Rapid reagentless quantification of alginate biosynthesis in Pseudomonas fluorescens bacteria mutants using FT-IR spectroscopy coupled to multivariate partial least squares regression

Abstract

Alginate is an important medical and commercial product and currently is isolated from seaweeds. Certain microorganisms also produce alginate and these polymers have the potential to replace seaweed alginates in some applications, mainly because such production will allow much better and more reproducible control of critical qualitative polymer properties. The research conducted here presents the development of a new approach to this problem by analysing a transposon insertion mutant library constructed in an alginate-producing derivative of the Pseudomonas fluorescens strain SBW25. The procedure is based on the non-destructive and reagent-free method of Fourier transform infrared (FT-IR) spectroscopy which is used to generate a complex biochemical infrared fingerprint of the medium after bacterial growth. First, we investigate the potential differences caused by the growth media fructose and glycerol on the bacterial phenotype and alginate synthesis in 193 selected P. fluorescens mutants and show that clear phenotypic differences are observed in the infrared fingerprints. In order to quantify the level of the alginate we also report the construction and interpretation of multivariate partial least squares regression models which were able to quantify alginate levels successfully with typical normalized root-mean-square error in predictions of only approximately 14%. We have demonstrated that this high-throughput approach can be implemented in alginate screens and we believe that this FT-IR spectroscopic methodology, when combined with the most appropriate chemometrics, could easily be modified for the quantification of other valuable microbial products and play a valuable screening role for synthetic biology.

Identification of genes affecting alginate biosynthesis in Pseudomonas fluorescens by screening a transposon insertion library

“…P. fluorescens was also grown in PIA medium [33], DEF4 medium [34] and DEF3 medium with low phosphate: KH 2 PO 4 0,14 mg/L, KCl 0.36 g/L, NH 4 Cl 2.21 g/L, citric acid · H 2 O 0.9 g/L, ferric citrate 0.02 g/L, H 3 BO 3 0.001 g/L, MnCl 2  · 4H 2 O 0.005 g/L, EDTA · 2H 2 O 0.0039 g/L, CuCl 2  · 2H 2 O 0.0005 g/L, Na 2 Mo 4 O 4  · 2H 2 O 0.0008 g/L, CoCl 2  · 6H 2 O 0.0008 g/L, Zn (CH 3 COO) 2  · 2H 2 O 0.0027 g/L, NaCl 1.56 g/L, MgSO 4  · 7H 2 O 0.57 g/L, MOPS 10 g/L. For precultures, 0.39 g/L yeast extract was added to the DEF4 medium.…”

“…Antibiotics used: ampicillin (Ap, 200 mg/L), tetracycline (Tc, 15 mg/L), apramycin (Am, 25 mg/L), kanamycin (Km, 50 mg/L). For growth in microtiter plates and micro bioreactors (BioLector®), half the concentrations of the media containing 7 g/L carbon source was used, and the cultures were incubated at 25 °C as detailed previously [34]. For some experiments adenine (0.8 mM), thiamine (0.05 mM), or tryptophan (2.5 mM) were added as medium supplements.…”

Phenotypic Characterisation of Shewanella oneidensis MR-1 Exposed to X-Radiation

“…The objective of the PLS model is to predict the classification of the new samples and in doing so, any separation between classes is displayed in score plots of the principal components [ 39 ]. Each model was cross-validated and an R 2 value generated, as described by Correa et al [ 40 ], in order to measure prediction performance.…”

“…DFA is a supervised method, whereby discrimination between groups is based on a priori knowledge of the experimental class structure. The algorithm finds linear combinations of the variables (canonical variates of the PCs fed into the algorithm) which maximize the ratio of between-group variance to within-group variance [ 40 , 43 ]. In order to quantify separation between control samples and treated samples at each growth phase, Euclidean distances between group centres were calculated from the scores plots of the first 3 DFs.…”

Quantification of alginate by aggregation induced by calcium ions and fluorescent polycations

“…Recently, Correa and coworkers have demonstrated the successful use of FT-IR spectroscopy to quantify alginate production by several Pseudomonas fluorescens strains. 6 The most commonly used carbazole assays involve heating the polysaccharides with concentrated sulfuric acid followed by an addition of an aromatic amine -carbazole. Carbazole-sulfuric acid method was first described by Dische 7 and further developed by Gurin and Hood 8 for the identification and estimation of hexoses and pentoses.…”