in acetogenic bacteria is used to reduce CO2 to acetyl coenzyme A (acetyl-CoA), a common biosynthetic intermediate, and eventually acetic acid. This WLP pathway for acetate synthesis consists of two separate branches, the methyl-branch and the carbonyl branch (Figure 1). In the carbonyl branch, CO2 is reduced to CO via the carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS). In the methyl-branch, the first reaction is CO2 reduction to formate. The formyl group is further reduced to methenyl-tetrahydrofolate (THF) and then to methyl-THF. The methyl group is eventually transferred via a corrinoid iron–sulfur protein (CoFeSP) to the CODH/ACS. This bifunctional enzyme fuses the bounded CO (from the carbonyl branch) and the methyl group (from the methyl branch) with CoA to form acetyl-CoA. (15) At this point, inert CO2 is successfully activated and C—C bonds are formed with low energy input through this elegant catalytic network.
The integration of fluorescent semiconductors with bacterial metabolic processes provides a unique optical tag and a noninvasive approach for studying microbial behavior and their metabolic processes. Linking bacteria to an optically addressable semiconductor nanoparticle could aid in spectroscopic studies to elucidate the electron transfer pathway of electrogenic bacteria among other biological responses to abiotic optical, electrical, and chemical stimuli. The first question to ask here is “where do the electrons come from?”. When the solar photons activate the semiconductor nanowires/nanoparticles, the photoexcited electrons will be generated at the semiconductor/electrolyte interface and are fed to the associated microorganisms across the cell membrane. In addition to direct electron transfer, these electrons can also react with water to generate H2 and reduce redox proteins on the cell membrane.
At the semiconductor/bacteria interface, reducing equivalents will be generated, transported, and can be oxidized by a hydrogenase (HydABC) to generate reduced ferredoxin and NADH, which are in turn used to generate NADPH. NADH, NADPH, and ferredoxin (Fd2–) are all electron carriers in the WLP pathway (Figure 1). (14) In the methyl-branch, the reduction of CO2 to methyl-THF requires both NADPH and NADH, and the reduced ferredoxin assists in CO2 reducing to CO. While it is still unclear how those solar-generated electrons that entered from membrane proteins are involved in CO2fixation catalytic network, it is believed that they play essential roles as reducing equivalents. Several pathways have been proposed for this initial charge transfer, implicating indirect electron transfer mechanisms (via H2 or uncharacterized soluble redox mediators) or direct mechanisms (unmediated transfer to membrane-bound enzymes, i.e., hydrogenases and cytochromes).
Fundamental to understanding the interface between inorganic and biological components such as CdS and S. Ovata is the mechanism of the extracellular electron transfer pathway that accomplishes CO2 reduction. While the mechanism and integral proteins for electron transfer from bacteria to the metal electrode (as in microbial fuel cells) have been largely elucidated, the basis for cathodic electron transfer in the reverse direction from metal/semiconductor to bacteria remains largely unexplored. Through the determination of the dominant electron pathways, it is possible to gain fundamental insight into this new and novel mode of charge transport and to design more efficient materials or biological systems to exploit these mechanisms for complex molecular synthesis. Studies of the interface between semiconductors and bacteria challenge the research community’s current technical and scientific capabilities and signify an unprecedented opportunity toward unraveling the powerful functions from these abiotic/biotic interfaces.
The study of the electron transfer mechanisms at this abiotic–biotic interface represents a significant technical challenge and opportunity. There are three major challenges one needs to overcome: (1) measurement under in vivobiological conditions, (2) low sample concentration and signal intensity due to the diffuse nature of biological whole cells, and (3) cell stability and sensitivity to high energy radiation sources. A few techniques meet these criteria, and new imaging and spectroscopic techniques need to be developed to meet these scientific challenges. For example, previously transient absorption spectroscopy (TAS) has been successfully applied to the characterization of hydrogenase-semiconductor nanocystal systems in vitro. (16) Similar experiments are possible on the whole bacterium-semiconductor system. TAS kinetics suggest that the photoexcited reducing equivalents may be taken up by hydrogenase or directly as electrons by membrane-bound proteins. (17)The biochemical activity of proteins involved with charge uptake can be correlated with charge carrier lifetimes through appropriate experimental design.
