Jochen Büchs

High-throughput screening for ionic liquids dissolving (ligno-)cellulose

The recalcitrance of lignocellulosic biomass poses a major challenge for its sustainable and cost-effective utilization. Therefore, an efficient pretreatment is decisive for processes based on lignocellulose. A green and energy-efficient pretreatment could be the dissolution of lignocellulose in ionic liquids. Several ionic liquids were identified earlier which are capable to dissolve (ligno-)cellulose. However, due to their multitude and high costs, a high-throughput screening on small scale is essential for the determination of the most efficient ionic liquid. In this contribution two high-throughput systems are presented based on extinction or scattered light measurements. Quasi-continuous dissolution profiles allow a direct comparison of up to 96 ionic liquids per experiment in terms of their dissolution kinetics. The screening results indicate that among the ionic liquids tested EMIM Ac is the most efficient for dissolving cellulose. Moreover, it was observed that AMIM Cl is the most effective ionic liquid for dissolving wood chips.

Introduction to advantages and problems of shaken cultures

Shaking bioreactors are the most frequently used reaction vessels in biotechnology and have been so for many decades. In spite of their large practical importance, very little is known about the characteristic properties of shaken cultures from an engineering point of view. The few publications available contain to some extent contradicting statements and conflicting advice concerning the correct operating conditions of shaking bioreactors. Depending on the investigated microbial system, the engineering parameters may more or less significantly influence the experimental results in a quantitative as well as in a qualitative manner. Unfortunately, these kind of interactions are often overlooked or ignored by scientists. Precise knowledge about the controlling hydrodynamic phenomena in shaking bioreactors and quantitative information about the physical parameters influencing the cultures are needed to assure reproducible and meaningful operating conditions. In this introduction, the state of the art of culturing microorganisms in shaking bioreactors is reviewed and some issues of their practical application in screening and process development projects are addressed.

Methods for intense aeration, growth, storage, and replication of bacterial strains in microtiter plates.

ABSTRACT 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 generaPseudomonas, Rhodococcus, andAlcaligenes, 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.

Online respiration activity measurement (OTR, CTR, RQ) in shake flasks

Abstract Online measurement of respiration activity (including oxygen transfer rate (OTR), carbon dioxide transfer rate (CTR), respiratory quotient (RQ)) of microbial cultures in stirred bioreactors with exhaust gas analysis has been state of the art for years. As much more experiments are conducted in shaking bioreactors compared to stirred bioreactors, Anderlei and Buchs [Biochem. Eng. J. 7 (2001) 157] developed a measuring device (OTR-Device) for online determination of the oxygen transfer rate in shake flasks under sterile conditions. In this paper, an extension of the OTR-Device, termed respiration activity monitoring system (RAMOS) is described, which allows additional measurement of the carbon dioxide transfer rate and the respiratory quotient in shaking bioreactors. Fermentations of the yeasts Saccharomyces cerevisiae and Pichia stipitis carried out with RAMOS are presented. These measurements show very clearly the differences in respiration activities between the Crabtree-positive yeast S. cerevisiae and the Crabtree-negative yeast P. stipitis . Furthermore, a fermentation of the bacterium Corynebacterium glutamicum is presented, showing the influence of an oxygen limitation on the metabolic activities of the culture. Also, a fermentation of a hybridoma cell line was carried out with RAMOS to elucidate the measuring sensitivity of the system. The new device provides the most important and characteristic parameters (OTR, CTR, RQ) representing biological cultures online, enabling users to draw conclusions on metabolisms of microorganisms already in shaking bioreactors.

Device for sterile online measurement of the oxygen transfer rate in shaking flasks.

The oxygen transfer rate (OTR) is the most suitable measurable parameter to quantify the physiological state of a culture of aerobic microorganisms since most metabolic activities depend on oxygen consumption. Online measurement of the oxygen transfer rate in stirred bioreactors is state of the art although technically difficult. However, the online determination of the oxygen transfer rate in shaking bioreactors under sterile conditions has not been possible until recently. A newly developed measuring device eliminates this deficit. Extremely useful information about cultivating conditions and the physiological state of microorganisms can be gained in early stages of research and bioprocess development from many reactors operated in parallel.

Characterisation of the gas-liquid mass transfer in shaking bioreactors.

The maximum gas-liquid mass transfer capacity of 250ml shaking flasks on orbital shaking machines has been experimentally investigated using the sulphite oxidation method under variation of the shaking frequency, shaking diameter, filling volume and viscosity of the medium. The distribution of the liquid within the flask has been modelled by the intersection between the rotational hyperboloid of the liquid and the inner wall of the shaking flask. This model allows for the calculation of the specific exchange area (a), the mass transfer coefficient (k(L)) and the maximum oxygen transfer capacity (OTR(max)) for given operating conditions and requires no fitting parameters. The model agrees well with the experimental results. It was furthermore shown that the liquid film on the flask wall contributes significantly to the specific mass transfer area (a) and to the oxygen transfer rate (OTR).

