Rhizobacteria Mediate the Phytotoxicity of a Range of Biorefinery-Relevant Compounds

Abstract

Advances in engineering biology have expanded the list of renewable compounds that can be produced at scale via biological routes from plant biomass. In most cases, these chemical products have not been evaluated for effects on biological systems, defined in the present study as bioactivity, that may be relevant to their manufacture. For sustainable chemical and fuel production, the industry needs to transition from fossil to renewable carbon sources, resulting in unprecedented expansion in the production and environmental distribution of chemicals used in biomanufacturing. Further, although some chemicals have been assessed for mammalian toxicity, environmental and agricultural hazards are largely unknown. We assessed 6 compounds that are representative of the emerging biofuel and bioproduct manufacturing process for their effect on model plants (Arabidopsis thaliana, Sorghum bicolor) and show that several alter plant seedling physiology at submillimolar concentrations. However, these responses change in the presence of individual bacterial species from the A. thaliana root microbiome. We identified 2 individual microbes that change the effect of chemical treatment on root architecture and a pooled microbial community with different effects relative to its constituents individually. The present study indicates that screening industrial chemicals for bioactivity on model organisms in the presence of their microbiomes is important for biologically and ecologically relevant risk analyses. Environ Toxicol Chem 2019;38:1911-1922. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Keywords: Biofuels; Ionic liquids; Microbiome; Plants; Toxicology screening.

Figure 1

Schematic of an advanced biomass to biofuel production pipeline. Bioenergy crops are engineered for improved biomass composition (green‐shaded box). Biomass is chemically pretreated and enzymatically hydrolyzed to release sugars and lignin (orange‐shaded box). Engineered microbes then convert these components into the products. The role of the compounds tested in the present study are marked on the schematic: protocatechuate (1), p‐coumarate (2), [Ch][Lys] (3), [C₂C₁im][OAc] (4), α‐pinene (5), and d‐limonene (6; see Table 1 for details).

Figure 2

Screening biofuel‐related compounds reveals distinct effects on Arabidopsis root growth. (A) Representative images of Arabidopsis plants 6 d after transfer to test (containing the chemical) or control medium, (B) primary root growth, and (C) lateral root numbers after 6 d of treatment. Scale bar = 1 cm. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (Kruskal‐Wallis); n > 21.

Figure 3

Inoculation with individual rhizobacterium influences Arabidopsis root growth. (A) Representative images of Arabidopsis, (B) primary root growth, and (C) lateral root numbers 6 d after inoculation. See Table 2 for details of microbes. Scale bar = 1 cm. *p < 0.05, **p < 0.01, and ****p < 0.0001 (Kruskal‐Wallis), n > 21. A. rhizogenes = Agrobacterium rhizogenes.

Figure 4

Inoculation with select rhizobacteria ameliorates ionic liquid toxicity in Arabidopsis. (A) Representative images of Arabidopsis, (B) primary root growth, and (C) lateral root numbers 6 d after 0.1 mM [Ch][Lys] treatment and/or bacterial inoculation. (D) Representative images of Arabidopsis, (E) primary root growth, and (F) lateral root number 6 d after 1 mM [C₂C₁im][OAc] treatment and/or bacterial inoculation. For (B), (C), (E), and (F) chemical treatment– or root inoculation–only controls from Figure 2 or Figure 3, respectively, have been repeated for clarity. **p < 0.01 and ****p < 0.0001 (Kruskal‐Wallis), n > 21. (G) Representative fluorescent microscopy images of Arabidopsis roots following propidium iodide staining. (I) Control, (II) 0.1 mM [Ch][Lys], (III) inoculation with Agrobacterium rhizogenes, (IV) 0.1 mM [Ch][Lys] and Agrobacterium rhizogenes. In (A) and (D) scale bar = 1 cm, and in (G) scale bar = 200 µm. Arrowheads indicate the ectopic formation of root hairs.

Figure 5

Agrobacterium rhizogenes partially restores root growth of [Ch][Lys]‐treated sorghum. (A) Representative images, (B) sorghum lateral root growth, and (C) crown root growth 8 d after [Ch][Lys] treatment and/or Agrobacterium rhizogenes inoculation. Scale bar = 2 cm. **p < 0.01 and ****p < 0.0001 (Kruskal‐Wallis), n > 30.

Figure 6

Composition of a minimal rhizobacterial community controls restoration of root growth under [Ch][Lys] stress. (A) Representative images of Arabidopsis, (B) primary root growth, and (C) lateral root numbers 6 d after 0.1 mM [Ch][Lys] treatment and/or inoculation with either the 14‐member rhizobacterial pool (pool A) or the same pool without Flavobacterium sp. and Paenibacillus sp. (pool B). Scale bar = 1 cm. In (B) and (C) chemical treatment– or root inoculation–only controls, from Figure 2 or Figure 3, respectively, have been repeated for clarity. **p < 0.01, ***p < 0.001, and ****p < 0.0001 (Kruskal‐Wallis), n > 21.