Impact of redox-mediators in the degradation of olsalazine by marine-derived fungus, Aspergillus aculeatus strain bpo2: Response surface methodology, laccase stability and kinetics.

The indiscriminate disposal of olsalazine in the environment poses a threat to human health and natural ecosystems because of its cytotoxic and genotoxic nature. In the present study, degradation efficiency of olsalazine by the marine-derived fungus, Aspergillus aculeatus (MT492456) was investigated. Optimization of physicochemical parameters (pH. Temperature, Dry weight) and redox mediators {(2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), p-Coumaric acid and 1-hydroxybenzotriazole (HOBT)} was achieved with Response Surface Methodology (RSM)-Box-Behnken Design (BBD) resulting in 89.43% removal of olsalazine on 7th day. The second-order polynomial regression model was found to be statistically significant, adequate and fit with p < 0.0001, F value=41.87 and correlation coefficient (R2=0.9826). Biotransformation was enhanced in the redox mediator-laccase systems resulting in 99.5% degradation of olsalazine. The efficiency of ABTS in the removal of olsalazine was more pronounced than HOBT and p-Coumaric acid in the laccase-mediator system. This is attributed to the potent nature of the electron transfer mechanism deployed during oxidation of olsalazine. The pseudo-second-order kinetics revealed that the average half-life (t1/2) and removal rates (k1) increases with increasing concentrations of olsalazine. Michaelis-Menten kinetics affirmed the interaction between laccase and olsalazine under optimized conditions with maximum removal rate, Vmax=111.11 hr-1 and half-saturation constant, Km=1537 mg L-1. At the highest drug concentration (2 mM); 98%, 95% and 93% laccase was remarkably stabilized in the enzyme-drug degradation system by HOBT, ABTS and p-Coumaric acid respectively. This study further revealed that the deactivation of laccase by the redox mediators is adequately compensated with enhanced removal of olsalazine.

Biodegradation of fluorene by the newly isolated marine-derived fungus, Mucor irregularis strain bpo1 using response surface methodology.

Fluorene, a low molecular weight polycyclic aromatic hydrocarbon (PAH), is of immense environmental interest because of its carcinogenicity, teratogenicity, mutagenicity, toxicity and persistence to microbial degradation. Existentially, there is paucity of information on PAH degradation by fungi isolated from marine environment. Therefore, this study investigated fluorene degradation efficiency of marine derived filamentous fungus, Mucor irregularis strain bpo1 (GenBank Accession Number: MK373020). Response Surface Methodology (RSM) using Box-Behnken Design (BBD) was successfully deployed in the optimization of process parameters (pH-7, temperature-32.5 °C, substrate concentration-100 mg L-1 and dry weight-2 g) resulting in 81.50% fluorene degradation on 5th day. The design and regression model were found to be statistically significant, adequate and appropriate with p < 0.0001, F value= 202.39, and predicted coefficient of determination (R2 =0.9991). Optimization of the vital constituents of the mineral salt medium (MSM) used for the study using RSM-Central Composite Design (CCD) resulted in 79.80% fluorene degradation rate. Enhanced fluorene degradation efficiency (82.50%) was recorded when the optimized process variables were subjected to growth-linked validation experiments. The enzyme activities revealed 87%, 59% and 31% induction of laccase, manganese peroxidase and lignin peroxidase respectively. Four metabolites; 9H-fluoren-9-one, benzene-1,2-dicarboxylic acid, 2-hydroxybenzoic acid and phenol obtained after the experiment were characterized and confirmed with GC-MS analysis. The findings revealed the promising potentials of M. irregularis in PAH degradation and by extension green remediation technology.

Synergistic effect of biological and advanced oxidation process treatment in the biodegradation of Remazol yellow RR dye.

The current study investigated the efficiency of synergistic biological and Advanced Oxidation Process (AOPs) treatment (B-AOPs) using Aeromonas hydrophila SK16 and AOPs-H2O2 in the removal of Remazol Yellow RR dye. Singly, A. hydrophila and AOPs showed 90 and 63.07% decolourization of Remazol Yellow RR dye (100 mg L-1) at pH 6 and ambient temperature within 9 h respectively. However, the synergistic B-AOPs treatments showed maximum decolorization of Remazol Yellow RR dye within 4 h. Furthermore, the synergistic treatment significantly reduced BOD and COD of the textile wastewater by 84.88 and 82.76% respectively. Increased levels in laccase, tyrosinase, veratryl alcohol oxidase, lignin peroxidase and azo reductase activities further affirmed the role played by enzymes during degradation of the dye. UV-Visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), high-performance liquid chromatography (HPLC) and gas chromatography-mass spectroscopy (GC-MS) confirmed the biotransformation of dye. A metabolic pathway was proposed based on enzyme activities and metabolites obtained after GC-MS analysis. Therefore, this study affirmed the efficiency of combined biological and AOPs in the treatment of dyes and textile wastewaters in comparison with other methods.

