*Article* **The Hyperproduction of Polyhydroxybutyrate Using** *Bacillus mycoides* **ICRI89 through Enzymatic Hydrolysis of Affordable Cardboard**

**Fady Abdelmalek <sup>1</sup> , Alexander Steinbüchel <sup>1</sup> and Marian Rofeal 1,2,\***


**Abstract:** Bioplastics are contemplated as remarkable substitutes for conventional plastics to accommodate green technological advancements. However, their industrial production has not been fully implemented owing to the cost of carbon resources. From another perspective, valorizing different paper mill wastes has become a prominent research topic. These materials may serve as an affording sustainable feedstock for bioplastic production. Adjustment of cardboard waste hydrolysate as suitable fermentation media for production of bacterial polyhydroxyalkanoates (PHAs) has been investigated. Cardboard samples were defibered and dried before enzymatic hydrolysis. The enzymatic degradation of commercial cellulase was monitored over 15 days. Interestingly, 18.2 ± 0.2 g/L glucose yield was obtained from 50 g cardboard samples using a 1.5% (*v*/*v*) enzyme concentration. The samples exhibited maximum weight loss values of 69–73%. Meanwhile, five soil samples were collected from local sites in Lodz, Poland. A total of 31 bacterial isolates were screened and cultured on Nile blue plates. Analysis of the 16S rRNA gene sequence of the most potent producer revealed 100% similarity to *Bacillus mycoides*. Cardboard hydrolysates whole medium, modified MSM with cardboard hydrolysate and nitrogen depleted MSM with cardboard hydrolysate were utilized for PHA production, followed by PHA productivity and cell dry weight (CDW) estimation compared to glucose as a standard carbon source. An impressive PHA accumulation of 56% CDW was attained when the waste hydrolysate was used as a carbon source. FTIR and NMR analysis of the isolated PHA indicated that functional groups of the polymer were related to PHB (polyhydroxybutyrate). Thermal analysis demonstrates that PHB and PHB-CB (PHB produced from cardboard hydrolysate) have degradation temperatures of 380 and 369 ◦C, respectively, which reflect the high thermal stability and heat resistance compared to the same properties for a standard polymer. This is the first demonstration of full saccharification of corrugated cardboard paper waste for high-level production of PHA. In addition, the attained PHB productivity is one of the highest levels achieved from a real lignocellulosic waste.

**Keywords:** polyhydroxyalkanoates; poly(3-hydroxybutrate); enzymatic hydrolysis; lignocellulosic waste; cardboard waste; waste management; *Bacillus* sp.

#### **1. Introduction**

It is acknowledged that there has been a growing need for bioplastics and biodegradable polymers in numerous fields owing to their diversified merits [1]. Polyesters are in high demanded in several sectors, such as pharmaceutical nanodelivery, food safety, and biomedical applications [2,3]. PHAs are efficient substitutes for petrochemical plastics from fossil fuels. They have been exploited in 3D printing inks, tissue engineering scaffolds, smart packaging materials, and biocompatible implants [4]. Moreover, modified PHA

**Citation:** Abdelmalek, F.; Steinbüchel, A.; Rofeal, M. The Hyperproduction of Polyhydroxybutyrate Using *Bacillus mycoides* ICRI89 through Enzymatic Hydrolysis of Affordable Cardboard. *Polymers* **2022**, *14*, 2810. https://doi.org/10.3390/polym 14142810

Academic Editor: Shashi Kant Bhatia

Received: 6 June 2022 Accepted: 8 July 2022 Published: 10 July 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

variants have become potent components as antimicrobial and antiviral agents, as well as targeting anticancer ligands [5,6]. Nevertheless, elevated production costs continue to inhibit scale-up. From an environmental perspective, various nations have urgent policies to turn to natural plastics manufacturing to mitigate the widespread usage of synthetic plastics [7]. Almost 4.8–12.7 million tons of plastic wastes are expected to enter the ocean by 2025, with no expected measures to manage this situation. PHAs are microbial polyesters accumulated inside bacterial cells under nitrogen limitations and excess carbon supplementation. The bioprocess technology for these polymers is still facing significant hindrance due to the negative effect of high-level production economics [8].

