Improvement of biohydrogen production and intensification of biogas formation

Kornel L. Kovacs*, Akos T. Kovacs, Gergely Maroti, Zoltan Bagi, Gyula Csanadi, KatalinPerei, Balazs Balint, Judit Balogh, Andras Fulop, Lıvia S. Meszaros, Andras Toth, Reka David,Dora Latinovics, Andras Varga & Gabor RakhelyDepartment of Biotechnology, University of Szeged, and Institute of Biophysics, Biological Research Centre,Hungarian Academy of Sciences, Szeged, Hungary (*author for correspondence: phone: +36-62-544-351;fax: +36-62-544-352; e-mail: kornel@nucleus.szbk.u-szeged.hu)

Received 12 July 2004; accepted 9 December 2004

Key words: biogas, biohydrogen, biotechnology, hydrogenase, phototroph

Abstract

H2 is considered as the ultimate cleanest energy carrier to be generated from renewable sources. Thisminireview intends to point out that in addition to this function, biologically produced hydrogen isimportant for environmental biotechnological applications. The purple sulphur phototrophic bacterium,Thiocapsa roseopersicina BBS contains several NiFe hydrogenases. These enzymes can be used e.g., as fuelcell H2 splitting catalyst or in photoheterotrophic H2 production. Microorganisms that supply H2 in situfacilitate the biodegradation of organic material and concomitant biogas production. Fast, efficient, andeconomic treatment of organic waste, sludge, manure is achieved and generation of significant amount ofrenewable fuel from waste is intensified. The technology has been field tested under mesophilic and ther-mophilic conditions with positive results.

1. Catalysts of biohydrogen production

Understanding the molecular fundamentals ofhydrogen production and utilisation in biologicalsystems is a goal of supreme importance for basicand applied research (Cammack et al. 2001). Twogroups of enzymes participate in biologicalhydrogen metabolism: nitrogenases and hydrog-enases. Nitrogenase produces H2 as the byproductof N2 fixation. The latter requires reducing powerand in situ generated H2 is an excellent reductant.In order to maintain the reaction equilibrium inthe desired direction, nitrogenase produces H2 inexcess, which then diffuses out of the cell. Nitro-genases usually work very slowly and need muchchemical energy to carry out the nitrogen fixationfunction. The key enzyme in biological H2

metabolism is hydrogenase. This unique class ofenzymes catalyses the formation and decomposi-

tion of the simplest molecule occurring in biology:H2. The seemingly uncomplicated task is solved bysophisticated macromolecular machinery.Hydrogenases are metalloenzymes harbouring Niand Fe, or only Fe atoms, arranged in an excep-tional structure. This study focuses on thehydrogenases with NiFe active centres. Like mostredox metalloenzymes, hydrogenases areextremely sensitive to inactivation by oxygen, hightemperature, CO, CN and various environmentalfactors. These properties are not favourable formost biotechnological applications, includingbiohydrogen production, water denitrification,bioconversion of biomass, and other bioremedia-tion uses.

Their physiological function vary: hydrogen-ases serve as redox safety valves to dispose ofexcess reducing power, or generators of chemi-cal energy by taking up and oxidising H2, or

Reviews in Environmental Science & Bio/Technology (2004) 3: 321–330 � Springer 2005DOI: 10.1007/s11157-004-7460-2

maintaining a reducing environment for reactionsof crucial importance, such as the fixation ofatmospheric nitrogen. In some organisms, thenumerous functions are performed by the sameenzyme, but more frequently, a separate, special-ized hydrogenase carries out each in vivo bio-chemical function.

In metal-containing biological catalysts, it isthe protein matrix, surrounding the metal cen-tres, which provides the unique environment forthe Fe and Ni atoms and allows hydrogenases tofunction properly, selectively, and effectively. TheNiFe hydrogenases are composed of at least twodistinct polypeptides (heterodimer), containinghighly conserved metal binding domains. Thelarge subunit harbours the active centre, fastenedto the protein by 4 cysteine ligands. The Featom ligates 2 CN and 1 CO diatomic moleculesand it is fixed to the Ni atom via sulphurbridges. The small subunit contains 2–3 Fe4S4clusters, which are 15 angstroms apart, and thus,form a conducting wire inside the protein tofacilitate the transport of electrons between theactive centre and the protein surface. A majorgoal for hydrogenase basic research is tounderstand the intimate protein–metal interac-tion in this complex structure (Cammack et al.2001).

