Organic acids and alcohols quantification by HPLC/RID in sugarcane vinasse: analytical method validation and matrix effect assessment

Bernardes, Alan Nascimento; Godoi, Leandro Augusto Gouvêa de; Camiloti, Priscila Rosseto; Gomes, Absai da Conceição; Sanchez, Bruna Larissa Sandy; Torres, Audálio Rebelo; Botta, Lívia Silva

doi:10.1080/03067319.2019.1666833

“…HPLC coupled to refractive index detection (HPLC-RID) is a generally accessible method for quickly identifying central metabolic precursors and end-products. Briefly, RID measures how analytes change the deflection of light by the mobile phase 12 . RID signals corresponding to target analytes in samples can then be quantified by comparisons with RID signals of standard solutions.…”

Section: Introductionmentioning

confidence: 99%

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Dinglasan

¹

,

Reeves

²

,

Hettich

³

et al. 2021

JoVE

View full text Add to dashboard Cite

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the cell's survival requirements. Alternatively, carrying out DBTL cycles in cell-free environments can accelerate this process and alleviate concerns with host compatibility. A promising approach to cell-free metabolic engineering (CFME) leverages metabolically active crude cell extracts as platforms for biomanufacturing and for rapidly discovering and prototyping modified proteins and pathways. Realizing these capabilities and optimizing CFME performance requires methods to characterize the metabolome of lysate-based cell-free platforms. That is, analytical tools are necessary for monitoring improvements in targeted metabolite conversions and in elucidating alterations to metabolite flux when manipulating lysate metabolism. Here, metabolite analyses using high-performance liquid chromatography (HPLC) coupled with either optical or mass spectrometric detection were applied to characterize metabolite production and flux in E. coli S30 lysates. Specifically, this report describes the preparation of samples from CFME lysates for HPLC analyses using refractive index detection (RID) to quantify the generation of central metabolic intermediates and by-products in the conversion of low-cost substrates (i.e., glucose) to various high-value products. The analysis of metabolite conversion in CFME reactions fed with 13 C-labeled glucose through reversed-phase liquid chromatography coupled to tandem mass spectrometry (MS/ MS), a powerful tool for characterizing specific metabolite yields and lysate metabolic flux from starting materials, is also presented. Altogether, applying these analytical methods to CFME lysate metabolism enables the advancement of these systems as alternative platforms for executing faster or novel metabolic engineering tasks.

show abstract

“…HPLC coupled to refractive index detection (HPLC-RID) is a generally accessible method for quickly identifying central metabolic precursors and end-products. Briefly, RID measures how analytes change the deflection of light by the mobile phase 12 . RID signals corresponding to target analytes in samples can then be quantified by comparisons with RID signals of standard solutions.…”

Section: Introductionmentioning

confidence: 99%

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Dinglasan

¹

,

Reeves

²

,

Hettich

³

et al. 2021

JoVE

View full text Add to dashboard Cite

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the cell's survival requirements. Alternatively, carrying out DBTL cycles in cell-free environments can accelerate this process and alleviate concerns with host compatibility. A promising approach to cell-free metabolic engineering (CFME) leverages metabolically active crude cell extracts as platforms for biomanufacturing and for rapidly discovering and prototyping modified proteins and pathways. Realizing these capabilities and optimizing CFME performance requires methods to characterize the metabolome of lysate-based cell-free platforms. That is, analytical tools are necessary for monitoring improvements in targeted metabolite conversions and in elucidating alterations to metabolite flux when manipulating lysate metabolism. Here, metabolite analyses using high-performance liquid chromatography (HPLC) coupled with either optical or mass spectrometric detection were applied to characterize metabolite production and flux in E. coli S30 lysates. Specifically, this report describes the preparation of samples from CFME lysates for HPLC analyses using refractive index detection (RID) to quantify the generation of central metabolic intermediates and by-products in the conversion of low-cost substrates (i.e., glucose) to various high-value products. The analysis of metabolite conversion in CFME reactions fed with 13 C-labeled glucose through reversed-phase liquid chromatography coupled to tandem mass spectrometry (MS/ MS), a powerful tool for characterizing specific metabolite yields and lysate metabolic flux from starting materials, is also presented. Altogether, applying these analytical methods to CFME lysate metabolism enables the advancement of these systems as alternative platforms for executing faster or novel metabolic engineering tasks.

show abstract

Effect of food industry by-products on bacterial cellulose production and its structural properties

Heydorn

¹

,

Lammers

²

,

Gottschling

³

et al. 2023

View full text Add to dashboard Cite

The advances in bioprocess design, genetic engineering, and media optimization have enabled enhanced bacterial cellulose (BC) production and its application for diverse purposes. Following the requirements of a bioeconomy, numerous approaches were conducted to investigate alternative carbon or nitrogen sources from industrial by-products for BC biosynthesis. They can, however, not only affect the BC production but also its properties. Beet molasses, vinasse, and waste beer fermentation broth (WBFB) have thus been investigated in single and combined approaches for their BC production potential and effects on structural properties using Komagataeibacter xylinus DSM 2325. Therefore, the composition of each complex component was initially analyzed for total organic carbon (TOC), total bound nitrogen (TNb), sugars, organic acids, and alcohols. The polymer properties were characterized via gel permeation chromatography and X-ray diffraction. In dynamic shake flask cultivations, the exchange of Hestrin-Schramm (HS) medium components for a combination of all three complex substrates on a TOC- or TNb-based quantity resulted in the highest BC concentration (8.2 g L−1). Comparable concentrations were achieved when combining molasses and WBFB (8.1 g L−1). Each investigated complex component led to differing degrees of polymerization (DPn: 2751−4601) and BC crystallinities (26−58%) in comparison to HS medium. Beet molasses and vinasse were found to decrease the polymer crystallinity but induce higher DPn whereas the opposite occurred for WBFB. This study thus highlights beneficial effects of food industry by-products for BC biosynthesis and elucidates concomitantly occurring structural polymer alterations to enable further studies dealing with alternative substrates for structurally tailored BC production.

show abstract

Organic acids and alcohols quantification by HPLC/RID in sugarcane vinasse: analytical method validation and matrix effect assessment

Cited by 4 publications

References 26 publications

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Effect of food industry by-products on bacterial cellulose production and its structural properties

Contact Info

Product

Resources

About