In this context, the usage of raw and waste materials has often been investigated in an attempt to identify economical strategies for bioplastic production. A strong example of this is a recent project which valorized marine algal biomass hydrolysate as promising feed stock for sustainable PHA synthesis. A simple acid pre-treatment for three marine algal biomass was performed in search of affordable sources of carbon to support microbial metabolism. A reasonable content of PHA was successfully produced using 3 different algal hydrolysates media from *Halomonas pacifica* ASL10 and *Halomonas salifodiane* ASL11 [9]. Since the issue of PHA commercialization has not been resolved, affordable alternative carbon sources, such as the use of agricultural waste [10], household waste [11], fats, oils, industrial by-products [12], glycerol, sugars, wastewater, and lignocellulosic materials [13], have been considered.

One of the most promising sustainable feedstocks for microbial polymer synthesis is lignocellulosic materials. Recently, a 70% yield of CDW (cell dry weight) poly(3 hydroxybutyrate) (P(3HB)) was obtained using *Burkholderia sacchari* DSM 17165 to promote enzymatically hydrolyzed wheat straw as a substitute medium [14]. Pretreatment of rice straw with dilute aqueous acid as an abundant sugar resource to provide a carbon source for producing high-value products, such as PHB using *B. megaterium* [15]. Mechanical conversion of biomass into smaller particles and fibers is commonly used to disrupt the lignocellulosic matrix and to gain access to the carbohydrates. Chemical pretreatment is frequently used to assist lignin decomposition and removal [16]. These procedures can be expensive, and they constitute a major impediment to broad carbohydrate production from lignocellulosic sources. These simple sugars can be used for biofuel production or other goods, such as biodegradable and compostable plastics, after the carbohydrate polymers are broken down to monomers [14].

Among these materials, cardboard is a yet undiscovered substance for affordable glucose supplementation. It is mainly composed of cellulose 56.1%, hemicellulose 10.4%, and lignin 12.8% [17]. They have been successfully used to obtain fermentable sugars, such as glucose [18,19]. Cardboard and other lignocellulosic stocks might undergo pretreatment, such as alkaline or paper hydrolysis, to ameliorate the efficiency of the enzymatic hydrolysis of cellulose. However, owing to the potential risks related to the formation of toxic compounds in alkaline or acidic hydrolysis, commercial cellulases digestion could be the optimum and most economical approach for the generation of glucose or fermentable sugars [15].

*Bacillus* sp. Has been widely used in industry and in academia due to the stability of its replication and plasmid maintenance, as well as its importance and supremacy in PHA production [20]. Furthermore, *Bacillus* sp. has a significant advantage over other bacterial species to produce PHAs due to the absence of a lipopolysaccharide layer, which makes extraction easier, as well as its ability to grow in low-cost raw materials and a high growth rate compared to other bacteria [21]. *Bacillus* sp. metabolism has proven to be rich, generating high-value products, such as lipopeptides, biosurfactants, antiviral proteins, and enzymes [22,23]. Thus, thorough screening of possible *Bacillus* strains from stressprone environments, improved PHA synthesis methodologies, and the addition of low-cost carbon sources may all contribute to make the entire process more cost-effective [20].

This study investigates a successful bioconversion of cardboard to one of the most important polyesters, PHB. The saccharification of cardboard samples was conducted via enzymatic hydrolysis. A set of experiments was performed to optimize the fermentation process of cardboard hydrolysate as a feedstock for a locally isolated *Bacillus* strain. This represents the first attempt to valorize cardboard as a reliable source of glucose for microbial PHAs synthesis.

#### **2. Materials and Methods**

#### *2.1. Materials and Chemicals*

A commercial cellulase enzyme NewCell Conc L (NewEnzymes, Portugal) was kindly provided by Professor Stanislaw Bielecki, Lodz University of Technology, Poland. The enzyme preparation was composed of Cellulase and, 2-Benzisothiazolin-3(2H)-one (>1%). Dinitrosalicylic acid (DNS) was purchased from Sigma-Aldrich, Germany. The glucose detecting kit was obtained from Biomaxima, Poland. All other chemicals and media components were purchased from Pol-Aura, Poland.

