Lyophilized Emulsions of Thymol and Eugenol Essential Oils Encapsulated in Cellulose.

Efforts to tap into the broad antimicrobial, insecticidal, and antioxidant activities of essential oils (EOs) are limited due to their strong odor and susceptibility to light and oxidation. Encapsulation of EOs and subsequent drying overcome these limitations and extend their applications. This study characterized freeze-dried (lyophilized) emulsions of eugenol (EU) and thymol (TY) EOs, encapsulated by chemically unmodified cellulose, a sustainable and low-cost resource. High-resolution scanning electron microscopy showed successful lyophilization. While the observed "flake-like" structure of the powders differed significantly from that of the emulsified microcapsules, useful properties were retained. Fourier transform infrared spectroscopy confirmed the presence of EOs in their corresponding powders and thermo-gravimetric analysis demonstrated high encapsulation efficiency (87-88%), improved thermal stability and resistance to evaporation, and slow EO release rates in comparison to their free forms. The lightweight and low-cost cellulose encapsulation, together with the results showing retained properties of the dried powder, enable the use of EOs in applications requiring high temperatures, such as EO incorporation into polymer films, that can be used to protect agricultural crops from microbial infections.

Cellulose-coated emulsion micro-particles self-assemble with yeasts for cellulose bio-conversion.

In the quest for alternative renewable energy sources, a new self-assembled hybrid configuration of cellulose-coated oil-in-water emulsion particles with yeast was formed. In this research, the addition of yeasts (S. cerevisiae) to the micro-particle emulsion revealed a novel self-assembly configuration in which the yeast cell is connected to surrounding cellulose-coated micro-particles. This hybrid configuration may enhance the simultaneous saccharification and fermentation process by substrate channeling. Glucose produced by hydrolysis of the cellulose shells coating the micro-particles, catalyzed by cellulytic enzymes attached to their coating, is directly fermented to ethanol by the yeasts to which the particles are connected. The results indicate ethanol yield of 62%, based on the cellulose content of the emulsion, achieved by the yeast/micro-particle hybrids. The functionality of this hybrid configuration is expected to serve as a micro-reactor for a cascade of biochemical reactions in a "one-pot" consolidated process transforming cellulose to valuable chemicals, such as biodiesel.

Lipase Catalyzed Transesterification of Model Long-Chain Molecules in Double-Shell Cellulose-Coated Oil-in-Water Emulsion Particles as Microbioreactors.

Lipase-catalyzed transesterification is prevalent in industrial production and is an effective alternative to chemical catalysis. However, due to lipases' unique structure, the reaction requires a biphasic system, which suffers from a low reaction efficiency caused by a limited interfacial area. The use of emulsion particles was found to be an effective way to increase the surface area and activity. This research focuses on cellulose as a natural surfactant for oil-in-water emulsions and evaluates the ability of lipase, introduced into the emulsion's aqueous phase, to integrate with the emulsion microparticles and catalyze the transesterification reaction of high molecular weight esters dissolved in the particles' cores. Cellulose-coated emulsion particles' morphology was investigated by light, fluorescence and cryogenic scanning electron microscopy, which reveal the complex emulsion structure. Lipase activity was evaluated by measuring the hydrolysis of emulsified p-nitrophenyl dodecanoate and by the transesterification of emulsified methyl laurate and oleyl alcohol dissolved in decane. Both experiments demonstrated that lipase introduced in the aqueous medium can penetrate the emulsion particles, localize at the inner oil core interface and perform effective catalysis. Furthermore, in this system, lipase successfully catalyzed a transesterification reaction rather than hydrolysis, despite the dominant presence of water.

Structural Insights into Cellulose-Coated Oil in Water Emulsions.

Cellulose is a renewable biopolymer, abundant on Earth, with a multi-level supramolecular structure. There has been significant interest and advancement in utilizing natural cellulose to stabilize emulsions. In our research, we develop and examine oil in water emulsions surrounded by unmodified cellulose as microreactors for the process of transformation of cellulose into valuable chemicals such as biodiesel. This study presents morphological characterization of cellulose-coated emulsions that can be used for such purposes. Cryogenic-scanning electron microscopy imaging along with light microscopy and light scattering reveals a multi-layer inner structure: an oil core surrounded by a porous cellulose hydrogel shell, coated by an outer shell of regenerated cellulose. Measurements of small-angle X-ray scattering provide quantification of the nano-scale structure within the porous cellulose hydrogel inner shell of the emulsion particle. These characteristics are relevant to utilization of cellulose-coated emulsions in various applications such as controlled release and as hosts for enzymatic biotechnological reactions.

Encapsulation of Thymol and Eugenol Essential Oils Using Unmodified Cellulose: Preparation and Characterization.

