Biocatalytic and Bioelectrocatalytic Approaches for the Reduction of Carbon Dioxide using Enzymes

Abstract In the recent decade, CO2 has increasingly been regarded not only as a greenhouse gas but even more as a chemical feedstock for carbon‐based materials. Different strategies have evolved to realize CO2 utilization and conversion into fuels and chemicals. In particular, biological approaches have drawn attention, as natural CO2 conversion serves as a model for many processes. Microorganisms and enzymes have been studied extensively for redox reactions involving CO2. In this review, we focus on monitoring nonliving biocatalyzed reactions for the reduction of CO2 by using enzymes. We depict the opportunities but also challenges associated with utilizing such biocatalysts. Besides the application of enzymes with co‐factors, resembling natural processes, and co‐factor recovery, we also discuss implementation into photochemical and electrochemical techniques.


Introduction
Alreadyi n1 896, Svante Arrhenius discussed in his work the impacto fa tmospheric carbon dioxide (CO 2 )o nt he greenhouse effect. According to his calculations,A rrhenius stated even back then ac orrelation between the CO 2 content in the atmosphere and an increasei nt he Earthst emperature. [1] Nowadays,c oncerns regardingg reenhouse gases,p articularly CO 2 ,a nd global warminga ffect politics,e conomy,a nd society.I nc omparison to other greenhouse gases such as methane (CH 4 )a nd water vapor, CO 2 has the highesti mpacto n global warming, as its atmospheric residence time is the highest, and moreover, its content in the atmosphere is second only to water vapor. [2,3] CO 2 is generated from the combustiono ff ossil carbon (e.g.,o il, gas,c oal) and biomass in which energy is released. Owing to the finite reserve of fossil-C,a nother issue is now rising:t he conveniencet or ecycle carbon more than to release it into the atmosphere or dispose of it underground. On the basis of these facts,p rimarily the utilization of CO 2 and substitution of fossil fuels as energy carriers have become some of the most discussed topics and have especially drawn attention from the scientific community. [4,5] 2. CO 2

as Chemical Feedstock
To reduce atmospheric CO 2 ,t wo approaches comprising different techniques are generally considered. In the carbon capturea nd sequestration( CCS) approach, CO 2 is stored in deep rock cavitiesu nder sea and land. [6] Practice of course is not ubiquitous nor accepted by the public everywhere and does not utilize CO 2 as such.D ifferently,i nt he carbonc apture and utilization (CCU) approach, CO 2 is regarded as ac arbon feedstock and startingm aterial for artificial fuels and chemicals.W ith this strategy,b oth issues,t hat is,d epletion of fossil fuels and reduction of CO 2 in the atmosphere, are taken into account. In this work, we will therefore address CCU as ak ey target to substitute fossil fuels and to reduce the atmospheric CO 2 content at the same time. [7][8][9][10][11][12] From ac hemical point of view,C O 2 is ah ighly stable molecule in which the carbon atom is in a + 4o xidation state ( Figure 1). [13] Any conversion of CO 2 into as pecies in which the carbona tom maintains the + 4o xidation state is an exergonic process (right part of Figure1); conversely,any conversion into am olecule in which the carbon atom has al ower oxidations tate (+ 2o re ven lower, left part of Figure 1) requires energy.F uels fall into this latter category (lower part of Figure 1). Noteworthy, to produce energy-rich chemicals, energy and hydrogen are necessary. [14] Thel atter must be In the recent decade,C O 2 has increasingly been regarded not only as ag reenhouse gas but even more as ac hemical feedstock for carbon-based materials.D ifferent strategies have evolved to realize CO 2 utilization and conversion into fuels and chemicals.I np articular, biological approaches have drawn attention, as natural CO 2 conversion serves as am odel for many processes. Microorganisms and enzymes have been studied extensivelyf or redox reactions involving CO 2 .Int his review,w efocus on monitoring nonlivingb iocatalyzedr eactions for the reduction of CO 2 by using enzymes. We depict the opportunities buta lso challenges associated with utilizing such biocatalysts.B esides the application of enzymes with co-factors,r esembling naturalp rocesses,a nd cofactor recovery,w ea lso discuss implementation into photochemical and electrochemical techniques. generated from water by using perennial energies such as sun, wind, hydropower, and geothermal( SWHG) energies. This is am ust. Therefore, perennial SWHG energiesm ust be used to convert large volumes of CO 2 into energy-rich products such as fuels. Table 1d epicts possible reduction reactions of CO 2 into variousp ossible products througho ne,t wo,o rm ore electron-transfer reactions.F or each reaction, standard thermodynamic reduction potentials are reported for aqueouss olutions at pH 7. They give an idea of the required energy input for the conversion of CO 2 into acertain product.
