trends in biotechnology 27 (2009) 287-297

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Advances in fermentative biohydrogenproduction: the way forward?Patrick C. Hallenbeck and Dipankar Ghosh

Departement de Microbiologie et Immunologie, Universite de Montreal, CP 6128 Succursale Centre-ville, Montreal, Quebec H3C

3J7, Canada

A significant effort is underway to develop biofuels as

replacements for non-renewable fossil fuels. Among the

various options, hydrogen is an attractive future energy

carrier due to its potentially higher efficiency of conver-

sion to usable power, low generation of pollutants and

high energy density. There are a variety of technologies

for biological hydrogen production; here, we concentrate

on fermentativehydrogen production and highlight some

recently applied approaches, such as response surfacemethodology, different reactor configurations and organ-

isms that have been used to maximize hydrogen pro-

duction rates and yields. However, there are significant

remaining barriers to practical application, such as low

yields and production rates, and we discuss several

methods, including two stage processes and metabolic

engineering, that are aimed at overcoming these barriers.

Introduction

The joint challenges of anthropogenic climate change and

dwindling fossil fuel reserves are driving intense research

into alternative energy sources. One attractive avenue is to

use a biological process to produce a biofuel. Among the

various biofuel options, biohydrogen gas is an attractivefuture energy carrier due to its potentially higher efficiency

of conversion to usable power, low to non-existent gener-

ation of pollutants and high energy density. Biological

hydrogen production processes can be classified into three

major categories: biophotolysis of water using algae and

cyanobacteria; photodecomposition of organic compounds

by photosynthetic bacteria (photofermentation); and fer-

mentative hydrogen production from organic wastes or

energy crops (Box 1) [1]. However, low yields and pro-

duction rates have been major barriers to the practical

application of biohydrogen technologies.

Intensive research on biohydrogen is underway, and in

the last few years several novel approaches have beenproposed and studied to surpass these drawbacks. Here

we concentrate mainly on fermentative hydrogen pro-

duction, for which there has been a virtual explosion of

research articles lately (but not hydrogen, not yet!), and

many recent reviews on specific aspects are available [27].

We discuss novel techniques, and improvements to exist-

ing techniques, that have been used in attempts to increase

hydrogen production rates and yields. Some of the dis-

cussed techniques have permitted large increases in hydro-

gen production rates, and this advance has brought

fermentative hydrogen production to the point where pro-

duction rates with real substrates (wastes) under realistic

conditions are approaching practical levels. Of course, it is

too premature to be able to exactly define what might be a

practical level for biohydrogen production, but a rough

yardstick can be obtained by examining a detailed process

analysis of lignocellulosic ethanol production [8], where

design metrics centered around a plant capable of produ-

cing 50 to 100 kJ/l/h of ethanol; the hydrogen equivalent

would be 5 to 10 l/l/h.The major remaining stumbling block is incomplete sub-

strate conversion and the consequent low yields. Traditional

techniques, such as the manipulation and optimization of

bioprocess parameters or of the organisms used, have failed

to achieve more than 25% conversion of substrate to H2.

Indeed, this is a direct consequence of the thermodynamics

of known metabolic processes, a fact pointed out theoreti-

cally over thirty years ago [9] and now amply demonstrated

in practice (for an example, see [10]). In addition to H2,

hydrogen-fermenting microbes make other products to

satisfy their metabolic needs and to further growth; these

include acetate, which permits ATP synthesis, and a variety

of reduced products (for example, ethanol, butanol and

Review

Glossary

ANN (artificial neural network): a computational statistical modeling process

based on the connectivity of biological neurons. It is nonlinear and adaptive

(i.e. it learns).

COD (chemical oxygen demand): commonly used to determine the amount of

organic compounds in water.

CSTR (continuous [flow] stirred tank reactor): the simplest reactor for

continuous operation. Essentially, it is a chemostat.

DOE (design of experiments): a structured method used in determining the

relationship between a series of variables in a process and the output of that

process. It should predict the important parameters and the relationship

between them.

FD (factorial design): a statistical method that shows the relationship between

a response and two or more experimental factors that are varied simulta-

neously.

HRT (hydraulic retention time): the residence time of the liquid in a

continuously fed reactor.

MEC (microbial electrohydrogenesis cell): a bioelectrochemical cell, basically a

modified microbial fuel cell (MFC), in which an applied voltage drives H2evolution.

OLR (organic loading rate): the concentration of the substrate, usually

expressed in g/l, that is fed to the reactor.

Reverse micelle (also called inverted micelle): a micelle formed by amphiphilic

surfactants in which the polar heads are oriented inwards encapsulating a

polar (water) phase and the hydrophobic tails are in contact with a nonpolar

solvent at the exterior.

RSM (response surface methodology): a computational method that explores

the relationship between variables that are subject to experimental manipula-

tion and the output from the system, in this case hydrogen.

UASB (upflow anaerobic sludge blanket): a type of reactor in which the

biomass (functioning as a catalyst) is immobilized, thus rendering microbial

growth independent of the liquid phase flow rate through the reactor.Corresponding author: Hallenbeck, P.C. ([email protected]).

