Growth of Chlamydomonas reinhardtii in acetate-free medium when co-cultured with alginate-encapsulated, acetate-producing strains of Synechococcus sp. PCC 7002
© Therien et al.; licensee BioMed Central Ltd. 2014
Received: 5 June 2014
Accepted: 2 October 2014
Published: 18 October 2014
The model alga Chlamydomonas reinhardtii requires acetate as a co-substrate for optimal production of lipids, and the addition of acetate to culture media has practical and economic implications for algal biofuel production. Here we demonstrate the growth of C. reinhardtii on acetate provided by mutant strains of the cyanobacterium Synechococcus sp. PCC 7002.
Optimal growth conditions for co-cultivation of C. reinhardtii with wild-type and mutant strains of Synechococcus sp. 7002 were established. In co-culture, acetate produced by a glycogen synthase knockout mutant of Synechococcus sp. PCC 7002 was able to support the growth of a lipid-accumulating mutant strain of C. reinhardtii defective in starch production. Encapsulation of Synechococcus sp. PCC 7002 using an alginate matrix was successfully employed in co-cultures to limit growth and maintain the stability.
The ability of immobilized strains of the cyanobacterium Synechococcus sp. PCC 7002 to produce acetate at a level adequate to support the growth of lipid-accumulating strains of C. reinhartdii offers a potentially practical, photosynthetic alternative to providing exogenous acetate into growth media.
With the ongoing increase in global energy demand, the development of alternative energy sources has been at the forefront of recent modern research. Of the various alternative energies, bioenergy has been of interest because of its many potential benefits over the currently used petroleum, natural gas, and coal. These benefits include carbon neutrality, renewability, low environmental toxicity, and reduction of dependence on foreign energy sources. Biodiesel and bioethanol, which together currently account for the major proportion of biofuels, are mainly produced from higher plants . These fuels can replace diesel and gasoline, respectively, in conventional engines without modification. Of these two biofuels, biodiesel has been considered the most viable option in the United States . However, the production of biodiesel from crops grown on arable land is controversial due to the potential implications of the competition with agricultural food production ,. One alternative to using food crops as oil sources is to produce biofuels using microalgae and/or cyanobacteria, which can be grown in areas not suitable for crop growth, such as deserts ,. Microalgae can be used to assimilate CO2 and produce multiple fuel molecules, including hydrogen, starch, and lipids -. Starch can be fermented to hydrogen or ethanol, while lipids can be converted to biodiesel -.
Some lipid-accumulating microalgae, such as Chlamydomonas reinhardtii, are able to grow photoautotrophically on sunlight and CO2, chemotrophically on acetate, or photomixotrophically in a combination of these two growth modes ,. Optimal lipid production by C. reinhardtii is observed under photomixotrophic conditions in the presence of acetate, which could present economic and practical challenges for large-scale production of algal-based biofuels ,. An alternative is to co-culture lipid-producing C. reinhardtii with an acetate-producing cyanobacterium, such as certain Synechococcus sp., that can naturally produce acetate during photosynthesis and/or fermentation -.
In this study, we present results of co-culturing wild-type and a lipid-accumulating (sta6) strain of C. reinhardtii, and an acetate-accumulating strain (glgA1) of Synechococcus sp. PCC 7002. We demonstrate sustained photomixotrophic growth of C. reinhardtii with acetate produced by Synechococcus 7002.
Results and discussion
Effect of temperature on growth of Synechococcus sp. PCC 7002 and C. reinhardtii
Effect of media composition on growth of C. reinhardtii and Synechococcus sp. PCC 7002
Effect of acetate on growth of C. reinhardtii and Synechococcus sp. PCC 7002
We have developed media formulations and growth conditions that support co-cultures of wild-type and the acetate-producing glgA1 strain of the Synechococcus sp. PCC 7002 and wild-type and lipid-accumulating sta6 mutant of C. reinhardtii (Additional file 2: Figure S1). A temperature of 30°C supported the growth of both organisms; however, Synechococcus sp. PCC 7002 overtook the culture when both organisms were present as free cells. Alginate encapsulation was found to be an effective way to slow the growth of Synechococcus sp. PCC 7002 while at the same time allowing the production of sufficient acetate to stimulate growth and lipid storage of C. reinhardtii. The encapsulation of Synechococcus sp. PCC 7002 also provides a potential mechanism for separating and harvesting C. reinhardtii for lipids and recycling of the Synechococcus sp. PCC 7002 cells. The results reported here provide a strong proof of concept for supplanting the provision of costly medium components through co-culturing that can be optimized through metabolic engineering and implementation of enhanced acetate-producing cyanobacteria.
