Fourier Transform Infrared Photoacoustic Spectroscopy (FT-IR-PAS) Combined with Two-Trace Two-Dimensional Correlation (2T2D) Analysis for Studying Surface Changes in Poly(3-hydroxybutyrate- co -3-hydroxyhexanoate) (PHBH) Films Under Marine Microbial Degradation

Materials and Methods

The PHBH film (thickness, 100  µm; 3HH content, 6 mol%) was provided by Kaneka Co., Ltd. (Osaka, Japan). All reagents were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan) unless otherwise noted.

Marine Microbial Degradation

Marine bacterial strains capable of degrading PHBH, designated Halioxenophilus aromaticivorans NBRC 116177, Cellvibrionaceae bacterium strain H, and Arenicellaceae bacterium strain K, were isolated from biodegradable plastic specimens immersed in the coastal waters of Japan, as reported previously. ⁸ PHBH films (approx. 10  mg) were incubated in basal artificial seawater inoculated with these strains under aerobic conditions at 25 °C and 130  rpm for 35 days. Blank samples were incubated under identical conditions without microbial inoculation (control experiment). After incubation, the residual films were sonicated in Milli-Q water to remove the biofilms, rinsed with Milli-Q water, and dried in a desiccator at room temperature. The dried specimens were weighed to determine the degree of degradation (DD) according to the following equation:

Measurements

The FT-IR-PAS was performed using a Tensor II spectrometer (Bruker, Germany) connected to a PA301 photoacoustic detector and optical microphone DSP module (Gasera, Inc., Finland). In this system, IR light is focused onto sample placed in an aluminum dish, and the PA signal is detected using a cantilever sensor in conjunction with a readout interferometer. This setup provides superior sensitivity compared with conventional microphone-based PA detection. FT-IR-PAS spectra in the wavenumber range of 4000–500 cm^–1 were recorded as an average of 64 scans at a resolution of 8 cm^–1 with a scanner velocity of 1.6 kHz.

Data Analysis

All calculations were performed using in-house programs in Matlab (The MathWorks, Inc., USA).

Marine Microbial Degradation of PHBH Films

Marine microbial degradation of bioplastics typically involves microbial adhesion to the bioplastic surface, biofilm formation, and secretion of extracellular enzymes within the biofilm. In this study, biofilm formation on the PHBH film surface was confirmed after 35 days of incubation with all three marine microorganisms (NBRC 116177, strain H, and strain K). The biofilms were easily removed by ultrasonic treatment, followed by rinsing with pure water. The resulting PHBH film residues were dried and then subjected to subsequent characterized.

The extent of degradation of the PHBH films by treatment with marine microorganisms was quantified using the DD value, common index representing the extent of weight loss of biodegradable plastics, as defined by Eq. 1 (Table I). As summarized in Figure 2, the DD values of each PHBH residue varied depending on the microorganism species used despite incubation for a defined period (35 days). In the control experiment, the recovered PHBH films (blank sample) retained their initial weight, and their DD values were negligible, indicating that abiotic degradation of the PHBH films under the present conditions was insignificant. Therefore, the DD values determined for each PHBH residue reflected microbial degradation. So, differences in the DD values between microbial species are thought to indicate species-dependent degradation kinetics.

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Figure 2.

DD values of PHBH residues degraded by marine microorganisms (NBRC 116177, strain H, and strain K) and blank samples (B). Data are shown as mean ± standard deviation (n = 3).

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Table I.

List of DD values of PHBH residues degraded by marine microorganisms.

Microorganism	Code	Wbefore (mg)	Wafter (mg)	DD (%)
NBRC 116177	N-1	10.6	8.8	17.0
	N-2	10.8	9.1	15.7
	N-3	10.6	8.8	17.0
Strain H	H-1	10.4	8.5	18.3
	H-2	10.7	8.9	16.8
	H-3	10.7	8.9	16.8
Strain K	K-1	10.5	9.2	12.4
	K-2	10.4	9.1	12.5
	K-3	10.6	9.3	12.3
*	B-1	10.5	10.5	0
	B-2	10.7	10.7	0
	B-3	10.1	10.1	0

W_before: Weight of the PHBH film before the control experiment. W_after: Weight of the blank PHBH film after the control experiment. *Without microorganisms (blank).

