Genomic insights into clostridia in bioenergy production: Comparison of metabolic capabilities and evolutionary relationships.

Bacteria from diverse genera, including Acetivibrio, Bacillus, Cellulosilyticum, Clostridium, Desulfotomaculum, Lachnoclostridium, Moorella, Ruminiclostridium, and Thermoanaerobacterium, have attracted significant attention due to their versatile metabolic capabilities encompassing acetogenic, cellulolytic, and C1-metabolic properties, and acetone-butanol-ethanol fermentation. Despite their biotechnological significance, a comprehensive understanding of clostridial physiology and evolution has remained elusive. This study reports an extensive comparative genomic analysis of 48 fully sequenced bacterial genomes from these genera. Our investigation, encompassing pan-genomic analysis, central carbon metabolism comparison, exploration of general genome features, and in-depth scrutiny of Cluster of Orthologous Groups genes, has established a holistic whole-genome-based phylogenetic framework. We have classified these strains into acetogenic, butanol-producing, cellulolytic, CO2-fixating, chemo(litho/organo)trophic, and heterotrophic categories, often exhibiting overlaps. Key outcomes include the identification of misclassified species and the revelation of insights into metabolic features, energy conservation, substrate utilization, stress responses, and regulatory mechanisms. These findings can provide guidance for the development of efficient microbial systems for sustainable bioenergy production. Furthermore, by addressing fundamental questions regarding genetic relationships, conserved genomic features, pivotal enzymes, and essential genes, this study has also contributed to our comprehension of clostridial biology, evolution, and their shared metabolic potential.

Kaolin-embedded cellulose hydrogel with tunable properties as a green fire retardant.

This study reports the synthesis of methylene bisacrylamide (MBA) crosslinked cellulose-kaolin (CMK) composite hydrogels. The internal structures of hydrogels were deduced using network parameters, viz. elastic modulus, average molecular weight, mesh size, and effective crosslink. Meanwhile, combustion behavior was investigated using the cone calorimeter test (CCT), limiting oxygen index (LOI) test, vertical flammability test (VFT), and open fire test (OFT). Our results revealed that kaolin addition improves the fire retardancy of hydrogels but reduces their swelling ability. Hydrogel having cellulose to MBA ratio of 1:2 and 2 % w/v kaolin (CM2K2) produced 63 % wt. char residue and the hydrogel-coated cotton fabric exhibited the lowest heat release rate (HRR) of 26.60 kJ/m2 and total heat release (THR) of 0.9 MJ/m2. The LOI of the cotton fabric surged from 20 % to 34.37 % after hydrogel coating. Kinetic analysis using the isoconversional model yielded the highest activation energy (216 kJ/mol) for the CM2K2 hydrogel, corroborating the increased LOI after kaolin addition. VFT and OFT validated the delay in the burning process and the formation of a char layer, which protected the underlying layer of cotton from burning. Overall, cellulose-kaolin hydrogels developed in this study are effective green fire retardant coatings for flammable materials.

Synthesis and characterization of poly(lactic acid)/clove essential oil/alkali-treated halloysite nanotubes composite films for food packaging applications.

In this study, nanocomposites of polylactic acid (PLA) with clove essential oil (CEO) and alkali treated halloysite nanotubes (NHNT) as fillers were synthesized by using simple solvent casting method. The treatment of halloysite nanotubes with NaOH increased the surface area from 50.16 m2⋅g-1 to 57.01 m2⋅g-1 and pore volume from 0.25 cm3⋅g-1 to 0.32 cm3⋅g-1. The as-synthesized nanocomposite films were characterized for physical, thermal, mechanical and water vapor barrier properties towards their use as food packaging material. The nanocomposite film PCOH0.5 (consisting 0.5 wt% NHNT and 200 µL CEO) possessed the best physical properties with percentage enhancements over PLA as: surface hydrophobicity (20.2 %), water vapor barrier (42.1 %), thermal stability (3.2 %), flexibility (682 %), tensile strength (20 %), elastic modulus (38 %), UV barrier property (62 %). In addition, a practical packaging test was performed on cut apples stored at room temperature for 6 days. The PCOH0.5 films showed substantially improved results (as compared to PLA) as follows: weight loss (40.5 %), mesophilic count (4.0 %), firmness (116.6 %), titratable acidity (110.8 %), pH (2.9 %) and total soluble solids (8.9 %). The results clearly indicate the efficiency of PLA/CEO/NHNT nanocomposite films as potential active food packaging material.

