*2.2. Efficacy of PHB-Producing Isolates*

### 2.2.1. Cultivation Media

A cultivation process was implemented so that isolates from each medium could be cultivated on the same isolation media for PHB production. Carbon and nitrogen sources for each medium were sterilized separately and were aseptically added before inoculation and after cooling of each medium.

For medium (A) [18], 20 g/L glucose (carbon source) and 2.0 g/L ammonium acetate (nitrogen source) were added to the sterilized medium. The pH was adjusted to 7. For to medium (B), 10 g/L fructose was added as a carbon source [19] and 1.2 g/L ammonium acetate (nitrogen source) was added to the sterilized medium; the pH was adjusted to 7.2. Finally, 30 g/L sucrose was used as a carbon source for medium (C) [20], and 1 g/L ammonium sulfate was added to the sterilized medium. The pH was adjusted to 6.8.

#### 2.2.2. Cultivation System

Batch cultivation on conical flasks was used in this experiment. Twenty ml of each medium was prepared from maintenance cultures to be used as an inoculum. Incubation was performed in an incubator shaker at 200 rpm at 30 ◦C for 48 h. The inoculum was aseptically transferred to 50 mL of the same medium and incubated under the same conditions for the same period mentioned above [27]. All experiments were conducted in three replications.

#### 2.2.3. Biomass Determination

A 50 mL sample of each culture was centrifuged at 5000× *g* for 10 min. Cell pellets were washed twice with sterile distilled water. The precipitated biomass was dried at 100 ◦C for 24 h or until a constant weight was reached. This value was used for determination of cell dry weight (CDW) [28].

#### *2.3. PHB Determination*

Ten ml of chloroform was added to the dried cell biomass obtained from the previous centrifuged cultures and incubated at 70 ◦C for 10 min, then centrifuged at 5000× *g* for 10 min. The resulting solution was separated and collected, and the chloroform was allowed to evaporate in order to obtain PHBs for further assessment. Then, the precipitate was dried at 100 ◦C for 24 h or until a constant weight was reached. This value was used for PHB–free cell dry weight determination [19]. The difference in weight between the previous two values was considered as PHB production (g/50 mL); this value was calculated by [29,30], as g/L:

$$\text{PHB (\%)}=\text{PHB weight (g/L)} \times 100/\text{total cell dry weight (g/L)}$$

Conversion coefficient (%) = PHB weight (g/L) × 100/utilized sugar weight (g/L)

#### *2.4. Determination of Utilized Sugars and Nitrogen*

Total carbohydrates were determined as glucose [31]. The total sugars remaining in the culture broth were estimated, and by subtracting this value from the amount of total sugars in the medium composition at the beginning of the experiment, the consumed sugars or utilized sugars were calculated. Total nitrogen was measured according to the Kjeldahl digestion method [32].

#### *2.5. The Relationship between PHB Production, CDW and Utilized Sugar (g/L)*

The correlation coefficient values (*r*) was calculated according the equation given in [33]. The correlation between PHB g/L and CDW g/L was calculated where *X* is the value of PHB g/L, *X* is the arithmetical mean of PHB g/L, *Y* is the value of CDW g/L, and *Y* is the arithmetical mean of the CDW g/L values. The value of CDW was replaced with the value of utilized sugar when the correlation coefficient was calculated between PHB g/L and utilized sugar.

$$r = \frac{\Sigma(X - \overline{X})\left(Y - \overline{Y}\right)}{\sqrt{\Sigma\left(X - \overline{X}\right)^2}\sqrt{\Sigma\left(Y - \overline{Y}\right)^2}}$$

If the value of the correlation coefficient (*r*) = +1, there is a perfect positive correlation; if (*r*) = −1, a perfect negative correlation; if *r* = 0, there is no correlation. If the value of the correlation coefficient is between *r* = 0.0–0.2, 0.2–0.4, 0.4–0.6, 0.6–0.8 or 0.8–1.0, this means a very weak correlation, weak correlation, moderate correlation, strong correlation or very strong correlation, respectively [33].

