Fungal Culture Supernatant Research Articles

Isolation of Endophytic Fungi from Adhatoda vasica and Assessing its Antibacterial Efficacy and Cytotoxic Impact on the A549 Cancer Cells

The current study investigated the anti-bacterial and anti-cancer properties of endophytic fungi obtained from medicinal plant Adhatoda vasica. We isolated and identified the endophytic fungi Humicola fuscoatra. by examining their morphological characteristics and microscopic structures. We then extracted their secondary metabolites using ethyl acetate. The ethyl acetate extracts of the fungus H. fuscoatra have shown promising effectiveness against human pathogens, including Klebsiella pneumoniae, Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. A strain of H. fuscoatra exhibited significant antibacterial activity. In addition, we have assessed the effects of ethyl acetate extracts from the fungus H. fuscoatra on the growth of the human lung cancer cell line A549 in vitro assays. Using ethyl acetate extracts from the fungus H. fuscoatra effectively suppressed the growth, survival, migration, and invasion of cancer cells. In this paper we studied in detail the identification of isolated endophytic fungi H. fuscoatra from Adhatoda vasica and characterized its active metabolite compounds. The ethyl acetate-extracted secondary metabolites from the fungal culture supernatant were analyzed using gas chromatography and mass spectrometry, which detected the presence of Hexadecanoic Acid, Hexadecane, Heptadecane, Octadecane, Octadecanoic acid, phytochemicals. The results suggest that the endophytic fungus H. fuscoatra found in medicinal plants has the potential to produce bioactive compounds. They could also be used as a biocontrol agent against bacterial pathogens and for therapeutic purposes in the A549 lung cancer cell line.

Isolation, identification, and antibacterial activity of endophytic fungus from Acanthus ilicifolius

Abstract Antimicrobial-resistant bacteria pose serious public health risks, necessitating bioprospecting for novel antimicrobial drugs. The endophytic fungi of the mangrove ecosystem are hotspots for discovering new bioactive chemical compounds. In this context, an investigation was designed to determine the isolation of the major endophytic fungi inhabiting the leaves of Acanthus ilicifolius, a mangrove plant with a long history of traditional use in the Chinese and Indian medical systems. Based on the morphological characterizations and molecular analysis of internal transcribed spacer rDNA sequence data, the study identified three unique endophytic fungal species, namely, Periconia macrospinosa, Coprinopsis cinerea, and Alternaria sp. Of these, P. macrospinosa was identified as the most dominant one, with the highest relative frequency (35.22%). The antibacterial activity of P. macrospinosa isolate (CMFRI/fPM-01) was evaluated by the well and diffusion method against six human pathogens, viz., Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus. The results demonstrated a high and wide spectrum of antimicrobial action of the isolate against all the tested human pathogens, with no significant difference (P > 0.05) in the activity between the pathogens. The antibacterial activity was further confirmed by determining the fungal culture supernatant's minimum inhibitory concentration and minimum bactericidal concentration. Although the studied fungi are known from other sources, this is the first report of P. macrospinosa and C. cinerea as endophytes in A. ilicifolius leaves. The outcomes also showed that the P. macrospinosa isolate could be used to discover effective antibacterial drugs against various human diseases.

Biosensor-guided detection of outer membrane-specific antimicrobial activity against Pseudomonas aeruginosa from fungal cultures and medicinal plant extracts.

