POLYHYDROXYBUTYRATE PRODUCTION UNDER NON-STERILE CONDITIONS BY HALOMONAS TITANICAE KHS3 Ht. KHS3

RODRÍGUEZ, AN; PONCE, B; MÁRQUEZ, V; DÍAZ BARRERA, A; HERRERA SEITZ, MK; STUDDERT, CA

Los Cocos, Córdoba

Congreso; XVII Congreso Anual de Samige (Sociedad Argentina de Microbiología General); 2022

Samige

Halomonas titanicae KHS3 (Ht KHS3) is a moderately halophilic bacterium isolated from the port of Mar del Plata. It has shown a wide metabolic versatility to grow in various nutritional conditions and different salt concentrations. These abilities represent a potential advantage for biotechnological uses, allowing the utilization of different industrial wastes as carbon sources in non-sterile conditions. Ht KHS3 accumulates polyhydroxybutyrate (PHB) when grown on glycerol as the only carbon source. In order to study the possibility to work under non-sterile conditions, batch cultures were carried out in a minimal medium with 25 g/l of glycerol as the only carbon source and 60 g/l of NaCl. The 1-l reactor was sanitized with sodium hypochlorite followed by alcohol before use. The growth was followed by OD600 and dry weight biomass. The PHB accumulation was quantified by HPLC. Daily monitoring of fuchsine staining was done to check bacterial morphology under the microscope. Ht KHS3 showed a linear growth up to 4.2 g/l of dry biomass and accumulated up to 1.4 g/l of PHB without apparent contamination after 264 hours of culture. In order to reduce cultivation time and associated costs, a two-stage culture scheme was set up. The goal of the first stage is to achieve biomass production under controlled conditions in a relatively short time. It was followed by a second stage to get PHB production minimizing the requirements of aeration and temperature control, and allowing the assessment/use of waste materials as carbon sources. For the first stage factorial experiments were carried out using the Design Expert 7.0.0 software. Screening was carried out using a fractional factorial design including eight factors: concentration of glycerol, ammonium, NaCl, Fe and yeast extract (YE), temperature, aeration and time. The biomass dry weight was evaluated as a response variable. Glycerol, ammonium, YE, time, and aeration had significant positive effects on biomass yields. In contrast, the temperature had a significant negative effect. NaCl and Fe concentration did not significantly affect biomass production. With these results, the experimental space was redefined and a central composite design was carried out for the optimization of glycerol, ammonium, and YE concentrations for biomass production. The desirability function was applied to find the combination of factors to generate the highest dry weight biomass. The optimal predicted was 1.04 g/l in 24 hours, and it was validated by triplicate. Since the remaining glycerol in the medium was still high (16 g/l), we reformulated the initial glycerol concentration and incubation time before the onset of the second stage. Starting from low glycerol concentration (0.55 g/l) different conditions for carbon source and nitrogen addition, aeration and salinity were assayed for the second stage in a new factorial design experiment. All these factors except for ammonium addition significantly affected PHB accumulation.