The problem is not simple to address, assome of the methods for scientific investigationprovide information on the metal atoms, withoutdirectly detecting the protein matrix aroundthem. Other modern techniques reveal details ofthe protein core, but do not expose the metalcentres within. A combination of the variousmolecular approaches is expected to uncover thefine molecular details of the catalytic action(Kovacs & Bagyinka 1990; Szilagyi et al. 2002).

A number of accessory gene-products governthe metal uptake, their attachment into the rightplace at the right time, the formation and liga-tion of the CN and CO groups, and the incor-poration and fixation of this labile inorganicstructure into the protein matrix. Our presentunderstanding suggests that the concerted actionof, at least, 15–20 such accessory proteins arenecessary for the formation of an active NiFehydrogenase (Cammack et al. 2001). Some of theparticipating proteins are hydrogenase pleio-tropic, called Hyp. They take part in the fabri-cation of every hydrogenase synthesised in the

cell. Others specifically work on one type ofNiFe. Sometimes, the accessory genes are neatlyarranged around the structural genes, but mostoften, they are scattered in the genome.

2. Limiting factors in biohydrogen production

2.1. Direct photolysis

The low solar energy density reaching the Earth’ssurface places severe economic limitations onpotential practical systems. In order to collectenough energy, large bioreactor surface area isneeded, which increases the cost for production ofhydrogen fuel. Photosynthetic energy conversionefficiency can be as high as 10%. This value is closeto the practical maximum value because the overallprocess is limited primarily by the efficacy of thedark reaction and not by the light energy captureprocess. Therefore, a great proportion of the ab-sorbed photon energy is wasted by the photosyn-thetic apparatus and dissipated as heat orfluorescence. Efficiencies close the 10% value havebeen demonstrated under low-light and low oxygenpartial pressure conditions (Greenbaum 1988).Unfortunately, these conditions are difficult tomaintain in a large-scale operation. The low oxygenpartial pressure is required by the apparentlyinherent oxygen sensitivity of the hydrogenasereaction. Approaches to overcome this probleminclude the use of O2 scavengers, but the involve-ment of irreversible or reversible O2 absorbersreduce the net efficiency. An increase of the respi-ratory activity (Rosenkranz & Krasna 1984) and asignificant research effort to increase oxygen toler-ance of hydrogenases are underway (Ghirardi et al.1997). Heterocystous cyanobacteria offer a possiblesolution of the problem (Lindberg et al. 2004). Theyare composed of two different cell types the vegeta-tive cells that carry out photosynthesis, includingO2

evolution, while H2 evolution takes place in theanaerobic environment of the heterocysts by thenitrogenase enzyme complex. However, the H2

evolving capacityofnitrogenases ismuch lower thanthat of the hydrogenases and the process requires alot of chemical energy, rendering the system eco-nomically unfeasible. In summary, practical directbiophotolysis systems should be highly resistant toO2 and even if the O2 inhibition problem could beovercome, the construction and maintenance costs

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of large photobioreactors should be solved beforelarge-scale applications are planned.

2.2. Indirect biophotolysis

Indirect biophotolysis (Benemann 1998) has theadvantage of separating in time the O2 and H2

evolution step. It involves a photosynthetic bio-mass production step and an anaerobic dark fer-mentation of the energy stored in biomass toproduce H2. If the oxygen and hydrogen evolutionsteps can be separated in time, the apparentlyinherent O2 sensitivity of the H2 evolving process iscircumvented. The separation takes place throughCO2 fixation and/or evolution.

