Cross-Feeding of a Toxic Metabolite in a Synthetic Lignocellulose-Degrading Microbial Community

Abstract

The recalcitrance of complex organic polymers such as lignocellulose is one of the major obstacles to sustainable energy production from plant biomass, and the generation of toxic intermediates can negatively impact the efficiency of microbial lignocellulose degradation. Here, we describe the development of a model microbial consortium for studying lignocellulose degradation, with the specific goal of mitigating the production of the toxin formaldehyde during the breakdown of methoxylated aromatic compounds. Included are Pseudomonas putida, a lignin degrader; Cellulomonas fimi, a cellulose degrader; and sometimes Yarrowia lipolytica, an oleaginous yeast. Unique to our system is the inclusion of Methylorubrum extorquens, a methylotroph capable of using formaldehyde for growth. We developed a defined minimal "Model Lignocellulose" growth medium for reproducible coculture experiments. We demonstrated that the formaldehyde produced by P. putida growing on vanillic acid can exceed the minimum inhibitory concentration for C. fimi, and, furthermore, that the presence of M. extorquens lowers those concentrations. We also uncovered unexpected ecological dynamics, including resource competition, and interspecies differences in growth requirements and toxin sensitivities. Finally, we introduced the possibility for a mutualistic interaction between C. fimi and M. extorquens through metabolite exchange. This study lays the foundation to enable future work incorporating metabolomic analysis and modeling, genetic engineering, and laboratory evolution, on a model system that is appropriate both for fundamental eco-evolutionary studies and for the optimization of efficiency and yield in microbially-mediated biomass transformation.

Keywords: Cellulomonas fimi; Methylorubrum extorquens; Pseudomonas putida; Yarrowia lipolytica; formaldehyde; lignocellulose; methylotrophy; microbial communities; synthetic ecology.

Figure 1

Conceptual model of interactions in the lignocellulose-degrading microbial consortium. Arrows indicate hypothesized interactions as described in the key. The colors and symbols used for compounds and species in this figure are the same as those used in data plots throughout the manuscript.

Figure 2

Vanillic acid inhibits the growth of C. fimi and P. putida substantially, but has only a minor effect on M. extorquens growth. Each organism was grown in pure culture in mineral medium and OD₆₀₀ monitored for up to 60 h with a range of vanillic acid concentrations (original growth curves are shown in Figure S2). C. fimi was provided with cellobiose and M. extorquens with methanol as growth substrates, whereas P. putida was able to use the vanillic acid as a growth substrate. Each point represents a biological replicate. Only growth rates for which R² > 0.9 are shown here. In the left panel, error bars denote the standard error of the fitted growth rate. For C. fimi and P. putida, lag time and growth rate are dependent on vanillic acid concentration.

Figure 3

C. fimi is the most formaldehyde-sensitive member of the consortium. (A–D) Each species was grown in pure culture in minimal medium with varying concentrations of formaldehyde, and growth monitored by OD₆₀₀. No growth was observed in C. fimi at concentrations of 0.5 mM or higher, whereas Y. lipolytica grew at up to 2 mM formaldehyde and P. putida at up to 3 mM within 30 h. Note that OD is shown here on a linear scale for ease of interpretability; note also that the color scale for formaldehyde concentrations is different between panels (A–C) and panel (D). Data from panel (D) are reproduced from [43]. (E) An alternative method of understanding formaldehyde tolerance is the enumeration of cells that are able to form colonies on agar medium containing formaldehyde. In M. extorquens, 100% of the plated population formed colonies at 1 mM and 1/10,000 formed colonies at 2 mM. P. putida cells also formed colonies at those concentrations but at a lower frequency. In C. fimi, no colonies were observed at 0.5 mM formaldehyde or higher. Error bars show the standard deviation of three replicate platings.

Figure 4

When P. putida grows on vanillic acid as a sole carbon substrate, formaldehyde accumulates in the medium to levels potentially toxic to C. fimi. When grown in coculture with P. putida, M. extorquens reduces formaldehyde accumulation. Shown here are the formaldehyde in the medium (top row) and total growth of the community (bottom row) from three separate experiments in which M. extorquens and P. putida were grown separately or together in minimal medium with vanillic acid as the sole carbon source: (A,B) 4 mM vanillic acid or protocatechuic acid; (C) 10 mM. Individual lines represent independent culture vessels. As M. extorquens cannot grow on vanillic acid, no activity was observed in the M. extorquens–only cultures. In cultures containing P. putida, formaldehyde was generated on vanillic acid during the exponential growth phase, and the duration and peak of the formaldehyde was lower in cultures containing M. extorquens. The data in panel (B) are from a larger experiment testing M. extorquens cultures from different pre-growth conditions and inoculation ratios; full results for that experiment are shown in Figure S4. All data shown here are from cultures initiated in stationary phase, and from M. extorquens pre-grown on methanol.

