Rhizopus Delemar Research Articles

The first report of rhinosinusitis by Rhizopus delemar in a patient with severe COVID-19 in Iran: a case report

BackgroundMucormycosis is a severe and fatal fungal infection in patients with coronavirus disease 2019 caused by Mucorales. Here we present a case of a 63-year-old man with coronavirus disease 2019 infection, along with rhinosinusitis mucormycosis caused by Rhizopus delemar.Case presentationA 63-year-old Iranian man suffering from a coronavirus disease 2019 infection with symptoms of cough, shortness of breath, and generalized body pain. On the basis of the clinical manifestations, such as headache, a history of black nasal discharge, nasal hypoesthesia, facial swelling, numbness, nasal obstruction, presence of necrotic lesions on the nasal passages on physical examination, and abnormal computed tomography scans of paranasal sinuses, the patient underwent surgical debridement. Direct microscopy of specimens obtained from the paranasal sinuses, and subsequent isolation and identification, revealed a rhinosinusitis mucormycosis caused by R. delemar. Despite therapeutic measures, such as sinus debridement surgery and antifungal therapy with amphotericin B injection (50 mg/day), the patient died after 35 days of hospitalization.ConclusionIn this report, we present the first documented case of human infection with R. delemar in a patient with coronavirus disease 2019 infection, who also exhibited rhinosinusitis mucormycosis. R. delemar appears to be an emerging agent of rhinosinusitis mucormycosis in this region. Furthermore, prompt diagnosis and the exploration of alternative antifungal treatments, beyond amphotericin B, may be crucial for effectively managing patients with R. delemar infections.

Dry gel spinning of fungal hydrogels for the development of renewable yarns from food waste

BackgroundRenewable materials made using environmentally friendly processes are in high demand as a solution to reduce the pollution created by the fashion industry. In recent years, there has been a growing trend in research on renewable materials focused on bio-based materials derived from fungi.ResultsRecently, fungal cell wall material of a chitosan producing fungus has been wet spun to monofilaments. This paper presents a modification for the fungal monofilament spinning process, by the development of a benign method, dry gel spinning, to produce continuous monofilaments and twisted multifilament yarns, from fungal cell wall, that can be used in textile applications. The fungal biomass of Rhizopus delemar, grown using bread waste as a substrate, was subjected to alkali treatment with a dilute sodium hydroxide solution to isolate alkali-insoluble material (AIM), which mainly consists of the fungal cell wall. The treatment of AIM with dilute lactic acid resulted in hydrogel formation. The morphology of the hydrogels was pH dependent, and they exhibited shear thinning viscoelastic behavior. Dry gel spinning of the fungal hydrogels was first conducted using a simple lab-scale syringe pump to inject the hydrogels through a needle to form a monofilament, which was directly placed on a rotating receiver and left to dry at room temperature. The resulting monofilament was used to make twisted multifilament yarns. The process was then improved by incorporating a heated chamber for the quicker drying of the monofilaments (at 30⁰C). Finally, the spinning process was scaled up using a twin-screw microcompounder instead of the syringe pump. The monofilaments were several meters long and reached a tensile strength of 63 MPa with a % elongation at break of 14. When spinning was performed in the heated chamber, the tensile strength increased to 80 MPa and further increased to 103 MPa when a micro-compounder was used for spinning.ConclusionThe developed dry gel spinning method shows promising results in scalability and demonstrates the potential for renewable material production using fungi. This novel approach produces materials with mechanical properties comparable to those of conventional textile fibers.

Metagenomic analysis of core differential microbes between traditional starter and Round-Koji-mechanical starter of Chi-flavor Baijiu.

