In situ multi-wavelength fluorescence spectroscopy as effective tool to simultaneously monitor spore germination, metabolic activity and quantitative protein production in recombinant Aspergillus niger fed-batch cultures

Abstract

The production of a mutant green fluorescent protein (S65TGFP), controlled by the maltose inducible glucoamylase promoter, was followed in situ in fed-batch cultures of recombinant Aspergillus niger using multi-wavelength fluorescence spectroscopy. Disturbance of quantitative product analysis by interfering fluorescence signals was resolved by using a set of defined combinations of excitation and emission wavelengths (lambda(ex)/lambda(em)). This technique resulted in excellent linearity between on-line signal and off-line determined S65TGFP concentrations. Spore germination was detectable in situ by monitoring the back scattered light intensity. Moreover, flavin-like fluorophores were identified as the dominating fungal host fluorophores. The time-dependent intensity of this fluorophore, potentially fungal flavin-containing oxidoreductase(s), did not correlate with the biomass concentration but correlated well with the fungal metabolic activity (e.g. respiratory activity). Other fluorophores commonly found in microbial cultures such NADH, pyridoxine and the aromatic amino acids, tryptophan, phenylalanine and tyrosine did not contribute significantly to the culture fluorescence of A. niger. Thus, multi-wavelength fluorescence spectroscopy has proven to be an effective tool for simultaneous on-line monitoring of the most relevant process variables in fungal cultures, e.g. spore germination, metabolic activity, and quantitative product formation.

On-line estimation of biomass, glucose and ethanol in Saccharomyces cerevisiae cultivations using in-situ multi-wavelength fluorescence and software sensors

“…MWF has been applied in combination with multivariate modelling for on-line monitoring of a number of microbial bioprocesses including cultivations of bacteria (Skibsted et al, 2001;Eliasson Lantz et al, 2006;Mortensen and Bro, 2006), filamentous fungi (Ganzlin et al, 2007;Haack et al, 2007) and yeasts (Haack et al, 2004;Hantelmann et al, 2006;Surribas et al, 2006a, b), generally yielding useful prediction models for relevant process variables, e.g., cell mass concentration. The method is limited to measurement of fluorescent compounds; therefore MWF has mainly been applied for biomass monitoring.…”

7.1 Concepts and Technologies for Advanced Process Monitoring and Control

“…As well as other spectroscopic methods, two-dimensional fluorescence spec- Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 7/6/15 12:02 PM troscopy provides an enormous amount of real-time data that need to be analyzed using multivariate chemometric techniques in order to extract relevant information from the spectra. In particular the application of principal component analysis (PCA) and partial least squares projection to latent structures (PLS), which is the regression extension of PCA [54], helped to pave the way for successful application of twodimensional fluorescence spectroscopy as a PAT tool in bioprocess monitoring. Multiple reports from the last recent years emphasize the suitability of two-dimensional fluorescence spectroscopy in different fields of biotechnological application comprising waste water treatment [55][56][57], food technology [58], as well as biopharmaceutical technology using fungi [54,59,60], yeast [50, 51 ,61], and bacteria [51,[62][63][64].…”

Evaluation of wavelength selection methods for 2D fluorescence spectra applied to bioprocesses characterization

“…spectroscopy have been highlighted in many recent studies (Boehl et al, 2003;Hantelmann et al, 2006;Ganzlin et al, 2007;Rhee and Kang, 2007;Ödman et al, 2009;Jain et al, 2011;Bogomolov et al, 2011;Rossi et al, 2012). In this technique, the fluorescence emission of metabolites present in the cultivation medium is monitored when they are excited with UV-Visible radiation in a specific wavelength range.…”

“…This non-invasive technique can be considered an evolution of the fluorescence sensor that could monitor only the fluorophore NAD(P)H and then would obtain information on cellular metabolism. Once the 2D fluorescence spectroscopy is capable of monitoring all fluorophores, it can yield information not only about cellular metabolism, but also about the cultivation medium (Scheper et al 1999, Ganzlin et al 2007.…”