In addition to kinetic insight, vibrational spectroscopy and X-ray spectroscopy will provide molecular-level insight into these new electron transfer mechanisms. For example, the structure of the Ni–Fe hydrogenase and its spectroscopic signatures throughout its catalytic cycle has been studied in vitro through IR, resonance Raman, and X-ray absorption spectroscopy (XAS). (18) However, such techniques have yet to be translated to more complicated whole-cell living systems. Utilizing the light-absorbing semiconductor as an electron source, time-resolved IR spectra can be compared to existing spectroscopic signatures of the Ni–Fe hydrogenase as it proceeds through its catalytic cycle. Greater chemical specificity of the initial steps of electron transfer and downstream metabolic processes can be potentially achieved by using XAS studies. By monitoring the oxidation states of the metal centers of critical metabolic enzymes, the interplay of semiconductor electron transfer and biocatalysis may be analyzed at the initial electron transfer step and throughout the following CO2 reduction pathway.
In addition to these spectroscopic-based investigations, several research groups are also probing these biohybrids using microbiological methods, including transcriptional and proteomic analyses. Proteomic and metabolomic characterization offers excellent insight into how increased genetic expression of specific genes activates specific metabolic pathways. For example, Zhang et al. studied these changes in the M. thermoacetica-CdS inorganic hybrid system using mass spectrometry and proteomic analysis. (19) The upregulation of electron transfer proteins flavoprotein, ferredoxin, and NADP dehydrogenase suggests the important roles they have in the overall photosynthetic process.
Besides these spectroscopic, transcriptional, and proteomic analyses, one can examine directly how the charge transfer occurs between a microorganism and a solid-state device. We have established a microbial electrochemical cell that comprises a single bacterium interfaced with a single nanowire photoelectrochemical platform. (20) The steady-state current transfer from a single electrochemically active bacterium can be directly monitored. Such time-resolved physical contact and the cathodic charge transfer can be correlated with the time-resolved photocurrent measurement under a specific electrochemical bias. With the same experimental platform, one can also apply electrochemical impedance spectroscopy (EIS) as a powerful tool to study charge transfer, mass transport, and the time constant in our nanowire array/bacteria platform. This suite of spectroscopic and photoelectrochemical techniques adapted to conduct measurements on whole cells will shed light on the mechanism of this unique inorganic–biological interface down to the molecular/atomic level. A deep molecular understanding of the interactions involving the semiconductor, the bacterium, and light will further enable a guided search through the vast microbial and enzymatic parameter space to increase the efficiency and performance of such hybrid solar-to-chemical platforms. Simultaneously, these insights will allow the translation of this electron transfer to engineered designer bacteria, expanding the repertoire of the synthetic biology toolbox.
We are setting to advance a new research frontier with these ongoing efforts and establish the body of new knowledge/language for the fundamental understanding of biotic–abiotic interfaces, their electron transfer, and energy transduction mechanism and finally the evolution of the powerful photosynthetic biohybrids. Through such studies, it is also expected that we will gain a fundamental understanding of the challenging problem of photoactivation of CO2, N2 to produce value-added chemicals, fertilizers, and fuels, such as n-butanol, polymer, and other complex natural products, with the energy input purely from sunlight. One could imagine fundamentally in the future that our chemical industry, the energy industry, and pharmaceutical industry can be powered entirely from renewable solar energy rather than mainly relying on traditional fossil fuel. In this way, with liquid sunlight produced from these photosynthetic biohybrids we are providing an ultimate carbon-neutral solution to fundamentally solve the CO2 emission, global warming, and climate change issues that we are facing on this planet Earth