Xiaoqu starter serves as the saccharifying and fermenting agent in the production of Cantonese soybean-flavor (Chi-flavor) Baijiu, and the complex microbial communities determine the flavor and quality of the product. Round-Koji-mechanical starter (produced by using an automated starter-making disk machine) is advantageous as it decreases operator influence, labor costs, and fermentation time, but the product quality is lower compared to traditional starter. Thus, two types of starters (traditional and Round-Koji-mechanical starter) from a Cantonese Baijiu factory were compared in a metagenomic analysis to investigate the differences in microbial community composition and core microbes. The results showed that several core microbes related to carbohydrate metabolism, amino acid metabolism and lipid metabolism, were differentially enriched in the traditional starter. Mucor lusitanicus and Rhizopus delemar were significantly positively correlated with the three key metabolic pathways. Saccharomyces cerevisiae, Cyberlindnera fabianii, Kluyveromyces marxianus, Lactobacillus fermentum, Mucor ambiguous, Rhizopus microspores, Rhizopus azygosporus, Mucor circinelloides, and Ascoidea rubescens were significantly positively correlated with two of the three key metabolic pathways. The results of this study provide a basis for understanding the differential core microbes in traditional and Round-Koji-mechanical starters of Chi-flavor Baijiu, and they also provide guidance for improving Round-Koji-mechanical starter.

Efficacy of an oral lipid nanocrystal formulation of amphotericin B (MAT2203) in the neutropenic mouse model of pulmonary mucormycosis.

Invasive mucormycosis (IM) is associated with high mortality and morbidity. MAT2203 is an orally administered lipid nanocrystal formulation of amphotericin B, which has been shown to be safe and effective against other fungal infections. We sought to compare the efficacy of MAT2203 to liposomal amphotericin B (LAMB) treatment in a neutropenic mouse model of IM due to Rhizopus arrhizus var. delemar or Mucor circinelloides f. jenssenii DI15-131. In R. arrhizus var. delemar-infected mice, 15 mg/kg of MAT2203 qd was as effective as 10 mg/kg of LAMB in prolonging median survival time vs placebo (13.5 and 16.5 days for MAT2203 and LAMB, respectively, vs 9 days for placebo) and enhancing overall survival vs placebo-treated mice (40% and 45% for MAT2203 and LAMB, respectively, vs 0% for placebo). A higher dose of 45 mg/kg of MAT2203 was not well tolerated by mice and showed no benefit over placebo. Similar results were obtained with mice infected with M. circinelloides. Furthermore, while both MAT2203 and LAMB treatment resulted in a significant reduction of ~1.0-2.0log and ~2.0-2.5log in Rhizopus delemar or M. circinelloides lung and brain burden vs placebo mice, respectively, LAMB significantly reduced tissue fungal burden in mice infected with R. delemar vs tissues of mice treated with MAT2203. These results support continued investigation and development of MAT2203 as a novel and oral formulation of amphotericin for the treatment of mucormycosis.

Transcriptomic analysis and carbohydrate metabolism-related enzyme expression across different pH values in Rhizopus delemar.

pH is one of the important factors affecting the growth and performance of microorganisms. We studied the pH response and plant growth-promoting (PGP) ability of Rhizopus delemar using cultivation experiments and transcriptomics, and verified the expression profiles using quantitative real-time PCR. pH affected the growth and PGP properties of R. delemar. At pH 7, the growth rate of R. delemar was rapid, whereas pH 4 and 8 inhibited mycelial growth and PGP ability, respectively. In the pot experiment, the plant height was the highest at pH 7, 56 cm, and the lowest at pH 4 and pH 5, 46.6 cm and 47 cm, respectively. Enzyme activities were highest at pH 6 to pH 7. Enzyme activities were highest at pH 6 to pH 7. Among the 1,629 differentially expressed genes (DEGs), 1,033 genes were up-regulated and 596 were down-regulated. A total of 1,623 DEGs were annotated to carbohydrate-active enzyme coding genes. The PGP characteristics, e.g., Phosphorus solubilization ability, of R. delemar were strongest at pH 7. The results provide useful information regarding the molecular mechanism of R. delemar pH response.

