Complex function by design using spatially pre-structured synthetic microbial communities: degradation of pentachlorophenol in the presence of Hg(ii)

Abstract

Naturally occurring microbes perform a variety of useful functions, with more complex processes requiring multiple functions performed by communities of multiple microbes. Synthetic biology via genetic engineering may be used to achieve desired multiple functions, e.g. multistep chemical and biological transformations, by adding genes to a single organism, but this is sometimes not possible due to incompatible metabolic requirements or not desirable in certain applications, especially in medical or environmental applications. Achieving multiple functions by mixing microbes that have not evolved to function together may not work due to competition of microbes, or lack of interactions among microbes. In nature, microbial communities are commonly spatially structured. Here, we tested whether spatial structure can be used to create a community of microbes that can perform a function they do not perform individually or when simply mixed. We constructed a core-shell fiber with Sphingobium chlorophenolicum, a pentachlorophenol (PCP) degrader, in the core layer and Ralstonia metallidurans, a mercuric ion (Hg(ii)) reducer, in the shell layer as a structured community using microfluidic laminar flow techniques. We applied a mixture of PCP and Hg(ii) to either a simple well-mixed culture broth (i.e. the unstructured community) or the spatially structured core-shell fibers. We found that without spatial structure, the community was unable to degrade PCP in the presence of Hg(ii) because S. chlorophenolicum is sensitive to Hg(ii). In contrast, with spatial structure in a core-shell fiber system, S. chlorophenolicum in a core layer was protected by R. metallidurans deposited in a shell layer, and the community was able to completely remove both PCP and Hg(ii) from a mixture. The appropriate size of the core-shell fiber was determined by the Damköhler number-the timescale of removal of Hg(ii) was on the same order of the timescale of diffusion of Hg(ii) through the outer layer when the shell layer was on the order of ~200 μm. Ultimately, with the ease of a child putting together 'Legos' to build a complex structure, using this approach one may be able to put together microorganisms to build communities that perform functions in vitro or even in vivo, e.g. as in a "microbiome on a pill".

Figure 1

Images from the previous literature illustrating the spatial structure of microbial communities involved in environmental remediation in soil and sludges. a) Scanning electron microphotograph of a thermophilic sludge granule. b) In situ hybridization of sections from a thermophilic sludge granule shows bacterial species (green) surrounding archaebacterial species (red). c) A higher magnification of b). Spatial structure is crucial to balance transport and reactions in these granules. Reprinted from Reference , with permission from the American Society for Microbiology (© 2009).

Figure 2

The spatially structured synthetic microbial community that performs simultaneous degradation of pentachlorophenol (PCP) and reduction of Hg(II). a) A schematic of the synthetic community. Sphingobium chlorophenolicum aerobically degrades the PCP molecules in the core fiber. Ralstonia metallidurans reduces mercuric ions, Hg(II), to non-toxic elemental mercury, Hg(0), in the shell. The chemical transformation from PCP to tetrachloro-p-hydroquinone, the key limiting step in PCP degradation, is highlighted. b) A schematic of the process by which the core-shell fiber system was constructed using a seven-barrel capillary. Cells were first suspended in a sodium alginate solution, then suspensions of S. chlorophenolicum (barrel 1) and suspensions of R. metallidurans (barrels 2–7) were extruded from the capillary into a CaCl₂ solution, forming a core-shell fiber system consisting of one core part and six shell parts with cells in a calcium alginate matrix (see Experimental section). c) An overlay of fluorescent and bright field microphotographs shows the spatial structure of a core-shell fiber system. This core-shell fiber system contained GFP-labeled Escherichia coli cells in the shell layer and RFP-labeled E. coli cells in the core layer. d) A schematic of the process to create fibers with seven independently controlled sections. Cells were first suspended in a solution of sodium alginate, then suspensions of RFP-labeled E. coli (barrel 1), Salmonella enterica (barrels 2, 4, and 6), and GFP-expressing Pseudomonas aeruginosa (barrels 3, 5, and 7) were extruded into a calcium alginate matrix (see Experimental section). e) An overlay of fluorescent and bright field microphotographs shows the spatial structure of a seven-part fiber system.

Figure 3

A well-mixed synthetic microbial community could not simultaneously degrade PCP and reduce Hg(II), but the same community performed both functions when it was spatially structured. Both communities were exposed to a mixture of PCP (120 µM) and Hg(II) (120 µM). PCP was added at time zero, Hg(II) ions were added at 1.5 h. a) Concentration of PCP (red circles) and Hg(II) ions (blue triangles) exposed to a well-mixed culture of S. chlorophenolicum and R. metallidurans as a function of time. The mixture did not degrade PCP but reduced Hg(II) ions. After 3 h, the level of Hg(II) was below the detection limit (0.37 µM). The cell density of each species was ~5×10⁸ CFU·mL⁻¹. The results of control experiments in the absence of cells are shown with open symbols. b) Concentration of PCP (red circles) and Hg(II) ions (blue triangles) exposed to core-shell fibers made of S. chlorophenolicum in the core and R. metallidurans in the shell as a function of time. Symbols are the same as in a). The structured community simultaneously degraded PCP and reduced Hg(II). After 7 h, the level of Hg(II) was below the detection limit. In the fiber, the cell density of S. chlorophenolicum was ~5×10⁸ CFU·mL⁻¹, the cell density of R. metallidurans was ~8.3×10⁷ CFU·mL⁻¹. In both a) and b), the error bars indicate standard errors (n = 2).

Figure 4

R. metallidurans cells neither degrade PCP (black diamonds) nor interfere with PCP degradation (red circles). Degradation of PCP was confirmed in both the pure culture of S. chlorophenolicum species (black triangles) and the well-mixed co-culture of R. metallidurans and S. chlorophenolicum species (red circles). In this experimental setup, no Hg(II) ions were added. The initial cell density of each species was adjusted to be ~10⁸ CFU·mL⁻¹. Error bars indicate standard errors (n = 2).

Figure 5

Different spatial organizations affect the efficiency of the functions in a synthetic bacterial community. The experimental setup ‘original’ indicates a spatially structured fiber containing S. chlorophenolicum cells in the core layer and R. metallidurans cells in the shell layer (black triangles); in contrast, ‘inverted’ represents a structured fiber constraining R. metallidurans cells in the core layer and S. chlorophenolicum cells in the shell layer (red inverted triangles). In this experiment, initial concentration of Hg(II) was adjusted at 100 µM in PM media from HgCl₂ stock solution (10 mM). The final cell density of the species constrained in a core layer was ~5×10⁸ CFU·mL⁻¹, whereas the initial cell density of the species constrained in a shell layer was ~8.33×10⁷ CFU·mL⁻¹ at 0 h. Error bars indicate standard errors (n = 2).