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REVIEW

Metabolic engineering for enhanced hydrogen production: a review Yogesh Goyal, Manish Kumar, and Kalyan Gayen

Abstract: Hydrogen gas exhibits potential as a sustainable fuel for the future. Therefore, many attempts have been made withthe aim of producing high yields of hydrogen gas through renewable biological routes. Engineering of strains to enhance theproduction of hydrogen gas has been an active area of research for the past 2 decades. This includes overexpression of hydrogen-producinggenes (native and heterologous), knockout of competitive pathways, creation of a new productive pathway,and creation of dual systems. Interestingly, genetic mutations in 2 different strains of the same species may not yield similarresults. Similarly, 2 different studies on hydrogen productivities may differ largely for the same mutation and on the samespecies. Consequently, here we analyzed the effect of various genetic modications on several species, considering a wide rangeof published data on hydrogen biosynthesis. This article includes a comprehensive metabolic engineering analysis of hydrogen-producing organisms, namely Escherichia coli, Clostridium , and Enterobacter species, and in addition, a short discussion on ther-mophilicand halophilic organisms. Also, apartfrom single-culture utilization, dual systems of various organisms and associateddevelopments have been discussed, which are considered potential future targets for economical hydrogen production. Addi-tionally, an indirect contribution towards hydrogen production has been reviewed for associated species.

Key words: hydrogen, metabolic engineering, Escherichia coli, Clostridium , Enterobacter , dual systems.

Rsum : Lhydrogne est un gaz qui prsente un bon potentiel pour devenir un combustible durable et de ce fait, plusieurstentatives ont t faites en vue dobtenir une production leve dhydrogne par des voies biologiques renouvelables. Lingnierie desouches an daugmenter la production dhydrogne a constitu un domaine de recherche actif au cours des 2 dernires dcennies.Ceci comprendla surexpression de gnes impliqus dans la production dhydrogne (natifs et htrologues), linactivation de sentierscomptitifs, la cration de nouveaux sentiers de production et les systmes mixtes. Fait intressant, des mutations gntiquesintroduites dans 2 souches diffrentes de la mme espce peuvent ne pas conduire a ` des rsultats similaires. De la mme faon, 2tudes de production dhydrogne diffrentes impliquant la mme mutation sur la mme espce peuvent diffrer sensiblement. Enconsquence, nous avons analys ici leffet de diffrentes modications gntiques sur diffrentes espces en prenant en considra-tion un vaste spectre de donnes publies sur la biosynthse de lhydrogne. Cet article comprend une analyse exhaustive delingnierie mtabolique dorganismes qui produisent de lhydrogne, notamment Escherichia coli, Clostridium et Enterobacter , et unecourte discussion sur les organismes thermophiles et halophiles. Aussi, en plus de lutilisation de cultures uniques, les systmesmixtesde diffrents organismes et lesdveloppements associsont tdiscuts. Ceux-cisont considrs comme descibles potentiellesfutures pour la production conomique dhydrogne. En outre, une contribution indirecte a ` la production dhydrogne par des

espces associes a t rsume. [Traduit par la Rdaction] Mots-cls : hydrogne, ingnierie mtabolique, Escherichia coli, Clostridium , Enterobacter , systmes mixte.

IntroductionHydrogen has gained attention throughout the world because

of its eco-friendly nature and highly efcient energy yield (Li et al.2010). Focus has been laid on developing techniques that couldreduce dependence on limited fossil fuel resources. At present,most of the hydrogen gas is being produced via steam reforming,coal gasication, and water electrolysis; however, steam reform-ingis thedominantoption (Das andVeziroglu 2001 ). Inadditiontothese techniques, biological processes, which are operated atroom temperatures and low pressures, are not only environmentfriendly and but also can be potentially used for recycling pur-poses by using up waste materials. However, these processes re-quire more research efforts to being economical ( Benemann 1997 ;Das and Veziroglu 2001 ; Kumar and Gayen 2012 ; Sasikala et al.1993). In this direction, processes like biophotolysis, photofer-mentation, and dark fermentation are some of the available re-newable methods, requiring no external energy source (Kapdanand Kargi 2006 ; Lalaurette et al. 2009 ; Levin et al. 2004 ; Manishand Banerjee 2008 ; Meher and Das 2008 ). Of these processes, it is

reported that production throughdark fermentationhas themax-imum yield and has a low energy requirement ( Hallenbeck andGhosh 2010 ; Lalaurette et al. 2009 ; Li et al. 2010 ). Production of hydrogen viafermentation on a large scale is betterin comparison with photosynthetic processes, as the latter incorporates 2 key problems, namely, impurity of oxygen in hydrogen and reexplosion-related issues ( Das and Veziroglu 2001 ; Maeda et al.2007 b; Yoshida et al. 2006 b). Also, high evolution rates for hy-drogen combined with constant throughput from organic sub-strates and versatility of the substrates makes it advantageous formass production (Brosseau andZajic1982 ; Dasand Veziroglu2001 ;

Pan et al. 2008 ; Tanisho et al. 1987 ). Apart from being used as afuel, hydrogen nds numerous other applications in processeslike hydrogenation, removal of trace oxygen, in glass and elec-tronics industries, and as a coolant in generators ( Das and Veziro-glu 2001 ; Ramachandran and Menon 1998 ; Veziroglu 1995 ).

Despite the relative easiness in dealing with engineering andother peripheral issues for select organisms, the hydrogen yieldis on a lower side and far from being economical production

Received 9 August 2012. Revision received 9 November 2012. Accepted 19 November 2012.

Y. Goyal and M. Kumar. Department of Chemical Engineering, Indian Institute of Technology, Gandhinagar, VGEC Complex, Chandkheda, Ahmedabad 382424 (Gujarat), India.K. Gayen. Department of Chemical Engineering, National Institute of Technology Agartala, Barjala, Jirania, West Tripura-799055, Tripura, India.Corresponding author: Kalyan Gayen (e-mail: [email protected] ).

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Can. J. Microbiol. 59 : 5978 (2013) dx.doi.org/10.1139/cjm-2012-0494 Published at www.nrcresearchpress.com/cjm on 26 November 2012.
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(Hallenbeck et al. 2009 ; Hallenbeck and Ghosh 2009 ). Theoret-ically, microorganisms like Escherichia coli and Enterobacter spe-cies can produce a maximum of only 2 mol of hydrogen gas, andobligate anaerobes like various Clostridium species can produce4 mol of hydrogen gas per 1 mol of glucose as substrate (underlow partial pressures) (Clark 1989 ; Hallenbeck and Ghosh 2010 ).Numerous approaches have been used to explore the possibil-ities of improving the hydrogen gas productivities ( Akhtar and

Jones 2008 b; Henstra et al. 2007 ). Unfortunately, genetic engi-neering technologies have shownlimited success for improvingmicrobial yields ( Akhtar and Jones 2008 b; Allers and Mevarech2005 ). The several studies being carried out, including knock-out of genes that encode uptake hydrogenases, competitiveenzymes, and negative regulatory elements, at the most provide yields that are closer to the theoretical maximum, but they are still very low ( Akhtar and Jones 2009 ; Maeda et al. 2008 ; Yoshida et al. 2006 b). Thus, some new pathways need to beengineered that are able to increase the yield and are alsoeconomical such that they are able to use low-cost substratesfor hydrogen production ( Akhtar and Jones 2009 ). There is apossibility of improving hydrogen yield up to 10 mol/mol glu-cose in aerobic and (or) anaerobic bacteria having hydrogenase(analysis done using theoretical explanations) ( Nath and Das

2004 ; Tanisho 2000 ; Tanisho et al. 1998 ). Also, though co-expression of genes and their deletion has resulted in an in-crease of hydrogen production, many times these operationsfail to improve product yield (Colletti et al. 2011 ).

For both facultative (partial oxygen environment) as well asobligate anaerobes, the pathway from glucose (as substrate) topyruvate remains the same. Pyruvate catabolism in both types of bacteria brings about the distinction in hydrogen-producing pathways. Whereas in obligate anaerobes pyruvate gets degraded toacetyl-coenzyme A (CoA), reduced ferredoxin, and carbon dioxidegas, in facultative anaerobes, pyruvate gets converted to formateand acetyl-CoA ( Hallenbeck 2009 ; Hallenbeck and Ghosh 2010 ).Creating a database of the availablehydrogen-producing strains isof utmost importance, as there have been several examples wherethe same bacterial communities result in different hydrogen pro-

duction rates and yield ( Hung et al. 2011 a or b). This review focuses on the application of metabolic and geneticengineering strategies to maximize the hydrogen yield for variouspromising microorganisms. Escherichia coli is the organism mostoften subjected to genetic engineering for the purpose of hy-drogen production precisely because of its comprehensive char-acterization. Hence, we have quantitatively analyzed publisheddata concerning genetically engineered E. coli for hydrogen pro-duction. Since, in the widely present species of Clostridium , hy-drogen production capabilities vary signicantly from strain tostrain, the effect of strain selection has been tabulated, whichshall help with making a better selection of strains. The effect of genetic engineering from the limited set of available publisheddata has also been analyzed for clostridia. In addition, a few En-terobacter species ( Enterobacter aerogenes and Enterobacter cloacae )have shown an innate ability to produce high hydrogen yields.Consequently, the analysis of the same, which includes the geneticengineering work done and various high hydrogen-producingstrains identied for these bacterial species, has been presented inthis review. Moreover, since many of these organisms have beenco-fermented with each other as well as with photo fermentativeorganisms to produce hydrogen, dual systems are discussed. Apart from the direct hydrogen-producing species, many spe-cies assist indirectly in hydrogen production, and their roleshave been identied.

