Background: Plastics are widely used
worldwide due to their characteristics of being inexpensive, capable of mass
production, lightweight, and highly durable. Although plastic, with its
excellent properties, has become an essential part of daily life, it poses a
significant threat because it does not decompose naturally in the environment
and can persist for decades due to its stable physical properties. This has
raised concerns about their impact on various ecosystems, including terrestrial
and marine environments. Polyhydroxyalkanoates (PHA), produced by various
bacteria and archaea, are biodegradable plastics with similar thermoplastic and
elastic properties to conventional plastics.
Aim: The aim of the study is to develop
a simple PHA quantification method using high-performance liquid chromatography
(HPLC) with alkaline hydrolysis pretreatment.
Methods: Analysis was conducted using an
HPLC (SHIMADZU, Kyoto, Japan) equipped with a UV-VIS detector (SHIMADZU,
SPD-10AV) and a separation column (SHIMADZU, SCR model). The PHBV produced by
Bacillus sp. CYR1 strain was quantified using both the developed method and GC
analysis, and the quantification results were compared. A de-gassed peracetic
acid solution (860 µL/L) was used as the mobile phase at a flow rate of 1.5
mL/min.
Results: By developing a simple PHA
quantification method using alkaline hydrolysis pretreatment combined with
HPLC, we attempted to quantify PHAs consisting of molecular chains longer than
PHB by applying a simplified pretreatment process with HPLC. The retention
times for standard reagents 2BE, 2PE, and 3HB were 11.1 minutes, 15.1 minutes,
and 6.4 minutes, respectively. Additionally, the formation of 2BE and 2PE was
observed from the PHA subjected to alkaline hydrolysis pretreatment, and the
generated calibration curves demonstrated linearity.
As P(3HB-co-3HV) is a copolymer of
3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV), 2BE and 2PE are produced
by the alkaline decomposition of P(3HB-co-3HV). For this, we used the equation
P(3HB) = α × 2BE and P(3HV) = β × 2PE, because the production ratio α of 2BE
from P(3HB) and the production ratio β of 2PE from P(3HV) are constant in the
reaction under the same decomposition conditions.
The bacteria Chromobacterium violaceum and Bacillus sp. CYR1 were used to
produce P(3HV) and P(3HB-co-3HV), respectively. The bacterium Bacillus sp. CYR1
produced 415 mg/L of P(3HB-co-3HV) when incubated with acetic acid and valeric
acid as the carbon sources, whereas the bacterium C. violaceum produced 0.198 g of P(3HV)/g dry biomass when
incubated with sodium valerate as the carbon source.
Since there is no standard sample for
P(3HV), it was produced in this study from C.
violaceum. From the ratio of the slopes of the calibration curve, the
production ratios were determined as α =3.26 and β =3.30. These results
indicate that short-chain-length PHA (scl-PHA) can be quantified easily and
with high sensitivity by measuring the 2BE and 2PE contents of actual samples
using the method proposed here.
Conclusion: It was confirmed that PHBV
can be accurately quantified using the quantification method developed in this
study. In the future, to assess the accuracy of the quantification results of
PHBV in dried cell biomass, it will be necessary to validate the quantification
method by performing GC analysis for comparison.
Author (s) Details
Young-Cheol Chang
Course of Chemical and Biological Engineering, Division of Sustainable and
Environmental Engineering, Muroran Institute of Technology, Hokkaido 050-8585,
Japan.
Please see the book here:- https://doi.org/10.9734/bpi/cmsrf/v1/4617