Ultrasound-assisted facile one-pot synthesis of ternary MWCNT/MnO2/rGO nanocomposite for high performance supercapacitors with commercial-level mass loadings.

Commercial application of supercapacitors (SCs) requires high mass loading electrodes simultaneously with high energy density and long cycle life. Herein, we have reported a ternary multi-walled carbon nanotube (MWCNT)/MnO2/reduced graphene oxide (rGO) nanocomposite for SCs with commercial-level mass loadings. The ternary nanocomposite was synthesized using a facile ultrasound-assisted one-pot method. The symmetric SC fabricated with ternary MWCNT/MnO2/rGO nanocomposite demonstrated marked enhancement in capacitive performance as compared to those with binary nanocomposites (MnO2/rGO and MnO2/MWCNT). The synergistic effect from simultaneous growth of MnO2 on the graphene and MWCNTs under ultrasonic irradiation resulted in the formation of a porous ternary structure with efficient ion diffusion channels and high electrochemically active surface area. The symmetric SC with commercial-level mass loading electrodes (∼12 mg cm-2) offered a high specific capacitance (314.6 F g-1) and energy density (21.1 W h kg-1 at 150 W kg-1) at a wide operating voltage of 1.5 V. Moreover, the SC exhibits no loss of capacitance after 5000 charge-discharge cycles showcasing excellent cycle life.

p-nitrophenol degradation by hybrid advanced oxidation process of heterogeneous Fenton assisted hydrodynamic cavitation: Discernment of synergistic interactions and chemical mechanism.

The present study has investigated p-nitrophenol (PNP) degradation by hybrid advanced oxidation process (AOP) of hydrodynamic cavitation with heterogenous Fe3O4 nanoparticles. 78.8 ± 1.2% of PNP degradation was obtained at optimum operational conditions: inlet pressure = 8 atm, pH = 3, initial concentration of PNP = 20 mg L-1, Fe3O4:H2O2 = 1:100. PNP degradation profiles were analyzed using a kinetic model based on the reaction network. The closest match between the simulated and experimental degradation profiles was obtained for the initial concertation of [H2O2] = 0.6 M, which was far higher than concentration of externally added H2O2. This was attributed to in-situ generation of H2O2 through transient cavitation. Intense shear and turbulence generated in cavitating flow caused surface leaching of Fe3O4 particles that released Fe2+/Fe3+ ions. The synergy in the hybrid AOP was in-situ Fenton reactions between leached Fe2+/Fe3+ ions and H2O2 present in the reaction mixture. The mechanism in •OH mediated oxidative degradation of PNP was further explored with Density Functional Theory (DFT) simulations. Both •OH addition on benzene ring and H-abstraction reactions were simulated to identify the possible pathways for the degradation. On the basis of activation free energy analysis, degradation pathways initiating with both •OH addition and H abstraction were determined to be feasible. The ortho-C of benzene ring was the most favourable site for •OH addition, while H atom of phenolic hydroxyl group was more susceptible (or more reactive) for H-atom abstraction route.

Investigations in physical mechanism of ultrasound-assisted antisolvent batch crystallization of lactose monohydrate from aqueous solutions.

Sonication is known to enhance crystallization of lactose from aqueous solutions. This study has attempted to reveal the mechanistic features of antisolvent crystallization of lactose monohydrate from aqueous solutions. Experiments were conducted in three protocols, viz. mechanical stirring, mechanical stirring with sonication and sonication at elevated static pressure. Mechanical stirring provided macroconvection while sonication induced microconvection in the system. Other experimental parameters were initial lactose concentration and rate of antisolvent (ethanol) addition. Kinetic parameters of crystallization were coupled with simulations of bubble dynamics. The growth rate of crystals, rate of nucleation, average size of crystal crop and total lactose yield in different protocols were related to nature of convection in the medium. Macroconvection assisted nucleation but could not give high growth rate. Microconvection comprised of microstreaming due to ultrasound and acoustic (or shock) waves due to transient cavitation. Sonication at atmospheric static pressure enhanced growth rate but reduced nucleation. However, with elimination of cavitation at elevated static pressure, sonication enhanced both nucleation and growth rate resulting in almost complete lactose recovery.