Power consumption in shaking flasks on rotary shaking machines: I. Power consumption measurement in unbaffled flasks at low liquid viscosity.

In this first article of a series a new method is introduced that enables the accurate determination of the power consumption in a shaking flask. The method is based on torque measurements in the drive and appropriate compensation of the friction losses. The results for unbaffled shaking flasks at low viscosities are presented after varying shaking frequency, flask size, filling volume, shaking diameter, and surface quality (hydrophilic and hydrophobic) of the inner flask walls. The order of magnitude of the values of power consumption in shaking flasks is equal to, or even higher than, the values typical for agitated tank bioreactors. A physically based model equation for shaking flasks is derived that introduces a modified power number and a resulting constant as the only fitting parameter. With this equation, the measured results are correlated with sufficient accuracy. For the first time, comprehensive data for the power consumption in unbaffled shaking flasks at low viscosity is available, giving a detailed picture of the influences of the different variables.

Validation of a high-throughput fermentation system based on online monitoring of biomass and fluorescence in continuously shaken microtiter plates

BackgroundAn advanced version of a recently reported high-throughput fermentation system with online measurement, called BioLector, and its validation is presented. The technology combines high-throughput screening and high-information content by applying online monitoring of scattered light and fluorescence intensities in continuously shaken microtiter plates. Various examples in calibration of the optical measurements, clone and media screening and promoter characterization are given.ResultsBacterial and yeast biomass concentrations of up to 50 g/L cell dry weight could be linearly correlated to scattered light intensities. In media screening, the BioLector could clearly demonstrate its potential for detecting different biomass and product yields and deducing specific growth rates for quantitatively evaluating media and nutrients. Growth inhibition due to inappropriate buffer conditions could be detected by reduced growth rates and a temporary increase in NADH fluorescence. GFP served very well as reporter protein for investigating the promoter regulation under different carbon sources in yeast strains. A clone screening of 90 different GFP-expressing Hansenula polymorpha clones depicted the broad distribution of growth behavior and an even stronger distribution in GFP expression. The importance of mass transfer conditions could be demonstrated by varying filling volumes of an E. coli culture in 96 well MTP. The different filling volumes cause a deviation in the culture growth and acidification both monitored via scattered light intensities and the fluorescence of a pH indicator, respectively.ConclusionThe BioLector technology is a very useful tool to perform quantitative microfermentations under engineered reaction conditions. With this technique, specific yields and rates can be directly deduced from online biomass and product concentrations, which is superior to existing technologies such as microplate readers or optode-based cultivation systems. In particular, applications with strong demand on high-throughput such as clone and media screening and systems biology can benefit from its simple handling, the high quantitative information content and its capacity of automation.

Effect of Oxygen Limitation and Medium Composition on Escherichia coli Fermentation in Shake-Flask Cultures

Shake‐flask cultures are widely used for screening of high producing strains. To select suitable strains for production scale, cultivation parameters should be applied that provide optimal growth conditions. A novel method of measuring respiratory activity in shake‐flask cultures was employed to analyze Escherichia coli fermentation under laboratory conditions. Our results suggest that the length of fermentation, choice of medium, and aeration do not normally satisfy the requirements for unlimited growth in shake flasks. Using glycerol rather than glucose as a carbon source greatly reduced the accumulation of overflow and fermentative metabolites when oxygen supply was unlimited. A rich buffered medium, Terrific Broth (TB), yielded 5 times more biomass compared to LB medium but also caused oxygen limitation in standard shake‐flask cultures at shaking frequencies below 400 rpm. These results were used to optimize the production of benzoylformate decarboxylase from Pseudomonas putida in E. coli SG13009, resulting in a 10‐fold increase in volumetric enzyme production. This example demonstrates how variation of medium composition and oxygen supply can be evaluated by the measurement of the respiratory activity. This can help to efficiently optimize screening conditions for E. coli.

Itaconic acid--a biotechnological process in change.

In the last years, itaconic acid has gained increasing interest as future bio-based platform chemical. To replace petrol-based compounds such as methacrylic acid in industry, the economic efficiency of the current biotechnological production processes with the fungus Aspergillus terreus has to be improved. The recent progress in understanding the biosynthesis, the regulation and the cellular transport of itaconic acid has facilitated the optimisation of existing processes as well as the construction of new microbial platforms. However, there is still need for further optimisation to increase the space-time yield, to achieve higher final concentrations and to use a broader range of low cost sustainable raw materials. Genetic engineering and process development need to apply intelligent screening platforms to obtain as much information as possible in small scale.