Prediction of intracellular storage polymers using quantitative image analysis in enhanced biological phosphorus removal systems

Abstract

The present study focuses on predicting the concentration of intracellular storage polymers in enhanced biological phosphorus removal (EBPR) systems. For that purpose, quantitative image analysis techniques were developed for determining the intracellular concentrations of PHA (PHB and PHV) with Nile blue and glycogen with aniline blue staining. Partial least squares (PLS) were used to predict the standard analytical values of these polymers by the proposed methodology. Identification of the aerobic and anaerobic stages proved to be crucial for improving the assessment of PHA, PHB and PHV intracellular concentrations. Current Nile blue based methodology can be seen as a feasible starting point for further enhancement. Glycogen detection based on the developed aniline blue staining methodology combined with the image analysis data proved to be a promising technique, toward the elimination of the need for analytical off-line measurements.

Polyhydroxyalkanoate granules quantification in mixed microbial cultures using image analysis: Sudan Black B versus Nile Blue A staining

“…For fluorescence image acquisition, and for each sample, 1 mL of fixed cell suspension was incubated at 55 C for 10 min with 1 drop of NBA according to the methodology described in Ostle and Holt [26], and further centrifuged at 4500 rpm for 5 min as described in Mesquita et al [21]. Pellet cells were washed with 0.9% NaCl and centrifuged using the same conditions.…”

“…Furthermore, additional procedures include more specialized staining techniques to detect intracellular storage compounds in MMC, such as PHA, poly-P and glycogen, as previously reviewed by Serafim et al [16] and Mesquita et al [17]. Nile Red, Nile Blue A (NBA), and Sudan Black B (SBB) are among the most used dyes for selective staining of PHA inclusions [18][19][20][21][22]. However, these staining procedures have been rarely applied in routine analysis, and QIA application has never been considered before as an alternative method to quantify intracellular storage compounds such as PHA.…”

A Critical Assessment of the Microorganisms Proposed to be Important to Enhanced Biological Phosphorus Removal in Full-Scale Wastewater Treatment Systems

“…Typically, FISH has been applied with microautoradiography (MAR; Nierychlo et al, 2015) and fluorescent staining methods (Serafim et al, 2002) to demonstrate in situ uptake of carbon and P and, PHA and polyphosphate storage, respectively. Raman-spectroscopy and novel staining methods should also allow in situ analyses of glycogen (Huang et al, 2007; Majed and Gu, 2010; Mesquita et al, 2013). Such methods have been applied to at least partially demonstrate the PAO phenotype in situ for “ Candidatus Accumulibacter spp.” (Hesselmann et al, 1999; Crocetti et al, 2000; Kong et al, 2004), Tetrasphaera spp.…”

Determination of Sudan I in duck feed by microscopic image processing and confocal Raman spectroscopy

“…With the improvement of commercial spectrometers, spectroscopy technology such as uorescence spectroscopy, 14 mid-and near-infrared spectroscopy, 15 terahertz spectroscopy, 16 and Raman spectroscopy, [17][18][19] have been used to detect adulteration. 24 In chemometrics, qualitative identication is performed via partial least-squares discriminant analysis (PLS-DA). It formally contains non-polar groups such as -N]Nand a benzene ring, which have obvious Raman spectral characteristic peaks.…”