Glucose Oxidation by Gluconobacter Oxydans: Characterization in Shaking Flasks

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Appl Microbiol Biotechnol (2003) 62:92–98
DOI 10.1007/s00253-003-1222-x


M. Silberbach · B. Maier · M. Zimmermann · J. Büchs

Glucose oxidation by Gluconobacter oxydans: characterization in shaking-flasks, scale-up and optimization of the pH profile Received: 20 September 2002 / Revised: 3 December 2002 / Accepted: 6 December 2002 / Published online: 26 February 2003  Springer-Verlag 2003

Abstract In this study, the advantage of a novel
measuring device for the online determination of oxygen
and carbon dioxide transfer rates in shaking-flasks is
reported for glucose oxidation by Gluconobacter oxydans.
In this fermentation process, this device was used for the
characterization of the oxidation pattern of different
strains. G. oxydans NCIMB 8084 forms 2,5-diketogluconate from d-glucose in a multi-stage process via three different membrane-bound dehydrogenases. This strain
was chosen for a scale-up of the process from shakingflasks to a 2-l stirred vessel. An enhancement of 2,5diketogluconate production was realized by controlling the pH at different levels during the fermentation.

A main characteristic of Gluconobacter sp. is its ability to rapidly oxidize many organic compounds to the corresponding acids and ketones (Asai et al. 1968). The accumulation of products in the medium was exploited to

establish several industrial processes using strains of this genus, e.g. the production of l-sorbose from d-sorbitol,
the production of dihydroxyacetone from glycerol and the
production of d-gluconate from d-glucose (Lusta and
Reshetilov 1998; Macauley et al. 2001). d-Glucose can be
oxidized by two different metabolic pathways: one
includes uptake, intracellular oxidation and further dissimilation via the pentose phosphate pathway; and the other consists of the direct oxidation by membrane-bound
pyrroloquinoline quinone (PQQ)-dependent glucose deM. Silberbach · B. Maier · J. Büchs ()) Department of Biochemical Engineering,
Aachen University of Technology,
Worringer Weg 1, 52056 Aachen, Germany
Tel.: +49-241-8025546
Fax: +49-241-8022265
M. Zimmermann
Department of Microbiology,
Aachen University of Technology,
Worringer Weg 1, 52056 Aachen, Germany

hydrogenase. The latter one was identified as the main
pathway during typical batch fermentations with G.
oxydans on media containing d-glucose (Levering et al.
1988; Pronk et al. 1989). Many strains of this genus show
the property of 2-keto-d-gluconate and/or 5-keto-d-gluconate production from d-gluconate by membrane-bound gluconate dehydrogenases (Asai 1968). Weenk et al.
(1984) described a characteristic oxidation pattern caused
by sequential production of ketogluconic acids from
glucose in pH-controlled batch fermentations. Beschkov
et al. (1995) investigated 5-keto-d-gluconate production
from d-glucose by G. oxydans NBIMCC 1043 by
membrane-bound NADP-independent dehydrogenases.
An intracellular, 5-keto-d-gluconate-producing enzyme
in G. oxydans DSM 3503, gluconate:NADP-5-oxidoreductase, was characterized by Klasen et al. (1995). During fermentation of G. oxydans subsp. melanogenes under
similar conditions, 2-ketogluconate formation was followed by its oxidation to 2,5-diketogluconate as the final product (Weenk et al. 1984).
The individual product yields vary widely among
different strains. Additionally, they depend on the medium and on the particular conditions used for cultivation (Asai 1968; Olijve and Kok 1979). The influence of pH
value on product formation is significant. De Ley and
Swings (1984) found a pH optimum at 5.5 for growth of
G. oxydans cells and Qazi et al. (1991) described one
optimal pH of 5.5 for the activity of the glucose
dehydrogenase and another below 3.5–4.0 for the activity
of gluconate- and ketogluconate-forming dehydrogenases.
In batch fermentations with G. oxydans, the pH of the
medium was often buffered by the addition of CaCO3
(e.g. Beschkov et al. 1995). As a...
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