Mechanistic investigations in ultrasound-assisted biodegradation of phenanthrene.

This study has addressed the biodegradation of polycyclic aromatic hydrocarbon, phenanthrene using Candida tropicalis. Optimization using central composite statistical design yielded optimum experimental parameters as: pH = 6.2, temperature = 33.4 °C, mechanical shaking = 190 rpm and % inoculum = 9.26% v/v. Sonication of biodegradation mixture at 33 kHz and 10% duty cycle in log phase (12 h per day for 4 days) resulted in a 25% enhancement in phenanthrene removal. Profiles of specific growth rate (µ) and specific degradation rate (q) versus initial substrate concentration were fitted to Haldane substrate inhibition model. Both µ and q showed maxima for initial concentration of 100 mg L-1. Kinetic analysis of degradation profiles showed higher biomass yield coefficient and smaller decay coefficient in presence of sonication. Expression of total intracellular proteins in control and test experiments were analyzed using SDS-PAGE. This analysis revealed overexpression of enzyme catechol 2,3-dioxygenase (in meta route metabolism) during sonication which is involved in ring cleavage of phenanthrene. Evaluation of cell viability after sonication by flow cytometry analysis revealed > 80% live cells. These effects are attributed to enhanced cellular transport induced by intense microturbulence generated by sonication.

Investigations in ultrasonic enhancement of ß-carotene production by isolated microalgal strain Tetradesmus obliquus SGM19.

Microalgae constitute relatively novel source of lipids for biodiesel production. The economy of this process can be enhanced by the recovery of ß-carotenes present in the microalgal cells. The present study has addressed matter of enhancement of lipids and ß-carotene production by microalgal species of Tetradesmus obliquus SGM19 with the application of sonication. As first step, the growth cycle of Tetradesmus obliquus SGM19 was optimized using statistical experimental design. Optimum parameters influencing microalgal growth were: Sodium nitrate = 1.5 g/L, ethylene diamine tetraacetic acid = 0.001 g/L, temperature = 28.5 °C, pH = 7.5, light intensity = 5120 lux, ß-carotene yield = 0.67 mg/g DCW. Application of 33 kHz and 1.4 bar ultrasound at 10% duty cycle was revealed to enhance the lipid and ß-carotene yields by 34.5% and 31.5%, respectively. Kinetic analysis of substrate and product profiles in control and test experiments revealed both lipid and ß-carotene to be growth-associated products. The intracellular NAD(H) content during late log phase was monitored in control and test experiments as a measure of relative kinetics of intracellular metabolism. Consistently higher NAD(H) concentrations were observed for test experiments; indicating faster metabolism. Finally, the viability of ultrasound-exposed microalgal cells (assessed with flow cytometry) was >80%.

Enzymatic hydrolysis of hemicellulose from pretreated Finger millet (Eleusine coracana) straw by recombinant endo-1,4-ß-xylanase and exo-1,4-ß-xylosidase.

This study focuses on enzymatic saccharification of hemicellulose part of the pretreated Finger millet straw (FMS) for production of xylose. The variation in the carbohydrate composition of FMS was analysed when subjected to different pretreatments. The recombinant endo-1,4-ß-xylanase (CtXyn11A) was most active on the FMS pretreated with 1% (w/v) NaOH combined with oven heating at 120 °C for 20 min, resulting in a total reducing sugar yield (TRS) of 32 mg/g pretreated biomass. The pretreatment aided in concentrating the holocellulose content from 69.3% of raw powdered FMS to 76.4%. The post-treatment solid biomass yield was 0.36 g/g raw biomass. The two-step optimization of hemicellulose saccharification from the above pretreated FMS with i) endo-1,4-ß-xylanase (CtXyn11A) at 55 °C and ii) exo-1,4-ß-xylosidase (BoGH43A) at 37 °C, both at pH 7.5 by Box-Behnken design yielded the TRS of 70 mg/g pretreated biomass. The percentage conversion of xylan to xylose by CtXyn11A and BoGH43A was 24.7%.

Homologous overexpression of hydrogenase and glycerol dehydrogenase in Clostridium pasteurianum to enhance hydrogen production from crude glycerol.