Pseudomonas aeruginosa responds to sub-lethal antimicrobial exposure by inducing the expression of lipopolysaccharide (LPS) surface modifications that mask antibiotic binding sites and contribute to repair and resistance of the outer membrane (OM). We exploit these membrane damage-responsive operons in a biosensor approach used to discover new antimicrobials that specifically target the OM. Chromosomal transcriptional luxCDABE reporters from the pmr (polymyxin resistance; aminoarabinose LPS modification) and speD2E2 (spermidine synthesis) operons are induced by validated outer membrane-acting agents including cationic antimicrobial peptides, cation chelators, ascorbic acid, detergents, and cell wall synthesis inhibitors cycloserine and bacitracin. To identify novel sources of OM-disrupting antimicrobials, we used these OM damage-responsive biosensors to screen a panel of fungal culture supernatants for novel antimicrobial and biosensor activity. Biosensor activity was used to determine the optimal time point of antimicrobial production from fungal supernatants and to guide the purification of active fractions after size-exclusion chromatography. Water and ethanol extracts of Chinese medicinal plants also proved to be a source of biosensor activity. The pathogen box is a 400-member drug library of potential antimicrobials, but none of these compounds induced our OM damage biosensors. This novel, sensitive, cell-based screening assay has potential for future discovery of lead compounds that specifically target the outer membrane, which is a significant barrier to antibiotic entry into Gram-negative bacteria.IMPORTANCENew approaches are needed to discover novel antimicrobials, particularly antibiotics that target the Gram-negative outer membrane. By exploiting bacterial sensing and responses to outer membrane (OM) damage, we used a biosensor approach consisting of polymyxin resistance gene transcriptional reporters to screen natural products and a small drug library for biosensor activity that indicates damage to the OM. The diverse antimicrobial compounds that cause induction of the polymyxin resistance genes, which correlates with outer membrane damage, suggest that these LPS and surface modifications also function in short-term repair to sublethal exposure and are required against broad membrane stress conditions.

Biochemical properties of Scedosporium aurantiacum extracellular elastase-like protease

Extracellular elastase-like protease is one of the key virulence proteases of Scedosporium aurantiacum. To date, little is known about this enzyme in terms of genetic information, structure, properties and virulence mechanism due to the difficulties in purification caused by its low secretion amount, high specific activity, uncompleted genome sequencing and annotation. This work investigated the gene, structure and enzymatic properties of this enzyme. The S. aurantiacum elastase-like protease from the fungal culture supernatant was analyzed through tandem mass spectrometry (MS/MS) approach, illustrating its primary structure. Bioinformatics tools were employed to predict the conserved domain and tertiary structure, the enzymatic properties were also studied. It turned out that S. aurantiacum extracellular elastase-like protease demonstrated well hydrolysis towards elastin and bovine achilles tendon collagen, with Vmax of 18.14 μg/s and 17.57 μg/s respectively, better than fish scale gelatin, with the lowest hydrolysis effect on casein. Its activity towards elastin was lower than that of the elastase from porcine pancreas, with values of Kcat/Km of 3.541 (μg/s) and 4.091 (μg/s), respectively. It was an alkaline protease, with optimal pH 8.2 and temperature 37 oC. Zn2+ promoted the enzymatic activity while Ca2+, Mg2+, Na+, elastatinal and PMSF inhibited its activity. Its sequence was similar to Paecilomyces lilacinus secreted serine protease (PDB Entry: c3f7oB_) with multiple conserved fractions each containing more than 7 amino acids, thus suitable for design of PCR primer. This study increased our knowledge on S. aurantiacum extracellular elastase-like protease in terms of structure and enzymatic properties, and may facilitate later studies on protein expression and virulence mechanism.

Structural and Functional Analysis of Peptides Derived from KEX2-Processed Repeat Proteins in Agaricomycetes Using Reverse Genetics and Peptidomics.

Bioactivities of fungal peptides are of interest for basic research and therapeutic drug development. Some of these peptides are derived from "KEX2-processed repeat proteins" (KEPs), a recently defined class of precursor proteins that contain multiple peptide cores flanked by KEX2 protease cleavage sites. Genome mining has revealed that KEPs are widespread in the fungal kingdom. Their functions are largely unknown. Here, we present the first in-depth structural and functional analysis of KEPs in a basidiomycete. We bioinformatically identified KEP-encoding genes in the genome of the model agaricomycete Coprinopsis cinerea and established a detection protocol for the derived peptides by overexpressing the C. cinerea KEPs in the yeast Pichia pastoris. Using this protocol, which includes peptide extraction and mass spectrometry with data analysis using the search engine Mascot, we confirmed the presence of several KEP-derived peptides in C. cinerea, as well as in the edible mushrooms Lentinula edodes, Pleurotus ostreatus, and Pleurotus eryngii. While CRISPR-mediated knockout of C. cinerea kep genes did not result in any detectable phenotype, knockout of kex genes caused defects in mycelial growth and fruiting body formation. These results suggest that KEP-derived peptides may play a role in the interaction of C. cinerea with the biotic environment and that the KEP-processing KEX proteases target a variety of substrates in agaricomycetes, including some important for mycelial growth and differentiation. IMPORTANCE Two recent bioinformatics studies have demonstrated that KEX2-processed repeat proteins are widespread in the fungal kingdom. However, despite the prevalence of KEPs in fungal genomes, only few KEP-derived peptides have been detected and studied so far. Here, we present a protocol for the extraction and structural characterization of KEP-derived peptides from fungal culture supernatants and tissues. The protocol was successfully used to detect several linear and minimally modified KEP-derived peptides in the agaricomycetes C. cinerea, L. edodes, P. ostreatus, and P. eryngii. Our study establishes a new protocol for the targeted search of KEP-derived peptides in fungi, which will hopefully lead to the discovery of more of these interesting fungal peptides and allow a further characterization of KEPs.

