**5. Physical characteristics of PHAs**

The PHA extracted from the 20 environmental samples that tested positive for *phaC* genes and/or the staining methods were analyzed using the FTIR spectroscopy method.

Pure bacterial overnight culture grown in LB media (10 ml) at 37°C was used for the PHA extraction and purification. Bacterial cells were pelleted at 4000 rpm for 25 min at RT using glass conical centrifuge tubes (Sigma, UK). The pellet was suspended in 10 ml of 0.1% sodium hypochlorite and centrifuged for 30 min at 4000 rpm. The supernatant was removed, and the pellet was washed with 5 ml of sterile water, 5 ml of acetone, and 5 ml of methanol consequently. The pellet was dissolved in 5 ml of chloroform and left overnight at RT to evaporate on a glass Petri dish (**Figure 6**) [31]. The PHA biopolymer was collected, weighed, and analyzed using a physical method.

The PHAs extracted from the isolates were analyzed using PerkinElmer®'s Spectrum 1™ FT-IR Spectrometer (USA); spectral range 4000–400 cm<sup>1</sup> with a spectral resolution of 4 cm<sup>1</sup> [32]. The characteristic absorption peaks were used to interpret the presence of specific functional groups in the extracted polymers. The yield of PHA in mg/10 ml extracted from the environmental pure bacterial isolates varied from 0.1 mg (24/2) to 8.8 mg (20/1). The main FTIR spectral peaks for the presumptive

*Bioplastics against Microplastics: Screening of Environmental Bacteria for Bioplastics… DOI: http://dx.doi.org/10.5772/intechopen.109756*

**Figure 6.** *PHA was obtained as a white powdery substance on a glass Petri dish.*

PHAs extracted from 20 studied bacterial isolates; from the PHA-producing bacterial strain (+ve), used as a positive control, along with peaks for the commercial PHB (Sigma, USA) are presented in **Table 3**.

According to **Table 3**, the spectra of the standard PHB show the peak at 1720.88 cm<sup>1</sup> , which corresponds to the C=O stretch of the ester group, and the peak at 1278.59 cm<sup>1</sup> , which corresponds to –CH group [33]. These peaks are similar to the published spectra peaks of PHB [34]. The positive PHA production bacterial strain (+ve) showed the peak at 1650.05 cm<sup>1</sup> , which corresponds to ester carbonyl group (C–O), and accompanying peak at 1275.33 cm<sup>1</sup> , which corresponds to –CH group.



**Table 3.**

*Functional groups identified by the FTIR method for PHA analysis.*

The results of the FTIR analysis of the PHA extracted from the 20 environmental samples showed a resemblance to the positive strain's characteristic absorption peaks, which indicates the presence of the PHA functional groups in the samples. As it was mentioned above, the positive-control strain was confirmed as a PHA producer by PCR and the staining methods. The strain 9/1 was tested positive by Sudan Black B, negative for the *phaC* gene by the PCR method, and it did not show any characteristic absorption peaks corresponding to PHA production. As a result, it was concluded that this strain was not a PHA producer.
