green infrastructure (e.g., bioretention). Particularly, fungal biotransformation is not well-studied in a stormwater context despite the known ability of certain fungi to remove recalcitrant contaminants. Here, we report the first study on fungal biotransformation of the TWCs acetanilide and hexamethoxymethylmelamine (HMMM). We found that the model white rot fungus, Trametes versicolor, removed 81.9% and 69.6% of acetanilide and HMMM, respectively, with no significant sorption to biomass. The bicyclic amine 1,3-diphenylguanidine was not removed. Additionally, we identified novel TWC metabolites using semi-untargeted metabolomics via high-resolution mass spectrometry. Key metabolites include multiple isomers of HMMM biotransformation products, melamine as a possible “dead-end” product of HMMM (verified with an authentic standard), and a glutamine-conjugated product of acetanilide. These metabolites have implications for environmental toxicity and treatment. Our discovery of the first fungal glutamine-conjugated product highlights the need to investigate amino acid conjugation as an important pathway in biotransformation of contaminants, with implications in other fields including natural products discovery.

Solubilization of struvite and biorecovery of cerium by Aspergillus niger

Cerium has many modern applications such as in renewable energies and the biosynthesis of nanomaterials. In this research, natural struvite was solubilized by Aspergillus niger and the biomass-free struvite leachate was investigated for its ability to recover cerium. It was shown that struvite was completed solubilized following 2 weeks of fungal growth, which released inorganic phosphate (Pi) from the mineral by the production of oxalic acid. Scanning electron microscopy (SEM) showed that crystals with distinctive morphologies were formed in the natural struvite leachate after mixing with Ce3+. Energy-dispersive X-ray analysis (EDXA), X-ray diffraction (XRD) and Fourier-transform infrared spectroscopy (FTIR) confirmed the formation of cerium phosphate hydrate [Ce(PO4)·H2O] at lower Ce concentrations and a mixture of phosphate and cerium oxalate decahydrate [Ce2(C2O4)3·10H2O] at higher Ce concentrations. The formation of these biogenic Ce minerals leads to the removal of > 99% Ce from solution. Thermal decomposition experiments showed that the biogenic Ce phosphates could be transformed into a mixture of CePO4 and CeO2 (cerianite) after heat treatment at 1000 °C. These results provide a new perspective of the fungal biotransformation of soluble REE species using struvite leachate, and also indicate the potential of using the recovered REE as biomaterial precursors with possible applications in the biosynthesis of novel nanomaterials, elemental recycling and biorecovery.Key points• Cerium was recovered using a struvite leachate produced by A. niger.• Oxalic acid played a major role in struvite solubilization and Ce phosphate biorecovery.• Resulting nanoscale mineral products could serve as a precursor for Ce oxide synthesis.

Submerged culture fermentation of Colletotrichum lindemuthianum DSM 12250 as biotechnological strategy for fungal chitin biotransformation

Chitin and its derived products have great potential in various industries, such as biomedical, pharmaceutical, food, among others, due to its biodegradability, biocompatibility, null toxicity, and versatility. At present, these compounds are obtained by extraction with physical, chemical, or enzymatic processes. The fungus Colletotrichum lindemuthianum DSM 12250 which produces the enzyme chitin deacetylase (ClCDA), is proposed as an alternative for the biotransformation of fungal chitin. Chitin with a % N-Acetylation of 63.6% was obtained from the dry mycelium of Aspergillus niger that, based on analysis of Fourier transform infrared spectroscopy (FT-IR) and Differential Scanning Calorimetry (DSC), is structurally similar to commercial chitin. During fermentation in submerged culture, chitin presence was not found to generate inhibition in microbial growth or the activity of the ClCDA. The maximum enzymatic activity was 0.910 ± 0.002 U/mL and deacetylation of 20.9% were achieved. This was possible due to the enzymatic machinery of the fungus that includes CDA, endo-chitinases, and N-acetyl hexosaminidases. These compounds contribute to decreasing the substrate complexity, obtaining chitosan with a DDA of 57.3%. Submerged culture fermentation of Colletotrichum lindemuthianum DSM 12250 with fungal chitin as substrate represents a potential alternative for integrating different steps in the extraction of chitin-derived products while contributing to a cleaner and more efficient production.

New derivatives of the iridoid specioside from fungal biotransformation.

