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.

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.

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.

The involvement of three signal transduction pathways in botryllid ascidian astogeny, as revealed by expression patterns of representative genes.

The patterning of the modular body plan in colonial organisms is termed astogeny, as distinct from ontogeny, the development of an individual organism from embryo to adult. Evolutionarily conserved signaling pathways suggest shared roots and common uses for both ontogeny and astogeny. Botryllid ascidians, a widely dispersed group of colonial tunicates, exhibit an intricate modular life form, in which astogeny develops as weekly, highly synchronized growth/death cycles termed blastogenesis, abiding by a strictly regulated plan. In these organisms both astogeny and ontogeny form similar body structures. Working on Botryllus schlosseri, and choosing a representative gene from each of three key Signal Transduction Pathways (STPs: Wnt/ß-catenin; TGF-ß, MAPK/ERK), we explored and compared gene expression at different stages of ontogeny and blastogenesis. Protein expression was studied via immunohistochemistry, ELISA and Western blotting. Five specific inhibitors and an activator for the selected pathways were used and followed to assess their impact during the blastogenic cycle and the development of distinctive phenotypes. Outcomes show that STPs are activated and function (while not necessarily co-localized) during both ontogeny and astogeny. Cellular patterns in blastogenesis, such as colony architecture, are shaped by these STPs. These results are further supported by administering Wnt agonist and anatagonist, TGF-ß receptor antagonists and inhibitors of Mek1/Mek2. Independent of their expression during ontogeny, some of the spatiotemporal patterns of STPs developed within short blastogenic windows. The results support the notion that while the same molecular machinery is functioning in Botryllus schlosseri astogeny and ontogeny, astogenic development is not an ontogenic replicate.

Repeated, long-term cycling of putative stem cells between niches in a basal chordate.

The mechanisms that sustain stem cells are fundamental to tissue maintenance. Here, we identify "cell islands" (CIs) as a niche for putative germ and somatic stem cells in Botryllus schlosseri, a colonial chordate that undergoes weekly cycles of death and regeneration. Cells within CIs express markers associated with germ and somatic stem cells and gene products that implicate CIs as signaling centers for stem cells. Transplantation of CIs induced long-term germline and somatic chimerism, demonstrating self-renewal and pluripotency of CI cells. Cell labeling and in vivo time-lapse imaging of CI cells reveal waves of migrations from degrading CIs into developing buds, contributing to soma and germline development. Knockdown of cadherin, which is highly expressed within CIs, elicited the migration of CI cells to circulation. Piwi knockdown resulted in regeneration arrest. We suggest that repeated trafficking of stem cells allows them to escape constraints imposed by the niche, enabling self-preservation throughout life.

Further portrayal of epithelial monolayers emergent de novo from extirpated ascidians palleal buds.

Astogeny in botryllid ascidians is executed by highly synchronized, repeated development and death cycles operating simultaneously on three coexisting asexually derived generations: zooids, primary buds, and secondary buds. In this study, we validated the fact that surgically removed blastogenic stage "D" primary buds cultured under in vitro conditions, away from any discrete colonial regulatory cues, exhibit intrinsic phenomena that are probably masked by astogenic controls. They produce de novo epithelial monolayers (EM), extending their lifespan from a few days to 1 mo and up to 5 mo when floating in the medium. Enhanced EM formation was documented when fibroblast growth factor (FGF) was added after at least 24 h incubation in FGF-free medium. Surprisingly, with no FGF administration, while intact isolated buds did not develop any EM, injured buds developed EM in half of the cases. Working on actin, PL10, FGF-R, P-MEK, MAP-kinase, and cadherin expressions, we documented that extirpated buds and monolayers are very active on the molecular/biochemical levels, revealing various cells and cellular organelle stains and rapid changes in the protein levels along a daily basis. Cells situated in the center of the monolayers stained differently for some proteins than peripheral cells. Cumulatively, results showed that flattened attached monolayers, as well as free-floating stage "D" buds, are highly active, not only exhibiting differential expressions of various proteins along incubation, but are also highly responsive to physical damages. These results establish a novel in vitro model system for epithelial cell development and senescence, revealing surprising rejuvenation and extended lifespan phenomena.