Mining genomic databases to identify novel hydrogen producers

Abstract

The realization that fossil fuel reserves are limited and their adverse effect on the environment has forced us to look into alternative sources of energy. Hydrogen is a strong contender as a future fuel. Biological hydrogen production ranges from 0.37 to 3.3 moles H(2) per mole of glucose and, considering the high theoretical values of production (4.0 moles H(2) per mole of glucose), it is worth exploring approaches to increase hydrogen yields. Screening the untapped microbial population is a promising possibility. Sequence analysis and pathway alignment of hydrogen metabolism in complete and incomplete genomes has led to the identification of potential hydrogen producers.

Full text

Mining genomic databases to identify
novel hydrogen producers
Vipin C. Kalia, Sadhana Lal, Rohit Ghai, Manabendra Mandal and Ashwini Chauhan
Institute of Genomics and Integrative Biology, (formerly Centre for Biochemical Technology), CSIR, Delhi University Campus,
Mall Delhi Road, 11 00 07, India
The realization that fossil fuel reserves are limited and
their adverse effect on the environment has forced us to
look into alternative sources of energy. Hydrogen is a
strong contender as a future fuel. Biological hydrogen
production ranges from 0.37 to 3.3 moles H
2
per mole of
glucose and, considering the high theoretical values of
production (4.0 moles H
2
per mole of glucose), it is
worth exploring approaches to increase hydrogen yields.
Screening the untapped microbial population is a pro-
mising possibility. Sequence analysis and pathway
alignment of hydrogen metabolism in complete and
incomplete genomes has led to the identification of
potential hydrogen producers.
At present, 90% of the world’s energy requirements are
fulfilled by fossil fuels, which are regarded as an endless
and cheap source. However, we now know that the Earth
possesses a finite amount of fossil fuels [1,2], and that their
indiscriminate use has caused extensive damage to human
health and to this planet [3]. There is now a global effort
focused on the development of non-polluting and sus-
tainable energy sources, which will replace fossil fuels
in the post-fossil fuel era. Among the alternative future
fuels (such as gasoline, ethanol, methanol, methane and
hydrogen), hydrogen appears to be the most promising
candidate because it burns to water, which can be re-used
in an environmentally friendly manner [3,4].
The ability to produce molecular hydrogen is widely
distributed among prokaryotes; it is also found in eukaro-
ytes [5–7]. However, most hydrogen-producing micro-
organisms are also able to consume it [8]. The production of
hydrogen by different microorganisms is closely linked to
their energy metabolism. Hydrogen production is one of
the specific mechanisms to dispose of excess electrons
through the activity of hydrogenase enzyme present in
hydrogen-producing microorganisms [2,8–15]. Biological
hydrogen production is limited by feedback inhibition,
partial pressure of hydrogen and the presence of hydrogen
consumers in a wide range of habitats [9]. The theoretical
maximum yield through fermentation is reported to be
4.0 moles H
2
per mole of glucose [16]. In practice, hydrogen
yield by pure or mixed cultures has been reported to
range from 0.37 to 3.3 moles H
2
per mole of glucose
[11,12,17– 19]. Considering these high theoretical values,
it is worth exploring approaches towards increasing the
yields. A challenging problem in establishing hydrogen as
a source of energy is the renewable and environmentally
friendly generation of large quantities of hydrogen gas.
Enzymes involved in hydrogen production
Hydrogen production and use by microorganisms involves
a specific enzyme called hydrogenase. Hydrogenase cata-
lyzes what is arguably the simplest chemical reaction:
2H
þ
þ2e
2
$H
2
. However, a survey of all presently
known enzymes capable of hydrogen evolution shows
that they contain complex metallo-centers as active sites
and that the active enzyme units are synthesized in
complex processes involving auxillary enzymes and pro-
tein maturation steps. At present three enzymes perform-
ing this reaction are known: nitrogenase, Fe-hydrogenase
and NiFe hydrogenase [20]. Nitrogenase is a two-
component protein system that uses Mg ATP (2ATP/e
2
)
and low-potential electrons derived from reduced ferre-
doxin or flavodoxin to reduce a variety of substrates. In the
absence of other substrates, nitrogenase continues to
turnover, reducing protons to hydrogen. The turnover of
this enzyme complex is extremely slow [20], and the
products of at least an additional 20 genes are necessary
for co-factor synthesis and insertion as well as metal
metabolism.
