Intensification of sorption-reduction coupled gold biorecovery process through microbial surface modification: effect on gold sorption and reduction.

Biorecovery is emerging as a promising approach to retrieve gold from various sources, while its efficiency is usually restricted by the limited functional groups on natural microbial biomass surface. This study aims to intensify Pycnoporus sanguineus boosted sorption-reduction coupled gold biorecovery process via microbial surface modification. Results showed that grafting polyallylamine hydrochloride onto P. sanguineus biomass surface increased amino group content on microbial biomass surface from 1.29 to 2.81 mmol/g. When applying modified biomass to gold biorecovery with initial gold concentrations of 1.0, 2.0 and 3.0 mM, biosorption equilibrium time shortened to the 12.5%, 37.5% and 41.7% of those obtained with pristine biomass, and sorption rate constants correspondingly increased to 11.2, 3.1 and 3.7 folds as well. Maximum sorption capacity increased 30% and the affinity between biomass and gold enhanced heavily after microbial surface modification. Meanwhile, microbial surface modification favored gold reduction and gold nanoparticles (AuNPs) formation. The change of microbial biomass morphology from smooth surface with some branched structure to layered stacking structure with many pores and the increase of amino group content on microbial biomass surface were the main impetus for the gold bioreocovery process intensification.

Effect of hexavalent chromium on the biodegradation of tetrabromobisphenol A (TBBPA) by Pycnoporus sanguineus.

The influence of Cr(VI) on the degradation of tetrabromobisphenol A (TBBPA) by a typical species of white rot fungi, Pycnoporus sanguineus, was investigated in this study. The results showed that P. sanguineus together with its intracellular and extracellular enzyme could effectively degrade TBBPA. The degradation efficiency of TBBPA by both P. sanguineus and its enzymes decreased significantly when Cr(VI) concentration increased from 0 to 40 mg/L. The subsequent analysis about cellular distribution of TBBPA showed that the extracellular amount of TBBPA increased with the increment of Cr(VI) concentration, but the content of TBBPA inside fungal cells exhibited an opposite variation tendency. The inhibition of TBBPA degradation by P. sanguineus was partly attributed to the increase of cell membrane permeability and the decrease of cell membrane fluidity caused by Cr(VI). In addition, the decline of H+-ATPase and Mg2+-ATPase activities was also an important factor contributing to the suppression of TBBPA degradation in the system containing concomitant Cr(VI). Moreover, the activities of two typical extracellular lignin-degrading enzymes of P. sanguineus, MnP and Lac, were found to descend with ascended Cr(VI) level. Cr(VI) could also obviously suppress the gene expression of four intracellular enzymes implicated in TBBPA degradation, including two cytochrome P450s, glutathione S-transferases and pentachlorophenol 4-monooxygenase, which resulted in a decline of TBBPA degradation efficiency by fungal cells and intracellular enzyme in the presence of Cr(VI). Overall, this study provides new insights into the characteristics and mechanisms involved in TBBPA biodegradation by white rot fungi in an environment where heavy metals co-exist.

Fungal co-culture increases ligninolytic enzyme activities: statistical optimization using response surface methodology.

The optimization of ligninolytic enzyme (LE) activities in a novel fungal co-culture between Pycnoporus sanguineus and Beauveria brongniartii were studied using a Plackett-Burman experimental design (PBED) and a central composite design (CCD). In addition, H2O2 role was analyzed. Laccase (EC. 1.10.3.2) and MnP (EC 1.11.1.14) activities of P. sanguineus increased 6.0- and 2.3-fold, respectively, in the co-culture with B. brongniartii. The H2O2 content was higher in the co-culture (0.33-7.12-fold) than in the P. sanguineus monoculture. The PBED revealed that yeast extract (YE), FeSO4, and inoculum amount were significant factors for laccase and MnP activities and H2O2 production in the co-culture, which increased by 8.2-, 5.2- and 1.03-fold, respectively. The YE and FeSO4 were studied using a CCD to optimize the studied response variables. Laccase activity was enhanced 1.5-fold by CCD, the optimal amount of YE was 0.366 g L-1. Quadratic term of FeSO4 modulated MnP activity and was associated with a 4.28-fold increase compared to the PBED. Both YE and its quadratic term significantly affected H2O2 production; however, the CCD did not enable an increase in H2O2 production. Pearson correlation indicated an increase in laccase (r2=0.4411, p = 0.0436) and MnP (r2=0.5186, p = 0.0198) activities following increases in H2O2 in the co-culture system.

Influence of co-existed tetrabromobisphenol A (TBBPA) and hexavalent chromium on the cellular characteristics of Pycnoporus sanguineus during their removal and reduction.

