Overcoming the Major Challenges to Algal Biohydrogen Production

Overcoming the Major Challenges to Algal Biohydrogen Production NSF/CBET-BSF: Overcoming the Major Challenges to Algal Biohydrogen Production Overview: Molecular hydrogen (H2) is a promising clean energy carrier and a valuable commodity used in many industrial technologies (e.g. fertilizer synthesis, petroleum refining). The current industrial method to produce hydrogen is steam reformation of natural gas, which produces carbon dioxide (and other pollutants) while consuming methane, a high-value fuel. Current technologies to produce hydrogen by reduction of protons are based on catalysts that use purified water, rare elements and/or have short lifespans. In contrast, hydrogen production by photosynthetic microbes represents an alternate strategy that makes use of sunlight and abundant resources (e.g. uncultivated soil, wastewater or seawater) and avoids competition with agriculture. Moreover, biological replication allows rapid and cheap production of solar bio-factories making H2. For industrial maturity, algal hydrogen production must be increased by ~5-fold. Two major challenges limit efficient biological H2 production: (1) inactivation of the hydrogen production catalyst (the hydrogenase enzyme) by molecular oxygen (O2), which is a byproduct of photosynthesis, and (2) limited electron flow from the photosynthetic apparatus to the hydrogenase. Intellectual merit: In order to address the first major challenge, our recent observation that a pool of active hydrogenase enzyme persists much longer than expected under aerobic conditions will be exploited. Our hypothesis is that the enzyme can be protected by local microoxic environments created by nearby O2 uptake mechanisms. Accordingly, several complementary ways to reduce O2 at the vicinity of the hydrogenase are being pursued, most of them involving the creation of chimeric proteins in which the hydrogenase is joined with a partner protein capable of reducing O2 or reactive oxygen species (e.g. glucose oxidase, flavodiiron protein). The most ambitious such chimera is a fusion of Photosystem I to hydrogenase, which the Redding group have shown is assembled and active in vivo. Together with the ferredoxin-hydrogenase chimera created by the Yacoby group, this represents a way to intercept a greater fraction of electrons from photosynthetic electron transfer, addressing the second major challenge (although this is not the focus of this project). Our plan is to test these chimeras in vitro and then express the most promising ones in vivo. Rapid molecular and spectroscopic tests will be used to identify limitations to light-driven hydrogen production in the engineered strains. Several genetic modifications can be utilized to rectify identified limitations in electron flow or H2 production activity. Complementary sets of expertise in the two research groups will be put to use in the creation, analysis, and optimization of the engineered algal cells. Together the two groups will determine the optimal way to arrange the various new components to make sustained high-level bio-hydrogen production a reality. Broader impacts: The PI will incorporate this project into his teaching, as well as involve undergraduate and graduate researchers in an inter-disciplinary team to work on this project. In his role as Director of the Center for Bioenergy and Photosynthesis, he is overseeing the creation of outreach programs in which talented ASU undergraduates are sent into local schools to talk about bioenergy, and sustainability in general, and do some hands-on learning experiences with the students there. For this particular project, the PI is partnering with sustainability scientists at the ASU Global Institute of Sustainability (GIOS) to develop a module on screening of bio-hydrogen production by algal strains developed in this project using a low-tech assay: the students will grow the algae on petri plates, overlay them with agar containing a bacterium that synthesizes a fluorescent protein when they detect H2, and then image them by visible fluorescence using their cell phones. The students will be integrated into the discovery process, as most of the strains they receive will be unknowns; their subsequent questions will drive active learning. Teachers from local schools will be trained in implementation of this module during a professional development workshop organized by GIOS personnel each year. It is expected that 100 teachers will be incorporated into the project; thus, this program has the potential to impact about 4000 local students. INTERN DCL: NSF/CBET-BSF: Overcoming the Major Challenges to Algal Biohydrogen Production In the wake of climate change, it is our moral imperative to find viable alternatives to our current fossil fuel-based economy. The biological process of photosynthesis harnesses the power of the sun and, in principle, could be employed to produce valuable molecules of interest with high efficiency and minimal carbon footprint. However, photosynthesis is intrinsically connected to the metabolism of the host organism and in order to become feasible for industry, the efficiency of energy conversion to new end products requires elimination of competing pathways. Our proof-of-principle Photosystem-I-hydrogenase chimera (Fig 1) have been shown to work in vivo linking photosynthesis to hydrogen production in green microalga Chlamydomonas reinhardtii (Kanygin, et al, submitted). This radically new system is designed to direct large fraction of photosynthetic current to proton reduction on catalytic site of hydrogenase. The primary aims of this proposal (which is designed to supplement our ongoing NSF award CBET-1706960) are to better understand (1) how this chimeric protein can withstand oxygen damage and (2) its capacity to redirect electron flux under controlled in vitro conditions.