0167-7799/$ see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2009.02.004 Available online 28 March 2009 287
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butyric acid) that permit the reoxidation of NADH, which is

necessary for continuing glycolysis (see Figures 1 and 2).

Types and relative proportions of products depends upon

the organism, environmental conditions (pH and the partial

pressure of hydrogen) and the oxidation state of the

substrate being degraded. Thus, fermentations are thought

to be limited to producing at most either 2 H2/glucose

(enteric bacterial type mixed acid fermentation) or 4 H2/

glucose (clostridia-type fermentation) [2]. Therefore, hydro-

gen yields are low, and two-thirds of the carbon and protons

Box 1. Basic biohydrogen production technologies

All biohydrogen technologies depend on either a hydrogenase or

nitrogenase for hydrogen evolution and derive energy either directly

from light energy or indirectly by consuming photosynthetically

derived carbon compounds. Each approach has positive and negative

aspects, and each has serious technical barriers that need to be

overcome before it could become practical.

Direct biophotolysis (Figure I)

In this process, an organism, for example a green alga orcyanobacterium, that carries out plant-type photosynthesis uses

captured solar energy to split water (producing O2) and reduce

ferredoxin, which can in turn reduce a hydrogenase or nitrogenase,

both of which are oxygen sensitive, producing H2.

Advantages

Abundant substrate (water); simple products (H2 and CO2).

Disadvantages

Low light conversion efficiencies; oxygen-sensitive hydrogenase;

expensive hydrogen impermeable photobioreactors required.

Photofermentation (Figure II)

In this process, a non-sulfur purple photosynthetic bacterium,

carrying out an anaerobic photosynthesis, uses captured solar energy

to produce ATP and high energy electrons (through reverse electron

flow) that reduce ferredoxin. ATP and reduced ferredoxin drive proton

reduction to hydrogen by nitrogenase. These organisms cannotderive electrons from water and therefore use organic compounds,

usually organic acids, as substrates.

Advantages

Complete conversion of organic acid wastes to H2and CO2; potential

waste treatment credits.

Disadvantages

Low light conversion efficiencies; high energy demand by nitrogen-

ase; expensive hydrogen impermeable photobioreactors required.

Dark fermentation (Figure III)

In this process, a variety of different microbes can be used

anaerobically to breakdown carbohydrate-rich substrates to hydrogen

and other products, principally acids (lactic, acetic, butyric, etc.) and

alcohols (ethanol, butanol, etc.). Product distribution can be different

dependent upon the microbe, oxidation state of the substrate and

environmental conditions (pH, hydrogen partial pressure).

Advantages

No direct solar input needed; variety of waste streams/energy crops

can be used; simple reactor technology.

Disadvantages

Low H2 yields; large quantities of side products formed.

Figure II. Photofermentation.

Figure III. Dark fermentation.

Figure I. Biophotolysis.

Review Trends in Biotechnology Vol.27 No.5

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in the substrate are excreted as other products and wasted.

Several approaches, including metabolic engineering and

various two-stage processes, are being actively investigated

in an attempt to solve this problem, and these are discussed

below. First, we examine some traditional techniques that

havebeen applied for biohydrogen production and discuss to

what extent they have, or might, contribute to increasing

biohydrogen production.

Techniques for improved biohydrogen

Reactor configurations

Possible improvements to biohydrogen production have

been sought through specialized bioreactor configurations

(seeTable 1). This has led to systems with more robust,

reliable performance that are stable over long periods of

time (months) and resistant to short-term fluctuations in

operational parameters. In addition, optimized volu-

metric production rates could be obtained. Biohydrogen

fermentations, as most other fermentations, can be car-

ried out in either batch or continuous mode. Batch mode

fermentations have been shown to be more suitable for

initial optimization studies [4,11], but any industrially

feasible process would most likely have to be performed on

a continuous or at least semi-continuous (fed or sequen-

cing batch) basis. Many studies have employed continuous

stirred tank reactors (CSTRs, see Glossary) with either

purified strains or microbial mixtures [4,5,11]. CSTRs,the

most commonly used continuous reactor systems, offer

simple construction, ease of operation and effective hom-

ogenous mixing. However, in these reactors, hydraulicretention time (HRT, seeGlossary) controls the microbial

growth rate and therefore HRTs must be greater than the

maximum growth rate of the organism(s), because faster

dilution rates cause washout. A second conceptual

category of continuous flow reactors, characterized by

the means of the physical retention of the microbial bio-

mass, overcomes this problem and offers several advan-

tages for a practical bioprocess. Because microbial growth

and the concentration of microbial biomass are rendered

independent of HRT, high cell concentrations can be

achieved, fostering high volumetric production rates,

and high throughput is possible, allowing the use (and

Figure 1. Hydrogen production during mixed-acid fermentation by Escherchia coli. The mixed-acid fermentation carried out by E. coliis schematically shown. Different

fermentation products can arise and their relative amounts can change depending upon the redox state of the substrate and the pH of the culture. Lactate formation and

formate degradation to CO2 and H2 are induced at acidic pH, which serves to relieve the acid stress. Succinate production from phosphoenolpyruvate is also possible but

usually represents only a minor fraction of total fermentation products. The relative proportions of products are balanced to maximize ATP production (via acetate

production from acetyl-CoA by theptaackApathway) while at the same time reoxidizing NADH through the formation of reduced products, such as ethanol, to provide the

NAD needed by the glycolytic pathway for further substrate utilization. Acetyl coenzyme A (acetyl-CoA) and formate are formed from pyruvate by pyruvateformate lyase.

