3-Hydroxyvalerate monomer

This method was in fact carried out around two decades ago [30, 31]. However, it was applied only in the fermentation of pure microbial cultures. In a recent report by Acros-Hernandez and coworkers [32], infrared spectroscopy was applied to quantify the PHA produced in microbial mixed cultures. Around 122 spectra from a wide range of production systems were collected and used for calibrating the partial least squares (PLS) model, which relates the spectra with the PHA content (0.03-0.58 w/w) and 3-hydroxyvalerate monomer (0-63 mol%). The calibration models were evaluated by the correlation between the predicted and measured PHA content (R ), root mean square error of calibration, root mean square error of cross validation and root mean square error of prediction (RMSEP). The results revealed that the robust PLS model, when coupled with the Fourier-Transform infrared spectrum, was found to be applicable to predict the PHA content in microbial mixed cultures, with a low RMSEP of 0.023 w/w. This is considered to be a reliable method and robust enough for use in the PHA biosynthesis process using mixed microbial cultures, which is far more complex. 

When propionic acid is present in the medium, 3-ketothiolase condenses one propionyl-CoA with one acetyl-CoA to form 3-ketovaleryl-CoA, which is polymerized after reduction to 3-hydroxyvalerate monomers by PHA synthase (Pathway III) [141]. Acetyl-CoA can be provided by a second substrate, but elimination of the carbonyl carbon of propionyl-CoA always takes place to some extent, so that both 3HB and 3HV units are synthesized when propionic acid is the sole carbon source. Still, the 3HV fraction in the copolymer rises with increasing ratio of propionic acid to acetyl-CoA-generating substrate [141]. With propionic acid alone as substrate, up to 40 mol% 3HV units are incorporated in the copolyester. 

Comonomers, such as 3-hydroxyvalerate (3HV, ethylene R-unit (-CH2-CH3) in Fig. 1) and 4-hydroxybutyrate, have been incorporated in the PHB chains using specific additives in the growth medium of the bacteria [21-25]. It has been shown by nuclear magnetic resonance (NMR) studies that poly(3HB-co-3HV) has a statistically random distribution of the monomer units throughout a range of compositions varying from 0 to 90 mol % 3HV [23-26]. 

During aging of PHVB in sterile water at pH 7 and 60 °C, 2-butenoic acid (crotonic acid), 2-pentenoic acid, 3-hydroxybutyric acid, 3-hydroxyvaleric acid, 3-hydroxybutyrate dimer, 3-hydroxybutyrate-3-hydroxyvalerate dimer and 3-hydroxyvalerate dimer were formed [99]. The weight loss was, however, only 2% after 200 days at 60 °C. Monomers, oligomers and derivatives, produced by dehydration at the OH-terminus were identified after alkaline hydrolysis of PHB [110]. In accordance CZE showed that the accelerated hydrolysis of PHB leads to the formation of hydroxyacid oligomers and a series of peaks formed by a side reaction leading to a C=C bond at the noncarboxylic acid end [111]. Kinetics of the abiotic hydrolysis of PHB in acid and alkaline media were monitored by following the forma-... 

Polymers derived from renewable resources (biopolymers) are broadly classified according to the method of production (1) Polymers directly extracted/ removed from natural materials (mainly plants) (e.g. polysaccharides such as starch and cellulose and proteins such as casein and wheat gluten), (2) polymers produced by "classical" chemical synthesis from renewable bio-derived monomers [e.g. poly(lactic acid), poly(glycolic acid) and their biopolyesters polymerized from lactic/glycolic acid monomers, which are produced by fermentation of carbohydrate feedstock] and (3) polymers produced by microorganisms or genetically transformed bacteria [e.g. the polyhydroxyalkanoates, mainly poly(hydroxybutyrates) and copolymers of hydroxybutyrate (HB) and hydroxyvalerate (HV)] [4]. 

The copolymerization of 3-hydroxybutyrate entities with 3-hydroxyoc-tanoate (HO), 3-hydroxyheptanoate (HH), or 3-hydroxyvalerate (HV) monomers modifies the physical and mechanical characteristics of the parent PHB, such as ductility and toughness to depress its processing temperature and embrittlement. Besides, copol5miers PHB-HV [7], PHB-HH [8], or PHB-HO [9] et al., have improved thermophysical and/or mechanical properties, and hence they expand the spectrum of constructional and medical materials/items. For predicting the behavior of PHB and its copolymers in an aqueous media, e.g., in vitro, in a living body or in a wet soil, it is essential to study kinetics and mechanism of hydrolytic destruction. 

Another terpolymer poly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-4HB-co-3HHx)] was found to have better thermal stability due to the introduction of 4HB and 3HHx monomers into P(3HB) (Xie and Chen 2008). It has lower crystalUnity and better flexibility compared to P(3HB) homopolymer and copolymers. Poly(3-hydroxybutyrate-C( -3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)] also showed superior properties over those of 3HB and its copolymer (Madden et al. 2000 Chanprateep and Kulpreecha 2006). Table 2.3 shows the comparison of different types of PHA polymer. 

In a study reported by Doi and co-workers [5], a fed-batch culture technique was applied for copolymer production using Alcaligenes eutrophus in which fructose and ammonium sulfate were fed at a rate of 0.76 and 0.15 g/h respectively. This feeding was then stopped and followed by pentanoic acid feeding at a rate of 0.6 g/h. At the end of the fermentation, 59 mol% of a 3-hydroxyvalerate (3HV) monomer was obtained the cell dry weight (CDW) and PHA content produced was 6.4 g/1 and 65 wt%, respectively. The periodic addition of methanol and n-amyl alcohol under nitrogen limitation to a culture of Paracoccus denitrificans in 3 1 fed-batch cultures resulted in the production of the poly[3-hydroxybutyrate (3HB)-co-3HV] copolymer. About 26% (w/w) containing 60 mol% of the 3HV monomer was obtained after 138 h [6]. 

Fig. 2 Common PHA monomer structures. Short-chain-length monomers 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV). Medium-chain-length monomers 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD), 3-hydroxydodecanoate (3HDD)...

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