Hydrogen production via dark fermentationDark fermentation is widely used to degrade organic substrates

to biohydrogen. However, hydrogen production via fermentative

metabolic pathways needs to be fully understood before any analysis or developments can be put into effect. Many facultative andobligateanaerobic bacteria are shownto produce hydrogen, whilesome aerobic species also produce hydrogen as a metabolic prod-uct ( Hallenbeck 2009 ). Biochemical reactions involved in the fer-mentation of proteins lead to formation of amino acids, whilelipids are hydrolyzed to glycerol and fatty acids instead of produc-ing hydrogen. This essentially leaves materials rich in carbohy-

drates for hydrogen production ( Hallenbeck 2009 ). In this review,to avoid overcrowding of the pathway map, focus has been main-tained on the hydrogen production using glucose as a substrate(Figs. 1 and 2). Hydrogen is produced differently in facultative (e.g., E. coli) and obligate anaerobes (e.g., various Clostridium species).However, the hydrogen-producing metabolic pathway is the sameup to the point of pyruvate production from glucose as the sub-strate ( Figs. 1 and 2). This process is called glycolysis and is accom-panied by ATP production and NAD + reduction ( Hallenbeck andGhosh 2010 ). Due to its commonality to both the pathways, pyruvate is thus considered a vital precursor for hydrogen production.

Figure 1 shows the breakdown of pyruvate into acetyl-CoA andformate, a characteristic pathway for facultative anaerobic bacte-ria. The responsible metabolic enzyme is pyruvate formate lyase(PFL). In addition to formate and acetyl-CoA formation, lactic acid

formation is also observed ( Fig. 1). Acetate and ethanol are formedfrom acetyl-CoA accompanied by synthesis of ATP and oxidationof NADH, respectively ( Hallenbeck 2009 ). Formate further de-grades to produce hydrogen gas from the formate hydrogen lyase(FHL) complex having NiFe hydrogenase thereby reducing theacidity of the medium. Here, hydrogen is derived only from for-mate and 2 mol formate/mol glucose is formed, maximum hy-drogen yield possible via this pathway is 2 mol/mol glucose.

Figure 2 shows hydrogen production from glucose via the pyruvate ferredoxin oxidoreductase (PFOR) pathway in obligate anaer-obes (like Clostridium species). Pyruvate is converted to acetyl-CoA and carbon dioxide. This step also leads to the formation of re-duced ferredoxin, which in turn fuels hydrogen production usingFeFe hydrogenase ( Hallenbeck and Ghosh 2010 ). In addition, re-duced ferredoxin can oxidize the NADH produced after reductionin theglycolytic pathway to produce hydrogen gas. However, sucha reaction is found to occur only under low partial pressures of hydrogen, and the corresponding pathway is not fully known(Hallenbeck 2009 ; Veit et al. 2008 ). Owing to this limitation, oxi-dation instead happens for other organic metabolite formation,like butyric acid, butanol, and acetone, in obligate anaerobes(Hallenbeck 2009 ). Now, 2 mol of hydrogen are produced via trans-fer of electrons from the reduced ferredoxin, while another 2 molcan potentially be produced from NADH, as 2 mol of NADH is pro-duced during glycolysis. This gives a maximum yield of 4 mol/molglucose for hydrogen. With such low theoretical yields, genetic engi-neering avenues need to be explored to improve the yield, whichindeed denes the feasibility of the production at a large scale.

Genetic engineering avenuesGenetic engineering provides a basis to increase the low yield

through increased ux for hydrogen production. There are nu-merous avenues to improve the metabolic yield of hydrogen viagenetic engineering. Examples include knockout of a competitivegene, overexpression of a homologous or heterologous gene, cre-ation of synthetic pathways, co-culturing, and identication of indirect hydrogen-producing organisms whose population dy-namics can be regulated ( Akhtar and Jones 2009 ; Colletti et al.2011; Hallenbeck and Ghosh 2010 ; Waks and Silver 2009 ). How-ever, it is important to note that the effect of genetic modicationfor an enzyme activity will be fruitful only when the quantity of that particular enzyme will be limiting (Hallenbeck and Ghosh2010). In addition, genetic engineering is not always fruitful, as

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undesirable effects are often encountered in the genetically mod-ied organisms ( Colletti et al. 2011 ).

Logically, elimination of competing pathways that produceother metabolites should result in an increased ux towards hy-drogen production. Figure 1 shows the various by-products thatare formed during dark fermentation as well as the genes encod-ing the enzymes responsible for their production. In this direc-tion, deletion of lactate dehydrogenase ( ldh) could be one option,as this would help drain pyruvate ux towards hydrogen produc-tion. However, deletion of the ldh gene is supposed to produce thedesired effect only when the medium is acidic, since acidic me-dium stimulates the formation of lactate ( Hallenbeck and Ghosh

2010). Thepresence of fumarate reductase( frdBC), a gene encodingenzymes for the bioconversion of phosphoenolpyruvate (PEP) tosuccinate with an intermediate product, oxaloacetate, reducespyruvate production and, in turn, hydrogen production ( Figs. 1and 2). In addition, NADH is consumed in butyrate formationfrom acetyl-CoA. Studies have indicated the presence of 6 genes(thl (thiolase), crt (crotonase), hbd ( -hydroxybutyryl-CoA dehy-drogenase), bcd (butyryl-CoA dehydrogenase), etf (electron trans-fer protein (ETF- )), and etf (ETF- )) encoding this reactionsenzymatic activity (Cai et al. 2011 ). Knockout of these genes may result in increased availability of NADH and, consequently, morehydrogen production, provided the reactions are made thermody-namically feasible. However, investigation of this manipulationthrough experiments still is a great opportunity.

Hydrogenases arethe keycontrolling enzymes in hydrogen pro-duction. Thus, another strategy could be to eliminate the uptakeactivity of the hydrogenases present, which are responsible forthe consumption of hydrogen produced via oxidation. For in-stance, Hyd-1 and Hyd-2 are shown to possess hydrogen uptakeactivity, which may result in decreased hydrogen yield. This, infact, has been carried out in practice in a lot of situations withsuccess ( Fan et al. 2009 ; Hallenbeck 2009 ; Redwood et al. 2008 ). Inaddition, Hyd-3 overexpression may lead to increased hydrogenproduction rates (and yield, too, in cases where overexpressioninhibits the other metabolite production), as it has been found topossess hydrogen-producing activity ( Hallenbeck and Benemann

2002 ; Rossmann et al. 1991 ). However, in one study, Hyd-3 hasshown some uptake activity and thus its role remains uncertain(Maeda et al. 2007 c ). As can be seen from Fig. 1, fhl encodes theproduction of hydrogen gas from formate, and thus, increasedexpression of these genes could improve yield. In addition, otherhydrogenases like Hyd-4 and many others, which have differentfunctions, are present. Identication and quantication of thesehydrogenases could be one vital step towards increased hydrogenproduction.

Apart from working on native pathways, research has beenextended on cloning of hydrogen-producing genes from varioushydrogen-producing organisms into host species (King et al.2006 ). These genes may include FeFe, NiFe hydrogenase from various strains, which are hydrogen producing. Also, synthetic

Fig. 1. Metabolic pathway for hydrogen production via pyruvate formate lyase (PFL) pathway (for facultative anaerobes). Here glucose is broken down to pyruvate through the glycolytic (EmbdenMeyerhofParnas) pathway. The desired pathway for hydrogen production isindicated by bold arrows ( ), while those pathways leading to other metabolites and gases are indicated by broken arrows ( ). Also, signiesmembrane. The nomenclature is as follows: PEP, phosphoenolpyruvate; Hyd-1, hydrogenase-1; Hyd-2, hydrogenase-2; fhl, formate hydrogenlyase; fdnG, -formate dehydrogenase-N; fdoG, -formate dehydrogenase-O; FocA, FocB, formate transporters; pta , phosphotransacetylase; ack ,acetate kinase; aceE, pyruvate dehydrogenase; poxB, pyruvate oxidase; NarL and Fnr, global transcription regulators; ldh, lactate dehy-drogenase; frdBC, fumarate reductase; Acetyl-CoA, acetyl coenzyme A; SleC, selenium metabolism.

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pathways can be constructed by expression of genes encodingPFOR and PFL enzymes ( Hallenbeck and Ghosh 2010 ). To add tothat, methods to drive ferredoxin reduction (which otherwise is athermodynamic limited process) could be explored for enhancedhydrogen yields and production rates (Hallenbeck and Benemann

2002 ). However, it is not always a feasible task, considering the bacterial cell viability issues, the absence of maturation factors,and the low level of synchronization in the native and syntheticpathway created on encoding these foreign genes ( Colletti et al.2011; Hallenbeck and Ghosh 2010 ). Usage of an articial operon isonesuch wayto eliminate some of theproblemsmentioned above( Akhtar and Jones 2008 b). Increasing cellular NADH for hydrogenproduction at reduced pressures is another option ( Hallenbeck and Ghosh 2010 ).

There has been identication of key transport proteins respon-sible for varying hydrogen yield via metabolic pathways. A trans-port protein may cause the ow of enzymes out of the cellresponsible for hydrogen production and (or) consumption ( Berkset al. 2003 ). Thus, identication of their quantitative effect com-prehensivelywillassistto improve theyield. We shall discuss latertheir effects on the hydrogen gas production and the develop-ments being done in this regard. In addition, recently, geneticengineering based on global transcription regulators has beenfound to be effective for the overall hydrogen yield (Fan et al.2009 ). Apart from these, dual systems of microorganisms (e.g., co-culturing of thedifferent species) and processes (e.g., combination of dark fermentation and photofermentation) have also improved theproductivity of hydrogen, often producing synergistic effects.