Pathways that have been eliminated with the aim of increasing hydrogen production are shown by dashed red crosses. Enzymes in the FHL (formate hydrogen lyase)

pathway that have been upregulated to increase hydrogen production are shown by green arrows. The standard designations for the genes encoding the enzymes for the

various steps shown are given in italics: adhE, alcohol dehydrogenase E; fdh, formate dehydrogenase; frdABCD, fumarate reductase A, B, C and D; fumBC, fumaratehydratase B and C; hyd1, (for convenience this stands for the operons necessary to express hydrogenase 1 activity); hyd2, (for convenience this stands for the operons

necessary to express hydrogenase 2 activity); hyd3, (for convenience this stands for the operons necessary to express hydrogenase 3 activity); ldhA, lactate dehydrogenase

A; mdh, malate dehydrogenase; pfl, pyruvateformate lyase; ptaackA, phosphotransacetylaseacetate kinase A.


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Figure 2. Different possible two-stage systems for the complete conversion of substrate. In the first stage (shown on the left) substrate is fermented to hydrogen and

various fermentation products, which vary with organism and conditions. The two types of fermentation pathways are shown within the box on the left. Glycolysis of

sugars produces pyruvate, from which hydrogen and other fermentation products are derived. Hydrogen is either produced from formate via an Ech (NiFe) hydrogenase

(H2ase) in enterobacterial-type fermentation (right-hand side of the box) or from reduced ferredoxin via an FeFe hydrogenase in Clostridia-type fermentations (left-hand side

of blue box). Additionally, hydrogen can be derived from NADH in Clostridia-type fermentations, although the molecular details are unclear and might involve an NADH-

dependent FeFe hydrogenase, at least in a few cases (as shown by the dashed pink box). In both cases, the formation of acetate provides ATP synthesis. As discussed in the

main text, acetate and the other fermentation products can then be fed into a second stage reactor (right panels), in which additional energy is extracted, either in the formof methane in the case of anaerobic digestion or hydrogen for photofermentations or MECs. In the latter case, additional energy is required for the second stage of the

process: either light for photofermentation or electricity for MECs. Anaerobic digestion as a second stage is shown on the upper right side. Anaerobic digestors comprise a

community of different organisms whose concerted action is to convert the various fermentation products of the first stage into methane. Archaea carry out

methanogenesis (blue hexagon) and can directly use the acetate produced in the first stage. Associated syntrophic bacteria (purple trapezoid) oxidize other fermentation

products from the first stage to substrates that can be used in methanogenesis, such as acetate, H 2and CO2. Photofermentation as a second-stage process is shown in the

middle right scheme. Usually, a pure culture of a purple non-sulfur photosynthetic bacterium is used to convert the organic acids produced in the first stage dark

fermentation to hydrogen. These organisms use the captured light energy to drive reverse electron flow (light blue cylinder), producing the necessary low potential

electrons as well as the ATP required for the nitrogenase (N 2ase), which in this case is the hydrogen-producing enzyme system (shown in red). A variation on this theme

would be inclusion of both metabolic types of organisms in a single photobioreactor. Indeed, it has been shown that glucose could be converted to hydrogen at a

respectable yield of 7 H2/glucose (60%) by an immobilized co-culture consisting of Lactobacillus, which converted glucose to lactate, and the photosynthetic bacterium

Rhodobacter sphaeroides, which converted the lactate to hydrogen [67]. A microbial electrohydrogenesis cell as a second stage is shown in the lower right scheme. Here,

bacteria in the anodic chamber (shown in green) degrade the fermentation products from the first stage and donate electrons to the anode. Additional voltage is added via a

power supply and hydrogen is evolved at the cathode in the cathodic chamber (shown in blue).


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treatment) of dilute waste streams with relatively small

volume reactors.

Indeed, many recent studies [3,5,1122] have shown

that high volumetric production rates of hydrogen can

be achieved in these reactors, and some examples are given

in Table 1. Physical retention of microbial biomass has

been accomplished by several different means, including

the use of naturally forming flocs or granules of self-

immobilized microbes, microbial immobilization on inertmaterials, microbial-based biofilms or retentive mem-

branes. In fact, there are so many variations that there

are almost as many different reactor types, with associated

acronyms (UASB, FBBAC, AFBR, ASBR, CIGSB, FBR), as

there are laboratories carrying out research in this area

(Table 1). Unfortunately, this also creates a situation in

which it is very difficult to ascertain whether the differ-

ences in the various studies are due to different reactor

configurations or to differences in operational parameters.

