Complete factorial design to adjust pH and sugar concentrations in the inoculum phase of Ralstonia solanacearum to optimize P(3HB) production

Abstract

Poly(3-hydroxybutyrate) (P(3HB)) is a biodegradable plastic biopolymer that accumulates as lipophilic inclusions in the cytoplasm of some microorganisms. The biotechnological process by which P(3HB) is synthesized occurs in two phases. The first phase involves cell growth in a complex culture medium, while the second phase involves polymer accumulation in the presence of excess carbon sources. As such, the efficiency of the second phase depends on the first phase. The aim of this study was to evaluate culture media with different concentrations of sucrose and glucose and different pH values in the inoculum phase of Ralstonia solanacearum RS with the intention of identifying methods by which the biomass yield could be increased, subsequently enhancing the yield of P(3HB). The culture medium was formulated according to the experimental planning type of central composite rotational design 22. The independent variables were pH and sugar concentration (sucrose and glucose), and the dependent variables were OD600nm, dry cell weight (DCW), and P(3HB) yield. The highest cell growth, estimated by the OD600nm (20.6) and DCW (5.35) values, was obtained when sucrose was used in the culture medium at a concentration above 35 g.L-1 in combination with an acidic pH. High polymer (45%) accumulation was also achieved under these conditions. Using glucose, the best results for OD600nm (12.5) and DCW (2.74) were also obtained at acidic pH but with a sugar concentration at the minimum values evaluated. Due to the significant accumulation of polymer in the cells that were still in the growth phase, the accumulating microorganism P(3HB) Ralstonia solanacearum RS can be classified as having type II metabolism in relation to the polymer accumulation phase, which is different from other Ralstonia spp. studied until this time.

Optimization of Ralstonia solanacearum cell growth using a central composite rotational design for the P(3HB) production: Effect of agitation and aeration

“…The decline phase in turn cannot be observed. Macagnan et al (2017) [16] reported that the stationary phase of the growth curve of R . solanacearum RS in YM medium [22], also expressed as OD 600nm , was within 22 to 28 h of culturing.…”

“…Although the Ralstonia spp. lineage is generally classified as belonging to group I [36–38], Macagnan et al (2017) [16] reported a P(3HB) production of 46.62% in the inoculum phase, without nutrient restriction, which is a characteristic of group II. Crochemore et al (2012) [34] described extensive P(3HB) accumulation—up to 70%, by the Pseudomonas strain CMM43 in the inoculum phase, suggesting that the accumulation occurred simultaneously with growth.…”

“…In the mathematical model tested, the dependent variables OD 600nm and DCW did not have predictive and significant results, thus it was not possible to generate response surfaces. Macagnan et al (2017) [16] evaluated the pH conditions in the inoculum phase to optimize the production process of P(3HB) by R . solanacearum RS, and found that a more acidic pH may indicate higher cell growth.…”

“…Then, the cell concentrates were resuspended in 0.89% (w/v) saline solution and centrifuged again. The DCW was obtained by drying the pellets in an oven at 56°C until constant weight [16].…”

“…solanacearum RS began its log phase of growth from 6 h of incubation, entering the stationary phase at 22 h. Glucose and sucrose were effective as carbon sources, and sucrose was the most appropriate source for cell growth, and the acidic pH assisted in a higher polymer yield, as a function of the carbon source used (sucrose or glucose). Thus, the microorganism was classified as having type II metabolism, once it did not require nutrient restrictions for the biopolymer accumulation [16].…”