Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Nov 25;11(1):5985.
doi: 10.1038/s41467-020-19823-5.

Photosynthetic hydrogen production by droplet-based microbial micro-reactors under aerobic conditions

Zhijun Xu  1 , Shengliang Wang  1 , Chunyu Zhao  1 , Shangsong Li  1 , Xiaoman Liu  1 , Lei Wang  1 , Mei Li  2 , Xin Huang  3 , Stephen Mann  4
Affiliations

Photosynthetic hydrogen production by droplet-based microbial micro-reactors under aerobic conditions

Zhijun Xu et al. Nat Commun. .

Abstract

The spontaneous self-assembly of multicellular ensembles into living materials with synergistic structure and function remains a considerable challenge in biotechnology and synthetic biology. Here, we exploit the aqueous two-phase separation of dextran-in-PEG emulsion micro-droplets for the capture, spatial organization and immobilization of algal cells or algal/bacterial cell communities to produce discrete multicellular spheroids capable of both aerobic (oxygen producing) and hypoxic (hydrogen producing) photosynthesis in daylight under air. We show that localized oxygen depletion results in hydrogen production from the core of the algal microscale reactor, and demonstrate that enhanced levels of hydrogen evolution can be achieved synergistically by spontaneously enclosing the photosynthetic cells within a shell of bacterial cells undergoing aerobic respiration. Our results highlight a promising droplet-based environmentally benign approach to dispersible photosynthetic microbial micro-reactors comprising segregated cellular micro-niches with dual functionality, and provide a step towards photobiological hydrogen production under aerobic conditions.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Schematic illustration showing the assembly, spatial organization and dual functionality of multicellular droplet-based living micro-reactors.
(i) Algal cell-based spheroids: a Large numbers of Chlorella algal cells (green spheres) are spontaneously captured within w/w dextran-in-PEG micro-droplets by emulsification in the presence of denatured BSA micro-particles (yellow rectangles). b Cell-containing emulsion droplets are hyperosmotically compressed (red arrows) by transfer to a concentrated PEG solution to produce robust multicellular spheroids comprising a closely packed aggregate of algal cells immobilized in a dextran/BSA hydrogel matrix. c Cell-mediated depletion of oxygen over time in the hydrogel matrix (yellow triangular network) generates hypoxic (interior, cyan) and aerobic (surface, green) micro-niches due to light shading of the algal cells in the core domain by the outer shell of Chlorella cells. Depending on the size of the spheroids, photosynthetic oxygen generation is decreased in the core such that respiration becomes dominant over photosynthesis resulting in the net depletion of cellular storage compounds, hypoxic conditions, hydrogenase activity and hydrogen production in daylight under air. Corresponding reactions: Shell domain: H2O → ½O2 + 2H+ + 2e− (PSII). Core domain: H2O → ½O2 + 2H+ + 2e− (PSII) and 2H+ + 2e → H2 (hydrogenase). (ii) Algal/bacterial hybrid spheroids: (d) Preparation of the droplets using mixtures of Chlorella and PEGylated E. coli cells (blue rods) results in a spatially segregated arrangement of photosynthetic algal cells enclosed by a thin oxygen-depleting layer of respiratory bacterial cells. e Hyperosmotic compression results in consolidation and immobilization of the two cellular micro-niches. f In daylight under air, the binary community acts synergistically to enhance the levels of hydrogen production produced by hypoxic photosynthesis in the core of the multicellular hybrid micro-reactor. Corresponding reactions: Shell domain: H2O → ½O2 + 2H+ + 2e− (PSII) and O2 → CO2 (respiration from E. coli). Core domain: H2O → ½O2 + 2H+ + 2e− (PSII) and 2H+ + 2e− → H2 (hydrogenase).
Fig. 2
Fig. 2. Capture of Chlorella cells within w/w emulsion droplets.
a Confocal bright-field images of a population of dextran-in-PEG micro-droplets and a single droplet (inset) stabilized by denatured BSA micro-particles. b, c Corresponding confocal fluorescence images of droplets displayed in (a) showing presence of FITC-dextran throughout the droplets (b, green fluorescence) and a thin shell of Nile red-stained BSA particles at the droplet surface (c, red fluorescence). (d) Overlay channel of (b, c). Scale bars in (a–d), 100 and 10 μm (insets). e Plot of red and green fluorescence intensities across a single droplet. Inset shows the corresponding 3D confocal scanning image; staining as in (d); scale bar, 10 μm (inset). f–i Optical microscopy images (f, h) and corresponding green fluorescence microscopy images (g, i) of w/w emulsion droplets prepared in the presence of low (f, g; 1.2 × 108 cells/mL) and high (h, i; 3.3 × 109 cells/mL) Chlorella cell densities. The algal cells were treated with fluorescein diacetate (FDA); viable cells gave rise to the hydrolytic release of fluorescein (green fluorescence). Scale bars in (f, g) and (h, i), 100 and 200 μm, respectively. j–n Confocal bright-field microscopy image (j) and corresponding red and green confocal fluorescence microscopy images (k, l), red/green overlay (m) and 3D image (n) of a single droplet showing discrete Chlorella cells captured within the dextran-rich interior. Samples were prepared at a low cell number density (6.9 × 107 cells/mL) using non-labelled dextran and Nile Red-stained BSA particles. The captured algal cells display red and green fluorescence due to intracellular chlorophyll and cell-mediated release of fluorescein, respectively. The Nile Red-stained BSA particles are observed as a red fluorescence ring at the w/w droplet interface. Scale bars in (j–m), 10 μm. All relevant experiments were performed independently at least three times with similar results. Source data underlying Fig. 2e are provided as a Source Data file.