Many microorganisms contain NiFe hydrogenase, which
might catalyze hydrogen oxidation or reduce protons to
hydrogen [15,21]. The NiFe hydrogenases are hetero-
dimeric proteins consisting of both small (S) and large (L)
subunits. The small subunit contains three iron-sulfur
clusters, two [4Fe-4S] and one [3Fe-4S]. The large subunit
contains a unique, complex nickel iron center with
co-ordination to 2CN and one CO, forming a biologically
unique metallo-center. Some nickel hydrogenases contain
selenium [NiFeSe] and are considerably more active than
normal [NiFe] enzymes and are catalytically as active
as [Fe] hydrogenases [22]. Selenium-containing hydro-
genases are synthesized if this trace element is available;
otherwise alternative hydrogenases are transcribed.
Hydrogenase, which is responsible for the production of
hydrogen in the fermentation process, has been found
to function together with formate dehydrogenase as a
coupled electron transfer system. The third group of
hydrogenase enzymes contains a unique complex, Fe–S
center, in which one of the Fe atoms is complexed with CO
and CN. The turnover number of these Fe hydrogenases is
a thousand times faster than that of nitrogenases. Major
Corresponding author: Vipin C. Kalia (vckalia@cbt.res.in).
Opinion TRENDS in Biotechnology Vol.21 No.4 April 2003
152
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laboratories in both Europe and worldwide are working on
solving the complicated puzzle of biohydrogen catalysis
and particularly the NiFe hydrogenase: understanding
the structure and function, assembly and regulation of
biosynthesis of these complex macromolecules [21].
Screening for hydrogen producers
The search for hydrogen-producing microbes usually
involves the classical route of isolation and characteriz-
ation by growing them on nutrient media. Several
methods are now available for detecting hydrogenase
activity in free-living cells, including amperometric
analysis, tritium uptake, gas chromatography and chemo-
lithotrophic growth [23]. Rapid screening of a large
number of individual colonies is a major hurdle in
this process.
The numerous reports of attempted purification of
hydrogenase from a wide range of organisms exemplify
the many problems associated with the isolation of the
enzyme. The purification of the enzyme from a particular
organism presents unique problems and thus there are
no general methods that can be applied to hydrogenase
isolation per se. There are two major problems in enzyme
purification: first, the hydrogenase has a tendency to be
intimately associated with the electron transport system
and some are membrane-bound; and second, the enzyme
is sensitive to inactivation by oxygen. Solubilization of
hydrogenase from the particulate fraction of crude cell
extracts has been achieved with several microorganisms
using a variety of reagents [5]. In view of the difficulties
faced in conventional methods, the search for hydrogen-
producers has been at a slow pace. In such a scenario,
we can either create mutations or look for untapped
genetic variability.
We can now exploit sequence and metabolic
pathway databases for screening microbes. The
National Center for Biotechnology Information (NCBI)
(http://www.ncbi.nlm.nih.gov) and the Kyoto Encyclopedia
of Genes and Genomes (KEGG) (http://www.genome.ad.jp)
databases greatly facilitate such analyses. Based on
genomic information, structural (or stoichiometric) ana-
lysis of the metabolic networks of the microorganisms
has been done. More and more microorganisms have been
fully sequenced and others have been partially sequenced
(see http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/genome_
table_cgi) by the rapidly developing genome projects, and
genes are recognized and annotated from the sequence
information using bioinformatics (see http://www.bioinfo.
de/isb/gcb01/talks/ma/main.html)[24,25]. From the anno-
tated sequence information, the enzymes that can be coded
by their genomes are known; for example, the relation
of hydrogenases to the redox proteins and enzymes has
been demonstrated by biochemical work, and perhaps in
the future through the compilation of sequence data. At
this time, the sequences of .100 hydrogenases are
available and the genomes of .80 microorganisms have
been sequenced [26].
Given that hydrogen metabolism has an important role
in many of these organisms, their genomes encode several
hydrogenases or their related proteins. The complete
genomic sequence of Escherichia coli contains the genes
encoding the previously known hydrogenases and their
accessory proteins [27], as well as the set of genes encod-
ing the fourth hydrogenases (Hyf) discovered by DNA
sequencing [28]. Genome sequences have proved useful for
generating information for microbes, which have not been
biochemically and genetically well-characterized. Several
genes encoding putative [NiFe]-hydrogenases have been
identified from the genomes of Archaeoglobus fulgidus [29]
and the bacterium Aquifex aeolicus [30]. Combing these
databases for functionally related hydrogen-producing
genes can lead to potential organisms, which have not
yet been exploited.
