Trends in Microbiology
ReviewMicrotiter plates as mini-bioreactors: miniaturization of fermentation methods
Section snippets
Progression from Erlenmeyer flasks to microtiter plates
The adoption of the shake flask in the 1880s as the vessel of choice for the growth of microorganisms in liquid cultures was pragmatic in the sense that the Erlenmeyer flask had been invented 20 years earlier, for use in chemical laboratories. More than a century later, the relatively large culture volumes in Erlenmeyer flasks gradually became obsolete for many research projects. Modern microbiologists showed a similar degree of pragmatism by adopting another existing vessel type – this time
Physicochemical effects of miniaturization
The size of the bulk of suspended cultures in 300 ml Erlenmeyer flasks is a factor 104 larger than that of an average microbial cell (5 cm versus 5 μm), whereas this factor is reduced to 103 in a 96-MTP well. It is reasonable to assume that the direct effects of this dimensional change on the physiology of an individual microbial cell are minimal. However, two indirect effects have significant consequences for an individual cell. One is the intrinsically higher ratio of the gas–liquid exchange
Well-closure systems
A crucial aspect of microbial growth in MTPs is the closure system of the individual wells, which should prevent cross-contamination during vigorous shaking, permit a limited degree of exchange of headspace air and limit evaporation. An identical physical condition in all wells is a further requirement for quantitative studies; the wells in the corners should have exactly the same characteristics as the wells in the middle of the microtiter plate. This is especially important for media
Gas–liquid oxygen-transfer rates
A sufficiently high exchange rate of headspace air is a prerequisite but not a guarantee that the cells growing in the wells are supplied with an adequate amount of oxygen; gas–liquid transfer is usually the chief limiting factor in this respect. Virtually all publications in this area have focused on orbital shaking as a mode to increase the gas–liquid exchange area. Stirring and bubbling are generally considered to be impractical for large numbers of cultures.
Methods for determining OTRs
To compare various types of miniature growth vessels and incubation conditions in quantitative terms, reliable methods for OTR determinations are essential. The cobalt-catalyzed oxidation of sulphite and the dynamic gassing-out method are used most commonly. The cobalt-catalyzed oxidation of sulphite method is suitable for volumes of less than 1 ml [11]. A minor disadvantage of this method is the high salt concentration applied (usually 0.5 M), which reduces the maximal solubility of oxygen to a
Cultivation in polypropylene square-well MTPs
Earlier literature on cultivation in MTPs 9, 15 focused on polypropylene MTPs with square deep-wells (8 × 8 mm in the horizontal plane, 40 mm deep, a well volume of 2.4 ml and culture volumes between 0.5 and 1 ml). OTRs were derived from the oxygen disappearance due to the cobalt-catalyzed oxidation of sulfite and from the oxygen-limited growth phase of growth curves of P. putida. The OTR values of these two methods were the same within an error margin of 15% if a proper correction factor (see
Cultivation in polystyrene round-well MTPs
Polystyrene round-well MTPs (especially 96- and 48-well plates) have received much attention in more recent years. The advantages of polystyrene MTPs include their transparency, which enables the direct reading of optical densities, the absence of toxic substances leaking out from polystyrene and the presence of two walls separating the wells rather than one, which further minimizes the risk of cross-contamination.
Hermann and Buchs [5] yielded a thorough insight into the influence of various
Mutant screenings
Open literature on the application of MTPs for mutant screenings is scarce and mainly limited to metabolic flux analysis studies in academic settings 18, 19, 20, 21, 22, 23, 24. The group of Uwe Sauer used 96-square deep-well MTPs to study glucose metabolism in 137 knockout mutants of B. subtilis using 13C tracer technology [18]. The application of small culture volumes in this research area is particularly appealing in the light of the high costs of 13C-labeled compounds. Culture volumes of 1.2
Discovery of new secondary metabolites
24-square well MTPs are often used in the discovery of new secondary metabolites (for reviews, see 26, 27). The relative ease with which high OTRs are achieved [13] ensures a high biomass and sufficient secondary metabolites formed by anabolic pathways requiring oxygenases. Furthermore, culture volumes of 2–4 ml enable a straightforward separation of cells, pellets or mycelia from the supernatant by centrifugation and adequate amounts of extracted compounds for multiple bioassays, such as those
Miscellaneous applications
Casey [28] described the successful use of MTPs to generate dose–response curves of test organisms (E. coli, B. subtilis and S. cerevisiae) to bioactive compounds formed by Streptomyces hygroscopicus. Diaz et al. [29] described a rapid assay for methionine using a methionine auxotroph E. coli strain growing in MTPs.
Doig et al.[30] used several microwell formats (96-round-, 96-deep square- and 24-round-well microtiter plates) for quantification of the kinetics of a Baeyer-Villiger oxygenase
Conclusions
In the past 7 years, MTPs have become a mature alternative to Erlenmeyer flasks for the batch cultivation of microorganisms and have helped to make the handling and screening of large numbers of strains and mutant libraries less time consuming. Orbital shaking is suitable to generate OTRs in MTPs equaling or even surpassing the OTRs achieved traditionally in Erlenmeyer flasks, if the right shaking conditions are used. The development of suitable cover systems has ruled out cross-contaminations.
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2021, Bioresource TechnologyCitation Excerpt :The MC1 static microplate strategy was not very efficient due to the difficulties of avoiding cross-contamination among the isolates; however, the isolates classification sequence was the same as the traditional shaker cultivation (SC), which suggests that the MC1 has potential and could be improved in the future. This strategy is also known as microtiter plate (MTP) and has been studied as mini-bioreactors for the production of several metabolites, suggesting well-closed systems with orbital shaking and headspace air to improve gas–liquid oxygen-transfer (Back et al., 2016; Duetz, 2007). In contrast, the MC2 and MC3 strategies in microtubes and cotton caps were efficient because they allowed the exchange of oxygen even though it was a static culture, and the isolates classification order was the same as SC.