*4.3. PHB Production from Cheap Alternative Carbon Sources*

Table 2 shows the effect of replacing glucose in medium (A) with three cheap alternative carbon sources, namely dried whey, treated sugar beet molasses, and treated date molasses, on PHB production from *B. paramycoides* strain MCCC 1A04098. Generally, in order to decrease the cost of PHB and increase the cell dry weight, PHB concentration, PHB%, and conversion coefficient, the addition of treated sugar beet molasses in the medium of *B. paramycoides* is recommended.

*Bacillus* sp. is capable of producing hydrolytic enzymes, which can degrade agroindustrial and other wastes that can be utilized as carbon sources for PHB production [43]. These results were similar to [46], where the highest PHB yield was observed in *B. mycoides* DFC1 (1.28 g/L) using wheat starch, and lower than [47], where a maximum dry cell weight of 4.35 g/L was obtained. In addition, these results were higher than those obtained in [59], where the maximum PHB% obtained by *B. subtilis* was 13.02% ± 1.67%. As for the attempts that have been made to reduce the cost of production, there have been many attempts, such as [1], who reported that PHB production from *B. aryabhattai* showed good polymer accumulation in basal medium with glycerol after 48 h of incubation and accumulated 1.79 g/L. Hence, glycerol may be a better option as it is cheaper than glucose and can be obtained as a byproduct from industries such as biodiesel production. In the same trend, the current study was better than [3], who studied the effect of different carbon sources on PHB production by *B. megaterium*. The highest production of PHB was observed with cane molasses as the sole carbon source (40.8, mg/L). The same results were obtained by [60], who reported that sugar beet molasses could be a suitable substrate for the production of PHB with *B. megaterium*. Cell dry weight was 16.7 g/L and PHB production was

0.6 g/L with batch cultivation. Sugarcane molasses was also used as a sole carbon source by [11,61,62], using *B. cereus* and *B. subtilis* to obtain a PHB% of 57.5%, 49.9% and 44.7%, respectively.

Fructose was replaced with alternative carbon sources in medium (B) for PHB production from *A. salinestris* strain NBRC 102611 (Table 2). In [52] it was found that there are more than of 300 bacterial species that can produce PHB, of which only a few have been used on a commercial scale. Molasses and whey have been reported to be excellent substrates for PHB production [35,57,63]. Similar results were obtained by [60], who reported that the use of sugar beet molasses in PHB production led to a two- to three-fold increase in biomass production compared to the control experiment. In [10] it was reported that the results obtained with beet molasses were the best compared with other sugars, especially unrefined sugar; in addition, it was proven that it is not only a source of carbon, but also contains some other nutrients that may interfere with the synthesis of PHB. Thus, to decrease the cost of PHB, the addition of sugar beet molasses in the medium of *A. salinestris* to increase cell dry weight, PHB concentration, PHB% and conversion coefficient appears very promising.

Sucrose was replaced with alternative carbon sources in medium (C) for PHB production from *Brevundimonas naejangsanensis* strain BIO-TAS2-2 (Table 2). Both the current study and [12] used date molasses as a novel carbon source for *B. naejangsanensis* and *Cupriavidus necator* for PHB production. The current findings were homogeneous with [58], who tested the ability of *B. vesicularis* to accumulate PHB and found that with acid-hydrolyzed wood (sawdust) as the substrate, PHB production was improved. In order to reduce the production cost by 50% and make it as economical as possible, more than 96% of sugars must be consumed, and the cells must contain more than 90% PHB with a yield not less than 64% of the DCW.

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

Multiple studies [64,65] have found a positive correlation between PHB production and CDW with r = 0.999, which was similar to our results where r = 0.987 (Table 3). The available studies on the correlation between PHB production and CDW were insufficient, with little information available; [64–66] published that there was a positive correlation between PHB and CDW. On the contrary, Aslim [67], reported there was a low correlation between CDW and the PHB contents of *Lactobacillus* cultures. Thus, there is a need for further study on this point. In this context, [68] reported that the correlation between PHB synthesis and nitrogen fixation was little discussed in purple non-sulfur bacteria species. In keeping with this trend, [65,69] studied the correlation between PHB production, pH, and viscosity, finding that it was decreased or that no correlation could be observed. However, there are no studies dealing with the correlation between PHB production and the amount of sugar consumed, which is the most important economic factor affecting polymer production.

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

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

Poly ß-hydroxybutyrate (PHB) Polymer extracted from the studied strains was characterized using IR spectra in the range of 600–4000 cm−1, as displayed in Figure 2. The results were obtained by [14], which observed that 1624–1724 cm−<sup>1</sup> was associated with the C=O stretching of the ester carbonyl bond. The bond at 1529 cm−<sup>1</sup> was characteristic of the stretching and deformation vibration of the C-H group, and those at 2960 and 3277 cm−<sup>1</sup> were characteristic of the stretching and deformation vibrations of the terminal OH groups. The PHB polymer functional groups were confirmed as C=O groups by FT-IR spectroscopy. In addition, analysis of the spectrum of PHB was observed at 3400, 1639, 3018, 2978, 2842, 1216, 1044 and 669–765 δ, respectively, for the (-OH) broad peak, (C=C) double bond, (≡C-H) acetylenic bond, (-CH3) methyl group, (-S-H) thiol weak adsorption, (≡C-O-C≡) ether,