Essential oils (EOs) are volatile natural organic compounds, which possess pesticidal properties. However, they are vulnerable to heat and light, limiting their range of applications. Encapsulation of EOs is a useful approach to overcome some of these limitations. In this study, a novel emulsification technique is utilized for encapsulation of thymol (TY) and eugenol (EU) (EOs) within microcapsules with an unmodified cellulose shell. Use of low cost materials and processes can be beneficial in agricultural applications. In the encapsulation process, unmodified cellulose was dissolved in 7% aqueous NaOH at low temperature, regenerated to form a dispersion of cellulose hydrogels, which was rigorously mixed with the EOs by mechanical mixing followed by high-pressure homogenization (HPH). Cellulose:EO ratios of 1:1 and 1:8 utilizing homogenization pressures of 5000, 10,000 and 20,000 psi applied in a microfluidizer were studied. Light microscopy, high-resolution cryogenic scanning electron microscopy (cryo-SEM) and Fourier transform infrared spectroscopy (FTIR) revealed successful fabrication of EO-loaded capsules in size range of 1 to ~8 µm. Stability analyses showed highly stabilized oil in water (O/W) emulsions with instability index close to 0. The emulsions exhibited anti-mold activity in post-harvest alfalfa plants, with potency affected by the cellulose:EO ratio as well as the EO type; TY showed the highest anti-mold activity. Taken together, this study showed potential for anti-fungal activity of cellulose-encapsulated EOs in post-harvest hay.

Cellulosic biofuel production using emulsified simultaneous saccharification and fermentation (eSSF) with conventional and thermotolerant yeasts.

BACKGROUND: Future expansion of corn-derived ethanol raises concerns of sustainability and competition with the food industry. Therefore, cellulosic biofuels derived from agricultural waste and dedicated energy crops are necessary. To date, slow and incomplete saccharification as well as high enzyme costs have hindered the economic viability of cellulosic biofuels, and while approaches like simultaneous saccharification and fermentation (SSF) and the use of thermotolerant microorganisms can enhance production, further improvements are needed. Cellulosic emulsions have been shown to enhance saccharification by increasing enzyme contact with cellulose fibers. In this study, we use these emulsions to develop an emulsified SSF (eSSF) process for rapid and efficient cellulosic biofuel production and make a direct three-way comparison of ethanol production between S. cerevisiae, O. polymorpha, and K. marxianus in glucose and cellulosic media at different temperatures. RESULTS: In this work, we show that cellulosic emulsions hydrolyze rapidly at temperatures tolerable to yeast, reaching up to 40-fold higher conversion in the first hour compared to microcrystalline cellulose (MCC). To evaluate suitable conditions for the eSSF process, we explored the upper temperature limits for the thermotolerant yeasts Kluyveromyces marxianus and Ogataea polymorpha, as well as Saccharomyces cerevisiae, and observed robust fermentation at up to 46, 50, and 42 °C for each yeast, respectively. We show that the eSSF process reaches high ethanol titers in short processing times, and produces close to theoretical yields at temperatures as low as 30 °C. Finally, we demonstrate the transferability of the eSSF technology to other products by producing the advanced biofuel isobutanol in a light-controlled eSSF using optogenetic regulators, resulting in up to fourfold higher titers relative to MCC SSF. CONCLUSIONS: The eSSF process addresses the main challenges of cellulosic biofuel production by increasing saccharification rate at temperatures tolerable to yeast. The rapid hydrolysis of these emulsions at low temperatures permits fermentation using non-thermotolerant yeasts, short processing times, low enzyme loads, and makes it possible to extend the process to chemicals other than ethanol, such as isobutanol. This transferability establishes the eSSF process as a platform for the sustainable production of biofuels and chemicals as a whole.

Partially Acetylated Cellulose Dissolved in Aqueous Solution: Physical Properties and Enzymatic Hydrolysis.

Cellulose acetate is one of the most important cellulose derivatives. The use of ionic liquids in cellulose processing was recently found to act both as a solvent and also as a reagent. A recent study showed that cellulose dissolution in the ionic liquid 1-ethyl-3-methylimidazoliumacetate (EMIMAc) mixed with dichloromethane (DCM) resulted in controlled homogenous cellulose acetylation; yielding water-soluble cellulose acetate (WSCA). This research investigated the properties of cellulose acetate prepared in this manner, in an aqueous solution. The results revealed that WSCA fully dissolves in water, with no significant sign of molecular aggregation. Its conformation in aqueous solution exhibited a very large persistence length, estimated as over 10 nm. The WSCA exhibited surface activity, significantly reducing the surface tension of water. Because of the molecular dissolution of WSCA in water, augmented by its amphiphilicity, aqueous solutions of WSCA exhibited an overwhelmingly high rate of enzymatic hydrolysis.

Structural Analysis of Cellulose-Coated Oil-in-Water Emulsions Fabricated from Molecular Solution.