Several conceptual schemes to achieve indirectbiophotolysis have been developed. These systemsuse algae in most cases and intend to exploit theircapability to produce high biomass yield per sur-face area photoautotrophically, to be able to carryout dark fermentation as well as photohetero-trophic growth. Detailed feasibility study of aconcept (Benemann 1998) that includes produc-tion of algal biomass, which is rich in easily fer-mentable storage carbohydrates, in open ponds;and subsequent anaerobic fermentation of thestored carbohydrates to yield 4H2 + 2acetate/glucose has been carried out. In the photofermen-tative step photoconversion of the acetates gives8H2/2acetate in a photobioreactor. The basic pro-cess can be supplemented with anaerobic digestion,i.e., biogas production, from the waste biomass andseparation of H2 and CO2 with a subsequentrecycling of CO2 to the algal growth ponds. Evenafter invoking the most up-to-date technologies,the process is estimated to be economically notfeasible, mostly due to the high production cost ofthe photobioreactors. A significant bioenergeticlimitation is the ultimate use of nitrogenase as H2

producing catalyst and nitrogenases are notoriousfor their extremely high chemical energy demand.Even if one assumes very high solar efficiencies andcarbohydrate content, the nitrogenase based H2

production suffers from high ATP requirement andlow turnover rate of the enzyme.

A H2 production process has been reported,which uses the green alga Chlamydomonas rein-hardtii (Melis et al. 2000). Separation of the pho-tosynthetic O2 evolution from H2 production intime is achieved via sulphur deprivation of the cells.Under these conditions, the biosynthesis of the

photosynthetic proteins is obstructed and the algastarts to respire the O2 evolved earlier photosyn-thetically. As O2 is consumed a whole array ofvarious fermentative pathways may turn on,including the degradation of the damaged proteinsand stored carbohydrates to yield H2. The disad-vantage of the concept is in the complexity of thedark anoxic reactions, which are difficult to under-stand and therefore difficult to control. A significantimprovement as compared to the nitrogenase basedsystems is, however, the use of Fe-hydrogenase forH2 production in Ch. reinhardtii as this enzyme hasseveral orders of magnitude higher H2 evolvingactivity than nitrogenase has (Posewitz et al. 2004).

2.3. Dark fermentation

Fermentative processes can use either biomassobtained in a photosynthetic solar energy conver-sion system, e.g., higher plants or microalgae con-taining starch or other fermentable storagecompounds, or various organic waste streams.Much is presently known about the molecularbiology of the H2 producing enzymes (Hallenbeck2001) and metabolic engineering to direct fermen-tation to H2 production is scientifically feasible(Keasling et al. 1998). Dark microbial H2 produc-tion is driven by the anaerobic metabolism of thekey intermediate, pyruvate. The theoretical maxi-mum of 4 moles of H2 +2 acetate per mole of glu-cose can be approached in thermophilic andhyperthermophilic systems (van Niel et al. 2002).The complete oxidation of glucose would yield astoichiometry of 12 moleH2 permole of glucose butin this case no energy is gained to support growthand metabolism of the producing organism. First,lower than the theoretical maximum yield is theconsequence of the fact that in nature fermentationserves to produce biomass and not hydrogen. Sec-ondly, even the low amount of H2 produced by thefermenting cells is considered as wasted energy bythe bacteria and therefore elaborated machineriesexist to recycle the evolved H2 in these cells. Theconsumed H2 contributes to the bioenergetic equi-librium of the cell. Thirdly, the H2 production yieldis negatively affected by the partial pressure of theproduct. Nevertheless, under carefully chosen con-ditions thermophiles produce up to 3.3–4 moles ofH2 per glucose demonstrating that higher hydrogenyields can be reached by extremophiles than usingmesophilic anaerobes (deVrije & Claassen 2003).

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The oxidative pentose phosphate pathway, asan alternative metabolic pathway exists forexample in microalgae, which can produce stoi-chiometric amount of H2 from glucose. However,this pathway does not work in practice as any suchreaction would not provide energy for cell growthand would thus be disadvantageous for the sur-vival of the strain. It is to be noted, that thepotential benefits of an in vitro system using thepentose phosphate pathway has been demon-strated (Woodward et al. 2000).