Iridoids are widely found from species of Bignoniaceae family and exhibit several biological activities, such as anti-inflammatory, antimicrobial, antioxidant, and antitumor. Specioside is an iridoid found from Tabebuia species, mainly in Tabebuia aurea. Thus, here fungus-mediated biotransformation of the iridoid specioside was investigated by seven fungi. The fungus-mediated biotransformation reactions resulted in a total of nineteen different analogs by fungus Aspergillus niger, Aspergillus flavus, Aspergillus japonicus, Aspergillus terreus, Aspergillus niveus, Penicillium crustosum, and Thermoascus aurantiacus. Non-glycosylated specioside was the main metabolite observed. The other analogs were yielded from ester hydrolysis, hydroxylation, methylation, and hydrogenation reactions. The non-glycosylated specioside and coumaric acid were yielded by all fungi-mediated biotransformation. Thus, fungus applied in this study showed the ability to perform hydroxylation and glycosidic, as well as ester hydrolysis reactions from glycosylated iridoid. KEY POINTS: • The biotransformation of specioside by seven fungi yielded nineteen analogs. • The non-glycosylated specioside was the main analog obtained. • Ester hydrolysis, hydroxylation, methylation, and hydrogenation reactions were observe.

Fungal Biotransformation of 2'-Methylflavanone and 2'-Methylflavone as a Method to Obtain Glycosylated Derivatives.

Methylated flavonoids are promising pharmaceutical agents due to their improved metabolic stability and increased activity compared to unmethylated forms. The biotransformation in cultures of entomopathogenic filamentous fungi is a valuable method to obtain glycosylated flavones and flavanones with increased aqueous solubility and bioavailability. In the present study, we combined chemical synthesis and biotransformation to obtain methylated and glycosylated flavonoid derivatives. In the first step, we synthesized 2′-methylflavanone and 2′-methylflavone. Afterwards, both compounds were biotransformed in the cultures of two strains of entomopathogenic filamentous fungi Beauveria bassiana KCH J1.5 and Isaria fumosorosea KCH J2. We determined the structures of biotransformation products based on NMR spectroscopy. Biotransformations of 2′-methyflavanone in the culture of B. bassiana KCH J1.5 resulted in three glycosylated flavanones: 2′-methylflavanone 6-O-β-d-(4″-O-methyl)-glucopyranoside, 3′-hydroxy-2′-methylflavanone 6-O-β-d-(4″-O-methyl)-glucopyranoside, and 2-(2′-methylphenyl)-chromane 4-O-β-d-(4″-O-methyl)-glucopyranoside, whereas in the culture of I. fumosorosea KCH J2, two other products were obtained: 2′-methylflavanone 3′-O-β-d-(4″-O-methyl)-glucopyranoside and 2-methylbenzoic acid 4-O-β-d-(4′-O-methyl)-glucopyranoside. 2′-Methylflavone was effectively biotransformed only by I. fumosorosea KCH J2 into three derivatives: 2′-methylflavone 3′-O-β-d-(4″-O-methyl)-glucopyranoside, 2′-methylflavone 4′-O-β-d-(4″-O-methyl)-glucopyranoside, and 2′-methylflavone 5′-O-β-d-(4″-O-methyl)-glucopyranoside. All obtained glycosylated flavonoids have not been described in the literature until now and need further research on their biological activity and pharmacological efficacy as potential drugs.

Fungal biotransformation of cedramber

Cedramber (cedrol methyl ether) (1) is a fragrance compound used in cosmetics such as soap, detergents, creams, lotions and perfumes. New cedrol derivatives are of interest in this context. Here we report the first biotransformation reaction of cedramber by Aspergillus niger ATCC 10549. The conversion afforded two known cedrol derivatives namely, 12β-hydroxy cedrol (2), and 3β-hydroxy cedrol (3), in 11.9, and 15.5% yields, respectively. The new metabolite chemical structures were characterized by spectroscopic techniques such as FT-IR, GC/MS and NMR analyses.

Optimization of (-)-cubebin biotransformation to (-)-hinokinin by the marine fungus Absidia coerulea 3A9.

The genus Absidia is widely used in the biotransformation of different classes of natural products. This study evaluates the ability of the Absidia coerulea 3A9 marine derived strain isolated from the ascidian Distaplia stilyfera to perform biotransformations by conducting assays with (-)-cubebin, as substrate. The experiment was optimized using the experimental design proposed by Plackett-Burman for seven factors and eight experiments, to establish the biotransformation conditions that would allow maximum production of biotransformed dibenzylbutyrolactone (-)-hinokinin. An analytical method based on Reverse-Phase-High Performance Liquid Chromatography (RP-HPLC) was developed to quantify the fungal biotransformation product. The factor that influenced the (-)-hinokinin peak area the most positively was the percentage of seawater (%seawater) given that its %relative standard deviation (%RSD) showed a 32.92% deviation from the real value.