#### *2.6. Identification of Bacterial Isolates Based on Molecular Biology*

The highest PHB-producing isolates were inoculated in suitable media and incubated overnight at 37 ◦C. The resulting bacterial suspension was pelleted at 10,000 rpm for 5 min and genomic DNA was extracted using a Pure Link genomic DNA mini kit. PCR amplification of 16s rDNA was performed using the isolated DNA. The PCR conditions were set as follows: initial denaturation at 95 ◦C for 5 min, followed by 25 cycles of denaturation at 95 ◦C for 40 s, annealing at 55 ◦C for 2 min and primer extension at 72 ◦C for 1 min, ending with the final elongation step at 72 ◦C for 7 min. PCR products were gel purified and sent for sequencing with 16S rRNA primer. The obtained sequences were trimmed to obtain a sequence with ladder 1–1.5 Kb DNA. Then, the sequences were BLAST search analyzed on the (www.ncbi.nlm.nih.gov, accessed on 1 September 2021) to identify the isolate [34].

### *2.7. PHB Production Using Cheap Alternative Carbon Sources*

Three different alternative carbon sources were used; dried whey and date molasses were purchased from Damietta City market, Damietta City, Egypt, and sugar beet molasses was purchased from the Sugar Beet Factory Kalabsho, Belkas, Dakahlia Governorate, Egypt. Molasses was pretreated according to the method in [35]. Total sugars and total nitrogen of all sources were determined. The values of carbon and nitrogen were considered during preparation of the cultivation media which already contain these sources.

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

IR was used to confirm the formation of PHB using a MATTISON 5000 FTIR spectrometer at Mansoura University, Faculty of Pharmacy, Chemistry Department, Spectral Analysis Unit. A 50 mm mortar and pestle were used. Hydraulic presses were performed using a PIKE die press. A SPECAC press instrument was used for preparation of the KBr sample discs of investigated drugs, pressing under a 7 mm die at a pressure of 2 tons for 1 min. A SHIMADZU CORPORATION balance BL-220H (Kyoto, Japan) was utilized throughout the work. IR spectra of the PHB polymer were determined at 2 cm−<sup>1</sup> spectral resolution by using a MATTSON 5000 FTIR spectrometer. An excitation wavelength at 3900 nm was provided and the laser power at the sample position was typically 500 nm. Spectra were obtained with a spectral resolution of 4 cm<sup>−</sup>1, and 1024 [36].

For GC-MS/MS analysis, the chemical composition was obtained after extraction and derivatized using BSTFA reagent. The derivatization product MTBSTFA-HB was analyzed on a Finnigan GCQ (Finnigan MAT, Austin, TX, USA), which is an ion trap MS with external ionization. A 30 m fused silica capillary column DB-5MS, I.D. 0.25 mm, film thickness 0.25 mm (J&W Scientific, Folsom, CA, USA) was used for separation. The samples were injected in splitless mode. The injection temperature was 250 ◦C, temperature

of ion source 200 ◦C and transfer line 275 ◦C. Helium velocity was 40 cm s−1. The oven temperature, initially at 60 ◦C, was gradually increased (20 ◦C min<sup>−</sup>1) to 250 ◦C and held at this temperature for 15.5 min. The MS/MS detection consisted of a sequence of three steps: (i) isolation of the "parent" ion of the detected compounds (immediate rejection of other ions except the "parent" ion); (ii) fragmentation of the "parent" ion; and (iii) analysis of the fragmentation products. These steps were realized using GCQ MS/MS software, version 2.0. The MS/MS measurements were performed at a collision energy of 0.8 V for parent ions of *m*/*z* 275 (for the analyte) and *m*/*z* 331 (for the internal standard). The product spectra in the mass range of *m*/*z* 100–331 were scanned. The retention time of the analyte was 8.34 ± 0.04 min and of the internal standard 10.2 ± 0.04 min [37].

#### *2.9. Statistical Analysis*

Data obtained throughout this study were analyzed by computer-assisted one-way ANOVA, using the software package stat graphics version 5.0 (costat). Least significance differences (LSDs) were calculated at level of significance *p* < 0.05 [38].