A Straightforward Approach to Synthesize 7-Aminocephalosporanic Acid In Vivo in the Cephalosporin C Producer Acremonium chrysogenum.

The pharmaceutical industry has developed various highly effective semi-synthetic cephalosporins, which are generated by modifying the side chains of the core molecule 7-aminocephalosporanic acid (7-ACA). In industrial productions, the 7-ACA nucleus is obtained in vitro from cephalosporin C (CPC) by chemical or enzymatic processes, which are waste intensive and associated with high production costs. Here, we used a transgenic in vivo approach to express bacterial genes for cephalosporin C acylase (CCA) in the CPC producer Acremonium chrysogenum. Western blot and mass spectrometry analyses verified that the heterologous enzymes are processed into α- and β-subunits in the fungal cell. Extensive HPLC analysis detected substrates and products of CCAs in both fungal mycelia and culture supernatants, with the highest amount of 7-ACA found in the latter. Using different incubation times, temperatures, and pH values, we explored the optimal conditions for the active bacterial acylase to convert CPC into 7-ACA in the culture supernatant. We calculated that the best transgenic fungal strains exhibit a one-step conversion rate of the bacterial acylase of 30%. Our findings can be considered a remarkable contribution to supporting future pharmaceutical manufacturing processes with reduced production costs.

β-1,3-d-Glucan and Galactomannan as Biomarkers for the Detection of Invasive Geotrichum and Magnusiomyces Infections: a Retrospective Evaluation.

Magnusiomyces and Geotrichum species are ascomycetous yeasts that can cause potentially life-threatening invasive fungal infections commonly referred to as geotrichosis. In this study, we aimed to estimate the incidence and mortality of these infections in a German tertiary care center. Furthermore, we evaluated the suitability of the fungal biomarkers galactomannan (GM) and β-1,3-d-glucan (BDG), which are both recommended as surrogate markers for Magnusiomyces capitatus infection by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) and the European Confederation of Medical Mycology (ECMM) joint clinical guidelines for the diagnosis and management of rare invasive yeast infections for detection of invasive geotrichosis. Cases meeting the inclusion criteria for invasive Magnusiomyces/Geotrichum infection were retrospectively identified. Serum samples and culture supernatants were analyzed with two commercially available fungal antigen tests (Platelia Aspergillus Ag EIA and Wako β-glucan test). For a control cohort, outpatient samples sent for lues testing were included. Thirty-eight cases of Magnusiomyces/Geotrichum infection were identified over an 11-year observation period. In the majority of cases, the fungus was isolated from intra-abdominal specimens of patients with a history of abdominal surgery/procedures (n = 32). All cases of fungemia occurred exclusively in haemato-oncologic patients (n = 14). Thirty-day survival was 42% in the fungemia and 43% in the intra-abdominal geotrichosis group. Serum samples were available for 23 patients (14 bloodstream and nine intra-abdominal infections). While BDG sensitivity was 65%, none of the sera was GM positive. This finding was supported by in vitro experiments analyzing fungal culture supernatants: M. capitatus secretes significant amounts of BDG but not GM. Specificity was 96% for BDG and 100% for GM. Magnusiomyces and Geotrichum infections are not limited to haemato-oncologic patients. Contrasting the current ESCMID/ECMM recommendation, our results indicate that GM is no suitable biomarker for the diagnosis of Magnusiomyces infection. Contrarily, BDG sensitivity is comparable to that of candidemia.