In any practical approach, however, the above potentials or energy inputs are expected to be higher. In fact, to perform CO 2 conversion, overpotentials have to be considered. To lower those energyb arriers,r eaction conditions such as high potentials,h igh pressures,a nd/or low temperatures [15,16] are required or catalysts have to be utilizedt op erform the reduction at ap otential as close as possible to the thermodynamic value. [17] However, whereas the incorporation of CO 2 into cyclic carbonates [18] (for application in cosmetics or adhesives) or polymers, [19] in whichC O 2 is an essential feedstock for industrial applications, is al ow-energy process,t he productiono f artificial fuels from CO 2 is ap rocess that requires ah igh energy input,e ven if it has great interest for the larger volume of fuels with respectt oc hemicals and materials. [20][21][22] Consideringt he possible products that can be generated from the reduction of CO 2 ,a lcohols specifically emerge as eminently advantageous.I nparticular, the C 1 and C 2 products methanol and ethanol are highly desired, as they meet the requirement of direct application as fuels.H owever,e ven thoughhigher alcoholswould provide higher energy densities for fuel applications,t he obtainment of mainly C 1 compounds such as methanol is thermodynamically favored from the chemical reduction of CO 2 (see Figure 2a nd Table 1). [23] Thec hallenge for this is to find techniquesa nd/or catalysts to lower the energy barrier for the reduction reaction and to enableo peration under rather mild reaction conditions.
To reduceCO 2 chemically,e lectron sources or donors (sacrificial) are required. Besides photochemical andp hotoelectrochemical approaches,w hich mimic artificial photosynthesis, [24,25] electrochemical techniques [26,27] have also aroused in-Stefanie Schlager joined the group of Linz Institute of Organic Solar Cells (LIOS) in 2010 and finished her studies in technical chemistry and chemical engineering at the Johannes Kepler University in Linz in 2011. The topic of her first diploma thesis was on organic Schottky diodes and was followed by her second diploma thesis and PhD at LIOS, where she focused on the application of semiconductor electrolyte interfaces for application in electrochemical CO 2 reduction as well as the biocatalytic and bioelectrocatalytic reduction of CO 2 .S ince the beginning of 2016, she has been ap ostdoctoral assistant at LIOS with her scientific work based on the further development of microbial electrosynthesis and electroenzymatic conversion processes.
