**Abstract**

Polyhydroxyalkanoates (PHAs) are biopolymers produced by numerous bacteria and can be used in the production of bioplastics. PHAs are synthesized by microorganisms by fermentation of carbon sources. Due to the different monomer structures of PHAs, there are many kinds of PHAs, and their corresponding material properties are also very different. Thus, the search for bacteria producing the PHAs is of great interest. In this study, the bacteria isolated from the environment were analyzed for the presence of PHA. PHA production was tested with staining methods Sudan Black B, Nile Blue, and Nile Red. The presence of a PHA synthase gene (*phaC*) was confirmed by PCR amplification. PHAs were extracted from the strains and characterized by the FTIR spectroscopy method. A biochip for a fast screening of environmental samples for the presence of PHA-producing bacteria was designed. The biochip contained 11 probes for coding class 1, 2, and 3 PHA synthase genes.

**Keywords:** polyhydroxyalkanoates (PHAs), PHA synthase gene, environmental bacteria, biochip, bioplastics

### **1. Introduction**

Plastics (polymeric materials) are highly functional materials that have become an essential part of the products we use in our daily life due to their ease of production and robustness. Around 360 million tons of plastics have been produced in 2020 around the world [1]. As more plastics are used, especially with their short-use life span, more waste surfaces. Plastic trash photodegrades into smaller fragments (microplastics). As it was emphasized in Ref. [1], the worldwide use of disposable face masks during the pandemic time and still now is an additional source of microplastics in the environment. Plastics are majorly manufactured from petrochemical feedstock, accounting for 80% of the total produced plastics. The building blocks for the polymerization of bioplastics are biopolymers cultivated from renewable production pathways, such as polymers from microorganisms [2, 3]. Bio-based

polymers have a lower carbon footprint than petrochemicals because they utilize biological materials and waste, which convert biocaptured CO2 into durable polymeric materials. It is a well-established fact that microorganisms are equipped with diverse metabolic activities that enable them to work as biorefineries for transforming a wide range of petrochemical and organic wastes into high-value specific products, such as polyhydroxyalkanoates biopolymers, while positively impacting the carbon cycle by consuming atmospheric CO2 [4]. The chemical structure of PHA is shown in **Figure 1**. PHA is a linear polyester that contains 3-hydroxy fatty acid monomers [5]. The most commonly produced PHA is poly 3 hydroxybutyrate (PHB), where the alkyl group is R = CH3. However, there are over 150 different monomers, such as polyhydroxyvalerate (PHV, R = C2H5), polyhydroxyhexanoate (PHH, R = C3H7), and polyhydroxyoctanote (PHO, R = C4H9). PHAs are high molecular weight linear polyesters, ranging in size from 50 to 10,000 kDa [2], characterized by a diversity of structures defined by the length of the carbon chain, referred to as short-chain length (SCL), medium-chain length (MCL) [6], and longchain length (LCL) [7] PHAs.

A very important property of biopolymers is hydrophobicity, which determines their solubility, biodegradability, and biocompatibility. As PHAs contain chains of hydrophobic groups with different lengths and structures, and at the same time PHAs are poorly hydrophilic due to the presence of carbonyl groups, the hydrophobicity-hydrophilicity balance is a very important point in the selection of appropriate bacteria that give the desired biomaterials [2]. PHAs can exist as homo or copolymers. PHB is a homopolymer, which has a linear isotactic structure, that is, highly crystalline making it brittle and unsuitable for many applications. This problem can be circumvented by forming a copolymer. The first commercially manufactured PHA, Biopol®, is copolymer produced from poly (3-hydroxybutyrateco-3-hydroxyvalerate (PHB/PHV) that had an increased side chain length making it less crystalline and more ductile [8]. These biopolymers have been produced commercially since the 1980s, and are currently marketed as Mirel®. The combinations in different proportions of the available PHAs create copolymer plastics with various properties [9]. PHAs are synthesized by various types of bacteria in the form of water-insoluble granules, and are stored as carbonosomes within the cell cytoplasm [10]. Bacteria capable of producing PHAs include species of *Alcaligenes*, *Bacillus*, *Burkholderia*, *Ralstonia*, *Pseudomonas*, *Aeromonas,* and *Thiococcus*, among others [11, 12]. PHA synthases and depolymerases, which are the catalyst enzymes for PHA production and degradation, respectively, are located at the membrane of the storage organelles and control the amount of PHA stored by the cell. There are eight major pathways for PHA synthesis. The main metabolite, acetyl-CoA, provides the various lengths of 3-hydroxyalkanoyl-CoA, which act as substrates for PHA production [13]. These pathways are intricately linked to central metabolic pathways

**Figure 1.** *Chemical structure of polyhydroxyalkanoates (PHAs).*

*Bioplastics against Microplastics: Screening of Environmental Bacteria for Bioplastics… DOI: http://dx.doi.org/10.5772/intechopen.109756*

*via* glycolysis, Kreb's cycle, β-oxidation, fatty acid synthesis, and others. Many enzymes and genes are involved in PHA synthesis. Bacterial cells normally grow when carbon (i.e., fructose and glucose) and nitrogen (ammonium) are present in the medium/environment. Polyhydroxybutyrate (PHB) is produced when the nitrogen source is depleted and carbon is present and will be utilized after refeeding cells with a nitrogen source [14].

The limiting step in the commercial development of non-petrochemical-based produced plastics is the biopolymer yield obtained after fermentation and the cost of its recovery. As the main drawback of bioplastics is the cost of the fermentation process, this has led to searches for cheaper carbon sources for fermentation, which has included activated sludge, paper and food wastes, wastewater, and various oil wastes [15]. Additionally, a lot of effort has been directed toward isolating PHAproducing bacteria from high carbon, low nitrogen, or phosphorus environments that might give greater yields in batch or continuous cultures [16]. The use of different carbon sources in fermentations and isolating producing strains from carbon-rich environments has led to the discovery of novel PHA polymers with different properties. In this study, the results of screening of environmental samples for PHAproducing organisms, isolation and characterization of the microorganisms, detection of genes, coding for enzymes involved in PHA synthesis, and physical characterization of the produced PHAs biopolymers are presented.
