Conversion of lignin model compounds by Pseudomonas putida KT2440 and isolates from compost

Abstract

Starting from mature vegetable compost, four bacterial strains were selected using a lignin-rich medium. 16S ribosomal RNA identification of the isolates showed high score similarity with Pseudomonas spp. for three out of four isolates. Further characterization of growth on mixtures of six selected lignin model compounds (vanillin, vanillate, 4-hydroxybenzoate, p-coumarate, benzoate, and ferulate) was carried out with three of the Pseudomonas isolates and in addition with the strain Pseudomonas putida KT2440 from a culture collection. The specific growth rates on benzoate, p-coumarate, and 4-hydroxybenzoate were considerably higher (0.26-0.27 h^-1) than those on ferulate and vanillate (0.21 and 0.22 h^-1), as were the uptake rates. There was no direct growth of P. putida KT2440 on vanillin, but instead, vanillin was rapidly converted into vanillate at a rate of 4.87 mmol (g_CDW h)^-1 after which the accumulated vanillate was taken up. The growth curve reflected a diauxic growth when mixtures of the model compounds were used as carbon source. Vanillin, 4-hydroxybenzoate, and benzoate were preferentially consumed first, whereas ferulate was always the last substrate to be taken in. These results contribute to a better understanding of the aromatic metabolism of P. putida in terms of growth and uptake rates, which will be helpful for the utilization of these bacteria as cell factories for upgrading lignin-derived mixtures of aromatic molecules.

Keywords: Aromatic compound conversion; Bacterial metabolism; Compost; Lignin; Pseudomonas.

Fig. 1

Enrichment cultures in the M9 medium supplemented with different sources of lignin-related products (indicated below the picture) after 6 days of incubation. Tubes in the bottom panel are inoculated with a mature compost wash; tubes in the upper part are non-inoculated controls

Fig. 2

Growth of P. putida KT2440 on six selected lignin model compounds as the only carbon source in the M9 medium. Duplicate experiments were performed, and the standard deviations are shown with an error bar. OD and the concentrations of model compounds are indicated with a black diamond and red square, respectively. The curve indicated with green triangle is the concentration of vanillate

Fig. 3

Growth of isolates C and Sigma on the mixture of vanillin, vanillate, and 4-hydroxybenzoate. OD is indicated with a black diamond line. The uptake of model compounds by these microorganisms was not measured as there was a lag phase (6 h) in their growth curves compared to isolates B and P. putida KT2440 (see Fig. 4)

Fig. 4

Growth of isolates B and P. putida KT2440 on the mixture of vanillin, vanillate, and 4-hydroxybenzoate. OD is indicated with a black diamond line. The concentration of model compounds is shown in violet circle (vanillin), gray open triangle (4-HBA), and green closed triangles (vanillate)

Fig. 5

Growth of isolates B and P. putida KT2440 on the mixture of benzoate, p-coumarate, and ferulate. OD is indicated with a black diamond line. The concentration of model compounds is shown in blue circle (benzoic acid), red closed square (p-coumarate), and pink open square (ferulate)

Fig. 6

Growth of isolates B and P. putida KT2440 on the mixture of vanillin, vanillate, and ferulate. OD is indicated with a black diamond line. The concentration of model compounds is shown in violet circle (vanillin), pink square (ferulate), and green triangle (vanillate)

Fig. 7

Main upper degradation pathways of ferulic acid, p-coumaric acid, and benzoates in Pseudomonas putida KT2440. The six model compounds used in this study are indicated in bold; names of the enzymes involved in each step of the pathway are shown in boxes. Fcs feruloyl-CoA synthase, Ech enoyl-CoA hydratase/lyase, Vdh vanillin dehydrogenase, PobA 4-hydroxybenzoate hydroxylase, VanAB vanillate O-demethylase complex, BenABC benzoate dioxygenase complex, BenD 1,6-dihydroxycyclohexa-2,4-diene-1-carboxylate dehydrogenase. Metabolic nodes that can undergo ring cleavage are shown in dashed boxes