2.4. Photofermentation

Photosynthetic bacteria have long been studied fortheir capacity to produce significant amounts ofhydrogen (Sasikala et al. 1993). The advantage oftheir use is in the versatile metabolic capabilities ofthese organisms and the lack of Photosystem II,which automatically eliminates the difficultiesassociated with O2 inhibition of H2 production.Phototrophic bacteria require organic or inorganicelectron source to drive their photosynthesis. Thisis a small disadvantage because they can utilize awide range of cheap compounds. The significantdisadvantages in these systems are due to mostly oftheir use of the nitrogenase enzyme as H2 genera-tion catalyst (see above), to the requirement forelaborate and expensive anaerobic photobioreac-tors covering large areas, and to the low photo-synthetic efficiencies. Phototrophic bacteria are,like microalgae and cyanobacteria, optimized forlow light conditions prevalent in their naturalhabitat. Improvements might be sought by geneticmanipulation of the ratio of antenna pigments toreaction centers, but the construction of inexpen-sive photobioreactors remains an engineeringchallenge. The slow and highly energy demandingnitrogenase enzyme can be replaced by the newlydiscovered bidirectional NiFe hydrogenases inpurple sulphur phototrophic bacteria (see belowthe Thiocapsa roseopersicina case). Catalysts likethe Hox hydrogenase of T. roseopersicina maygenerate a renewed interest and novel routes forH2 production with photosynthetic bacteria. Pho-tosynthetic bacteria have been shown to produceH2 from various organic acids and food processingand agricultural wastes. Thus these systems offerthe advantage of combining organic waste disposalproblems with the production of the clean fuel H2.

In a proposed integrated system dark fermentationand photofermentation is combined in order toachieve maximal conversion of the substrate tobiohydrogen (deVrije & Claassen 2003).

3. Biohydrogen production possibilities using

Thiocapsa roseopersicina BBS

T. roseopersicina is a phototrophic purple sulphurbacterium; the strain marked BBS has been iso-lated from the cold water of the North Sea. Itsanaerobic photosynthesis uses reduced sulphurcompounds (sulphide, thiosulfide), but it can alsogrow on organic compounds (sugar, acetate) in thedark. The bacterium contains a nitrogenase en-zyme complex, thus it is capable of fixing atmo-spheric N2, a process accompanied by H2

production (Vignais et al. 1995). Previous studiesin our laboratory have revealed that T. roseoper-sicina contains at least two membrane-associatedNiFe hydrogenases with remarkable similaritiesand differences. One of them (HydSL/HynSL (forrecent nomenclature change see Vignais et al.,2001)) shows extraordinary stability: it is muchmore active at 80 �C, than around 25–28 �C evenafter isolation from the membrane. It is to be no-ted that T. roseopersicina cannot grow above30 �C. HynSL of T. roseopersicina is also reason-ably resistant to oxygen inactivation and staysactive after removal from the membrane. Theother NiFe hydrogenase, HupSL, is very sensitiveto all these environmental factors and thus itresembles the NiFe hydrogenases known fromother microorganisms. The structural genes codingfor these enzymes have been cloned and sequenced(Colbeau et al. 1994; Rakhely et al. 1998). Thetranslated protein sequences indicate a significantsequence homology between the two NiFe hy-drogenases. Despite the pronounced differences instability, the two small subunits are identical in46% of their amino acids and the two large su-bunits show 58% sequence identity. In order tounderstand the physiological roles of these hy-drogenases, mutants lacking either or both of themhave been generated in our laboratory by markerexchange mutagenesis. Much to our surprise, thehydrogenase-deleted mutants grew just as avidly asthe wild type strain. The phenomenon was finallyunderstood when two additional NiFe hydrogen-ases were discovered in the cytoplasm of the bac-

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terium (Figure 1). According to our currentunderstanding, there are four distinct NiFehydrogenase molecular species in T. roseopersici-na, representing all hydrogenase forms thus fardescribed in various micro-organisms, in a singlecell (Kovacs et al. 2002).