Prof. N. S. Sariciftci studied at the University of Vienna, Austria, and earned his PhD degree in physics in 1989. After at wo-year postdoctoral stay at the University of Stuttgart, Germany,h ej oined the Institute for Polymers and Organic Solids at the University of California, Santa Barbara, CA, working in the group of Prof. Alan J. Heeger.S ince 1996 he has been the Ordinarius (chair) Professor for Physical Chemistry and the founding director of LIOS at the Johannes Kepler University in Linz. His major contributions are in the fields of photoinduced optical, magnetic resonance, and transport phenomena in semiconducting and metallic polymers and in organic and bioorganic semiconductors. In recent years, research on CO 2 utilization has attracted his increasing interest. Sariciftci is am ember of the Academy of Sciences in Austria (ÖAW)a nd has been awarded honorary doctorates and has received several prizes, among them the prestigious Wittgenstein Prize of Austria in 2012.  terest. All of these strategies,h owever, involve electron injection from an energy source or result from excitation from al ight source.E ither way,t he energy must be provided from perennial energy sources as discussed above,i ndirectly by drivinge lectrochemical processes or directly by irradiation. [28] Fort his purpose,b ioinspired materials,b ased on models from photosynthesis and other biological approaches,a nd biobasedm aterials have particularly gained high interest. Besides several approaches involving the use of organic and metal-organic compounds as catalysts in photochemical, photoelectrochemical, and electrochemical methods,t he direct application of biocatalysts such as enzymesa nd microorganisms is,a bove all, favoredf or utilization in CO 2 reduction. [29][30][31] Them ain advantages of biocatalysts, in comparison to synthetic catalysts,a re high selectivityt owards the products obtained and high yields. [32] Furthermore,a sn atural processes mainly proceed under ambient conditions,t he utilization of biocatalystse ases processesi nt erms of conditions and makest hem highly attractivef or possible large-scale applications. [33] Enzymes especially feature remarkablep otential for this purpose,a st hey are nonliving and, therefore,d o not require nutrients or have to be especially treated in contrast to microorganisms including algae.A lso,p rocessesi nvolving the use of living organisms underlie self-regeneration or replication,w hich is additionally dependent on environmental conditions such as nutrients,t emperature,a nd pH value.M oreover,m icrobial CO 2 conversioni sm ainly based on fermentation processes,a nd as such, those factors pose problems for scaling for biofuel production. Isolation of individual enzymes from living organisms would, therefore,b e an attractive alternative. [34,35] However, the properties of the enzyme as well as the desired reaction depend on the sourceo ft he enzymeorthe microorganism from which it was isolated, and therefore,t he microbial source has to be chosen accurately.F or the direct reduction of CO 2 ,d ehydrogenases have particularly gained high interest. Moreover, dehydrogenase enzymes are capable of converting CO 2 into alcohols directly under ambient conditionsand in aqueousenvironments. [36][37][38][39] In the following paragraphs,e nzymatic processes for CO 2 reduction to differentproducts and through various pathways will be discussed.

CO 2 reduction with co-enzymes
As this review focuses on reduction of CO 2 ,e mphasis will be put on dehydrogenase enzymes.
Dehydrogenase-catalyzed reactions can be performed either for reduction or for oxidation processes.N atural oxidation reactions preferably occur. [40,41] However, reactionk inetics can be influenced andr eaction equilibria can be shifted by providing the substrate to be converted in excess amounta nd further by addingt he corresponding redox equivalent of the co-factor, which is required for charge and proton transfer. [42] In this study,w em ainly focus on the reductive pathway of enzymatic reactions to convert CO 2 .
Fort he direct reduction of CO 2 there are two main possibilities involving the use of dehydrogenases,a ss hown in Scheme 1: first, CO 2 can be reduced to carbon monoxide (CO) by utilizing carbon monoxide dehydrogenase (Scheme1a). [37,43,44] Furthermore,c onversion of CO 2 into methanol is feasible by using athree-step enzymecascadeincluding formate dehydrogenase (FDH), formaldehyde dehydrogenase (F ald DH), and alcohol dehydrogenase (ADH) (Scheme1b). [36,45] Figure 2. Gibbs free energies of C 1 molecules. [13] Scheme 1. Reaction steps forthe enzyme-catalyzed reduction of a) CO 2 to CO with carbonm onoxidedehydrogenase and b) CO 2 to CH 3 OH through at hreestep cascade of dehydrogenases. Both kinds of reactionsr equire as acrificial co-factor that, in the case of reductions,s erves as the electrona nd proton donor and that is,t herefore,o xidized in the same step. Redox reactionsi nvolving the use of enzymes as catalysts occur through the formation of an intermediatec ompound, consisting of the enzyme, co-factor,a nd substratet ob er educed or oxidized. Within this intermediate state,c harge and proton transfer is performed between the co-factor and substrate over the (metal) active site of the enzyme. [46] Ther educed substratea nd oxidized co-factor, or vice versa, are then released again.
