Co-Production of Poly(3-hydroxybutyrate) and Gluconic Acid from Glucose by Halomonas elongata

Abstract

Polyhydroxyalkanoates (PHA) are biopolyesters regarded as an attractive alternative to petroleum-derived plastics. Nitrogen limitation and phosphate limitation in glucose cultivations were evaluated for poly(3-hydroxybutyrate) (P(3HB)) production by Halomonas elongata 1H9^T, a moderate halophilic strain. Co-production of P(3HB) and gluconic acid was observed in fed-batch glucose cultivations under nitrogen limiting conditions. A maximum P(3HB) accumulation of 53.0% (w/w) and a maximum co-production of 133 g/L of gluconic acid were attained. Fed-batch glucose cultivation under phosphate limiting conditions resulted in a P(3HB) accumulation of only 33.3% (w/w) and no gluconic acid production. As gluconic acid is a valuable organic acid with extensive applications in several industries, this work presents an interesting approach for the future development of an industrial process aiming at the co-production of an intracellular biopolymer, P(3HB), and a value-added extracellular product, gluconic acid.

Keywords: Halomonas elongata; gluconic acid; halophiles; polyhydroxyalkanoates.

Figure 1

Fed-batch culture of Halomonas elongata 1H9^T with glucose as a carbon source and under nitrogen limiting conditions. Cell biomass and P(3HB) production (a), glucose and ammonium utilization and gluconic acid production (b), and data acquired automatically during the cultivation, namely feed volume, agitation, and DO (% sat), (c).

Figure 2

Fed-batch culture of Halomonas elongata 1H9^T with glucose as a carbon source and under phosphate limiting conditions. Cell biomass and P(3HB) production (a); sugar utilization (b); feed volume, agitation, and DO% sat (c).

Figure 3

LC–HRMS analysis of a sample of the cultivation broth of Halomonas elongata 1H9^T on glucose under N-limiting conditions. (a) Total ion chromatogram acquired in the ESI negative mode. Two main extracted ion chromatograms were obtained: (b) for the deprotonated molecule of gluconic acid [C6H11O7]⁻, tR 1.4 min, m/z 195.0509 (∆ −0.5 ppm, mSigma 5.5); and (c) for the deprotonated molecule of 2-oxoglutaric acid [C5H5O5]^-, tR 1.9 min, m/z 145.0148 (∆ −3.5 ppm, mSigma 8.5). Each precursor ion was selected by the quadrupole mass analyzer, transferred to the collision cell where fragmentation occurred by collision-induced dissociations (CID) with nitrogen, and the fragmented ions were separated by the TOF mass analyzer, yielding the high-resolution tandem mass spectra. The assignment of the fragmented ions was based on their accurate mass measurements, and the fragmentation paths are proposed in Figures S1 and S2 (Supplementary Materials).

Figure 4

Pathways leading from glucose to 6-phosphogluconate in Halomonas elongata 1H9T. Abbreviations used are as follows: Gad, gluconate dehydrogenase; Gdhq, glucose dehydrogenase; Glk, glucokinase; GnuK, gluconokinase; KguD, 2-keto-6-phosphogluconate reductase; KguK, 2-ketogluconatekinase; Zwf, glucose-6-phosphate dehydrogenase.