*2.7. Characterization of the Produced Polymer*

#### (a) Fourier Transform Infrared (FTIR)

The refined polymers were subjected to FTIR analysis. The study was carried out in a range of 400–4000 cm*−*<sup>1</sup> using an FTIR Nicolet 6700 spectrophotometer and OMNIC 3.2 software (Thermo Scientific Products: Riviera Beach, FL, USA) [9].

#### (b) 1H and 13C NMR

Polymer samples (25 mg) were diluted in 1 mL deuterated chloroform (CDCl3) and analyzed using NMR. The investigation was carried out using a JEOL JNM ECA 500 MHz (JEOL, Japan) to determine the chemical structure of the produced polymers [31].

#### (c) TGA, DTG, and DTA

Thermal analysis was performed on the purified polymers using the DSC Q20 and TGA Q50 analyzers. The analysis was carried out in the presence of a 20 mL/min N2 flow. Using a sequential heating system (heating rate 10 ◦C/min, temperature range 0 to 600 ◦C), 10 mg of moisture-free distilled PHAs were heated. Following that, the polymer's degradation temperature (*Td*), glass transition temperature (*Tg*), enthalpy of fusion (DHm), and melting temperature (*Tm*) were measured [31].

(d) XRD

The diffractograms were used to investigate the crystalline structure of the polyesters. A copper tube with a wavelength of 1.5418 A◦ was used to record the spectrum, which was operated at 30 kV and 10 mA. A 2 mm diameter capillary tube was employed, each scan was recorded in step-by-step mode from 0 to 100◦ (2θ) with 5◦ intervals, and the intensities were recorded [5].

#### *2.8. Statistical Analysis*

The statistically significant differences across the studies were examined using a oneway ANOVA with the Tukey test (*p* < 0.05 confidence). The process monitoring assays were

performed in triplicate, and the results were reported as the mean value and its standard deviation. The data was analyzed using Prism 7 (GraphPad, Inc. San Diego, CA, USA).

### **3. Results and Discussion**

#### *3.1. Enzymatic Degradation of Cardboard Samples*

Owing to the high percentage of cellulose in cardboard, which can reach up to 59% [25], it could be a promising source of carbon to produce value-added products. The complexity of polysaccharides in such waste (mainly cellulose and hemicellulose) has made cellulase systems paramount in liberating fermentable sugars. From an environmental perspective, the industrial implementation of the current study could assist in tackling waste accumulation issues in Europe, especially Poland. Paper and cardboard waste comprised approximately 15% of municipal solid waste from 2012 to 2017, whereas plastics resamples were 18%, and this data is expected to witness a considerable rise in the next years [32]. The chemical composition of corrugated cardboard showed that our samples contained 58.2% cellulose, 9.6% hemicellulose, 10.0% lignin, and 22.2% other components, which were in good line with another investigation [33]. Enzyme-assisted cardboard hydrolysis seems to be an appropriate approach for supplying PHA producers with sufficient amounts of carbon source in an economic manner. To assess cellulase activity on cardboard digestion, representative experimental concentration profiles of glucose and reducing sugars obtained in this set of experiments are shown in Figure 1.

**Figure 1.** Enzymatic hydrolysis of cardboard, (**a**) Incubation time effect on the hydrolysis process, (**b**) presentation of the enzymatic hydrolysis process on cardboard fibers on the 1st and the 7th day and (**c**) colorimetric analysis of the enzymatic hydrolysis products, including reducing sugars and glucose.

Regarding the liberated glucose profile, the glucose concentration was directly proportional to incubation time till day 7 at pH 5.5 and 55 ◦C. Moreover, the highest released glucose concentration was reported to be 18.3 ± 0.2 g/L on day 7, which comprises approximately 62% of the total cellulose content in the investigated cardboard samples (Figure 1a), whereas it exhibited almost constant values from days 8 to 15 (data not shown). The reason for the positive correlation between the released glucose and incubation days could be

attributed to cellulase activity on the cellulosic portion of cardboard [34]. Cellulases work synergistically to hydrolyze cellulose as it is degraded from crystalline or amorphous cellulose to small, soluble cellobiose fragments and finally to glucose [35]. The constant glucose records after day 7 demonstrated a complete saccharification of cellulose and hemicellulose contents, where the major morphological changes in the fiber structure took place. It might also be attributed to the depletion of cellulosic components in cardboard as well as the generation of metabolic by-products. (Figure 1b) [33]. Therefore, the incubation period of 7 days has been selected as the enzyme incubation time in the following experiments.

The effect of different enzyme concentrations was assessed on different samples' weights in terms of cardboard degradation. Maximum weight loss values for all samples were observed at an enzyme concentration of 1.5% (*v*/*v*), which was found to be in the range of 69 to 73% (Table 1). The maximum weight loss values that resulted from the highest enzyme concentration supports the fact that cellulase catalyzes the polysaccharide decomposition by simply cleaving β-1,4-glycosidic linkages [36]. The enzymatic action of cellulase could be seen in Figure 1b, where the firm fibers of cardboard were transformed to powder-like structures after 7 days incubation at 55 ◦C.