Future studies could help to resolve this ambiguity by

directly comparing different reactor configurations or, at

the very least, by operating a novel configuration under

conditions used in previous studies. One study that specifi-

cally compared two different reactor types showed that

granules gave superior performance over a biofilm, with an

approximately threefold higher hydrogen production rate

[21]. In general, the major advance of these types of

reactors over a CSTR is their greatly increased volumetric

production rate, which in some cases can be up to an

astounding>50-fold larger (see Table 1). In addition, these

reactor types present a favorable environment for the de-

velopment and maintenance of mixed microbial consortia,

which have advantages that are discussed below. A poten-

tial problem with these types of reactors is the loss of

hydrogen through the formation of methane. Because cell

growth is no longer directly controlled by HRT in systems

using retained microbial biomass, slow growing methano-gens can flourish even at high rates of liquid throughput. It

should be noted that the hydrogen yields obtained with

these systems are no greater than those achieved with

CSTRs, and indeed there is no reason to think that the

reactor type would influence the yield.

The use of mixed microbial consortia

Most of the reactors described above depend upon the

formation of flocs or granules, which are macroscopic

aggregates of microbial cells. The ability to form these

particles is a rare trait in a pure culture, but it can be

easily selected for when a mixed culture is used as an

inoculum. There are also a number of other potential

advantages of using microbial consortia instead of pure

cultures. Industrial hydrogen fermentations will have to be

carried out under non-sterile conditions using readily

available complex feedstocks with only minimal pretreat-

ment. Microbial consortia address these issues as they

have been selected for growth and dominance under

non-sterile conditions. As a complex community they are

also likely to contain a suite of the necessary hydrolyticactivities, and they are potentially more robust to changes

in environmental conditions[23].

A large number of recent studies have examined hydro-

gen production using microbial consortia, and some

relevant examples are given in Table 1 (others can be

found elsewhere[24]). The use of microbial consortia has

indeed been proven useful in reactor systems that yield

high volumetric rates of hydrogen production, as discussed

above. However, there are also several issues associated

with their use. As complex communities, their composition

can vary over time, with changes in process parameters

and from reactor to reactor, as was shown by molecular

(16sRNA) studies[2528]. A possible way to overcome this

issue might be to construct designer consortia [29] with

the goal of creating a community of diverse members, each

contributing a unique and essential metabolic capacity.

The total community metabolic range would be greater

than any individual member, while at the same time

mutual interdependence would assure stable maintenance

of individual members. However, little is known about the

complex interactions that occur in natural consortia or how

stable synthetic microbial communities could be built[30].

Thus, much additional fundamental work might be

required before practically useful synthetic hydrogen-pro-

ducing consortia could become a reality. It seems that

spatial organization within a consortium might be import-

ant[31].

Improving biohydrogen production by metabolic

engineering of existing pathways

Metabolic engineering has recently received increased

attention in attempts to increase biohydrogen production,

in particular hydrogen yields [3240]. Improvements in

hydrogen production by existing pathways can be sought

by increasing the flux through gene knockouts of competing

pathways or increased homologous expression of enzymes

involved in the hydrogen-generating pathways. The

majority of studies employing these strategies have used

the laboratory workhorse for metabolic engineering,

Table 1. Available dark fermentation reactors

Microorganisms Substrate Type of reactor H2 rate (l H2/l/h) Refs

Sludge (wastewater treatment plant) M olasses Cont inuous stirred- tan k r eactor (CSTR) 0.20 [12]

Sludge (wastewater treatment plant) Glucose Ana erobic sequencing batch rea ctor (ASBR) 0. 23 [13]

Sludge (wastewater treatment plant) Sucrose Fixed bed bioreactor with activated carbon (FBBAC) 1.2 [14]

Activated sludge and digested sludge Glucose Anaerobic fluidized bed reactor (AFBR) 2.4 [15]

Sludge (wastewater treatment plant) Sucrose Upflow anaerobic sludge blanket reactor (UASB) 0.27 [16]

Anaerobic sludge Sucrose Polymethymethacrylate (PMMA) immobilized cells 1.8 [17]

Sludge (wastewater treatment plant) Sucrose Carrier-induced granular sludge bed (CIGSB) 9.3 [18]

Sludge (wastewater treatment plant) Sucrose Fluidized bed reactor (FBR) 1.4 [19]

Sludge (wastewater treatment plant) Glucose Anaerobic fluidi ze d bed reactor (AF BR) 7 .6 biofilm; 6.6 gra nul es [20]

Sludge (wastewater treatment plant) Sucrose Continuously stirred anaerobic bioreactor (CSABR) 15.0 [19]

Heat-treated soil Glucose Membrane bioreactor (MBR) 0.38 [21]


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Escherchia coli (Table 2, Figure 1). The rationale behind

usingE. coli has been: (i) its genome can be easily manipu-

lated; (ii) its metabolism is the best understood of all

bacteria; and (iii) it readily degrades a variety of sugars.

