Swati Khanna

Microbial conversion of glycerol: present status and future prospects.

Biodiesel has emerged as a potential alternate renewable liquid fuel in the past two decades. Total annual production of biodiesel stands at 6.96 million tons and 11.2 million tons in USA and Europe, respectively. In other countries, Asia and Latin America, biodiesel production has increased at unprecedented rate. Despite this, the economy of biodiesel is not attractive. An obvious solution for boosting the economy of the biodiesel industry is to look for markets for side products of the transesterification process of biodiesel synthesis. The main by-product is glycerol. However, this glycerol is contaminated with alkali/acid catalyst and alcohol, and thus, is not useful for conventional applications such as in toothpaste, drugs, paints and cosmetics. Conversion of this glycerol to value-added product is a viable solution for effective and economic utilization, which would also generate additional revenue for the biodiesel industry. Intensive research has taken place in area of conversion of glycerol to numerous products. The conventional catalytic route of glycerol transformation employs prohibitively harsh conditions of temperature and pressure, and thus, has slim potential for large-scale implementation. In addition, the selectivity of the process is rather small with formation of many undesired side products. The bioconversion processes, on the other hand, are highly selective although with slower kinetics. In this review, we have given an assessment and overview of the literature on bioconversion of glycerol. We have assessed as many as 23 products from glycerol bioconversion, and have reviewed the literature in terms of microorganism used, mode of fermentation, type of fermentor, yield and productivity of the process and recovery/purification of the products. The metabolic pathway of conversion of glycerol to various products has been discussed. We have also pondered over economic and engineering issues of large-scale implementation of process and have outlined the constraints and limitations of the process. We hope that this review will be a useful source of information for biochemists, biotechnologists, microbiologists and chemical engineers working in the area of glycerol bioconversion.

Physical features of ultrasound assisted enzymatic degradation of recalcitrant organic pollutants.

This work has attempted to provide answer to the interaction of sonolysis and enzymatic treatment on degradation of recalcitrant dyes in a combined treatment. The model system comprises of two dyes, acid red and malachite green as model pollutants, along with horseradish peroxidase as a model enzyme and ultrasound of 20 kHz frequency. A dual approach of coupling experimental results with simulations of cavitation bubble dynamics has been adopted. Utilization of oxidation potential of horseradish peroxidase has been found to be a function of convection level in the medium. Cavitation phenomenon is found to have an adverse effect on enzyme action due to generation of high amplitude shock waves, which denature the enzyme. Degradation of dye at high static pressure increases due to absence of cavitation and high energy interaction (or collisions) between enzyme and dye molecules, which are beneficial towards enzymatic oxidation of the latter. High intensity convection generated by ultrasound also obviates need for an external shielding agent such as PEG that prevents attachment of the phenoxy radicals to enzyme that blocks the active sites of the enzyme.

Mechanistic investigation of ultrasonic enhancement of glycerol bioconversion by immobilized Clostridium pasteurianum on silica support

Glycerol, the principal byproduct of biodiesel production, can be a valuable carbon source for bioconversion into diverse class of compounds. This article attempts to investigate the mechanistic aspects of ultrasound mediated bioconversion of glycerol to ethanol and 1,3‐propanediol (1,3‐PDO) by immobilized Clostridium pasteurianum cells on silica support. Our approach is of coupling experimental results with simulations of cavitation bubble dynamics and enzyme kinetics. In addition, the statistical analysis (ANOVA) of experimental results was also done. The glycerol uptake by cells was not affected by either immobilization or with ultrasonication. Nonetheless, both immobilization and ultrasonication were found to enhance glycerol consumption. The enhancement effect of ultrasound on glycerol consumption was most marked (175%) at the highest glycerol concentration of 25 g/L (271.7 mM). The highest glycerol consumption (32.4 mM) was seen for 10 g/L (108.7 mM) initial glycerol concentration. The immobilization of cells shifted the metabolic pathway almost completely towards 1,3‐PDO. No formation of ethanol was seen with mechanical shaking, while traces of ethanol were detected with ultrasonication. On the basis of analysis of enzyme kinetics parameters, we attribute these results to increased substrate‐enzyme affinity and decreased substrate inhibition for 1,3‐PDO dehydrogenase in presence of ultrasound that resulted in preferential conversion of glycerol into 1,3‐PDO. Biotechnol. Bioeng. 2013; 110: 1637–1645.

Sonoenzymatic decolourization of an azo dye employing immobilized horse radish peroxidase (HRP): a mechanistic study.

