Eco-friendly detoxification of hazardous Congo red dye using novel fungal strain Trametes flavida WTFP2: Deduced enzymatic biomineralization process through combinatorial in-silico and in-vitro studies.

Growing textile industry is a major global concern, owing to the presence of recalcitrant hazardous pollutants, like synthetic dyes in discharged effluents. To explore new bioresources for mycoremediation, a high laccase-producing novel white-rot fungus (WRF), Trametes flavida WTFP2, was employed. T. flavida is an underexplored member of Polyporales. Using bioinformatic tools, 8 different cis-acting RNA elements were identified in the 5.8 S ITS gene sequence, where CRISPR (CRISPR-DR15), sRNA (RUF1), and snoRNA (ceN111) are uniquely present. Molecular docking was adopted to predict the catalytic interaction of chosen toxic diazo colorant, Congo red (CR), with four dye-degrading enzymes (laccase, lignin peroxidase, azoreductase, and aryl alcohol oxidase). With 376.41 × 103 U/L laccase production, novel WRF exhibited dye-decolorization potential. WTFP2 effectively removed 99.48 ± 0.04% CR (100 mg/L) and demonstrated remarkable recyclability and persistence in consecutive remediation trials. Mycelial dye adsorption was not only substantial driver of colorant elimination; decolorization using active T. flavida was regulated by enzymatic catalysis, as outlined by in-vitro growth, induction of extracellular enzymes, and FESEM. Fifteen metabolites were identified using HRLCMS-QTOF, and novel CR degradation pathway was proposed. Furthermore, microbial and phyto-toxicity tests of metabolites suggested complete detoxification of toxic dye, making the process clean, green, and economically sustainable.

Cold-tolerant phosphate-solubilizing Pseudomonas strains promote wheat growth and yield by improving soil phosphorous (P) nutrition status.

It is well-known that phosphate-solubilizing bacteria (PSB) promote crop growth and yield. The information regarding characterization of PSB isolated from agroforestry systems and their impact on wheat crops under field conditions is rarely known. In the present study, we aim to develop psychrotroph-based P biofertilizers, and for that, four PSB strains (Pseudomonas sp. L3, Pseudomonas sp. P2, Streptomyces sp. T3, and Streptococcus sp. T4) previously isolated from three different agroforestry zones and already screened for wheat growth under pot trial conditions were evaluated on wheat crop under field conditions. Two field experiments were employed; set 1 includes PSB + recommended dose of fertilizers (RDF) and set 2 includes PSB - RDF. In both field experiments, the response of the PSB-treated wheat crop was significantly higher compared to the uninoculated control. In field set 1, an increase of 22% in grain yield (GY), 16% in biological yield (BY), and 10% in grain per spike (GPS) was observed in consortia (CNS, L3 + P2) treatment, followed by L3 and P2 treatments. Inoculation of PSB mitigates soil P deficiency as it positively influences soil alkaline phosphatase (AP) and soil acid phosphatase (AcP) activity which positively correlated with grain NPK %. The highest grain NPK % was reported in CNS-treated wheat with RDF (N-0.26%, P-0.18%, and K-1.66%) and without RDF (N-0.27, P-0.26, and K-1.46%), respectively. All parameters, including soil enzyme activities, plant agronomic data, and yield data were analyzed by principal component analysis (PCA), resulting in the selection of two PSB strains. The conditions for optimal P solubilization, in L3 (temperature-18.46, pH-5.2, and glucose concentration-0.8%) and P2 (temperature-17°C, pH-5.0, and glucose concentration-0.89%), were obtained through response surface methodology (RSM) modeling. The P solubilizing potential of selected strains at <20°C makes them a suitable candidate for the development of psychrotroph-based P biofertilizers. Low-temperature P solubilization of the PSB strains from agroforestry systems makes them potential biofertilizers for winter crops.

Molecular modeling and dynamics simulation of alcohol dehydrogenase enzyme from high efficacy cellulosic ethanol-producing yeast mutant strain Pichia kudriavzevii BGY1-γm.

