Building 3D Algae Spheroids with Water Emulsion to Produce Clean Hydrogen

Dec 4, 2020. By Kolemann Lutz

Millions of nanoscale, algae-based microbial factories could one day use photosynthesis to seed the atmosphere of Mars and Venus to produce vast quantities of clean hydrogen from carbon dioxide and water.

Carl Sagan imagined seeding the atmosphere of Venus with algae, which would convert water, nitrogen, and carbon dioxide into organic compounds, capturing a majority of the carbon dioxide from the atmosphere.

The biocompatibility of water-in-water (w/w) emulsion systems consists of droplets of water-solvated molecules in another continuous aqueous solution and holds great potential for designing and building multicellular aggregates for applications in biomedicine; printing tumor spheroids; assembling heterogeneous stem cell niches; and developing artificial models of cellular assembly. While micro-engineering and microfluidics--the science of manipulating and controlling fluids at a molecular level--has garnered much attention, water-dispersible microbial reactors are not as familiar.

Aqueous algae cells typically convert CO2 and produce oxygen via photosynthesis. In a new study, a team of international researchers packed approximately 10,000 chlorella algae cells into each sugary water droplet and exposed them to sunlight in the air on Earth’s surface to enable the microbes to generate hydrogen, rather than oxygen, from chlorophyll and sunlight.

The researchers discovered a breakthrough: photobiological production of hydrogen under aerobic room temperature conditions with water emulsion using minimal mass, energy, and local resources. After mixing BSA (Bovine serum albumin) hydrogel microparticle stabilizer with a solution of chlorella cells, the hydrophilic micro-droplets captured high loadings of photosynthetic algae cells by emulsification. This solution was then injected into a mixture of rapidly stirred PEG solution to produce 50% w/w emulsion.

Chlorella cells were hyperosmoticly compressed to mean sizes of 165, 92, and 22 microns (μm), generally within 110 seconds, in emulsion water droplets to form negatively charged, stable spheroids consisting of closely packed 3D assemblies. After the emulsion droplets containing packed aggregates of Chlorella cells were exposed to sunlight at 100 μE m-2 s-1, measurements indicated a linear increase in hydrogen concentration over a 96-hour period.

Increasing mean size of light-exposed spheroids from 22 to 92 um maximized hydrogen evolution in the core and decreased in dissolved O concentration, indicating that respiration became dominant over photosynthesis as the spheroid core became larger. The researchers believe the onset of the hypoxic reactions in the center of spheroids was due to a combination of partial shielding, which allowed less sunlight photons to reach algae cell and the diffusion of atmospheric oxygen into the core regions.

Using an oxygen detector and hydrogen detector the researchers used the total content of chlorophyll associated in the core and surface regions as a proxy to monitor gas concentrations in the air. The hydrogen production rate for algae cell spheroids was 3X lower than the rate determined under argon gas (.25 and .86 μmol H2 (mg chlorophyll)-1 hr-1 ), demonstrating that close to two-thirds of the chlorella cells were not contributing to hydrogen generation under the aerobic environment.

To increase hypoxic photosynthesis in the core, the researchers coated approximately a quarter of a million 10-micron-thick microbial reactors with a thin outer shell of respiring E. coli bacteria, which were then able to scavenge for oxygen and increase the number of algae cells for hydrogenase, an enzyme that catalyzes the oxygen gain or less of electrons of H2 , inside one milliliter of water. Following exposure to daylight under same conditions, the hybrid spheroid micro-reactors exhibited a reduced induction period from 0-12 hours and rapid onset of hydrogen production around 2.02 μmol, which is double the amount of hydrogen production of non-E.coli reactors containing only algae cells with an incubation time of 12-24 hours producing 1.98 μmol.

Hydrogen production of the hybrid spheroids was 2.2 times greater than the instantaneous biomass-to-fuel yield of chlorella algae cells in nature (.20 μmol of H2 per milligram of chlorophyll per hour). Because the bacteria-infused 3D microreactors were stabilized only by compression, non-covalent interactions, and hydrogelation of BSA microparticles, the spheroids disassembled over time and hydrogen production terminated within 72 hours after disassembly at 108 hours. Seeking to prolong the lifetime of the hybrid bioreactor, the researchers added aldehyde-functionalized dextra (Dex-CHO) into w/w dextran-rich emulsion droplets to chemically bind the BSA protein hydrogel matrix within core of spheroids. The resulting more stable spheroids extended hydrogen production to over 168 hours or one week, which is 2.3 times longer in duration than the hybrid microreactor.

“Our methodology is facile and should be capable of scale-up without impairing the viability of the living cells,” said Professor Xin Huang at the Harbin Institute of Technology in China. “It also seems flexible; for example, we recently captured large numbers of yeast cells in the droplets and used the microbial reactors for ethanol production.”

The researchers added, “it should be possible to combine our methodology with more complex bioengineering approaches involving sulfur deprivation, genetically modified oxygen-tolerant hydrogenases, or cellular surface modifications. However, hybrid algae-based micro-reactors have limited rates and biomass-to-fuel yields, which remains a challenge to compete with other synthetic hydrogen-producing systems.

Industrial algae cultivation can be used for many applications including the production of bioplastics, lipids, proteins, chemical feedstocks, ethanol, refined transportation fuels, methane, fertilizers, pharmaceuticals, and foods (omega-3 fatty acids), food colorants and dyes. Rotating, self replicating greenhouse blocks, a concept being developed by Daniel Tompkins, Founder at GrowMars, holds the potential to grow large quantities of algae at a rate of 5 g dry algae mass per liter per day. This approach could radically improve ISRU and be processed and filtered into chlorella algae feedstocks, which could be embedded with water emulsion and hybrid microreactor spheroids to produce clean affordable hydrogen from sunlight, CO2, and H2O.

Producing hydrogen on celestial bodies traditionally requires large amounts of energy and heavy electrolysis machinery. The assembly, spatial organization, and immobilization of synthetic algae systems offers an affordable method to produce considerable levels of hydrogen in situ. More research is required to understand the significance of alternative sunlight, gravity, pressure, temperature on algae performance and biomass-to-fuel yield.

More broadly, using this aqueous water emulsion droplet method could be used to improve biomedical applications (cell therapy and tissue engineering), controlling 3D assembly of living cells, and constructing protocell colonies and prototissues.

By harnessing the local materials of sunlight, water, and CO2, synthetic hydrogen production systems could enable the in-situ manufacturing of liquid rocket fuel for terrestrial life on other planets.

Reference: “Photosynthetic hydrogen production by droplet-based microbial micro-reactors under aerobic conditions” by Xu Z, Wang S, Li S, Liu X, Wang L, Li M, Huang X and Mann S, 25 November 2020, Nature Communications.

What we are up to

© 2021