Sequence analysis and pathway alignment of hydrogen
metabolism
Comparative analysis of metabolic pathways in different
genomes yields important information on their biotechno-
logical applications [31]. Sequence analysis of 84 complete
and 92 partially sequenced genomes (http://www.ncbi.nlm.
nih.gov/cgi-bin/Entrez/genome_table_cgi) coupled with
Fig. 1. Hydrogen production pathway from glyoxalate and dicarboxylate
metabolism. Source: Kyoto Encyclopedia of Genes and Genomes (KEGG)
(http://www.genome.ad.jp/kegg).
TRENDS in Biotechnology
Pyruvate
Formate
Acetyl CoA
Fdred Fdox
Formate
dehydrogenase
Hydrogenase H2
2H+
CO2
NAD+NADH
CoA
Fig. 2. (a –c). Domain structures of formate dehydrogenase (including hydro-
genase-3). (d– e) Domain structures of hydrogenase (hydrogenase-1) through
RPS– Blast (reverse position specific – basic local alignment search tool).
(a) gil3868719lformate dehydrogenase-N, nitrate-inducible, alpha subunit
[Escherichia coli K12] (1015 letters). (b) gil1787749lformate dehydrogenase-N,
nitrate-inducible, iron– sulfur beta subunit [E. coli K12] (294 letters). (c) gil1790325l
formate dehydrogenase, cytochrome b
556
(FDO) subunit [E. coli K12] (211
letters). (d) gil1787206lhydrogenase-1 small subunit [E. coli K12] (372
letters). (e) gil1787207lhydrogenase-1 large subunit [E. coli K12] (597 letters).
Source: National Center for Biotechnology Information (NCBI)
(http://www.ncbi.nlm.nih.gov).
150
TRENDS in Biotechnology
1 200 400 600 800 1015(a)
Molybdoptcrin
1 50 100 200 250 300 350372(d)
1 100 500200 400300 597(e)
Oxidored_q6
NiFeSe_Hases
1 5025 100 15075 125 175 200 211(c)
1 50 100 150 200 250 1294(b)
fer4 ThiC
Ni_hydr_CYTB
Opinion TRENDS in Biotechnology Vol.21 No.4 April 2003 153
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one pathway alignment is described here in an effort to
identify novel hydrogen-producers. KEGG has infor-
mation about microorganisms from a wide range of genera.
Because our basic interest is in searching new micro-
organisms that are involved in hydrogen production and
enzymes involved in these metabolic pathways, we chose
from the glyoxalate and dicarboxylate metabolic pathways
a small route for hydrogen production.
The two principal enzymes involved in hydrogen gene-
ration are formate dehydrogenase (EC 1.2.1.2) and hydro-
genase (Ni-dependent) (EC 1.18.99.1) (Fig. 1). Screening of
92 microbes reported in KEGG was performed for these
two enzymes. Amino acid sequences of formate dehydro-
genase (three subunits) and hydrogenase (two subunits) of
E. coli were collected from KEGG. The domain structures
for these sequences are shown in Figure 2. The domains
were identified using RPS-BLAST (reverse position
specific – basic local alignment search tool) [32]. The
same sequences (from KEGG) were used as queries
against the non-redundant and unfinished microbial
database using BLAST [32]. All the sequences found to
be annotated as being the same as the query sequences
were collected. For identifying a putative homolog in the
unfinished genome database all hits having an expect
value ,0.01 were taken. The goal of the analysis was to
identify the new microbes, which possess the hydrogenase
and formate dehydrogenase genes but have not been used
as hydrogen-producers yet (Table 1). The review of avail-
able information reveals some such lead microbes. All the
potential hydrogen-producing microbes being reported
here possess hydrogenase genes, except Cytophaga hutch-
insonii and Novosphingobium aromaticivorans, although
the latter have formate dehydrogenase. Similarly, the
KEGG and NCBI databases reveal some organisms such
as Methanococcus,Bacillus and Pyrococcus spp. to possess
only formate dehydrogenase but which have been reported
to produce hydrogen [8,10,15].
Conclusion
Biological hydrogen production is the most challenging area
of biotechnology with respect to environmental problems. In
the past 25 years hydrogen energy has moved in all fronts,
making roads in all areas of energy. In the next 20 years the
progress will be many fold greater, and the hydrogen energy
system will provide Earth with the energy system she
deserves, which is hospitable to life, clean and efficient.
