BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING Development of a fed-batch process for the production of a dye-linked formaldehyde dehydrogenase in Hyphomicrobium zavarzinii ZV 580 Valérie Jérôme & Markus Hermann & Frank Hilbrig & Ruth Freitag Received: 27 April 2007 / Revised: 18 September 2007 / Accepted: 18 September 2007 / Published online: 16 October 2007 # Springer-Verlag 2007 Abstract The dye-linked formaldehyde dehydrogenase (dlFalDH) from Hyphomicrobium zavarzinii ZV 580 pro- cesses formaldehyde in a highly selective manner and without need for NAD(P). The enzyme thus has consider- able potential for technical applications if the difficulties associated with its efficient production can be resolved. In this contribution, a fed-batch bioprocess is developed, which improves both the biomass production of H. zavarzinii ZV 580 (from 0.6 to 2 g l −1 dry mass) and the specific dlFalDH production (from 0.1 to 0.3 units g −1 biomass), resulting in an overall improvement of the productivity by more than an order of magnitude compared to the previously reported process (Klein et al., Biochem J 301:289–295, 1994). In particular, the process uses an automated feeding strategy controlled via the dissolved oxygen concentration. In addition, our results show that the growth of H. zavarzinii ZV 580 is rather sensitive toward increasing salt concentration in the culture medium. Growth is also inhibited by the presence of surfactant-based antifoam reagents. Adjustment of the pH via the addition of methylamine instead of NaOH, on the other hand, leads to an increase in biomass yield. Keywords Hyphomicrobium zavarzinii . Dye-linked formaldehyde dehydrogenase . Fed-batch culture . Fermentation optimization . Process development Introduction The Gram-negative bacterial strain Hyphomicrobium zavarzinii ZV 580 has first been described in 1955 (Zavarzin 1955) and elicited moderate interest since then (e.g., Matzen and Hirsch 1982; Moore and Duxbury 1981; Zavarzin 1960). H. zavarzinii ZV 580 is a ‘slow growing’ methylotrophic bacterium, (Hirsch and Conti 1964b) most noted for the facultative expression of a formaldehyde- oxidizing enzyme when grown on certain C1-sources. This enzyme belongs to the family of NAD(P)-independent aldehyde dehydrogenases. It is described as a tetramer, each subunit with a molecular weight of 54,000 carrying a— putatively covalently bound—cofactor. The molecular nature of this cofactor has not yet been elucidated but bears features of a protein-bound quinone (Klein et al. 1994), hence the name ‘dye-linked formaldehyde dehydro- genase’ (dlFalDH) given to this particular enzyme. dlFalDH produced by H. zavarzinii ZV 580 when grown, e.g., on methylamine hydrochloride, differs from other dye- linked aldehyde dehydrogenases of methylotrophic bacteria by its affinity and specificity towards formaldehyde. This, together with the absence of NAD(P) as electron acceptor in the catalytic reaction, makes dlFalDH particular suitable for several technical applications, for example, in the biosensor field, if enough material can be produced in a cost efficient manner. However, to create a viable produc- tion process, the biomass and the specific dlFalDH yield of the organism have to be maximized. Expression of the enzyme in recombinant form is not straightforward due to the necessity of the cofactor. To date, little effort has been made to optimize the cultivation parameters for H. zavarzinii ZV 580 (Matzen and Hirsch 1982). The only previously established produc- tion process was based on a batch mode fermentation Appl Microbiol Biotechnol (2007) 77:779–788 DOI 10.1007/s00253-007-1218-z V. Jérôme : M. Hermann : F. Hilbrig : R. Freitag (*) Chair for Process Biotechnology, University of Bayreuth, 95440 Bayreuth, Germany e-mail: bioprozesstechnik@uni-bayreuth.de