One of the major constraints limiting the use of abundantly available lignocellulosic biomass as potential feedstock for alcohol industry is the lack of C6/C5 co-sugar fermenting yeast. The present study explores a mutant yeast Pichia kudriavzevii BGY1-γm as a potential strain for bioconversion of glucose/xylose sugars of green biomass into ethanol under batch fermentation. The mutant strain having higher alcohol dehydrogenase activity (11.31%) showed significantly higher ethanol concentration during co-fermentation of glucose/xylose sugars (14.2%) as compared to the native strain. Based on 99% sequence similarity of ADH encoding gene from the mutant with the gene sequences from other yeast strains, the ADH enzyme was identified as ADH-1 type. The study reveals first three-dimensional model of ADH-1 utilizing glucose/xylose sugars from P. kudriavzevii BGY1-γm (PkADH mutant). The refined and validated model of PkADH mutant was used for molecular docking against the substrate (acetaldehyde) and product (ethanol). Molecular docking results showed that substrate and product exhibited a binding affinity of -4.55 and -4.5 kcal/mol with PkADH mutant. Acetaldehyde and ethanol interacted at the active site of PkADH mutant via hydrogen bonds (Ser42, His69 and Asp163) and hydrophobic interactions (Cys40, Ser42, His69, Cys95, Trp123 and Asp163) to form the stable protein-ligand complex. Molecular dynamics analysis revealed that PkADH-mutant acetaldehyde and PkADH-mutant ethanol complexes were more stable than PkADH mutant. MMPBSA binding energy confirmed that binding of substrate and product results in the formation of a lower energy stable protein-ligand complex.Communicated by Ramaswamy H. Sarma.

Lignolytic mushroom Lenzites elegans WDP2: Laccase production, characterization, and bioremediation of synthetic dyes.

A mycoremedial study was undertaken for decolourization of synthetic dyes using wood rot fungal culture Lenzites elegans WDP2. The culture was isolated from decaying wood as fruiting body, and identified on the basis of 5.8S ITS rRNA gene sequence analysis. Qualitative plate screening of culture showed extracellular laccase and lignin peroxidase production, while only laccase enzyme was produced in higher amount (156.793 Uml-1) in minimal salt broth medium containing glucose and veratryl alcohol. Laccase activity was increased up to 189.25 Uml-1 after optimization of laccase production by optimization of one variable at a time approach. Molecular characterization of laccase enzyme was done using SDS PAGE and Native PAGE based isozyme analyses. The culture was able to decolorize three synthetic dying compounds (congo red, Malachite green and brilliant green) in broth media, while showed very less decolourization in plate assay. The fungal culture varied in their dye decolourizing potential in broth culture, showing 92.77%, 21.27% and 98.8% maximum decolourization of brilliant green, malachite green and congo red respectively. The congo red dye was completely bio-absorbed by fungal culture within one month. The fungal decolourized broth also revealed the extracellular laccase activity; varied from 10 Uml-1 to 68.5 Uml-1 in all the three cases, supports the involvement of laccase enzyme in decolorization. Phase contrast microscopy clearly revealed bio-sorption of the dyes by fungal culture into the mycelium/spores in the photomicrographs.

Solid state fermentation and crude cellulase based bioconversion of potential bamboo biomass to reducing sugar for bioenergy production.

BACKGROUND: Lignocellulosic biomass from bamboo is an attractive feedstock for the bioethanol industry owing to its high cellulosic content and fast growth rate. In this study, powdery biomass was first enzymatically delignified and then saccharified using crude enzymes. RESULTS: The biological pretreatment decreased the lignin content of the biomass from an initial value of 295 to 137.7 g kg-1 , with a simultaneous increase in exposed cellulose content from 379.3 to 615.9 g kg-1 . For optimization of the saccharification, response surface methodology was adopted using a three-factor/three-level Box-Behnken design with crude fungal cellulase loading (FPU g-1 substrate), substrate concentration (% w/v) and saccharification temperature (°C) as the main process parameters. A maximum saccharification yield of 47.19% was achieved under the optimized conditions (cellulase enzyme 18.4 FPU g-1 substrate, substrate concentration 1.0% w/v, temperature 39.49 °C). Biological delignification and saccharification of the biomass were further confirmed through scanning electron microscopy analysis. CONCLUSION: It is evident from the study that bamboo, as a renewable energy bioresource, can be hydrolysed to reducing sugars by using crude laccase/cellulase enzymes of fungal origin with good saccharification yield. Thus crude enzyme preparations could be utilized efficiently for eco-friendly and cost-effective bioethanol production. © 2018 Society of Chemical Industry.

Lignocellulosic agriculture wastes as biomass feedstocks for second-generation bioethanol production: concepts and recent developments.

Production of liquid biofuels, such as bioethanol, has been advocated as a sustainable option to tackle the problems associated with rising crude oil prices, global warming and diminishing petroleum reserves. Second-generation bioethanol is produced from lignocellulosic feedstock by its saccharification, followed by microbial fermentation and product recovery. Agricultural residues generated as wastes during or after processing of agricultural crops are one of such renewable and lignocellulose-rich biomass resources available in huge amounts for bioethanol production. These agricultural residues are converted to bioethanol in several steps which are described here. This review enlightens various steps involved in production of the second-generation bioethanol. Mechanisms and recent advances in pretreatment, cellulases production and second-generation ethanol production processes are described here.

Purification and characterization of an acidothermophilic cellulase enzyme produced by Bacillus subtilis strain LFS3.