Insights into Metabolic Changes Caused by the Trichoderma virens-Maize Root Interaction.

The interactions of crops with root-colonizing endophytic microorganisms are highly relevant to agriculture, because endophytes can modify plant resistance to pests and increase crop yields. We investigated the interactions between the host plant Zea mays and the endophytic fungus Trichoderma virens at 5 days postinoculation grown in a hydroponic system. Wild-type T. virens and two knockout mutants, with deletion of the genes tv2og1 or vir4 involved in specialized metabolism, were analyzed. Root colonization by the fungal mutants was lower than that by the wild type. All fungal genotypes suppressed root biomass. Metabolic fingerprinting of roots, mycelia, and fungal culture supernatants was performed using ultrahigh performance liquid chromatography coupled to diode array detection and quadrupole time-of-flight tandem mass spectrometry. The metabolic composition of T. virens-colonized roots differed profoundly from that of noncolonized roots, with the effects depending on the fungal genotype. In particular, the concentrations of several metabolites derived from the shikimate pathway, including an amino acid and several flavonoids, were modulated. The expression levels of some genes coding for enzymes involved in these pathways were affected if roots were colonized by the ∆vir4 genotype of T. virens. Furthermore, mycelia and fungal culture supernatants of the different T. virens genotypes showed distinct metabolomes. Our study highlights the fact that colonization by endophytic T. virens leads to far-reaching metabolic changes, partly related to two fungal genes. Both metabolites produced by the fungus and plant metabolites modulated by the interaction probably contribute to these metabolic patterns. The metabolic changes in plant tissues may be interlinked with systemic endophyte effects often observed in later plant developmental stages.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Role of Protein in Fungal Biomineralization of Copper Carbonate Nanoparticles

Biomineralization processes are of key importance in the biogeochemical cycling of metals and other elements by microorganisms, and several studies have highlighted the potential applications of nanoparticle synthesis via biomineralization. The roles played by proteins in the transformation and biologically induced biomineralization of metals by microorganisms is not well understood, despite the interactions of protein and nanoparticles at mineral interfaces attracting much interest in various emerging fields for novel biomaterial synthesis. Here, we have elucidated the association and involvement of fungal proteins in the formation of biogenic copper carbonate nanoparticles (CuNPs) using a carbonate-enriched biomass-free ureolytic fungal culture supernatant. Proteomic analysis was conducted that identified the major proteins present in the culture supernatant. Of the proteins identified, triosephosphate isomerase (TPI) exhibited a strong affinity to the CuNPs, and the impact of purified TPI on CuNP formation was studied in detail. The combined use of scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) confirmed that TPI played an important role in controlling the morphology and structure of the nanomaterials. Fourier transform infrared spectroscopy (FTIR) was applied to examine conformational changes of the proteins to further clarity the interaction mechanisms with CuNPs during biomineralization. Such analyses revealed unfolding of proteins on the mineral surface and an increase in β sheets within the protein structure. These results extend understanding of how microbial systems can influence biomineral formation through protein secretion, the mechanisms involved in formation of complex protein/inorganic systems, and provide useful guidelines for the synthesis of inorganic-protein based nanomaterials.

Tobacco Growth Promotion by the Entomopathogenic Fungus, Isaria javanica pf185

Isaria javanica pf185 is an important entomopathogenic fungus with potential for use as an agricultural biocontrol agent. However, the effect of I. javanica pf185 on plant growth is unknown. Enhanced tobacco growth was observed when tobacco roots were exposed to spores, cultures, and fungal cell-free culture supernatants of this fungus. Tobacco seedlings were also exposed to the volatiles of I. javanica pf185 in vitro using I-plates in which the plant and fungus were growing in separate compartments connected only by air space. The length and weight of seedlings, content of leaf chlorophyll, and number of root branches were significantly increased by the fungal volatiles. Heptane, 3-hexanone, 2,4-dimethylhexane, and 2-nonanone were detected, by solid-phase micro-extraction and gas chromatography-mass spectrophotometry, as the key volatile compounds produced by I. javanica pf185. These findings illustrate that I. javanica pf185 can be used to promote plant growth, and also as a biocontrol agent of insect and plant diseases. Further studies are necessary to elucidate the mechanisms by which I. javanica pf185 promotes plant growth.

