Saccharification of sunflower stalks using lignocellulases from a fungal consortium comprising Pholiota adiposa and Armillaria gemina.

Lignocellulases from Armillaria gemina and Pholiota adiposa are efficient in hydrolyzing aspen and poplar biomass, respectively. In the present study, lignocellulosic enzymes obtained from a fungal consortium comprising P. adiposa and A. gemina were used for the saccharification of sunflower stalks. Sunflower stalks were thermochemically pretreated using 2 % NaOH at 50 °C for 24 h. The saccharification process parameters including substrate concentration, enzyme loading, pH, and temperature were optimized using response surface methodology to improve the saccharification yield. The highest enzymatic hydrolysis (84.3 %) was obtained using the following conditions: enzyme loading 10 FPU/g-substrate, substrate 5.5 %, temperature 50 °C, and pH 4.5. The hydrolysis yield obtained using the enzymes from the fungal consortium was equivalent to that obtained using a mixture of commercial enzymes Celluclast and Novozyme ß-glucosidase. Addition of up to 500 ppm of heavy metal ions (As, Cu, Fe, Mn, Ni, Pb, and Zn) during saccharification did not significantly affect the saccharification yield. Thus, the biomass grown for phytoremediation of heavy metals can be used for the production of reducing sugars followed by ethanol fermentation.

Characterization of a novel endo-ß-1,4-glucanase from Armillaria gemina and its application in biomass hydrolysis.

A novel endo-ß-1,4-glucanase (EG)-producing strain was isolated and identified as Armillaria gemina KJS114 based on its morphology and internal transcribed spacer rDNA gene sequence. A. gemina EG (AgEG) was purified using a single-step purification by gel filtration. The relative molecular mass of AgEG by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 65 kDa and by size exclusion chromatography was 66 kDa, indicating that the enzyme is a monomer in solution. The pH and temperature optima for hydrolysis were 5.0 and 60 °C, respectively. Purified AgEG had the highest catalytic efficiency with carboxymethylcellulose (k(cat)/K(m) = 3,590 mg mL⁻¹ s⁻¹) unlike that reported for any fungal EG, highlighting the significance of the current study. The amino acid sequence of AgEG showed homology with hydrolases from the glycoside hydrolase family 61. The addition of AgEG to a Populus nigra hydrolysate reaction containing a commercial cellulase mixture (Celluclast 1.5L and Novozyme 188) showed a stimulatory effect on reducing sugar production. AgEG is a good candidate for applications that convert lignocellulosic biomass to biofuels and chemicals.

Characterization of a ß-1,4-mannanase from a newly isolated strain of Pholiota adiposa and its application for biomass pretreatment.

A highly efficient ß-1,4-mannanase-secreting strain, Pholiota adiposa SKU0714, was isolated and identified on the basis of its morphological features and sequence analysis of internal transcribed spacer rDNA. P. adiposa ß-1,4-mannanase was purified to homogeneity from P. adiposa culture supernatants by one-step chromatography on a Sephacryl gel filtration column. P. adiposa ß-1,4-mannanase showed the highest activity toward locust bean gum (V max = 1,990 U/mg protein, K m = 0.12 mg/mL) ever reported. Its internal amino acid sequence showed homology with hydrolases from the glycoside hydrolase family 5 (GH5), indicating that the enzyme is a member of the GH5 family. The saccharification of commercial mannanase and P. adiposa ß-1,4-mannanase-pretreated rice straw by Celluclast 1.5L (Novozymes) was compared. In comparison with the commercial Novo Mannaway(®) (113 mg/g-substrate), P. adiposa ß-1,4-mannanase-pretreated rice straw released more reducing sugars (141 mg/g-substrate). These properties make P. adiposa ß-1,4-mannanase a good candidate as a new commercial ß-1,4-mannanase to improve biomass pretreatment.

Characterization of a novel xylanase from Armillaria gemina and its immobilization onto SiO2 nanoparticles.

