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.

Microbial production, ultrasound-assisted extraction and characterization of biopolymer polyhydroxybutyrate (PHB) from terrestrial (P. hysterophorus) and aquatic (E. crassipes) invasive weeds.

This study reports synthesis of biodegradable poly(3-hydroxybutyrate) (PHB) polymer from two invasive weeds, viz. P. hysterophorus and E. crassipes. The pentose and hexose-rich hydrolyzates obtained from acid pretreatment and enzymatic hydrolysis of two biomasses were separately fermented using Ralstonia eutropha MTCC 8320 sp. PHB was extracted using sonication and was characterized using FTIR, 1H and 13C NMR and XRD. PHB content of dry cell mass was 8.1-21.6% w/w, and the PHB yield was 6.85×10-3-36.41×10-3% w/w raw biomass. Thermal properties of PHB were determined by TGA, DTG and DSC analysis. PHB obtained from pentose-hydrolyzate had glass transition temperatures of 6°-9°C, while PHB from hexose-rich hydrolyzate had maximum thermal degradation temperatures of 370°-389°C. These thermal properties were comparable to the properties of commercial PHB. Probable causes leading to differences in thermal properties of pentose and hexose-derived PHB are: extent of crystallinity and presence of impurity in the polymer matrix.

Mechanistic investigation in ultrasound induced enhancement of enzymatic hydrolysis of invasive biomass species.

This study has assessed four invasive weeds, viz. Saccharum spontaneum (SS), Mikania micrantha (MM), Lantana camara (LC) and Eichhornia crassipes (EC) for enzymatic hydrolysis prior to bioalcohol fermentation. Enzymatic hydrolysis of pretreated biomasses of weeds has been conducted with mechanical agitation and sonication under constant (non-optimum) conditions. Profiles of total reducible sugar release have been fitted to HCH-1 model of enzymatic hydrolysis using Genetic Algorithm. Trends in parameters of this model reveal physical mechanism of ultrasound-induced enhancement of enzymatic hydrolysis. Sonication accelerates hydrolysis kinetics by ∼10-fold. This effect is contributed by several causes, attributed to intense micro-convection generated during sonication: (1) increase in reaction velocity, (2) increase in enzyme-substrate affinity, (3) reduction in product inhibition, and (4) enhancement of enzyme activity due to conformational changes in its secondary structure. Enhancement effect of sonication is revealed to be independent of conditions of enzymatic hydrolysis - whether optimum or non-optimum.

Mechanistic analysis of ultrasound assisted enzymatic desulfurization of liquid fuels using horseradish peroxidase.

This study has attempted to gain physical insight into ultrasound-assisted enzymatic desulfurization using system comprising horseradish peroxidase enzyme and dibenzothiophene (DBT). Desulfurization pathway (comprising DBT-sulfoxide and DBT-sulfone as intermediates and 4-methoxy benzoic acid as final product) has been established with GC-MS analysis. Intrinsic fluorescence and circular dichroism spectra of ultrasound-treated enzyme reveal conformational changes in secondary structure (reduction in α-helix and ß-conformations and increase in random coil content) leading to enhancement in activity. Concurrent analysis of desulfurization profiles, Arrhenius and thermodynamic parameters, and simulations of cavitation bubble dynamics reveal that strong micro-convection generated by sonication enhances enzyme activity and desulfurization kinetics. Parallel oxidation of DBT by radicals generated from transient cavitation gives further boost to desulfurization kinetics. However, random motion of enzyme molecules induced by shock waves reduces frequency factor and limits the ultrasonic enhancement of enzymatic desulfurization.