On the downside is the fact that its metabolism restricts

hydrogen yields to 2 H2/glucose[2].

Nevertheless, the use ofE. coli has served as a proof of

principal that metabolic engineering can be used to achieve

the maximal yields of hydrogen predicted by theory (see

Table 2 and Figure 1 for the genes that have been tar-geted). The inactivation of pathways that drain the pyr-

uvate pool, the source of electrons (via formate) for proton

reduction, is expected to increase hydrogen production,

and modest increases in H2yields were found when ldhA

(lactate dehydrogenase) [3336] or frd (fumarate

reductase) [35,36] were inactivated. In E. coli, formate,

which is formed from pyruvate, is degraded to H2and CO2by the FHL (formatehydrogen lyase) pathway, and

manipulations that led to an increase in the expression

of enzymes, such as FdhH (formate dehydrogenase H) and

Hyd3 (hydrogenase 3), involved in this pathway also

increased H2 yields [3336]. Moreover, the total amount

of hydrogen produced can be increased by inactivating the

hydrogenases Hyd1 and Hyd2, which are potentially able

to oxidize it [2]. These effects are additive, and mutated

strains of E. coli harboring multiple relevant mutations

demonstrated H2yields that were close to the theoretical

maximum of 2 H2/glucose for the metabolic pathways that

exist in this organism [2]. Although less studied so far,

similar metabolic engineering strategies could be applied

to other organisms. However, it would not seem possible to

surpass present metabolic limits (2 to 4 H2/glucose) by this

approach.

Other attempts

Modeling and optimization have been carried out in

attempts to improve biohydrogen production. Rates andyields of hydrogen production, as for many other biopro-

cesses, are a function of several variables, including pH,

temperature, substrate concentration and nutrient avail-

ability, among others. Although many studies have

reported the effects of varying individual parameters

one-at-a-time (for a partial compilation, see [11]), modeling

and analysis could be used to determine the optimal values

of the important relevant parameters. A variety of model-

ing methods have been developed that are broadly

applicable to a spectrum of diverse fields, including engin-

eering, biology, environmental science, food processing and

industrial processing, and their application in biohydrogen

production is the subject of a very recent review [41].

Although the metabolic pathways and fluxes are relatively

well understood for a pure culture fermenting a defined

substrate, this type of analysis can nevertheless have some

value for modeling fermentations of complex substrates by

microbial consortia that involve multiple metabolic types

with unknown interactions.

One type of treatment, principal component analysis

[42], was recently used to assess the effects of pH, HRT and

mixing on hydrogen production [43]. Two main types ofwidely used modeling approaches that are pertinent to

BioH2 production are artificial neural networks (ANNs,

seeGlossary) and design of experiments (DOE, see Glos-

sary). ANNs were successfully used to model the results of

hydrogen production and chemical oxygen demand (COD,

see Glossary) removal with an upflow anaerobic sludge

blanket reactor (UASB, see Glossary) [44] and an expanded

granular sludge bed (EGSB) reactor[45]. DOE uses stat-

istical modeling to analyze the relationship between a set

of controllable independent experimental factors and to

propose an appropriate design for their experimental ver-

ification[46]. A common and powerful DOE approach is

afforded by factorial design (FD) combined with response

surface methodology (RSM, seeGlossary) [47]. FD inves-

tigates how responses of multiple factors depend on each

other and permits the identification of the most important

parameters that control the process and the degree of

interaction among them. FD approaches can be coupled

to experimental systems by RSM, which uses a response

function to fit the obtained experimental data to the theor-

etical design.

Several recent studies investigating dark fermentative

hydrogen production have applied FD and RSM with the

aim of optimizing hydrogen production (Table 3). Their

results demonstrate that this type of analysis can indeed

be used to optimize various aspects of a biohydrogen

process, including pretreatment strategies [48,49], con-ditions for spore germination [50], micronutrient formu-

lations [5153], substrate composition [34], carbon/

nitrogen (C/N) and carbon/phosphate (C/P) [53]. The most

useful application has been to determine the optimal oper-

ating conditions with regard to HRT [13,55,56], pH [5761]

and substrate concentration, that is, the organic loading

rate (OLR, seeGlossary)[5661]. Although all mesophilic

fermentations will require similar conditions, there are

sufficient differences in some of the parameters to justify

the need for optimization of a particular system.

In addition, these methods could prove helpful in establish-

ing process parameters for novel consortia or complex

Table 2. Targets for modification of existing pathways in Escherchia coli

Gene Function Mode of action Effect on H2productiona Refs

ldhA Lactate dehydrogenase Inactivation eliminates a drain on the pyruvate pool Modest increase (2030%) [3336]

frdBC Fumarate reductase Inactivation eliminates side reaction in EMP thereby increasing

pyruvate pool

Modest increase (2030%) [35,36]

hycA Inhibitor of fhlA expression Inactivation increases synthesis of FhlA, increase in FHL complex No increase [35]

fhlA Activator of Fhl expression Introduction of constitutive allele (FhlA*), increase in FHL complex Small increase (510%) [33,34]

hyd1, hyd2 Uptake hydrogenases Inactivation prevents H2oxidation Modest increase (35%) [33,34]

hyd3 Ech hydrogenase H2 evolving, FHL H2ase Large increase over low

basal level

[37]

aLaboratory E. colistrains can vary widely in their unmodified hydrogen production capacity, possibly because some carry unrecognized mutations. All the strains reported

here, with the exception of that used in [37], had relatively high native hydrogen evolution. Thus, in most cases, the reported increases are quantitatively significant.