sulfoxide and (F-Cl) haloalkanes [41]. The same results were obtained by [70], who showed that the bands of PHB between 3300 and 3700 cm−<sup>1</sup> were ascribed to O-H groups in the phenolic and aliphatic structures. The band at 2923 cm−<sup>1</sup> was assigned to C-H stretching in aromatic methoxyl groups, or in methyl and methylene groups of the side chain. The band at 1645 cm−<sup>1</sup> was attributed to unconjugated C=O stretching. The bands centered at 1508 and 1593 cm−<sup>1</sup> were associated with aromatic structural vibrations. The bands between 1400 and 1450 cm−<sup>1</sup> originated from C-H deformation coupled with the vibration from the aromatic ring. The bands at 1217 and 1128 cm−<sup>1</sup> indicated guaiacyl and syringyl groups, respectively. Moreover, Rathika [11], reported that, the broad transmittance peak at 3281 cm−<sup>1</sup> could be ascribed to the stretching of O–H groups. Peaks observed at 2924 and 2852 cm−<sup>1</sup> were associated with the C-H stretching of bonds of methyl (CH3) and methylene (CH2) groups. The intense absorption peak at 1722 cm−<sup>1</sup> was the characteristic carbonyl (C=O) stretching of ester groups in the extracted PHAs.

The 1H NMR spectrum showed the expected resonances for PHB as demonstrated by the methine group (-CH-) between 5.22 and 5.28 ppm, a methylene group (-CH2-) between 2.45 and 2.62 ppm, and the methyl group (-CH3) between 1.26 and 1.28 ppm, as in standard PHB. Scans of 13C NMR showed peaks at 169.13, 67.62, 40.81 and 19.76 ppm, which represent the carbonyl carbon (-C-), ester (-O-CH-) group, methylene (-CH2-) and the methyl (-CH3) groups, as shown in the standard. These results confirm the material as a homopolymer of 3-hydroxybutyrates, i.e., poly-3-hydroxybutyrate [1].

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

Our observation in Figures 3–5 were in agreement with [11], who detected PHAs extracted from *B. subtilis* RS1 cultured and processed in a medium of sugarcane molasses using GC–MS and revealed significant peaks at retention times 20.9, 23.1, and 23.8 min, corresponding to methyl esters of pentadecanoic acid and hexadecanoic acid, respectively. Furthermore, the results revealed that hexadecanoic acid methyl ester is the predominant monomer of PHA produced from *B. subtilis* RS1. In addition, Ramya [14] reported that the bioplastics produced by *B*. *cereus* RBL6 were characterized by GC–MS. *B*. *cereus* RBL6 was reported to produce methyl-3-hydroxy hexadecanoic acid, which belongs to the monomer chains of the medium-chain-length PHA class.

#### **5. Conclusions**

Our study aimed to use cheap and available alternative carbon sources (whey, sugar beet molasses and date molasses) to produce PHB using *Bacillus paramycoides*, *Azotobacter salinestris* and *Brevundimonas naejangsanensis*. The results showed that the addition of sugar beet molasses in the medium of *A. salinestris* increased the cell dry weight (CDW), PHB concentration, PHB% and conversion coefficient. In addition, statistical analysis indicated that the correlation coefficient values between PHB g/L and CDW g/L varied between r = 0.987 and r = 0.457 (very strong positive correlation and moderate positive correlation); however, these values were r = −0.161 and r = 0.897 when the correlation was calculated between PHB g/L and utilized sugar g/L. In addition, the IR of the produced PHB from *B. paramycoides* and *A. salinestris* showed similar bands, which confirmed the presence of PHB; however, *B. naejangsanensis* showed weak bands. The chemical composition obtained by GC-MS of the PHB extract represented 2, 4-ditert-butylphenol for *B. paramycoides*, and isopropyl ester of 2-butenoic acid for both of *A. salinestris* and *Brevundimonas naejangsanensis*.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/polym13213801/s1: Table S1. Morphological and physiological characteristics of bacterial isolates on medium (A); Table S2. Morphological and physiological characteristics of bacterial isolates on medium (B); Table S3. Morphological and physiological characteristics of bacterial isolates on medium (C).

**Author Contributions:** Conceptualization, S.M.E.-K., A.E.-D.O., M.E. and D.F.I.A.; methodology, S.M.E.-K., A.E.-D.O., S.E.-N., A.A.S., H.F.A.E.-S., H.A.H.E.-Z. and D.F.I.A.; software, S.M.E.-K., A.E.-D.O., H.A.H.E.-Z. and D.F.I.A.; validation, S.M.E.-K., A.E.-D.O., M.E. and D.F.I.A.; formal analysis, S.M.E.-K., A.E.-D.O., H.F.A.E.-S., H.A.H.E.-Z. and D.F.I.A.; investigation, S.M.E.-K., A.E.-D.O., H.A.H.E.-Z. and D.F.I.A.; resources, S.M.E.-K., A.E.-D.O., H.F.A.E.-S. and H.A.H.E.-Z. and D.F.I.A.; data curation, S.M.E.-K., A.E.-D.O., S.E.-N., M.E. and D.F.I.A.; writing—original draft preparation, S.M.E.-K., A.E.-D.O. and D.F.I.A.; writing—review and editing, S.M.E.-K., A.E.-D.O. and D.F.I.A.; visualization, S.M.E.-K., A.E.-D.O. and D.F.I.A.; supervision, S.M.E.-K., funding acquisition, M.E. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data that supports the findings of this study are contained within the article or supplementary material and available from the corresponding author upon reasonable request.

**Acknowledgments:** The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University for funding this work through the Research Groups Project under grant number R.G.P. 1/112/42. All the authors are grateful for the support provided by Faculty of Agriculture, Damietta University, Faculty of Agriculture, Mansoura University and Soils, Water and Environment Research Institute (SWERI), Agriculture Research Center (ARC), Egypt.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

