Modeling the bioconversion of starch to P(HB-co-HV) optimized by experimental design using Bacillus megaterium BBST4 strain.

Poly(hydroxybutyrate-co-hydroxyvalerate) (P(HB-co-HV)) is a prominent biopolymer as a potential candidate for use in the biomedical area. Several Bacillus spp. strains show promising characteristics in the use of several carbon sources and are an interesting alternative for the production of P(HB-co-HV). Sewage from the agricultural and food processing industries can be used to obtain abundantly starch as a carbon source for PHA production. The aim of the present study was to optimize by response surface methodology and desirability, the production of PHA by a Bacillus megaterium strain using starch as the sole carbon source. Two optimal conditions were determined without sporulation and were used to perform new experiments to calibrate and validate a mechanistic model, developed to simulate the dynamics of PHA and biomass production. The developed model successfully represents the kinetics of the microorganism. Employing different characterization techniques, it was determined that the PHA produced by the strain is a copolymer composed of different HB:HV proportions. Using starch as the sole carbon source in a minimal salt medium, this work shows the first reports in the literature of: 1) a mathematical model for predicting growth kinetic and PHA production for B. megaterium strain and 2) a Bacillus spp. producing P(HB-co-HV) copolymer.

Improved intracellular PHA determinations with novel spectrophotometric quantification methodologies based on Sudan black dye.

The presence of intracellular polyhydroxyalkanoates (PHAs) is usually studied using Sudan black dye solution (SB). In a previous work it was shown that the PHA could be directly quantified using the absorbance of SB fixed by PHA granules in wet cell samples. In the present paper, the optimum SB amount and the optimum conditions to be used for SB assays were determined following an experimental design by hybrid response surface methodology and desirability-function. In addition, a new methodology was developed in which it is shown that the amount of SB fixed by PHA granules can also be determined indirectly through the absorbance of the supernatant obtained from the stained cell samples. This alternative methodology allows a faster determination of the PHA content (involving 23 and 42 min for indirect and direct determinations, respectively), and can be undertaken by means of basic laboratory equipment and reagents. The correlation between PHA content in wet cell samples and the spectra of the SB stained supernatant was determined by means of multivariate and linear regression analysis. The best calibration adjustment (R2 = 0.91, RSE: 1.56%), and the good PHA prediction obtained (RSE = 1.81%), shows that the proposed methodology constitutes a reasonably precise way for PHA content determination. Thus, this methodology could anticipate the probable results of the above mentioned direct PHA determination. Compared with the most used techniques described in the scientific literature, the combined implementation of these two methodologies seems to be one of the most economical and environmentally friendly, suitable for rapid monitoring of the intracellular PHA content.

Novel spectrophotometric technique for rapid determination of extractable PHA using Sudan black dye.

Classical techniques employed to determine the amount of extractable poly(hydroxyalkanoate)s (PHAs) from cells, are laborious and destructive. Sudan black staining is commonly used in the laboratory to investigate the presence of intracellular PHA. The aim of the present study was to develop a low-cost alternative technique to achieve a quick determination of extractable intracellular PHA. This methodology employs a basic laboratory spectroscopy equipment and Sudan black dye for spectra determination. The correlation between the content of PHA in cell samples taken directly from the culture flask and its spectra was determined using partial least square regression analysis and simple linear regression analysis. The best fit obtained for calibration correlation analysis (R2=0.944, RSE: 1.24%), together with the good extractable PHA predictions (RSE=0.51%) demonstrate that the proposed methodology constitutes a fast way with high potential for the determination of extractable PHA. Based on its simplicity and flexibility, its application would be suitable in routine monitoring and rapid quantification in large-scale processes involving PHA metabolism.