Little is known about HupUV, which appearsto be a hydrogen sensor protein (Cammack et al.2001). The gene cluster encoding for the cyto-plasmic NAD+ reducing NiFe hydrogenase(HoxEFUYH) was identified and sequenced. Thetranslated gene products showed the highest sim-ilarity to the corresponding subunits of thecyanobacterial bidirectional soluble hydrogenases.The five genes (hoxEFUYH) were localized on asingle transcript according to RT-PCR. The Hoxhydrogenase was proven to be a bi-directionalhydrogenase: it produced H2 under nitrogenase-repressed conditions and it recycled the hydrogenproduced by the nitrogenase in cells fixing N2

(Figures 2 and 3). This is the first example indi-cating that such kind of a hydrogenase is presentin phototrophic bacteria (Rakhely et al. 2004).The Hox enzyme opens the possibility for biohy-

drogen production via a new and highly efficientmechanism.

This makes T. roseopersicina one of the bestcandidates for studies of NiFe hydrogenasestructure–function relationships and assembly.The outstanding situation allows us to addressspecific questions concerning the assembly of eachof these enzymes and the regulation of their bio-synthesis. Answers to these questions will be ofdirect relevance in designing an optimal catalystfor biological hydrogen production and/or util-isation and to protein engineering of scientificallyintriguing and biotechnologically important redoxenzymes in general. From the fragmented infor-mation available, there is no clear answer as towhy T. roseopersicina needs so many distincthydrogenases. Our working hypothesis links thisabundance of various NiFe hydrogenases to thefact that this bacterium should be able to performvarious metabolic activities (photoautotrophic,photoheterotrophic, heterotrophic metabolism) inorder to survive in its natural habitat (Imhoff2001). Having numerous hydrogenases at handincreases the chances of survival for the bacterium

Figure 1. Hydrogenases in Thiocapsa roseopersicina. The core hydrogenase heterodimer, harbouring the active center and iron-sulphurclusters, is indicated with black colour, structural gene products associated with the heterodimer are white. The Hyn and Hup arehydrogen-uptake enzymes and are associated with the photosynthetic membrane. The bidirectional Hox and sensor HupUV arelocated in the cytoplasm. Hyn may interact with the photosynthetic apparatus and with sulphur metabolism, the physiological functionof Hup is to recycle excess H2 produced by the nitrogenease complex. Note, that the orientation of the membrane bound hydrogenasesallows them to produce ATP during the H2 consumption reaction.

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Figure 2. Hydrogen production in the absence of NiFe hydrogenases in T. roseopersicina. HypF is an accessory protein that is requiredfor the biosynthesis of any NiFe hydrogenases. Under nitrogen fixing growth conditions the nitrogenase enzyme complex forms H2,which is released into the environment as the hydrogen-uptake hydrogenases are missing.

Figure 3. Hydrogen production by the Hox NiFe hydrogenase in T. roseopersicina. In the absence of active Hyn and Hup (mutation inthe corresponding structural genes or deletion of the HupK accessory gene), the soluble Hox enzyme generates H2, which is releasedinto the environment because the uptake-type hydrogenases cannot recycle the H2. This hydrogen production is independent from thenitrogenase system and therefore does not consume much of ATP.

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and increases our chances to understand basicphenomena of hydrogenase catalysis.

Random transposon mutagenesis wasemployed to trace the accessory proteins neededfor the proper assembly of NiFe hydrogenases inT. roseopersicina. One of the mutations was in agene showing high sequence similarity to HypFproteins in other organisms. The reconstructedhypF gene was able to complement the hypF defi-cient mutant of T. roseopersicina BBS. Heterolo-gous complementation experiments, using hypF)

strains of T. roseopersicina, Escherichia coli, andRalstonia eutropha, and various hypF genes wereperformed. Heterologous complementation wassuccessful in all of the cases tested. RT-PCR datasuggested that HypF has no effect on the tran-scriptional regulation of the structural genes ofhydrogenases in this organism (Fodor et al. 2001).Inactivation of hydrogen uptake activity of allNiFe hydrogenases in the hypF deficient mutantresulted in a 60-fold increase in hydrogen evolu-tion capacity of T. roseopersicina under nitrogenfixing conditions (Figure 2).