Biocorrosion of copper metal by Aspergillus niger

Several geoactive fungi were investigated for their biocorrosion impact on metallic copper, to further understanding of the potential roles that fungi may have in the biotransformation of such substrate, and the mechanisms involved. Copper metal showed little toxicity and test fungi were able to grow in direct or indirect contact with copper and to colonize copper sheet. A. niger was able to biodeteriorate copper metal through proton- and ligand-mediated dissolution mechanisms, leading to significant mass loss and surface etching. The formation of a secondary copper oxalate (moolooite) biomineral crust together with cuprite deposition lead to alteration of surface topography and visual appearance, highlighting the significance of oxalate excretion in effecting fungal metal biotransformations. The metal transforming influence of fungal colonization may have some implications for biodeterioration, protection and preservation of cultural relics and artefacts as well as certain components of the built environment.

Extraction and purification of limonene-1,2-diol obtained from the fungal biotransformation of limonene

R-(+)-limonene is the main component of the orange peel oil, which is available in large amounts, at low cost, and can be employed in biotransformation processes as a precursor of different value-added aroma compounds. Limonene-1,2-diol is one of the oxyfunctionalyzed counterparts of limonene that can be obtained from the biotransformation process, but its biological properties and applications are still little investigated in literature. Thus, we aimed to study the recovery and purification of limonene-1,2-diol obtained from fungal biotransformation, which is an essential step for further investigations. First, liquid-liquid extractions were performed to select the most appropriate solvent, extraction time, and extracting material (whole fermented broth or supernatant) for recovering limonene-1,2-diol. Subsequently, sequential extractions with different solvents were considered for recovering larger amounts of limonene-1,2-diol as well as for removing the residual limonene. Finally, Open Column Chromatography procedures were evaluated for purifying limonene-1,2-diol originating from a limonene:limonene-1,2-diol mixture. According to the results, ethyl acetate was the most adequate solvent for recovering limonene-1,2-diol, and equilibrium was quickly reached (20 s in vortex). The extraction of the supernatant was repeated twice with hexane, followed by two sequential extractions with ethyl acetate, and was found to be the most suitable process for recovering limonene-1,2-diol in higher yields and purities. In addition, a silica-gel column chromatography was used for separating limonene from limonene-1,2-diol, but this procedure was deemed unnecessary, since better recovery and purification yields were obtained from the liquid-liquid extraction. The processes described in our study may be useful for researchers working with limonene-1,2-diol or other oxidized monoterpenoid as well as with downstream processing of biotransformation products.

Biotransformation of fruit residues via solid state bioprocess using Lichtheimia ramosa

Lichtheimia ramosa is a promising candidate fungus for solid state bioprocesses (SSB) due its rapid colonization. Fruit wastes present ideal conditions for fungal growth and biotransformation, which can promote the release of products of biotechnological interest. Thus, the aim of this work was to evaluate the mycelial growth and the enzymatic activities of L. ramosa in pineapple (Ananas comosus), orange (Citrus sinensis), mango (Mangifera indica), passion fruit (Passiflora edulis) and grape (Vitis vinifera) fruit wastes via SSB, and their influence in the generation of molecules with potential use. The SSB was evaluated in terms of capacity of biotransformation of the substrate (composition, protein enrichment, and fatty acid profile) and production of amylase, carboxymethylcellulase (CMCase), xylanase and lipase enzymes. Main cultivations were carried out at 30 °C for 30 days and every 5 days samples were taken and analyzed for microbiological content, proximal composition and enzymatic profile. Fatty acids were determined at day 0 (baseline) and at the end of the cultivations. L. ramosa grew well in all substrates up to the 25th day, except for on the orange residue, upon which development was slightly lower at this time. Protein enrichment was found in all substrates as follows: passion fruit (309.54%), pineapple (294.89%), mango (263.45%), orange (65.60%) and grapes (19.17%). Regarding enzymes, lipase was not synthetized in any of the substrates, though amylase, CMCase and xylanase were observed at different levels. The fatty acid profile varied from raw to cultivated substrates, indicating that L. ramosa can act in the synthesis and conversion of these acids. It was concluded that L. ramosa and the studied substrates are viable for SSB.

Fungal Biotransformation of Clobazam–Induction, Inhibition and Kinetic Studies

Fungal Biotransformation of Clobazam–Induction, Inhibition and Kinetic Studies

Biotransformation strategies for steroid estrogen and androgen pollution.