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substrates (waste streams). Finally, it should be noted that

although these methods can quickly optimize a particular

fermentation, they cannot further improve the yield over

what could be obtained by more classical methods of

optimization, as can been seen by the results reported in

Table 3.

Reverse micelles have also been proposed as a possible

means to increase hydrogen production. Reverse micelles,

essentially self-assembling nano-structures in which an

amphiphilic surfactant is used to entrap a hydrophilic

biological catalyst, have been used primarily to investigate

their ability to increase enzyme stability, enhance enzyme

kinetics and promote partitioning of substrates or products

between the organic and aqueous phase [62,63]. The encap-

sulation of whole cells in reverse micelles has been much

less studied, and the possible benefits to whole-cell-

mediated catalysis are much less evident. Several studies

have examined hydrogen production by different co-cul-

tures in reverse micelles in small-scale batch studies[64

66]. However, the reasons for the higher yields observed

are unclear. Much work, including very substantial scale

up, remains before this system could be exploited as apractical biohydrogen-producing system that could func-

tion continuously on a long-term basis. At any rate, it is

difficult to see how the use of reverse micelles could further

improve yields or surpass the improvements in volumetric

production rates already achieved by reactor configur-

ations with retained microbial biomass (see above).

Moving towards complete substrate conversion

However, even with the improvements noted above, hydro-

gen yields are restricted by the existing metabolic path-

ways to either 2 H2/glucose (Enterobacteracae) or, at most,

4 H2/glucose (Clostridia) at very low hydrogen partial

pressures. The techniques already discussed have not,

and cannot, increase yields beyond these limits. These

yields are, however, practically unacceptable for several

reasons. For one, they are not competitive with the pro-

duction of other biofuels, such as biomethane or bioethanol,

from the same starting materials, because the efficiency of

substrate conversion to these biofuels is 80% or greater.

Furthermore, incomplete substrate conversions during

hydrogen fermentations (two-thirds of the substrate is

used to make other products; see Box 1 and Figures 1

and 2) lead to the production of a large amount of side

products (i.e. COD), which subsequently need to be dis-

posed of. Therefore, development of a practical biohydro-

gen fermentation process requires either the introduction

of additional pathways that would allow near stoichio-

metric conversion (12 H2/glucose) within the microbial

cell or the development of a two-stage system that would

allow nearly complete energy recovery by introducing a

second process to generate energy, preferably hydrogen,

from the fermentation side products that are produced in

the first hydrogen-producing dark fermentation stage.

Metabolic engineering of new hydrogen-producing

pathways

One approach to surmounting the metabolic barrier is to

construct in E. coli an alternative hydrogen-producing

pathway from pathways found in other organisms; for

example, the E. coli construct could express highly active

FeFe hydrogenase (hydA), an enzyme that is normally

responsible for hydrogen evolution in the fermenting Clos-

tridia (Figure 2). However, the insertion of additional

genes would be required because E. coli does not possess

the genes hydE, hydF and hydG, which are required for

FeFe hydrogenase maturation, nor any obvious pathways

Table 3. Application of FD and RSM for biohydrogen production

Bioprocess aspect Parameters studied Results RefsOptimized value H2 yield (mol H2/mol hexose) H2 rate (l/l/day)

a

Pretreatment Temp, cavitation, alkalinization Alkalinizat ion most ef fective 1.55 0.63 [48]

Temp, pH, enzyme/substrate 35 8C, 7.0, 2.5% 0.98 2.1 [49]

Seed spore germination pH, [substrate] 5.5, 5 g/l 0.66 2.2 [50]

Micronutrient formulation [Fe2+] 3 mg/l 1.72 3.2 [51]

180 mg/l 2.2 n/a [52]

260 mg/l 2.6 0.58 [53]

Substrate composition Food waste/sewage 87:13 0.9 3.0 [54]

C/N, C/P 74, 450 n/a n/a [53]

Fermenter operation HRT 48 h n/a n/a [55]

16 h 1.6 2.9 [56]

8 h 0.77 5 [13]

pH 7.5b, 7.5c, 6.0d 1.8b, 1.5c, 1.6d 3.3b, 5.2c, 4.9d [57]

6.1 n/a n/a [58]

6.5 n/a n/a [59]

5.5 1.74 0.5 [60]

7.2 2.05 6.8 [61]

Substrate concentration (g/l) 14.5e 1.6 2.95 [56]

5b, 5c, 15d 1.8b, 1.5c, 1.6d 3.3b, 5.2c, 4.9d [57]

21c n/a n/a [58]

18c n/a n/a [59]

25c 1.74 0.5 [60]

28c 2.05 6.8 [61]

n/a could not be calculated either due to the use of a complex substrate, or to lack of sufficient information.aNote that inTable 1the volumetric rates are per hour, whereas here they are per day.bLactose.cGlucose.dCheese whey powder.eSucrose.