As mentioned previously, the microbial degradation of biodegradable plastics proceeds through the action of extracellular enzymes secreted into the biofilms. These enzymes cannot penetrate the bulk of the plastic; therefore, they act only on the outermost surface, cleaving the polymer chains into oligomers or monomers. In line with this general understanding, gel permeation chromatography (GPC) analysis of PHBH residues showed no significant change in the molecular weight distribution compared to the original PHBH film (Figure S1, Supplemental Material), indicating that enzymatic scission was confined to the surface layer. This interpretation is reasonable because GPC reflects bulk properties, and confined surface scission has little impact on the overall molecular weight distribution. Furthermore, the oligomers and monomers generated by enzymatic scission are likely to dissolve and diffuse into the surrounding biofilm rather than remain in the PHBH residues, which explains why they were not detected by GPC analysis. Consequently, our subsequent investigation focused on the surface of PHBH film residues, where traces of enzymatic action were expected to remain. Indeed, the subsequent FT-IR-PAS analysis provided further insights into the details of microbial degradation.

FT-IR-PAS Spectra of PHBH Samples

To examine the structural changes on the surface of the PHBH films during microbial degradation, we employed FT-IR-PAS. Unlike conventional ATR-based IR spectroscopy, FT-IR-PAS avoids the application of mechanical pressure, which can cause sample fractures or spectral distortions and enables non-contact acquisition of surface-sensitive IR spectra. These features are particularly suitable for analyzing the brittle or rough surfaces of PHBH films after biodegradation.

The FT-IR-PAS spectra of PHBH samples exhibited characteristic peaks corresponding to their chemical structures. Figure 3 compares representative FT-IR-PAS spectra of original PHBH film, PHBH film residues after the microbial degradation, and the blank sample, with particular attention to the peak observed in the range of 1675–1775 cm^–1, which is attributed to the C=O stretching vibration, ν(C=O). An important characteristic of this peak is its high sensitivity to the local environment surrounding the C=O moiety, thereby serving as an indicator of the molecular order of the PHBH chains. Previous reports on structural analysis of PHBH using ATR-IR spectroscopy have attributed the lower-wavenumber component around 1723 cm^–1 to crystalline state, where C=O stretching vibrations are highly restricted by hydrogen bonding. Conversely, the higher-wavenumber component around 1740 cm^–1 corresponds to the amorphous state, where restrictions on C=O stretching vibrations due to hydrogen bonding are minimal. Consistently, the FT-IR-PAS spectra of PHBH samples showed distinct peaks for crystalline and amorphous phases around 1726 cm^–1 and 1740 cm^–1, respectively.^17–19 The difference in the crystalline peak position between previous reports (∼1723 cm^–1) and this study (∼1726 cm^–1) can be reasonably attributed to differences in measurement principles and spectral resolution (2 or 4 cm^–1 for typical ATR-IR study, and 8 cm^–1 for this study). Other vibrational regions were also inspected, but their FT-IR-PAS intensities were insufficient for reliable analysis. The C=O stretching region (1675–1775 cm^–1) exhibited comparatively strong and well-resolved signals, enabling clear differentiation of crystalline and amorphous components. Although all the FT-IR-PAS spectra shown in Figure 3 exhibit distinct peaks for the crystalline and amorphous phases, their overall profiles appear very similar, making extracting information on subtle and detailed structural differences caused by microbial degradation from the raw spectra alone difficult. Therefore, we applied a side-by-side comparison using two-trace two-dimensional correlation (2T2D) analysis, which is a robust approach for revealing hidden spectral variations.

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Figure 3.

Comparison of the ν(C=O) band on FT-IR-PAS spectra of the original PHBH film, PHBH film residues (H-1, N-1, and K-1) and the blank sample (B-1). The peak component at approximately 1726 cm^–1 is associated with the crystalline phase and that at approximately 1743 cm^–1 is associated with the amorphous phase of PHBH chains, respectively.

2T2D Correlation Analysis of FT-IR-PAS Spectra

Based on the observations described in the previous section, we performed 2T2D correlation analysis on the peak observed in the range of 1675–1775 cm^–1 in the FT-IR-PAS spectra. 2D correlation spectroscopy is a powerful technique that enhances spectral resolution by spreading overlapping bands onto a two-dimensional correlation map, where synchronous and asynchronous plots reveal subtle variations in peak positions and intensities. In the 2T2D approach, two sets of FT-IR-PAS spectra, namely, those of the PHBH film before microbial degradation and those of the PHBH residues after degradation, were directly correlated, amplifying minor differences that were not apparent from direct inspection of the raw spectra.

