Environmental Engineering Reference
In-Depth Information
cysteine and methionine and is therefore essential to the synthesis of proteins as well,
including the rapidly-recycled D1 protein of PS II. In the absence of reduced sulfur, PS II
function diminishes significantly, resulting in decline of O2 production as well, even under
light exposure. Algal aerobic respiration is not strongly affected by the sulfur deprivation over
relatively short time periods (<100 hours) and therefore proceeds, diminishing O
2
concentrations sufficiently to induce the Fe-hydrogenase activity. Because PS I and the
electron-transport proteins, cytochromes b6 and f, are not significantly affected, the cell can
maintain an ATP-generating proton gradient across the thylakoid membrane by using PS I to
activate electrons released to the plastoquinone pool (Figure 16) by the degradation of starch,
proteins, and lipids via a NADH reductase complex. H2 production then follows, as a means
of removing the spent electrons from the photosynthetic electron transport chain [11], for
approximately 100h [52].
Commercialization of this process is being actively investigated [44]. Significant effort
has also been directed toward the modification of Fe-hydrogenases to yield varieties with
greater O
2
tolerance. This work has been encouraged by the discoveries of greater O
2
tolerance among some hydrogenases, particularly those of the bacterium Desulfovibrio
vulgaris and the alga Pandorina morum [37], as well as the discovery of an Fe-hydrogenase
sequence in the genome of the bacterium Shewanella oneidensis, a facultative rather than
strict anaerobe (53). In addition, the solution of the crystal structures of both monomeric and
dimeric Fe-hydrogenases [54, 55]. Flynn, Ghirardi, and colleagues employed traditional
chemical mutagenesis and screening with H2-sensitive tungsten oxide films to isolate mutants
with up to 10 times greater O
2
-tolerance of H2 production [51, 29, 56-59] as well as
increased rates of H2 production [60]. This success has inspired further efforts to alter one of
the Chlamydomonas Fe-hydrogenases to diminish substantially its sensitivity to O
2
. Toward
this end, further random chemical mutagenesis, error-prone PCR-mediated mutagenesis, and
site- directed mutagenesis are currently underway [61].
At least two other research groups are also investigating directed evolution as an
approach for generating greater O
2
tolerance in algal Fe-hydrogenases. Because gene
shuffling requires a diverse pool of parental Fe-hydrogenase genes, it is fortunate that
numerous potential parental genes exist, representing genera among the archaea, eubacteria,
fungi, algae, protists, and higher eukaryotes as well as monomeric and dimeric forms. Even
among the multimeric enzymes, genes for subunits that show significant homology to the
monomeric forms are considered valid potential parents in generating the monomeric Fe-
hydrogenase mutants in Chlamydomonas.
While most known Fe-hydrogenases are highly O
2
-sensitive, experiencing irreversible
inactivation, that of Desulfovibrio vulgaris (Hildenborough) appears to be only reversibly
inactivated by O
2
, with the result that this sequence is highly attractive as a parent [62, 63], as
is that of Pandorina morum [37]. Fe-hydrogenases in general show very highly conserved
active site structures and sequences, such that the three-dimensional active site structures of
the enzymes from the distantly related Clostridium and Desulfovibrio bacteria can be
superimposed with a calculated deviation of only approximately 1 angstrom [23]. This feature
indicates that a relatively high diversity of parental origins for the Fe-hydrogenases may
result in functional progeny.
The rate at which photosynthetically-generated O
2
must be removed from an H2-
producing algal bioreactor depends directly on the O
2
-tolerance of the algal H2 production
pathway. A thorough cost-benefit analysis of algal H2-producing bioreactor operation