The role of the HupK (HoxV) in the matura-tion process of the NiFe hydrogenases isunknown. Conserved regions could be recognizedat the N- and C- termini, while the middle portionof the proteins appeared variable. The highesthomology was found at the C-terminus and,remarkably, this region showed significant identityto the HupL (hydrogenase large subunit) proteinsas well, although half of the conserved cysteineswere missing. In-frame deletion mutagenesis wasused to determine the specificity of the HupKprotein. The physiological effects of the mutationon the hydrogenase enzyme activities were testedin H2 uptake activity assays of each individualNiFe hydrogenase enzyme in T. roseopersicina.Approximately 10% of both membrane-associatedHynSL and HupSL activities were preserved incomparison to the wild type. On the contrary, thesoluble fraction retained almost all of its activity;around 75% of Hox activity was detectable in theHupK deleted strain, with respect to the wild type.Homologous complementation with the hupK genefully restored the hydrogenase activity of the cells.This has further proven the selectivity of HupK,which is important for the formation of bothfunctionally intact membrane-associated NiFehydrogenases, but it is not involved in the matu-ration of the soluble Hox enzyme in this bacte-

rium. Although our study did not uncover theprecise function of HupK, this was the first dem-onstration that this protein made a selectionamong the various NiFe hydrogenases in the cell,and participated in the biosynthesis of the mem-brane bound ones (Maroti et al. 2003). The HupKmutant strain displayed a remarkable H2 produc-tion capacity due to the lack of uptake hydroge-nase activity (HupSL and/or HynSL) anduninhibited expression of the bi-directional Hox-YH (Figure 3). A set of modular broad host rangeexpression vectors with various affinity tags (6His,FLAG, StrepII) were created. The vectors aresmall, and mobilizable by conjugation. Such vec-tors would be particularly useful in the purificationof proteins and protein complexes from variousbacteria (Fodor et al. 2004).

4. Utilisation of hydrogen metabolism in biogas

production

Hydrogen evolution by intact bacterial cells isfrequently observed in the laboratory. In microbialecosystems the role of these microorganisms iscreation and maintenance of anaerobic, reductiveenvironment as well as supplying the universalreducing agent, molecular hydrogen. Gaseoushydrogen is usually not released from the naturalecosystems unless there is an excess of reductivepower which needs to be disposed of in order toensure the optimal metabolic and redox equilib-rium in the population. H2 generated in vivo byhydrogen forming bacteria is utilized by hydrogenconsuming members of the microbiological com-munity. Hydrogen is transferred to the recipientmicro-organism very effectively by interspecieshydrogen transfer. The molecular details of thisprocess are not fully understood, but its signifi-cance in safeguarding the optimum performanceof the entire ecosystem and the delicate regulatorymechanisms should be appreciated. In the mixedpopulation bacterial systems the advantages ofinterspecies hydrogen transfer are exploited(Kovacs & Polyak 1991).

Decomposition of wastes anaerobically to formbiogas is one of the earliest applications of bio-technology. Let us turn first briefly to the micro-biology of the biogas formation (Ahring 2003). Itis well known that three distinct microbe popula-tions take part in the anaerobic digestion process.

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These microbe populations are the polymerdegrading, so-called hydrolyzing bacteria, theacetogens and the methanogens (Angelidaki et al.2003). The first group, the polymer degradersattack the macromolecules using extracellularenzymes and producing intermediers. Because ofits abundance in Nature, cellulose is the mainsubstrate for hydrolyzing bacteria. The acetogensthen use these sugars and oligosaccharides andproduce organic acids, like acetate, succinate,formate, propionate, and CO2. The third group isthe methanogens. These microorganisms belong tothe Archaebacteria and thus possess uniquemolecular and cellular properties. They producemethane using acetate, hydrogen and carbondi-oxide. The major part of the carbon flow takesplace between the acetogens and methanogens(Pind et al. 2003). The products of the first twogroups of microorganisms, responsible for hydro-lyzing and fermenting the organic material to H2

