# Biosynthesis of Polyhydroxyalkanoates from Defatted _Chlorella_ Biomass as an Inexpensive Substrate
## 1 Introduction  
Petrochemical-based materials have gained particular attention in various fields due to their excellent properties, such as good mechanical resistance and high thermal stability. However, being non-biodegradable, the accumulation of these materials may produce adverse effects on environmental and public health [1]. Recently, biodegradable polymers derived from microbial fermentation have been investigated as promising alternatives to help reduce plastic waste disposal in the environment. Among a variety of bioplastics, polyhydroxyalkanoates (PHAs) produced through microbial fermentation have received particular interest owing to their low toxicity, biocompatibility, complete biodegradability, and competitive properties compared with conventional plastics [2]. Despite their excellent properties and environmental benefits, the industrial production of PHA is somehow restricted due to the use of highly purified substrates that may entail high costs. Therefore, many studies have investigated the use of residue raw materials such as rice bran [3], lignocellulosic biomass [4], waste cooking oils [5], and coffee waste [6] as inexpensive fermentation substrates to reduce the PHA production cost. Among a variety of waste materials, biomass from macro- and microalgae has shown great potential as a promising candidate for the industrial production of bioplastics due toits high carbohydrate content and negligible lignin amount giving rise to high reducing sugar recovery without any costly lignin removal [7]. Moreover, the utilization of algal biomass as a substrate for PHA production may reduce food waste utilization, which may entail ethical issues related to the interference with human nutrition and animal feeding. Dilute acid hydrolysis has been widely used for the pretreatment of algal biomass owing to its ability to enhance the conversion efficiency of carbohydrates into reducing sugars [8]. Previously, high fermentable sugar contents of 29 g/L and 52 g/L were respectively reported by [PERSON] et al. [9] and [PERSON] et al. [10] as a result of subjecting either _Laminaria japonica_ or defatted _Chlorella_ biomass to dilute acid hydrolysis using 94 mM of sulfuric acid (H\({}_{2}\)SO\({}_{4}\)) and 0.3 N of hydrochloric acid (HCl), respectively. _Chlorella_ (chlorophyte, Trebouxiophyceae) is considered to be one of the most cultivated microalgae in the world, providing more than 2000 tons of annual biomass production, and has been widely used as feed and food raw material owing to its high content of proteins, short-chain polyunsaturated fatty acids, vitamins, and minerals [11]. In the last few years, defatted _Chlorella_ biomass (DCB) has received a particular interest as an excellent feedstock for bioethanol production [12; 13] owing to its high content of carbohydrates either present in the form of hemicellulose and cellulose in the cell wall or accumulated in the form of starch in the chloroplast [14]. Recently, our research group has claimed the potential use of _Chlorella_ biomass as a promising substrate for the production of PHA owing to its high carbohydrate content (69.5%) and has reported a maximum PHA production of 3.62 g/L (39.4% of dry cell weight) when _Paracoccus_ sp. LL1 was cultivated in a minimal medium supplemented with 20 g/L of DCB-hydrolysate-derived sugars [10]. Among a variety of PHA-accumulating bacteria, _Bacillus megaterium_ ALA2, _Cupriavidus necator_ KCTC 2649, and _Haloferax mediterranei_ DSM 1411 strains have shown great potential in producing high PHA contents through the bioconversion of algae biomass [7; 9; 15]. In this work, a dilute acid pretreatment using 0.3 N of HCl was performed to extract reducing sugars from 10% (\(w/v\)) of DCB. The resulting HCl DCB hydrolysate was investigated as a sustainable substrate to assess the ability of three bacterial strains, namely _B. megaterium_ ALA2, _C. reactor_ KCTC 2649, and _H. mediterranei_ DSM 1411 to produce PHA in shake flasks. Along with PHA, the ability of _H.