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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 1 H- 13 C HMBC, 1 H- 13 C HSQC, and 1 H- 1 H 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, 1 H, 13 C and 31 P 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.

Background: Biological routes for utilizing both carbohydrates and lignin are important to reach the ultimate goal of bioconversion of full carbon in biomass into biofuels and biochemicals. Recent biotechnology advances have shown promises toward facilitating biological transformation of lignin into lipids. Natural and engineered  Rhodococcus strains (e.g.,  R. opacus PD630 , R. jostii  RHA1, and R. jostii  RHA1 VanA - ) have been demonstrated to utilize lignin for lipid production, and co-culture of them can promote lipid production from lignin., Results: In this study, a co-fermentation module of natural and engineered  Rhodococcus strains with significant improved lignin degradation and/or lipid biosynthesis capacities was established, which enabled simultaneous conversion of glucose, lignin, and its derivatives into lipids. Although  Rhodococci  sp. showed preference to glucose over lignin, nearly half of the lignin was quickly depolymerized to monomers by these strains for cell growth and lipid synthesis after glucose was nearly consumed up. Profiles of metabolites produced by Rhodococcus  strains growing on different carbon sources (e.g., glucose, alkali lignin, and dilute acid flowthrough-pretreated poplar wood slurry) confirmed lignin conversion during co-fermentation, and indicated novel metabolic capacities and unexplored metabolic pathways in these organisms. Proteome profiles suggested that lignin depolymerization by  Rhodococci sp. involved multiple peroxidases with accessory oxidases. Besides the β-ketoadipate pathway, the phenylacetic acid (PAA) pathway was another potential route for the in vivo ring cleavage activity. In addition, deficiency of reducing power and cellular oxidative stress probably led to lower lipid production using lignin as the sole carbon source than that using glucose., Conclusions: This work demonstrated a potential strategy for efficient bioconversion of both lignin and glucose into lipids by co-culture of multiple natural and engineered Rhodococcus strains. In addition, the involvement of PAA pathway in lignin degradation can help to further improve lignin utilization, and the combinatory proteomics and bioinformatics strategies used in this study can also be applied into other systems to reveal the metabolic and regulatory pathways for balanced cellular metabolism and to select genetic targets for efficient conversion of both lignin and carbohydrates into biofuels., Competing Interests: The authors declare that they have no competing interests.

Background: When suspended in cell culture medium, nano-objects composed of soluble metals such as silver can dissolve resulting in ion formation, altered particle properties (e.g. mass, morphology, etc.), and modulated cellular dose. Cultured cells are exposed not just to nanoparticles but to a complex, dynamic mixture of altered nanoparticles, unbound ions, and ion-ligand complexes. Here, three different cell types (RAW 264.7 macrophages and bone marrow derived macrophages from wild-type C57BL/6 J mice and Scavenger Receptor A deficient (SR-A (-/-) ) mice) were exposed to 20 and 110 nm silver nanoparticles, and RAW 264.7 cells were exposed to freshly mixed silver ions, aged silver ions (ions incubated in cell culture medium), and ions formed from nanoparticle dissolution. The In Vitro Sedimentation, Diffusion, Dissolution, and Dosimetry Model (ISD3) was used to predict dose metrics for each exposure scenario., Results: Silver nanoparticles, freshly mixed ions, and ions from nanoparticle dissolution were toxic, while aged ions were not toxic. Macrophages from SR-A (-/-) mice did not take up 20 nm silver nanoparticles as well as wild-types but demonstrated no differences in silver levels after exposure to 110 nm nanoparticles. Dose response modeling with ISD3 predicted dose metrics suggest that amount of ions in cells and area under the curve (AUC) of ion amount in cells are the most predictive of cell viability after nanoparticle and combined nanoparticle/dissolution-formed-ions exposures, respectively., Conclusions: Results of this study suggest that the unbound silver cation is the ultimate toxicant, and ions formed extracellularly drive toxicity after exposure to nanoparticles. Applying computational modeling (ISD3) to better understand dose metrics for soluble nanoparticles allows for better interpretation of in vitro hazard assessments.