In the case of most reactions involving the use of carbon monoxide dehydrogenase,f erredoxin serves as the electron and proton donor. Differently for formate,f ormaldehyde, and alcohol dehydrogenases,n icotinamide adenine dinucleotide (NADH) is the corresponding co-enzyme.E ach of the three steps in the cascade represents at wo-electronr eduction and, therefore,needs one NADH molecule for each step to donate electronsa nd protons. Three molecules of NADH are therefore oxidized in the reduction of CO 2 to CH 3 OH. In natural processes,t he oxidized forms of the co-factors are regeneratedi ns ubsequent redox reactions to complete ar eversible cycle of reductions and oxidations.F or approaches performed in vitro,h owever, those co-factors are sacrificial and have to be delivered after the reaction or have to be regenerated in additional processes.
One of the first works involving the use of adehydrogenase enzymef or the conversiono fC O 2 was done by Rusching et al. They report CO 2 reduction to formatew ith NADH as the co-factor. In their work, they perform homogeneous catalysis by dissolvingt he enzymeins olutiona nd determine the formate generated by 14 Clabeling with 14 CO 2 . [47] In adifferent work, Schuchmanna nd co-workers deal with CO 2 hydrogenation by using ah ydrogen-dependent CO 2 reductase (HDCR) isolated from the acetogenic bacterium Acetobacterium woodii.I nt his hydrogenation reduction to formate,f ormate dehydrogenase plays ak ey role. [48] Consideringt he importance of the microbial sourceo ft he enzyme, Alissandratos has presented results on improved catalytic properties for CO 2 reduction by using af ormated ehydrogenase expressed from the Clostridium carboxidivorans strain P7 T . [49] However, an importantp ointf or the efficient and sustainable use of enzymes in experimental approaches is the fact of denaturation of enzymes,d ue to their delicate nature,i fu sed as homogenousc atalystsi ns olution.
One possibility to improve thermal stability is the application of thermophilic enzymes,a sd iscussed by Hondae tal. [50] Moreover, apart from thermal stabilization, it hasb een found that suitable immobilization of enzymes prevents their degradation and enables further reusabilityo ft he catalyst and their easier separation from products. [51] However, ac rucial thing here is to find appropriate materials that do not limit mass transport of substratet ot he catalyst active site and releaseo ft he product from the system. Heichal-Segal and co-workers first investigated an alginate-silicateh ybrid matrix for the immobilization of glucosidase.T hey report promising resultst hat show that the activity is not leached and chemical and thermal denaturation of the enzymes can be avoided. [52] This matrix has also turned out to be highly suitable for dehydrogenases.O bert and Dave show the application of alginate-silicate hybrid gel for the three-step dehydrogenase cascade (se Scheme 1) to reduce CO 2 to methanol. [36] Moreover the groups of Xu and Lu have investigated such hybrid gels for the conversion of CO 2 into formate by using formate dehydrogenase and have also extended the cascadet om ethanol generation. They describe an optimized constitution between alginate and silicate and reveal the importance of silicate for cross-linkingi nt he gel. As as ilicate source,t hey use tetramethoxysilane [Si(OCH 3 ) 4 ]. [45,53] This, however, may lead to methanol releasei fn ot sufficiently hydrolyzed in the liquid phase for the subsequent bead-formation procedure.D ifferently,A resta et al. uset etraethoxysilane [Si(OC 2 H 5 ) 4 ]insteadtoa void such interferences. [54] In ad ifferent study,L uo et al. show the sequential and coimmobilization of all three dehydrogenases for the reduction of CO 2 to methanol. In their work,t hey present immobilization by noncovalentb indingt of lat-sheet polymeric membranes. They report not only promisingr esults for both immobilizationt echniques but even better methanol yield from sequential immobilization. [55] All these results emphasize that immobilizationo fe nzymeso ffers the possibility for the efficient use of enzymes owing to enabled reusability, reproducibility,a nd improved stability.
Even thoughCO 2 reduction reactionswith dehydrogenases together with the correspondingc o-factors providep roducts in high yields and high selectivity, such reactions are limited to laboratory-scale applications owing to the high costs of the co-factor supply and regeneration.
