Abstract
BackgroundAnalyses of substrate and metabolites are often bottleneck activities in high-throughput screening of microbial bioprocesses. We have assessed Fourier transform infrared spectroscopy (FTIR), in combination with high throughput micro-bioreactors and multivariate statistical analyses, for analysis of metabolites in high-throughput screening of microbial bioprocesses. In our previous study, we have demonstrated that high-throughput (HTS) FTIR can be used for estimating content and composition of intracellular metabolites, namely triglyceride accumulation in oleaginous filamentous fungi. As a continuation of that research, in the present study HTS FTIR was evaluated as a unified method for simultaneous quantification of intra- and extracellular metabolites and substrate consumption. As a proof of concept, a high-throughput microcultivation of oleaginous filamentous fungi was conducted in order to monitor production of citric acid (extracellular metabolite) and triglyceride lipids (intracellular metabolites), as well as consumption of glucose in the cultivation medium.ResultsHTS FTIR analyses of supernatant samples was compared with an attenuated total reflection (ATR) FTIR, which is an established method for bioprocess monitoring. Glucose and citric acid content of growth media was quantified by high performance liquid chromatography (HPLC). Partial least square regression (PLSR) between HPLC glucose and citric acid data and the corresponding FTIR spectral data was used to set up calibration models. PLSR results for HTS measurements were very similar to the results obtained with ATR methodology, with high coefficients of determination (0.91–0.98) and low error values (4.9–8.6%) for both glucose and citric acid estimates.ConclusionsThe study has demonstrated that intra- and extracellular metabolites, as well as nutrients in the cultivation medium, can be monitored by a unified approach by HTS FTIR. The proof-of-concept study has validated that HTS FTIR, in combination with Duetz microtiter plate system and chemometrics, can be used for high throughput screening of microbial bioprocesses. It can be anticipated that the approach, demonstrated here on single-cell oil production by filamentous fungi, can find general application in screening studies of microbial bioprocesses, such as production of single-cell proteins, biopolymers, polysaccharides, carboxylic acids, and other type of metabolites.
Highlights
Analyses of substrate and metabolites are often bottleneck activities in high-throughput screening of microbial bioprocesses
Reference measurements of intra‐ and extracellular metabolites Measurements of substrate and extracellular metabolite in the growth media were performed by high performance liquid chromatography (HPLC) (Fig. 1), while lipid accumulation in the biomass was measured by gas chromatography (GC) as previously reported [7]
HPLC measurements of organic acids and alcohols have shown significant citric acid production for P. glabrum cultivations, while productions of extracellular metabolites were negligible during the cultivation of M. circinelloides and U. isabellina
Summary
Analyses of substrate and metabolites are often bottleneck activities in high-throughput screening of microbial bioprocesses. Micro-bioreactors, usually in the form of multi-well microtiter plates, enable highthroughput parallel cultivation of microorganisms with culture volumes ranging from milliliter to nanoliter [1,2,3,4,5,6,7] Application of such systems saves valuable time and decrease costs in the development of bioprocesses. Screening of oleaginous microorganisms requires measurements of accumulation of intracellular lipids, as well as changes in chemical composition of the growth media This is obtained by tedious lipid extraction methods followed by gas chromatography (GC), while substrate consumption and release of extracellular metabolites is usually monitored by high performance liquid chromatography (HPLC) and by biochemical assays [1, 7, 8]. Chromatographies are powerful methods for the analysis of metabolites, different mobile-solid phase configurations are needed for different type of analytes based on their molecular weight, solubility, polarity, and other parameters, often the change of a configuration or instrumentation is needed
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