Silica ecosystem for synergistic biotransformation

Abstract

Synergistical bacterial species can perform more varied and complex transformations of chemical substances than either species alone, but this is rarely used commercially because of technical difficulties in maintaining mixed cultures. Typical problems with mixed cultures on scale are unrestrained growth of one bacterium, which leads to suboptimal population ratios, and lack of control over bacterial spatial distribution, which leads to inefficient substrate transport. To address these issues, we designed and produced a synthetic ecosystem by co-encapsulation in a silica gel matrix, which enabled precise control of the microbial populations and their microenvironment. As a case study, two greatly different microorganisms: Pseudomonas sp. NCIB 9816 and Synechococcus elongatus PCC 7942 were encapsulated. NCIB 9816 can aerobically biotransform over 100 aromatic hydrocarbons, a feat useful for synthesis of higher value commodity chemicals or environmental remediation. In our system, NCIB 9816 was used for biotransformation of naphthalene (a model substrate) into CO2 and the cyanobacterium PCC 7942 was used to provide the necessary oxygen for the biotransformation reactions via photosynthesis. A mathematical model was constructed to determine the critical cell density parameter to maximize oxygen production, and was then used to maximize the biotransformation rate of the system.

Figure 1. Silica gel matrix optimization based on its optical and mechanical properties, and the post-encapsulation activity of PCC 7942 and NCIB 9816.

Four different α values (0.5: Highest Si alkoxide, to 0.12: Lowest Si alkoxide in gel formulation) and two different nanoparticle sizes (HS40: 12 nm, TM40: 22 nm) were tested. (a) Schematic of the biotransformation system illustrating the silica gel encapsulated bacteria, and the transport of substrates between cells (b) Optical transmittance of the gels at 680 nm and 1 cm pathlength, (c) Stress at failure, (d) Elastic modulus, (e) Oxygen generation rate of encapsulated PCC 7942 (in PBS) and oxygen consumption rate of encapsulated NCIB 9816 during biotransformation of naphthalene in saturated naphthalene solution (All error bars indicate standard deviation with n ≥ 3).

Figure 2. Modeling the oxygen generation rate of the silica gel encapsulated PCC 7942.

Light attenuation in the matrix by the silica gel material and encapsulated cells was characterized using UV-Vis spectroscopy. Modeling results were experimentally verified by measuring oxygen generation rate of the encapsulated cells at varying cell density. (a) Light attenuation in cells suspended in PBS (red) and silica gel encapsulated cells (blue), (b) Schematic for the model in two different gel geometries with encapsulated cells, (c) Schematic: Experiment setup (Oxygraph) used for oxygen generation or consumption rate measurements with encapsulated cells, Image: Silica gel samples with encapsulated PCC 7942. Oxygen (synthesized via photosynthesis) bubbles are clearly visible on the supporting wires (indicated with white arrows), (d) Experimental measurements of oxygen generation rate of silica gel encapsulated PCC 7942 (black diamonds), and model results with (red curve) and without (blue curve) light back-scattering effects (All error bars indicate standard deviation, n > 3).

Figure 3. Synergistic biotransformation by silica gel co-encapsulated NCIB 9816 and PCC 7942.

(a) Confocal images of silica gel co-encapsulated PCC 7942 (red) and NCIB 9816 (green) cells. Both species are homogeneously distributed in the silica gel matrix and positioned in micron-scale proximity. (b) Cell densities of PCC 7942 (ρ_C) and NCIB 9816 (ρ_N) optimized for the experimental setup of the biotransformation experiment. curve indicates the optimal operation conditions where the system has neither an oxygen deficit or surplus. The maximum biotransformation rate is achieved at ρ_C = ρ_cr and corresponding ρ_N on the curve c) Schematic of the experiment setup used for biotransformation of naphthalene. Four cases were tested: I) No cells (Negative control), II) NCIB 9816 (Oxygen is limited to the dissolved oxygen in solution), III) NCIB 9816 with headspace (Supplemental oxygen is provided via the air in the headspace), IV) NCIB 9816 with PCC 7942 (Oxygen is provided by the co-encapsulated PCC 7942) (d) Results of the naphthalene biotransformation experiment. NCIB 9816 with co-encapsulated PCC 7942 achieved the highest biotransformation ratio (All error bars indicate standard deviation, n = 3).