Fore conomically favorable use and subsequent potential large-scale utilization, the framework conditions of the enzymes,i ng eneral, have to be considered.F or this,t he whole reaction mechanism, including parameters such as temperature,p ressure, pH, solvent, and co-factors,h as to be taken into account. Specifically,t he role of the co-factors is crucial and has ap articular impacto nc ost and efficiency of the enzymatic processes.A sd iscussed earlier in this study,c o-enzymes and co-factors are usually sacrificial for redox reactions and are therefore depleted. Nature has developed their recycling by using other enzymes that may regenerate the reduced forms.
Nevertheless,t oa void co-factor loss in technicali mplementations and to improve the efficiency of these redox processes,s everal approaches have been developed. Some of them focus on co-factor regeneration or substitution, which will be discussed in the following paragraphs.

Co-enzyme regeneration
One strategy to make enzymatic CO 2 reduction feasible and attractive for large-scale applicationi sc o-factor substitution or regeneration.D epending on the desired reaction,e ither the oxidized or the reduced form of the co-factor is required. Therefore,i nt he case of regenerations trategies,a dditional redox processes are established, for which the co-factor is reduced or oxidized to its initial state and then becomes reusable for the actualr edox reaction. Aksu et al. have published results showing the regenerationo fo xidized nicotinamide co-factors.T hey comparer esults for NADH and its phosphorylated derivativeN ADPH. They couple an alcohol dehydrogenasec atalyzed oxidation reaction to al accaseo xidation with simple O 2 that, moreover,p rovides the electron for reduction and, therefore,a llowsr egeneration of the cofactor. Forb othd erivatives,t hey find similar rates and turnover numbers over 300. [56] Zhang et al. show an approacht o substitute NAD(P)Hc o-factors with an artificialf luoro-containing co-factor based on as implified structure. [57] Also, Paul and co-workers report work on simplified synthetic cofactorsc omprising structures of NAD(P)H. [58] Work on cofactor regenerationh as been presented, for example,b yP almore and co-workers. Thet opic of their study is the regeneration of the oxidized form NAD + . [59] However, for the CO 2 reduction route,t he reducedf orm of the co-factor,n amely,N ADH,i sn ecessary.T he group of Cazelles has investigateds uch reactionsf or the opposite redox pathway. In their work, they screen three different possibilities for regenerating NADH from NAD + ,w hich is obtained from the dehydrogenase-catalyzed reduction of CO 2 to methanol. Their approaches comprise two techniques involving the useo fp hosphite dehydrogenase and glycerol dehydrogenase as enzymes and one photocatalytic approach with the useofachloroplast-containing photosystem. [60] Theg roup of Omanovic et al. has presented as tudy on converting NAD + into NADH by using an electrochemical approachw ith ar uthenium-modified glassy carbone lectrode. [61] Furthermore,t hey modifyag lassy carbon electrode with Pt and Ni for the same purpose ( Figure 3). [62] Radical formationfrom NAD + ,however, is critical, as this could give rise to the formationo fd imers. Desired reduction reactions might not be performed quantitatively or might be hindered. Therefore,f ormation of dimersh as to be prevented and either solution or electrodes urface reactions may play ak ey role in preventing the coupling of radicals to afford dimers; this addresses the reaction towards the reduction of NAD + to the active NADH isomer. Very recent results of this group show the utilization of barea nd Ir-Ru oxide modified Ti electrodes for the electrochemical regeneration of NADH for over 80 %r ecovery. [63,64] Another electrochemical approach has been presented by Minteer and co-workers.T hey use dehydrogenase enzymes coupled to an electrode for the reduction of CO 2 and subsequent regenerationo fN ADH. They report improved efficiencies if another enzyme, carbonic anhydrase,isa dded. [65] It is not only metals such as Ti,R u, Ni, andP tt hat have turnedo ut to serve as suitable catalysts to regenerate NADH. Moreover, besides the electrochemical