Degeneration of solventogenic Clostridium strains monitored by Fourier transform infrared spectroscopy of bacterial cells

The study of microbial heterogeneity at the single-cell level is a rapidly growing area of research in microbiology and biotechnology due to its significance in pathogenesis, environmental biology, and industrial biotechnologies. However, the tools available for efficiently and precisely probing such heterogeneity are limited for most bacteria. Here we describe the development and application of flow-cytometric (FC) and fluorescenceassisted cell-sorting techniques for the study of endospore-forming bacteria. We show that by combining FC light scattering (LS) with nucleic acid staining, we can discriminate, quantify, and enrich all sporulationassociated morphologies exhibited by the endospore-forming anaerobe Clostridium acetobutylicum. Using FC LS analysis, we quantitatively show that clostridial cultures commonly perform multiple rounds of sporulation and that sporulation is induced earlier by the overexpression of Spo0A, the master regulator of endospore formers. To further demonstrate the power of our approach, we employed FC LS analysis to generate compelling evidence to challenge the long-accepted view in the field that the clostridial cell form is the solvent-forming phenotype.

mentioning

confidence: 99%

Development and Application of Flow-Cytometric Techniques for Analyzing and Sorting Endospore-Forming Clostridia

Tracy

Gaida

Papoutsakis

2008

“…Transition from acidogenesis to solventogenesis is a prerequisite for a successful ABE fermentation process (7,8,18,25,34). However, when C. acetobutylicum is growing at or close to its maximum growth rate or when the metabolic rate of C. acetobutylicum is very high, excess acid instead of solvent is produced and the switch from acidogenesis to solventogenesis fails to occur (22). This phenomenon is termed "acid crash" (13).…”

mentioning

confidence: 99%

Formic Acid Triggers the “Acid Crash” of Acetone-Butanol-Ethanol Fermentation by Clostridium acetobutylicum

Wang

Zhang

Dong

et al. 2011

109

Solvent production by Clostridium acetobutylicum collapses when cells are grown in pH-uncontrolled glucose medium, the so-called "acid crash" phenomenon. It is generally accepted that the fast accumulation of acetic acid and butyric acid triggers the acid crash. We found that addition of 1 mM formic acid into corn mash medium could trigger acid crash, suggesting that formic acid might be related to acid crash. When it was grown in pH-uncontrolled glucose medium or glucose-rich medium, C. acetobutylicum DSM 1731 containing the empty plasmid pIMP1 failed to produce solvents and was found to accumulate 0.5 to 1.24 mM formic acid intracellularly. In contrast, recombinant strain DSM 1731 with formate dehydrogenase activity did not accumulate formic acid intracellularly and could produce solvent as usual. We therefore conclude that the accumulation of formic acid, rather than acetic acid and butyric acid, is responsible for the acid crash of acetone-butanolethanol fermentation.

“…Analysis by FT-IR spectroscopy also includes the measurement of proteins and nucleic acids which are not naturally included in the metabolome definition. FT-IR spectroscopy has previously been successfully applied to study bioprocesses (industrial fermentations) with respect to the detection of degenerate variants in solventogenic clostridia (40), the monitoring of ␣ 2 interferon production by Escherichia coli (27), and gibberellic acid production by the fungus Gibberella fujikuroi (26). These studies focused on analyzing the actual bacteria or monitoring the production of a specific metabolite.…”

mentioning

confidence: 99%

High-Throughput Metabolic Fingerprinting of Legume Silage Fermentations via Fourier Transform Infrared Spectroscopy and Chemometrics

Johnson

Broadhurst

Kell

et al. 2004

Silage quality is typically assessed by the measurement of several individual parameters, including pH, lactic acid, acetic acid, bacterial numbers, and protein content. The objective of this study was to use a holistic metabolic fingerprinting approach, combining a high-throughput microtiter plate-based fermentation system with Fourier transform infrared (FT-IR) spectroscopy, to obtain a snapshot of the sample metabolome (typically low-molecular-weight compounds) at a given time. The aim was to study the dynamics of red clover or grass silage fermentations in response to various inoculants incorporating lactic acid bacteria (LAB). The hyperspectral multivariate datasets generated by FT-IR spectroscopy are difficult to interpret visually, so chemometrics methods were used to deconvolute the data. Two-phase principal component-discriminant function analysis allowed discrimination between herbage types and different LAB inoculants and modeling of fermentation dynamics over time. Further analysis of FT-IR spectra by the use of genetic algorithms to identify the underlying biochemical differences between treatments revealed that the amide I and amide II regions (wavenumbers of 1,550 to 1,750 cm ؊1 ) of the spectra were most frequently selected (reflecting changes in proteins and free amino acids) in comparisons between control and inoculant-treated fermentations. This corresponds to the known importance of rapid fermentation for the efficient conservation of forage proteins.