Experimental and geochemical simulation of nickel carbonate mineral precipitation by carbonate-laden ureolytic fungal culture supernatants

In the biomineralization of NiCO3, the secondary structure of extracellular proteins changed from α-helices to β structures.

Biotransformation of lanthanum by Aspergillus niger

Lanthanum is an important rare earth element and has many applications in modern electronics and catalyst manufacturing. However, there exist several obstacles in the recovery and cycling of this element due to a low average grade in exploitable deposits and low recovery rates by energy-intensive extraction procedures. In this work, a novel method to transform and recover La has been proposed using the geoactive properties of Aspergillus niger. La-containing crystals were formed and collected after A. niger was grown on Czapek-Dox agar medium amended with LaCl3. Energy-dispersive X-ray analysis (EDXA) showed the crystals contained C, O, and La; scanning electron microscopy revealed that the crystals were of a tabular structure with terraced surfaces. X-ray diffraction identified the mineral phase of the sample as La2(C2O4)3·10H2O. Thermogravimetric analysis transformed the oxalate crystals into La2O3 with the kinetics of thermal decomposition corresponding well with theoretical calculations. Geochemical modelling further confirmed that the crystals were lanthanum decahydrate and identified optimal conditions for their precipitation. To quantify crystal production, biomass-free fungal culture supernatants were used to precipitate La. The results showed that the precipitated lanthanum decahydrate achieved optimal yields when the concentration of La was above 15 mM and that 100% La was removed from the system at 5 mM La. Our findings provide a new aspect in the biotransformation and biorecovery of rare earth elements from solution using biomass-free fungal culture systems.

Bio-Synthesis of Gold Nanoparticles by Fusarium oxysporum and Assessment of Their Conjugation Possibility with Two Types of β-Lactam Antibiotics without Any Additional Linkers

In the present study, biosynthesis of gold nanoparticles (GNPs) by Fusarium oxysporum was carried out and their conjugation possibility with two β-lactam antibiotics was evaluated. F. oxysporum was cultured and the fungal culture supernatant was subjected to the 1 mmol final concentration of chloroauric acid solution. The produced GNPs were analyzed using visible spectrophotometer, X-ray diffraction analysis (XRD) and transmission electron microscope (TEM). After the purification of GNPs, they were subjected to penicillin G and ceftriaxone without any additional linkers. Finally, the mixture was analyzed using visible spectrophotometer, Fourier transform infrared spectroscopy (FTIR) and TEM and subjected to antibacterial activity test using the well diffusion method. Results confirmed the presence of GNPs in the F. oxysporum culture supernatant after the addition of chloroauric acid. TEM results showed that GNPs were spherical and amorphous with sizes around 10–25 nm and XRD confirmed the presence of GNPs in the fungal culture supernatant. After the incubation of GNPs with the antibiotics, FTIR results demonstrated the successful linking of GNPs with the corresponded antibiotics and TEM images showed that GNPs sizes were bigger than the pure ones (around 50–100 nm). Finally the antibacterial activity test indicated that absolutely, the antibacterial properties of the GNPs-β-lactam antibiotics was lowered or not changed in contrast to the pure antibiotics. The present study showed that GNPs had high tendency of conjugation with antibiotics but unlike the previous researches, linking of the antibiotics to GNPs always cannot improve their antibacterial activity based on the antibiotics that were used. The high conjugation affinity of GNPs made them a good candidate as detoxification agent in diverse areas of medicine or environmental sciences.

