Medium-chain-length poly(hydroxyalkanoate)s (PHAmcl) are biodegradable and renewable biopolymers with elastomeric qualities. Here we report on the preparation and characterization of composite materials using thermally reduced graphene (TRG) nanoparticles as filler with three PHAmcl polymers. The matrices vary with respect to chain packing length, capacity for noncovalent bonding with the TRG surface, and the presence of covalent cross-linking. Results show that the addition of up to 2.5 vol % TRG to PHAmcl increases the melting temperature by 1–3 °C, the modulus by 200–590%, and the electrical conductivity by >7 orders of magnitude. Additionally, we use rheology and microscopy to characterize the composites. We discuss our results in light of polymer entanglement theory and the effects of polymer structure, filler loading volume, and the role of graphene-polymer interfacial forces. We extend our discussion by comparing the modulus enhancements of PHAmcl composites to those reported in other studies in which layered carbon nanofillers are combined with structurally related biopolyesters including: polylactide, polylactide-co-polyglycolide, polycaprolactone, and two other PHA copolymers.

Elementary mode analysis is a useful metabolic pathway analysis tool to identify the structure of a metabolic network that links the cellular phenotype to the corresponding genotype. The analysis can decompose the intricate metabolic network comprised of highly interconnected reactions into uniquely organized pathways. These pathways consisting of a minimal set of enzymes that can support steady state operation of cellular metabolism represent independent cellular physiological states. Such pathway definition provides a rigorous basis to systematically characterize cellular phenotypes, metabolic network regulation, robustness, and fragility that facilitate understanding of cell physiology and implementation of metabolic engineering strategies. This mini-review aims to overview the development and application of elementary mode analysis as a metabolic pathway analysis tool in studying cell physiology and as a basis of metabolic engineering.

Hydrolases show good catalytic activity in deep eutectic solvents, despite the presence of urea, which can denature enzymes, or alcohols, which can interfere with hydrolase-catalyzed reactions.

A fully evolved metabolic network can be described as a weighted sum of elementary modes where the usage probabilities of modes are distributed according to the Boltzmann distribution law (Srienc and Unrean, 2010). An organism presumably achieves the fully evolved state through adaptive changes in the kinetics of rate-controlling enzymes. Metabolic control analysis identifies reactions catalyzed by such enzymes. Comparison of the experimentally determined metabolic flux distributions of Thermoanaerobacterium saccharolyticum AS411 with the predicted flux distribution of a fully evolved metabolic network identified phosphoglucose isomerase (PGI) as the enzyme with the greatest flux control, the rate-controlling enzyme. The analysis predicts that an increased activity of PGI would enable the metabolic network to approach the fully evolved state and result in a faster specific growth rate. The prediction was confirmed by experimental results that showed an increased specific activity of PGI in a culture of strain AS411 that adaptively evolved over 280 generations. Sequencing of the gene confirmed the occurrence of a group of mutations clustered in the subunit binding domain of the dimeric enzyme. The results indicate that the evolutionary path is predictable as the strain AS411 adapted toward the fully evolved state by increasing the PGI activity. This experimental finding confirms that enzymes with predicted highest metabolic flux control are the targets of adaptive metabolic pathway evolution.Highlights► Metabolic flux control coefficients have been computed from predicted flux distribution. ► Phosphoglucose isomerase (PGI) is identified as the growth rate controlling enzyme. ► Adaptive evolution experiments result in faster growth due to beneficial mutations in PGI.

We have developed highly efficient ethanologenic Escherichia coli strains that selectively consume pentoses and/or hexoses. Mixed cultures of these strains can be used to selectively adjust the sugar utilization kinetics in ethanol fermentations. Based on the kinetics of sugar utilization, we have designed and implemented an immobilized cell system for the optimized continuous conversion of sugars into ethanol. The results confirm that immobilized mixed cultures support a simultaneous conversion of hexoses and pentoses into ethanol at high yield and at a faster rate than immobilized homogenous cells. Continuous ethanol production has been maintained for several weeks at high productivity with near complete sugar utilization. The control of sugar utilization using immobilized mixed cultures can be adapted to any composition of hexoses and pentoses by adjusting the strain distribution of immobilized cells. The approach, therefore, holds promise for ethanol fermentation from lignocellulosic hydrolysates where the feedstock varies in sugar composition.

Mechanical testing of solvent cast films consisting of short-chain-length (SCL) polyhydroxyalkanoate (PHA) films suggested that films consisting of block copolymers retained more elasticity over time with respect to films of similar random copolymers of comparable composition. Two experimental techniques, wide angle X-ray scattering (WAXS) and uniaxial extension, were used to quantitatively investigate the structure–property relationship of bacterially synthesized PHA block copolymers of poly(3-hydroxybutyrate) (PHB) homopolymer and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) random copolymer (PHBV) segments. Uniaxial testing experiments yielded the Young's modulus, ultimate tensile strength, and the elongation until fracture of the films. Percent crystallinity was determined by deconvolution of amorphous and crystalline scattering peaks obtained from WAXS. Two PHBV films containing either 8% 3-hydroxyvalerate monomer (3HV) or 29% 3HV exhibited a quick transition to brittle behavior, decreasing to less than 20% percent elongation at fracture within a few days after annealing. Conversely, the block copolymer samples remained higher than 100% elongation at fracture a full 3 months after annealing. Because block copolymers covalently link polymers that would otherwise form thermodynamically separate phases, the rates and degrees of crystallization of the block copolymers are less than the random copolymer samples. These differences translate into materials that extend the property space of biologically synthesized SCL PHA.

Ring-opening polymerization of five lactones catalyzed by Candida antarctica lipase B in ionic liquids yielded poly(hydroxyalkanoates) of moderate molecular weights up to Mn = 13,000. In the ionic liquid 1-butyl-3-methylimidazolium bis(trifluoromethane)-sulfonimide and with a low weight ratio of enzyme to lactone (1:100) we obtained polymers from β-propiolactone, δ-valerolactone, and ɛ-caprolactone with degrees of polymerization as high as 170, 25, and 85, respectively; oligomers from β-butyrolactone and γ-butyrolactone with degrees of polymerization of 5; and a copolymer of β-propiolactone and β-butyrolactone with a degree of polymerization of 180. Water-immiscible ionic liquids were superior to water-miscible ionic liquids. Reducing the water content of the enzyme improved the degree of polymerization by as much as 50% for β-propiolactone and ɛ-caprolactone.

Detailed knowledge of mammalian cell culture proliferation kinetics is important to determine fed-batch strategies for industrial bioreactor operations. In particular, predicting the end of exponential proliferation in batch culture is a critical process parameter during culture scale-up. Using automated flow cytometry we show that an increase in the non-viable sub-population in CHO cell culture can predict the onset of stationary phase by approximately 40 h. This enables a completely automated culture scale-up process as well as a reliable and reproducible control of fed-batch additions during culture expansion. It is shown that the automated scale-up results in a significantly higher total cell count in the reactor than manual scale up initiated in stationary growth phase. During individual, subsequent culture expansions, a significant variation in the proliferation rate was observed despite control of bulk culture parameters. Thus, automated flow cytometry is critical to uncovering useful process parameters that enable new control strategies. Such improved process supervision derived from knowledge-based data analysis is central to the FDA's Process Analytical Technology (PAT) initiative and is expected to result in better and higher quality products.

Hydrolases show good catalytic activity in deep eutectic solvents, despite the presence of urea, which can denature enzymes, or alcohols, which can interfere with hydrolase-catalyzed reactions.