Simultaneous TBBPA removal and Cr(VI) reduction by Pycnoporus sanguineus together with the effect of these co-existed pollutants on the fungal cellular characteristics were investigated in this study, aiming at illuminating the mechanism involved in the interactions between contaminants and microbial cells. The results revealed that Cr(VI) reduction and TBBPA removal declined from 92.5%, 75.4-30.6%, 44.8% when Cr(VI) concentration increased from 5 to 40mg/L, respectively. The removal efficiencies for Cr(VI) and TBBPA reached 61.4% and 94% separately under the optimum concentration of TBBPA at 10mg/L. Subsequent analyses indicated that the negative effect of Cr(VI) of high concentrations on Cr(VI) reduction and TBBPA removal was mainly attributed to the inhibition of fungal growth, intracellular proteins synthesis, cell viability and ATP enzyme activity. Compared with the moderate impact of TBBPA, the cell membrane of P. sanguineus was impaired severely and the surface morphology and intracellular structure changed dramatically in the presence of high concentration of Cr(VI) (above 10mg/L). This study also suggested that high level of TBBPA (15 and 20mg/L) promoted the synthesis of intracellular proteins and improved ATP enzyme activity within the first 48h of the reaction for enhancing the transportation and transformation of TBBPA.

A High Redox Potential Laccase from Pycnoporus sanguineus RP15: Potential Application for Dye Decolorization.

Laccase production by Pycnoporus sanguineus RP15 grown in wheat bran and corncob under solid-state fermentation was optimized by response surface methodology using a Central Composite Rotational Design. A laccase (Lacps1) was purified and characterized and the potential of the pure Lacps1 and the crude culture extract for synthetic dye decolorization was evaluated. At optimal conditions (eight days, 26 °C, 18% (w/w) milled corncob, 0.8% (w/w) NH4Cl and 50 mmol·L(-1) CuSO4, initial moisture 4.1 mL·g(-1)), the laccase activity reached 138.6 ± 13.2 U·g(-1). Lacps1 was a monomeric glycoprotein (67 kDa, 24% carbohydrate). Optimum pH and temperature for the oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) were 4.4 and 74.4 °C, respectively. Lacps1 was stable at pH 3.0-8.0, and after two hours at 55-60 °C, presenting high redox potential (0.747 V vs. NHE). ABTS was oxidized with an apparent affinity constant of 147.0 ± 6.4 µmol·L(-1), maximum velocity of 413.4 ± 21.2 U·mg(-1) and catalytic efficiency of 3140.1 ± 149.6 L·mmol(-1)·s(-1). The maximum decolorization percentages of bromophenol blue (BPB), remazol brilliant blue R and reactive blue 4 (RB4), at 25 or 40 °C without redox mediators, reached 90%, 80% and 60%, respectively, using either pure Lacps1 or the crude extract. This is the first study of the decolorization of BPB and RB4 by a P. sanguineus laccase. The data suggested good potential for treatment of industrial dye-containing effluents.

Evaluation of productivity and antioxidant profile of solid-state cultivated macrofungi Pleurotus albidus and Pycnoporus sanguineus.

The aim of this study was to investigate the production profile of Pleurotus albidus and Pycnoporus sanguineus on different waste substrates containing natural phenolics, and also to investigate whether phenolic-rich substrates can improve the phenolic content of these macrofungi. The medium formulated with Pinus sp. sawdust (PSW) made possible the highest yields (2.62±0.73%) of P. sanguineus. However, the supplementation of PSW with apple waste (AW) resulted in better P. albidus yields (23.94±2.92%). The results indicated that the substrate composition affected macrofungi production, also the chemical composition and the presence of phenolic compounds in the production media influence phenolic content and antioxidant activity in macrofungi.

Cell-based bioreporter assay coupled to HPLC micro-fractionation in the evaluation of antimicrobial properties of the basidiomycete fungus Pycnoporus cinnabarinus.