Comparison of the bioaccumulation and biosorption of copper ions by Rhizopus delemar and Candida lipolytica

In this study, the bioaccumulation of Cu(II) ion by Rhizopus delemar and Candida lipolytica was investigated and the bioaccumulation and biosorption of Cu(II) ions by Candida lipolytica compared. The specific growth rate and the maximum microorganism concentration of both microorganisms increased with increasing initial molasses concentration whereas decreased with increasing initial Cu(II) ion concentrations up to 250 mg/L. Accoding to Monod equation, the maximum specific growth rate and saturation constant for C. lipolytica and R. delemar were determined as 0.335 h−1-32.330 g/L and 0.406 h−1-24.182 g/L, respectively. The bioaccumulation efficiencies of R. delemar and C. lipolytica were determined as 74.18% and 67.30% in the presence of 50 mg/L Cu(II), respectively. At pH 4.0, the maximum biosorption rate (4.72 mg/g min) and the biosorbed Cu(II) ion concentration (49.0 mg/L) were obtained for C. lipolytica and these values increased with increasing Cu(II) concentration. Cu(II) biosorption by C. lipolytica fitted well to the Langmuir isotherm model and pseudo-second-order kinetic model and maximum adsorption capacity was found as 114.942 mg/g. The bioaccumulated Cu(II) was found to be 21.5 mg/g whereas the amounts of biosorbed Cu(II) were calculated as 53 mg/g by C. lipolytica at a concentration of 100 mg/L of Cu(II).

Mycobiota Associated with Dacryodes edulis H. J. Lam fruits sold In Rumokoro Market, Port Harcourt, Nigeria

Dacryodes edulis H. J. Lam is a common fruit in Nigeria used to eat roasted or boiled corn. It can also be eaten alone by softening with hot water, hot ash, or by roasting on low heat. It is called “ube” in the eastern part of Nigeria. D. edulis fruits are prone to infection by fungal pathogens in the field, in transit and during storage. Proper identification of these pathogens is important in disease prevention and control. This study was carried out to isolate and identify the fungal species associated with D. edulis fruits. Fruits were obtained from Rumokoro market, Rivers State, Nigeria in January 2020. Isolation of fungi was done by Potato Dextrose (PDA) method and identification was carried out by molecular method. Fungal DNA was extracted using the Zymo Fungal/Bacterial DNA Miniprep Kit. Polymerase chain reaction amplification of the internal transcribed spacer (ITS) region was carried out using the primer pair: ITS4 and ITS5. BLAST search was carried out on the National Centre for Biotechnology Information (NCBI) database and the fungi were identified as: Rhizopus delemar, Aspergillus oryzae and A. welwitschiae. The Phylogenetic tree was constructed to show the evolutionary relationship among the isolates and other fungal species retrieved from GenBank. The sequences of the isolates have been deposited in GenBank under the accession numbers: ON965489, ON965490 and ON965491 for Rhizopus delemar, Aspergillus oryzae and A. welwitschiae respectively. The molecular method used in this study enabled the isolates to be identified at the species levels.

Microbial removal of nutrients from anaerobic digestate: assessing product-coupled and non-product-coupled approaches.

Although anaerobic digestate contains >90% water, the high nutrient content of digestate makes it economically and technically intractable to treatment by existing wastewater treatment technologies. This study separately assessed the feasibility of nutrient removal from digestate by Rhizopus delemar DSM 905 and a culture of phosphate-accumulating organisms (PAOs). With Rhizopus delemar DSM 905, we investigated concomitant nutrient removal from digestate-supplemented medium and fumaric acid production, as a potentially economical strategy for digestate treatment. Following the cultivation of R. delemar DSM 905 in a fermentation medium containing 25% (v/v) digestate, the concentrations of Al, Cr, Cu, Fe, K, Mg, Mn, Pb, and Zn reduced 40, 12, 74, 96, 12, 26, 23%, ~18, and 28%, respectively. Similarly, the concentrations of total phosphorus, total nitrogen, phosphate (PO4-P), ammonium (NH4-N), nitrate (NO3-N), and sulfur decreased 93, 88, 97, 98, 69, and 13%, respectively. Concomitantly, cultures supplemented with 25 and 15% (v/v) digestate produced comparable titers of fumarate (~11 and ~ 17 g/L, respectively) to the digestate un-supplemented control cultures. With PAOs, we assessed the removal of total phosphorus, total nitrogen, PO4-P, and NH4-N, of which the concentrations reduced 86, 90%, ~99, and 100%, respectively in 60% (v/v) digestate. This study provides additional bases for microbial removal of excess nutrients from anaerobic digestate, with the potential to engender future water recovery from this waste stream that is currently largely recalcitrant to treatment.