This study reports engineering of a hypertransformable variant of C. pasteurianum for bioconversion of glycerol into hydrogen (H2). A functional glycerol-triggered hydrogen pathway was engineered based on two approaches: (1) increasing product yield by overexpression of immediate enzyme catalyzing H2 production, (2) increasing substrate uptake by overexpression of enzymes involved in glycerol utilization. The first strategy aimed at overexpression of hydA gene encoding hydrogenase, and the second one, through combination of overexpression of dhaD1 and dhaK genes encoding glycerol dehydrogenase and dihydroxyacetone kinase. These genetic manipulations resulted in two recombinant strains (hydA++/dhaD1K++) capable of producing 97% H2 (v/v), with yields of 1.1 mol H2/mol glycerol in hydA overexpressed strain, and 0.93 mol H2/mol glycerol in dhaD1K overexpressed strain, which was 1.5 fold higher than wild type. Among two strains, dhaD1K++ consumed more glycerol than hydA++ which proves that overexpression of glycerol enzymes has enhanced glycerol intake rate.

Physical insights of ultrasound-assisted ethanol production from composite feedstock of invasive weeds.

Invasive weeds ubiquitously found in terrestrial and aquatic ecosystems form potential feedstock for lignocellulosic ethanol production. The present study has reported a bioprocess for production of ethanol using mixed feedstock of 8 invasive weeds found in India. The feedstock was subjected to pretreatment comprising dilute acid hydrolysis (for hydrolysis of hemicellulosic fraction), alkaline delignification and enzymatic hydrolysis of cellulosic fraction. Pentose-rich and hexose-rich hydrolyzates obtained from pretreatment were fermented separately using microbial cultures of S. cerevisiae and C. shehatae. Fermentation mixture was sonicated at 35 kHz at 10% duty cycle. The time profiles of total reducing sugars, ethanol and biomass was fitted to a kinetic model using Genetic Algorithm. Sonication boosted the kinetics of fermentation 2-fold. The net bioethanol yield of the process was ∼220 g/kg raw biomass (with contributions of 86.8 and 133 g/kg raw biomass from pentose and hexose fermentations, respectively). Comparative evaluation of parameters of kinetic model under control and test conditions revealed several beneficial influences of sonication on both pentose and hexose fermentation systems such as faster transport of nutrients, substrate and products across cell membrane, rise in Monod saturation constant for substrate with concurrent reduction in substrate inhibition, and reduction of energy requirements for cell maintenance. Flow cytometry analysis of native and ultrasound-treated cells revealed no adverse influence of sonication on cell viability.

Mechanistic investigations in biobutanol synthesis via ultrasound-assisted ABE fermentation using mixed feedstock of invasive weeds.

This study reports an ultrasound-assisted Acetone-Butanol-Ethanol (ABE) fermentation process using Clostridium acetobutylicum MTCC 11,274 and mixed feedstock consisting of eight highly invasive weeds. Composite (pentose + hexose) hydrolyzate was fermented with sonication at 35 kHz and 10% duty cycle (test) and mechanical agitation at 150 rpm (control). Net solvent yield with sonication was 0.288 g/g raw biomass in 92 h against yield of 0.168 g/g raw biomass in 120 h with mechanical agitation. Butanol yield in test and control fermentation was 0.233 and 0.149 g/g total fermentable sugar, respectively. Substrate and metabolites profiles in test and control fermentation were analyzed using biokinetic model. Sonication enhanced kinetics of metabolic reactions with rise in substrate affinity of enzymes (reduced saturation constants) and greater resistance to substrate inhibition. Flow cytometry analysis of cells exposed to sonication revealed high cell viability with no adverse effect on physiology.

Ultrasound-assisted biodiesel production using heterogeneous base catalyst and mixed non-edible oils.