Isolation and risk assessment of Geotrichum spp. in the white shrimp (Litopenaeus vannamei Boone, 1931) from culture ponds

The present study was done in order to identify the fungus invading some of the supralittoral ponds used for shrimp aquaculture in the CIBNOR facilities in La Paz, Baja California Sur (BCS), México during the summer season. From the walls and bottoms of the ponds, two strains of Geotrichum spp. were isolated and morphologically identified. Fungal adhesion towards hemocytes and primary cultures of various white shrimp (Litopeneaus vannamei) tissues (gill, tegument, and gut) was analyzed to determine infectivity. Extracellular protease, lipase, and amylase activity were evaluated as virulence factors. Survival of shrimp postlarvae (PL8) exposed to fungal culture supernatant or to their filaments was also investigated. The results showed that shrimp tegument cells and hemocytes were very susceptible to Geotrichum spp. invasion, and that this fungus provokes great mortality of post-larvae. Hence, Geotrichum spp. could be considered an opportunistic pathogen that might represent a serious health risk to shrimp in culture.

Paracoccidioides brasiliensis induces cytokine secretion in epithelial cells in a protease-activated receptor-dependent (PAR) manner.

Paracoccidioides brasiliensis is one of the etiological agents of the human systemic mycosis paracoccidioidomycosis. Protease-activated receptors (PARs) are expressed in many cell types and comprise a family of G protein-coupled receptors (PAR-1, PAR-2, and PAR-4), which may be activated by proteases secreted by several pathogens. In the present study, we showed that the pathogenic fungus P. brasiliensis secretes components that promote interleukin (IL)-6 and IL-8 secretion by the lung epithelial cell line A549. Cytokine secretion was reduced by antagonistic peptides for PAR-1 and PAR-2, but not for PAR-4. P. brasiliensis proteases were isolated from fungal culture supernatants in a p-aminomethylbenzamidine-Sepharose column. The obtained fractions were tested for enzymatic activity against fluorescence resonance energy transfer (FRET) peptides derived from sequences that spanned the activation sites of human PARs. The eluted fraction, termed PbP, contained protease activities that were able to hydrolyze the FRET peptides. PbP also induced IL-6 and IL-8 secretion in A549 epithelial cells, which was reduced upon heat inactivation of PbP, incubation with antagonistic peptides for PAR-1 and PAR-2, and the protease inhibitors aprotinin, leupeptin, and E-64. Together, these results show for the first time that P. brasiliensis yeasts secrete proteases that activate PARs in lung epithelial cells, leading to cytokine secretion.

Effect of Paecilomyces lilacinus, Trichoderma harzianum and Trichoderma virens fungal extracts on the hatchability of Ancylostoma eggs

BackgroundAncylostoma species have demanded attention due to their zoonotic potential. The use of anthelmintics is the usual method to prevent environmental contamination by Ancylostoma eggs and larvae. Nematophagous fungi have been widely used in their biological control due to the fungus ability to capture and digest free nematode forms. AimsThe aim of this study was to evaluate the effect of four different fungal extracts of Paecilomyces lilacinus (n=2), Trichoderma harzianum (n=1) and Trichoderma virens (n=1) isolates on the hatchability of Ancylostoma eggs. MethodsFungal extracts consisted of fungal broth culture supernatant without filtration (crude extract) and filtered broth (filtered extract), macerated mycelium (crude macerate), and macerated mycelium submitted to filtration (filtered macerate). The Ancylostoma eggs were obtained from the feces of naturally infected dogs. In vitro assays were performed in five replicates and consisted of four treatments and one control group. ResultsThe activity of the fungal extracts of each evaluated fungus differed (p<0.05) from those of the control group, showing significant ovicidal activity. The hatching of the eggs suffered reduction percentages of 68.43% and 47.05% with P. lilacinus, and 56.43% with T. harzianum, when crude macerate extract was used. The reduction with the macerate extract of T. virens was slightly lower (52.25%) than that for the filtered macerate (53.64%). ConclusionsThe results showed that all extracts were effective in reducing the hatchability of Ancylostoma eggs. The ovicidal effect observed is likely to have been caused by the action of hydrolytic enzymes secreted by the fungi.

Evaluation of bioactive secondary metabolites from endophytic fungus Pestalotiopsis neglecta BAB-5510 isolated from leaves of Cupressus torulosa D.Don.

