*3.8. Analysis and Characterisation of the Purified Polymer* 3.8.1. FTIR

The chemical structure of the purified biopolymers was determined using FTIR. The IR spectra of polymer generated in a medium containing glucose as a carbon source revealed three prominent absorption bands at 1730, 765, and 710 cm−<sup>1</sup> due to ester, CH group, and carbonyl group, respectively. These bands also appeared in the IR spectra of the polyester produced from cardboard hydrolysate medium as a carbon source at 1733, 769, and 708 cm<sup>−</sup>1, respectively (Figure 4). The existence of -CH bonding is shown by the typical bands at 2955 and 2965 cm<sup>−</sup>1, while the C=O and ester groups are represented by the bands at 1730 and 1733 cm−1. The bands found at 1080 and 1090 cm−<sup>1</sup> correspond to the C-O bonding for PHB derived from glucose and cardboard, respectively. The bands of the aforementioned polyester samples are quite similar to the bands of standard PHB, which affirms the high purity of the generated polymer. These findings are consistent with those reported by a recent study of PHB produced by *B. megaterium* MTCC 453 [51]. Because of the FTIR data, it is apparent both polymers produced from glucose (PHB) and cardboard hydrolysate (PHB-CB) as carbon sources are PHB, which is the common homopolymer of PHAs.

**Figure 4.** FTIR spectra of polymers isolated from *B. mycoides* ICRI89 grown in MSM containing either glucose (PHB) or cardboard hydrolysate (PHB-CB) as carbon sources.

## 3.8.2. NMR

PHB samples were characterized using 1H NMR and 13C NMR spectroscopic methods. The 1H and 13C NMR spectra of PHB produced from *B. mycoides* ICRI89 by microbial fermentation of glucose and cardboard hydrolysate are shown in Figure 5. PHB characteristic peaks were detected in both PHB and PHB-CB, such as δ = 5.21 and 5.23 ppm, which correspond to –CH doublet, δ = 2.50 and 2.51 ppm for –CH2 multiplet, and δ = 1.21 and 1.22 of –CH3 doublet for the PHB and PHB-CB, respectively. The large peaks at δ = 7.3 ppm (Figure 5a,c) indicate the solvent (CD3Cl), while the small peaks at δ = 1.61 and 1.63 ppm are due to the minor H-O contamination of the solvent. These findings were identical to those obtained using the PHB standard. Hence, we determined that the polyesters produced by *B. mycoides* ICRI89 strain cultivated in glucose and cardboard hydrolysate as the carbon source were PHBs [52]. 13C NMR analysis also confirmed these findings. The functional groups C=O (170.5 and 170.3), CH (65.9 and 65.7 ppm), CH2 (42.55 and 41.9 ppm), and CH3 (19.5 and 19.6 ppm) peaks were assigned for PHB and PHB-CB, and they were similar to the PHB previously obtained from *Bacillus* sp. [53].

**Figure 5.** 1HNMR and 13CNMR analysis of PHB and PHB-CB, (**a**) 1HNMR for PHB, (**b**) 13CNMR for PHB, (**c**) 1HNMR for PHB-CB and (**d**) 13CNMR for PHB-CB.

#### 3.8.3. TGA and DTG

TGA profiles of PHB and PHB-CB synthesized by *B. mycoides* ICRI89 are depicted in Figure 6a. The TGA curve represents the weight loss of the synthesized PHB in two phases for the two generated polyesters generated. The first step of mass loss occurred at temperatures ranging from 100 to 180 ◦C. For PHB and PHB-CB, the mass loss was approximately 1.5 and 1.3% of total mass, respectively. This loss is caused by the evaporation of physically adsorbed solvents, such as methanol, chloroform, and others that have formed on the polymer surface. Furthermore, the second or major step of polymer degradation started after 200 ◦C, which occurs after the melting point of PHB. The decomposition process involves a molecular weight decrease, which includes chain scission and hydrolysis. The random chain scission process, which involves the breakage of C=O and C-O bonds in ester moieties by β-scission, destruction of crystalline areas, and depolymerization, is responsible for the rapid heat breakdown of PHB at this stage [54]. The second stage of weight loss occurred when the temperature increased further, as hydrolysis, chain scission, and the synthesis of crotonic acid all contribute to the deterioration process. From the analysis of the initial and the maximum degradation temperatures of main step weight loss and residual mass percentage for PHB and PHB-CB, the maximum degradation temperatures for PHB and PHB-CB were found to be 380 and 369 ◦C, respectively. As a result, it can be inferred that both forms of PHB exhibited greater thermal stability when compared to standard PHB, which was found to have a decomposition temperature of 285 ◦C [55]. Furthermore, the residual mass of PHB and PHB-CB is less than 1.5%. The second stage of degradation for PHB produced from *Bacillus* sp. ranges between 237 and 320 ◦C which is lower than our records. This implies that the synthesized PHBs have higher degrees of thermal stability than the PHB produced by *Bacillus* sp. N-2 [56].

