CONICET | Buscador de Institutos y Recursos Humanos

Halomonas titanicae KHS3 (Ht KHS3) is a moderately halophilic bacterium isolated from Mar del Plata harbor. It has shown a wide metabolic versatility to grow in different nutritional conditions and salt concentrations. This ability raises the potential for the utilization of different industrial wastes as carbon sources in non-sterile conditions. Ht KHS3 accumulates polyhydroxybutyrate (PHB) when grown in a minimal medium with a high salt concentration and glycerol as carbon source. A two-stage process was evaluated in order to reduce production costs due to long cultivation times. A first stage of biomass production (with sterility, shorter cultivation time, commercial carbon source) and a second stage for PHB accumulation with lower requirements (no sterility, waste carbon sources, less agitation). Statistical experimental design was used for the optimization of biomass production. Optimized conditions were 48 hours incubation time, minimal medium LM2 with 60 g/l NaCl, 0.06% Yeast Extract (YE) and 5 g/l of glycerol as the carbon source, 28°C and 200 rpm orbital agitation. Cultures obtained from this first stage had a high biomass (2.5 g/l) and exhausted carbon and nitrogen sources. In order to optimize the second stage, a factorial fractional design was carried out using the Design Expert 7.0.0 software. The screening included five factors: concentration of glycerol, ammonium and NaCl, aeration and agitation. The percentage of PHB accumulation was evaluated as a response variable. Concentration of glycerol, agitation and aeration had significant positive effects on PHB accumulation. In contrast, the NaCl concentration had a significant negative effect. Ammonium concentration did not significantly affect PHB accumulation. This stage was done under unsterile conditions and Ht KHS3 accumulated PHB without apparent contamination for 7 days. Based on these results, no ammonium nor NaCl were added to the culture. The experimental space was redefined and a Box-Behnken design was carried out for the optimization of glycerol concentration, agitation and aeration for PHB production. In this design the PHB productivity (grams of PHB per liter of culture) was evaluated as a response variable. The desirability function was applied to find the combination of factors to generate the highest PHB productivity. The optimal predicted was 2.3 g/l in 7 days with 12 g/l of glycerol, 163 rpm and 51,7% of volume. It was validated by triplicate. Therefore, the experimental design led to an optimized two-stage accumulation process. The first controlled stage in 48 h reduces the potential time in a bioreactor. Besides, the low agitation in the second stage reduces significantly energy costs. This design allowed to study the use of waste carbon sources for PHB production like beer bagasse, hydrocarbon-rich wastes, landfill waste, dairy industry waste, further contributing to the reduction of production costs and the increase of added value to the whole process.