Crude glycerol – a by‐product of the large scale production of diesel oil from rape – is examined for its possible use as a cheap feedstock for the biotechnological synthesis of poly(3‐hydroxybutyrate) (PHB). The glycerol samples of various manufacturers differ in their contamination with salts (NaCl or K2SO4), methanol or fatty acids. At high cell density fermentation these pollutants could possibly accumulate to inhibiting concentrations. The bacteria used were Paracoccus denitrificans and Cupriavidus necator JMP 134, which accumulate PHB from pure glycerol to a content of 70 % of cell dry mass. When using crude glycerol containing 5.5 % NaCl, a reduced PHB content of 48 % was observed at a bacterial dry mass of 50 g/L. Furthermore the PHB yield coefficient was reduced, obviously due to osmoregulation. The effect of glycerol contaminated with K2SO4 was less pronounced. The molecular weight of PHB produced with P. denitrificans or C. necator from crude glycerol varies between 620000 and 750000 g/mol which allows the processing by common techniques of the polymer industry.

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An increased usage of poly‐β‐hydroxyalkanoates (PHA), for instance as bulk biodegradable and biocompatible plastics, will require a cheaper production and downstream processing. If the synthesis of this intracellular biopolyester could be combined with the production of another valuable intracellular product, the economic balance of the process could be improved. It was found that the moderately halophilic bacterium Halomonas elongata simultaneously synthesizes PHA and a protector molecule, called ectoine. Whereas the synthesis of PHA is a response to the shortage of nutrients, the production of ectoine counteracts osmotic imbalances. This behavior is in so far surprising as the conditions of a bi‐factorial stress initiate the fast simultaneous synthesis of ectoine and PHA. In the presence of 100 g/L NaCl, Halomonas elongata accumulated up to 50 % w/w PHA and up to 14 % ectoine within 2–3 days under so far non‐optimized conditions. Furthermore, it was found that other Halomonas species (e.g. Halomonas halodenitrificans and own isolates of Halomonas halodeneurihalina and Halomonas salina) were able to produce both ectoine and PHA.

Two distinct crotonyl-CoA hydratases of Methylobacterium rhodesianum MB 126 were separated by column chromatography on DEAE-Sepharose CL-6B. The two enzymes were further purified by chromatography on red 120 agarose and Mono Q. Enzyme A was specific for L(+)-hydroxybutyryl-CoA. It had an apparent molecular weight of 160 000 with two identical subunits of 43 000 and 34 000. The apparent K, values for L(+)-hydroxybutyryl-CoA and crotonyl-CoA were 83 and 90 pM, respectively. Enzyme B was specific for D(-)-hydroxybutyryl-CoA. It had an apparent molecular weight of 39 000 with identical subunits of 12 500. It showed sigmoidal kinetics for crotonyl-CoA, and the Hill coefficient was about 2.5. The apparent K, value for D(-)-hydroxybutyryl-CoA was 0.5 mM. The possible contribution of a sequence including P-ketothiolase, NADH-linked L(+)-specific acetoacetyl-CoA reductase, and two stereospecific crotonyl-CoA hydratases to PHB synthesis in methylotrophic serine-pathway bacteria is discussed. Resume: Deux crotonyl-CoA hydratases distinctes prksentes chez Methylobacterium rhodesianum MB 126 ont Ct C s6parCes par chromatographie sur colonne avec du DEAE-Sepharose CL-6B. Ces deux enzymes ont ensuite Ct C purifiCes par chromatographie sur red 120 agarose et Mono Q. L'enzyme A Ctait spCcifique du L(+)-hydroxybutyryl-CoA. Elle a un poids molCculaire estimC B 160 000 et comprend deux sous-unit& identiques de 43 000 et 34 000. Les K, de la L(+)-hydroxybutyryl-CoA et de la crotonyl-CoA dtaient de 83 et 90 pM, respectivement. L'enzyme B Ctait spdcifique du D(-)hydroxybutyryl-CoA. Son poids molCcules est CvaluC B 39 000 et elle contient deux sous-unit& identiques de 12 000. Elle prCsente une cinCtique de type sigmo'ide envers le crotonyl-CoA, le coefficient de Hill Ctant d'environ 2,5. La valeur du K, pour le D(-)hydroxybutyryl-CoA est de 0,5 mM. Concemant la synthkse de PHB chez les bactdries mCthylotrophes utilisant le sentier de la sCrine, nous spCculons sur le r61e possible d'une sCquence d'dvtnements impliquant une P-~Ctothiolase, une rCductase like au NADH spCcifique du ~(+)acCtoacCtyl-CoA ainsi que deux crotonylCoA hydratases stCrCospCcifiques.

Different substrate mixtures of acetic acid and valeric acid were used to synthesize copolymers of poly([R]‐3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) (PHB/HV) with Paracoccus denitrificans under N‐limited conditions. A correlation between the substrate ratio and HV content was found in batch experiments, which seemed to be suitable to produce a number of defined copolymers. In fed‐batch fermentation, such correlation could only be found with carbon substrate mixtures of very restricted composition. Due to the individual substrate consumption rates with this technique, a polymer with 16.5 mol.‐% HV content [w/w] could be reproducibly synthesized. However, under N‐limited chemostatic cultivation conditions it was possible to produce a spectrum of definitely composed copolymers (3.0 %–46.3 mol.‐% HV) from different mixtures of acetic acid and valeric acid.