Journal Article

FTIR spectroscopy as a unified method for simultaneous analysis of intra- and extracellular metabolites in high-throughput screening of microbial bioprocesses

Kohler, Achim, Shapaval, Volha, Kosa, Gergely, Zimmermann, Boris

2017

Overview

Background Analyses 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. Results HTS 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. Conclusions The 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.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Analysis / Applied Microbiology / Biomass / Biopolymers / Bioreactors / Biotechnology / Carbohydrates / Carboxylic acids / Chemistry / Chemistry and Materials Science / Chemometrics / Chromatography / Citric acid / Citric Acid - metabolism / Cultivation / Culture media / Culture Media - analysis / E coli / Enzymology / Fourier transform infrared spectroscopy / Fourier transforms / Fungi / Fungi - metabolism / Genetic Engineering / Glucose / Growth media / High performance liquid chromatography / High-throughput screening / High-Throughput Screening Assays / Infrared spectroscopy / Intracellular / Laboratory equipment / Least-Squares Analysis / Lipid metabolism / Lipids / Liquid chromatography / Metabolites / Methods / Microbial Genetics and Genomics / Microbiology / Microcultivation / Microorganisms / Mucor - metabolism / Multivariate Analysis / Nutrients / Oleaginous fungi / Partial least squares regression / Penicillium / Penicillium - metabolism / Polysaccharides / Proof of Concept Study / Proteins / Regression analysis / Saccharides / Screening / Spectroscopy, Fourier Transform Infrared - methods / Statistical analysis / Statistical methods / Studies / Substrates / Triglycerides - metabolism