Growth of microorganisms
Inoculum cultures of both Synechococcus sp. PCC 7002 and C. reinhardtii (25 ml) were prepared in standard growth media for each organism in 50-ml Erlenmeyer flasks oscillating at 100 rpm at a constant illumination of 100 μmol photons m-2-s-1 with warm white fluorescent light. Wild-type and glgA1 knockout mutant strains of Synechococcus sp. PCC 7002 were grown in medium A+ (pH = 8.0)  (Additional file 1: Table S1) and were incubated at 38°C while sparging with 1% v/v CO2 in air. Wild-type C. reinhardtii and sta6 mutant strains were grown in TAP media (pH = 7.0) incubated at 30°C. The optical density was monitored at 750 nm (ThermoSpectronic Bio Mate 3 spectrophotometer) for Synechococcus sp. PCC 7002 and C. reinhardtii strains. Cultures were harvested by centrifugation for 10 min at 8,000 × g, washed in the medium to be used, centrifuged again, and finally resuspended in the appropriate medium and used to inoculate different culture conditions for co-culturing. Optimal co-culturing conditions were determined by first growing wild-type Synechococcus sp. PCC 7002 and C. reinhardtii strains individually at 30°C, 34°C, and 38°C on TAP medium (standard C. reinhardtii growth medium) or A+ medium (standard Synechococcus sp. PCC 7002 growth medium). The formulation of the TAP media, A+ medium, and modified derivatives of each can be found in the supplemental data (Additional file 1: Table S1). For the study, standard TAP medium was modified by eliminating acetic acid and supplementing with 1 g NaNO3 L-1 and 4 μg vitamin B12 L-1, and this formulation was termed co-culture medium. A+ medium was supplemented with 7.5 g NH4Cl L-1 and 1 ml glacial acetic acid L-1, and the concentration of NaNO3 and NaCl was reduced by 50%. Co-cultures were inoculated with a mixture of seed cultures grown in the appropriate culture media, and the optical densities were monitored at both 600 nm and 750 nm. The cultures were monitored for bacterial contamination by microscopic examination, and cell numbers were determined by direct cell counting with a hemocytometer.
Synechococcus sp. PCC 7002 cultures were harvested at an OD650 nm ranging from 0.8-1.0 (ThermoSpectronic Bio Mate 3 spectrophotometer) via centrifugation and resuspended in 1/5 the original media volume, yielding a concentrated cell suspension. Alginate beads were made by dissolving 3 g of sodium alginate directly in 100 ml of concentrated culture media and adding it dropwise with a syringe and needle into a 1% (w/v) solution of CaCl2 in growth media from a height of at least 30 cm . The bead size was controlled by needle gauge: 22 G yields beads approximately 2 mM in diameter and 18 G yields beads of 3 mM, while 4-mm beads are made directly from a 10-ml syringe without a needle. Beads were formed immediately and were allowed to harden further in the CaCl2 solution for 15 min. After hardening, the beads were removed from the CaCl2 solution and added to fresh growth media. To prevent leakage of cells, a cell-free layer of alginate was used to coat the beads. To produce the coating, the beads were submerged in a 3% (w/v) alginate solution in growth medium followed by transfer to a 1% (w/v) solution of CaCl2 in growth medium to allow hardening of the cell-free alginate layer.
Acetate concentrations were determined by 1H-NMR . Samples were centrifuged for 10 min at 14,000× g and 500 μl of the supernatant was transferred to an NMR tube to which 50 μl of D2O was added. Spectra were collected on a Bruker DRX500 NMR spectrometer operating at a frequency of 500.13 MHz with a 1D NOESY pulse sequence and a 100-ms mixing time at 32 K. Typically, 64 4.28-s scans were collected for each sample with a pre-saturation pulse to suppress the water signal.
JBT and OAZ contributed equally in conducting the experiments. JWP, DAB, MCP, OAZ, and JBT designed the experiments, analyzed the data, and wrote the article. All authors read and approved the final manuscript.
The authors would like to thank Dr. Scott Busse, Interim Director of Montana State University NMR facility, for assistance in measuring the acetate concentrations. The project was supported by a grant from DOE Energy Efficiency and Renewable Energy (DE-EE0003136) to JWP and DAB, and AFOSR (FA9550-14-1-0147) to MCP and JWP.
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