Strains and Culture Medium

Methylocystis parvus OBBP (Biopolis S.L., Valencia, Spain) and a Methylocystis-enriched consortium obtained according to a previous study were used as methanotrophic PHA producers.³² The mixed consortium used was obtained from Sphagnum and dominated by the genus Methylocystis (>50%).³² Both strains were grown in a mineral salt medium (NMS) containing macronutrients (g L^–1): 0.2 CaCl₂·2H₂O, 1.0 KNO₃, and 1 mL of a trace element solution composed of (mg L^–1) 0.38 Fe-EDTA, 0.4 Na₂MoO₄·2H₂O, 0.3 Na₂EDTA·2H₂O, 1.0 CuSO₄·5H₂O, 0.5 FeSO₄·7H₂O, 0.4 ZnSO₄·7H₂O, 0.015 H₃BO₃, 0.01 and 0.03 CoCl₂, 0.02 MnCl₂·4H₂O, and NiCl₂·6H₂O. The addition of 10 mL of a buffer solution containing 72 g L^–1 Na₂HPO₄·12H₂O and 26 g L^–1 KH₂PO₄ was required to set the initial pH at 6.8.

The culture medium described above, deprived of KNO₃, was used for the PHBV production stage. During this phase, pure valerate and a mixture of VFAs (36.5% acetate, 31.7% propionate, 21.9% butyrate, and 9.9% valerate) were supplied as cosubstrates for producing PHBV. It is worth noting that the composition of the VFA mixture aimed to replicate a mixture that can be recovered from the digestate produced during the anaerobic digestion (AD) of food waste, in which the specific operating conditions of temperature (T: 35 °C) and pH (5–7) allowed to incorporate up to 10% of valeric acid, which is a pivotal and well-established precursor for 3-HV inclusion.⁴¹ Both cosubstrates were supplied to represent 15% and 30% of the total carbon (C_tot) present in the bottles, respectively. The fraction of carbon supplied through the cosubstrate was determined based on previous experimental studies.⁴² More specifically, it was demonstrated that a concentration of cosubstrate around 15% is sufficient for inducing 3-HV production, while 30% can be set as the upper tolerance limit for the biomass involved, at which the copolymer formation does not further increase.

Rhodococcus opacus DSM 43205 (Leibniz Institute, Germany), used as a microbial partner during M. parvus cultivation, was grown in M9 mineral salt medium consisting of (g L^–1) 7.52 Na₂HPO₄·2H₂O, 3 KH₂PO₄, 0.5 NaCl, 0.5 NH₄Cl, 4 C₆H₁₂O₆, 0.246 MgSO₄·7H₂O, 0.044 CaCl₂·2H₂O, and vitamins (1 mL of biotin and 1 mL of thiamine solutions). A trace element solution (10 mL L^–1) containing (g L^–1) 5 EDTA, 0.83 FeCl₃·6H₂O, 0.084 ZnCl₂, 0.013 CuCl₂·2H₂O, 0.01 CoCl₂·2H₂O, 0.01 H₃BO₃, and 0.0016 MnCl₂·4H₂O was also added. Rhodococcus opacus DSM 43205 was selected based on its ability to degrade a wide range of organic compounds, including some secondary byproducts of the metabolism of M. parvus that could potentially inhibit its growth.