Ruud A. Weusthuis

Properties, Modifications and Applications of Biopolyesters

Poly(hydroxyalkanoates) (PHAs), of which poly(hydroxybutyrate) (PHB) is the most common, can be accumulated by a large number of bacteria as energy and carbon reserve. Due to their biodegradability and biocompatibility these optically active biopolyesters may find industrial applications. A general overview of the physical and material properties of PHAs, alongside with accomplished applications and new developments in this field is presented in this chapter. The properties of PHAs are dependent on their monomer composition and therefore it is of great interest that recent research has revealed that, in addition to PHB, a large variety of PHAs can be synthesized microbially. The monomer composition of PHAs depends on the nature of the carbon source and microorganism used. PHB is a typical highly crystalline thermoplastic whereas medium chain length PHAs are elastomers with low melting points and a relatively lower degree of crystallinity. By (chemical) modification of the PHAs, the ultimate properties of the materials can be adjusted even further, when necessary. Applications that have been developed from PHB and related materials (e.g. Biopol) can be found in very different application areas and cover packaging, hygienic, agricultural and biomedical products. Recent application developments based on medium chain length PHAs range from high solid alkyd-like paints to pressure sensitive adhesives, biodegradable cheese coatings and biodegradable rubbers. Technically, the prospects for PHAs are very promising. When the price of these materials can be further reduced, application of biopolyesters will also become economically very attractive.

Microbial production of bulk chemicals: development of anaerobic processes

Innovative fermentation processes are necessary for the cost-effective production of bulk chemicals from renewable resources. Current microbial processes are either anaerobic processes, with high yield and productivity, or less-efficient aerobic processes. Oxygen utilization plays an important role in energy generation and redox metabolism that is necessary for product formation. The aerobic productivity, however, is relatively low because of rate-limiting volumetric oxygen transfer; whereas the product yield in the presence of oxygen is generally low because part of the substrate is completely oxidized to CO₂. Hence, new microbial conversion processes for the production of bulk chemicals should be anaerobic. In this opinion article, we describe different scenarios for the development of highly efficient microbial conversion processes for the anaerobic production of bulk chemicals.

Development of environmentally friendly coatings and paints using medium-chain-length poly(3-hydroxyalkanoates) as the polymer binder

Unsaturated medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHAs) produced by Pseudomonas putida from linseed oil fatty acids (LOFA) and tall oil fatty acids (TOFA), were used as the polymer binder in the formulation of high solid alkyd-like paints. The relatively high concentration of unsaturated alkyl side chains incorporated into the PHA resins resulted in oxidative drying PHA paints having excellent coating properties. The homogeneously pigmented PHA coatings yielded high-gloss, smooth and strong films upon curing and showed an excellent flexibility, a good adhesion to different substrates, cohesive film properties and resistance to chipping.

NADPH-generating systems in bacteria and archaea.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is an essential electron donor in all organisms. It provides the reducing power that drives numerous anabolic reactions, including those responsible for the biosynthesis of all major cell components and many products in biotechnology. The efficient synthesis of many of these products, however, is limited by the rate of NADPH regeneration. Hence, a thorough understanding of the reactions involved in the generation of NADPH is required to increase its turnover through rational strain improvement. Traditionally, the main engineering targets for increasing NADPH availability have included the dehydrogenase reactions of the oxidative pentose phosphate pathway and the isocitrate dehydrogenase step of the tricarboxylic acid (TCA) cycle. However, the importance of alternative NADPH-generating reactions has recently become evident. In the current review, the major canonical and non-canonical reactions involved in the production and regeneration of NADPH in prokaryotes are described, and their key enzymes are discussed. In addition, an overview of how different enzymes have been applied to increase NADPH availability and thereby enhance productivity is provided.

Xylose metabolism in the fungus Rhizopus oryzae: effect of growth and respiration on l(+)-lactic acid production

The fungus Rhizopus oryzae converts both glucose and xylose under aerobic conditions into chirally pure l(+)-lactic acid with by-products such as xylitol, glycerol, ethanol, carbon dioxide and fungal biomass. In this paper, we demonstrate that the production of lactic acid by R. oryzae CBS 112.07 only occurs under growing conditions. Deprivation of nutrients such as nitrogen, essential for fungal biomass formation, resulted in a cessation of lactic acid production. Complete xylose utilisation required a significantly lower C/N ratio (61/1) compared to glucose (201/1), caused by higher fungal biomass yields that were obtained with xylose as substrate. Decreasing the oxygen transfer rate resulted in decline of xylose consumption rates, whereas the conversion of glucose by R. oryzae was less affected. Both results were linked to the fact that R. oryzae CBS 112.07 utilises xylose via the two-step reduction/oxidation route. The consequences of these effects for R. oryzae as a potential lactic acid producer are discussed.