The bacterial species E. coli, Clostridia , and E. aerogenes/ E. cloacaeareknown for hydrogen production. Beforediscussing the studieson genetic modications of species, it is necessary to be ac-quainted with the benecial characteristics of individual species. They are listed as follows:

Escherichia coli: To date, it is the best characterized bacterium(Maeda et al. 2007 a). Numerous studies have been conducted onthe engineering of E. coli for improving the yield of hydrogen(Penfold et al. 2006 ). However, specicity of substrate from theindigenous pathway is found to be very low. In addition, the

heterologous expression is a daunting task owing to the pres-ence of multiple membrane-bound proteins and a unique acidat its active site (Sawers 2005 ; Vignais and Colbeau 2004 ; Waksand Silver 2009 ). Nevertheless, this is overshadowed by thehuge amount of research on this bacterium that has helped toincrease yield immensely. Also, these strategies adopted for E. coli shall serve as benchmarks for research carried on otherspecies that are not fully characterized.

Clostridium : Clostridium is a diverse microbial genus and is widely found in nature. Clostridia have the capability to pro-duce hydrogen gas as a fermentation product by removing theexcess reducing powers during growth on biomass materials(Kaji et al. 1999 ; Kumar and Gayen 2011 ; Morimoto et al. 2005 ).

Enterobacter aerogenes / E. cloacae: Enterobacter typically ourisheson basic media under both oxygen containing and anoxic con-ditions ( Kumar et al. 2001 ). Enterobacter aerogenes is a fast grow-ing, high hydrogen-producing microorganism t for industrialhydrogen production and has uninhibited growth in total hy-drogen atmosphere ( Ito et al. 2004 ; Lu et al. 2010 ; Nakashimadaet al. 2002 ; Yokoi et al. 1998 ; Zhao et al. 2009 ).

Hydrogen-producing strains

Escherichia coli

Native hydrogenase directly responsible for hydrogen productionHydrogenase/dehydrogenase enzymes form an integral part

of the hydrogen production mechanism in E. coli. Escherichia colican express the hydrogenases Hyd-1, Hyd-2, Hyd-3, and Hyd-4,

Fig. 2. Metabolic pathway for hydrogen production via the pyruvate ferredoxin oxidoreductase (PFOR) pathway (for strict anaerobes). Glucoseis broken down to pyruvate through the glycolytic (EmbdenMeyerhofParnas) pathway. The desired pathway for hydrogen production isindicated by bold arrows ( ), while the pathways leading to other metabolites and gases are indicated by broken arrows ( ). Thenomenclature is as follows: PEP, phosphoenolpyruvate; pta , phosphotransacetylase; ack , acetate kinase; aceE, pyruvate dehydrogenase; poxB,pyruvate oxidase; ldh, lactate dehydrogenase; frdBC, fumarate reductase; Acetyl-CoA, acetyl coenzyme A.



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of which Hyd-4 has not yet been characterized ( Redwood et al.2008 ). Hyd-1 is an uptake hydrogenase encoded by the hyaoperon and is expressed to recycle the fermentative hydrogenproduced from formate ( Redwood et al. 2008 ; Sawers et al.1985). Redwood et al. (2008 ) found that strains devoid of Hyd-2showed increased production rates while strains without Hyd-1showed no signicant change in hydrogen production. Conse-quently, Hyd-2 hydrogenase knockout resulted in a high yield

in the experiments carried out by Fan et al. (2009 ). However,compared with the single mutant, the double mutant failed toimprove on the hydrogen yield ( Table 1 ) (Fan et al. 2009 ;Redwood et al. 2008 ). The FHL complex (formed by Hyd-3 andencoded by hyc operon) is responsible for hydrogen productionfrom formate ( Rossmann et al. 1991 ). Also, Maeda et al. (2007 c )later showed that Hyd-3 possesses hydrogen uptake activity . Inaddition, deletion of Hyd-3 resulted in no hydrogen gas produc-tion ( Table 1 a ) (Redwood et al. 2008 ). Further, contrary to earlierresults and previously held belief, hycA deletion did not affectthe hydrogen production ( Penfold et al. 2003 ; Penfold andMacaskie 2004 ; Redwood et al. 2008 ; Yoshida et al. 2006 b).

In E. coli, formate is the direct metabolic precursor for hydrogenproduction, which eventually cleaves into hydrogen and CO 2 by the FHL complex (Sawers 2005 ; Waks and Silver 2009 ). Penfold

et al. (2003 ) found that if HycA-repressing agents are absent, FHLsystems activity increases resulting in improvement of hydrogenproduction ( Table 1 b) . However, it was found that the net hy-drogen production by E. colimight actually have reduced owing tothe other activities that recycle some hydrogen and also the oxi-dation of some formate by the respiratory FHL. This was followed by analysis of hydrogen gas production while working on twin-arginine translocation (Tat) system, which is a transporter of re-spiratory hydrogenases ( Table 1 b) (Penfold et al. 2006 ). However,FHL overexpression did not affect hydrogen gas productivity (yield wise) in the experiments carried out by complying withprevious studies ( Table 1 c ) (Sode et al. 1999 ). Interestingly, a combination of fhlA overexpression and disruption of hycA did lead toa 2.8-fold increase in hydrogen production rate as compared withthe wild-type strain having no overexpression and knockout( Table 1 d) ( Yoshida et al. 2006 b). It should be noted that there wasno improvement observed where a combination of both thesegenetic mutations was used.

Also, formate dehydrogenases encoded by fdnG ( -formate dehy-drogenase-N) and fdoG ( -formate dehydrogenase-O) consume for-mate, and thus, deletion of these may result in increase inhydrogen production ( Maeda et al. 2007 a). While fdoG-deletedstrain resulted in increased hydrogen gas production, focA (for-mate transporter) and fdnG mutations failed to increase hydrogengas production by very much in the hyaB-, hybC-, and hycA-deleted(hydrogenase-encoding genes) strain in the closed system (Maedaet al. 2007 a). For low partial pressure systems, both fdoG and fdnGlead to an increase in production rates but not as much as has been observed earlier using formate as substrate ( Maeda et al.2007 a , 2008 ). In addition, deleting both fdoG and fdnG led to adecrease in hydrogen production, while not affecting the hy-

drogen yield, signifying the importance of the presence of eitherof the 2 in increasing hydrogen production ( Maeda et al. 2007 a). focB (formate transporter) mutations along with narG ( -nitratereductase A) resulted in an increase in hydrogen production incomparison with the wild-type strain under closed systems(Maeda et al. 2007 a). In an unusual result, there was no signicantimprovement in hydrogen production on expressing fhlA, whilethe production in fact decreased when overexpression of fhlA wasdone by adding isopropylthiogalactoside ( Maeda et al. 2007 a ; Yoshida et al. 2006 b).

Heterologous hydrogenase responsible for hydrogen productionResearch has gone into incorporating hydrogenase-encoding

genes from a foreign microorganism into the host hydrogen-

producingspecies to enhance hydrogen production.Heterologousoverexpression of hydA (hydrogenase-encoding gene) from E. cloa-cae IIT-BT-08 in E. coli BL-21 resulted in a dramatically high yield of 3.12 mol/mol glucose, which was higher than that from E. cloacaeIIT-BT-08 itself ( Table 1 e) (Chittibabu et al. 2006 ). Expression of HupSL hydrogenase isolated from Rhodobacter sphaeroides into E. coliresulted in a 20-fold increase in thehydrogen gasproduction( Table 1 f ) (Lee et al. 2010 ). There is no developed notion that could

help to understand the increased production of hydrogen gason expression of HupSL ( Lee et al. 2010 ). Still, such a study brings into light the novel methods that could be implementedto increase the hydrogen production using fermentative microor-ganisms harboring hydrogen-producing genes from photosyn-thetic bacteria.

Expression of bidirectional hydrogenase from cyanobacteriumin E. coli resulted in increased hydrogen yields, which, in turn,generated many other ndings ( Maeda et al. 2007 b). Hydrogenuptake was shown to be inhibited by the expression of bidirec-tional hydrogenase, HoxEFUYH (from a Clostridium and Sybechocystissp. strain PCC 6803), as the hydrogen yield was found to be 41times higher than the wild-type strain after 18 h of fermentation(Maeda et al. 2007 b). Interestingly, mutation in Hyd-3 did not giveany benet on overexpressing HoxEFUYH, signifying the need of

Hyd-3 for hydrogen production (Maeda et al. 2007 b). Conse-quently, HoxEFUYH was responsible for restricting the hydrogenuptake by Hyd-1 and Hyd-2 but not by Hyd-3 ( Maeda et al. 2007 b).

Transporter enzymesHydrogen gas yield largely depends on the functioning of trans-

porter proteins, which are often responsible for transporting var-ious enzymes catalyzing an increase or decrease in hydrogen yields. As discussed earlier, since respiratory hydrogenases areresponsible for altering hydrogen gas production, analyses on thehydrogen gas production while working on the twin-argininetranslocation (Tat) system, a transporter of respiratory hy-drogenases, have been done (Penfold et al. 2006 ). TatA, TatB, and TatC proteins form a large oligomeric complex of Tat transporter(Berks et al. 2003 ; Penfold et al. 2006 ). Penfold et al. found thatknockouts of tatC and tatAE ( tatABCD tatE ) increased hydrogenproduction by almost 2-fold in both the cases ( Table 1 c ) (Penfoldet al. 2006 ; Wexler et al. 2000 ). The inactivity of respiratory hy-drogenase (FdH N, FdHO ) and uptake hydrogenase (Hyd-1 andHyd-2), which rely on Tat, leads to such a rise in production rate(Penfold et al. 2006 ). However, the growth rate decreased, whichcan be reasoned on the basis of disruption of the outer membraneand separation of the cell in the engineered strains (Tat knockout)(Redwood et al. 2008 ; Stanley et al. 2001 ). Also, no increase inhydrogen production was observed for the tatC and hycA double-deleted strain (Penfold et al. 2006 ).

