Frederik van Keulen

Enhanced bioproduction of poly-3-hydroxybutyrate from wheat straw lignocellulosic hydrolysates

Polyhydroxyalkanoates (PHAs) are bioplastics that can replace conventional petroleum-derived products in various applications. One of the major barriers for their widespread introduction in the market is the higher production costs compared with their petrochemical counterparts. In this work, a process was successfully implemented with high productivity based on wheat straw, a cheap and readily available agricultural residue, as raw material. The strain Burkholderia sacchari DSM 17165 which is able to metabolise glucose, xylose and arabinose, the main sugars present in wheat straw hydrolysates (WSHs), was used. Results in shake flask showed that B. sacchari cells accumulated about 70%gpoly(3-hydroxybutyrate)(P(3HB))/g cell dry weight (CDW) with a yield of polymer on sugars (YP/S) of 0.18g/g when grown on a mixture of commercial C6 and C5 sugars (control), while these values reached about 60%gP(3HB)/g CDW and 0.19g/g, respectively, when WSHs were used as carbon source. In fed-batch cultures carried out in 2L stirred-tank reactors (STRs) on WSH, a maximum polymer concentration of 105 g/L was reached after 61 hours of cultivation corresponding to an accumulation of 72% of CDW. Polymer yield and productivity were 0.22 gP(3HB)/g total sugar consumed and 1.6g/L hour, respectively. The selected feeding strategy successfully overcame the carbon catabolite repression (CCR) phenomenon observed with sugar mixtures containing hexoses and pentoses. This is the first work describing fed-batch cultivations aiming at PHA production using real lignocellulosic hydrolysates. Additionally, the P(3HB) volumetric productivities attained are by far the highest ever achieved on agricultural waste hydrolysates.

Development of a reaction system for the selective conversion of (−)-trans-carveol to (−)-carvone with whole cells of Rhodococcus erythropolis DCL14

Abstract The present article addresses the development of a microbial reaction system for the transformation of carveol to carvone, using whole cells of Rhodococcus erythropolis DCL14. This strain contains a NAD-dependent carveol dehydrogenase (CDH) when grown on limonene or on cyclohexanol. When a mixture of (−)- cis and (−)- trans -carveol is supplied, only (−)- trans -carveol is converted. Thus, besides (−)-carvone, pure (−)- cis -carveol can be obtained as product. Initial experiments were performed batchwise using an aqueous system. (−)- Trans -carveol conversion rate gradually decreased during successive reutilisation batches. After the third reutilisation, activity was completely lost. Cells grown on cyclohexanol showed a slightly higher activity as compared to cells grown on (+)-limonene. A production of 4.3 μmol (−)-carvone formed per mg protein was achieved. A significant improvement with respect to initial reaction rate and productivity was obtained with aqueous–organic two-phase systems. Using a 5 to 1 buffer/ iso -octane system, a 40% increase in the initial rate and a 16-fold increase of the production was observed. A further improvement resulted from increasing the volume of solvent (1 to 1 buffer/dodecane ratio). An initial reaction rate of 26 nmol/(min∗mg protein) was observed, while production increased to 208 μmol (−)-carvone formed per mg protein. As in the single-phase system, reaction rate gradually decreased along the successive cell reutilisation batches. Addition of co-substrates for the regeneration of NAD did not prevent this decay. A simple downstream process was developed for the recovery of carvone and cis -carveol.

Production and Recovery of Limonene-1,2-Diol and Simultaneous Resolution of a Diastereomeric Mixture of Limonene-1,2-Epoxide with whole Cells of Rhodococcus Erythropolis DCL14

Cells of Rhodococcus erythropolis DCL14 present a limonene epoxide hydrolase activity when grown on terpenes, which enables them to convert cis-limonene-1,2-epoxide to limonene-1, 2-diol. Trans-limonene-1,2-epoxide is only converted when no cis is present. An organic/aqueous system was developed to overcome the low solubility and instability of limonene-1,2-epoxide in the aqueous phase. The presence of the organic solvent allowed high epoxide concentrations to be used which resulted in high limonene-1,2-diol production rates. Relatively cheap solvents were tested without any significant loss of epoxide hydrolase activity. Using a 500 ml fed-batch mechanically stirred reactor it was possible to produce 197.2 g of diol per g of protein and to obtain a yield of 98.2% and 67.9% for the trans-epoxide and the diol, respectively. Production of 72.4 g of diol per g of protein was obtained using a magnetically stirred reactor with an external loop for product separation. In this case, trans-epoxide and diol yields were 98.5% and 94.1%, respectively. A downstream process, based both on the preference of the substrate for organic solvents and that of the product for the aqueous phase, allowed the recovery of limonene- 1,2-diol, as well as of the trans-epoxide, with a purity higher than 99%.