**Table 1.** Weight loss (%) of different cardboard samples after enzymatic hydrolysis. Mean and standard deviations were used to show the values. The presence of different superscript letters within the same column indicates significance (*p* < 0.05).


The hydrolysis products, such as released glucose and reducing sugars, were determined in 50 g cardboard samples. The highest enzyme concentration (1.5% (*v*/*v*)) released 18.3 ± 0.3 g glucose, which was the maximum value compared with the lower cellulase concentrations. Moreover, the glucose percentage per 50 g cardboard sample was determined to be 36.5 ± 0.4%, which represents 18.3 ± 0.3 g glucose produced from a 50 g cardboard sample. This glucose content was estimated to be approximately 85.1 ± 0.1% of reducing sugars per 50 g sample (Figure 1c). Such data were consistent with the obtained glucose values measured at 36–63% (g/g of cellulose sample) using commercial enzymes cellulase CTec2 and hemicellulase HTec [33]. These results support the fact that the yielded carbon source would provide a sufficient supplement for PHA production.

#### *3.2. Molecular Identification of the PHA Producer and Phylogenetic Analysis*

The screened soil bacterial isolates resulted in 9 PHA producers, which were confirmed by orange color fluorescence on Nile blue plates and Sudan black staining. The bacterial isolate with the highest PHA productivity was selected for further molecular identification. The 16S rRNA study of the PHA producing strain exhibited a significant degree of similarity to the *Bacillus* sp. genera. A sequence comparison using BLAST showed strain ICRI89's close relationship to *Bacillus mycoides* (formerly known as *Bacillus weihenstephanensis* UT11) with 100% similarity. The 16S rRNA sequence of the newly isolated strain was submitted to GenBank, NCBI as *Bacillus mycoides* ICRI89. A neighbor-joining dendrogram with several *Bacillus* sp. as an outer group shows the phylogentic position of *B. mycoides* ICRI89 (Figure 2).

**Figure 2.** Neighbor-joining phylogenetic analysis based on 16S rRNA gene sequences showing the position of strain *B. mycoides* ICRI89. These phylogenetic relationships were identified by MEGA 11 sequence alignment editor (version 11.0.11).

#### *3.3. Nucleotides Accession Numbers*

Consensus sequences were generated with the MEGA 11 sequence alignment editor (version 11.0.11), and the sequences were then evaluated with the BLASTN software (NCBI) [37]. The 16S sequence was submitted to GenBank with the accession number ON231789.

#### *3.4. MSM Supplemented with Glucose as a Carbon Source*

*B. mycoides* ICRI89 was cultivated in 1 L MSM in 2 L flasks with 2% (*w*/*v*) glucose and 0.05% (*w*/*v*) (NH4)2SO4 at 37 ◦C, pH = 7, 160 rpm for 7 days. The biomasses from PHA production were collected, lyophilized, and weighed [31]. The lyophilized mass of *B. mycoides* ICRI89 was 4.27 g/L, and the generated polyester was recovered from the cells. The pure polymers produced were 2.63 ± 0.1 g/L, corresponding to 61.7% CDW (Figure 3). These results are highly similar to those of a previous investigation [38] in which *B. megaterium* accumulated a maximum PHA weight of 2.74 g/L with glucose as the sole carbon source. When compared to other carbon sources, such as arabinose, starch, lactose, lactic acid, glycerol, or sodium acetate, *Bacillus* sp. can efficiently metabolize glucose for greater PHA synthesis. As a result, when glucose was added as a carbon source, it produced the highest PHA content when compared to other carbon sources [38]. The maximum PHA yield in the current study was detected upon using (NH4)2SO4 as a nitrogen source. PHA synthesis was comparable when various nitrogen sources were used, including, with

a minor difference, protease peptone, glycine, potassium nitrate, urea, and ammonium chloride. Previously, ammonium sulphate was shown to be the optimum nitrogen source for *B. mycoides* RLJ B017 [39] and *B. Megaterium* [40]. On the other hand, the current findings showed a higher PHA productivity than those of *Bacillus* sp. AZR-1, which used glucose for PHA production. *Bacillus* sp. AZR-1 produced CDW of 1.88 g/L with a PHA content up to 40%. It was shown to be extremely effective in exploiting soluble starch as a precursor for PHA accumulation, having a CDW of 0.87 g/L and a PHA content of 0.19 g/L, resembling 22% CDW. This amount is approximately half that when glucose was utilized as a carbon source, which is logical given that the starch hydrolysis process makes starch less useful when compared to simple monosaccharides, such as glucose [41]. This suggests that for growth and subsequent PHA synthesis, in general, these bacteria prefer simple monomers, such as glucose. Our isolate *B. mycoides* ICRI89 appears to have the most active metabolic machinery for synthesis of PHA. In many studies, there has been a positive correlation between the amount of glucose used as a carbon source and PHA production [42].