The deletion of tatC gene does not have a signicant effect onhydrogen productionusingsucrose as substrateas compared withthe control strain ( Penfold and Macaskie 2004 ). A probable reasongiven was that due to an excess of formate present, formate nolonger remained the rate-limiting step ( Penfold and Macaskie

2004 ). However, the addition of plasmid pUR400, which encodessucrose assimilation genes Scr K , Y , A, B, and a tetracycline resis-tance cassette did result in a signicant increase in hydrogen gasevolution ( Table 1 g ) (Penfold and Macaskie 2004 ).

Metabolite formation enzymesDuring the synthesis of any product, it is understandable that

side reactions will occur, which decrease the yield of the requiredproduct. In the metabolic pathway, disruption of succinate- andlactate-producing pathways shall lead to an increased yield of hydrogen gas ( Fig. 1) ( Yoshida et al. 2006 a). Consequently, Yoshidaet al. showed an approximately 2-fold increase in hydrogen gas yield on disruption of ldhA and frdBCpathways, whichproduce themetabolites lactate and succinate, respectively ( Table 1 c ). This

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yield obtained is 90% of the theoretical yield of hydrogen fromglucose ( Yoshida et al. 2007 ). An increase in specic productionrate as well as volumetric production rate was also observed in theengineered strains ( Yoshida et al. 2006 b). Deletion of succinate-and lactate-producing pathways has shown a similar effect in pre- vious studies (i.e., increase in hydrogen production) (Maeda et al.2007 a ; Yoshida et al. 2006 b). Another study suggested that knock-outs of dld, lldD, ldhA (lactate dehydrogenases), 2 of which are

membrane-boundavoproteins, may help in increasinghydrogenproduction ( Maeda et al. 2007 a). Also, pyruvate is consumed by pyruvate dehydrogenase and pyruvate oxidase encoded by aceEand poxB respectively ( Maeda et al. 2007 a). Deletion of aceE re-sulted in an increased hydrogen production and yield. However,deletion of poxB was found to be inefcient in increasing hy-drogen production, primarily because it is more affecting underaerobic conditions, while E. coli grows under anaerobic conditionto produce hydrogen gas ( Abdel-Hamid et al. 2001 ; Maeda et al.2007 a).

Synthetic pathwaysOwing to the inherent limitation of the native pathway for

hydrogen production in E. coli, which limits its yield, efforts have been made to increase the yield via injection of synthetic pathways for hydrogen production. Akhtar and Jones (2009 ) con-structed a synthetic pyruvate to the H 2 pathway in E. coli toenhance hydrogen production. This included overexpression of the proteins YdbK from E. coli; [4Fe4S]-ferredoxin from Clostridium pasteurianum ; HydA, HydE, HydF, and HydG from Clostridiumacetobutylicum . A 9-fold (or more) increase in the hydrogen gas yield on simultaneous co-express ion of YdbK, and CpFdx(C. pasteurianum [4Fe4S]-ferredoxin in MCS1with MCS2 deleted),and Hyd from C. acetobutylicum as compared with the wild-typestrain was observed ( Akhtar and Jones 2009 ). However, on individ-ual co-expression of either YdbK or CpFdx, the yield did notchange much from the wild-type strain ( Table 1 h) ( Akhtar and Jones 2009 ). In addition, along with the above changes, heterolo-gous co-expression of AmyE from Bacillus subtilis helpedto producehydrogen in starch based minimal media for E. coli ( Akhtar and Jones 2009 ). The deletion of iscR can be instrumental in improvinghydrogen gas productivity, while overexpression of the isc operondecreased the hydrogen gas productivity due to the presence of hydrogenase genes from the accompanying plasmid ( Table 1 i)( Akhtar and Jones 2008 a). Deletion of iscR in the strain co-expressing the 3 metabolic components YdbK, CpFdx, and HydA lead to a 2-fold increase in the yield as compared with the strain without the gene deletion ( Table 1 h) ( Akhtar and Jones 2009 ). How-ever, there was little effect in hydrogen gas productivity whenthere was no expression of YdbK under anoxic conditions. A study suggested the possibility of reduction of ferredoxin as the rate-limiting step instead of hydrogenase activity and that YdbK isinvolved in catalyzing this rate-limiting step. Here, E. coli BL21having virtually zero yield for hydrogen production could pro-duce appreciable amounts of hydrogen on introduction of syn-thetic pathways ( Akhtar and Jones 2009 ). Akhtar and Jones

proposed a general model that involves engineering of high hy-drogen-producing strains and consequent transfer of the hy-drogen-producing pathway to another species that has relatively less hydrogen-producing capabilities in the order of yield ( Akhtarand Jones 2008 b, 2009 ).

Other enzymes and global transcription factors A comprehensive study done by Fan et al. (2009) provides in-

sights into the effect of various knockouts and expressions, in-cluding expressions of global transcriptional regulators ( Table 1 j).Deletion of focA(formate transportgene)resulted in an increase in yield of hydrogen gas (Fan et al. 2009 ). Simultaneously, deletion of this gene caused the accumulation of formate, as it is involved inthe transportation of formate inside the cell ( Fan et al. 2009 ; T

a b l e 1 ( c o n c l u d e d ) .

S . N o . S t r a i n

K n o c k o u t

O v e r e x p r e s s i o n

Y i e l d o r p r o d u c t i o n

P r o d u c t i o n

R e f e r e n c e

j

W 3 1 1 0

, w i l d t y p e

0 . 5 4 m o l ( m o l g l u c o s e )

1

9 . 8

m o l H 2 ( m g d r y m a s s ) 1

F a n e t a l . 2

0 0 9

Z F 1 , m u t a n t

f o c A

0 . 6 3 m o l ( m o l g l u c o s e )

1

1 4 . 8

5 m o l H 2 ( m g d r y m a s s ) 1

F a n e t a l . 2

0 0 9

Z F 2 , m u t a n t

h y b C

0 . 7 0 m o l ( m o l g l u c o s e )

1

1 2 . 1 4

m o l H 2 ( m g d r y m a s s ) 1

F a n e t a l . 2

0 0 9

Z F 3 , m u t a n t

n a r L

0 . 9 6 m o l ( m o l g l u c o s e )

1

1 4 . 3

9 m o l H 2 ( m g d r y m a s s ) 1

F a n e t a l . 2

0 0 9

Z F 4 , m u t a n t

P p c

0 . 7 3 m o l ( m o l g l u c o s e )

1

1 1 . 2

0 m o l H 2 ( m g d r y m a s s ) 1

F a n e t a l . 2

0 0 9

Z F 5 , m u t a n t

f o c A + h y b C

8 . 6 5

m o l H 2 ( m g d r y m a s s ) 1

F a n e t a l . 2

0 0 9

Z F 6 , w i l d t y p e + p l a s m i d

1 . 0 7

m o l H 2 ( m g d r y m a s s ) 1

F a n e t a l . 2

0 0 9

Z F 7 , m u t a n t o n Z F 6

p f n r

6 . 2 3

m o l H 2 ( m g d r y m a s s ) 1

F a n e t a l . 2

0 0 9

Z F 1 1

, m u t a n t o n Z F 6

p s e l C

1 . 8 1

m o l H 2 ( m g d r y m a s s ) 1

F a n e t a l . 2

0 0 9

Z F 1 3


f o c A

p f n r

1 0 . 3

2 m o l H 2 ( g

d r y m a s s ) 1

F a n e t a l . 2

0 0 9

Z F 1 0


p m o d E

1 . 2 7

m o l H 2 ( m g d r y m a s s ) 1

F a n e t a l . 2

0 0 9

N o t e : T h e m a i n m o t i v e i s t o d i r e c t t h e u x t o w a r d s h y d r o g e n . E

f f e c t o f t h e m u t a t i o n s i s s t u d i e d o n h y d r o g e n y i e l d p e r m o l e o f s u b s t r a t e a n d o n h y d r o g e n p r o d u c t i o n , w h i c h h a s v a r i o u s u n i t s a s d e s c r i b e d i n t h e

r e s p e c t i v e p u b l i c a t i o n .

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Sawers 2005 ). Also, deletion of ppc , the gene encoding phos-phoenolpyruvate carboxylase (PEPC), resulted in an increase inthe molar yield. This is because the carboxylation of PEP is oneof the most important steps in succinate production, which ishindered on ppc gene deletion ( Fan et al. 2009 ). Further, NarL(global transcription regulator) is a repressor for structuralgenes of FHL, PFL, and nickel transporters mik operon (Fan et al.2009 ). Inactivation of NarL gave a 2-fold increase in molar yield

of hydrogen without compromising with the growth character-istics (relative to the wild-type strain) (Fan et al. 2009 ). Interest-ingly, an increase of 63% was observed when the selC gene (forselenium metabolism) was overexpressed in the wild-typestrain as compared with the control strain ZF6. This providesnew insights into exploring ways to improve the cofactor pro-duction in the FHL enzyme system, which in turn improves thehydrogen yield ( Fan et al. 2009 ). Overexpression of fnr , anotherglobal transcription regulator resulted in a 5.5-fold increase inspecic hydrogen gas yield. Moreover, this overexpressionalong with focA knockout resulted in very high specic hy-drogen gas production in both M9 and TYP media ( Fan et al.2009 ). Other global regulators like ArcAB and IhfAB were foundto have no signicant impact on hydrogen yield ( Fan et al.2009 ).

Since, currently, the theoretical yield from FHL pathway is noteconomically feasible, many authors have tried to enhance theper mole yield by adapting a dual system approach, often involv-ing E. coli (Redwood and Macaskie 2006 ; Waks and Silver 2009 ;Keasling et al. 1998 ). A detailed discussion of the same is doneunder a separate heading dual systems.