Figure 5

Vitamin and amino acid supplements have minimal effect on growth rates of each of the consortium members, except for C. fimi. Each species was grown in pure culture in minimal medium with a range of concentrations of methionine, thiamine, or biotin. For the methionine experiment, all cultures contained 25 µg/L thiamine and 10 µg/L biotin; for the thiamine experiment, 10 mg/L methionine and 10 µg/L biotin; for the biotin experiment, 10 mg/L methionine and 25 µg/L thiamine.

Figure 6

Higher carbon concentrations in model lignocellulose medium result in slower growth and higher final yield; C. fimi is more strongly inhibited than P. putida. Each species was grown in pure culture in Model Lignocellulose medium (Table 1) with either 1× carbon (4 mM cellobiose, 5 mM xylose, 4 mM vanillic acid), or 0.25 times or 3 times those concentrations.

Figure 7

P. putida inhibits C. fimi growth. (A–E) Results from an experiment in which each organism was grown in pure culture and in combinations, in minimal medium containing cellobiose and a low concentration of vanillic acid. The combination of species in each culture tube is indicated by the color and shape of the points. (A) Growth of the entire community as measured by optical density. Cultures without P. putida grew most rapidly and reached the highest final OD. No increase in OD was attributed to P. putida because 2 mM vanillic acid supports very little growth. (B) Cellobiose concentrations over time. C. fimi is the only organism capable of consuming cellobiose, and consumption was most rapid in cultures lacking P. putida. (C) Formaldehyde concentrations and (D) Vanillic acid concentrations over time. Formaldehyde peaked and disappeared, and vanillic acid was consumed, within 12 h, indicating activity by P. putida despite fact that the change in OD was not measurable. (E) Viable cells from each species, as measured by colony counts, at the beginning of the experiment (“inoc.” = inoculum), and at the end (78 h; each panel is titled with the species present in that culture tube). While no growth substrate was explicitly included to support M. extorquens, it increased slightly in abundance by the end of the experiment and the greatest increase was in the presence of C. fimi and absence of P. putida. Replicate culture tubes are shown along the x-axis; points indicate replicate measurements from each tube. (F) Results from a similar experiment, testing only a subset of the species combinations in Model Lignocellulose medium. Only optical density is shown here.

Figure 8

There are multiple potential explanations for the inhibitory effect of P. putida on C. fimi growth. (A) Growth of individual species and combinations (denoted by color and shape of plot symbol) on Model Lignocellulose medium in which vanillic acid is replaced by protocatechuic acid, which does not result in formaldehyde production. Cultures with P. putida still show slower growth than those without. (B,C) Effect of increasing the iron or supplements (methionine, thiamine, and biotin) in the medium. In both cases, higher concentrations support more growth. (D) Growth of C. fimi alone on either Model Lignocellulose or P. putida spent medium: a lower final yield is reached on spent medium. (E,F) Growth of the model community (P. putida, C. fimi, M. extorquens) on combinations of different iron (panels), methionine (color scale), and thiamine + biotin (color scale) concentrations.

Figure 9

A methionine-overproducing strain of M. extorquens can support the growth of C. fimi without the addition of methionine to the medium. Top: growth curves of C. fimi with different strains of M. extorquens (symbols), on MP medium with glucose and supplements (panels). Without succinate, M. extorquens growth is negligible. For C. fimi alone, growth with methionine is much greater than without. However, when succinate is present to enable M. extorquens growth, the culture of C. fimi + the methionine excreter reaches substantially higher growth than by C. fimi + WT M. extorquens, suggesting a benefit to C. fimi from the methionine. Bottom: colony counts of each species (symbols) from the endpoint of an experiment in which the three-species consortium was grown on Model Lignocellulose medium with either of the two strains of M. extorquens (x-axis), and supplemented with either nothing, methanol to support M. extorquens growth, or both methanol and methionine (panels). C. fimi grows only when methionine is present or when both methanol and the methionine-excreting M. extorquens strain are present. P. putida does not show the same reliance on methionine or M. extorquens.

Figure 10

Revised conceptual model of interactions in model lignocellulose-degrading microbial consortium. To our original model, we have added competition between P. putida and C. fimi for iron and methionine, and inhibitory effects of vanillic acid on C. fimi and P. putida and high concentrations of iron on C. fimi. We have found little evidence for inhibitory effects of formaldehyde (at the concentrations produced here) on any of the members besides C. fimi; there is evidence that C. fimi, but not P. putida, supports M. extorquens growth, likely through organic acid production. In addition, we have added the ability for M. extorquens to support C. fimi through the production of methionine.