Enhanced catalytic activities of different lignocellulases were obtained from Armillaria gemina under statistically optimized parameters using a jar fermenter. This strain showed maximum xylanase, endoglucanase, cellobiohydrolase, and ß-glucosidase activities of 1,270, 146, 34, and 15 U mL(-1), respectively. Purified A. gemina xylanase (AgXyl) has the highest catalytic efficiency (k (cat)/K (m) = 1,440 mg mL(-1) s(-1)) ever reported for any fungal xylanase, highlighting the significance of the current study. We covalently immobilized the crude xylanase preparation onto functionalized silicon oxide nanoparticles, achieving 117 % immobilization efficiency. Further immobilization caused a shift in the optimal pH and temperature, along with a fourfold improvement in the half-life of crude AgXyl. Immobilized AgXyl gave 37.8 % higher production of xylooligosaccharides compared to free enzyme. After 17 cycles, the immobilized enzyme retained 92 % of the original activity, demonstrating its potential for the synthesis of xylooligosaccharides in industrial applications.

Transcriptome-wide marker gene expression analysis of stress-responsive sulfate-reducing bacteria.

Sulfate-reducing bacteria (SRB) are terminal members of any anaerobic food chain. For example, they critically influence the biogeochemical cycling of carbon, nitrogen, sulfur, and metals (natural environment) as well as the corrosion of civil infrastructure (built environment). The United States alone spends nearly $4 billion to address the biocorrosion challenges of SRB. It is important to analyze the genetic mechanisms of these organisms under environmental stresses. The current study uses complementary methodologies, viz., transcriptome-wide marker gene panel mapping and gene clustering analysis to decipher the stress mechanisms in four SRB. Here, the accessible RNA-sequencing data from the public domains were mined to identify the key transcriptional signatures. Crucial transcriptional candidate genes of Desulfovibrio spp. were accomplished and validated the gene cluster prediction. In addition, the unique transcriptional signatures of Oleidesulfovibrio alaskensis (OA-G20) at graphene and copper interfaces were discussed using in-house RNA-sequencing data. Furthermore, the comparative genomic analysis revealed 12,821 genes with translation, among which 10,178 genes were in homolog families and 2643 genes were in singleton families were observed among the 4 genomes studied. The current study paves a path for developing predictive deep learning tools for interpretable and mechanistic learning analysis of the SRB gene regulation.

Classification of bacterial nanowire proteins using Machine Learning and Feature Engineering model.

Nanowires (NW) have been extensively studied for Shewanella spp. and Geobacter spp. and are mostly produced by Type IV pili or multiheme c-type cytochrome. Electron transfer via NW is the most studied mechanism in microbially induced corrosion, with recent interest in application in bioelectronics and biosensor. In this study, a machine learning (ML) based tool was developed to classify NW proteins. A manually curated 999 protein collection was developed as an NW protein dataset. Gene ontology analysis of the dataset revealed microbial NW is part of membranal proteins with metal ion binding motifs and plays a central role in electron transfer activity. Random Forest (RF), support vector machine (SVM), and extreme gradient boost (XGBoost) models were implemented in the prediction model and were observed to identify target proteins based on functional, structural, and physicochemical properties with 89.33%, 95.6%, and 99.99% accuracy. Dipetide amino acid composition, transition, and distribution protein features of NW are key important features aiding in the model's high performance.

Immobilization of Pholiota adiposa xylanase onto SiO2 nanoparticles and its application for production of xylooligosaccharides.

Enhanced yields of different lignocellulases were obtained under statistically-optimized parameters using Pholiota adiposa. The k (cat) value (4,261 s(-1)) of purified xylanase under standard assay conditions was the highest value ever reported. On covalent immobilization of the crude xylanase preparation onto functionalized silicon oxide nanoparticles, 66 % of the loaded enzyme was retained on the particle. Immobilized enzyme gave 45 % higher concentrations of xylooligosaccharides compared to the free enzyme. After 17 cycles, the immobilized enzyme retained 97 % of the original activity, demonstrating its prospects for the synthesis of xylooligosaccharides in industrial applications.

Application of Thermostable Xylanase of Bacillus pumilus in Textile Processing.