#### *2.2. Cardboard Sample Preparation*

Corrugated cardboard specimens were obtained from local public solid waste containers. The contents of cellulose, hemicellulose, and lignin were detected according to the Van Soest and Robertson method [24]. The specimens were cut into pieces before being suspended in water at 60 ◦C for 24 h at a solid concentration of 50 g/L and defibered for 1 min using an IKA T50 Ultra Turrax Mixer. Samples that had been defibered were filtered and air dried for further examination [25].

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

Enzymatic hydrolysis tests were conducted using a commercial cellulase enzyme preparation. The specific activity of 65 ± 0.3 FPU/mL and enzyme protein concentration of 122 ± 0.3 mg/mL were determined using filter paper activity and Bradford methods [26,27]. The hydrolysis process took place in Erlenmeyer flasks at 55 ◦C, 150 rpm. The pH was preserved at 5.5 using 0.05 N citric acid–sodium citrate buffer.

#### (a) Determination of optimum incubation time

To determine the optimum incubation time for the enzyme activity, 50 g samples of defibered cardboard were incubated with 1.5% (*v*/*v*) cellulase enzyme in 1 L at different time intervals (1–15 days). The released glucose concentrations were determined by calorimetric kit at 540 nm.

(b) Weight loss

Different sample weights (20, 50, 80, and 100 g) were used for enzyme activity assessment using different enzyme concentrations at 55 ◦C, 150 rpm for 7 days incubation. The remaining cardboard was filtrated, dried at 70 ◦C for 24 h and weighed [25]. The following equation was used to calculate the proportion of cardboard consumption:

$$(\text{W0} - \text{Wt}) \times 100/\text{W0}$$

where W0 is the sample's dry weight at zero time and Wt is the sample's dry weight after 7 days incubation.

(c) Determination of reducing sugars

The quantity of reducing sugars released in the enzyme-cardboard mixture was determined by DNS reagent technique [28,29]. A total of 1 g dinitro salicylic acid was mixed with 10 mL distilled water and agitated for 10 min. A volume of 500 μL supernatant was mixed with 500 μL dinitrosalicylic acid reagent, left for 15 min at 28 ◦C, boiled for 5 min, then rapidly cooled, and the optical density was measured at 540 nm.

(d) Determination of liberated glucose

The enzymatic hydrolysis processes were stopped after enzymatic hydrolysis by keeping the solution at 4 ◦C for 2 h. The supernatants were recovered by centrifugation at 4 ◦C for 10 min at 8000 g. The glucose colorimetric kit (Biomaxima, Poland) was used to determine the glucose content of the hydrolysis liquors [30].

#### *2.4. Collection of Samples, Growth Conditions, and Screening of PHA Producing Isolates*

Soil samples were collected from several places in Lodz, Poland. In brief, samples were collected in sterile containers and transported directly to the lab, where they were serially diluted and inoculated on PHA screening plates. The screening medium is a Linko medium containing 2% glucose and 0.02 mg/L Nile blue stain as an indicator (Sigma-Aldrich-Darmstadt, Germany). Under UV light, the PHA synthesizing colonies appear orange [31]. Pure cultures of PHA-producing bacteria were then grown on Nutrient agar plates (pH 7.0 at 37 ◦C for 24 h) for future usage. Sudan black-B (SBB) stain (Sigma-Aldrich-Darmstadt, Germany) was used to perform a confirmatory screening test for PHA generating isolates. The accessibility of the C/N ratio in the medium plays a key role in PHA production. The use of a high carbon-containing medium promotes PHA accumulation. Thus, the medium employed was previously reported by [31] with minor adjustments. The production medium contained (in g/L) glucose (20), (NH4)2SO4 (0.5), KH2PO4 (2.0), Na2HPO4 (2.0), MgSO4·7H2O (0.5), Na2HCO3 (0.5), and CaCl2 (0.01) and a 100 mL trace element solution containing ZnSO4·7H2O (0.01), MnCl2·4H2O (0.003), H3BO4 (0.003), CuCl2·2H2O (0.001), and NICI2·6H2O (0.002). The incubation was carried out at 37 ◦C, pH 7 and 160 rpm.