Metabolic Engineering of TCA Cycle for Production of Chemicals

The tricarboxylic acid (TCA) cycle has been used for decades in the microbial production of chemicals such as citrate, L-glutamate, and succinate. Maximizing yield is key for cost-competitive production. However, for most TCA cycle products, the maximum pathway yield is lower than the theoretical maximum yield (Y(E)). For succinate, this was solved by creating two pathways to the product, using both branches of the TCA cycle, connected by the glyoxylate shunt (GS). A similar solution cannot be applied directly for production of compounds from the oxidative branch of the TCA cycle because irreversible reactions are involved. Here, we describe how this can be overcome and what the impact is on the yield.

Cultivation of the oleaginous yeast Cryptococcus curvatus in a new reactor with improved mixing and mass transfer characteristics (Surer

Cultivation and lipid production using the yeast Cryptococcus curvatus has proven to be efficient in a fed-batch fermentation using a stirred tank reactor. Scale up of this reactor however results in changing mixing and mass-transfer characteristics. In this paper we report cultivation of the yeast in a new type of reactor (Surer®), which can easily be scaled up. A high cell density (91 gl−1) and a lipid production rate of 0.42 g lipid l−1h−1 were obtained.

Dilute H2SO4-catalyzed hydrothermal pretreatment to enhance enzymatic digestibility of Jatropha curcas fruit hull for ethanol fermentation

Dilute sulfuric acid pretreatment of the Jatropha curcas fruit hull at high temperatures (140°C to 180°C) performed in a 110-mL stainless steel reactor was investigated to enhance the enzymatic digestibility of its lignocellulosic components. Carbohydrates accounted for 43% of the dry matter of the J. curcas fruit hull biomass. The goal of the study was to optimize the pretreatment conditions (acid concentration, time, and temperature) in order to obtain the highest sugar yield after subsequent enzymatic hydrolysis. A Box-Behnken design was applied to the experimental setup in order to reduce the number of experiments. The optimal pretreatment conditions are 30-min incubations at a temperature of 178°C with a sulfuric acid concentration of 0.9% (w/v). Using these pretreatment conditions for a fruit solid loading of 9.52% followed by a 24-h enzymatic hydrolysis resulted in a liberation of 100% of all pentoses present (71% yield and 29% degradation to furfural) and 83% of the hexoses (78% yield and 5% degradation to 5-hydroxymethylfurfural). The simultaneous saccharification and fermentation experiment showed that acid-pretreated fruit hull can be used as a substrate for Saccharomyces cerevisiae to produce ethanol.

Ethyl acetate production by the elusive alcohol acetyltransferase from yeast

Ethyl acetate is an industrially relevant ester that is currently produced exclusively through unsustainable processes. Many yeasts are able to produce ethyl acetate, but the main responsible enzyme has remained elusive, hampering the engineering of novel production strains. Here we describe the discovery of a new enzyme (Eat1) from the yeast Wickerhamomyces anomalus that resulted in high ethyl acetate production when expressed in Saccharomyces cerevisiae and Escherichia coli. Purified Eat1 showed alcohol acetyltransferase activity with ethanol and acetyl-CoA. Homologs of eat1 are responsible for most ethyl acetate synthesis in known ethyl acetate-producing yeasts, including S. cerevisiae, and are only distantly related to known alcohol acetyltransferases. Eat1 is therefore proposed to compose a novel alcohol acetyltransferase family within the α/β hydrolase superfamily. The discovery of this novel enzyme family is a crucial step towards the development of biobased ethyl acetate production and will also help in selecting improved S. cerevisiae brewing strains.

Identifying inhibitory effects of lignocellulosic by-products on growth of lactic acid producing micro-organisms using a rapid small-scale screening method

Sugars obtained from pretreated lignocellulose are interesting as substrate for the production of lactic acid in fermentation processes. However, by-products formed during pretreatment of lignocellulose can inhibit microbial growth. In this study, a small-scale rapid screening method was used to identify inhibitory effects of single and combined by-products on growth of lactic acid producing micro-organisms. The small-scale screening was performed in 48-well plates using 5 bacterial species and 12 by-products. Large differences were observed in inhibitory effects of by-products between different species. Predictions can be made for growth behaviour of different micro-organisms on acid pretreated or alkaline pretreated bagasse substrates using data from the small-scale screening. Both individual and combined inhibition effects were shown to be important parameters to predict growth. Synergy between coumaric acid, formic acid and acetic acid is a key inhibitory parameter in alkaline pretreated lignocellulose, while furfural is a key inhibitor in acid pretreated lignocellulose.