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
. 2017 Jan 23:11:4.
doi: 10.1186/s13036-017-0048-5. eCollection 2017.

Synthetic photosynthetic consortia define interactions leading to robustness and photoproduction

Stephanie G Hays  1   2 , Leo L W Yan  3   4 , Pamela A Silver #  1   2 , Daniel C Ducat #  3   5
Affiliations

Synthetic photosynthetic consortia define interactions leading to robustness and photoproduction

Stephanie G Hays et al. J Biol Eng. .

Abstract

Background: Microbial consortia composed of autotrophic and heterotrophic species abound in nature, yet examples of synthetic communities with mixed metabolism are limited in the laboratory. We previously engineered a model cyanobacterium, Synechococcus elongatus PCC 7942, to secrete the bulk of the carbon it fixes as sucrose, a carbohydrate that can be utilized by many other microbes. Here, we tested the capability of sucrose-secreting cyanobacteria to act as a flexible platform for the construction of synthetic, light-driven consortia by pairing them with three disparate heterotrophs: Bacillus subtilis, Escherichia coli, or Saccharomyces cerevisiae. The comparison of these different co-culture dyads reveals general design principles for the construction of robust autotroph/heterotroph consortia.

Results: We observed heterotrophic growth dependent upon cyanobacterial photosynthate in each co-culture pair. Furthermore, these synthetic consortia could be stabilized over the long-term (weeks to months) and both species could persist when challenged with specific perturbations. Stability and productivity of autotroph/heterotroph co-cultures was dependent on heterotroph sucrose utilization, as well as other species-independent interactions that we observed across all dyads. One destabilizing interaction we observed was that non-sucrose byproducts of oxygenic photosynthesis negatively impacted heterotroph growth. Conversely, inoculation of each heterotrophic species enhanced cyanobacterial growth in comparison to axenic cultures. Finally, these consortia can be flexibly programmed for photoproduction of target compounds and proteins; by changing the heterotroph in co-culture to specialized strains of B. subtilis or E. coli we demonstrate production of alpha-amylase and polyhydroxybutyrate, respectively.

Conclusions: Enabled by the unprecedented flexibility of this consortia design, we uncover species-independent design principles that influence cyanobacteria/heterotroph consortia robustness. The modular nature of these communities and their unusual robustness exhibits promise as a platform for highly-versatile photoproduction strategies that capitalize on multi-species interactions and could be utilized as a tool for the study of nascent symbioses. Further consortia improvements via engineered interventions beyond those we show here (i.e., increased efficiency growing on sucrose) could improve these communities as production platforms.