Fig. 3
Fig. 3. Formation of Chlorella cell-based spheroids.
a Time sequence of optical microscopy images of a single dextran-in-PEG micro-droplet containing entrapped Chlorella cells and undergoing shrinkage after immersion in hyperosmotic PEG solution at 0, 30, 70 and 110 s; scale bar, 50 μm. b Corresponding plots of brightness line profiles before (blue; t = 0 s) and after (red; t = 110 s) hyperosmotic compression. Scale bar, 50 μm (inset). c Plots of diameters of Chlorella-loaded droplets prepared at different spinning rates, before (navy blue) and after (deep cyan) hyperosmotic compression. Data are presented as mean values ± SD, error bars indicate standard deviations. d SEM image of a single multicellular spheroid; scale bar, 10 μm. e, f Confocal microscopy fluorescence images of Chlorella cell-based spheroids after FDA staining showing viable algal cells after hyperosmotic compression within the spheroids (e); red fluorescence is from intracellular chlorophyll (f); scale bars, 75 μm. g Sequence of fluorescence microscopy images showing slow diffusion of fluorescein into a single Chlorella cell-based spheroid recorded at t = 0, 5, 10 and 15 min after addition of the dye; scale bar, 30 μm. All relevant experiments were performed independently at least three times with similar results. Source data underlying Fig. 3b, c are provided as a Source Data file.
Fig. 4
Fig. 4. Hypoxic and aerobic photosynthesis in algal cell micro-reactors under air.
a Time-dependent measurements of dissolved oxygen concentration in suspensions of native Chlorella cells (black squares), Chlorella-loaded w/w dextran-in-PEG emulsion droplets (blue triangles), and Chlorella multicellular spheroids with a mean size of 22 μm (grey spheres) or 92 μm (red inverted triangles). All samples were in sealed vials and exposed to daylight at an intensity of 100 μE m−2 s−1. Data are presented as mean values ± SD, error bars indicate standard deviations (n = 3). b Time-dependent changes in ATP concentration of native free Chlorella cells and Chlorella cells within closely packed spheroids (mean size, 92 μm). Data are presented as mean values ± SD, error bars indicate standard deviations (n = 5). c Time-dependent measurements of hydrogen concentration in suspensions of native Chlorella cells (orange squares), Chlorella-loaded w/w dextran-in-PEG emulsion droplets (blue triangles (superimposed on orange squares)), and Chlorella multicellular spheroids with a mean size of 92 μm (red inverted triangles). All samples were in sealed vials and exposed to daylight at an intensity of 100 μE m−2 s−1. Data are presented as mean values ± SD, error bars indicate standard deviations (n = 3). d Time-dependent production of hydrogen for Chlorella multicellular spheroids prepared with mean sizes of 22 (blue), 92 (red) or 165 μm (black). Data are presented as mean values ± SD, error bars indicate standard deviations (n = 3). Source data are provided as a Source Data file.
Fig. 5
Fig. 5. Synergistic hydrogen production in Chlorella/E. coli hybrid micro-reactors in air.
a–c Bright-field image (a), and blue and red confocal fluorescence images (b, c) of a single w/w dextran-in-PEG emulsion droplet with captured red algal and surface-adsorbed bacterial cells. PEGylated E. coli cells are labelled with Atto425 (blue fluorescence); red fluorescence is from chlorophyll present within the Chlorella cells; scale bars, 25 μm. Samples were prepared at 200 rpm; mean droplet size = 116 μm. d–f Confocal bright field image of a single Chlorella/E. coli multicellular spheroid (d) and corresponding red (e) and blue (f) confocal fluorescence images. PEGylated E. coli cells are labelled with Atto425 (blue fluorescence) and are located specifically at the spheroid surface while the Chlorella cells (red fluorescence, chlorophyll) are closely packed throughout the hybrid spheroid. g Overlay image of (e) and (f) showing the core-shell spatial organization. Scale bars in (d–g), 20 μm. Samples were prepared at 200 rpm; mean spheroid size = 90.8 μm. h Time-dependent measurements of hydrogen concentration in mixed suspensions of native Chlorella and E. coli cells (blue circles), Chlorella spheroids and free E. coli cells (blue triangles), Chlorella/E. coli multicellular spheroids (blue squares) and Dex-CHO crosslinked Chlorella/E. coli multicellular spheroids (blue inverted triangles); corresponding decrease in oxygen content for the algal/bacterial cell spheroids is also shown (red squares). All samples were in sealed vials and exposed to daylight at an intensity of 100 μE m−2 s−1. Data are presented as mean values ± SD, error bars indicate standard deviations (n = 3). i Time-dependent plots of hydrogen production for Chlorella cell-based spheroids (grey columns) and Chlorella/E. coli hybrid core-shell spheroids (purple columns) at different time periods. Data are presented as mean values ± SD, error bars indicate standard deviations (n = 3). j Time-dependent plots of hydrogen production for Chlorella spheroids (black) and Chlorella/E. coli hybrid core-shell spheroids (red). Data are presented as mean values ± SD, error bars indicate standard deviations (n = 3). k Plots of average hydrogen production rate for Chlorella multicellular spheroids (red) and Chlorella/E. coli hybrid spheroids (blue) and for the natural instantaneous biomass-to-fuel yield (grey). Production rates for the spheroids were determined between time points of 24 and 72 h. Data are presented as mean values ± SD, error bars indicate standard deviations (n = 3). All relevant experiments were performed independently at least three times with similar results. Source data underlying Fig. 5h–k are provided as a Source Data file.
See this image and copyright information in PMC