Desizing of cotton and micropoly fabrics was done using thermostable xylanase from Bacillus pumilus ASH. Micropoly fabric showed better desizing than cotton under same conditions. Violet scale readings from the TEGEWA test after enzymatic desizing for 90 min at pH 7.0 and at 60°C showed the readings falling in the range of 4-5, indicating good desizing efficiency. During bioscouring the weight loss values and liberation of reducing sugars were highest when EDTA was used along with xylanase. The weight loss value of 1.5% was observed for dry cotton fabric after 1 h in case of agitated system at pH 7.0 and at an optimal enzyme dosage of 5 IU/g. The weight loss values and the liberation of reducing sugars were higher in case of cotton fabrics. Wetting time of fabrics was lowered significantly after 60 min of bioscouring using xylanase. Increase in temperature or concentration of surfactant led to further reduction in the wetting time. The whiteness values of fabrics after bioscouring were 0.9% higher than the chemically scoured fabrics indicating good efficacy of xylanase during the scouring process.

Molecular regulation of conditioning film formation and quorum quenching in sulfate reducing bacteria.

Sensing surface topography, an upsurge of signaling biomolecules, and upholding cellular homeostasis are the rate-limiting spatio-temporal events in microbial attachment and biofilm formation. Initially, a set of highly specialized proteins, viz. conditioning protein, directs the irreversible attachment of the microbes. Later signaling molecules, viz. autoinducer, take over the cellular communication phenomenon, resulting in a mature microbial biofilm. The mandatory release of conditioning proteins and autoinducers corroborated the existence of two independent mechanisms operating sequentially for biofilm development. However, both these mechanisms are significantly affected by the availability of the cofactor, e.g., Copper (Cu). Generally, the Cu concentration beyond threshold levels is detrimental to the anaerobes except for a few species of sulfate-reducing bacteria (SRB). Remarkably SRB has developed intricate ways to resist and thrive in the presence of Cu by activating numerous genes responsible for modifying the presence of more toxic Cu(I) to Cu(II) within the periplasm, followed by their export through the outer membrane. Therefore, the determinants of Cu toxicity, sequestration, and transportation are reconnoitered for their contribution towards microbial adaptations and biofilm formation. The mechanistic details revealing Cu as a quorum quencher (QQ) are provided in addition to the three pathways involved in the dissolution of cellular communications. This review articulates the Machine Learning based data curing and data processing for designing novel anti-biofilm peptides and for an in-depth understanding of QQ mechanisms. A pioneering data set has been mined and presented on the functional properties of the QQ homolog in Oleidesulfovibrio alaskensis G20 and residues regulating the multicopper oxidase properties in SRB.

Antibacterial and antifungal studies of macrocyclic complexes of trivalent transition metal ions with their spectroscopic approach.

A new series of complexes of the type [M(C24H16N4)X]X2, where M = Cr(III), Fe(III), and Mn(III), X = Cl-, NO3-, and CH3COO-, has been synthesized by template condensation of 1,8-diaminonaphthalene and glyoxal in the presence of trivalent metal salts in methanolic medium. The complexes have been characterized with the help of elemental analysis, conductance measurements, magnetic measurements, and electronic, NMR, IR, and mass spectral studies. On the basis of these studies, a five-coordinate square pyramidal geometry for all of these complexes has been proposed. All the synthesized metal complexes were also tested for their in vitro antimicrobial activities against some bacterial strains, viz. Bacillus subtilis, Bacillus stearothermophilus (gram-positive bacteria), Escherichia coli, and Pseudomonas putida (gram-negative bacteria), and some fungal strains, viz. Aspergillus flavus and Aspergillus niger. The results obtained were compared with standard antibiotics: chloramphenicol, streptomycin, and the antifungal drug cyclohexamide. Some of the tested complexes showed remarkable antimicrobial activities.

Vitamin-C-enabled reduced graphene oxide chemistry for tuning biofilm phenotypes of methylotrophs on nickel electrodes in microbial fuel cells.

This study reports the use of multi-layered reduced graphene oxide (rGO) coating on porous nickel foam (NF) electrodes for enhancing biofilm growth of Rhodobacter Sphaeroides spp fed with methanol in microbial fuel cells (CH3OH-MFCs). Electrochemical methods were used to assess the methylotrophic activity on rGO/NF electrodes. The power density and current density offered by rGO/NF (1200 mW m-2 and 680 mA m-2) were 220-fold and 540-fold higher compared to bare NF (5.50 mW m-2 and 1.26 mA m-2), respectively. Electrochemical impedance spectroscopy results show that rGO/NF suppresses charge transfer resistance to CH3OH oxidation by 40-fold compared to the control. This improved performance is due to the ability of rGO coatings to decrease the wetting contact angle (improve the hydrophilicity) of NF from 1280 to 00. A preliminary cost analysis was carried out to assess the viability of rGO/NF electrodes via vitamin-C-enabled graphene oxide chemistry for CH3OH-MFCs applications.

