Comparative performance of medium-chain-length Polyhydroxyalkanoates production 1 between Pseudomonas aeruginosa and Pseudomonas putida

Non-degradable polymer waste enlarged over increasing of human population. This badly 26 affected on environmental pollution and biosphere changing. Polyhydroxyalkanoates (PHAs), an eco- 27 friendly biopolymer, were substituted for conventional- or petrochemical-derived polymer. They are 28 reserve carbon energy produced from diversity of microorganisms in granular under limited nutrient for 29 bacterial growth (e.g. nitrogen and phosphorus). Pseudomonas aeruginosa TISTR 1287 was used to 30 catalyze emulsified palm oil to produce valuable PHAs. The highest yield of PHAs production (0.65 31 g/L, 38.0%) was obtained in MSM supplemented with 0.75% (v/w) emulsified palm oil after 72-hr 32 cultivation, which was a few lower than that produced by Pseudomonas putida TISTR 1522 (0.95 g/L, 33 40.15%) cultivated in 1% (w/v) fatty acid salt for after 24-hr cultivation. The intracellular PHAs were 34 detected by staining with Nile red. The characters of intracellular PHAs examined by Transmission 35 Electron Microscope (TEM) exhibited that PHAs accumulate in white and roundish-shaped granules 36 with 0.2-0.5 m diameter inside the cells, about 2-3 granules per rod-shaped bacterium cell. These 37 optimizations were successfully demonstrated high content of intracellular PHAs accumulation in P. 38 aeruginosa TISTR 1287 by utilization of emulsified palm oil. 39


Introduction 44
The overpopulation of human has led to the accumulation of enormous amounts of non-45 degradable petrochemical polymer waste causing environmental pollution and dramatically changing 46 of biosphere [1][2][3]. To reduce these problems, an environment-friendly polymer was developed to 47 substitute for petrochemical-derived polymers. Polyhydroxyalkanoates (PHAs) are biopolymer 48 produced in granular by a variety of bacteria under unbalance nutrient condition as carbon or reserve 49 material [1,[4][5]. They have attracted attention as alternative plastic to replace petrochemical polymers 50 with favorable properties of their complete biodegradability and production from a low-cost, renewable 51 feedstock such as palm oil [6][7]. The high carbon content and abundance of palm oil are promising of 52 3 feedstock that can be converted into valuable PHAs by bacteria. Pseudomonas aeruginosa and 53 Pseudomonas putida are promising microorganisms that accumulate high PHAs content and capably 54 utilize a number of feedstock including plant oil [7][8][9][10]. The fatty acids in plant oil were favorable 55 carbon source for bacteria growth and catabolized via -oxidation to produce PHAs [8,10]. The PHAs 56 have a broad range of advantageous applications, for instance, a nanoparticle for drug carrier, a part of 57 bioplastic element for molded plastic or single-used polymer, and fertilizer coating film which was 58 applied in agriculture [12]. 59 Palm oil (Morakhot olein palm oil), a plant oil, available in local market was used as a sole 70 carbon source for PHAs production. The composition of fatty acid in palm oil was determined as 71 described previously [8]. Gum arabic was discovered as an effective emulsifier for palm oil, with no 72 effect on bacterial growth [10]. In this study, for PHAs production by P. aeruginosa TISTR 1287, 73 bacteria secretes lipase, palm oil was emulsified using Gum arabic as emulsifying agent. Emulsified 74 palm oil was sterilized, mixed with culture medium and adjusted pH to 6.90 [6, 10, 13]. In comparison 75 to our pervious study, fatty acid salt obtained from saponified palm oil was used as a carbon source to 76 produce PHAs by P. putida TISTR 1522, due to its lack of secreted lipase [11].