**Figure 3.** PHA content (g/L), cell dry weight (CDW) (g/L), and PHA productivity (%) in four different models.

#### *3.5. Cardboard Hydrolysate as a Whole Medium*

Enzymatically hydrolyzed cardboard was employed as a complete medium to evaluate our new strain's potential to accumulate PHA. When hydrolyzed cardboard was utilized as a PHA production medium for *B. mycoides* ICRI89, the highest PHA concentration was 0.4 ± 0.1 g/L, with 33.3% CDW PHA productivity (Figure 3). The PHA content witnessed an 84% reduction in comparison with that of glucose as a carbon source. In the same manner, there was an apparent decline in the yielded CDW to be 1.2 g/L compared to 4.27 g/L for the glucose standard medium. The major explanation for the low PHA concentration might be the lack of certain minerals and salts. Furthermore, the existence of KH2PO4 in the production medium at concentrations less than 0.1 g/L could reduce cellular proliferation, whilst concentrations higher than 0.1 g/L might improve PHA productivity. Thus, phosphate limitation (KH2PO4 and K2HPO4) was discovered to have an essential role in PHA build-up [43]. Enzymatically hydrolyzed waste paper was recently used as a sustainable feed stock for PHA synthesis by *B. Sacchari*. The bacterial strain accumulated 3.63 g/L PHA, which comprises approximately 44.2% CDW. The relatively higher yield compared to our study could be reasoned to performing enzymatic digestion for paper waste using an enzyme mixture of cellulase, β-glucosidase, and hemicellulose [44]. Such a

mixture may have played a critical role in having a better saccharification of lignocellulosic biomass, including paper waste. That's why it is recommended to examine the effect of different enzyme cocktails on the saccharification of different kinds of cardboard in future studies.

### *3.6. Modified MSM with Cardboard Hydrolysate*

The filtrate of cardboard hydrolysate (1 L) was supplied with all components of MSM except glucose, where the cardboard hydrolysate would be the carbon source. The concentration of carbon source was adjusted to be almost 2% (*v*/*v*)**,** which represents the content of reducing sugar in cardboard hydrolysate. The PHA generating isolate *B. mycoides* ICRI89 had a maximum PHA concentration of 2.1 ± 0.1 g/L, which corresponds to 65.6% CDW (Figure 3). This experiment yielded almost the same PHA weights of that when glucose was used as the only carbon source. These findings are consistent with a previous study aiming at increasing PHA bioproduction using wheat straw lignocellulosic hydrolysates [14]. Since *B. sacchari* DSM 17165 can metabolize its primary carbohydrates, such as glucose, xylose, and arabinose, it was employed to generate PHA from wheat straw hydrolysates. When grown on a mixture of commercial C6 and C5 sugars as a control production medium, the *B. sacchari* cell weight of 6.0 g/L accumulated approximately 4.4 g/L PHA, resembling 70% CDW with a polymer on sugar yield of 0.18 g/g, whereas when wheat straw hydrolysates were used as the carbon source, these values reached approximately 4.4 g/L PHA, presenting 60% CDW [14]. Even though PHAs offer several environmental benefits, their high production costs limit their widespread application. One of the grand challenges facing these polymers scale-up is the high-cost feed stock, especially carbon sources, which comprise nearly the majority of the final cost [45,46]. Paper rejects, including cardboard, could be promising sources for essential and simple sugars for bacterial metabolism supporting polyester accumulation. Different cardboard rejects treatment procedures may provide more glucose and xylose per kilogram. For example, sodium hydroxide treatment of cardboard yielded approximately 373 g glucose and 61 g xylose per kilogram of rejects, which is regarded as an economical technique for PHA scale-up [47].

## *3.7. Nitrogen Depleted MSM with Cardboard Hydrolysate*

Under nutrient-restricted environments, microbial polyesters accumulate with surplus carbon and polymerize as inclusion bodies [48]. To mimic such conditions, 1 L filtrate of cardboard hydrolysate was supplied with all components of MSM except glucose and (NH4)2SO4, where the cardboard hydrolysate would be the carbon and nitrogen sources. The concentration of carbon source was adjusted to be almost 2% (*v*/*v*), which represents the content of reducing sugar in cardboard hydrolysate. After 7 days of incubation at 37 ◦C and 160 rpm, *B. mycoides* ICRI89 used this hydrolysate to generate a PHA content of 0.3 ± 0.1 g/L (30% CDW) (Figure 3). The hydrolysate contains not only carbon but also nitrogen. The amount of nitrogen in the hydrolysate is insufficient to support biomass growth. A recent study [49] reported a low 0.003% nitrogen content in corrugated carboard acidic hydrolysate. Since high nitrogen concentrations directly increase cell biomass rather than PHA formation [50], PHA generation and cell proliferation were severely hindered.

When all the preceding data are compared, it is clear that the PHA concentration achieved by employing modified MSM with cardboard hydrolysate was extremely near to that obtained when glucose was used as the only carbon source. Sugar consumption in the cardboard hydrolysate medium was the primary cause of PHA production and build-up. Thus, polymers extracted from cells grown on glucose and cardboard hydrolysate medium as a carbon source were used for further characterization.
