# Valorization of Agri-Food Waste into PHA and Bioplastics: From Waste Selection to Transformation
## 3 Production of Bioplastics from Volatile Fatty Acids
### Microorganisms Responsible for the Transformation of VFA into PHA
#### 3.1.2 Mechanism of PHA Accumulation. Accumulating Microorganisms  
The mechanism of PHA accumulation has been extensively studied. There are various naturally occurring pathways for PHA formation. Numerous genetic studies have been conducted to investigate the regulatory mechanisms that control PHA formation in response to different growth conditions. Several studies using PHA mutants with alterations in genes other than the p b genes have provided insights into the fundamental role of cell physiology and central metabolism. These studies not only provide important knowledge for the design and engineering of recombinant organisms for PHA production [135]. Figure 4[138] illustrates the different pathways (A-G) to produce short-chain-length PHA (scl-PHA) using glucose as the carbon source:  
A: Represents the tricarboxylic acid cycle. B: Synthetic pathway to produce acetyl-CoA monomers. In this pathway, LDH refers to lactate dehydrogenase and PCT to propionyl-CoA transferase. C: Pathways derived to produce 3-hydroxybutyl-CoA (3 HB-CoA) from _Ralstoniaeutropha_ for., PhaA (ketothiolase), and PhaB (ketoreductase) are involved. D: Pathway for the formation of 3-hydroxyvaleryl-CoA (3 HIV-CoA) involving IlvA (threonine deaminase) and BktB (ketothiolase). E: 3 HB-CoA pathway production from fatty acid biosynthesis. Key enzymes are AccA (acetyl-CoA carboxylase), FabD (malonyl-CoA:ACPTransacylase), FabH (3-ketoacyl-CoA-CoA synthase III), and FabG (3-ketoacyl-CoA-CoA reductase). F: Alternative pathway for 3 HV synthesis involving the enzymes Sbm (bella dormant mutase), YgfG (methylmalonyl-CoA decarboxylase), and BktB (ketothiolase). G: The 4-hydroxybutyl-CoA production pathway involving SucD (succinate dehydrogenase), 4 HbD (4-hydroxybutyrate dehydrogenase), and the transferases Cat2 and Cat1 (4-hydroxybutyrate transferase). The study by [PERSON] and [PERSON] [135] provided a comprehensive account of the mechanism of polyhydroxyalkanoate synthesis in bacteria, with a particular focus on poly(3-hydroxybutyrate) (PHB) and other short- and medium-chain PHAs. The synthesis process of scl-PHA can be summarized as follows: The synthesis process commences with the generation of precursors derived from acetyl-CoA. This metabolite is essential and has its origin in a variety of carbon sources, including sugars, fatty acids (of which VFA is a prominent example), and other organic compounds. In circumstances where specific nutrients (such as nitrogen or phosphorus) are scarce, but carbon is plentiful, bacteria redirect surplus acetyl-CoA toward PHA synthesis as a means of storing energy and carbon. The initial stage of PHB biosynthesis entails the condensation of two acetyl-CoA molecules, resulting in the formation of acetoacetyl-CoA. The enzyme \(\beta\)-ketoacyl-CoA thiolase is responsible for catalyzing this step, which is a common process in many bacteria and marks the commencement of the PHA production pathway. This enzyme plays a pivotal role in the regulation of substrate allocation toward PHA synthesis, rather than other metabolic pathways. Subsequently, the formation of 3-hydroxybutyl-CoA is catalyzed by the enzyme acetoacetyl-CoA reductase, which is dependent on the cofactor NADPH (nicotinamide adenine dinucleotide phosphate (reduced form). This is a pivotal stage, as the reduction determines the structure of the monomer unit that will be incorporated into the PHA polymer. It is a selective step for the formation of 3-hydroxybutyrate (3 HB) monomers, which constitute the fundamental building block of PHB. The availability of NADPH is crucial for this reaction, indicating that the cellular environment must possess sufficient NADPH regeneration capacity to sustain the process. Subsequently, the polymerization of 3-hydroxybutyl-CoA into a PHA chain occurs and is catalyzed by a key enzyme: PHA polymerase. This enzyme binds to the 3-hydroxybutyl-CoA monomer and catalyzes the formation of ester bonds, resulting in the linear polymerization of the monomer and the formation of a very long chain.