Number range CHAPTERCombinatorial Mutagenesis of a Bidirectional
Hydrogenase in Chlamydomonas reinhardtii

Dianne Ahmann

[Introduction]{.underline}. Molecular hydrogen, H~2~, is a promising
future energy source due to its clean combustion and to its potential
for sustainable production. Among the numerous technologies that are
being explored for H~2~ production, microbiological mechanisms that
employ photosynthetic pathways are of particular interest because of
their potentially low energy requirements. The green alga Chlamydomonas
reinhardtii is a model organism for these efforts, generating H~2~ by
means of an Fe-hydrogenase-mediated system that has the highest
potential energy efficiency of any known photosynthetic H~2~-generating
mechanism.

Unique scientific breakthrough. A substantial obstacle to the
development of biological sources of H~2~, including both algae and
bacteria, is the absence of a rapid, convenient, high-throughput screen
for H~2~ production in aqueous media. The great need for such a process
was articulated in 2002 by microbial H~2~ production scientists at an
international convention hosted by the U.S. Air Force and the National
Renewable Energy Laboratory. In addition, the Genomes to Life program of
the Department of Energy is presently contemplating the development of a
center concentrating on screening of biological H~2~ production, for
which this assay is ideally suited.

During the course of our TSE work, we became limited by this obstacle
ourselves and so developed a rapid colorimetric assay for H~2~ in
aqueous media that is amenable to development into a high-throughput
screen. The assay involves a blue dye, resazurin, that is colorless when
completely reduced by H~2~ and pink when reoxidized. Cells are induced
to produce H~2~ by incubation in darkness and anoxia. Wild-type algal
cells attain a clear green color in the assay, while mutants remain
purplish.

The assay has now been thoroughly investigated: optimal conditions have
been identified, artifacts have been quantified or eliminated, and
extensive testing for accuracy and reproducibility has been
accomplished. Most beneficially, the assay has proven itself amenable to
high-throughput screening.

[Environmental Impact]{.underline}. Hydrogen is heralded as a
clean-burning fuel, in contrast to fossil fuels, because its combustion
with oxygen yields water rather than atmospheric pollutants. However,
H~2~ is presently synthesized primarily from fossil fuels. Steam
reforming of natural gas, catalytic decomposition of natural gas,
partial oxidation of heavy oil, and coal gasification top the list of
currently mature technologies for H~2~ production. Clearly,
environmentally benign methods for H~2~ production are urgently needed
before the promise of H~2~ to deliver clean energy can be realized.

The ideal technology would use an abundant source of hydrogen atoms,
such as water, in combination with an abundant source of energy, such as
sunlight or municipal wastes, to produce H~2~. As a result, numerous
water-splitting technologies are currently under active investigation.
Each of these requires a highly efficient catalyst to overcome the
activation energy involved in converting the H atoms of water into H~2~.
In electrolysis, photolysis, magnetolysis, and other abiotic methods,
heavy metals such as platinum and palladium are used. These present
their own environmental problems, both in their extraction from ores in
which they represent only a tiny fraction (\<\<1%) as well as in their
inherent toxicity. In biocatalytic H~2~ production, however, the
catalytic function is fulfilled by enzymes that have been evolved over
thousands of years for high efficiency. Biological H~2~ production is
thus an important member of the set of technologies we will need to
achieve a truly sustainable H~2~ energy economy.

The benefit of the color assay described above will come in the form of
facilitating commercial development of H~2~-producing organisms:
bacteria, cyanobacteria (blue-green algae), and green algae. Each of
these produces H~2~ using different energy sources and has its
advantages in certain settings; for example, some of the H~2~-producing
bacteria convert energy of municipal wastes into H~2~, while green algae
use only sunlight energy.

Nature did not intend for microbes to yield copious amounts of energy to
their environments (or to humans) in the form of H~2~, however. Rather,
H~2~ is produced as a waste product only when the organisms have no
other way to dispose of excess electrons and cannot use the H~2~
effectively themselves. Typically, therefore, H~2~ is produced only
under anoxic conditions and in some cases only transiently.

To develop H~2~-producing organisms into robust industrial catalysts,
the hydrogenase enzymes involved in H~2~ production, and the proteins
that regulate their synthesis and activity in response to environmental
conditions, must be altered substantially. The tools to modify enzymes
at the genetic level exist and are well-developed, but they generate
large libraries consisting of millions of mutants that must be screened
for the desired trait: improved H~2~ production.

Current tools to measure H~2~ production in aqueous media are gas
chromatography, Clark-type electrodes, and tungsten oxide films. These
approaches are slow (up to 100 samples/day/person), slow and tedious,
and unreliable, respectively. The assay we have discovered will make the
automation of H~2~ detection in liquid culture possible, with speed that
surpasses that of the currently available methods by several hundred- or
possibly several thousand-fold.

As a result, the field of biological H~2~ production will be opened both
to the molecular optimization of known hydrogenases as well as to the
?biological prospecting? for new organisms that produce H~2~.