Dataset Information

Online monitoring applying the anaerobic respiratory monitoring system reveals iron(II) limitation in YTF medium for Clostridium ljungdahlii.

ABSTRACT: Online monitoring of microbial cultures is an effective approach for studying both aerobic and anaerobic microorganisms. Especially in small-scale cultivations, several parallel online monitored experiments can generate a detailed understanding of the cultivation, compared to a situation where a few data points are generated from time course sampling and offline analysis. However, the availability of small-scale online monitoring devices for acetogenic organisms is limited. In this study, the previously reported anaerobic Respiration Activity MOnitoring System (anaRAMOS) device was adapted for online monitoring of Clostridium ljungdahlii (C. ljungdahlii) cultures with fructose as the carbon source. The anaRAMOS was applied to identify conversion of different carbon sources present in commonly used YTF medium. An iron(II) deficiency was discovered in this medium for C. ljungdahlii. Addition of iron(II) to the YTF medium reduced the cultivation time and increased biomass yield of C. ljungdahlii cultures by 50% and 40%, respectively. The measurement of the carbon dioxide transfer rate was used to calculated the iron(II) contained in complex components. By demonstrating the application of the anaRAMOS device for medium optimization, it is proven that the described online monitoring device has potential for use in process development.

SUBMITTER: Mann M

PROVIDER: S-EPMC7837299 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Online monitoring applying the anaerobic respiratory monitoring system reveals iron(II) limitation in YTF medium for <i>Clostridium ljungdahlii</i>.

Mann Marcel M Wittke Darina D Büchs Jochen J

Engineering in life sciences 20201105 1-2

Online monitoring of microbial cultures is an effective approach for studying both aerobic and anaerobic microorganisms. Especially in small-scale cultivations, several parallel online monitored experiments can generate a detailed understanding of the cultivation, compared to a situation where a few data points are generated from time course sampling and offline analysis. However, the availability of small-scale online monitoring devices for acetogenic organisms is limited. In this study, the pr ...[more]

PMID: 33531887

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Project description:One of the biggest limitations in the study and engineering of anaerobic Clostridium organisms is the lack of strong fluorescent reporters capable of strong and real-time fluorescence. Recently, we developed a strong fluorescent reporter system for Clostridium organisms based on the FAST protein. Here, we report the development of two new strong fluorescent reporter systems for Clostridium organisms based on the HaloTag and SNAP-tag proteins, which produce strong fluorescent signals when covalently bound to fluorogenic ligands. These new fluorescent reporters are orthogonal to the FAST ligands and to each other, allowing for simultaneous labeling and visualization. We used HaloTag and SNAP-tag to label the strictly anaerobic organisms Clostridium acetobutylicum and Clostridium ljungdahlii We have also identified a new strong promoter for protein expression in C. acetobutylicum, based on the phosphotransacetylase gene (pta) from C. ljungdahlii Furthermore, the HaloTag and the SNAP-tag, in combination with the previously described FAST system, were successfully used to measure cell populations in bacterial mixed cultures and showed the simultaneous orthogonal labeling of HaloTag and SNAP-tag together with the FAST protein reporter. Finally, we show the expression of recombinant fusion protein of FAST and the ZapA division protein (from C. acetobutylicum) in C. ljungdahlii. The availability of multiple strong fluorescent reporters is a major addition to the genetic toolkit of Clostridium and other anaerobes that will lead to better understanding of these unique organisms.IMPORTANCE Up to this point, assays and methods involving fluorescent reporter proteins were unavailable or limited in Clostridium organisms and other strict anaerobes. Green fluorescent protein (GFP), mCherry, and flavin-binding proteins (and their derivatives) have been used only in a few clostridia with limited success and yielded low fluorescence compared to aerobic microbial systems. Recently, we reported a new strong fluorescent reporter system based on the FAST protein as a first step in expanding the fluorescence-based reporters for Clostridium and other anaerobic microbial platforms. Additional strong orthogonal fluorescent proteins, with distinct emission spectra are needed to allow for (i) multispecies tracking within the growing field of microbial cocultures and microbiomes, (ii) protein localization and tracking in anaerobes, and (iii) identification and development of natural and synthetic promoters, ribosome-binding sites (RBS), and terminators for optimal protein expression in anaerobes. Here, we present two new strong fluorescent reporter systems based on the HaloTag and SNAP-tag proteins.

Dataset Information

Online monitoring applying the anaerobic respiratory monitoring system reveals iron(II) limitation in YTF medium for Clostridium ljungdahlii.

Publications

Online monitoring applying the anaerobic respiratory monitoring system reveals iron(II) limitation in YTF medium for <i>Clostridium ljungdahlii</i>.

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