Optimization of PHB production by Halomonas titanicae KHS3 applying experimental design

MARQUEZ, V.; HERRERA SEITZ, M. K.; RODRÍGUEZ, A.N.; STUDDERT, C.A.

Congreso; LVII Reunión Anual de SAIB- XVI Reunión Anual de SAMIGE; 2021

Polyhydroxyalkanoates are biodegradable polymers, considered potential substitutes for conventional petroleum-derived plastics. Halomonas titanicae KHS3 (Ht KHS3) is a moderately halophilic bacterium isolated from seawater of the port of Mar del Plata which has shown a wide metabolic versatility to grow in various nutritional conditions. Glycerol is the main by-product of the biodiesel industry, and therefore, the ability of Ht KHS3 to grow and accumulate polyhydroxybutyrate (PHB) with glycerol as the only carbon source was studied in our group. In order to simultaneously study the variables affecting the PHB accumulation, factorial experiments were carried out, using the Design Expert 7.0.0 software. A first screening was carried out using a Plackett-Burman design including nine factors: concentration of glycerol, ammonium, NaCl, Mg, phosphates, and Fe; initial pH, volume, and culture time. The volumetric yield of PHB was evaluated as a response variable. The concentration of glycerol, phosphates and Fe, and the initial pH, had significant positive effects on the PHB yield, whereas ammonium concentration showed a significant negative effect on this response. NaCl concentration did not significantly affect the PHB yield. We redefined the experimental space, establishing values for volume, growth time and NaCl (in the upper range to limit contamination risk) and for a more detailed analysis a fractional factorial design was used. This second analysis identified significant positive effects for glycerol, Fe, and the initial pH on the production of PHB and were involved in significant interactions. Again, ammonium had a significant negative effect on PHB yield. Phosphates and Mg did not have a significant effect. Finally, for optimization, a central composite design was carried out, fixing values for phosphates and Mg within the range studied and varying glycerol, ammonium, initial pH, and Fe. As a result, it was possible to find an optimum in the production of PHB. The desirability function was applied, using the same software, to find numerically the combination of factors to generate the highest PHB yield. To validate this result, cultures were performed in triplicate in the predicted optimal condition. The result showed a PHB value slightly higher than that predicted by the program. In this work, main effects, and interactions of multiple variables on PHB production could be systematically explored and it was possible to optimize the production of PHB by Ht KHS3 using glycerol as the sole carbon source. The observed negative effect of ammonium concentration on PHB yield might be due to the fact that PHB accumulation initiates after the nitrogen source is seriously exhausted. Therefore, at this moment we are using the same type of design to make a production in two stages, the first of biomass production and in the second favoring the production of PHB, in order to improve yields and reduce costs.