In vivo quantification of polyhydroxybutyrate (PHB) in the alphaproteobacterial methanotroph, Methylocystis sp. Rockwell

Abstract

Methane is a common industrial by-product that can be used as feedstock for production of the biopolymer polyhydroxybutyrate (PHB) by alphaproteobacterial methanotrophs. In vivo assessment of PHB production would shed light on the biosynthesis process and guide design of improved production strategies, but it is currently difficult to perform efficiently. In this study, the alphaproteobacterial methanotroph Methylocystis sp. Rockwell was grown on methane with three different nitrogen sources (ammonium, nitrate, and atmospheric nitrogen), and biomass samples were harvested at defined time points during lag, exponential, and stationary growth phases. PHB cell content was analyzed at these sampling points via a standard gas chromatography-flame ionization detector method, which requires hydrolysis of PHB and esterification of the resulting monomer under acidic conditions, and a novel, rapid, cost-effective approach based on fixation and staining of bacterial cells via Nile Blue A fluorescent dye enabling differential staining of cell membranes and intracellular PHB granules for single-cell analysis through fluorescence microscopy. Overall, the two PHB quantification approaches were in agreement at all stages of growth and in all three growing conditions tested. The PHB cell content was greatest with atmospheric nitrogen as a nitrogen source, followed by ammonium and nitrate. Under atmospheric nitrogen and ammonium conditions, PHB cell content decreased with growth progression, while under nitrate conditions PHB cell content remained unchanged in all growth phases. In addition to presenting a rapid, efficient method enabling in vivo quantification of PHB production, the present study highlights the impact of nitrogen source on PHB production by Methylocystis sp. Rockwell. Key points• A novel fluorescence microscopy method to quantify PHB in single cells was developed • The microscopy method was validated by the derivation/gas chromatography method • Methylocystis sp. Rockwell synthesizes PHB granules without nutrient stress Keywords Polyhydroxybutyrate • Methylocystis sp. Rockwell • Methanotroph • Fluorescence microscopy • Granule visualization • Gas chromatography Marina Lazic and Ravindra Gudneppanavar contributed equally to this work.

Resource availability governs polyhydroxyalkanoate (PHA) accumulation and diversity of methanotrophic enrichments from wetlands

“…For example, others have reported 22.2% PHA from a rice field soil enrichment ( Kulkarni et al, 2022 ), 39% from an activated sludge enrichment ( Myung et al, 2015 ), 43.1% from a pure culture Methylocystis hirsuta fed with biogas ( López et al, 2018 ), and 25.9% from a pure culture of Methylocystis sp. Rockwell in nitrogen-free media ( Lazic et al, 2022 ). The oxygen limitation experienced by our high resource enrichments could have contributed to the limited PHA production as this has been shown to negatively affect methanotroph PHA production ( Zaldívar Carrillo et al, 2018 ).…”

“…PHA granules were stained with Nile Blue A and visualized with epifluorescence microscopy ( Lazic et al, 2022 ). Cells collected after a PHA accumulation cycle were diluted and stained with 10 µL of 30-fold diluted SYBR Green solution and 10 µL Nile Blue A solution (0.05% (w/v) Nile Blue A in ethanol) per milliliter.…”

“…The percent area was converted to a percent volume by assuming a volume equal to that of a sphere with the measured cross-sectional area. Finally, the percent volume was converted to a percent mass using the 1.099 PHA to biomass density ratio described by ( Lazic et al, 2022 ). To calculate the percentage of cells harboring PHA, the number of cells with detectable PHA signal was divided by the image cell count based on SYBR Green DNA staining.…”

Methylosinus trichosporium OB3b bioaugmentation unleashes polyhydroxybutyrate-accumulating potential in waste-activated sludge

“…For cultivation, the ammonia mineral salt medium (AMS) was chosen due to its superior ability to accumulate polyhydroxybutyrate (PHB) compared to nitrate mineral salt medium (NMS) [32][33][34] In 160 mL serum bottles, 43 mL of AMS medium was distributed and sealed with butyl rubbers and aluminum caps. After autoclaving, the media were supplemented with 0.5 mL of 10x vitamin stock, 0.5 mL of 0.4 M of phosphate buffer solution, and 1 mL of 0.5 mM CuCl 2 •2H 2 O solution.…”

Monitoring Phenotype Heterogeneity at the Single-Cell Level within Bacillus Populations Producing Poly-3-hydroxybutyrate by Label-Free Super-resolution Infrared Imaging

“…At the single-cell level, microbial populations display tremendous cell-to-cell variations in phenotypic traits due to random stochastic fluctuations in biochemical processes as well as use these adaptive strategies for population growth and survival. − The development of phenotypic heterogeneity within microbial populations may occur due to intrinsic factors such as cell-growth phase and variable gene expression as well as extrinsic external factors such as heat, aeration, or acidity, which cause significant environmental perturbations. ,, Metabolic heterogeneity is a major issue in the context of industrial bioprocessing as it may negatively affect the overall productivity; therefore, monitoring phenotypic instability at the single-cell level is crucial to the development of robust and high-yield bioprocesses. ,− Flow cytometry is the most common tool to probe microbial phenotypic heterogeneity in bioprocesses, and it has previously been used to quantify PHB. − Advanced imaging methods such as transmission electron microscopy (TEM) and fluorescence-based microscopy are often employed to analyze the formation of PHB granules within cells. − Recently, optical photothermal infrared (O-PTIR) spectroscopy has emerged as a tool for single-cell microbial metabolomics with potential applications for monitoring bacterial phenotype heterogeneity including intracellular PHB content. − In contrast to TEM, O-PTIR signatures enable biochemical analysis by measuring the infrared spectrum of a single bacterium, which provides important molecular information on small molecules, intermediates, and products of microbial metabolism. Compared to fluorescence-based methods, O-PTIR is a label-free technique and therefore does not rely on commercially available fluorescent probes that are susceptible to photobleaching or may even interfere with cell metabolism .…”