Production of poly (3-hydroxybutyrate-co-4-hydroxybutyrate) by Burkholderia sacchari using wheat straw hydrolysates and gamma-butyrolactone

Burkholderia sacchari DSM 17165 is able to grow and produce poly(3-hydroxybutyrate) both on hexoses and pentoses. In a previous study, wheat straw lignocellulosic hydrolysates (WSH) containing high C6 and C5 sugar concentrations were shown to be excellent carbon sources for P(3HB) production. Using a similar feeding strategy developed for P(3HB) production based on WSH, fed-batch cultures were developed aiming at the production of the copolymer P(3HB-co-4HB) (poly(3-hydroxybutyrate-co-4-hydroxybutyrate)) by B. sacchari. The ability of this strain to synthesize P(3HB-co-4HB) was first shown in shake flasks using gamma-butyrolactone (GBL) as precursor of the 4HB units. Fed-batch cultures using glucose as carbon source (control) and GBL were developed to achieve high copolymer productivities and 4HB incorporations. The attained P(3HB-co-4HB) productivity and 4HB molar% were 0.7g/(Lh) and 4.7molar%, respectively. The 4HB incorporation was improved to 6.3 and 11.8molar% by addition of 2g/L propionic and acetic acid, respectively. When WSH were used as carbon source under the same feeding conditions, the values achieved were 0.5g/(Lh) and 5.0molar%, respectively. Burkholderia sacchari, a strain able to produce biopolymers based on xylose-rich lignocellulosic hydrolysates, is for the first time reported to produce P(3HB-co-4HB) using gamma butyrolactone as precursor.

Modelling the biokinetic resolution of diastereomers present in unequal initial amounts

Abstract The enantiomeric ratio ( E ) is commonly used to evaluate enzyme-catalysed kinetic resolutions. Chen et al. (1982) proposed a model for the enantiomeric ratio, which relates the extent of substrate conversion and the enantiomeric excess. The model, however, does not apply to cases where the substrate initially contains unequal amounts of enantiomers. This paper describes the adaptation of Chens model to cases where the reagent consists of a mixture of diastereomers present in unequal initial amounts and gives product as a single diastereomer. Diastereomeric ‘resolutions’ of (+)-limonene-1,2-epoxide and of (−)-carveol were used to validate this model.

Biotransformations in the Flavour Industry

Bacteria and fungi synthesise or degrade a vast number of natural and xenobiotic substrates. Biotransformations and bioconversions occur, if a single substrate structure is altered by an identifiable redox, hydrolysis, or addition type reaction, or by a sequence of these reactions. Generally competing with chemosynthesis, biocatalysts possess inherent advantages: They functionalise chemically inert carbons, modify one functionality in a multifunctional molecule selectively or specifically, introduce chirality, resolve racemates, and operate under ambient conditions. The production of natural flavour and aroma ingredients could benefit from these characteristics. The biotransformation of volatile terpenes particularly well demonstrates perspectives, but also current problems. Solvent tolerant bacteria hold much promise for a lipophilic biotechnology. Genetic engineering will help to create tailor-made biocatalysts. Downstreaming of products, based on in situ solvent extraction or adsorption, is required for an efficient bioprocess. Cost calculations show that high-yielding biotransformations have commercial potential.

Upgrading wheat straw to HOMO and co-polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are biodegradable and thus environmentally friendly thermoplastics that are synthesized by various microbial strains as intracellular storage materials. These polyesters present a broad range of properties varying from very crystalline to more elastomeric polymers and find applications from agriculture to medicine. Despite their versatility, they are still not competitive due to the high production costs, of which the C-source accounts for circa 30%. To decrease raw materials costs, lignocellulosic agro-industrial residues rich in cellulose and hemicelluloses can be used as the C-source after being processed to yield simple sugars. Wheat straw lignocellulosic hydrolysates (LCH) were prepared (biorefinery.de GmbH) by pre-treating this residual biomass using the AFEX process followed by enzymatic hydrolysis. A hydrolysate rich in glucose and xylose and with low titres of inhibitory compounds is produced that can be used as carbon source for PHA production. Burkholderia sacchari DSM 17165 was selected for its ability to use both hexoses and pentoses. Polymer production was optimized in fed-batch cultivations in stirred-tank reactors (STR). Polymer concentration, volumetric productivity and polymer cell content of respectively 84 g/L, 1.6 g L-1h-1 and 68 % (w/w) were attained [1]. Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB-co-4HB) copolymers exhibit attractive thermal and mechanical properties due to the 4HB monomer. Synthesis of this monomer was achieved upon the addition of gamma-butyrolactone (GBL) as co-substrate to fed-batch cultures. Using a DOstat feeding strategy for LCH and a continuous addition of GBL, the maximum attained P(3HB-co-4HB) productivity and 4HB molar % were 0.5 g/(L.h) and 5.0 molar %, respectively [2]. Extraction of P(3HB) from the cells usually involves the use of halogenated solvents to attain high recovery yields and purities. However, the use of these solvents causes health and environmental hazards. To lessen this drawback green solvents were tested and high recovery yields and purities were achieved. Lignocellulosic agricultural residues can thus be ugraded with high yields and productivities to value-added products using the biorefinery concept.