and acetate, should be simultaneously used by themethanogens. In particular a sensitive balanceexists between the acetogens and methanogens(Zeeman & Lettinga 1999). The biogas formationbecomes thermodynamically possible whenhydrogen is kept below a certain concentration.This is why H2 is barely detectable in biogas. Atthe same time, the biological activity of methano-gens require a good supply of hydrogen in order tocarry out the redox reaction. The relationshipbetween the acetogens and methanogens is a syn-trophic one and the process is called interspecieshydrogen transfer (Thiele & Zeikus 1988). Thisphenomenon makes possible that the acetogensprovide enough hydrogen without raising the H2

concentration in the system. The syntrophic part-ners become physically attached to each other andthe microbial consortia tend to form flocs orgranules to facilitate interspecies hydrogen transfer(Lettinga & Haandel 1993). The often macroscopicstructures endow the biogas production with sig-nificantly increased yield (Ahring 2003). The phe-nomenon is exploited particularly efficiently inwastewater and wastewater sludge treatment(Gray 2004). The conditions can only be metwithin a narrow range of hydrogen concentrations(Hofman-Bang et al. 2003).

It is widely recognized that H2 has an impor-tant role in the anaerobic fermentation. The reg-ulatory roles of hydrogen levels and interspecieshydrogen transfer optimize the concerted action of

the entire population. The concentration of eitheracetate or hydrogen, or both together, can bereduced sufficiently to provide a favorable free-energy change for propionate oxidation. Duringanaerobic biodegradation hydrogen concentrationis reduced to a much lower level than that ofacetate. The acetate concentration in an anaerobicdigester tends to range between 10

)4

and 10)1

M,H2 ranges between 10

)8

and 10)5

M, or about fourorders of magnitude less. In addition, the hydro-gen partial pressure can change rapidly, perhapsvarying by an order of magnitude or more within afew minutes. This is related to its rapid turnoverrate. The energy available to the acetate-usingmethanogens is independent of hydrogen partialpressure, whereas that of the hydrogen-producingand hydrogen-consuming species is very much afunction of it.

The low concentration of free hydrogen withinthe digester has implications for the rate of H2

turnover in the system. In a typical system theturnover rate of the hydrogen pool is 6.8 · 10

6

/dayor about 80/s. The interspecies H2 transport istherefore a very rapid, delicate, and rate limitingstep. For processes on this microscale, diffusion isthe main mechanism for hydrogen transportbetween species. The calculations clearly indicatethat the optimal distance between interactinghydrogen producing and consuming species isabout 10 lm under practical conditions. This dis-tance is equal to about 10 bacterial widths.Clearly, the bacteria must indeed be close together,otherwise interspecies hydrogen transfer will be therate limiting step in the overall process. This is thecase in an ideal, completely mixed or continuousstirred tank reactor where the individual bacterialspecies are dispersed uniformly throughout thesystem, and the distribution of reactants, inter-mediates, products of biotransformation, andbacterial species is homogeneous throughout thereactor (Miron et al. 2000).

An unexplored possibility to exploit the linkbetween biohydrogen and biogas production is theutilization of the acetate (volatile organic acids)generated during dark hydrogen fermentation(deVrije & Claassen 2003) discussed above assubstrate for methanogenesis. Organic matter isnot completely oxidized to CO2 under the O2

limited anaerobic conditions of biohydrogen pro-duction. Intermediary compounds, like acetic acidor lactate may therefore accumulate. These par-

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tially reduced carbonaceous compounds need to beutilized for making a positive balance in estimatingthe energy balance from organic matter. Acetateand other organic acids, generated as a byproductof the anaerobic biohydrogen fermentation, can befurther metabolized to methane in biogas pro-ducing systems. The drawbacks of incompletedark biohydrogen fermentation thus may be cou-pled to the production of an increased rate andamount of biogas from organic residues. Theeconomic advantages of such systems are obvious.

Acknowledgements

The work has been supported by EU 5th Frame-work Programme projects (QLK5-1999-01267,QLK3-2000-01528, QLK3-2001-01676) and bydomestic sources (OTKA, FKFP, OMFB, OMKFHAT). International collaboration within theEU network COST Action 841 is greatly appreci-ated.

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