Clostridium

Knockouts and insertion of genesRecently, research to improve the yield of hydrogen gas has

been extended through gene knockouts and overexpression inClostridium . In this direction, investigation on engineering of classof Clostridium is being done by Kaji et al. (1999) , where hydrogengas production vanished when hydA gene was disrupted in Clos-tridium perfringens . The study suggested that this particular gene

was responsible for production of hydrogen gas from C. perfringens(Kaji et al. 1999 ). On the other hand, Morimoto et al. (2005) re-ported a 1.7 times increase in hydrogen gascontent in comparison with the parent clone Clostridium paraputricum M-21 on overex-pression of hydA ( Table 2 a). This was accompanied by considerablereduction in the lactic acid formation (Morimoto et al. 2005 ). Inthe same study, authors conjectured that the overexpression of hydA led to over oxidation of NADH to NAD + , thus disrupting theconsequent productionof lactic acid frompyruvic acid ( Morimotoet al. 2005 ). However, there were contrasting results reported by Klein et al. (2010) who stated that overexpression of hydrogenasedidnot improve the yield of hydrogen production( Table 2 b). Kleinet al. did a comparative study on C. acetobutylicum and C. butyricumand reported a huge increase in specic hydrogenase activity forC. butyricum but only slight increase in the case of C. acetobutylicum .

The authors reported that the maximum hydrogen gas produc-tion (volume) was comparable for the overexpressed and wild-type strain of C. acetobutylicum. They proposed that the enzymeresponsible for transfer of electrons to oxidized ferredoxin is nota limiting factor for hydrogen gas production.Several alternatives were suggested, which are listed as follows ( Klein et al. 2010 ):

There could be any other step in the metabolic pathway for hydrogen production, which is limiting hydrogen gasproduction.

NAD+ regeneration could be rate-limiting step. A combination of disruption of acetate kinase gene and over-

expression of hydA maylead to higher hydrogen gasproduction(Liu et al. 2006 b).

The overall dynamics is thermodynamically limited. For exam-ple, constant removal of hydrogen gas might drive the forwardreaction.

It was found that butyric acid formation was immune to hydAoverexpression (Morimoto et al. 2005 ). Hence, research shouldaddress ways to inhibit the butyric acid formation, which shallincrease the hydrogen gas.

Nakayama et al. (2008) reported the enhancement of hydrogenproduction by 3.1 times as a result of decreased hydrogen uptakeactivity in Clostridium saccharoperbutylacetonicum N1-4 on down-regulation of hupCBA (hydrogenase genes), as compared with the wild-type strain ( Table 2 c ). This, thus, reects upon its uptakeactivity. On deletion of the ack (acetate kinase) mutant from Clos-tridium tyrobutyricum , hydrogen yield rose by 50% of the value ob-tained by the wild-type strain, providing insights that eventhough, theoretically, deletion of the acetate-producing gene may not increase hydrogen ux, it may actually affect hydrogen yield( Table 2 d) (Hallenbeck and Ghosh 2010 ; Liu et al. 2006 b). This wasaccompanied by a decrease in acetate kinase activity and an in-crease in hydrogenase and phosphotransacetylase activities (Liuet al. 2006 b). A similar study done by the same group showed aslight decrease in hydrogen production by using C. tyrobutyricumPPTA-Em having pta (encoding phosphotransacetylase) geneknockout ( Table 2 e) (Liu et al. 2006 a). This provides insights intotrying different gene knockouts to nd out the effect on hy-drogen-uptake and (or) hydrogen-producing activity.

Strain selection and process integrationSurprisingly, there has not been enough study on genetically

engineered clostridia. However, the yield and production of hy-drogen varies largely from strain to strain and study of the sameprovides an invaluable tool forchoosing theappropriate strain forhydrogen production. Also, as we will see later, analysis of all thediverse species of clostridia becomes allthe more importantwhen we take into account the indirect participation of some of thenon-hydrogen-producing clostridia for hydrogen production(Hung et al. 2011 a). For instance, the wild-type strain Clostridium beijerinckii Fanp3 showed a fairly high yield comparatively ( Ta-

ble 2 f ) (Pan et al. 2008 ). In a study, however, the maximum yieldfrom an isolated strain was found out to be 2.91 mol/mol sucrose, which is relatively low owing to the amount of hydrogen andcarbon atoms present in it as compared with glucose (Chen et al.2005 a). In addition, hydrogen gas production rate was found to berelatively high but at a different pH ( Table 2 f ) (Chen et al. 2005 a). Thus, we see that there is a wide variation in the hydrogen pro-duction, which is strain and condition specic. Since there arenumerous examples of using various clostridia, we have tried topresent most of the published results in tabular form ( Table 2 f ).

The hydrogen yield was found to be astonishingly high(9.95 mol/mol glucose) when fermentation of Clostridium thermo-cellum was followed by microbial electrolysis cells of the remain-ingmetabolites. This approach (2-step processes) can be employedto improve hydrogen yield, especially for various Clostridium spe-

cies, which are available widely in diverse forms, and is discussedlater under section 4.5 ( Lalaurette et al. 2009 ).

Enterobacter aerogenes and Enterobacter cloacae

Knockout and insertion of genes Working on these species can be very advantageous, since a

slight increase in yield with genetic engineering leads to yields very close to the theoretical maximum. Enterobacter aerogenes and Enterobacter cloacae are facultative anaerobes. Characterization of hydrogenases in hydrogen-producing strains from various Entero- bacter species has been performed (Dutta et al. 2009 ; Mishra et al.2002 , 2004 ). Ren et al. (2005) did quantitative comparisons of hy-drogen-evolving and hydrogen-uptake activities, which triggersthe need for the development of strategies of controlling uptake



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Table 2. Effects of gene knockout and overexpression in various Clostridium species and strains.

S. No. Species Strain Knockout Overexpression Yield or production Production

a C. paraputricum M-21, wild type 1.4 mol(mol glucose) 1C. paraputricum M-21 pJIR751, mutant hydA 2.4 mol(mol glucose) 1

b C. acetobutylicum DSM 792, wild type 1.79 mol(mol glucose) 1 1810 mLC. acetobutylicum DSM 792 [pSOS], mutant thl promoter 1.77 mol(mol glucose) 1 1740 mLC. acetobutylicum DSM 792 [pSOShydACa], mutant hydA 1.81 mol(mol glucose) 1 1650 mLC. acetobutylicum DSM 792 (pSOShydACb), mutant hydA 1.80 mol(mol glucose) 1 1710 mL

c C. saccharoperbutylacetonicum N1-4 pNAK1, wild type 500 mL HC. saccharoperbutylacetonicum N1-4 pAntihup, mutant hupCBA operon 1546 mL H

d C. tyrobutyricum Wild type 1.44 mol(mol glucose) 1C. tyrobutyricum PAK-Em, mutant ack 2.16 mol(mol glucose) 1

e C. tyrobutyricum ATCC 25755, wild type 1.35 mol(mol glucose) 1

C. tyrobutyricum PAK-Em, mutant ack 2.61 mol(mol glucose) 1C. tyrobutyricum ATCC 25755 PPTA-Em, mutant pta 1.08 mol(mol glucose) 1

f C. beijerinkii Fanp3, wild type 2.52 mol(mol glucose) 1 39 mL HC. butyricum CGS5, wild type 2.9 mol(mol sucrose) 1 209 mL HC. thermolacticum Wild type 3 mol(mol lactose) 1 2.58 mmC. acetobutylicum ATCC 824, Wild type 0.9 mol(mol glucose) 1 8.9 mmoC. paraputricum M-21, wild type 1.9 mol(mol GlcNAc) 1C. tyrobutyricum JM1, wild type 223 mL(g glucose) 1 7.2 L H2

TERI BH05, wild type 2.3 mol(mol glucose) 1 22.3 mmClostridium sp. DMHC-10, wild type 3.35 mol(mol glucose) 1Clostridium sp. R-1, wild type 3.5 mol(mol cellobiose) 1C. beijerinckii RZF-1108, wild type 1.97 mol(mol glucose) 1 2209 mL

Note: Hydrogen yield of various strain is being shown due to heavy dependence on strain selection. Effect of the mutations and variety in strains is studied on hydrogen yield per mole production which has various different units as mentioned in the respective studies.

This species was found to be similar to a Clostridium species in the corresponding study.

P u b l i s h e d b y NR C R e s e ar c h P r e s s

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activity and enhancing evolving activity. Remarkably, unlike E. coli, E. aerogenes has been found to operate v ia 2 pathways,(i) pyruvate to formate and ( ii) NA DH oxidation ( Vos et al. 1983 ;Zhang et al. 2009 ; Zhao et al. 2009 ). Research on metabolic engi-neering for hydrogen using this species has been started very recently; pr evious researc h was mostly based o n altering cultur econditions ( Lu et al. 2010 ; Nath and Das 2004 ; Zhao et al. 2009 ).Still, published information on the metabolic pathways for hy-

drogen producti on using E. aerogenes is not well characterized(Zhao et al. 2009 ). The effect of deleting the genes hycA and hybO encoding FHL