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for reducing this hydrogenase, which functions with

reduced ferredoxin. At a minimum, two more genes would

probably be required to drive significant hydrogen evol-

ution: fdxA (clostridial-type ferredoxin) and porA (pyru-

vate ferredoxin/flavodoxin oxidoreductase). Recently, an

artificial operon approach [38] was used to construct a

ferredoxin-dependent NAD(P)HH2 pathway in E. coli[39]. Two compatible plasmids were used to introduce

six genes (hydF, -E, -G and -A, fdxA and yumC, whichencodes an NAD(P)H:ferredoxin oxidoreductase) under the

control of IPTG-inducible T7 promotors. Hydrogen pro-

duction, which was sensitive to variation in hydrogen

partial pressures as expected, was observed [39]. However,

the levels of hydrogen produced were quite low and insig-

nificant compared to what would be observed with a wild-

type E. coli. However, the host strain, BL21(DE3), is

incapable of hydrogen metabolism, providing a proof of

principle for the introduction of a de novo hydrogen-produ-

cing pathway. Additional strategies, including increasing

the activity of FeFe hydrogenase and modifying the metab-

olism to generate higher levels of reduced nucleotides,

might bring further improvements. This area clearly needsfurther research efforts if present metabolic limits are to be

surpassed, which would constitute a pivotal breakthrough.

Some suggestions, based on computational modeling, of

how to achieve this goal have recently been presented and

include greatly increasing glucose oxidation through the

pentose phosphate pathway with conversion of the gener-

ated NADPH to NADH by transhydrogenase [40].

Additionally, it has been suggested that limited respiration

could be used to completely dissimilate the substrate-

derived pyruvate, producing energy to drive reverse elec-

tron flow and produce more than 4 H2/glucose)[1,2], but

this has never been demonstrated experimentally.

Hybrid two-stage systems

The basic principle of a two-stage process is as follows: (i) in

the first stage, the fermentation of the substrate to hydro-

gen and organic acids takes place in a process that has been

optimized using one or several of the advanced technol-

ogies described above; (ii) then, in the second stage,

additional gaseous energy, either methane or (more pre-

ferably) hydrogen, is extracted from the effluent of the first

stage reactor. Three different two-stage systems that are

theoretically capable of complete energy extraction have

been proposed and are under active investigation in small

laboratory-scale experiments (Figure 2). One approach is

to use a different reactor for the second stage that is

operated under different conditions, such as higher pHand longer HRT, than the first reactor, thus favoring

methanogenesis. Despite the disadvantage of generating

two different gas streams, hydrogen and methane, in

practical terms this might be useful because hydrogen-

methane mixtures are cleaner fuels for internal combus-

tion engines than methane alone in that they produce less

NOX[68]. Although in the long term the goal should be to

produce only a hydrogen stream, this hybrid two-stage

system, producing both hydrogen and methane, is nearly

ready to be put into practice and has already been scaled up

to the pilot plant stage [69]. Such a two-stage system might

offer several advantages over a traditional simple methane

fermentation, including an effective solubilization of sub-

strates such as organic solid wastes and increased toler-

ance to high OLR. Several recent studies have reported the

successful operation of such two-stage systems using

actual wastes [6971], including a pilot plant-scale fermen-

tation of kitchen waste that was able to generate relatively

high production rates of hydrogen (5.4 m3/m3/day) and

methane (6.1 m3/m3/day) while at the same time achieving

removal of 80% of the COD. The efficiency of this process isdemonstrated by the fact that methane yields were twofold

higher than a comparable single-stage process [69].

Another possible process for increasing the overall

energy extraction is the use of photofermentation in the

second stage, with the aim of recovering additional hydro-

gen from the products of a dark hydrogen-generating

fermentation. In this reaction, non-sulfur purple photosyn-

thetic bacteria capture light energy and use it to quanti-

tatively convert organic acids to hydrogen. A great amount

of research has been performed in this area and has been

recently reviewed[72,73]. The main conclusions were that

a large numberof these organisms are capable of degrading

a variety of organic acids to hydrogen, sometimes achievinghigh yields with pure substrates. Indeed, in principle,

photofermentations are capable of completely converting

organic compounds into hydrogen, even against relatively

high hydrogen partial pressures, because hydrogen evol-

ution is driven by ATP-dependent nitrogenase and the

requisite ATP is formed via the photosynthetic capture

of light energy. In fact, in practice, nearly stoichiometric

conversion of some organic acids to hydrogen can be

obtained. Some recent studies, in which the second stage

was actually fed effluent from a hydrogen-producing reac-

tor, have demonstrated the feasibility of such a two-stage

system, although yields were well below stoichiometric

[7476]. However, low light conversion efficiencies coupled

with low volumetric rates of production translate to a

requirement for impossibly large surface areas. Although

further research could very well help to increase yields, the

technical hurdles to the practical application of such pro-

cesses are severe given the rather low photosynthetic

efficiencies of these organisms at moderate to high light

intensities and the requirement for high cost photobior-

eactors that are transparent and hydrogen-impermeable.