Dectin-3-targeted antifungal liposomes efficiently bind and kill diverse fungal pathogens.

DectiSomes are anti-infective drug-loaded liposomes targeted to pathogenic cells by pathogen receptors including the Dectins. We have previously used C-type lectin (CTL) pathogen receptors Dectin-1, Dectin-2, and DC-SIGN to target DectiSomes to the extracellular oligoglycans surrounding diverse pathogenic fungi and kill them. Dectin-3 (also known as MCL, CLEC4D) is a CTL pathogen receptor whose known cognate ligands are partly distinct from other CTLs. We expressed and purified a truncated Dectin-3 polypeptide (DEC3) comprised of its carbohydrate recognition domain and stalk region. We prepared amphotericin B (AmB)-loaded pegylated liposomes (AmB-LLs) and coated them with this isoform of Dectin-3 (DEC3-AmB-LLs), and we prepared control liposomes coated with bovine serum albumin (BSA-AmB-LLs). DEC3-AmB-LLs bound to the exopolysaccharide matrices of Candida albicans, Rhizopus delemar (formerly known as R. oryzae), and Cryptococcus neoformans from one to several orders of magnitude more strongly than untargeted AmB-LLs or BSA-AmB-LLs. The data from our quantitative fluorescent binding assays were standardized using a CellProfiler program, AreaPipe, that was developed for this purpose. Consistent with enhanced binding, DEC3-AmB-LLs inhibited and/or killed C. albicans and R. delemar more efficiently than control liposomes and significantly reduced the effective dose of AmB. In conclusion, Dectin-3 targeting has the potential to advance our goal of building pan-antifungal DectiSomes.

Identification of lncRNA and weighted gene coexpression network analysis of germinating Rhizopus delemar causing mucormycosis

ABSTRACT Rhizopus delemar, an opportunistic fungal pathogen, causes a highly fatal disease, mucormycosis. Spore germination is a crucial mechanism for disease pathogenesis. Thus, exploring the molecular mechanisms of fungal germination would underpin our knowledge of such transformation and, in turn, help control mucormycosis. To gain insight into the developmental process particularly associated with cell wall modification and synthesis, weighted gene co-expression network analysis (WGCNA) was performed including both coding and non-coding transcripts identified in the current study, to find out the module of interest in the germination stages. The module-trait relationship identified a particular module to have a high correlation only at the resting phase and further analysis revealed the module to be enriched for protein phosphorylation, carbohydrate metabolic process, and cellular response to stimulus. Moreover, co-expression network analysis of highly connected nodes revealed cell wall modifying enzymes, especially those involved in mannosylation, chitin-glucan crosslinking, and polygalacturonase activities co-expressing and interacting with the novel lncRNAs among which some of them predicted to be endogenous target mimic (eTM) lncRNAs. Hence, the present study provides an insight into the onset of spore germination and the information on the novel non-coding transcripts with key cell wall–related enzymes as potential targets against mucormycosis.

Nano-combination for Reviving the Activity of Fluconazole against Rhizopus delemar.