Keywords: Microbial communities; Photoproduction; Synthetic biology; Synthetic consortia.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Axenic characterizations of candidate strains. a This schematic shows the engineered microbial community design. CscB + S. elongatus (green) capture light and CO2 via photosynthesis. Fixed carbon is secreted as sucrose (black arrows) when induced with IPTG in the presence of osmotic pressure (NaCl). This secreted carbon then supports the growth of B. subtilis (blue), S. cerevisiae (purple), or E. coli (orange) with the final goal of production of target compounds from those heterotrophs. Axenic cscB + S. elongatus was grown in CoBBG-11 with (solid line) and without IPTG (dashed line) to induce sucrose secretion. Cell density (b) and sucrose levels in culture supernatants (c) were measured. Error bars are standard deviation of 8 biological replicates. For characterization of cyanobacteria in CoYBG-11 see Additional file 1: Figure S1. d Heterotroph growth in isolation was characterized via growth rate in co-culture buffer supplemented with 2% sucrose. Error is standard deviation of ≥ 3 replicates
Fig. 2
Fig. 2
S. elongatus supports microbial communities in batch culture. Batch cultures of cscB + S. elongatus (green) in co-culture with B. subtilis (blue), S. cerevisiae (purple), or E. coli (orange) were grown in constant light. CscB + S. elongatus cells/mL were determined by flow cytometry every 12 hours for co-cultures containing B. subtilis (a; green), S. cerevisiae (b, d; green), and E. coli (e, f; green). Co-cultures with uninduced (dashed lines) or induced cscB expression (solid lines) were tested. Heterotroph viability was monitored by colony forming unit (CFU) for all B. subtilis (a; blue), S. cerevisiae (b strain W303, d strain W303Clump; purple), and E. coli (e; strain W, f; strain W ΔcscR; orange) co-cultures. Data for a, b, d, e, and f, are representative, same-day experiments where error bars are the standard error in 3 biological replicates. Additional replicates in Additional file 1: Figure S2. c Axenic heterotroph growth was tested in defined media with varying concentrations; the range of sucrose that cscB + S. elongatus can secrete in 48 hours is denoted by a green box. Average OD600 is shown as a metric of growth for ≥ 6 biological replicates. OD600 was correlated to viable colony forming units (CFU) in Additional file 1: Figure S3. No contaminants/heterotrophic colonies grew from axenic cyanobacteria controls
Fig. 3
Fig. 3
Microbial interactions. Engineered consortia demonstrate un-engineered interactions that can be classified into two categories: negative effects that cyanobacteria have on heterotrophs (a) and positive effects heterotrophs have on cyanobacteria (e). B. subtilis 3610 (b), W303Clump S. cerevisiae (c), and E. coli W ΔcscR (d) were co-cultured with various concentrations of S. elongatus and heterotroph CFU/mL were determined after 12 hours of cultivation in either light or dark. Ratios of CFU in light compared to CFU in dark are reported (b-d). Additional strains were tested in Additional file 1: Figure S4. P-values of two-tailed t-tests with Welch’s correction are denoted with asterisks: * 0.01 to 0.05, ** 0.001 to 0.01, *** 0.0001 to 0.001, **** <0.0001. Positive effects of heterotrophs on cyanobacteria (e) were observed in liquid (f), evidenced by the number of cyanobacteria cells measured in co-cultures relative to axenic controls after 48 hours in constant light. These co-cultures were inoculated with two orders of magnitude fewer cscB + S. elongatus (~1.7x106cells/mL) than the co-cultures depicted in Fig. 2 (~1.7x108cells/mL), and 1 mM IPTG was added to all cultures to induce sucrose export. Thick horizontal lines represent the average measurement for each condition while thin horizontal lines represent one standard deviation from the mean. Positive effects of heterotrophs on cyanobacteria in previous liquid batch experiments is summarized in Additional file 1: Figure S5. The influence of heterotrophs on cyanobacterial growth on solid media (g) was determined by plating a dilute lawn of cscB + S. elongatus on CoBBG-11 agar plates. The cyanobacterial lawn was overlaid with the specified strain in ten-fold serial dilutions of heterotroph and in constant light with or without IPTG
Fig. 4
Fig. 4
Co-cultures persist through time and perturbation. Representative continuous co-cultures of E. coli W ΔcscR/cscB + S. elongatus (a) and W303Clump S. cerevisiae/cscB + S. elongatus (b) were cultured in photobioreactors with 1 mM IPTG. E. coli-containing consortia were grown in constant light while S. cerevisiae communities were exposed to 16:8 hour light/dark photo periods (grey spaces represent darkness). Optical density of the entire culture (black points) as well as counts for the individual cell types were tracked (green S. elongatus, orange E. coli W ΔcscR, purple W303Clump S. cerevisiae). Additional photobioreactor cultures for E. coli W ΔcscR and W303Clump S. cerevisiae are presented in Additional file 1: Figure S6 and S7, respectively. Extended W303Clump S. cerevisiae/cscB + S. elongatus co-cultures are presented in Additional file 1: Figure S8. Recovery of B subtilis 3610/cscB + S. elongatus (c) or E. coli W ΔcscR /cscB + S. elongatus (d) batch cultures following dilution at 24 hours was monitored by viable colony counts and flow cytometry for heterotrophs and cyanobacteria, respectively. Perturbations on to solid media are presented in Additional file 1: Figure S9
Fig. 5
Fig. 5
Photoproduction of enzymes and metabolites from co-culture. Flexible functionalization of co-cultures was accomplished via the addition of heterotrophs capable of producing target compounds (a). Alpha-amylase is naturally produced and secreted by B. subtilis strain 168. Supernatants from 24 hour cultures of B. subtilis 168 alone or in co-culture with cscB + S. elongatus were tested for enzymatic activity (b). Western blots also reveal the presence of alpha-amylase in co-cultures containing IPTG (c). E. coli is capable of making PHB when carrying the pAET41 plasmid. Batch co-cultures of E. coli (with or without pAET41 to enable PHB production) and cscB + S. elongatus were cultivated for one week with or without IPTG to induce sugar. PHB content of the total culture was analyzed (d). Filled circles represent measured values; hollow circles placed on the x-axis represent cultures in which no PHB was formed or was produced at levels below the detection limit. Thick horizontal lines represent the average measurement for each condition while thin horizontal lines represent one standard deviation from the mean
See this image and copyright information in PMC

References

    1. Hays SG, Ducat DC. Engineering cyanobacteria as photosynthetic feedstock factories. Photosynth Res. 2015;123:285–95. doi: 10.1007/s11120-014-9980-0. - DOI - PMC - PubMed
    1. Möllers KB, Cannella D, Jørgensen H, Frigaard N-U. Cyanobacterial biomass as carbohydrate and nutrient feedstock for bioethanol production by yeast fermentation. Biotechnol Biofuels. 2014;7:64. doi: 10.1186/1754-6834-7-64. - DOI - PMC - PubMed
    1. Aikawa S, Joseph A, Yamada R, Izumi Y, Yamagishi T, Matsuda F, Kawai H, Chang J-S, Hasunuma T, Kondo A, Ducat DC, Way JC, Silver PA, Lopez PJ, Desclés J, Allen AE, Bowler C, Rosenberg JN, Oyler GA, Wilkinson L, Betenbaugh MJ, Melis A, Dismukes GC, Carrieri D, Bennette N, Ananyev GM, Posewitz MC, Harun R, Jason WSY, Cherrington T, et al. Direct conversion of Spirulina to ethanol without pretreatment or enzymatic hydrolysis processes. Energy Environ Sci. 2013;6:1844. doi: 10.1039/c3ee40305j. - DOI
    1. Markou G, Angelidaki I, Georgakakis D. Carbohydrate-enriched cyanobacterial biomass as feedstock for bio-methane production through anaerobic digestion. Fuel. 2013;111:872–879. doi: 10.1016/j.fuel.2013.04.013. - DOI
    1. John RP, Anisha GS, Nampoothiri KM, Pandey A. Micro and macroalgal biomass: A renewable source for bioethanol. Bioresour Technol. 2011;102:186–93. - PubMed