The common steroid hormones are estrone (E1), 17β-estradiol (E2), estriol (E3), 17α-ethinylestradiol (EE2), and testosterone (T). These steroids are reported to contaminate the environment through wastewater treatment plants. Steroid estrogens are widespread in the aquatic environment and therefore pose a potential risk, as exposure to these compounds has adverse impacts on vertebrates. Excessive exposure to steroid estrogens causes endocrine disruption in aquatic vertebrates, which affects the normal sexual life of these animals. Steroid pollutants also cause several health problems in humans and other animals. Microbial degradation is an efficient method for removing hormone pollutants from the environment by remediation. Over the last two decades, microbial metabolism of steroids has gained considerable attention due to its higher efficiency to reduce pollutants from the environment. The present review is focused on the major causes of steroid pollution, concentrations of these pollutants in surface water, groundwater, drinking water, and wastewater, their effect on humans and aquatic animals, as well as recent efforts by various research groups that seek better ways to degrade steroids by aerobic and anaerobic microbial systems. Detailed overview of aerobic and anaerobic microbial biotransformation of steroid estrogens and testosterone present in the environment along with the active enzyme systems involved in these biotransformation reactions is described in the review article, which helps readers to understand the biotransformation mechanism of steroids in depth. Other measures such as co-metabolic degradation, consortia degradation, algal, and fungal steroid biotransformation are also discussed in detail.

Micro-analytical Evidence of Copper-Based Pigment and Fungal Contamination of Medieval Mural Paintings in Beram, Croatia.

The aim of the present study was to map the painting materials, degradation processes, and biological features present on the mural painting in the church of St. Mary in Beram (Croatia) to study their possible interaction and produce information helping the preservation of this valuable painting. The research was conducted on micro samples of painting materials taken from different sites along the painting and the characterization of the present fungal species was carried out. The painting samples, together with observable patinas and degradation products, were studied by optical microscopy (OM), scanning electron microscopy, energy-dispersive spectroscopy (SEM/EDS), Fourier-Transform Infrared spectroscopy, and powder X-ray diffraction. Fungal diversity was studied using cultivation methods followed by OM and SEM analyses in addition to molecular analysis. The results contribute to the characterization of the original painting materials, successively added materials and occurred interventions, to the understanding of degradation progressions and fungal biotransformation processes. A mineral, cumengite, a copper-based pigment extremely rarely used in art, was found. Its occurrence together with barium sulfate, gypsum, and calcium oxalate possibly produced by microbiological activity was studied and information was added regarding the composition of painting materials in St. Mary church mural cycle.

Assessment of headspace solid-phase microextraction (HS-SPME) for control of asymmetric bioreduction of ketones by Alternaria alternata

The aim of this study was to assess the effectiveness of headspace solid-phase microextraction (HS-SPME) compared to liquid–liquid extractions using with methylene chloride (CH2Cl2) for control of fungal biotransformation of ketones of varying volatility. The proposed method was successfully applied. The best way to extract all the components of the mixture (alcohols, aldehydes) in quantities similar to the extraction of methylene chloride was the use of fibres coated with a combination of nonpolar material. SPME fibre assembly polydimethylsiloxane/divinylbenzene (PDMS/DVB) was most suitable for the extraction of the products mixture obtained after biotransformation of acetylcyclohexane and acetophenone. On the other hand, the best results were obtained for 2-acetylthiophene, α,α,α-trifluoroacetophenone and their derivatives using divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fibre. In addition, our study showed that Alternaria alternata is a good biocatalyst for bioreduction of ketones to alcohols according to Prelog's rule.

Sclerotinia sclerotiorum Circumvents Flavonoid Defenses by Catabolizing Flavonol Glycosides and Aglycones.

Flavonols are widely distributed plant metabolites that inhibit microbial growth. Yet many pathogens cause disease in flavonol-containing plant tissues. We investigated how Sclerotinia sclerotiorum, a necrotrophic fungal pathogen that causes disease in a range of economically important crop species, is able to successfully infect flavonol-rich tissues of Arabidopsis (Arabidopsis thaliana). Infection of rosette stage Arabidopsis with a virulent S. sclerotiorum strain led to the selective hydrolysis of flavonol glycosidic linkages and the inducible degradation of flavonol aglycones to phloroglucinol carboxylic and phenolic acids. By chemical analysis of fungal biotransformation products and a search of the S. sclerotiorum genome sequence, we identified a quercetin dioxygenase gene (QDO) and characterized the encoded protein, which catalyzed cleavage of the flavonol carbon skeleton. QDO deletion lines degraded flavonols with much lower efficiency and were less pathogenic on Arabidopsis leaves than wild-type S. sclerotiorum, indicating the importance of flavonol degradation in fungal virulence. In the absence of QDO, flavonols exhibited toxicity toward S. sclerotiorum, demonstrating the potential roles of these phenolic compounds in protecting plants against pathogens.