Rhizopus delemar, the main causative pathogen for the lethal mucormycosis and a severe threat during the COVID-19 pandemic, is resistant to most antifungals, including fluconazole, a known selective antifungal drug. On the other hand, antifungals are known to enhance fungal melanin synthesis. Rhizopus melanin plays an important role in fungal pathogenesis and in escaping the human defense mechanism, thus complicating the use of current antifungal drugs and fungal eradication. Because of drug resistance and the slow discovery of effective antifungals, sensitizing the activity of older ones seems a more promising strategy. In this study, a strategy was employed to revive the use and enhance the effectiveness of fluconazole against R. delemar. UOSC-13, a compound synthesized in-house to target the Rhizopus melanin, was combined with fluconazole either as is or after encapsulation in poly (lactic-coglycolic acid) nanoparticles (PLG-NPs). Both combinations were tested for the growth of R. delemar, and the MIC50 values were calculated and compared. The activity of fluconazole was found to be enhanced several folds following the use of both combined treatment and nanoencapsulation. The combination of fluconazole with UOSC-13 caused a 5-fold reduction in the MIC50 value of fluconazole. Furthermore, encapsulating UOSC-13 in PLG-NPs enhanced the activity of fluconazole by an additional 10 folds while providing a wide safety profile. Consistent with previous reports, the encapsulation of fluconazole without sensitization showed no significant difference in activity. Collectively, sensitization of fluconazole represents a promising strategy to revive the use of outdated antifungal drugs back in the market.

Tunable Fungal Monofilaments from Food Waste for Textile Applications.

A fungal biorefinery is presented to valorize food waste to fungal monofilaments with tunable properties for different textile applications. Rhizopus delemar is successfully grown on bread waste and the fibrous cell wall is isolated. A spinnable hydrogel is produced from cell wall by protonation of amino groups of chitosan followed by homogenization and concentration. Fungal hydrogel is wet spun to form fungal monofilaments which underwent post-treatments to tune the properties. The highest tensile strength of untreated monofilaments is 65MPa (and 4% elongation at break). The overall highest tensile strength of 140.9MPa, is achieved by water post-treatment. Moreover, post-treatment with 3% glycerol resulted in the highest elongation % at break, i.e., 14%. The uniformity of the monofilaments also increased after the post-treatments. The obtained monofilaments are compared with commercial fibers using Ashby's plots and potential applications are discussed. The wet spun monofilaments are located in the category of natural fibers in Ashby's plots. After water and glycerol treatments, the properties shifted toward metals and elastomers, respectively. The compatibility of the monofilaments with human skin cells is supported by a biocompatibility assay. These findings demonstrate fungal monofilaments with tunable properties fitting a wide range of sustainable textiles applications.

The regulatory effects of microbial community on the formation of higher alcohols and volatile flavor components in Hongqu rice wine brewing

The contents of higher alcohols (HAs) and volatile components produced by microbial metabolism largely determine the drinking comfort and flavor quality of Hongqu rice wine. The purpose of this study was to compare the differences of volatile flavor profile and microbial community in the brewing process of Hongqu rice wines with low and high HAs content (LHA and HHA). The results showed that the main HAs in Hongqu rice wine were isobututyl alcohol, isoamyl alcohol, phenylethyl alcohol, and the total content of HAs in HHA (749.07 ± 20.47 mg/L) was much higher than that in LHA (407.61 ± 20.80 mg/L). Multivariate statistical analysis and visualization showed that the volatile flavor characteristics of HHA and LHA were significantly different, and the contents of ethyl esters, medium- and long-chain fatty acids in HHA were higher than those in LHA. Microbiome analysis based on metagenomic sequencing indicated that fermentation starter has significant effect on the composition of microbial community during Hongqu rice wine brewing. Microbial species such as Cronobacter sakazakii, Weissella paramesenteroides, Klebsiella pneumoniae, Aspergillus niger, Millerozyma farinosa and Rhizopus delemar were significantly enriched in HHA brewing, while Pediococcus pentosaceus, Lactobacillus curvatus, Leuconostoc pseudomesenteroides and Leuconostoc mesenteroides were significantly enriched in LHA brewing. Furthermore, the potential relationships between the key microbial species and characteristic volatile components were revealed through correlation heatmap analysis. Bioinformatical analysis revealed that the abundances of microbial genes encoding enzymes involved in HAs metabolism were higher in HHA than LHA, but the gene abundance of phenylacetaldehyde dehydrogenase in LHA was significantly higher than that in HHA, which could limit the formation of HAs during Hongqu rice wine brewing. Collectively, this study provides important scientific data for enhancing the flavor quality of Hongqu rice wine, and lays a solid foundation for the healthy development of Hongqu rice wine industry.