CONTEXT: Identification of bioactive components from complex natural product extracts can be a tedious process that aggravates the use of natural products in drug discovery campaigns. OBJECTIVE: This study presents a new approach for screening antimicrobial potential of natural product extracts by employing a bioreporter assay amenable to HPLC-based activity profiling. MATERIALS AND METHODS: A library of 116 crude extracts was prepared from fungal culture filtrates by liquid-liquid extraction with ethyl acetate, lyophilised, and screened against Escherichia coli using TLC bioautography. Active extracts were studied further with a broth microdilution assay, which was, however, too insensitive for identifying the active microfractions after HPLC separation. Therefore, an assay based on bioluminescent E. coli K-12 (pTetLux1) strain was coupled with HPLC micro-fractionation. RESULTS: Preliminary screening yielded six fungal extracts with potential antimicrobial activity. A crude extract from a culture filtrate of the wood-rotting fungus, Pycnoporus cinnabarinus (Jacq.) P. Karst. (Polyporaceae), was selected for evaluating the functionality of the bioreporter assay in HPLC-based activity profiling. In the bioreporter assay, the IC50 value for the crude extract was 0.10 mg/mL. By integrating the bioreporter assay with HPLC micro-fractionation, the antimicrobial activity was linked to LC-UV peak of a compound in the chromatogram of the extract. This compound was isolated and identified as a fungal pigment phlebiarubrone. DISCUSSION AND CONCLUSION: HPLC-based activity profiling using the bioreporter-based approach is a valuable tool for identifying antimicrobial compound(s) from complex crude extracts, and offers improved sensitivity and speed compared with traditional antimicrobial assays, such as the turbidimetric measurement.

Growth inhibition and antioxidative response of wood decay fungi exposed to plant extracts of Casearia species.

UNLABELLED: Ligninolytic fungi take part in critical processes in ecosystems such as nutrient recycling; however, some fungal species can be pathogenic to forest and urban trees and deteriorate wood products. The tropical flora is an important source of antimicrobial compounds environmentally safer than traditional wood preservatives. Therefore, this study aimed to evaluate the inhibitory activity of ethanol plant extracts of Casearia sylvestris and Casearia decandra on the white-rot wood decay basidiomycetes Trametes villosa and Pycnoporus sanguineus. In addition, the effect of the extracts on the fungal antioxidative metabolism was studied. Among the different substances present in the extracts, the phytochemical analyses identified a clerodane diterpenoid (C. sylvestris) and cinnamic acid, hydroquinone and ß-sitosterol (C. decandra). The extracts inhibited the fungi up to 70% and caused hyphal morphology changes. The extracts triggered oxidative stress process as indicated by the increased levels of the antioxidant enzymes catalase and glutathione reductase. Therefore, the Casearia extracts are a potential source of natural biocides to control wood decay fungi, and one of the mechanisms of action is the oxidative stress. SIGNIFICANCE AND IMPACT OF THE STUDY: The Casearia plant extracts exhibited important antifungal activity on wood decay fungi and triggered oxidative stress process, an inhibitory mechanism rarely studied in filamentous fungi exposed to plant extracts. Therefore, a starting point was provided for the development of natural compounds-based products as an alternative to chemical fungicides. In addition, subsidies were given to further studies in order to elucidate in more detail how compounds present in extracts of native tropical plants affect the physiology of fungi.

Modeling of growth and laccase production by Pycnoporus sanguineus.

Production of extracellular laccase by the white-rot fungus Pycnoporus sanguineus was examined in batch submerged cultures in shake flasks, baffled shake flasks and a stirred tank bioreactor. The biomass growth in the various culture systems closely followed a logistic growth model. The production of laccase followed a Luedeking-Piret model. A modified Luedeking-Piret model incorporating logistic growth effectively described the consumption of glucose. Biomass productivity, enzyme productivity and substrate consumption were enhanced in baffled shake flasks relative to the cases for the conventional shake flasks. This was associated with improved oxygen transfer in the presence of the baffles. The best results were obtained in the stirred tank bioreactor. At 28 °C, pH 4.5, an agitation speed of 600 rpm and a dissolved oxygen concentration of ~25 % of air saturation, the laccase productivity in the bioreactor exceeded 19 U L(-1 )days(-1), or 1.5-fold better than the best case for the baffled shake flask. The final concentration of the enzyme was about 325 U L(-1).

Simultaneous production of cellulase and reducing sugar through modification of compositional and structural characteristic of sugarcane bagasse.

This study examined the potential of untreated and alkali-pretreated sugarcane bagasse (SCB) in cellulase, reducing sugar (RS) and fungal biomass production via solid state fermentation (SSF) using Pycnoporus sanguineus. The impact of the composition, structure and cellulase adsorption ability of SCB on the production of cellulase, RS and fungal biomass was investigated. From the morphological and compositional analyses, untreated SCB has relatively more structural changes with a higher percentage of depolymerisation on the cellulose, hemicellulose and lignin content compared to alkali-pretreated SCB. Thus, untreated SCB favoured the production of cellulase and fungal biomass whereas alkali-pretreated SCB yielded a higher amount of RS. The composition and morphology of untreated SCB did not encourage RS production and this suggested that RS produced during SSF might be consumed in a faster rate by the more abundantly grown fungus. Besides that, alkali-pretreated SCB with higher cellulase adsorption ability could have adsorbed the cellulase produced and resulted in a lower cellulase titre. In short, the production of specific bioproducts via SSF is dependent on the structure and composition of the substrate applied.