Valorization of methane from environmental engineering applications: A critical review.

Wastewater and waste management sectors alone account for 18% of the anthropogenic methane (CH4) emissions. This study presents a critical overview of methanotrophs ("methane oxidizing microorganisms") for valorizing typically discarded CH4 from environmental engineering applications, focusing on wastewater treatment plants. Methanotrophs can convert CH4 into valuable bioproducts including chemicals, biodiesel, DC electricity, polymers, and S-layers, all under ambient conditions. As discarded CH4 and its oxidation products can also be used as a carbon source in nitrification and annamox processes. Here we discuss modes of CH4 assimilation by methanotrophs in both natural and engineered systems. We also highlight the technical challenges and technological breakthroughs needed to enable targeted CH4 oxidation in wastewater treatment plants.

Characterization of a novel Lytic Polysaccharide Monooxygenase from Malbranchea cinnamomea exhibiting dual catalytic behavior.

A novel Lytic Polysaccharide Monooxygenase (LPMO) family AA9 (PMO9A_MALCI) protein from thermophilic fungus Malbranchea cinnamomea was cloned and expressed in Pichia pastoris. The expressed protein was purified to homogeneity using ion exchange and hydrophobic interaction chromatography. SDS-PAGE analysis showed PMO9A_MALCI to be ~27 kDa protein. High performance anion exchange chromatography and mass spectrometry confirmed that purified protein was active against an array of cellulosic (avicel, carboxy methyl cellulose) and hemicellulosic (birch wood xylan, wheat arabinoxylan and rye arabinoxylan) substrates, releasing both oxidized and unoxidized cello-oligosaccharide and xylo-oligosaccharide products respectively. Presence of double oxidized products during mass spectrometric analysis as well as in-silico analysis confirmed that the expressed protein belongs to Type 3 LPMO family. Molecular dynamic simulations further confirmed the sharing of common amino acid residues conserved for catalysis of both cellulosic and hemicellulosic substrates which further indicates that both substrates are equally preferred. Enzymatic cocktails constituted by replacing a part of commercial cellulase CellicCTec2 with PMO9A_MALCI (9:1/8:2) led to synergistic improvement in saccharification of acid and alkali pretreated biomass. This is the first report on heterologous expression of LPMO from M. cinnamomea, exhibiting catalysis of cellulose and pure xylan.

Potential application of alkaline pectinase from Bacillus subtilis SS in pulp and paper industry.

Pectinase production from Bacillus subtilis SS was optimized under solid-state fermentation (5,943 U/g of dry bacterial bran). The pectinase produced was stable in neutral to alkaline pH range at 70 degrees C; therefore, the suitability of this pectinase in pulp and paper industry was investigated. The enzyme pretreatment process was optimized, and a pectinase dose of 5 IU/g of oven-dried pulp (10% consistency) at pH 9.5 temperature 70 degrees C after 150 min of treatment gave the best pretreatment to the pulp. An increase of 4.3% in brightness along with an increase of 14.8 and 65.3% in whiteness and fluorescence, respectively, whereas a 15% decrease in the yellowness of the pretreated pulp were observed. There was a 5.85% reduction in kappa number and 6.1% reduction in permanganate number along with a reduction in the chemical oxygen demand value. Significant characteristics showed by pectinase open new possibilities of application of this cellulase-free enzyme in the pulp and paper industry by reducing the negative environmental impact of chemicals apart from improving the properties of paper.

Producing methane, methanol and electricity from organic waste of fermentation reaction using novel microbes.

Residual solid and liquid streams from the one-pot CRUDE (Conversion of Raw and Untreated Disposal into Ethanol) process were treated with two separate biochemical routes for renewable energy transformation. The solid residual stream was subjected to thermophilic anaerobic digestion (TAD), which produced 95 ±â€¯7 L methane kg-1 volatile solid with an overall energy efficiency of 12.9 ±â€¯1.7%. A methanotroph, Methyloferula sp., was deployed for oxidation of mixed TAD biogas into methanol. The residual liquid stream from CRUDE process was used in a Microbial Fuel Cell (MFC) to produce electricity. Material balance calculations confirmed the integration of biochemical routes (i.e. CRUDE, TAD, and MFC) for developing a sustainable approach of energy regeneration. The current work demonstrates the utilization of different residual streams originated after food waste processing to release minimal organic load to the environment.