Enhancing the Hydrolytic Activity of a Lipase towards Larger Triglycerides through Lid Domain Engineering.

Lipases have valuable potential for industrial use, particularly those mostly active against water-insoluble substrates, such as triglycerides composed of long-carbon chain fatty acids. However, in most cases, engineered variants often need to be constructed to achieve optimal performance for such substrates. Protein engineering techniques have been reported as strategies for improving lipase characteristics by introducing specific mutations in the cap domain of esterases or in the lid domain of lipases or through lid domain swapping. Here, we improved the lipase activity of a lipase (WP_075743487.1, or LipMRD) retrieved from the Marine Metagenomics MarRef Database and assigned to the Actinoalloteichus genus. The improvement was achieved through site-directed mutagenesis and by substituting its lid domain (FRGTEITQIKDWLTDA) with that of Rhizopus delemar lipase (previously R. oryzae; UniProt accession number, I1BGQ3) (FRGTNSFRSAITDIVF). The results demonstrated that the redesigned mutants gain activity against bulkier triglycerides, such as glyceryl tridecanoate and tridodecanoate, olive oil, coconut oil, and palm oil. Residue W89 (LipMRD numbering) appears to be key to the increase in lipase activity, an increase that was also achieved with lid swapping. This study reinforces the importance of the lid domains and their amino acid compositions in determining the substrate specificity of lipases, but the generalization of the lid domain swapping between lipases or the introduction of specific mutations in the lid domain to improve lipase activity may require further investigation.

Sequencing the Genomes of the First Terrestrial Fungal Lineages: What Have We Learned?

The first genome sequenced of a eukaryotic organism was for Saccharomyces cerevisiae, as reported in 1996, but it was more than 10 years before any of the zygomycete fungi, which are the early-diverging terrestrial fungi currently placed in the phyla Mucoromycota and Zoopagomycota, were sequenced. The genome for Rhizopus delemar was completed in 2008; currently, more than 1000 zygomycete genomes have been sequenced. Genomic data from these early-diverging terrestrial fungi revealed deep phylogenetic separation of the two major clades-primarily plant-associated saprotrophic and mycorrhizal Mucoromycota versus the primarily mycoparasitic or animal-associated parasites and commensals in the Zoopagomycota. Genomic studies provide many valuable insights into how these fungi evolved in response to the challenges of living on land, including adaptations to sensing light and gravity, development of hyphal growth, and co-existence with the first terrestrial plants. Genome sequence data have facilitated studies of genome architecture, including a history of genome duplications and horizontal gene transfer events, distribution and organization of mating type loci, rDNA genes and transposable elements, methylation processes, and genes useful for various industrial applications. Pathogenicity genes and specialized secondary metabolites have also been detected in soil saprobes and pathogenic fungi. Novel endosymbiotic bacteria and viruses have been discovered during several zygomycete genome projects. Overall, genomic information has helped to resolve a plethora of research questions, from the placement of zygomycetes on the evolutionary tree of life and in natural ecosystems, to the applied biotechnological and medical questions.

Identification and Molecular Characterization of Rhizopus delemar from Eastern Ghats of State of India and its Biotechnological Applications

