Quantitative Determination of the Biodegradable Polymer Poly(β-hydroxybutyrate) in a Recombinant Escherichia coli Strain by Use of Mid-Infrared Spectroscopy and Multivariative Statistics

Abstract

Fourier transform infrared (FTIR) spectroscopy in combination with the partial least squares (PLS) multivariative statistical technique was used for quantitative analysis of the poly(␤-hydroxybutyrate) (PHB) contents of bacterial cells. A total of 237 replicate spectra from 34 samples were obtained together with gas chromatography-determined reference PHB contents. Using the PLS regression, we were able to relate the infrared spectra to the reference PHB contents, and the correlation coefficient between the measured and predicted values for the optimal model with a standard error of prediction of 1.49% PHB was 0.988. With this technique, there are no solvent requirements, sample preparation is minimal and simple, and analysis time is greatly reduced; our results demonstrate the potential of FTIR spectroscopy as an alternative to the conventional methods used for analysis of PHB in bacterial cells.Many prokaryotic microorganisms synthesize polyhydroxyalkanoates (PHAs) as carbon and energy reserves (2,5,8,25,26), and poly(␤-hydroxybutyrate) (PHB) is the most common PHA. The general chemical structure of these compounds is shown in Fig. 1.The composition of the alkyl side chain (the R group) and the number of methylene units together determine the identity of the monomer. For PHB, the number of methylene units is 1 and the R group is CH 3 . A number of organisms have been developed in an effort to optimize the yield of PHB, and recently accumulations of PHB at levels up to 80% of dry cell weight have been obtained under optimum growth conditions (25). PHB is an attractive alternative to the environmentally unfriendly petrochemically derived plastics because its copolymer with polyhydroxyvalerate is a biodegradable molecule with properties similar to those of polyolefins, such as polypropylene and polyethylene. For this reason industrial fermentation of PHB has been the focus of a number of studies (1,38,39), while other studies have been directed towards the use of PHB-producing organisms in the breakdown of wastewater (32, 42).The most common method available at present for analysis of PHAs in bacterial cells is gas chromatography (GC) (6,22,31). The GC method involves hydrolysis and subsequent methanolysis or propanolysis of the PHAs in whole cells in the presence of sulfuric acid and chloroform. This method is timeconsuming and laborious and involves extensive use of solvents. Other methods of PHA analysis include gravimetry, infrared (IR) spectroscopy of chemically extracted PHB (23), fluorimetry (10), and cell carbon analysis (37). To aid in the development of more efficient fermentation processes and to monitor production, rapid feedback on the state of fermentation in terms of the PHA content of cells is required.Fourier transform infrared (FTIR) spectroscopy is a routine chemical technique used to study molecular structure, but when it is applied to a large collection of intact microbial cells, the resulting spectra reflect the total biochemical composition of the cells (28). IR spectroscopy can thus provid...

Formation of Polyhydroxyalkanoate in Aerobic Anoxygenic Phototrophic Bacteria and Its Relationship to Carbon Source and Light Availability

“…At about 1,540 cm Ϫ1 , the deformation vibration peak of the NOH bond of amides associated with proteins becomes apparent. It is usually called the amide II band and may also contain contributions from CAN stretches (21). The two peaks at about 1,650 and 1,540 cm Ϫ1 have disappeared in the sample spectrum.…”

“…The peak at about 1,382 cm Ϫ1 is the symmetric deformation peak of -CH3 and -CH 2 groups and the symmetric stretch of COO bonds of COO Ϫ groups (46); the positions of these assignments could also vary with contributions from PHA (46). The peaks between 1,200 cm Ϫ1 and 900 cm Ϫ1 are due to the vibration of COOOC bonds (43,46), with contributions from PHA (21).…”

“…At about 1,455 cm Ϫ1 , the asymmetric stretching peak of -CH 3 and -CH 2 groups (46) emerges. The positions of these assignments could vary with contributions from PHA (21). The peak at about 1,382 cm Ϫ1 is the symmetric deformation peak of -CH3 and -CH 2 groups and the symmetric stretch of COO bonds of COO Ϫ groups (46); the positions of these assignments could also vary with contributions from PHA (46).…”

Generation and Utilization of Microbial Biomass Hydrolysates in Recovery and Production of Poly(3-hydroxybutyrate)

“…Figure 4 compares the FTIR spectra of the purified PHB granules and the original oven-dried PHB-containing cell mass. The peaks of amide I band at ~1650 cm -1 and amide II band at ~1540 cm -1 (N-H bend) are characteristic infrared radiation absorption of the proteins in cell mass [25,26]. They disappeared in the spectrum of purified PHB granules.…”

Enhanced Agarose and Xylan Degradation for Production of Polyhydroxyalkanoates by Co-Culture of Marine Bacterium, Saccharophagus degradans and Its Contaminant, Bacillus cereus

“…Absorbance spectra were recorded between 4000 and 400 cm −1 wave number values using a Nicolet model Magna-IR 200 FT-IR spectrometer. The diagnostic signals for PHAs (peak at 1728-1742 cm −1 ) were taken as per Kansiz et al [38].…”