approach, photochemical techniques have also evolved. Theg roups of Dibenedetto and Aresta show the regeneration of NADH by using sodium dithionite for chemical conversion and zinc sulfide (ZnS) derivatives as ab lank material or modified with Ru for photochemical applications. [54] In other work,t hey show the photochemical regeneration of NADH by using aR h-bipyridinec atalyst. In both approaches,t hey investigate NADH regeneration coupled to CO 2 reduction to methanol by using encapsulated enzymes. [66] Also,O ppelt et al. focus on the utilization of Rh-based complexes for the regenerationo fN ADH and NADH derivatives.T hey regenerate BNADH, as implified NADH derivative,with the aid of atin porphyrin complex, ethylenediaminetetraacetic acid (EDTA) or triethanolamin (TEOA),a nd aR hc omplex. Moreover, they show the immobilization of aR h-complex-based polymer on glass beads for the photoregeneration of NADH (Figure 4). [67,68]

Enzymatic Electrocatalysis for CO 2 Reduction
Co-factorr egeneration represents one strategy towards reducingt he costs of enzymatic processes.C o-factor recovery is indeed af easible and promising approach to make enzyme-catalyzed reactions less expensive and more efficient. However, an even more practical method would be the substitution of co-factors as electron and proton suppliers. Ad ifferent idea, gaining particular interest, is waiving the use of co-enzymesa nd co-factors.A ss uch substances are responsible for the donation of electronsa nd protonsi nt he case of reduction reactions,c o-factor substitution can only becomea ffordable if techniques that take over the task of providing charges are found. Fort his purpose,p hoto-and electrochemical strategies have primarily been found to be  suitable.M oreover, to developt his idea further, renewable energies could play an important role as energy sources for such methods.A tb est, the combination of biocatalyzed CO 2 reduction to af uel, driven by ar enewable energys ource, could provide as ustainable technique for energys torage. In some work, photochemical investigationsh ave been made by utilizing light sources to deliver charges. [30] In 1984, Parkinsona nd Weaver presented results on photoelectrochemical approaches with the use of ap -type potentiostatically driven InP photocathodei natwo-compartment cell withoutt he requiremento fa ny co-factor.B yu sing incident light, they report the photogeneration of methylviologen (MV + ), which can serve as ar eduction equivalent. CO 2 is reduced to formic acid with FDH as the catalyst, whereas MV + is oxidized but subsequently regenerated at the semiconductore lectrode. [69] Mandler and Willner describe the photoreductiono fC O 2 to formate by using visible light and Pd colloids as the catalyst. [70] Also, Woolertonetal. report on the photoreduction of CO 2 .T hey use enzyme-modified metal nanoparticles to catalyze the reduction of CO 2 .Anadditional photosensitizer is oxidized by transferring the electronsf or the reduction,a nd it is then regenerated in anextrastep. [71][72][73] Baran et al. have recently published worko np hotocatalytic CO 2 reduction by using as ynthesized p-type CuI semiconductor. They attribute the favorable photoreduction of CO 2 to the fact that the conduction band edges are lower than those of n-type semiconductors. [74] However, whereas photoelectrochemical and photochemical methods require in situ incident light, electrochemical techniquesw ould be flexible on the sourceo fe nergy (e.g., sun or wind).
Electroenzymatic processes,w ithouta ny sacrificial electron donors or mediators,e ntail direct electron injection into the enzymes.T he challenge is to introduce charges to the active site of the enzymei namanner similar to that in photochemical approaches.T herefore,t he naturally occurring intermediate state with the co-factor, which is required for charge transfer, would be mimicked. Scheme 2d emonstrates the possible reduction route with direct electron injection from an electrodei nstead of co-factors as the electron and proton donors.
Consequently,i ne lectroenzymaticp rocessese lectrons are providedd irectly from an electrode and from an external energy source and protonsh ave to be delivered from aqueous electrolyte solutions.