The humid climatic condition of the Koraput district of the eastern ghat of Odisha, India favors the growth of a diverse range of microbial and fungal strains, which not only lay scathing consequences on the environment but also prove to be beneficial in many industrial and biotechnological sectors. Hence, proper exploration of their roles and impacts is the necessity for an hour. With this goal, the present study reports on the identification and molecular characterization of the fungal strain collected from Koraput district of the eastern ghat of Odisha. The fungal strain was cultured, isolated, and identified using Internal transcribed spacer (ITS) 18S rRNA-specific primers. The estimation of fungal biomass was carried out by taking the dry weight of fungal mycelia after 48, 72, 96, and 120 hours of inoculation, which showed an increase in biomass (in w/w) exponentially. The fungal isolate (CAR02) was identified as Rhizopus delemar with GenBank accession number OQ683833. The SDS PAGE protein analysis showed more intensified and concentrated multiple fungal protein bands ranging from 13 to 97 kDa. ELISA (Enzyme- Linked Immunosorbent Assay) was used to estimate the antigenicity of the isolated R. delemar proteins from soil samples of Koraput. The proteins of Pythium species with an absorbance value of 1.5 shared 50-60% of similar antigenic sequences with the R. delemar proteins. Hence, the findings of our investigation suggests that the obtained antigenic sequences could further be used to isolate its epitopic specific regions that are responsible for the rot infections, against which antibodies could be developed. Further study is required to explore the rot causing fungal diversity in the soil of eastern ghats which will be helpful in designing novel disease diagnostic strategies in agriculture sectors. Furthermore, this identified fungus can also be used to synthesize various enzymes that will aid in the production of commercially significant by-products.

Identification of potential anti-mucor agents by targeting endothelial cell receptor glucose-regulated protein-78 using in silico approach

Mucormycosis is a fungal infection of the sinuses, brain and lungs that is the cause of approximately 50% mortality rate despite the available first-line therapy. Glucose-Regulated Protein 78 (GRP78) is already reported to be a novel host receptor that mediates invasion and damage of human endothelial cells by Rhizopus oryzae and Rhizopus delemar, the most common etiologic species of Mucorales. The expression of GRP78 is also regulated by the levels of iron and glucose in the blood. There are several antifungal drugs in the market but they pose a serious side effect to the vital organs of the body. Therefore, there is an immediate need to discover effective drug molecules having increased efficacy with no side effects. With the help of various computational tools, the current study was attempted to determine potential antimucor agents against GRP78. The receptor molecule GRP78 was screened against 8820 known drugs deposited in DrugBank library using high-throughput virtual screening method. Total top 10 compounds were selected based on the binding energies greater than the reference co-crystal molecule. Furthermore, molecular dynamic (MD) simulations using AMBER were performed to calculate the stability of the top-ranked compounds in the active site of GRP78. After extensive computational studies, we propose that two compounds (CID439153 and CID5289104) have inhibitory potency against mucormycosis and can serve as potential drugs that can form the basis of treating mucormycosis disease. Communicated by Ramaswamy H. Sarma

Bioconversion of Carrot Pomace to Value-Added Products: Rhizopus delemar Fungal Biomass and Cellulose

Carrot pomace (CP) which is generated in a large volume in the juice production process, is rich in cellulose, hemicellulose, sugars, pectin, and minerals. However, in many previous investigations, only cellulose was purified and utilized while other components of CP were discarded as waste. Here, CP was valorized into fungal biomass and cellulose with the aim of utilizing all the CP components. Enzymatic pretreatments were applied to solubilize the digestible fraction of CP including hemicellulose, pectin, sucrose, and other sugars for fungal cultivation, while cellulose remained intact in the solid fraction. The dissolved fraction was utilized as a substrate for the cultivation of an edible fungus (Rhizopus delemar). Fungal cultivation was performed in shake flasks and bench-scale bioreactors. The highest fungal biomass concentration was obtained after pretreatment with invertase (5.01 g/L) after 72 h of cultivation (36 and 42% higher than the concentrations obtained after hemicellulase and pectinase treatments, respectively). Invertase pretreatment resulted in the hydrolysis of sucrose, which could then be taken up by the fungus. Carbohydrate analysis showed 28–33% glucan, 4.1–4.9% other polysaccharides, 0.01% lignin, and 2.7–7% ash in the CP residues after enzymatic pretreatment. Fourier transform infrared spectroscopy and thermogravimetric analysis also confirmed the presence of cellulose in this fraction. The obtained fungal biomass has a high potential for food or feed applications, or as a raw material for the development of biomaterials. Cellulose could be purified from the solid fraction and used for applications such as biobased-textiles or membranes for wastewater treatment, where pure cellulose is needed.

