Exploring Lemon Industry By-Products for Polyhydroxyalkanoate Production: Comparative Performances of Haloferax mediterranei PHBV vs. Commercial PHBV

Abstract
This study investigates the valorisation of lemon industry by-products as carbon sources
to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) using the halophilic
archaeon Haloferax mediterranei. The resulting polymer (HFX PHBV) was supplemented
with nucleating agents (orotic acid, boron nitride, and theobromine) and compared with a
commercial PHBV grade (Enmat Y1000) under identical conditions. Fermentation strategies
were optimised by varying the lemon by-product concentration, inoculum size, and nutrient
stoichiometry (C:N:P ratios), followed by scaleup in a 2 L bioreactor. A 11% (v/v) lemon
by-product combined with a 5% (v/v) inoculum yielded the highest productivity under
minimal medium conditions (2.127 g/L PHBV), while enriched media further enhanced
the polymer accumulation (up to 3.250 g/L PHBV). A comparative characterisation of HFX
PHBV and Enmat Y1000, using NMR, TGA, MFR, DSC, Raman spectroscopy, XRD, and
DMA, revealed that HFX PHBV exhibited lower crystallinity, increased flexibility, and a
high hydroxyvalerate content (27.4%), which conferred improved ductility. Investigation
of nucleating agents demonstrated that orotic acid was the most effective at enhancing
the crystallisation kinetics. Overall, this study demonstrates an efficient PHBV production
process based on waste valorisation, yielding a biopolymer with competitive physicochemical
properties relative to a commercial standard, and provides integrated solutions to the
global challenges of plastic pollution and food waste.