Enhancement of polyhydroxyalkanoate production by co-feeding lignin derivatives with glycerol in Pseudomonas putida KT2440

Abstract

Background Efficient 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. 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 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. 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 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.

Lignin Aromatics to PHA Polymers: Nitrogen and Oxygen Are the Key Factors for Pseudomonas

“…Together with ongoing important metabolic engineering efforts for strain development on improving lignin aromatic uptake, balancing of carbon flux and avoiding mcl-PHA reassimilation, 8,11,19,20,51,55,56 we encourage future research on linking aspects like: media formulation (i.e., optimization of nutrients like nitrogen amount to ratio of real mixed lignin fractions), 2 oxygen fine-tuned feeding strategies (i.e., DO stat fed batch cultivations) 59 to improve mcl-PHA concentrations and minimize diauxic aromatic uptake profiles during different nitrogen availability (i.e., "feast and famine" stages), synergistic cocarbon feeding strategies to balance metabolic energy 17,21,60 and ensuring final product quality (i.e., mcl-PHA material properties). 16 The outcomes of this in depth bioprocess understanding may also benefit the production of other value compounds than mcl-PHA from lignin aromatics.…”

“…Furthermore, limited methods for analysis of the heterogeneous aromatic fractions and complex catabolic repression phenomena make a detailed physiological study on mcl- PHA production from liquors obtained after lignin depolimerization very challenging. Therefore, much of the physiological research thus far focused on the evaluation of single lignin-derived aromatic compounds like benzoate, , vanillate, ferulate, or cofeeding some of them with glycerol . Thus, detailed studies toward a physiological understanding of the uptake and conversion dynamics of defined aromatic mixtures into mcl- PHA are necessary.…”

“…Therefore, much of the physiological research thus far focused on the evaluation of single lignin-derived aromatic compounds like benzoate, 15,19 vanillate, 14 ferulate, 20 or cofeeding some of them with glycerol. 21 Thus, detailed studies toward a physiological understanding of the uptake and conversion dynamics of defined aromatic mixtures into mcl-PHA are necessary. In particular, knowledge on how the native funnelling capacity of P. putida is affected under nutrient limited conditions (i.e., when triggering mcl-PHA accumulation) is required, as hierarchical aromatics consumption is likely to heavily affect productivities.…”

Endophytes in Lignin Valorization: A Novel Approach

“…On the other hand, alkali pre-treatment of lignocellulose is considered to be a promising alternative as it can process at low temperature and pressure conditions. Typically, NaOH and KOH are used which breaks the lignin-carbohydrate bonds ( Xu et al, 2021 ). The biggest hindrance in this method is the larger retention time (hours or days).…”

“…As already mentioned, microbial PHA synthesis is positively correlated to diverse nutrient deficiencies, the biofunneling capacity of Pseudomonas strains to yield mcl -polyhydroxyalkanoate ( mcl -PHA) was enhanced after subjected to nitrogen and oxygen limitations ( Ramírez-Morales et al, 2021). Recently, conversion of lignin into PHA’s by Psuedomonas putida KT2440 was enhanced by the addition of glycerol to the lignin derivatives, such as, vanillin, vanillic acid and benzoic acid ( Xu et al, 2021 ).…”