PNNL

Abstract

Background: The efficient utilization of all available carbons from lignocellulosic biomass is critical to the economic efficiency of a bioconversion process that produces bioplastic polyhydroxyalkanoates (PHA). Previous research has mainly focused on different fermentation strategies, including sequential feeding xylose only as the growth stage substrate and octanoic acid-only as the PHA producing substrate, feeding glycerol as the sole carbon substrate, and co-feeding lignin and glucose. However, the metabolic responses that enable Pseudomonas putida to utilize mixed carbon sources to generate reducing power and PHA remain unclear. This study developed a new strategy of co-feeding glycerol and lignin derivatives such as benzoate, vanillin, and vanillic acid in Pseudomonas putida KT2440 for the first time, which simultaneously improved both cell biomass and PHA production. Results Co-feeding lignin derivatives (i.e. benzoate, vanillin, and vanillic acid) and glycerol to P. putida KT2440 was shown for the first time to simultaneously increase cell dry weight (CDW) by 9.4 %-16.1 % and PHA content by 29.0 %-63.2 %, respectively, compared with feeding glycerol alone. GC-MS results revealed that the addition of lignin derivatives to glycerol decreased the distribution of long chain monomers (C10 and C12) by 0.4 %-4.4 % and increased the distribution of short chain monomers (C6 and C8) by 0.8 %-3.5 %. The 1H -13C HMBC, 1H-13C HSQC, and 1H-1H COSY NMR analysis confirmed that the PHA monomers (C6-C14) were produced when glycerol was fed to the bacteria alone or together with lignin derivatives. Moreover, investigation of the glycerol/benzoate/nitrogen ratios showed that the PHA enhancement is not due to the influence of the carbon to nitrogen ratio. Furthermore, in the 1H, 13C and 31P NMR metabolite analysis, NADH/NADPH and mass spectrometry-based quantitative proteomics measurements suggested that the addition of benzoate stimulated oxidative-stress responses, enhanced glycerol consumption, and altered the intracellular NAD(P)+/NAD(P)H ratios by up-regulating the proteins involved in energy generation and storage processes, including the Entner-Doudoroff (ED) pathway, the reductive TCA route, trehalose degradation, fatty-acid ß-oxidation, and PHA biosynthesis. Conclusions: This work demonstrated an effective co-carbon feeding strategy to improve PHA content/yield and convert lignin derivatives into value-added products in P. putida KT2440. Co-feeding lignin breakdown products with other carbon sources, such as glycerol, has been demonstrated as an efficient way to utilize biomass to increase PHA production in P. putida KT2440. Moreover, the involvement of aromatic degradation favors further lignin utilization, and the combination of proteomics and metabolomics with NMR sheds light on the metabolic and regulatory mechanisms for cellular redox balance and potential genetic targets for a higher biomass carbon conversion efficiency.