Δπ=0 reverse osmosis enriches a high osmotic pressure solution from a low-titre fermentation broth to a saturated solution or salt form using RO and NF membranes

Diverse biotechnology products are produced by microbial or eukaryotic cell fermentations in aqueous solutions. Removal of water is inevitable to enrich the product into a concentrated solution or into solid forms (such as crystals). The theoretical minimum energy required to remove 1 m3 of water is 716 kWh for thermal methods and 1 kWh for reverse osmosis (RO). In practice, the thermal methods equipped with heat energy recycling needs about 25 kWh to remove 1 m3 of water, and the RO methods needs about 4 kWh since extra energy (3 kWh) is required to operate pumps and other facilities in a plant. In general, membrane processes need less energy than thermal processes since there is no phase change in the separation processes and do not damage heat-sensitive biotechnology products. While both RO and NF membranes are permeable to water, RO membrane retains NaCl molecules and NF membrane is permeable to NaCl molecules, which is useful to remove inorganic salts from the products. Unlike thermal processes, the application of the membrane processes is limited by high osmotic pressure as the product solution is enriched by removing water. Chang et al. (2013) proposed a concept of osmotic pressure-free reverse osmosis (Δπ=0 RO) that overcomes this limitation and allows concentration of any solution with high osmotic pressure to its saturation point and further to crystal form. Δπ=0 RO, a two-component system, is distinct from 3-component forward osmosis and does not require the third component (draw component or extraction solvent) that must be separated from the aqueous solution at the end. This presentation will compare (1) ways of Δπ=0 RO technologies in desalination, and, furthermore (2) dewatering and desalination of high osmotic solutions of NaCl (343 bar), volatile fatty acids (400 – 600 bar), and fuel ethanol (6000 bar) with thermal separation methods in terms of energy consumption and potential of Δπ=0 RO technology. Chang et al. (2017), US patent 14,764,975(2015, 07,30), registration in progres

Role of Myosin Va in the Plasticity of the Vertebrate Neuromuscular Junction In Vivo

Background: Myosin Va is a motor protein involved in vesicular transport and its absence leads to movement disorders in humans (Griscelli and Elejalde syndromes) and rodents (e.g. dilute lethal phenotype in mice). We examined the role of myosin Va in the postsynaptic plasticity of the vertebrate neuromuscular junction (NMJ). Methodology/Principal Findings: Dilute lethal mice showed a good correlation between the propensity for seizures, and fragmentation and size reduction of NMJs. In an aneural C2C12 myoblast cell culture, expression of a dominant-negative fragment of myosin Va led to the accumulation of punctate structures containing the NMJ marker protein, rapsyn-GFP, in perinuclear clusters. In mouse hindlimb muscle, endogenous myosin Va co-precipitated with surface-exposed or internalised acetylcholine receptors and was markedly enriched in close proximity to the NMJ upon immunofluorescence. In vivo microscopy of exogenous full length myosin Va as well as a cargo-binding fragment of myosin Va showed localisation to the NMJ in wildtype mouse muscles. Furthermore, local interference with myosin Va function in live wildtype mouse muscles led to fragmentation and size reduction of NMJs, exclusion of rapsyn-GFP from NMJs, reduced persistence of acetylcholine receptors in NMJs and an increased amount of punctate structures bearing internalised NMJ proteins. Conclusions/Significance: In summary, our data show a crucial role of myosin Va for the plasticity of live vertebrate neuromuscular junctions and suggest its involvement in the recycling of internalised acetylcholine receptors back to th

Protocols for RecET-based markerless gene knockout and integration to express heterologous biosynthetic gene clusters in Pseudomonas putida

Pseudomonas putida has emerged as a promising host for the production of chemicals and materials thanks to its metabolic versatility and cellular robustness. In particular, P.\ua0putida KT2440 has been officially classified as a generally recognized as safe (GRAS) strain, which makes it suitable for the production of compounds that humans directly consume, including secondary metabolites of high importance. Although various tools and strategies have been developed to facilitate metabolic engineering of P.\ua0putida, modification of large genes/clusters essential for heterologous expression of natural products with large biosynthetic gene clusters (BGCs) has not been straightforward. Recently, we reported a RecET-based markerless recombineering system for engineering P.\ua0putida and demonstrated deletion of multiple regions as large as 101.7\ua0kb throughout the chromosome by single rounds of recombineering. In addition, development of a donor plasmid system allowed successful markerless integration of heterologous BGCs to P.\ua0putida chromosome using the recombineering system with examples of - but not limited to - integrating multiple heterologous BGCs as large as 7.4\ua0kb to the chromosome of P.\ua0putida KT2440. In response to the increasing interest in our markerless recombineering system, here we provide detailed protocols for markerless gene knockout and integration for the genome engineering of P.\ua0putida and related species of high industrial importance

CRISPR technologies for bacterial systems: Current achievements and future directions

Throughout the decades of its history, the advances in bacteria-based bio-industries have coincided with great leaps in strain engineering technologies. Recently unveiled clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) systems are now revolutionizing biotechnology as well as biology. Diverse technologies have been derived from CRISPR/Cas systems in bacteria, yet the applications unfortunately have not been actively employed in bacteria as extensively as in eukaryotic organisms. A recent trend of engineering less explored strains in industrial microbiology—metabolic engineering, synthetic biology, and other related disciplines—is demanding facile yet robust tools, and various CRISPR technologies have potential to cater to the demands. Here, we briefly review the science in CRISPR/Cas systems and the milestone inventions that enabled numerous CRISPR technologies. Next, we describe CRISPR/Cas-derived technologies for bacterial strain development, including genome editing and gene expression regulation applications. Then, other CRISPR technologies possessing great potential for industrial applications are described, including typing and tracking of bacterial strains, virome identification, vaccination of bacteria, and advanced antimicrobial approaches. For each application, we note our suggestions for additional improvements as well. In the same context, replication of CRISPR/Cas-based chromosome imaging technologies developed originally in eukaryotic systems is introduced with its potential impact on studying bacterial chromosomal dynamics. Also, the current patent status of CRISPR technologies is reviewed. Finally, we provide some insights to the future of CRISPR technologies for bacterial systems by proposing complementary techniques to be developed for the use of CRISPR technologies in even wider range of applications

Revisiting statistical design and analysis in scientific research

Statistics is essential to design experiments and interpret experimental results. Inappropriate use of the statistical analysis, however, often leads to a wrong conclusion. This concept article revisits basic concepts of statistics and provides a brief guideline of applying the statistical analysis for scientific research from designing experiments to analyzing and presenting the data