Culture medium and cultivation condition 81
Cells were grown in Nutrient Broth (NB) and induced for PHAs production in Mineral Salts 82 Medium (MSM) [9]. The MSM was mixed with emulsified palm from previous experiment before using 83 for PHAs production by P. aeruginosa TISTR 1287. Experiments were set up in an orbital shaker 84 incubator (180 rpm, 30°C). Cells were harvested by centrifugation at 9,000 rpm for 20 min. 85 To obtain cell mass for PHAs production, P. aeruginosa TISTR 1287 was grown overnight in 86 50 mL NB (first-subculture) before transferring to 500 mL NB (second subculture). During late-87 exponential growth, bacterial cells were harvested by centrifugation and washed with MSM. The 88 standard curve between Colony Forming Unit (CFU) and cell dry weight (CDW) content was used to 89 calculate the amount of initial bacterial cells (10 11 CFU mL-1) for PHAs production in MSM 90 supplemented with emulsified palm oil [10]. For P. putida TISTR 1522, cell mass was obtained as 91 described above. 92 93

Optimization for PHAs production 94
The influence of various parameters on PHAs production by P. aeruginosa TISTR 1287 and P.

PHAs extraction 116
PHAs extraction was performed as described previously [7]. The PHAs-accumulating cells 117 were harvested, washed with 0.85% (w/v) NaCl solution and dried at 80 °C for 5 hr. The dried cells 118 (0.4 g) were soaked in 95% ethanol, placed in Cellulose Extraction Thimble and extracted by 119 dichloromethane for 5 hr using Sohxlet extractor. The dried weight product (PHAs) was determined 120 after incubation of pellet at 70 °C for 2 hr. 121

Monitoring of bacterial growth 123
In the growth curve of P. aeruginosa TISTR 1287 and P. putida TISTR 1522, late-exponential 124 growth of bacterial cells exhibited between 10-24 hr and 5-11 hr, respectively (Fig 1a). Moreover, the 125 first-and second-subculture showed both late-exponential growths after 24 hr cultivation for P. 126 aeruginosa TISTR 1287, and after18 and 30 hr cultivation for P. putida TISTR 1522 (Fig 1b, c). The 127 initial amount of bacterial cells that calculated from standard curve between CFU and CDW content of 128 P. aeruginosa TISTR 1287 and P. putida TISTR 1522 (Fig 1d, e) was grown in MSM supplemented of 129 different concentration of emulsified palm oil or fatty acid salt for PHAs production. concentration and then slightly increased over cultivation time [7], which showed the pH values in the 145 ranging of 6.34-6.40 for P. aeruginosa TISTR 1287 and 6.22-6.47 for P. putida TISTR 1522 (Fig 2). and PHAs accumulation which was 0.75% (w/v) emulsified palm oil for P. aeruginosa TISTR 1287 160 (this study) and 1.00% (w/v) fatty acid salt for P. putida TISTR 1522 [7]. Therefore, this emulsified 161 palm oil concentration was selected for further optimization for PHAs production.  (Table 1). This observation was noticeable that PHAs 174 accumulation and CDW content increased when the pH value decreased (Fig 4), consistent with 175 previous result that PHAs production by P. putida TISTR 1522 in MSM supplemented with 1% fatty 176 acid salt which obtained maximum value at 1.23 g/L with CDW content of 3.23 g/L after 60 hr 177 cultivation [7]. Accordingly, P. putida KT2442 and P. aeruginosa ATCC 9027 produce PHAs during 178 exponential growth [19][20], and PHAs production relied on the nutrient depletion on medium [6, 9, 11, 179 13-14]. These result show that emulsified palm oil and fatty acid salt can be a good carbon source for 180 PHAs production using P. aeruginosa TISTR 1287 and P. putida TISTR 1522. Comparison of our 181 PHAs production yield with that from other reports reveals that our PHAs production from P. 182 aeruginosa TISTR 1287 was higher than previously reported data using other P. aeruginosa strains, 183 and comparable or higher than previously reported data using other P. putida strains ( Table 2) We confirm that the manuscript has been read and approved by all named authors. 256 We confirm that the order of authors listed in the manuscript has been approved by all named authors. 257 258

Funding 259
Funding was received for this work.

Competing Interests 266
The authors declare that they have no known competing financial interests or personal relationships that 267 could have appeared to influence the work reported in this paper. 268 269

Availability of data and materials 270
All data generated or analyzed during this study are included in this published article.