**Figure 6.** Characterization of PHB and PHB-CB demonstrating (**a**) TGA, (**b**) DTG, (**c**) DTA, and (**d**) XRD.

The rate of mass loss of a polymer sample with relation to temperature was investigated using differential thermogravimetric (DTG) analysis (Figure 6b). The DTG curve peaks reflect the thermal stability of PHB in relation to the temperature at which the highest breakdown rate of the polymer matrix occurs. The DTG characteristic curves, as the TGA curves, revealed three distinct phases. The mass loss rate in the first phase was approximately 0.16 to 0.20 mass%/min until 190 to 200 ◦C, and the amount of residue is quite high in both PHB and PHB-CB samples. The maximum degradation temperature for PHB in the second stage of degradation was approximately 266 ◦C, with a maximum mass loss rate of 34%/min. PHB-CB's maximum degradation temperature was approximately 268 ◦C, with a maximum mass loss rate of 32%/min. It has previously been reported that PHB standard has a degradation temperature of roughly 236 ◦C, with a maximum mass loss rate of 30%/min [51]. According to the results of the foregoing investigation, the PHB and PHB-CB produced by *B. mycoides* ICRI89 indicate strong thermal stability or resistance to heat deterioration.

#### 3.8.4. DTA

Differential Thermal Analysis (DTA) aids in determining breakdown heat (Figure 6c). This experiment was carried out to assess the cross-linking capabilities and the heat stability of the generated polymer. Due to the existence of cross-linking events during PHB degradation, an exothermic peak is found in the DTA thermogram. The curing temperatures, which are 331 and 325 ◦C for both PHB and PHB-CB, are the temperatures at which cross-linkage occurs. It is the most essential attribute that appears to be a major impediment to the commercial application of PHB, generating thermal instability due to a lack of cross-link capacity [57].

#### 3.8.5. XRD

The XRD spectra (Figure 6d) presents X-ray diffraction of PHB samples from both pure polyesters, PHB and PHB-CB. The observed peaks in XRD spectra for PHB are 2θ = 3.69◦, 13.26◦, 16.22◦, 22.91◦, and 25.14◦, while the observed peaks in XRD spectra for PHB-CB are 2θ = 3.61◦, 13.49◦, 16.35◦, 22.01◦, and 25.22◦. The peaks at 2θ = 13.26◦,13.49◦,16.22◦, and 16.35◦, which are the most intense and scattering peaks, each indicate an orthorhombic

unit cell. The relatively weaker peaks observed at 2θ = 22.91◦ and 22.01◦ correspond to α-PHB crystal, while the minor spectra observed at 2θ = 25.14◦ and 25.22◦ denote PHB's partly crystalline nature. The polymer matrix adopts a regular helicoidal shape with two antiparallel chains in the rhombic unit cell inside the crystalline domain [51–58]. Pradhan et al. [44] showed that the diffractogram of the produced PHBs in the current study is nearly equivalent to that of PHB produced from *Bacillus* sp. [51] Collectively, more efforts should be made to recycle environmental waste [59].

#### **4. Conclusions**

The current study proposes corrugated cardboard waste hydrolyzed by commercial cellulase as a cheap and readily available substrate for microbial PHB synthesis. This would remove one of the major barriers facing PHB scaling-up. The enzyme concentration affected the reducing sugars, reaching a satisfactory yield of 21.3 ± 0.1 g/L at an enzyme concentration of 1.5% (*v*/*v*) at 55 ◦C. Moreover, the newly isolated *B. mycoides* ICRI89 accumulated 2.1 ± 0.2 g/L of PHAs when grown on modified MSM containing cardboard hydrolysate, which was highly close to that produced when grown on MSM supplemented with glucose. It is important to note that the polymers purified from B. mycoides ICRI89 cells were almost entirely composed of PHB, according to FTIR, NMR, and XRD findings.

## **5. Future Prospects**

The present research has several future prospects, including the industrial fermentation of cardboard and paper waste in humongous bioreactors, using several enzyme mixtures for promoting polyester synthesis. In addition, the methods of PHB purification from bacterial cells have to be improved to obtain pure polymer batches at a large scale.

**Author Contributions:** F.A., A.S. and M.R. conceived and designed the research. F.A. and M.R. conducted the experiments. F.A. and M.R. contributed to analyzing the data. F.A. and M.R. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was conducted as a part of the International Research Agendas PLUS program of the Foundation for Polish Science, co-financed by the European Union under the European Regional Development Found (MAB PLUS/2019/11).

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The accession number(s) can be found on the GenBank. Available online: https://www.ncbi.nlm.nih.gov/, ON231789 (Accessed on 17 May 2022).

**Acknowledgments:** The authors gratefully acknowledge Stanisław Bielecki, Institute of Molecular and Industrial Biotechnology, Lodz University of Technology for providing the cellulase enzyme.

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

#### **References**