Novel cobiomass degradation of NSAIDs by two wood rot fungi, Ganoderma applanatum and Laetiporus sulphureus: Ligninolytic enzymes induction, isotherm and kinetic studies.

A novel study on biodegradation of 30 mg L-1 of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) mixture (celecoxib, diclofenac and ibuprofen) by two wood-rot fungi; Ganoderma applanatum (GA) and Laetiporus sulphureus (LS) was investigated for 72 h. The removal efficiency of celecoxib, diclofenac and ibuprofen were 98, 96 and 95% by the fungal consortium (GA + LS). Although, both GA and LS exhibited low removal efficiency (61 and 73% respectively) on NSAIDs. However, 99.5% degradation of the drug mixture (NSAIDs) was achieved on the addition of the fungal consortium (GA + LS) to the experimental set-up. Overall, LS exhibited higher degradation efficiency; 92, 87, 79% on celecoxib, diclofenac and ibuprofen than GA with 89, 80 and 66% respectively. Enzyme analyses revealed significant induction of 201, 180 and 135% in laccase (Lac), lignin peroxidase (LiP) and manganese peroxidase (MnP) by the fungal consortium during degradation of the NSAIDs respectively. The experimental data showed the best goodness of fit when subjected to Langmuir (R2 = 0.980) and Temkin (R2 = 0.979) isotherm models which suggests monolayer and heterogeneous nature exhibited by the mycelia during interactions with NSAIDs. The degradation mechanism followed pseudo-second-order kinetic model (R2 = 0.987) indicating the strong influence of fungal biomass in the degradation of NSAIDs. Furthermore, Gas Chromatography-Mass Spectrometry (GCMS) and High-Performance Liquid Chromatography (HPLC) analyses confirmed the degraded metabolic states of the NSAIDs after treatment with GA, LS and consortium (GA + LS). Hence, the complete removal of NSAIDs is best achieved in an economical and eco-friendly way with the use of fungi consortium.

Biodegradation of a monochlorotriazine dye, cibacron brilliant red 3B-A in solid state fermentation by wood-rot fungal consortium, Daldinia concentrica and Xylaria polymorpha: Co-biomass decolorization of cibacron brilliant red 3B-A dye.

Efficient decolorization of cibracron brilliant red 3B-A dye by novel white rot fungal consortium was studied in static and shaking conditions using solid state fermentation technology. Daldinia concentrica (DC) and Xylaria polymorpha (XP) consortium showed dye removal efficiency than the individual strains within 5 days. The enzymes analysis revealed significant inductions in laccase (84%), lignin peroxidase (78%) and manganese peroxidase (65%) by the fungal co-culture (DC + XP), Xylaria polymorpha (XP) and Daldinia concentrica (DC) respectively. Enhanced decolorization was recorded when the medium was supplemented with glucose and ammonium nitrate as carbon and nitrogen sources respectively. The GCMS and HPLC analysis of metabolites suggest the different fates of biodegradation of cibracron brilliant red 3B-A dye by DC, XP and DC + XP consortium. The isotherm and kinetic studies revealed the goodness of fit of the experimental data when subjected to Freundlich and pseudo-second order models respectively. Phytotoxicity studies revealed that the biodegradation of the cibracron brilliant red 3B-A dye by the DC + XP consortium and individual strains has also led to the detoxification of the pollutant. This study revealed the effectiveness of white rot fungi in the eco-friendly remediation of dye polluted environment.

Relative profile analysis of molecular markers for identification and genetic discrimination of loaches (Pisces, Nemacheilidae).

Genus Nemacheilus, Nemachilichthys and Schistura belong to the family Nemacheilidae of the order Cypriniformes. The present investigation was undertaken to observe genetic diversity, phylogenetic relationship and to develop a molecular-based tool for taxonomic identification. For this purpose, four different types of molecular markers were utilized in which 29 random amplified polymorphic DNA (RAPD), 25 inter-simple sequence repeat (ISSR) markers, and 10 amplified fragment length polymorphism (AFLP) marker sets were screened and mitochondrial COI gene was sequenced. This study added COI barcodes for the identification of Nemacheilus anguilla, Nemachilichthys rueppelli and Schistura denisoni. RAPD showed higher polymorphism (100%) than the ISSR (93.75-100%) and AFLP (93.86-98.96%). The polymorphic information content (PIC), heterozygosity, multiplex ratio, and gene diversity was observed highest for AFLP primers, whereas the major allele frequency was observed higher for RAPD (0.5556) and lowest for AFLP (0.1667). The COI region of all individuals was successfully amplified and sequenced, which gave a 100% species resolution.

Phylogenetic analysis, genetic diversity and relationships between the recently segregated species of Corynandra and Cleoserrata from the genus Cleome using DNA barcoding and molecular markers.