In the present investigation, a microorganism hydrolyzing carboxymethylcellulose (CMC) was isolated and identified as Bacillus subtilis strain LFS3 by 16S rDNA sequence analysis. The carboxymethylcellulase (CMCase) enzyme produced by the B. subtilis strain LFS3 was purified by (NH4)2SO4 precipitation, ion exchange and gel filtration chromatography, with an overall recovery of 15 %. Native-PAGE analysis of purified CMCase revealed the molecular weight of enzyme to be about 185 kDa. The activity profile of CMCase enzyme showed the optimum activity at temperature 60 °C and pH 4.0, respectively. The enzyme activity was induced by Na⁺, Mg²âº, NH4⁺, and EDTA, whereas strongly inhibited by Hg²âº and Fe³âº. The purified enzyme hydrolyzed CMC, filter paper, and xylan, but not p-nitrophenyl ß-D-glucopyranoside and cellulose. Kinetic analysis of purified enzyme showed the K(m) value of 2.2 mg/ml. Thus, acidophilic as well as thermophilic nature makes this cellulase a suitable candidate for current mainstream biomass conversion into fuel and other industrial processes.

Effect of abiotic stress on phosphate solubilization by biocontrol fungus Trichoderma sp.

This study was undertaken to explore the role of Trichoderma sp. in phosphate (P) solubilization and antagonism against fungal phytopathogens. All fungal isolates (SE(6), KT(6), KT(28), and BRT(11)) and a standard culture of T. harzianum (Th-std) were able to antagonize two fungal phytopathogens (Sclerotium rolfsii and Rhizoctonia solani) of chickpea (Cicer arietinum L.) wilt complex. Transmission electron microscopic studies (TEM) further confirmed ultra-cytological changes in the sclerotia of S. rolfsii parasitized by Trichoderma sp. All fungal cultures exhibited production of NH(3) and siderophore, but only BRT(11), SE(6), and Th-std could produce HCN. Among all the cultures tested, isolate KT(6) was found to be most effective for solubilization of ferric phosphate releasing 398.4 µg ml(-1) phosphate while isolates BRT(11) and SE(6) showed more potential for tricalcium phosphate (TCP) solubilization releasing 449.05 and 412.64 µg ml(-1) phosphate, respectively, in their culture filtrates. Part of this study focused on the influence of abiotic stress conditions such as pH, temperature, and heavy metal (cadmium) on phosphate (TCP) solubilizing efficiency. Two selected cultures KT(6) and T. harzianum retained their P solubilizing potential at varying concentrations of cadmium (0-1000 µg ml(-1)). Isolate KT(6) and standard culture of T. harzianum released 278.4 and 287.6 µg ml(-1) phosphate, respectively, at 1000 µg ml(-1)cadmium. Maximum solubilization of TCP was obtained at alkaline pH and at 28°C temperature. Isolate BRT(11) was found most alkalo-tolerant releasing 448.0 µg ml(-1) phosphate at pH 9.

Phosphate solubilization potential and phosphatase activity of rhizospheric Trichoderma spp

Trichoderma sp., a well known biological control agent against several phytopathogens, was tested for its phosphate (P) solubilizing potential. Fourteen strains of Trichoderma sp. were isolated from the forest tree rhizospheres of pinus, deodar, bamboo, guava and oak on Trichoderma selective medium. The isolates were tested for their in-vitro P-solubilizing potential using National Botanical Research Institute Phosphate (NBRIP) broth containing tricalcium phosphate (TCP) as the sole P source, and compared with a standard culture of T. harzianum. All the cultures were found to solubilize TCP but with varying potential. The isolate DRT-1 showed maximum amount of soluble phosphate (404.07 µg.ml-1), followed by the standard culture of T. harzianum (386.42 µg.ml-1) after 96 h of incubation at 30+1(0)C. Extra-cellular acid and alkaline phosphatases of the fungus were induced only in the presence of insoluble phosphorus source (TCP). High extra-cellular alkaline phosphatase activity was recorded for the isolate DRT-1 (14.50 U.ml-1) followed by the standard culture (13.41 U.ml-1) at 72h. The cultures showed much lesser acid phosphatase activities. Under glasshouse conditions, Trichoderma sp. inoculation increased chickpea (Cicer arietinum) growth parameters including shoot length, root length, fresh and dry weight of shoot as well as roots, in P-deficient soil containing only bound phosphate (TCP). Shoot weight was increased by 23 percent and 33 percent by inoculation with the isolate DRT-1 in the soil amended with 100 and 200 mg TCP kg-1 soil, respectively, after 60 d of sowing. The study explores high P-solubilizing potential of Trichoderma sp., which can be exploited for the solubilization of fixed phosphates present in the soil, thereby enhancing soil fertility and plant growth.