For degradation of biorefractory pollutants, enzymatic treatments and sonochemical treatments have shown high potential. A combined technique of sono-enzymatic treatment is of special interest as it has shown enhancement effect than the individual techniques. This work has attempted to give a mechanistic insight into the interaction of sonochemical and enzymatic treatments using immobilized horseradish peroxidase (HRP) enzyme on the decolourization of acid red dye (an azo dye). In order to segregate the effect of ultrasound and cavitation, experiments were conducted at elevated static pressure. The kinetic parameters of HRP, viz. Vmax and Km were marginally affected by immobilization. There was a minor change in pH optima and temperature optima for immobilized HRP (6.5, 25°C) from free HRP (7.0, 20-25°C). Though the specific activity of free enzyme (0.272U/mg) was found to be higher than the immobilized enzyme (0.104U/mg), immobilized enzyme exhibited higher stability (up to 3 cycles) and degradation potential than free enzyme in all experiments. The results revealed that the coupling of sonication and enzymatic treatment at high pressure in presence of polyethylene glycol (PEG) yielded the highest decolourization of acid red (61.2%). However, the total decolourization achieved with combined technique was lesser than the sum of individual techniques, indicating negative synergy between the sonochemical and enzymatic techniques.

Mechanistic insight into sono-enzymatic degradation of organic pollutants with kinetic and thermodynamic analysis

In this paper, we have attempted to get a physical insight into process of sono-enzymatic treatment for degradation of recalcitrant organic pollutants. Decolourization of an azo dye has been used as model reaction with different experimental protocols that alter characteristics of ultrasound and cavitation phenomena in the system. Experimental data is analyzed to determine kinetic and thermodynamic parameters of decolorization process. The trends observed in kinetic and thermodynamic parameters of decolourization are essentially manifestations of the dominating mechanism of the decolorization of the textile dye (or nature of prevalent chemical reaction in the system), viz. either molecular reaction due to enzyme or radical reaction due to transient cavitation. The activation energy for sonochemical protocol is negative, which indicates instantaneity of the radical reactions. The frequency factor is also low, which is attributed to high instability of radicals. For enzymatic and sono-enzymatic protocols, activation energy is positive with higher frequency factor. Enthalpy change for sonochemical protocol is negative, while that for enzymatic and sono-enzymatic protocols is positive. The net entropy change for sonochemical protocol is more negative than enzymatic or sono-enzymatic protocol due to differences in prevalent chemical mechanism of dye decolorization. Due to inverse variations of frequency factor and activation energy, marginal rise in reaction kinetics is seen for sono-enzymatic protocol, as compared to enzymatic treatment alone. Due to inverse variations of enthalpy and entropy change, net Gibbs energy change in all experimental protocols shows little variation indicating synergism of the mechanism of ultrasound and enzyme.

Effect of fermentation parameters on bio-alcohols production from glycerol using immobilized Clostridium pasteurianum: an optimization study.

This article addresses the issue of effect of fermentation parameters for conversion of glycerol (in both pure and crude form) into three value-added products, namely, ethanol, butanol, and 1,3-propanediol (1,3-PDO), by immobilized Clostridium pasteurianum and thereby addresses the statistical optimization of this process. The analysis of effect of different process parameters such as agitation rate, fermentation temperature, medium pH, and initial glycerol concentration indicated that medium pH was the most critical factor for total alcohols production in case of pure glycerol as fermentation substrate. On the other hand, initial glycerol concentration was the most significant factor for fermentation with crude glycerol. An interesting observation was that the optimized set of fermentation parameters was found to be independent of the type of glycerol (either pure or crude) used. At optimum conditions of agitation rate (200 rpm), initial glycerol concentration (25 g/L), fermentation temperature (30°C), and medium pH (7.0), the total alcohols production was almost equal in anaerobic shake flasks and 2-L bioreactor. This essentially means that at optimum process parameters, the scale of operation does not affect the output of the process. The immobilized cells could be reused for multiple cycles for both pure and crude glycerol fermentation.

Physical and Chemical Mechanisms of Ultrasound in Biofuel Synthesis

Physical and chemical mechanisms ultrasound-assisted processes as related to the synthesis of biofuels are reviewed. Ultrasound and its secondary effect of cavitation have physical and chemical effects on a reaction system, which can contribute to enhancement of the kinetics and yield. In this chapter, a mechanistic insight into the ultrasound assisted biofuels synthesis is given by coupling simulations of cavitation bubble dynamics with experimental data. The physical effect of ultrasound and cavitation is through intense micro-convection in the system that gives marked improvements in the mass transfer of the system. The chemical effect is through generation of highly reactive radicals through transient cavitation that induce or accelerate chemical reactions. Chemical effects include thermal decomposition of the solvent vapor molecules in the cavitation bubble resulting in generation of smaller molecular species that also affect chemistry of the process. Raising the static pressure of the reaction system above ultrasound pressure amplitude in the system helps to discriminate between physical and chemical effects of ultrasound and cavitation. Biofuels systems considered in this chapter are the pretreatment of biomass, biodiesel synthesis with acid/base and homogeneous/heterogeneous catalysts, extraction of microalgal lipids, bioconversion of crude glycerol from biodiesel industry to value added products and desulfurization of the fuel. Among the physical effects of ultrasound and cavitation, micro-streaming by ultrasound has a greater influence on reactions than shock waves generated by cavitation bubbles. In some cases, chemical effects of transient cavitation are revealed to have adverse influence on a reaction. Many biofuels systems are limited by their intrinsic characteristics that restrict the effect of ultrasound and cavitation on the reaction system.