References

    1. Elbert DL. Bottom-up tissue engineering. Curr. Opin. Biotechnol. 2011;22:674–680. doi: 10.1016/j.copbio.2011.04.001. - DOI - PMC - PubMed
    1. Morimoto Y, Hsiao AY, Takeuchi S. Point-, line-, and plane-shaped cellular constructs for 3D tissue assembly. Adv. Drug Deliv. Rev. 2015;95:29–39. doi: 10.1016/j.addr.2015.09.003. - DOI - PubMed
    1. Salehi SS, Shamloo A, Hannani SK. Microfluidic technologies to engineer mesenchymal stem cell aggregates-applications and benefits. Biophys. Rev. 2020;12:123–133. doi: 10.1007/s12551-020-00613-8. - DOI - PMC - PubMed
    1. Tavana H, et al. Nanolitre liquid patterning in aqueous environments for spatially defined reagent delivery to mammalian cells. Nat. Mater. 2009;8:736–741. doi: 10.1038/nmat2515. - DOI - PMC - PubMed
    1. Gobbo P, et al. Programmed assembly of synthetic protocells into thermoresponsive prototissues. Nat. Mater. 2018;17:1145–1153. doi: 10.1038/s41563-018-0183-5. - DOI - PubMed

Publication types

LinkOut - more resources