In the present study, the ultrasound-assisted biodiesel production from mixed feedstock of non-edible oils in presence of KI impregnated ZnO as a catalyst in batch reactor was investigated. The production was optimized by using two approaches (1) feedstock optimization and (2) process parameters optimization. Various non-edible oils at optimum volumetric ratio were blended and used as feedstock for transesterification reaction. Biodiesel yield was optimized by Box-Behnken statistical design. The maximum triglyceride conversion of 92.35 ±â€¯1.08% was achieved at optimized conditions of catalyst loading = 7% (w/w); alcohol/oil molar ratio = 11.68:1 and reaction temperature = 59 °C. Transesterification process with mechanical agitation was used as base case for identification of role of sonication in the process. The transesterification process was analysed for kinetic behaviour using pseudo first order kinetics and Eley-Rideal mechanism based model. Overall activation energy of transesterification process for mechanically agitated and ultrasound-assisted systems was calculated as 135.4 and 123.65 kJ/mol, respectively. However, the sum of activation energies of three reaction steps of Eley-Rideal mechanism (64.69 kJ/mol and 46.63 kJ/mol, for mechanically agitated and ultrasound-assisted system, respectively) was much lower. This discrepancy is attributed to mass transfer limitations in the system, even in presence of sonication.

Ultrasonic enhancement of xylitol production from sugarcane bagasse using immobilized Candida tropicalis MTCC 184.

This study investigates ultrasonic enhancement of xylitol production from sugarcane bagasse using C. tropicalis MTCC 184 immobilized on PU foam. Initial xylitol yield of 0.53 g/g xylose improved to 0.65 g/g of xylose (in 36 h fermentation) after optimization of medium and fermentation parameters. Optimum values of experimental parameters for maximum xylitol were: yeast extract = 5.78 g/L, (NH4)2SO4 = 3.22 g/L, KH2PO4 = 0.58 g/L, MgSO4·7H2O = 0.57 g/L and temperature = 29.3 °C, initial pH = 6.2, agitation rate = 151 rpm and initial xylose concentration = 20.9 g/L. Application of 37 kHz sonication @10% duty cycle during fermentation at optimum conditions resulted in marked intensification of fermentation kinetics. Xylitol yield of 0.66 g/g of xylose has been obtained in ultrasound-assisted fermentation in just 15 h. Fitting of time profiles of substrates and products to kinetic model has highlighted actual physical mechanisms underlying 2-fold faster kinetics induced by sonication.

Mechanistic investigations in ultrasound-assisted xylitol fermentation.

This study has investigated ultrasound-assisted xylitol production through fermentation of dilute acid (pentose-rich) hydrolysate of sugarcane bagasse using free cells of Candida tropicalis. Sonication of fermentation mixture at optimum conditions was carried out in ultrasound bath (37 kHz and 10% duty cycle). Time profiles of substrate and product in control (mechanical shaking) and test (mechanical shaking + sonication) fermentations were fitted to kinetic model using Genetic Algorithm (GA) optimization. Max. xylitol yield of 0.56 g/g and 0.61 g/g of xylose was achieved in control and test fermentations, respectively. The biomass yield also increased marginally (∼17%) with sonication. However, kinetics of fermentation increased drastically (2.5×) with sonication with 2× rise in xylose uptake and utilization by the cells. With comparative analysis of kinetic parameters in control and test experiments, this result was attributed to enhanced permeability of cell membrane that allowed faster diffusion of nutrients, substrates and products across cell membrane, higher enzyme-substrate affinity, dilution of toxic components and reduced inhibition of intracellular enzymes by substrate.

Comparative analysis of pretreatment methods on sorghum (Sorghum durra) stalk agrowaste for holocellulose content.

This study compares different types of pretreatment methods, such as thermal pretreatment at 120 °C, autoclaving, microwaving and ultrasonication in the presence of water, dilute acid (1% H2SO4) or dilute alkali (1% NaOH) on Sorghum stalk with respect to the holocellulose and Acid Detergent/Insoluble Lignin content. Among all the methods, pretreatment with 1% NaOH along with autoclaving at 121 °C and 15 psi for 30 min was the most effective method for Sorghum stalk. Fourier Transform Infra-Red spectroscopy analysis of this pretreated biomass showed the removal of lignin and Field Emission Scanning Electron Microscope analysis displayed enhanced surface roughness. The enzymatic hydrolysis of raw and best pretreated Sorghum stalk using recombinant endo-ß-1,4-glucanase (CtCel8A) and ß-1,4-glucosidase (CtBgl1A) both from Clostridium thermocellum gave glucose yields, 22.4 mg/g raw biomass and 34 mg/g pretreated biomass, respectively, resulting in 1.5-fold increase of glucose yield after the pretreatment.

Discernment of synergism in pyrolysis of biomass blends using thermogravimetric analysis.