Simultaneous hydrolysis and fermentation of unprocessed food waste into ethanol using thermophilic anaerobic bacteria.

The one-pot CRUDE (Conversion of Raw and Untreated Disposal into Ethanol) process was developed for simultaneous hydrolysis and fermentation of unprocessed food waste into ethanol using thermophilic (growing at 65°C) anaerobic bacteria. Unlike existing waste to energy technologies, the CRUDE process obviates the need for any pre-treatment or enzyme addition. A High-Temperature-High-Pressure (HTHP) distillation technique was also applied that facilitated efficient use of fermentation medium, inoculum recycling, and in-situ ethanol collection. For material balancing of the process, each characterized component was represented in terms of C-mol. Recovery of 94% carbon at the end confirmed the operational efficiency of CRUDE process. The overall energy retaining efficiency calculated from sugars to ethanol was 1262.7kJdryweightkg-1 of volatile solids using HTHP. These results suggest that the CRUDE process can be a starting point for the development of a commercial ethanol production process.

Metal accumulation by sunflower (Helianthus annuus L.) and the efficacy of its biomass in enzymatic saccharification.

Accumulation of metal contaminants in soil as a result of various industrial and anthropogenic activities has reduced soil fertility significantly. Phytoextraction of metal contaminants can improve soil fertility and provide inexpensive feedstock for biorefineries. We investigated the hyperaccumulation capacity of sunflower (Helianthus annuus) biomass by cultivating these plants in various concentrations of metal contaminants. Sunflowers were grown in soils contaminated with various levels of heavy metals (10-2,000 mg/kg dry soil). The degree of metal uptake by different parts of the biomass and the residual concentration in the soil were estimated through inductively coupled plasma mass spectrometry. An almost 2.5-fold hyperaccumulation of Zn2+ was observed in the leaf and flower biomass compared with the concentration in the soil. For the subsequent saccharification of biomass with hyperaccumulated contaminants, a fungal lignocellulosic consortium was used. The fungal consortium cocktail retained more than 95% filter paper activity with 100 mM Ni2+ ions even after 36 h. The highest saccharification yield (SY, 87.4%) was observed with Ni2+ as the contaminant (10 mg/kg dry wt), whereas Pb2+ (251.9 mg/kg dry wt) was the strongest inhibitor of biomass hydrolysis, resulting in only a 30% SY. Importantly, the enzyme cocktail produced by the fungal consortium resulted in almost the same SY (%) as that obtained from a combination of commercial cellulase and ß-glucosidase. Significant sugar conversion (61.7%) from H. annuus biomass hydrolysate occurred, resulting in the production of 11.4 g/L of bioethanol. This is the first study to assess the suitability of phytoremediated sunflower biomass for bioethanol production.

Correction: Metal accumulation by sunflower (Helianthus annuus L.) and the efficacy of its biomass in enzymatic saccharification.

[This corrects the article DOI: 10.1371/journal.pone.0175845.].

Simultaneous pretreatment and saccharification: green technology for enhanced sugar yields from biomass using a fungal consortium.

Two different biomasses were subjected to simultaneous pretreatment and saccharification (SPS) using a cocktail of hydrolytic and oxidizing enzymes. Application of a novel laccase as a detoxifying agent caused the removal of 49.8% and 32.6% of phenolic contents from the soaked rice straw and willow, respectively. Hydrolysis of soaked substrates using a newly developed fungal consortium resulted in saccharification yield of up to 74.2% and 63.6% for rice straw and willow, respectively. A high saccharification yield was obtained with soaked rice straw and willow without using any hazardous chemicals. The efficiency of each step related to SPS was confirmed by atomic force microscopy. The suitability of the developed SPS process was further confirmed by converting the hydrolysate from the process into bioethanol with 72.4% sugar conversion efficiency. To the best of our knowledge, this is the first report on the development of a less tedious, single-pot, and eco-friendly SPS methodology.