repressor and up take hydrogenase has been previously studied(Zhao et al. 2009 ). The study not only revealed the increase inhydrogen gas production and yield but also individu al contribu-tions of the 2 hydrogen-producing pathways ( Table 3 a) (Zhao et al.2009 ). While the FHL pathway resulted in an increase in hydrogen yields for all mutants in the wild-type strain, the NADH pathway showed no improvement for some and slight impro vement forthe s train having double knockout of hycA and hybO (Zhao et al.2009 ). However, the cell growth rate decreased, which may be anareaof con cern andshouldbe addressedby further research (Zhaoetal. 2009 ). Heterologous overexpression of hydA in E. aerogenes ledto an enormous increase i n the yiel d and cumulative productionof hydrogen from glucose ( Table 3 b) (Zhao et al. 2010 ). This opensup thepossibility of exploringa possible relationshipbetween theroute being followed and the knockout(s) and overexpressions being done. The addition of formate demo nstrated enhancementinhy drogen producti on using E. aerogenes (Kurokawa and Tanisho2005 ; Seol et al. 2008 ). There is very little knowledge on whetherhydrogen production c an be improved by regulation of the FHLpathway ( Lu et al. 2009 ). In addition, the NADH pathway is consid-ered to be more sensitive to hydrogen gas production than is theformate pathway. To understand these aspects, a study on over-expression of genes fdhF (encoding formate dehydrogenase) and fhlA (encoding formate hydrogen lyase activator protein), whichencode formate dehydrogenase and FHL activator prot ein from E. coli, respectively, has been performed (Lu et al. 2009 ). Overex-pression of both genes individually led to an increased hy-drogen yield in most of the strains except for mutant Oreecting upon the vari ed effect of var ious expressions ondifferent strain ( Table 3 c ) (Lu et al. 2009 ). Similar tre nds wereobserved for the hydrogen production values ( Table 3 c ). Interest-ingly, the decrease in hydrogen yield in the mutant O was attrib-uted to the decrease in yield from the FHL pathway, wher eas yieldfrom the NADH pathway remained essentially constant ( Lu et al.2009 ). Removing the other metabolite concentration has very of-ten resulted in an increased hydrogen production in E. coli.Similarly, lactate production from the metabolic path way of E. aerogenes is found to consu me most of the NADH present (Zhanget al. 2009 ; Zhao et al. 2009 ). ldh deletion could possibly increaseNADH, leading to increased hydrogen production. Consequentdeletion of ldhA resulted in an increase in hydrogen gas yield buta slight decrease in th e total producti on rate. No lactate wa s pro-duced by this strain (Lu et al. 2010 ). Kumar et al. (2001) did a

comprehensive study on E. aerogenes IIT-BT where the alcohol-forming pathways were initially defected. The result was a de-crease in the production in hydrogen gas. It was found that mostof the metabolic ux was directed towards the formation of acidsratherthan hydrogen.On blocking of theacid-producingpathway along with the alc ohol-pro ducing pathway, h ydrogen gas yieldincreased 1.5-fold ( Table 3 f ) (Kumar et al. 2001 ). In another study,the yield increased massivel y by 86.8% on the overex pression of fdh1 in an ldh-deleted strain ( Table 3 d) (Lu et al. 2010 ). This is animportant nding with respect to analysis of various factors con-trolling hydrogen yield and in fact cellular metabolism in E. aerogenes.

In the quest to understand whether global en ergy regulationcould lead to an increased hydrogen metabolism, Lu et al. (2011 b)

studied the effect of polyphosphate kinase (PPK) in E. aerogenes.PPK is a key enzyme in the conservation and release of energy aspolyphosphates. Compared with the wild-type strain, overexpres-sion of PPK in E. aerogenes showed varied effect in hydrogen atdifferent oxygen levels. At 2.1% oxygen levels, the maximum in-crease in hydrogen yield (mainly vi a NADH pathway incre ased) was observed though not very high ( Table 3 e) (Lu et al. 2011 b). Inaddition, it was found that there was an adjustment of cellular

oxidation states combined with mo re electron o w for hydrogenproduction on PPK overexpression ( Lu et al. 2011 b). However, theincrease in yield for other oxygen levels was signicantly low andin fact decreased slightly for 8% oxygen environment. Nonethe-less, this study provides insights into a possible area in metabolicengineering not explored much till now.

A study reported that disposal of excess reduci ng equivalentslead to hydrogen gas production in E. aerogenes ( Tanisho et al.1989). NADH oxidizes to form ethanol, butanediol, lactic acid, and butyric acid from glucose. If, somehow, the production of thesecompounds can be blocked, hydrogen gas production will in-crease on t he account of increase in NADH concentration (Kumaret al. 2001 ). In fact, a detailed study of t he effects of NADH andNAD+ has been done (Zhang et al. 2009 ). Results suggested a de-creased hydrogen production from NADH pathway and increasedhydrogen production from the formate pathway on addition of NADH in the cultivation media. The total effect was a reduction of hydrogen gas production.However, NAD + addition yielded resultsopposite to those obtained by NADH addition (Zhang et al. 2009 ).

Strain selection and multiple systems There have been numerous efforts using different strains to

produce hydrogen gas in Enterobacter species ( Table 3 g ). A numberof hydrogen-producing strains have been identied that inher-ently produce very high hydrogen yield. In addition, E. aerogeneshas been found to be immune to the negative effects of presenceof other species. Consequently, E. aerogenes has beenputto use forhydrogen production, with a fairly decent amoun t of su ccess when used in combination with Clostridium species ( Table 4 ) and with a photofer mentation bacter ia, makinga 3-system implemen-tation possible ( Yokoi et al. 2001 ).

Other microorganisms

Thermophilic bacterial species Thermophilic bacteria invite considerable attention owing to

their benets, such as high hydrogen production rate at elevatedtemperatures, high molar yield, comparatively less array of endproducts, inn ate ability to convert biomass into useful fuels, etc.(Li et al. 2010 ). Elevated temperatures are particularly very impor-tant because they help to overcom e the thermodynami c barrierand improve the reaction kinetics ( Nguyen et al. 2008 b). Severalthermobacterium species have been id entied that could poten-tially be used for hydrogen productio n (Kongjan et al. 2009 , 2011;Li et al. 2010 ). A recent study done by Li et al. (2010) suggests that by modifying the metabolic pathway to direct ux towards hy-drogen pathway production, the yield increases considerably.

Knockout of ldh from Thermoanaerobacterium aotearoense resultedin no metabolite presence of lactate after 48 h of cultivation. There was a 2-fold increase in the yield of hydrogen and a 2.5-foldincrease in its production rate, as compared with the wild-typestrain). There are many other instances of exploration of effects of this genre of bacteria ( Ciranna et al. 2011 ; Ngo et al. ). Thus, they form a vital part of the research to make biohydrogen productionfeasible and can be genetically engineered for its enhancement.Organisms operating at extremely high temperatures (hyperther-mophilic, examples include Thermtoga maritima , Caldicellulosiruptor saccharolyticus, Thermot oga neapolitana ) have show n potential forhydro gen production ( Nguyen et al. 2008 a , 2008 b; van Niel et al.2002 ). In fact, Thermotoga maritima MSB8 and DSM 3109 reporte d a yield of 4 mol hydrogen/mol glucose ( Schroder and Selig 1994 ).



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Halophilic bacterial speciesHalophilic microorganisms are organisms capable of surviving

under high salt concentrations and have been extensively used toproduce hydrogen. Also, the ability of halophilic microorganismsto survive under highly saline concentrations and their resistanceto heavy metals make them suitable for situations where glycerolis a by-product (e.g., biodiesel industry) (Kivisto et al.2010 ). Intheirrecent study on the halophilic bacteria Halanaerobium saccharolyti-

cum subsp. saccharolyticum and H. saccharolyticum subsp. senegalesis,Kivisto et al. (2010) found that the production of hydrogen fromsubspecies senegalesis was higher than from subspecies saccharolyti-cum using glycerol as substrate. This is due to the formation of 1,3-propanediol in the subspecies saccharolyticum , a path which isnot a hydrogen-producing route; however, no 1,3-propanediolwasformed from subspecies senegalensis. Also, the absence of produc-tion of 1,3-propanediol even from subspecies saccharolyticum whileusing glucose as substrate resulted in yields from both the sub-species to be comparable (Kivisto et al. 2010 ). The results obtainedprovide new insights into using halophilic bacteria to producehydrogen especially from glycerol.

Indirect contribution to hydrogen productionSince most of the genetic engineering research for hydrogen

has been done for organisms that are sequenced (e.g., E. coli,Clostridium ), many other organisms have been left out ( Hung et al.2011a). A study doneby Hunget al. (2011 a) explores the potential of various other microorganisms to be used for hydrogen produc-tion in varied capacities, such as contribution via biomass reten-tion, anaerobic environment maintenance, degradation of organic matter, and blocking hydrogen consumption ( Hung et al.2011a). Species like Megasphaera cerevisiae , Ethanoligenens harbinense , Anaerotruncus , and those belonging to the Veillonellaceae family have been shown to be hydrogen producing (Castell et al. 2009 ;Hung et al. 2011 a). Streptococcus sp. has been found to contributeheavily towards granular sludge formation, which is imperativefor optimization of hydrogen production (Hung et al. 2011 b). Inaddi-tion, Streptococcus sp. along with Klebsiellasp. and Bacillussp. have thecapabilities to be used as catalysts to have obligate anaerobic envi-

ronments for high hydrogen species like Clostridium (Hung et al.2011a , 2011 b). Species like Bacillus sp., Bidobacterium sp., Klebsiellaoxytoca, and many more have shown substrate degradation trendsthat can be instrumental in bio-hydrogenproduction ( Cheng et al.2008 ; Hung et al. 2011 a , 2011 b). This strategy can potentially beused in dual systems and is discussed further there. Most clos-tridia species that are not direct hydrogen producers have beenleft out. However, they may be indirectly responsible for hy-drogen production by degradation of cellulose materials (e.g.,Clostridium cellulosi and Clostridium stercorarium ) (Hunget al. 2011 a; Nissilet al. 2011 ).

Dual systems With respect to metabolic hydrogen production, there have

been immense initiatives to use dual systems in varied capacity

(Chen et al. 2008 ; Nath et al. 2008 ; Redwood et al. 2009 ; Redwoodand Macaskie 2006 ; Tao et al. 2007 ; Waks and Silver 2009 ). Variousapproaches include the following:

One microbial species to produce an intermediate substrate orsimpler molecule (e.g.,formate) and, consequently, anotherspecies to produce hydrogen (Cheng et al. 2008 ; Waks andSilver 2009 ).

Dark fermentation followed by photofermentation ( Chenet al. 2008 ; Redwood et al. 2009 ; Redwood and Macaskie2006 ; Tao et al. 2007 ).

