*3.5. The Correlation Coefficient Value between PHB Production, CDW and Utilized Sugar (g/L) of All Tested Isolates and Strains*

The correlation coefficient was calculated according the equation in [33]. Statistical analysis indicated there was a very strong positive correlation (r = 0.987) between PHB g/L and CDW g/L in the *A. salinestris* strain and a strong positive correlation (r = 0.457) in the isolates grown on medium (B). A very weak negative correlation (r = −0.161) between PHB g/L and utilized sugar g/L in the isolates grown on medium (B) was observed, with the highest value between them being r = 0.897 (a very strong positive correlation) in the case of *B. naejangsanensis* (Table 3).


**Table 3.** The correlation analysis between PHB, dry cell weight and utilized sugar.

Medium (A) [15]; Medium (B) [16]; Medium (C) [17].

#### *3.6. The Physical Properties of PHB Polymer*

#### 3.6.1. Fourier Transform Infrared Spectroscopy (FTIR)

Poly ß-hydroxybutyrate (PHB) Polymer extracted from *B. paramycoides* was characterized using IR spectra in the range of 600–4000 cm−1, as displayed in Figure 2a. The IR spectra of PHB produced show obvious peaks at 1262 and 1725 cm−1, corresponding to specific rotations around carbon atoms specific to –CH and C=O stretching of the ester group present in the molecular chain [36]. Strong peaks at 1034 and 1097 indicate the presence of C-O stretching. Additionally, the absorption bands at 2926 and 2963 cm−<sup>1</sup> are due to C–H stretching vibrations of methyl and methylene groups, which confirms PHB formation. PHB extracted from *A. salinestris*, as shown in Figure 2b, is similar to that extracted from *B. paramycoides*. Furthermore, the IR spectra of the extract from *A. salinestris* display similar bands at 1032, 1098 1262, 1725, 2925 and 2963 cm−<sup>1</sup> which confirm the presence of PHB, as shown in Figure 2b. On the other hand, the IR spectra of PHB extracted from *Brevundimonas naejangsanensis* illustrate weak bands at 1264 and 1748 cm<sup>−</sup>1, indicating lower PHB concentration in comparison with the other obtained extracts, as shown in Figure 2c. In addition, all peaks at 3400 to 3600 cm−<sup>1</sup> correspond to specific amino groups.

#### 3.6.2. Gas Chromatography–Mass Spectrometry (GC-MS) Analysis

Chemical composition was obtained after extraction and derivatized using BSTFA reagent. Figure 3a shows a peak at retention time 15.91 min, which represents Polyhydroxybutyrate (PHB) from *B. paramycoides*. The molecular ion peak in the mass spectrum at *m*/*z* 104 agrees well with the molecular formula for PHB. In addition, Figure 3b shows a peak at 20.08 min, which represents 2, 4-ditert-butylphenol. The area under the peak denotes the PHB content, which was determined to be 4.85%.

Figure 4a shows the GC-MS chromatogram of the PHB extracted from *A. salinestris*; the peak at retention time 16.07 min represents the isopropyl ester of 2-butenoic acid (Figure 4b), confirming the present of Polyhydroxybutyrate (PHB). The area under the peak denotes the PHB content, which was determined to be 10.73% (Figure 4b). Figure 5a shows the GC-MS chromatogram of the PHB extracted from *Brevundimonas naejangsanensis*, showing a peak at retention time 16.07 min which represents the isopropyl ester of 2 butenoic acid (Figure 5b), confirming the presence of Polyhydroxybutyrate (PHB). The area under the peak denoting the PHB content was determined to be 0.85%, with very low mass spectrum matching.

**Figure 2.** *Cont*.

**Figure 2.** IR spectrum of PHB extracted from *B. paramycoides* (**a**), *A. salinestris* (**b**), and *Brevundimonas naejangsanensis* (**c**).

**Figure 3.** *Cont*.

**Figure 3.** (**a**) Total GC-MS chromatogram of PHB extracted from *Bacillus paramycoides.* (**b**): The mass spectrum of isopropyl ester of 2-butenoic acid of PHB extracted from *Bacillus paramycoides*.

**Figure 4.** (**a**): Total GC-MS chromatogram of PHB extracted from *Azotobacter salinestris*. (**b**): The mass spectrum of isopropyl ester of 2-butenoic acid of PHB extracted from *Azotobacter salinestris*.

**Figure 5.** (**a**): Total GC-MS chromatogram of PHB extracted from *Brevundimonas naejangsanensis.* (**b**): The mass spectrum of isopropyl ester of 2-butenoic acid of PHB extracted from *Brevundimonas naejangsanensis*.

#### **4. Discussion**

#### *4.1. Screening the Best PHB-Producing Bacterial Isolates*

Significant differences were observed between the bacterial isolates in most cases. Generally, the best isolate which yielded the highest PHB production with reasonable sugar utilization was No. A6 (Table 1). Isolates No. A1, A2 and A5 produced more than 20% PHB. These results were similar to the results obtained by [39], who isolated three bacteria from the roots of beans (*Vicia faba*) grown in Qalyubia Governorate, Egypt to produce PHB using the flask culture technique. These isolates collected more than 20% of PHB, and were identified as *Pseudomonas fluorescens* S48, *B. megaterium* 7A and *B. megaterium* UBF19. Four isolates (A1, A2, A6 and A10) were higher than those found by [18], who reported that the highest value of PHB in *B. megaterium* was 0.27 g/L. Furthermore, all of these isolates were

higher than [40], who produced PHB by batch fermentation of *B. subtilis* from glucose and reached only 0.077 g/L.

In addition, all tested isolates grown on medium (B) produced PHB (Table 1). It was observed that there were significant differences between bacterial isolates. Thus, the best isolate with the highest value of PHB (0.93 g/L) was No. P3. Multiple previous studies [19,41] produced PHB from *A. chroococcum* in amounts ranging from 2.0 to 3.0 g/L, higher than in the current study; however, [10] produced only about 1.0 g/L from *A. vinelandii*, lower than this study. In [42], the production of PHB was studied using the shaking culture method with *A. chroococcum* resulting in a PHB% of 46.80%. On the other hand, PHB was produced from all isolates grown on medium (C) in the present study (Table 1).