Table 1. Potential hydrogen-producing microorganisms and their unique properties
Organisms Hydrogenase Formate
dehydrogenase
Unique properties
Autotroph
Aquifex aeolicus þþ Hyperthermophilic (958C), limited genome size (1.55 £10
6
bp), that is
only one-third the size of Escherichia coli genome, biosynthetic rather
than degradative
Chemolithoautotroph
Oligotropha carboxidovorans þ2Converts CO !CO
2
, poison-eating bacteria, pollutant indicator, CO
sensor
Chemotrophs: facultative anaerobes
Magnetococcus sp. MC1
a
þþ Only magnetotactic bacteria occurring in pure culture, novel aerotactic
sensory mechanism
Wolinella succinogenes þþ Biodegradation of industrial wastewaters (perchlorates), remediation
of contaminated soil and ground water, reduction of selenate and
selenite to element selenium, production of succinic acid for synthesis
of polyesters
Acidithiobacillus ferrooxidans
a
þ2Leaching of insoluble metal sulfides, desulfurization of coal and
decontamination of industrial wastes
Helicobacterpylori þ2Most common chronic bacterial infection of humans, highly acid-
resistant, valuable in drug discovery and vaccine development
Strict anaerobes
Desulfomicrobium baculatum þ2Fresh water sulphate reducer, accumulates S-sulphate up to 120-fold
Desulfitobacterium dehalogenans þ2Bioremediation by dehalogenating bacteria, environment pollution
reducer, e.g. chlorinated phenols, ethenes, polychlorinated biphenyls
(PCBs)
Desulfitobacterium hafniense
a
þ2
Methylococcus capsulatus
a
þ2Ability to convert methane into biomass, increases nutritive value of
animal feed, development of novel ingestible vaccines
Aerobes
Burkholderia fungorum
a
þþ Pollutant-degrading capabilities, beneficial plant root colonizers, role in
global C-cycle, commercially important for bioremediation, reliable aid
in distinguishing the pathogenic members from the environmentally
useful strains
Cytophaga hutchinsonii
a
2þCellulose (the most abundant biopolymer on Earth) utilization as
renewable resources
Novosphingobium aromaticivorans
a
2þDegrades aromatic hydrocarbons including toluene, p-cresol, xylene,
naphthalene, biphenyl, dibenzothiophene and fluorine. Ability to grow
in a wide range of environments including soil, both marine and fresh
waters, marine life and from plants
Hydrogenase, EC 1.18.99.1; formate dehydrogenase, EC 1.2.1.2
a
Unfinished genomes
Opinion TRENDS in Biotechnology Vol.21 No.4 April 2003
154
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Hence hydrogen energy system will enhance our quality of
life and help to preserve our biosphere [3].
In view of the urgent need for using H
2
as an energy
source, it is of fundamental, ecological and biotechno-
logical interest to look for hydrogen-producing hydro-
genases and the physiological capabilities of the organism
harboring them. Although quite a few hydrogen producers
have been reported so far, the progress in the search for
new organisms for this metabolism has slowed down. In the
past decade, very few new organisms have been reported
(e.g. Caldicellulosiruptor saccharolyticus,Gloeocapsa alpi-
cola,Rubrivivax gelatinosus and Thermotoga elfii), and
there has been little significant improvement in the H
2
yields, which ranges up to 3.3 moles per mole of glucose
[19,33,34]. Metabolic pathways exist in microbes [e.g. the
oxidative pentose pathway (PPP)], which produced essen-
tially stoichiometric amounts of hydrogen from glucose-6-
phosphate (G6P) in vitro [35]. However, in practice no such
high-yielding dark hydrogen fermentations in microbes
are known [20]. Enzymatic production of G6P is feasible on
an industrial scale by using the thermostable enzymes.
Aquifex aeolicus is hyperthermophilic, which may be a
good candidate for such enzymes, particularly because
hydrogen formation becomes thermodynamically more
feasible at elevated temperatures.
Alternatively, we need hydrogen-producing microbes
that can utilize biological wastes that are cheap and
renewable [20]. The organisms reported in Table 1 have
the ability to utilize a wide range of industrial waste-
waters, leach insoluble metal sulfides, have magnetotactic
ability, dehalogenate, and degrade environmental pollu-
tants. Anaerobic treatment of wastes for biogas production
including hydrogen has the dual advantage of energy
generation and waste stabilization [10–12,36– 39].
The future of biological hydrogen production depends
not only on research advances (i.e. improvement in
efficiency through computational genomics, genetically
engineering microorganisms and/or the development of
bioreactors) but also on economic considerations (the cost
of fossil fuels), social acceptance and the development of
hydrogen energy systems. Biomass-derived hydrogen is
likely to become a competitive future fuel [40,41]. Thus,
processes that are presently conceptual in nature, or
at a developmental stage in the laboratory, need to be
encouraged, tested for their feasibility and otherwise
applied toward commercialization.
Acknowledgements
We are thankful to the Director of the Institute of Genomics and
Integrative Biology, CSIR for providing the necessary facilities and moral
support.
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