Molecular and morphological characterization of fungi isolated from nutmeg (Myristica fragrans) in North Sulawesi, Indonesia

Abstract. Arifah F, Aini LQ, Muhibuddin A. 2023. Molecular and morphological characterization of fungi isolated from nutmeg (Myristica fragrans) in North Sulawesi, Indonesia. Biodiversitas 24: 441-453. Nutmeg (Myristica fragrans Houtt) is one of the predominant commodities from Indonesia exported to the EU market. However, it is frequently rejected due to mycotoxin (especially aflatoxin) content exceeding the aflatoxin standard set by the EU. The high levels of aflatoxin were hypothesized to be produced by a toxigenic fungus infecting nutmeg during and after harvest. The study aims to determine and identify fungal contaminants in nutmeg kernels and provide information on the types of mycotoxins produced. Ten fungal morphotypes were isolated from nutmeg kernels collected from North Minahasa, North Sulawesi, Indonesia, and were grouped into four genera (Aspergillus, Lasiodiplodia, Rhizopus, and Penicillium) based on morphological characters. Ten fungal morphotypes were identified, i.e., Aspergillus flavus (Groups I and II), A. tamarii (Groups I and II), A. niger, A. aculeatus, A. ochraceus, Lasiodiplodia sp., Rhizopus delemar, and Penicillium sp. Isolates of A. flavus and A. tamarii were divided into two groups based on slight differences in their colony appearances. Molecular identification using homology of 18S rDNA sequences and ITS regions supports morphological identification except for isolates of A. flavus (Groups I and II), Lasiodiplodia sp., and Penicillium sp. These isolates were identified as A. nomiae and A. aflatoxiformans (A. autswikcii), L. theobromae, and P. citrinum. The maximum likelihood phylogenetic tree showed that isolates from the same species were grouped in the same clade. The L. theobromae was the most dominant type of fungus in the nutmeg kernels (55.61%), followed by A. niger (23.8%) and A. tamarii (10.53%). It is the first report of A. aflatoxiformans or A. autswikcii in nutmeg kernels, and no incidence of L. theobromae in nutmeg kernels from Indonesia previously.

Microbiology Profile of COVID-19-Associated Rhino-Orbital Mucormycosis Pathogens in South India.

This study describes the microbiological and histopathological features of patients with COVID-19-associated rhino-orbital mucormycosis (ROM) seen at the L V Prasad Eye Institute between May and August 2021. Diagnosed clinically and radiologically, 24 patients with ROM were included in the study. Deep nasal swabs or endoscopically collected nasal swabs or orbital tissues were submitted for microbiological evaluation and invitro susceptibility testing by microbroth dilution for natamycin, amphotericin B, caspofungin, posaconazole, ketoconazole, and voriconazole. Cultures were processed by 28S ribosomal DNA polymerase chain reaction and molecular sequencing. A portion of orbital tissues was also sent for histopathological evaluation. The age of the patients ranged from 27 to 75 (mean 48.58 ± 14.09) years and the majority (79%) were male. Nineteen patients were known to be diabetic prior to developing ROM and 18 patients had recovered from active COVID-19 infection. Thirteen patients had a history of hospitalization during COVID-19 infection and eight received steroids. Of the 24 samples, microbiological evaluation identified Rhizopus arrhizus in 12, Rhizopus microsporus in 9, Lichtheimia ramosa in 2, and Rhizopus delemar in 1. Twelve isolates were tested for antifungal susceptibility and all were susceptible to natamycin and amphotericin B. The susceptibility to posaconazole was high, with minimum inhibitory concentration (MIC) < 2 µg/mL for 10/12 (84%) isolates, whereas the MIC of other drugs varied. Histopathological examination of tissues showed acute fulminant disease, granuloma formation, and vascular invasion by the fungal pathogens in these specimens. Rhizopus arrhizus was predominantly associated with ROM and most isolates were susceptible to amphotericin B and posaconazole. Further studies are needed to corroborate the findings and explain possible underlying links.