### *2.5. Identification and Characterization of PHA Producing Isolates*

The Genomic Mini kit (A&A biotechnology, Pomeranian Voivodeship, Poland) was used to extract genomic DNA from the selected bacterial strain, with minor adjustments to the first step: bacterial cells were treated with lysozyme and incubated at 37 ◦C for 20 min. The PCR was carried out using a MJ Mini Gradient Thermal Cycler (Bio-Rad, Hercules, CA, USA). The 16S rRNA gene was amplified using universal primers 27F and 1492R (5 - AGAGTTTGATCCTGGCTCAG-3 5 -GGTTACCTTGTTACGACTT-3 ). Each PCR reaction contained 40 pmol of each primer, 1.5 U of RedTaq ReadyMix DNA polymerase (Sigma-Aldrich, St. Louis, MO, USA) and 20 ng of template DNA, and it was built up to 50 μL with PCR grade water. A 1.0% (*w*/*v*) agarose gel electrophoresis in 0.5 TBE buffer was used to identify PCR products (Sigma-Aldrich). The Big Dye Terminator Ready Reaction Cycle Sequencing kit was used to purify PCR products and extract gene nucleotide sequences (Applied Biosystems, Foster City, CA, USA). The PCR products were examined using an Applied Biosystems model 3730 Genetic Analyzer. The nucleotide sequences of the 16S rRNA gene were proofread, assembled, and aligned in Vector NTI Express Software (Life Technologies, Thermo Fisher Scientific Inc., Waltham, MA, USA), and they were compared with sequences available in the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA), using the BLASTN algorithm (Version 2.2.30+) [31].

### *2.6. PHA Production Extraction and Purification*

#### (a) MSM supplemented with glucose as a carbon source

The isolated PHA producer was cultured in Nutrient broth overnight at 37 ◦C and 160 rpm before being transferred to Mineral Salt Medium (MSM) to produce PHA [9]. The medium was supplemented with 20 g (2% *w*/*v*) glucose and 0.5 g (0.05% (*w*/*v*) (NH4)2SO4 to maintain a higher C/N ratio. The production cultures were incubated at 37 ◦C, 160 rpm for 7 days.

#### (b) Cardboard hydrolysate as a whole medium

The production media were prepared by filtrating 1 L of the cardboard hydrolysate medium to remove the cardboard residues. The filtrate was then kept at 4 ◦C for 2 h to stop the enzyme activity. The filtrate medium was sterilized by filtration using 200 nm bacterial filters (Alchem, Poland), then inoculated with the PHA producing strain and incubated at 37 ◦C, 160 rpm for 7 days.

(c) Modified MSM with cardboard hydrolysate

PHA production was studied in a synthetic medium in which 1 L filtrate of cardboard hydrolysate 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*).

#### (d) Nitrogen depleted MSM with cardboard hydrolysate

The filtrate of cardboard hydrolysate (1 L) was supplied with all components of MSM except glucose and (NH4)2SO4, where the cardboard hydrolysate would be the source of carbon and nitrogen. The concentration of carbon source was adjusted to be almost 2% (*v*/*v*), as previously mentioned.

#### (e) Extraction of PHA

The whole volume of the fermentation medium was centrifuged at 4 ◦C in a cooling centrifuge (4500 rpm for 15 min), and the cell pellets were freeze dried. The dry pellet weight was measured, and the bacterial cell wall was disrupted by treating it with hot acetone (50 ◦C) for 20 min. The suspension was centrifuged at 4500 rpm for 15 min before being dried to eliminate excess acetone. PHA was dissolved with chloroform at 37 ◦C for 48 h, while shaking at 160 rpm. The precipitation of PHA was done using cold methanol and water (7:3) [31]. The percentage of PHA yield was determined by the following equation:

$$\text{PHA yield} \%= \mathsf{W\_{PHA}}/\mathsf{W\_{Cells}} \text{ \* 100}$$

where WPHA resembles the weight of the polymer recovered from the freeze-dried cells weight (WCells).