In 1993, Pantanoa nd Kuhr presented their work on dehydrogenase-modified carbon-fiber microelectrodes.T hey use these electrodes to electrochemically monitor the formation of NADH, which serves as an electron mediator. [75] Similarly,S rikanthe tal. present an approachf or electrochemical CO 2 reduction to formatec atalyzedb yf ormate dehydrogenasei mmobilized on an electrode.B esides CO 2 reduction,N ADH is regenerated in the electrochemical system at the same time ( Figure 5). [76] Also,t he group of Yoneyama et al. have realized the potential in using enzymes as electrocatalysts.T he focus of their work is on the electrochemical fixation and conversion of CO 2 .E specially,i nt heir later work they show,a so ne of the first groups,t he electrochemical addressing of dehydrogenasee nzymes withoutt he requiremento fa ny co-factor. Scheme 2. Reaction steps fordirect electron injection in the electroenzymatic reduction of a) CO 2 to CO with carbonm onoxide dehydrogenase and b) CO 2 to CH 3 OH throughathree-step cascade of dehydrogenases withouta ny co-factors. They present the reduction of CO 2 to methanol with methylviologen asa nelectron shuttle. [77,78] Methylviologen also serves as as upportingm ediatorf or electron transfer in the work of Shin et al. They investigate the electrochemical application of carbonm onoxide dehydrogenase.A sahighly interesting result, the electroenzymatic reduction of CO 2 to CO is performed at low overpotentials at 0.57 Vv ersus the normal hydrogen electrode (NHE). [79] Wang and co-workers also focus on investigating carbon monoxide dehydrogenase.I nt heir study,t hey screen two different carbon monoxide dehydrogenases by using proteinfilm electrochemistry.M easurements arep erformed in the presenceo fC O 2 ,a nd the influence of the differenti nhibitors is shown by comparing both dehydrogenases. [80] Furthermore, calculations on the potential of such electroenzymatic approaches with carbon monoxide dehydrogenase and the role of the metals in the reduction of CO 2 to CO have been published by Hansen et al. [81] Referring to methylviologen as an electron shuttle for electroenzymatic reactions, Amao and Shuto describe as imilar approach. Differently,t hey couple formate dehydrogenase directly to methylviologen with al ong alkyl chain,w hich is then linked to an indium tin oxide (ITO) electrode for an artificialp hotosynthesis approach, also comprising CO 2 reduction to formate (Figure6). [82] Direct electron transfer without any shuttle has been studied by Razumiene et al. Fort heir experiments,t hey use pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase immobilized on carbone lectrodes.A ni ncrease in the oxidative current is only observed if ethanol is added to the electrochemical system,w hichp roves direct electron injection and electroenzymatic oxidation without any co-factor or shuttle. [83] Periasamy and co-workers have also investigated ethanol oxidationb yu sing alcohol dehydrogenase in an electrochemical system. In their approach, alcohol dehydrogenase is immobilized on glassy carbon, and the electrodei sf urther coated with toluidine blue and Nafion to prevent leaching of the ADH. [84] An electrochemical approach to address multienzymec ascades insteado fs ingle enzymes has been presented, for example,b yt he group of Minteer et al. They show direct electron transfer from enzymesf or biofuel cells.F urthermore, they also show direct electron injection into dehydrogenase enzymes (i.e., FDH,F ald DH, and ADH) for methanol production coupled to NADH regeneration,a sp reviously discussed. [65,85] Important work toward direct electron injection into enzymesf or the heterogeneous,e lectroenzymatic reduction of CO 2 without the requirement of any co-factors or mediators was done by Reda et al. They demonstrate adsorption of tungsten-containing formated ehydrogenase on glassy carbon.U sing this enzyme electrode, they describeC O 2 reduction to formatea tr eduction potentials below À0.8 V versus Ag/AgClt oy ield faradaic efficiencies of 97 %a nd higher. Furthermore,t hey suggest an electron-transfer mechanism among the electrode,t he enzyme,a nd CO 2 for the subsequent reduction reaction. [86] Similarly,B assegoda et al. use formate dehydrogenase with am olybdenum active site for the reversible conversion of formate and CO 2 .T hey suggest that the molybdenum-containing FDH is even more electrochemically active than at ungsten-containing FDH (Figure 7). [87] Following the idea of catalytice lectrodes for heterogeneous electrochemical CO 2 reduction, Schlagere tal. have recently described the immobilization of dehydrogenases encapsulated in an alginate-based matrix on ac arbon felt electrode ( Figure 8).
