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

Date Published: January 29, 2019

Publisher: Public Library of Science

Author(s): Mariane Igansi Alves, Karine Laste Macagnan, Camila Rios Piecha, Matheus Marques Torres, Izadora Almeida Perez, Sônia Maria Kesserlingh, Rosane da Silva Rodrigues, Patrícia Diaz de Oliveira, Angelita da Silveira Moreira, Vijai Gupta.


The intracellular accumulation of polyhydroxyalkanoates (PHAs) normally occurs after cell growth, during the second fermentation stage and under nutrient-limited conditions in the presence of a carbon excess. However, some microorganisms are able to accumulate PHAs as poly(3-hydroxybutyrate) [P(3HB)] during the first fermentation stage, the cell growth phase, without nutrient limitation, once they have been reported to utilize type II metabolism during the polymer accumulation phase. This study evaluated the effect of aeration and agitation on cell growth and P(3HB) accumulation in Ralstonia solanacearum RS, performed in a bioreactor for 24h at 32°C. A 22 central composite rotational design (CCRD) was used, with agitation (150 to 250 rpm) and aeration (0.3 to 1 vvm) as independent variables and optical density (OD600nm), dry cell weight (DCW), and P(3HB) yield as dependent variables. A significant polymer accumulation, until 70% of P(3HB), was observed, proving that R. solanacearum RS exhibited metabolism type II, regardless of the aeration process. The best results were obtained for 1 vvm and 250 rpm (+1, +1), with values of OD600nm (18.04) and DCW (4.82 g.L-1).

Partial Text

In the last six decades, 8.3 billion metric tons of plastics have been produced, most of which disposable single-use plastics [1]. It is estimated that 91% of all plastics produced is not recycled, and 6.3 billion metric tons has become plastic waste [1,2]. For years, scientists have been investigated ways to reduce these numbers to prevent the volume of plastics that end up in the world’s oceans, causing damage to marine mammals, birds, and fish, among others. By the middle of the century, there will be more plastics in the oceans than fish––a chilling prognosis [2]. In this context, the use biodegradable polymers may be an effective alternative to reduce the excessive amount of plastic waste in the environment, thus leading to a lower environmental impact [3,4].

The growth curves in terms of OD600nm of the R. solanacearum RS strains developed in both the shaker and the bioreactor are shown in (Fig 1A, 1B and 1C).

In growth curves, OD600nm exhibited similar values at 20 h in both the shaker and bioreactor, with the highest OD600nm observed in the bioreactor. Higher DCW values (7.6 g.L-1) and lower P(3HB) yield were observed over time. The sugar residuals decreased during the process, and nitrogen also decreased within 30 h, increasing afterwards. According to the present results, the best aeration and agitation conditions in the inoculum phase was 1 vvm and 250 rpm (pH 5.9, OD600nm 18.04, DCW 4.82 g.L-1, and 42.87% yield P(3HB). In relation to the P(3HB) accumulation by R. solanacearum RS in the initial phase, it can be said that the microorganism belongs to group II, once it has no metabolic requirements for the biopolymer production. Further studies should be performed to evaluate whether the growth and P(3HB) accumulation in the microorganism studied can be improved by a continuous approach.




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