Sugar Beet Molasses as a Potential C-Substrate for PHA Production by Cupriavidus necator

Abstract

To increase the availability and expand the raw material base, the production of polyhydroxyalkanoates (PHA) by the wild strain Cupriavidus necator B-10646 on hydrolysates of sugar beet molasses was studied. The hydrolysis of molasses was carried out using β-fructofuranosidase, which provides a high conversion of sucrose (88.9%) to hexoses. We showed the necessity to adjust the chemical composition of molasses hydrolysate to balance with the physiological needs of C. necator B-10646 and reduce excess sugars and nitrogen and eliminate phosphorus deficiency. The modes of cultivation of bacteria on diluted hydrolyzed molasses with the controlled feeding of phosphorus and glucose were implemented. Depending on the ratio of sugars introduced into the bacterial culture due to the molasses hydrolysate and glucose additions, the bacterial biomass concentration was obtained from 20-25 to 80-85 g/L with a polymer content up to 80%. The hydrolysates of molasses containing trace amounts of propionate and valerate were used to synthesize a P(3HB-co-3HV) copolymer with minor inclusions of 3-hydroxyvlaerate monomers. The introduction of precursors into the medium ensured the synthesis of copolymers with reduced values of the degree of crystallinity, containing, in addition to 3HB, monomers 3HB, 4HB, or 3HHx in an amount of 12-16 mol.%.

Keywords: Cupriavidus necator; hydrolysis; properties of PHA; sugar beet molasses; synthesis.

Figure 1

Indicators of the culture of Cupriavidus necator B-10646 when grown on molasses hydrolysate and Schlegel’s medium: 1-, 2-, 3-, 10-, 20-, 30-fold dilution of molasses hydrolysate, respectively; 4-complete Schlegel medium; 5-with 50% nitrogen content. Biomass concentration (X, g/L), polymer (% of CDW).

Figure 2

Production parameters of bacterium Cupriavidus necator B-10646 when grown on hydrolyzed sugar beet molasses in 2 L glass flasks under different scenarios of its use: (a)—20-fold dilution of molasses hydrolysate; (b)—30-fold dilution of molasses hydrolysate with the addition of phosphorus and glucose to the culture (indicated by arrows).

Figure 3

Production parameters of the bacterial culture Cupriavidus necator B-10646 when grown on hydrolyzed sugar beet molasses in the 8 L fermenter under different versions of feeding the culture with nutritive elements: (a)—20-fold dilution of molasses hydrolysate; culture feeding with phosphorus at the first stage, and feeding with phosphorus + glucose at the second stage; (b)—20-fold dilution of molasses hydrolysate with feeding the culture with phosphorus and glucose at the first and second stages (indicated by arrows).

Figure 4

Production parameters of Cupriavidus necator B-10646 culture when cultivated in the 8 L fermenter using 20-fold dilution of molasses hydrolysate, concentrated inoculum (5 g/L), and a larger amount of glucose and phosphorus supplementation as a source of carbon, nitrogen, and mineral elements at the first stage with feeding with phosphorus and glucose; at the second stage—in a nitrogen-free medium with culture fed with glucose and phosphorus (feeds are shown by arrows): the concentration of the total cell biomass in the culture (X, g/L) and the polymer in the cells (PHA% of CDW); specific cell growth rate (µX h⁻¹) and specific polymer synthesis rate (µPHA, h⁻¹).