Figure 4 shows the 2T2D asynchronous correlation spectrum between the original PHBH film and PHBH film residue after microbial degradation by strain NBRC 116177. This spectrum was constructed using the FT-IR-PAS spectrum of the original PHBH film as the reference spectrum, r(v), and that of the PHBH residue treated with strain NBRC 116177 as the sample spectrum, s(v). These two spectra are displayed along the right and top margins of the correlation map to facilitate direct comparison and navigation. The negative cross-peaks within the 2D correlation spectrum are shaded for clarity. The theoretical basis of the 2T2D correlation analysis lies in the principle that spectral bands originating from the same chemical or physical source cannot vary independently; thus, their intensity ratios remain constant across conditions. Consequently, the appearance of the correlation peaks in an asynchronous correlation spectrum provides strong evidence that the two bands associated with the coordinates of the cross-peak arise from different sources.^15,16

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Figure 4.

Asynchronous correlation spectrum between the original PHBH film and PHBH residues after microbial degradation by strain NBRC 116177 (N-1). This spectrum was constructed using the FT-IR-PAS spectrum of the original PHBH film as reference spectrum r(v) and that of N-1 as the sample spectrum s(v), respectively. Negative cross-peaks within the 2D correlation spectrum are shaded for clarity.

This approach enables the identification of subtle spectral variations that are otherwise difficult to discern using conventional visual inspection. For example, in Figure 4, the positive asynchronous correlation peak at (1726 cm^–1, 1743 cm^–1) reveals that the signal intensity arising from the crystalline component in s(v) is more pronounced than that arising from the amorphous component in r(v). This indicates that the surface of the PHBH film residue after degradation by strain NBRC 116177 showed a higher proportion of crystalline regions than amorphous regions compared to the original PHBH film. The interpretation from the viewpoint of the microbial degradation mechanism will be addressed later.

Similarly, 2T2D analyses were conducted on the FT-IR-PAS spectra of other PHBH residues using the original PHBH film as the reference spectrum. As in the case of the PHBH film residue degraded by strain NBRC 116177 (Figure 4), the resulting asynchronous correlation spectra also showed a specific cross-peak at (1726 cm^–1, 1743 cm^–1) (Figure S2 and Figure S3, Supplemental Material). Figure 5a compares the asynchronous correlation intensities at (1726 cm^–1, 1743 cm^–1) calculated for the FT-IR-PAS spectra of PHBH film residues across microbial species. Blank samples showed negligible values, indicating minimal structural changes during the control experiment. In contrast, other PHBH residues exhibited significant correlation intensities, suggesting that surface structural change increased the proportion of crystalline regions relative to amorphous regions during microbial degradation. One plausible explanation for these observations is that, within the set of microbial species investigated here, the amorphous regions on the PHBH film surface consistently underwent predominant erosion, irrespective of species differences. This explanation is consistent with the general understanding that when biodegradable plastics are degraded by the corresponding enzymes, the less ordered and more accessible amorphous regions are generally more susceptible to degradation than the densely packed crystalline regions.^20,21

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Figure 5.

(a) Asynchronous correlation intensity at (1726 cm^–1, 1743 cm^–1) calculated with a 2T2D correlation scheme using the FT-IR-PAS spectra of PHBH samples treated with each microorganism. Data are shown as mean ± standard deviation (n = 3). (b) A plot of DDs versus asynchronous correlation intensity at (1726 cm^–1, 1743 cm^–1) calculated with PHBH samples.

The observed differences in asynchronous correlation intensities at (1726 cm^–1, 1743 cm^–1) for PHBH residues in Figure 5a could arise from species-specific degradation mechanisms or variations in degradation kinetics. To clarify this relationship, the asynchronous correlation intensities at (1726 cm^–1, 1743 cm^–1) were plotted against the corresponding DD values for each PHBH residue (Figure 5b). Notably, the asynchronous correlation intensity exhibited a positive association with the DD values, indicating that weight loss due to microbial degradation is related to changes in the asynchronous correlation intensities. Taken together, these findings suggest that the variations in the asynchronous correlation intensities shown in Figure 5(a) are more likely to reflect differences in degradation kinetics rather than microbial species-specific mechanistic differences.

Finally, considering the above observations, a possible surface change in the PHBH films due to microbial degradation is illustrated in Figure 6. Extracellular enzymes secreted within the biofilm preferentially degraded the amorphous regions of the PHBH film surface at a faster rate than the crystalline regions. As degradation progressed, the relative proportion of crystalline domains gradually increased. Such localized changes on the PHBH film surface could be elucidated by combining FT-IR-PAS with 2T2D correlation analysis, highlighting the capability of this methodology to detect subtle structural variations during microbial degradation. Further investigations are currently underway, and the findings will be reported elsewhere.

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Figure 6.

Schematic illustration of the proposed surface changes in PHBH films during marine microbial degradation, as inferred from FT-IR-PAS and 2T2D correlation analysis of films degraded by the microorganisms used in this study. Extracellular enzymes preferentially degrade amorphous regions, leaving crystalline domains enriched on the surface as degradation progresses.