Environmental Engineering Reference
In-Depth Information
and do not reduce acetylene to ethylene [29]. Instead of ethylene, the alterna-
tive nitrogenases release small amounts of ethane (C
2
H
6
) from acetylene. The
production of ethane can be used as an indicator for the presence of alterna-
tive nitrogenases. In addition, the V nitrogenase may be less affected by lower
temperatures [29].
Nitrogenase genes are found in a wide diversity of prokaryotes that possess
equally diverse physiological capabilities [98]. The phylogeny of N
microorganisms based on 16S rRNA was reviewed by Young [136]. How-
ever, rRNA phylogenies donot provideuseful tools foridentifyingN
2
-fixing
2
-
fix
ing microorganisms, since the nitrogenase genes are not distributed evenly
throughout closely related taxa. For example, some species of
Klebsiella
fix N
2
while others do not. The most extensive phylogenetic analyses of diazotrophic
prokaryotes are based on
nifH
genes (encoding Component II). There are now
thousands of partial and full length
nifH
sequences in GenBank, largely derived
by PCR amplification of DNA from environmental samples [142].
nifH
gene
phylogeny appears concordant with ribosomal RNA phylogeny [136, 142].
Several microorganisms have multiple copies of nitrogenase genes;
Azotobac-
ter vinelandii
and
A. chroococcum
each contain conventional, alternative and
second alternative nitrogenases [10, 29]. The evolution of the different nitro-
genases, which require different metals, may reflect the chemical evolution of
the biosphere.
There are three major clusters of
nifH
genes that encode active nitrogenases
(termed Clusters I-III). The Cluster I
nifH
genes contain the cyanobacteria, most
of the proteobacteria, and the
Bacillus
group of the
Firmicutes
(gram positive
organisms) (Fig. 1). Cluster II contains second alternative nitrogenases, which
contain Fe instead of Mo or V as a component of the cofactor of Component I
(dinitrogenase) (Fig. 1).
Cluster III contains the nitrogenases found in methanogens, sulfate redu-
cers (including delta proteobacteria and
Firmicutes
), the clostridia group of
the Firmicutes, and genes from other anaerobic microorganisms such as green
sulfur bacteria (
Chlorobium
spp.). The Cluster III nitrogenases are presumed
to contain Mo, and protein structure studies have shown that the nitrogenase
in
Clostridium
is structurally similar to Cluster I nitrogenases [53, 114]. The
Cluster III nitrogenases are particularly interesting and relevant to discussions
of anoxic water columns, since this cluster contains
nif
genes from anaerobic
microorganisms, including sulfate reducers and Archaea. These prokaryotes
may have played an important role in the evolution of N
2
fixation, and are
present in modern day anoxic water columns.
The final groups of
nifH
sequences are in a deeply divergent group of
sequences (termed Cluster IV, or designated Clusters IV and V in [105]). These
deeply divergent groups of sequences contain genes that are not involved in N
2
