**3. Biosynthesised Plastics**

In recent years, PHA polymers have emerged as one of the most promising biodegradable materials. Unlike commonly used fossil-based plastics or PLA, which requires the additional step of lactic acid polymerisation, PHAs are the product of bacterial metabolism and have a function of cytoplasmic inclusions. These materials, thanks to the over 150 monomer units, can have a variety of polymer properties that can compete with commonly used plastics, such as polyethene or polypropylene [6]. Monomer composition is connected to the substrate specificity of PHA synthase, hence the type of derived PHA is highly connected to the microorganism used for its production. For example, biosynthesis of short-chain length PHAs (SCL-PHAs) consisting of poly-3-hydroxybutyrate P(3HB) homopolymers is a three-step process regulated by 3-ketothiolase, acetoacetyl-CoA reductase and the SCL PHA synthase [7,8]. Bacteria, such as *Aeromonas caviae* and *Pseudomonas stutzeri*, are proven producers of PHA synthases with wide substrate specificity. Moreover, these enzymes have been identified after recombinant expression in *Ralstonia eutropha*, previously PHA negative. These PHA synthases can produce copolymers of SCL- and medium-chain length PHAs (MCL-PHA) [9]. It has been reported that combinatorial mutations in *P. aeruginosa*, *P. oleovorans*, and *P. putida* resulted in their ability to synthesise PHAs with 3-hydroxybutyrate (3HB), 3-hydroxyhexanoate (3HHx), 3-hydroxyocatanoate (3HO), 3-hydroxydecanoate (3HD), or 3-hydroxydodecanoate (3HDD) monomers [10–13]. These structures are presented in Table 1. The development of recombinant strains and using genetic engineering techniques can lead to improved mechanical and thermal characteristics of PHA materials. These properties are highly dependent on various factors during upstream and downstream processes. Modification of PHAs chemical structure, such as the introduction of functional groups or producing blends and copolymers, can also affect the quality of these materials [14]. Traditional chemical synthesis techniques have been successfully used for creating block copolymers with PHA materials. For example, block copolymers, including PHB blocks balancing with other materials (poly(6 hydroxyhexanoate), poly(3-hydroxyoctanoate), monomethoxy-terminated poly(ethylene glycol) (mPEG), and poly(ethylene glycol) (PEG)), have been reported. These structures are presented in Table 2 [8,15].

**Table 1.** Chemical structures of monomers described as units of PHA copolymer producing strains.

**Table 2.** Chemical structures of polymers commonly found as building blocks in PHA related block copolymers.

PHAs can also be derived from various substrates, including industrial waste streams [16], food waste [17], supplemented solid biodiesel waste, plant oils [7]. Besides these, seaweeds were found to be great feedstock for PHAs productions [18]. These materials can be produced using different strategies, including batch, fed-batch and continuous processes, and various conditions could be used for their conduction. Batch cultivations are easy and simple to operate, but production yields are very low. On the other hand, fed-batch cultivation can provide higher product and cell concentrations (no substrate inhibition) [19].

Continuous cultivation are also considered to be a viable strategy, providing required concentrations of limiting substrates, such as fatty acids and their derivatives [20]. Nevertheless, large scale application is still impractical with this type of production. Another strategy used for PHA production is solid state fermentation (SSF)—microbial cultivation on solid support made of appropriate substrates. SSF can be performed with inexpensive cultivation media, such as substrates, based on agro-industrial residues. This strategy provides disposing of waste, while valuable compounds are being produced at the same time [21]. There are few additional advantages of SSF over submerged fermentation: Easier aeration, higher substrate concentration, as well as reduced downstream processing steps. However, keeping conditions constant during the process is the biggest drawback to the market-ready production of PHAs using this strategy [22].

Bacteria from the genus *Komagataeibacter* (former *Gluconacetobacter*) synthesises another interesting, biosynthesised bioplastic—bacterial cellulose (BC). The BC production is a multi-step, strictly regulated process that involves regulatory proteins, few enzymes and catalytic complexes. The formation of 1,4-β-glucan chains is the first step in BC synthesis. This process, together with chains' assembly and crystallisation, occurs intracellularly. The second step involves extracting cellulose chains from the cells and their assembly in fibrils [4,23]. The yield and properties of the resulting material highly depend on the used bacterial strain and conditions of the conducted process, including medium composition, aeration, shaking, etc. Morphological and physical properties of the resulting material are in correlation to the cultivation broth composition. BC production can be improved using genetically modified producing strains, isolating novel strains with the ability to produce BC, and investigating process parameters and their significance. Florea et al. isolated *Komagataeibacter rhaeticus* strain able to produce a high yield of BC, while growing in low nitrogen level medium [24]. In order to increase BC production in limited oxygen conditions, genetically engineered strains have been developed [25]. Hungund et al. reported using ethyl methanesulfonate and ultraviolet radiation for *Gluconacetobacter xylinus* NCIM 2526 strain improvement that resulted in a significantly higher yield of BC (30%) [26].

Different strategies can be used to produce BC, including aerated submerged cultivation, static culture and airlift bioreactors [27]. Effects of these conditions on cellulose mechanical properties are presented in Figure 1.

Tensile strength, polymerisation degree and crystallinity index are highly influenced by BC structure. Produced in static conditions, BC forms pellicles on the surface of the medium. This gelatinous membrane can vary in thickness up to few centimetres depending on substrates' availability. Produced like this, BC has a significantly higher crystallinity index and tensile strength than the BC produced in agitated culture. Supply of air in agitated cultures will result in pellets with a higher crystallinity index. Crucial factors in BC production are the design of the reactor and proper control of conditions and process. These parameters highly affect the yield and quality of the resulting material. No matter which strategy is used, pH, oxygen supply, and temperature are essential conditions that should be carefully monitored. These parameters are mostly strain-dependent, but it was shown that the optimal pH value for cell growth and BC production is usually between 4.0 and 7.023, while the optimal temperature is in the range 28–30 ◦C [28].

Despite the great mechanical properties of the resulting material, static culture cannot provide uniformity of the cultivation broth; hence, cells are not equally exposed to nutrients and the thickness of the BC layer can be uneven. Additionally, productivity achieved in this strategy is very low, and it demands an extensive period of cultivation [29,30]. To improve the productivity of this process, the fed-batch strategy was developed, and a constant BC production rate was achieved for 30 days [31]. Submerged cultivations with agitation provide uniformity of nutrients, especially oxygen, resulting in higher yields in comparison to the static cultivations and making the production process cost-effective. Products of agitated fermentation depend on applied agitation speed and may include various forms of cellulose: Spheres, pellets, fibrous suspension [32]. As previously mentioned, higher productivity is the biggest advantage of submerged compared to static cultivation, but drawbacks, such as products' shape consistency and limited mechanical properties, are issued to overcome [33]. Another problem that can limit BC yield in aerated cultivation is a synthesis of gluconic acid. Due to the high agitation rates and hydrostatic stresses, the production of the secondary, protective metabolites is favoured over cellulose. Nevertheless, submerged cultivation of BC was implemented in different types of bioreactors, such as airlift, stirred tank and rotating disk. Production of this material on a large scale is still an issue, and designing new or improving existing equipment for this purpose is an important subject of research [34].

**Figure 1.** Young's modulus, crystallinity index and degree of polymerisation of BC depending on cultivation conditions: a—with shaking, b—without shaking, c—with additional oxygen supply.