Besides the immobilization of alcohol dehydrogenase alone for the conversion of an aldehyde into the corresponding alcohol, co-immobilization of all three dehydrogenase enzymesfor the reduction of CO 2 to methanol has also been investigated ( Figure 9). Both approaches deliver promisingr esults with faradaic efficiencies of 10 %f or the conversion of CO 2 into methanol and evenh igher faradaic yields of up to 40 %f or the single immobilized enzyme.M oreover, all experiments are performed without the addition of any cofactor or electron mediator. [88,89]   Specifically,a pproaches requiring no additional mediators or electron shuttles are of high interest. Reduced costs owing to such simplified processesc ombined with heterogeneous electrocatalysis to enable electrode reusability make such processes attractive for large-scale applications.S tudies on optimizing electroenzymatic processes for application in renewable energys torage and CO 2 reduction are enormously evolving and pave the way towards sustainable fuel generation. Moreover, the feasibility of immobilizing enzymes on electrodes and subsequent directe lectron injection offers the possibility for applications other than fuel generation, such as in the food and pharmaceutical industries.

Summary and Outlook
In this work, an approacht owards CO 2 utilization was presented. CO 2 represents ac arbon source and carbon feedstock that can be reduced to valuable chemicals and fuels.I nt he last decades,m uch work has been presented that discusses the feasibility of recycling CO 2 and therefore substituting fossil carbons ources such as oil, gas, coal, and biomass.I n particular, approaches mimicking natural processes have drawn attention, and techniques for the photocatalytic and electrocatalytic conversion of CO 2 have been developed.
However, most synthetic catalysts such as organic, metallic, and organometallic compounds that have been used to enable the reduction of CO 2 to energy-rich chemicals often do not yield high efficiencies or provide high selectivity to the desired products.
Therefore,t he use of not only bioinspired but even biobased catalysts such as enzymes has especially gained interest in science. Biocatalysts and enzymes are known from natural CO 2 reduction processes,f or which they yield high efficiencies and selectivityu nder mild reaction conditions (e.g.,a mbient temperature and pressure, aqueous media at neutral pH). Nevertheless,s uch processes require electron and protond onors or co-factors.I nn ature,s uch substancesa re regenerated in coupled reactions and, therefore,c losed cycles. Fort he purpose of CO 2 reduction in laboratory or industrialapproaches,however, such co-factors and equivalents are sacrificial and are,t herefore,n ot practical because of their high costs.
Herein, we have given an overview of studies in which enzymesw ere used as catalysts for CO 2 reduction.W es howed different strategiesf ocusing on co-factor regenerationo nt he one hand and substitution of such co-factors on the other hand. Differentc atalysts to recover the co-factors,s uch as nicotinamide adenined inucleotide, as well as photochemical and electrochemical approaches for direct electron injection into enzymes without the requirement of any co-factors were shown. Such strategies make enzymes appealing for the purpose of CO 2 conversion, as processes can be eased and costs can be reduced remarkably.W ed isplayedr ecent results in this increasinglye volving field, representing approaches with high potential towardC O 2 utilization and renewable energy storage at the same time and making enzymatic processes attractive for large-scale applications. Figure 8. Preparation procedure for the immobilization of enzymes on ac arbon felt electrode. a) Encapsulation of dehydrogenases in alginate-silicate hybrid gel. b) Resulting enzyme containingg el bead after precipitation. c) Blank carbon felt electrode that is soaked withthe alginate-silicate mixture and precipitated in CaCl 2 .d )Alginate-covered carbon felt electrodea fter precipitation. Figure 9. Scheme for the setup of an electrochemical cell for electroenzymatic CO 2 reduction. Electrons are injected directly into the enzymes, which are encapsulated in alginate-silicate hybrid gel (green) and immobilized on ac arbon felt working electrode.C O 2 is reduced to methanol at the working electrode. Oxidation reactions take place at the counter electrode. [89] Energy Technol.