Integrated Environmental Biotechnology - Environmental Biotechnology: Theory and Application - page 257

like iron, sulphur and molybdenum which is a component of the nitrogen fixing
enzymes. It also provides its nitrogen-fixing symbiots with an oxygen free envi-
ronment, as described later in this discussion. The analogous chemical process
has an enormous energy requirement to achieve the necessary high temperature,
in the region of 500 ◦ C, and a pressure in excess of 200 atm. This is part of
the reason why manufacture of fertilisers for agricultural purposes, which is in
effect the industrial equivalent of the biological process, is a drain on natural
resources. Together with the unwelcome leaching of surplus fertiliser into water-
ways, causing algal blooms among other disturbances, this makes the widespread
application of fertiliser recognised as a potential source of environmental dam-
age. It is understandable to see a drive towards the engineering of plants both
to increase the efficiency of nitrogen fixation, which is estimated at being 80%
efficient, and to extend the range of varieties, and especially crop species, which
have this capability. It is also worth noting that unlike superficially applied fer-
tilisers which may exceed locally the nitrogen requirement and so leach into the
surrounding waterways, nitrogen fixation by bacteria occurs only in response to
local need and so is very unlikely to be a source of pollution.
The following brief description of the required interactions between plants
and microbes, the example used here being Rhizobium , should serve to illustrate
why such a goal is very difficult to achieve. Firstly, the plant is invaded by a
member of the Rhizobium family of free living soil bacteria. There is a specific
relationship between plant and bacterium such that only plants susceptible to that
particular member of the group may be infected. Genes involved in the infection
process and nodule formation called nod genes are coded for by the bacterium.
The nod genes are activated by a mixture of flavonoids released by the plant
into the region around the roots and thus the plant signals to the bacterium its
receptiveness to be infected.
After infection through the root hairs, the multiplying bacteria find their way
into the cells of the inner root cortex. They are drawn into the cell by endocy-
tosis shown in Figure 10.5 and so are present within the cell bounded by plant
cell membrane. This structure then develops into nodules containing the nitro-
gen fixing bacteria. Several changes to the plant then ensue including the syn-
thesis of proteins associated with the nodule, the most abundant of which is
leghaemoglobin, which may reach levels of up to 30% of the total nodule pro-
tein. The genes coding for this protein are partly bacterial and partly plant in
origin and so exemplify the close symbiosis between the two organisms. The
expression of leghaemoglobin is essential for nitrogen fixation, since it is respon-
sible for the control of oxygen levels. The enzymes for fixation are coded for
by a plasmid of Rhizobium and are referred to as the nif gene cluster. There
are two components each comprising a number of genes. One component is
nitrogenase reductase the function of which is to assimilate the reducing power
used by the second component, nitrogenase , to reduce nitrogen to ammonium
ion. Expression of the nif genes is highly regulated in all organisms studied to
date. In addition to the nif and the nod genes, Rhizobium also carries additional
genes which are involved in the fixation of nitrogen called the fix genes. Once