Differential gene expression in Pycnoporus coccineus during interspecific mycelial interactions with different competitors.

Fungi compete against each other for environmental resources. These interspecific combative interactions encompass a wide range of mechanisms. In this study, we highlight the ability of the white-rot fungus Pycnoporus coccineus to quickly overgrow or replace a wide range of competitor fungi, including the gray-mold fungus Botrytis cinerea and the brown-rot fungus Coniophora puteana. To gain a better understanding of the mechanisms deployed by P. coccineus to compete against other fungi and to assess whether common pathways are used to interact with different competitors, differential gene expression in P. coccineus during cocultivation was assessed by transcriptome sequencing and confirmed by quantitative reverse transcription-PCR analysis of a set of 15 representative genes. Compared with the pure culture, 1,343 transcripts were differentially expressed in the interaction with C. puteana and 4,253 were differentially expressed in the interaction with B. cinerea, but only 197 transcripts were overexpressed in both interactions. Overall, the results suggest that a broad array of functions is necessary for P. coccineus to replace its competitors and that different responses are elicited by the two competitors, although a portion of the mechanism is common to both. However, the functions elicited by the expression of specific transcripts appear to converge toward a limited set of roles, including detoxification of secondary metabolites.

Fungal pretreatment of switchgrass for improved saccharification and simultaneous enzyme production.

This study investigates fungal pretreatment of switchgrass involving solid state fermentation (SSF) to improve saccharification and simultaneously produce enzymes as co-products. The results revealed that the fungus Pycnoporus sp. SYBC-L3 can significantly degrade lignin and enhance enzymatic hydrolysis efficiency. After a 36-d cultivation period, nearly 30% reduction in lignin content was obtained without significant loss of cellulose and hemicellulose, while a considerable amount of laccase, as high as 6.3 U/g, was produced. After pretreatment, pores on switchgrass surface were observed using scanning electron microscopy (SEM). The enzymatic hydrolysis efficiency for the switchgrass with 36-d pretreatment was about 50% greater than the untreated one. Our results suggest that solid state fungal cultivation may be a good method for switchgrass pretreatment, which can simultaneously achieve high efficiency of enzymatic hydrolysis and production of some useful enzymes for other industrial utilization.

Evaluation of different lignocellulosic substrates for the production of cellulases and xylanases by the basidiomycete fungi Bjerkandera adusta and Pycnoporus sanguineus.

Agricultural waste products are potential resources for the production of a number of industrial compounds, including biofuels. Basidiomycete fungi display a battery of hydrolytic enzymes with prospective use in lignocellulosic biomass transformation, however little work has been done regarding the characterization of such activities. Growth in several lignocellulosic substrates (oak and cedar sawdust, rice husk, corn stubble, wheat straw and Jatropha seed husk) and the production of cellulases and xylanases by two basidiomycete fungi: Bjerkandera adusta and Pycnoporus sanguineus were analyzed. Growth for P. sanguineus was best in rice husk while corn stubble supported the highest growth rate for B. adusta. Among the substrates tested, cedar sawdust produced the highest cellulolytic activities in both fungal species, followed by oak sawdust and wheat straw. Xylanolytic activity was best in oak and cedar sawdust for both species. We found no correlation between growth and enzyme production. Zymogram analysis of xylanases and cellulases showed that growth in different substrates produced particular combinations of protein bands with hydrolytic activity.

Mutation of a gene in the fungus Leptosphaeria maculans allows increased frequency of penetration of stomatal apertures of Arabidopsis thaliana.

Leptosphaeria maculans, a pathogen of Brassica napus, is unable to invade most wild-type accessions of Arabidopsis thaliana, although several mutants are susceptible. The infection pathway of L. maculans via a non-invasive inoculation method on A. thaliana lms1 (undefined), pmr4-1 (defective in callose deposition), and pen1-1 and pen2-1 (defective in non-host responses to several pathogens) mutants is described. On wild types Col-0 and Ler-0, hyphae are generally arrested at stomatal apertures. A T-DNA insertional mutant of L. maculans (A22) that penetrates stomatal apertures of Col-0 and Ler-0 five to seven times more often than the wild-type isolate is described. The higher penetration frequency of isolate A22 is associated with an increased hypersensitive response, which includes callose deposition. Complementation analysis showed that the phenotype of this isolate is due to T-DNA insertion in an intronless gene denoted as ipa (increased penetration on Arabidopsis). This gene is predicted to encode a protein of 702 amino acids with best matches to hypothetical proteins in other filamentous ascomycetes. The ipa gene is expressed in the wild-type isolate at low levels in culture and during infection of A. thaliana and B. napus.