Coculturing of dark fermentation and photofermentation bacteria or simple cocul turing irrespective of dark, photo bacteria ( Ding et al. 2009 ; Lu et al. 2007 ).

Fermentation followed by electrohydrogenesis by microbialelectrolysis cells (Logan et al. 2009 ; Oh and Logan 2005 ).

Waks and Silver (2009) tried to produce hydrogen via overpro-duction of formate from S. cerevisiae followed by a 2-step process by using E. coli as catalyst in the formate-containing medium. Theprocess uses S. cerevisiae PSY3649 with the knockout of FDH1 andFDH2 and overexpression of PFL and AdhE. Under anaerobicconditions, FDH1 and FDH2 deletion eliminates the consump-tion of formate thereby facilitating a format e-containing media(Overkamp et al. 2002 ; Waks and Silver 2009 ). The formate secre-tion increases by 4.5-fold on the overexpression of PFL; remark-ably, it showed a steep increase in the formate production when acombination of PFL and AdhE was used. However, this processproduces only 1.5% of the theoretical maximal yield of formatefrom galactose, compared with E. coli, w hich has an efciency of 33% formate form ation from glucose ( Waks and Silver 2009 ; Yoshida et al. 2007 ). Nonetheless, the 2-step process promises to be a faster means of producing hydrogen directly from formateand opens up new insights into dual-or ganism systems for hy-drogen production ( Waks and Silver 2009 ). Also, there are reportsof a cocultured bacteria in hydrogen production from Bidobacte-rium sp. that helps in breaking down starch into smaller mole-cules making it co nvenient for utilization by Clostridium species

(Cheng et al. 2008 ). There are s e veral other exam ples existing forthis strategy ( Hung et al. 2011 a ; Nissil et al. 2011 ).

For the process of dark fermentation followed by photofermen-tation, the organic products of the rst process are being de-gradedinto hydrogen gas, andnear stoichiometric yields for som eorganic acids is practically feasi ble (Hallenbeck and Ghosh 2009 ).Redwood and Macaskie (2006 ) tried producing hydrogen in a2-step process of fermentation by E. coli followed by photofermen-tation of the residue liquor by Rhodobacter sphaeroides . While there was no hydrogen production from the second step because of thepresence of xed nitrogen sources, a synthetic medium havingthe same concentrations as that of an E. coli-subjected liquor devoid of ni trogen sources produced 124 m L of hydrogen per cultureper day ( Redwood and Macaskie 2006 ). In addition, a study revealed 2-fold increase in hydrogen yield from sucrose when the

fermented liquor was subjected to photofermentat ion, an d hy-drogen pr oduction also increased by a huge amount ( Table 4 ) ( Taoetal. 2007 ). The highlightingpointof a similar study done by Chenet al. (2008) was the stable hydrogen production rate and contin-uous yield for 10 consecutive days, indicating the commercialfeasibility of such a process. This gives an insight into the im-mense potential for hydrogen using this 2-step process. Compre-hensive data covering the yield a nd production rates (whereverapplicable) is presented in Table 4 . Also, there have been instances where co-culturing of 2 different bacteria or yeast has resulted inan incre ased yield due to a synergistic effect of the combination( Table 4 ) (Lu et al. 2007 ).

Use of waste streams, which are rich in carbohydrates (e.g.,cereals), can be used for hydrogen production via fermentationpathways, which may well be economically feasible. Here by H +

and e can be used to produce hydrogen by increasing the cathodepotential in a microbial fuel cell (M FC) (Logan et al. 2009 ; Oh andLogan 2005 ; Rupprecht et al. 2006 ). However, currently reported volumetric rates are found to be of an order or 2 less than that by dark fermentations and thus questions the use of such a system(Hallenbeck and Ghosh 2009 ).

Quantication of the hydrogen-producing metabolicpathways

Various attempts have been made towards quantication of metabolic pathways of different hydrogen-producing organisms. These studies were performed by using systems biology ap-proaches, mainly metabolic ux analysis (MFA). MFA is widely considered to be a vital tool to calculate the ux in a metabolic

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network. However, the metabolic network of hydrogen-producingorganisms has not been well understood at prese nt because of lim-ited resear ch being carried out in this direction ( Cai et al. 2011 ; Ohet al. 2008 ). Theoretically, 12 mol hydrogen per mol glucose can begenerated during the fermentation process. However, theoretical yield is reduced to 4 and 2 mol hydrogen per mol of glucose with thesynthe sis of a sole by-prod uct, namely acetate and butyrate, respec-tively ( Cai et al. 2010 , 2011).

Fermentation proles of various studies on facultative organ-isms have been employed for the ux analysis of metabolic network of organism. MFA techniques have not been implementedmuch for obligate anaerobes as yet owing to their highly sensi tivemetabolic pathways with respect to the physical conditions (Caiet al. 2011 ). Studies based on pathway analysis of obligate anaer-obes postulated that anaerobes have a decision-making regula-tory system for choosing either the EntnerDoudoroff pathway orthe EmbdenMeye rhof pathway as a primary sub-p athway of glu-cose consumption ( Cai etal. 2011 ; Fuhrer etal. 2005 ). These kind of regulatory systems illustrate the complexity of pa thway duri n gthe conversion of glucose to synthesize pyruvate (Figs. 1 and 2). Analysis has also highlighted the dependency of hydrogen pro-ductionon NADH as pre cursor in various sp ecies such as E. coliand Rhodobacter sphaeroides (Fuhrer et al. 2005 ). Recently, it was re-ported that EmbdenMeyerhof pathway was 9 times mo re robustthan the pentose phosphatepathway for C. butyricum W5 (Cai etal.2010). During the investigation, it was reported that the ux towards hydrogen production is impaired with the synthesis of lac-tate and ethanol. Various levels of lactate, ethanol, and hydrogen were predicted using MFA with different growth media. The pre-dicted yiel d of hydrogen was also veried experimentally (Caiet al. 2010 ). Similarly, the concentrations of various intermediatemetabolites and hydrogen were found sensitive to the pH of themedium. Pathway analysis has shown that theeffectof pH is m oresignicant at 3 n on-rig id nodes,PEP, pyruvate,and acetyl-CoA (Caiet al. 2010 , 2011) (Fig. 2 ). Low pH was favorable for the formation of oxaloacetate at the PEP node, while the ux percentage to lactatedecreased on decreasing t he pH value from 7.0 to 6.0 at the pyruvate node ( Cai et al. 2010 ). The effect of pH was not profound forthe ux of the formation of acetate and butyrate, but it affec tedthe ethano l production considerably at the acetyl-CoA node (Caiet al. 2010 ). In the metabolic pathway of C. butyricum W5, a pH of 6.0 was found optimum for signicant production of hydrogen. Additionally, initial glucose concentration was not a ver y signi-cant factor in hydrogen production for C. butyricum W5 (Cai et al.2010). On an average, 170190 mmol/L ux was directed to the PEPnode, which consequently, resulted in hydrogen, acetate, bu-tyrate, and ethanol production of 130160, 1733, 2836, and47 mmol/L, resp ectively, from 100 m mol/Lglucose taken initially C. butyricum W5 (Cai et al. 2010 , 2011).

To date, wild-type E. coli is the one of o rganisms of intere st for various pat h way quantication studies (Kabir et al. 2005 ; Kimet al. 2009 ; Manish et al. 2007 ). Concentrations of 17, 63, and51 mmol/L hydrogen, acetate, and ethanol, respectively, were ob-tained from 140 mM PE P, starting with 10 0 mmol/L glucosefor the

E. coli wild-type strain ( Kabir et al. 2005 ). Another study on engi-neered E. coli suggested production of 148165 mmol/L hydr ogenfrom 100 mmol/L glucose (concentrations rescaled to 100) ( Kimet al. 2009 ).

In a recent study on E. aerogenes, MFA reported production of 45.1, 43.2, 76.9, and 89.7 mmol/L acetate, ethanol, lactate, andhydrogen from 100 mmol/L glucose, and via the formate andNADH pathways, H 2 production was 88.4 and 1.4 mmol/L, respec-tively ( Lu et al. 2011 a). However, a mutant strain reported a 16-foldincrease in hydrogen from t he NADH path way and a mild increasefrom the formate pathway ( Lu et al. 2011 a).

A study on Citrobacter amalonaticus Y19, using 100 mmol/L glu-cose as substrate, yielded the incredibly high concentration of 817 mmol/L hydrogen through the pentose phosphate pathway

and by utilization of NAD(P)-linked hydrogenase (Ho et al. 2010 ).Other stoichiometric analysis has been performed on Klebsiella pneu-monia and C. butyricum where it was reported that a maximumhydrogen yield of 6.68 mol/mol glucose in K. pneumonia wouldresult in all moles of acetyl-CoA entering into the tricarboxylicacid cycle cycle and that molecular oxygen oxidizes 53% of thereducing equivalents (Chen et al. 2005 b). Also, a maximum hy-drogen yield of 3.26 mol/mol glucose was predicted for C. butyri-

cum if all moles of acetyl-CoA entered the acetate pathway (Chenet al. 2005 b). Remarkably, networkquantication-based studiessuggested the nature of association of hydrogen production with biomass growth varies organism to organism. For instance, in K. pneumonia the high production of hydrogen was detected at low biomass growth, unlike C. butyricum for which the high hydrogenconcentration was observed at high biomass growth ( Chen et al.2005 b). Thus, these types of predictions provide insightful infor-mation to maintain the signicant ux ow towards maximiza-tion of molar hydrogen yield. The ux analysis approach was alsoimplemented on thermophiles to quantify the hydrogen andother metabolites over the different stresses of pH and redoxpotential (Sridhar and Eiteman 2001 ).

