Miniaturized growth systems for heterogeneous culture collections are not only attractive in reducing demands for incubation space and medium but also in making the parallel handling of large numbers of strains more practicable. We report here on the optimization of oxygen transfer rates in deep-well microtiter plates and the development of a replication system allowing the simultaneous and reproducible sampling of 96 frozen glycerol stock cultures while the remaining culture volume remains frozen. Oxygen transfer rates were derived from growth curves of Pseudomonas putida and from rates of oxygen disappearance due to the cobalt-catalyzed oxidation of sulfite. Maximum oxygen transfer rates (38 mmol liter ؊1 h ؊1 , corresponding to a mass transfer coefficient of 188 h ؊1 ) were measured during orbital shaking at 300 rpm at a shaking diameter of 5 cm and a culture volume of 0.5 ml. A shaking diameter of 2.5 cm resulted in threefold-lower values. These high oxygen transfer rates allowed P. putida to reach a cell density of approximately 9 g (dry weight) liter ؊1 during growth on a glucose mineral medium at culture volumes of up to 1 ml. The growth-and-replication system was evaluated for a culture collection consisting of aerobic strains, mainly from the genera Pseudomonas, Rhodococcus, and Alcaligenes, using mineral media and rich media. Cross-contamination and excessive evaporation during vigorous aeration were adequately prevented by the use of a sandwich cover of spongy silicone and cotton wool on top of the microtiter plates.The general practice during screening of large microbial strain collections for new enzyme activities or bioactive compounds is that each strain is handled individually. The labor and time involved in separately reviving the stock cultures on agar plates and the subsequent inoculation of tubes or Erlenmeyer flasks are major limiting factors in the progress of such screening projects. This bottleneck might be alleviated if large numbers of microbial strains could be handled in parallel rather than in sequence, preferably in a format that is compatible with current standards in the manipulation of large numbers of liquid samples, e.g., in 96-or 384-well microtiter plates.Until now, the use of microtiter plates for the growth and maintenance of microbial strains has been mainly limited to clonal libraries in Escherichia coli (9,11,20) and yeasts (2, 18). For this purpose, the apparent drawbacks of growth in microtiter plates-low aeration rates and small working volumesare generally less relevant since E. coli and yeasts can grow anaerobically. Furthermore, small amounts of biomass generally suffice as the use of high-copy-number vectors ensures high levels of the desired gene or gene product. The resulting mixed aerobic-anaerobic growth, however, can lead to undesirable variations in biomass levels from well to well, especially since oxygen diffusion rates for an individual well may be affected by its position in the microtiter plate. Such well-to-well variations are especially troublesome in...

Résumé -Diversité des systèmes alcane hydroxylase dans l'environnement -La première étape dans la dégradation aérobie des alcanes par les bactéries, les levures et les champignons est catalysée par des oxygénases, une classe d'enzymes capables d'introduire des atomes d'oxygène issus de l'oxygène moléculaire dans le substrat alcane. Ces enzymes jouent un rôle important dans la biodégradation du pétrole et dans la biodégradation cométabolique de composés tels que le trichloroéthylène et les éthers-carburants. De plus, ce sont des biocatalyseurs très utiles qui peuvent également servir de modèles pour caractériser une réaction chimique difficile : l'activation régio-et stéréospécifique de la liaison C-H. Plusieurs autres classes d'enzymes catalysent l'oxydation des alcanes. Les souches de levures capables de dégrader les alcanes contiennent plusieurs alcanes hydroxylases appartenant à la superfamille des P450, alors que différentes bactéries contiennent des enzymes similaires au système alcane hydroxylase membranaire de Pseudomonas putida GPo1. Les alcanes à courte chaîne sont probablement oxydés par des alcanes hydroxylases solubles similaires aux méthanes monooxygénases. La présence dans l'environnement de ces oxygénases a été étudiée dans des échantillons de sols et aquifères uniquement pour les alcanes hydroxylases associés aux membranes. Abstract -Diversity of Alkane Hydroxylase Systems in the Environment

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