Six endophytic fungi were isolated from Cupressus torulosa D.Don and identified phenotypically and genotypically. The fungal cultures were further grown and the culture was extracted by two organic solvents methanol and ethyl acetate. The screening was carried out using the agar well diffusion method against human pathogen such as Escherichia coli, Salmonella typhimurium, Bacillus subtilis and Staphylococcus aureus. Isolated strain of Pestalotiopsis sp. was showing prominent antibacterial activity. The crude methanol and ethyl acetate extract of Pestalotiopsis sp. showed MIC of 6.25 mg/mL for S. typhimurium and S. aureus which showed its efficacy as a potent antimicrobial. The phytochemical screening revealed the existence of a diverse group of secondary metabolites in the crude extracts of the endophytic fungi that resembled those in the host plant extracts. On the basis of phenotypic characteristics and rDNA sequencing of the ITS region of the endophyte was identified as P. neglecta which turned out to be a promising source of bioactive compounds. There is little known about endophytes from C. torulosa D.Don. In this paper we studied in detail the identification of isolated endophytic fungi P. neglecta from C. torulosa D.Don and characterization of its active metabolite compounds. The partially purified second fraction (PPF) extracted from the fungal culture supernatant was subjected to gas chromatography followed by mass spectrometry which revealed the presence of many phytochemicals. These results indicate that endophytic fungi P. neglecta isolated from medicinal plants could be a potential source for bioactive compounds and may find potential use in pharmaceutical industry.

Multi-stage laccase extraction and separation using aqueous two-phase systems: Experiment and model

This work presents results of experimental and model investigation of continuous multi-stage enzyme extraction using aqueous two-phase systems for the first time. The aqueous two-phase system comprised polyethylene glycol 3000 and phosphate with additional sodium chloride buffered to pH 7. Two different laccases served as model enzymes. One of the laccases was directly taken from fungal culture supernatant, while the other laccase was solubilized lyophilisate. The modeling is based on an equilibrium stage approach. Equilibrium data were taken from single-stage experiments and approximated by different correlation equations. The model describes densities, phase equilibrium, enzyme activity partitioning between the phases. Moreover it allows to consider activity changes due to the aqueous two-phase system. Eight multi-stage mixer-settler experiments under varying operation conditions were performed to validate the proposed model; whereas the total throughput of all multi-stage extraction experiments was about 350gh−1. The average relative deviation of modeled activities from experimentally measured activities was 23%. Therefore, the model is able to calculate the behavior of the phases as well as the partitioning of the two enzymes between the two phases for a multi-stage process based on single-stage data.

High throughput automated colorimetric method for the screening of l-lactic acid producing microorganisms

Lactic acid is a valuable and fully degradable organic acid with promising applications in poly-lactic acid production (Taskila S and Ojamo, 2013 [1]). Despite their efficiency, the cost of the current lactic acid bio-processes is still an obstacle to this application (Miller et al., 2011 [2]). To ameliorate lactic acid producing strains, researchers are using mutations and metabolic engineering techniques, as well as medium optimization. All these studies necessitate a good and high throughput screening method. Currently, researchers mostly use HPLC methods which often necessitate sample preparation, are not stereospecific and do not allow high throughput. To help optimizing l-lactic acid production, we developed a high throughput colorimetric method inspired by the blood l-lactic acid detection method used for diagnosis (Lin et al., 1999 [3]).•Two sequential enzymatic reactions using l-lactate oxidase, peroxidase and ABTS (2,2′-azino-di-[3-ethylbenzthiazoine-sulfonate]), a chromogenic peroxidase substrate, are used to quantify l-lactate between 13.8 and 90mg/l.•The accuracy of the method was ascertained before automation.•The method was successfully applied for the direct determination of l-lactate content in fungal culture supernatants.

Corrigendum: Overcoming Bottlenecks of Enzymatic Biofuel Cell Cathodes: Crude Fungal Culture Supernatant Can Help to Extend Lifetime and Reduce Cost

Corrigendum: Overcoming Bottlenecks of Enzymatic Biofuel Cell Cathodes: Crude Fungal Culture Supernatant Can Help to Extend Lifetime and Reduce Cost