Abstract
BackgroundEfficient utilization of all available carbons from lignocellulosic biomass is critical for economic efficiency of a bioconversion process to produce renewable bioproducts. However, the metabolic responses that enable Pseudomonas putida to utilize mixed carbon sources to generate reducing power and polyhydroxyalkanoate (PHA) remain unclear. Previous research has mainly focused on different fermentation strategies, including the sequential feeding of xylose as the growth stage substrate and octanoic acid as the PHA-producing substrate, feeding glycerol as the sole carbon substrate, and co-feeding of lignin and glucose. This study developed a new strategy—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.ResultsCo-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 benzoate acted as an independent factor in PHA synthesis. Furthermore, 1H, 13C and 31P NMR metabolite analysis 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+/NADH and NADPH/NADP+ 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 β-oxidation, and PHA biosynthesis.ConclusionsThis 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 break-down 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 favours 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.
Highlights
Efficient utilization of all available carbons from lignocellulosic biomass is critical for economic effi‐ ciency of a bioconversion process to produce renewable bioproducts
Pseudomonas putida are natural producers of mediumchain-length PHA (mcl-PHA) due to their unique metabolic versatility to synthesize polymers when grown on various carbon sources, such as glucose, glycerol, fatty acids, and lignin derivatives [29, 39]
Results indicated that the addition of 0.5 g L−1 aromatic monomer stimulated cell growth as the cell dry weight at 72 h increased by 16.1%, 9.4% and 15.0%, respectively, compared to when feeding glycerol alone
Summary
Efficient utilization of all available carbons from lignocellulosic biomass is critical for economic effi‐ ciency of a bioconversion process to produce renewable bioproducts. As a natural soil resident, Pseudomonas putida is capable of divergent carbon substrate utilization, and thrives in versatile nutritional environments [5, 6] Multiple carbon sources, such as carbohydrates, organics acids, and aromatic compounds can be broken down and transformed into building blocks and energy equivalents for cell growth in Pseudomonas putida through cyclic central metabolism pathways, including the Embden–Meyerhof–Parnas (EMP), Entner–Doudoroff (ED), and pentose phosphate (PP) pathways as well as the tricarboxylic acid cycle (TCA) [6,7,8]. Using biomass-derived lignin and glycerol to produce PHA is an effective way to potentially reduce carbon source costs, and to increase carbon conversion efficiency
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