Cleome is the largest genus in the family Cleomaceae and it is known for its various medicinal properties. Recently, some species from the Cleome genus (Cleome viscosa, Cleome chelidonii, Cleome felina and Cleome speciosa) are split into genera Corynandra (Corynandra viscosa, Corynandra chelidonii, Corynandra felina), and Cleoserrata (Cleoserrata speciosa). The objective of this study was to obtain DNA barcodes for these species for their accurate identification and determining phylogenetic relationships. Out of 10 screened barcoding regions, rbcL, matK and ITS1 regions showed higher PCR efficiency and sequencing success. This study added matK, rbcL and ITS1 barcodes for the identification of Corynandra chelidonii, Corynandra felina, Cleome simplicifolia and Cleome aspera species in existing barcode data. Corynandra chelidonii and Corynandra felina species belong to the Corynandra genus, but they are not grouped with the Corynandra viscosa species, however clustered with the Cleome species. Molecular marker analysis showed 100% polymorphism among the studied plant samples. Diversity indices for molecular markers were ranged from He=0.1115-0.1714 and I=0.2268-0.2700, which indicates a significant amount of genetic diversity among studied species. Discrimination of the Cleome and Corynandra species from Cleoserrata speciosa was obtained by two RAPD primers (OPA-4 and RAPD-17) and two ISSR primers (ISSR-1 and ISSR-2). RAPD and ISSR markers are useful for the genetic characterization of these studied species. The present investigation will be helpful to understand the relationships of Cleome lineages with Corynandra and Cleoserrata species.

Analysis of genetic variability in endemic medicinal plants of genus Chlorophytum from the Indian subcontinent using amplified fragment length polymorphism marker.

The genus Chlorophytum consists of medicinally important species like Chlorophytum borivilianum, C. tuberosum and C. attenuatum. Uncontrolled harvest of this plant from wild habitat due to its high commercial value made the species of this genus be listed in the Red Data Book of Indian plants as an endangered species. In India, approximately nineteen species of Chlorophytum are found; out of these, only C. borivilianum is cultivated commercially. The objective of this study was to measure genetic diversity, population structure and phylogenetic relationship among the species using Amplified Fragment Length Polymorphisms (AFLP). Fifteen pairs of primer (out of 64 primer pairs screened) were used to analyse the genetic diversity in eighteen species of genus Chlorophytum. Cluster analysis, estimation of the gene flow among the species and of the phylogeographic distribution of this genus were carried out using an AFLP data matrix. A high level of genetic diversity was observed on the basis of the percentage of polymorphic bands (99.91%), Shannon's information index (0.3592) and Nei's gene diversity (0.2085) at species level. Cluster analysis of UPGMA dendrogram, principal component analysis and Bayesian method analysis resolved these species in three different clusters, which was supported by morphological information. The Mantel test (r=0.4432) revealed a significant positive correlation between genetic and geographic distances. The collected data have an important implication in the identification, authentication, and conservation of the species of the genus Chlorophytum.

Characterization of modular bifunctional processive endoglucanase Cel5 from Hahella chejuensis KCTC 2396.

Cel5 from marine Hahella chejuensis is composed of glycoside hydrolase family-5 (GH5) catalytic domain (CD) and two carbohydrate binding modules (CBM6-2). The enzyme was expressed in Escherichia coli and purified to homogeneity. The optimum endoglucanase and xylanase activities of recombinant Cel5 were observed at 65 °C, pH 6.5 and 55 °C, pH 5.5, respectively. It exhibited K m of 1.8 and 7.1 mg/ml for carboxymethyl cellulose and birchwood xylan, respectively. The addition of Ca(2+) greatly improved thermostability and endoglucanase activity of Cel5. The Cel5 retained 90 % of its endoglucanase activity after 24 h incubation in presence of 5 M concentration of NaCl. Recombinant Cel5 showed production of cellobiose after hydrolysis of cellulosic substrates (soluble/insoluble) and methylglucuronic acid substituted xylooligosaccharides after hydrolysis of glucuronoxylans by endo-wise cleavage. These results indicated that Cel5 as bifunctional enzyme having both processive endoglucanase and xylanase activities. The multidomain structure of Cel5 is clearly distinguished from the GH5 bifunctional glycoside hydrolases characterized to date, which are single domain enzymes. Sequence analysis and homology modeling suggested presence of two conserved binding sites with different substrate specificities in CBM6-2 and a single catalytic site in CD. Residues Glu132 and Glu219 were identified as key catalytic amino acids by sequence alignment and further verified by using site directed mutagenesis. CBM6-2 plays vital role in catalytic activity and thermostability of Cel5. The bifunctional activities and multiple substrate specificities of Cel5 can be utilized for efficient hydrolysis of cellulose and hemicellulose into soluble sugars.