Moreover, tracer-based techniques were also used to quantify the metabolic ux distribution in the synthesis of hydrogen pro-

duction using 13

C isotopes ( Tang et al. 2008 ). The quanticationstudies for metabolic networks mainly contribute to detect theux ow for the synthesis of hydrogen and by-products. On the basis of this gap-lling information, opportunity is provided tomolecular and systems biologists to manipulate the genome of anorganism to maximize the ux ow to a branch of network thatcarries maximum hydrogen synthesis. Also, we can acquire thelocations of the nodes in the metabolic pathway that are neces-sary for bacterial growth. During the genetic manipulation of anorganism, the identication of these nodes promotes the devel-opment of engineering strategies to nd the higher yielded pathway without interfering in the growth of the organism. There areseveral other advantages resulting from pathway quantication,such as optimization of media and identication of the vital in-termediates of the network.

DiscussionHydrogen is the most energy efcient fuel, and numerous ef-

forts have been made to produce it via biological processes, whichneed to be implemented on an industrial scale. The parameters totest the feasibility of hydrogen gas via biological processes arechiey yield, production, and production rates of hydrogen. While Clostridium spp. give relatively high yield, E. coli is fast grow-ing and biologically and physiologically well characterized, thusmaking it an attractive organism to work with for hydrogen pro-duction (Redwood et al. 2008 ). Also, compared with obligate an-aerobes like clostridia, facultative anaerobes are able to cope withthe hydrogen production activity under accidental oxygen dam-age and thus are better in this regard (Nath and Das 2004 ). Inaddition, Enterobacter species are found to utilize 2 pathways for

hydrogen production. Thus, different hydrogen-producing organ-isms have different properties that make them favorable targetsof future research for hydrogen production. Furthermore, hy-drogen yield is low partly due to inefcient pathway specicity andpartlydue to incomplete oxidationof the substrate moleculesin the case of hydrogen ( Akhtar and Jones 2009 ). Thus, we mustresort to modifying the metabolic pathways and in fact developnovel pathways for hydrogen production to direct metabolic uxtowards hydrogen production to makeit economically acceptable.

Metabolic engineering of fermentative pathways has led to anincrease in hydrogen yield and production rates. In some studies,knockout and overexpression of various genes have inuencedsufcient increase in the production and yield of hydrogen. En-deavors of this type included overexpression of cellulases and



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hydrogenase involved in hydrogen production, and knockout of uptake hydrogenases (Hyd-1, Hyd-2, and Hyd-3) and other metab-olite (e.g., succinate, lactate) producinggenes. In addition to theseendeavors, deletion of transporter proteins and more recently mutation in global transcription regulators have also been inves-tigated. Table 5 summarizes the effect of various genetic modi-cations on organisms viz E. coli, E. aerogenes and E. cloacae, andspecies of Clostridium . The effect of various genetic modications

is being analyzed on the basis of percentage increase or decreasein the overall hydrogen yield and production rates from the strain without that particular modication. Accordingly, wide varia-tions in the effects were observed not only for the same geneknockout in different organisms but also for the same organism.For instance, tat , hycA and hyd-2 mutations in E. coli ( Table 5 a), and fdhF and fhlA mutations in Enterobacter species ( Table 5 b) leads toan increase in hydrogen production and a decrease in hydrogen yields. Various gene knockouts were found to behave differently for different species ( Table 5 ). For example, hydrogen production was found to be more sensitive for E. colithan species of Clostridiumto HycA (formate hydrogenase) knockout ( Tables 5 a and 5 b). Inter-estingly, gene knockout of Src encoding sucrose transport pro-duced an unexpectedly high increase in production in E. coli( Table 5 a). However, this is a result of the exceedingly low hy-drogen production from the wild-type strain ( Table 1 g ). Similarly,pfnr (global transcription regulator) produced a very high changein production, which can be attributed to low initial productionrate of ZF6 ( Table 1 j). Insertion of a plasmid resulted in a substan-tial decrease in yield ( Table 5 a). This is an important area of con-cern especially when we look to develop synthetic pathways forhydrogen production. As can be seen, less data are available onmutations for clostridia ( Table 5 ). It shall be noted that a range of percentage change in yield and productivity is not available forthose mutations that dont have too many published data points.Consequently, the tabulated data for the same will be more valid,provided multiple researches are carried out for these mutations.

Dual systems involving 2 organisms have yielded positive re-sults and opens up wide applications for hydrogen production.For example, producing formate from industrially robust specieslike S. cerevisiae and consequently using E. coli for hydrogen pro-duction is one such option ( Waks and Silver 2009 ). In addition,studies have also been done on using a dark fermentative and aphotofermentative organism together to maximize hydrogen yield and production ( Redwood and Macaskie 2006 ). The presenceof one peripheral microbial species in a dominant hydrogen-producing species can result in either decreased or increasedhydrogen production (Hung et al. 2011 a). Also, since population dy-namics of organisms can result in diminution of hydrogen-producing organisms or an increase in hydrogen-consumingorganisms, this phenomenon needs to be regulated (Hussy et al.2003 ). Secondly, there exist cases where peripheral species interfere with the hydrogen-producing species for substrate consumption(Hung etal. 2011 a; Jo et al.2007 ). Research inworking outpossibilitiesto minimize this interference shall help to improve hydrogen yieldsa great deal. Thirdly, there exists many unidentied organisms ac-companying a dark fermentation medium, whose roles are notclear, and these can also be explored not only for theirhydrogen-producing tendencies but also for their side effects (posi-tive or negative) (Hung et al. 2011 a). Fourthly, there have been in-stanceswhena non-hydrogen-producingstrainhas resultedin a highhydrogen yield by metabolically engineering them, thus, invitingconsiderable attention. However, genetic engineering endeavors aresometimes associated withvariousnegativeimpacts,like decrease incell density, slowness in thegrowth rates of theorganism, incompat-ibility of heterologous pathways with the native pathways, metabolic imbalance, and accumulation of other undesired metabolites(Colletti et al. 2011 ; Lu et al. 2010 ; Zhao et al. 2009 ).

Future perspectivesHydrogen illustrates better efcacy and less pollution impacts

over its consumption along with various limitations for its eco-nomic production through microbes. When we look at hydrogenas a clean and energy-efcient fuel, we also have to look at it fromthe economics point of view. There have been several attempts togo into in-depth analysis of the ways to increase hydrogen produc-tion. However, endeavors for economic analysis of microbialmethods of hydrogen production can also provide the specictargets for scaling up the process to commercial production ( Nathand Das 2004 ). Only cost-efcient fermentative hydrogen gas pro-duction shall make all the research being carried out fruitful.Focus has to be put on species that can be easily engineered, asmost promising microorganisms today are not genetically tracta- ble ( Jones 2008 ). In addition, other high hydrogen-producing spe-cies cannot be ignored owing to their other advantages, such asfast growth, high immune systems, etc. The use of industrial waste products like that from the pulp and paper industry and of cellulosic materials shall help bring down the cost of the produc-tion. In fact, there have been numerous genetic engineering works done in this respect to improve the cellulose consumptionand consequently the fermentation yield ( Guedon et al. 2002 ). Theoutcomes will benet us 2-fold in the decomposition of waste

material as well as in production of biofuels. Computational sys-tems biology has been used widely to maximize production ef-ciency, and these in silico methods are bound to have a very instrumental role if hydrogen production is to be extended to theindustrial level ( Jones 2008 ). One advantage that computationalmethods hold is the ability to develop metabolic networks modelsforspecies whose characterization has notbeen done ( Jones 2008 ).

Being restricted by the low metabolic yield and productionfrom the native pathway, effort is required on the metabolic en-gineering front to improve these parameters. Metabolic engineer-ing thus shall dictate the economic feasibility of biologicalhydrogen production to a large extent. Nevertheless, the processconditions (like pH, temperature, partial pressure over the headspace, culturing media) also need to be improved, as they shallhelp to increase the hydrogen yield. Once both metabolic engi-

neering of species and process conditions complement eachother, hydrogen production via fermentative processes may beseen as an economically acceptable option. Identication of anoptimal set of conditions along with genetic engineering applica-tion is of utmost importance.

ConclusionsHydrogen turns out to be an ideal fuel, provided its economics

becomes feasible. A shift in hydrogen production from conven-tional methods to biological processes requires rigorous researchin increasing the yield and production rates from the latter pro-cesses before it becomes economically feasible. The known pathways for hydrogen production are marred by thermodynamic andmetabolic constraints that restrict the yield and productivities to values that can be scaled up immensely on working on metabolic

pathways, either synthetic or native. Thus, metabolic engineeringof microbialspecies is a very prominentarea of research to realizethis objective in addition to optimization of physical and processconditions. Adequate understanding of cells at the molecularlevel provided the signicant space to manipulate the genometowards higher yield of desired metabolites. Therefore, this tech-nique is powerful to engineer the genome of hydrogen-producingspecies, as a low yield of hydrogen is one the main hurdles in itseconomic production. Furthermore, quantication of roles of un-explored enzymes in the hydrogen-producing pathway shows in-direct benets, and their identication can allow geneticengineers to modify their expression for causing high yield of hydrogen. The current available data give a wide range of effectsof gene knockout and overexpression on the yield and productiv-

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ity, which brings in high subjectivity and difcult decision mak-ing. More research in this area is needed to attain dependableresults. Negative effects of mutations need to be mitigated by careful analysis of their effects and exploration of ways to negatethese side effects. Dual systems also shall drive major research inthe coming years to increase hydrogen productivity and yields.Despite all the attempts made up to now, the maximum yieldobtained still stands very low, and it may be a long way until we

reach yields that may allow industrial applications. Nevertheless,the ux of research directed towards genetic engineering strate-gies recently hints at attaining this earlier than expected.

AcknowledgementKalyan Gayen acknowledges nancial support from Depart-

ment of Science and Technology, Government of India.

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