Another approach employs microbial electrohydrogen-

esis cells (MECs), in which electricity applied to a microbial

fuel cell provides the necessary energy to convert organic

acids, which are typical side products of a hydrogen fer-

mentation, to hydrogen [7782]. This area has been

thoroughly covered in an excellent recent review [83], soonly a summary of the major points is given here. This

technology represents an elegant application of thermo-

dynamic principles to the manipulation of microbial

metabolism for use in the biotechnological production of

fuels. MECs are in fact quite versatile; not only is it

possible to select the appropriate bacteria during operation

but also other substrates, such as wastewater, can be used.

Thus, because of the versatility of the microbial community

in the anodic chamber, MECs are not restricted to using

fermentation products as substrate but can also, at least in

principle, completely decompose glucose or glucose-con-

taining substrates, such as cellulose, to hydrogen [80].


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Here, the mixed community carries out dark fermentation

in situ, and the resulting fermentation products are used

by the electrogenic members of the community. The elec-

trogenic bacteria in the bioelectrochemical cells catabolize

their substrate using the electrode (anode) as terminal

electron acceptor. Supplementary voltage (>200 mV) is

added to that already generated from the substrate

(acetate 300 mV) to drive hydrogen evolution at the

cathode. Thus, in principle, a second stage MEC after aninitial fermentative hydrogen stage could completely con-

vert a substrate to hydrogen, achieving 12 H2/glucose with

only a small investment of electricity. In the six years since

MECs were first described, several innovations have been

developed and consequently their performance has been

dramatically improved. Currently reported volumetric

rates (6 m3H2/m3 reactor/day) are much higher than

those originally obtained. However, these rates are still

almost two orders of magnitude lower than those obtained

with dark fermentations and, moreover, have only been

observed at high applied voltages (800 mV). It remains to

be seen whether or not sufficiently high volumetric rates

can be achieved with nominal electrical inputs. Thus,substantial challenges to the practical implementation of

this promising technology remain, including the replace-

ment of expensive platinum for the cathode, the increase of

current densities and the reduction of the electrical input

required.

Outlook and future directions

Many say that hydrogen is the fuel of the future; some add

and it always will be! Indeed, in spite of a large amount of

research in the past and at present, major hurdles remain

to be overcome before a feasible practical process can be

demonstrated for any approach to biological hydrogen

production. Here we have concentrated on recent progress

in hydrogen production by dark fermentation. As shown

here, there have been substantial recent improvements in

both the yield and volumetric production rates of hydrogen

fermentations. Yet, to be practical, yields must consider-

ably extend past the present metabolic limitation of 4 H2/

glucose. Thus, the major outstanding question is Can a

practical biological process be created to extract nearly all

the H2from the substrate (12 H2/glucose)? Attempting to

address this challenge should be the focus of future biohy-

drogen research.

Thermodynamics[9]and metabolic constraints suggest

that it would be impossible to find an organism capable of

the complete conversion of sugar-based substrates to

hydrogen by fermentation and that human interventionis needed to solve this problem. Several possibilities exist,

including further metabolic engineering or various two-

stage systems, as discussed here. However, there are out-

standing questions that must be answered for each of these

different approaches if they are to succeed. For example,

with regard to metabolic engineering, can novel pathways

be introduced that are capable of the nearly complete

proton extraction from the substrate and its reduction to

H2? Although already demonstrable on a pilot scale,

coupled hydrogen fermentation and methane digestion

(which is one of the two-stage systems discussed here) does

not produce a pure hydrogen stream and is therefore less

desirable. In order for the other two-stage systems dis-

cussed here to move forward, major impediments must be

overcome. For a hybrid system using photofermentation,

the key questions are: (i) can more efficient photosynthetic

bacteria be created?; and (ii) can materials scientists

develop sufficiently low cost transparent and hydrogen-

impermeable photobioreactors? For a hybrid system using

MECs, a different outstanding question arises: can MECs

be developed that have sufficient current densities, requirelower voltages and use inexpensive cathodes? Solving the

outstanding problem of increasing hydrogen yields could

lead to the development of systems capable of the complete

conversion of waste streams and energy crops to hydrogen,

a potential sustainable fuel of the future.

AcknowledgementsBiohydrogen research in the laboratory of P.C.H. is supported by NSERC

(Natural Sciences and Engineering Council of Canada) and NRCan

(Natural Resources Canada). D.G.s stay as a visiting scholar from the

Indian Institute of Technology, Khargpur was supported by a GSEP

(Graduate Student Exchange Program) grant from the Department of

Foreign Affairs and International Trade, Canada.

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