Microscale Quantitative Analysis of Polyhydroxybutyrate in Prokaryotes Using IDMS

Poly(3-hydroxybutyrate) (PHB) is an interesting biopolymer for replacing petroleum-based plastics, its biological production is performed in natural and engineered microorganisms. Current metabolic engineering approaches rely on high-throughput strain construction and screening. Analytical procedures have to be compatible with the small scale and speed of these approaches. Here, we present a method based on isotope dilution mass spectrometry (IDMS) and propanolysis extraction of poly(3-hydroxybutyrate) from an Escherichia coli strain engineered for PHB production. As internal standard (IS), we applied an uniformly labeled 13C-cell suspension, of an E. coli PHB producing strain, grown on U-13C-glucose as C-source. This internal 13C-PHB standard enables to quantify low concentrations of PHB (LOD of 0.01 µg/gCDW) from several micrograms of biomass. With this method, a technical reproducibility of about 1.8% relative standard deviation is achieved. Furthermore, the internal standard is robust towards different sample backgrounds and dilutions. The early addition of the internal standard also enables higher reproducibility and increases sensitivity and throughput by simplified sample preparation steps.


Fragmentation of Derivatized 3-HB
Two major fragments were identified by GC-MS for PHB analyses: m/z 131 and m/z 145. The fragment m/z 131 was used for the quantification, the mass of the internal standard was m/z 134. The retention time of the fragments was 4.53 minutes. Figure S1. Chemical structure of derivatized 3-HB and observed fragments. Table S1. Preparation of a standard calibration (external) using the following standard concentrations (13 points).

Complete degradation of the polymer
The standard method suggests a time for derivatization of two hours. Thus, was desired to confirm whether a complete degradation of the polymer could be achieved in less time. The (PHB)1 concentration at different time points 30, 60, 120, 150, and 180 minutes was tested. One milliliter samples were taken from one shake flask in triplicate, the peak areas obtained were corrected with the IS-13 C-PHB. The concentration in the broth was 2.925 ± 0.044. The t-test (p < 0.05) indicated no significant differences between the time points measured and the concentration in the broth.

Homogeneity
During derivatization, vortexing is an important step for the complete mixing of the sample. The standard method recommends to vortex every 30 minutes during derivatization. Hence, was determined if the mixing is enough with the boiling itself. Three samples from one shake flask were measured without vortexing during derivatization. The concentrations were corrected with the IS-13 C-PHB. The results show that there is no need for vortexing during derivatization. However, this was only tested with E. coli, is possible that other species might not deliver the same result if not vortexed.

Biomass quantification:
In order to know the content of PHB in biomass. Cell dry weight was compared by two methods: filtration and freeze drying.
To determine biomass content by filtration, a 5 mL sample in triplicate was taken from one shake flask. Through freeze drying, first was determined the weight of IS-13 C-PHB and PAA only, as a blank. Then the biomass was measured subtracting such value to obtain the actual value of cell dry weight per one milliliter. Each vial was weighted before adding sample and after freeze drying.
There was no significant difference between the two methods used for the biomass determination. Table S6. Comparison of biomass determination in g/L through freeze drying and filtration.

Calibration using Benzoic Acid as Internal Standard
The stock solutions of the internal standards PAA and BA were prepared in a concentration of 0.02 mmol/mL. A volume of 50 µL of each internal standard was added to the samples. The step in which the internal standards were added in the samples differs, BA was added after freeze drying and PAA before freeze drying. The calibration lines obtained were analyzed with and without the addition ofIS-13 C-PHB. The internal standard BA was added to compare with the IS proposed in this study. The relative and the absolute error of the data obtained with BA, was calculated with the presence of IS-13 C-PHB that was 0.0045 and 0.0004, respectively.

Calibration using Phenyl Acetic Acid as Internal Standard
The calibration lines obtained were analyzed in the same way as with BA with and without the presence of the IS-13 C-PHB. The purpose of adding the internal standard PAA was to have a similar correction as the 13 C-PHB, because was added before freeze drying. The relative and the absolute error with the presence of IS-13 C-PHB was calculated that was 0.0137 and 0.00005, respectively. Therefore, such internal standards were not reliable for quantification with GC-IDMS. Figure S4. Measured 12 C/PAA ratio and linear regression line. Both standard concentration and measured ratio are in log scale.
The internal standards PAA and BA showed lower reproducibility and when spiked with a known amount of (PHB)1 the recoveries were overestimated with BA and underestimated with PAA. The overestimation using BA comes from adding the internal standard after the freeze drying. Therefore, the correction does not take into account losses of the first steps.