This study reports pyrolysis kinetics of biomass blends using isoconversional methods, viz. Friedman, FWO and KAS. Blends of three biomasses, viz. saw dust, bamboo dust and rice husk, were used. Extractives and volatiles in biomass and minerals in ash had marked influence on enhancement of reaction kinetics during co-pyrolysis, as indicated by reduction in activation energy and increase in decomposition intensity. Pyrolysis kinetics of saw dust and rice husk accelerated (positive synergy), while that of bamboo dust decelerated after blending (negative synergy). Predominant reaction mechanism of all biomass blends was 3-D diffusion in lower conversion range (α ≤ 0.5), while for α ≥ 0.5 pyrolysis followed random nucleation (or nucleation and growth mechanism). Higher reaction order for pyrolysis of blends of rice husk with saw dust and bamboo dust was attributed to catalytic effect of minerals in ash. Positive ΔH and ΔG was obtained for pyrolysis of all biomass blends.

Ultrasound-assisted synthesis and characterization of magnetite nanoparticles and poly(methyl methacrylate)/magnetite nanocomposites.

Poly(methyl methacrylate)/magnetite (PMMA/Fe3O4) nanocomposites were prepared with a two-step technique involving sonication. Fe3O4 nanoparticles were synthesized by ultrasound-assisted co-precipitation, and then PMMA/Fe3O4 (1-5 wt%) nanocomposites were synthesized via ultrasound-assisted in-situ emulsion polymerization. Best physical properties of the nanocomposites for different Fe3O4 loadings were: tensile strength (2 wt%) = 40.28 MPa, Young's modulus (2 wt%) = 2.4 GPa, percentage elongation (2 wt%) = 2.24%, glass transition temperature (2 wt%) = 122.5 °C, thermal inflection point (2 wt%) = 383 °C, electrical conductivity (5 wt%) = 2.0 × 10-13 S/cm, coercivity = 59.85 Oe (2 wt%), magnetic saturation (5 wt%) = 5.12 emu/g, magnetic remanence (5 wt%) = 0.56 emu/g, and electromagnetic interference shielding effectiveness (5 wt%) = 1.45 dB. This unique combination of physical properties at relatively low Fe3O4 loading is attributed to ultrasound-mediated uniform dispersion of the nanofiller in the polymer matrix.

Investigations in sonication-induced intensification of crude glycerol fermentation to dihydroxyacetone by free and immobilized Gluconobacter oxydans.

This study reports crude glycerol fermentation by G. oxydans for dihydroxyacetone (DHA) production, and intensification of fermentation with sonication. Fermentation was carried out using both free and immobilized cells (on polyurethane foam support) for initial glycerol concentrations of 20, 30 and 50 g/L. Sonication at 20% duty cycle enhanced glycerol consumption by 60-84% with no significant change in cell morphology. Lesser DHA yield in crude glycerol fermentation was attributed to possible formation of inhibitory products. Slight reduction in DHA yield for initial glycerol concentration of 50 g/L was attributed to substrate inhibition. Higher DHA productivity was obtained for immobilized cells. Circular dichroism analysis of intracellular proteins obtained from ultrasound-treated G. oxydans revealed significant reduction in α-helix and ß-sheet content. These conformational changes in protein structure could augment activity of intracellular glycerol dehydrogenase, which is manifested in terms of enhanced metabolism of glycerol by G. oxydans.

Investigations in ultrasound-induced enhancement of astaxanthin production by wild strain Phaffia rhodozyma MTCC 7536.

This work reports ultrasound-induced enhancement of astaxanthin production in batch fermentation using wild strain of P. rhodozyma MTCC 7536. The methodology adopted in this study comprises of statistical optimization of the medium and fermentation parameters, followed by application of sonication at optimized conditions. P. rhodozyma fermentation at conditions of 20 g/L glucose, pH 4.4, temperature 21 °C, 4% v/v inoculum, shaking at 205 rpm with nitrogen sources of (NH4)2SO4 and yeast extract yielded 6.8 mg/L or 1360 µg/g DCW astaxanthin in 84 h. Application of 33 kHz and 140 kPa sonication at 10% duty cycle in final 12 h of fermentation enhanced the astaxanthin yield to 8.6 mg/L or 1728 µg/g DCW, which is higher than several mutant strains reported in literature. These results are essentially manifestations of intense microturbulence generated by sonication in fermentation mixture.