Natural cellulose has been used as a coating to stabilize oil-in-water (o/w) emulsions by exploiting the amphiphilic character of the cellulose chains molecularly dissolved in the ionic liquid 1-ethyl-3-methylimidazolium acetate. Its cellulose coating exhibits a continuous amorphous structure which differs significantly from the cellulose particle stabilization used in Pickering emulsions. The structure of these cellulose-coated o/w emulsion particles, in particular the cellulose coating shell characteristics (thickness, porosity, and composition), is studied by using a combination of direct imaging methods such as cryogenic electron microscopy and fluorescence microscopy with small-angle neutron scattering measurements. This work suggests a unique multicompartment structure of the emulsion particles: an oil core, surrounded by an inner shell composed of a porous cellulose gel, encapsulated by a dense outer cellulose shell, a few nanometers in thickness. The thickness of the inner cellulose shell varies significantly. The nanoscale emulsion droplets exhibit a thickness of 10 ± 3 nm, whereas the larger micron-sized droplets exhibit a thicker inner cellulose shell of 500-750 nm. It is also inferred that the cellulose shells contain water rather than oil.

Enhanced hydrolysis of cellulose hydrogels by morphological modification.

Cellulose is one of the most abundant bio-renewable materials on earth, yet the potential of cellulosic bio-fuels is not fully exploited, primarily due to the high costs of conversion. Hydrogel particles of regenerated cellulose constitute a useful substrate for enzymatic hydrolysis, due to their porous and amorphous structure. This article describes the influence of several structural aspects of the cellulose hydrogel on its hydrolysis. The hydrogel density was shown to be directly proportional to the cellulose concentration in the initial solution, thus affecting its hydrolysis rate. Using high-resolution scanning electron microscopy, we show that the hydrogel particles in aqueous suspension exhibit a dense external surface layer and a more porous internal network. Elimination of the external surface layer accelerated the hydrolysis rate by up to sixfold and rendered the process nearly independent of cellulose concentration. These findings may be of practical relevance to saccharification processing costs, by reducing required solvent quantities and enzyme load.

Cellulose gel dispersion: From pure hydrogel suspensions to encapsulated oil-in-water emulsions.

Cellulose hydrogel particles were fabricated from molecularly-dissolved cellulose/IL solutions. The characteristics of the formed hydrogels (cellulose content, particles' size and porosity) were determined as a function of cellulose concentration in the precursor solutions. There is a significant change in the hydrogel structure when the initial cellulose solution concentration increases above about 7-9%wt. These changes include increase of the cellulose content in the hydrogel, and decrease in its pore size. The finest cellulose particle dispersions can be obtained using low concentration cellulose/IL solutions (cellulose concentration in dispersion less than 2%wt.) or hydrogels (concentration less than 1%wt.) in a dispersing medium consisting of IL with no more than 20%wt. water. Stable paraffin oil-in-water emulsions are achieved by mixing oil and water with cellulose/IL solutions. The optimal conditions for obtaining the finest particles (about 20µm in diameter) are attained using cellulose solutions of concentration between 0.7 and 4%wt. at temperature of 70°C and oil/cellulose mass ratios between 1 and 1.5.

True molecular solutions of natural cellulose in the binary ionic liquid-containing solvent mixtures.

Evidence is presented for the first time of true molecular dissolution of cellulose in binary mixtures of common polar organic solvents with ionic liquid. Cryogenic transmission electron microscopy, small-angle neutron-, X-ray- and static light scattering were used to investigate the structure of cellulose solutions in mixture of dimethyl formamide and 1-ethyl-3-methylimidazolium acetate. Structural information on the dissolved chains (average molecular weight ∼ 5 × 10(4)g/mol; gyration radius ∼ 36 nm, persistence length ∼ 4.5 nm), indicate the absence of significant aggregation of the dissolved chains and the calculated value of the second virial coefficient ∼ 2.45 × 10(-2)mol ml/g(2) indicates that this solvent system is a good solvent for cellulose. More facile dissolution of cellulose could be achieved in solvent mixtures that exhibit the highest electrical conductivity. Highly concentrated cellulose solution in pure ionic liquid (27 wt.%) prepared according to novel method, utilizing the rapid evaporation of a volatile co-solvent in binary solvent mixtures at superheated conditions, shows insignificant cellulose molecular aggregation.

Cellulose as a novel amphiphilic coating for oil-in-water and water-in-oil dispersions.

The amphiphilic character of cellulose molecules provides the opportunity to use it as a novel eco-friendly emulsifying agent for formation of stable oil-in-water or water-in-oil dispersions. This may be done by mixing water, oil and cellulose solution in an ionic liquid. A more practical alternative is to form first a hydrogel from the cellulose/ionic liquid solution by coagulation with water and applying it into the sonicated water/oil or oil/water mixtures. The dissolution/regeneration process affords higher mobility to the cellulose molecules so an encapsulating coating can be formed at the water-oil interface. A solid-state dispersion was obtained by drying liquid dispersions, which can be repeatedly dissolved in excess water reforming a sustainable dispersion. The damp dispersion can be blown under reduced pressure, yielding a nanoporous foam ("aerocellulose"). The n-eicosane based solid dispersion as well as the aqueous dispersion possess a very high effective heat-absorption capacity. X-ray diffraction patterns indicate that the encapsulating cellulose shell is indeed in the amorphous state. Small-angle diffraction patterns of n-eicosane dispersions exhibit two sharp reflections. One is due to the n-eicosane triclinic crystal bulk phase and the other at somewhat smaller angles is interpreted as due to less ordered phase, possibly due to interactions with the encapsulating cellulose.