**1. Introduction**

Since petroleum-based plastics have become a major cause of environmental pollution, degradable plastics have attracted a great deal of attention worldwide for their unique properties, such as thermoplasticity. However, production of these vital plastics is limited due to their high cost of production. Hence, studies looking for bacterial strains with better yields and improving their culture conditions are of the greatest importance in order to reduce the costs of PHB production [1]. The slow growth of the PHB market since the 1980s is due to the high cost of production for the amount of PHB produced. Currently, only about 1% of plastic materials on the market are biodegradable, and with the increase in environmental pollution, there is a need to increase the production of PHB [2]. PHB and

**Citation:** El-Kadi, S.M.; Elbagory, M.; EL-Zawawy, H.A.H.; EL-Shaer, H.F.A.; Shoukry, A.A.; El-Nahrawy, S.; Omara, A.E.-D.; Ali, D.F.I. Biosynthesis of Poly-ß-Hydroxybutyrate (PHB) from Different Bacterial Strains Grown on Alternative Cheap Carbon Sources. *Polymers* **2021**, *13*, 3801. https:// doi.org/10.3390/polym13213801

Academic Editor: Edina Rusen

Received: 4 October 2021 Accepted: 29 October 2021 Published: 3 November 2021

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**Copyright:** © 2021 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/).

different biodegradable polyesters are promising tools for the development of eco-friendly and fully biodegradable plastics. PHBs are a class of biopolymers produced by different microbial species. Unlike plastics derived from petrochemicals, PHB is biodegradable and biocompatible in nature. PHBs have advanced applications in the medical sector (surgical pins and sutures, wound dressings, bone replacements), the packaging industry (packaging films, bags, cosmetics containers, shampoo bottles), and agriculture (plastic mulch, as a carrier for long-term doses of herbicides, fungicides and insecticides) [3–6]. PHB is produced using various raw materials such as agricultural and food industry wastes, which contributes to its economic feasibility. Research is currently concerned with reducing the cost of production [7]. The use of food industrial byproducts in the production of PHB is considered one of the growing areas of modern biotechnology; these products can be used as a nutrient-rich medium at no additional cost. Whey and molasses are very important byproducts that are rich in nutrients and which bacteria can convert into PHB [8]. The use of molasses as a potential low-cost substrate for PHB biopolymer production has been studied by [9]. The use of molasses, which is a cheap substrate available in Egypt, may help reduce the cost of producing such biopolyesters [3]. Beet molasses has been shown to be an excellent starting material for PHB production by *Azotobacter vinelandii*. The substrate cost for PHB production from beet molasses in fed-batch culture was one-third of that using glucose [10]. The results obtained by [11] indicate that by using *Bacillus subtilis* RS1, agro-industry wastes such as sugarcane molasses can be used as an inexpensive substrate for PHB production. Date molasses and date seed oil have also been used as the sole carbon source for PHB biosynthesis [12]. Processes are currently underway to improve PHB production via genetic modification of some strains in order to develop desirable and suitable properties for some diverse applications [7]. Pavan [13] aimed to conduct an economic assessment of PHB production process using molasses. The alternative sources showed equally low production costs (USD 4.28 per kg) and better economic indicators among the evaluated scenarios. In recent years, using renewable raw materials for PHB production as degradable bioplastics has been a good method of environment consideration. Low-cost carbon substrates have the potential to reduce PHB production cost and increase sustainability [14]. According to [11], the global PHB market is expected to reach USD 93.5 million by 2021. In the Middle East and Arab countries, Date palm (*Pheonix dactylifera* L.) is an important commercial crop. Egypt is one of the largest date producers among Arab and world countries. Egyptian date production in 2020 was 1.9 million tons, which represented 21.2% of world production. Date soft types are highly perishable and easily attacked by microbial enzymes; thus, date and date molasses are considered good materials for fermentation processes [15–17]. The main reason for the low prevalence of bioplastic products is the high cost of production.

Therefore, this study aims to produce PHB using efficient PHB-producing bacteria on different available alternative carbon sources (whey, sugar beet molasses and date molasses) and to study their properties by infrared (IR) spectroscopy and gas chromatography GC-MS/MS mass spectrometry analysis.

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

#### *2.1. Bacterial Isolation*

Four different soil samples were obtained from Damietta and Dakahlia Governorates (New Damietta city, Kafer El-Batekh, Kafer Saad, and Kalabshou), Egypt, were used for bacterial isolation. From these soil samples, 30 bacterial isolates were randomly isolated on three different selective media (10 for each medium): medium (A) [18], medium (B) [19] and medium (C) [20], for PHB production. These isolates were identified in the Agricultural Microbiology Department, Faculty of Agriculture, Damietta University, Damietta, Egypt. Morphological and physiological characteristics of bacterial isolates such as shape, Gram stain, spore stain, capsule stain and motility were microscopically studied, as were indole and catalase tests, starch hydrolysis and casein hydrolysis [21–23].

Isolates obtained from each of the three previously-mentioned media (10 randomly picked from each medium) were maintained on slants of Nutrient agar medium [24], Modified Ashby's medium [25], and Nutrient-rich medium [26], incubated at 5 ◦C, and sub-cultured monthly.
