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SUMMARY AND CONCLUSION
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CHAPTER: 6
SUMMARY AND CONCLUSION
Biological nitrogen-fixation is the single-most important factor that
sustains our agriculture. Nitrogen fixation depends upon the availability of
energy released from breakdown of cellulosic materials by cellulolytic
microflora. This is a cooperative process. In this process a good quantity of
nitrogen is used up by cellulolytic microflora, and thus actual nitrogen gain
through nitrogen fixation becomes marginal. Some reports suggest that there
exist in nature some nitrogen-fixing bacteria which unlike the conventional
nitrogen-fixers are self-sustaining with respect to their energy and nitrogen
demand.
The present work is limited to the isolation o f such self-sustaining
nitrogen-fixing bacteria from little studied soils o f Assam w ith a view to
ascertain their capability to fix nitrogen and at the same time to decompose
cellulose also. Such non-conventional nitrogen-fixation by cellulolytic
bacteria or cellulose breakdown by nitrogen-fixing bacteria will assume
great importance in future as the poor organic matter content o f our
agriculture field will not be able to sustain high energy expensive co­
operative nitrogen-fixation that involves both free-living nitrogen-fixing
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bacteria and celluloytic bacteria. Bacteria that naturally combine both these
abilities will have greater advantage in that such a system will ensure more
economy in the use o f energy and nitrogen when both are limited. Not much
significant progress has been made in isolation and characterization o f such
types of self-sustaining bacteria since the first discovery o f such
microorganisms by Waterbury in 1983 and subsequent work o f Leschine in
1988, Halsall et a i in 1985 and 1986. The present work is an humble effort
to contribute a little more to the study o f such type o f self-sustaining
nitrogen-fixing bacteria from soil o f Assam.
Not much literature on the subject is available. Major techniques o f
isolation and various media had been formulated through trial. The result
obtained in the laboratory condition may lead to subjective judgement which
may differ in scale in actual situation in soil where various factors interplay.
The outcome o f the present investigation has been summerised below:
1. Soil samples from forest land, rice field, wheat field, grass land, tea
gardens, vegetable gardens, sugarcane fields, sawmill areas etc. spread
over Assam were collected for isolation o f bacteria having dual function.
Rhizosphere soil samples o f rice, wheat and some grasses were also
collected for the same purpose.
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2. A total o f 52 nitrogen-fixing isolates were purified using nitrogen-free
sucrose (NFS) medium. Again 21 microoaerphilic isolates having
nitrogen-fixing ability were selected using semisolid nitrogen-free malic
acid (NFM) medium. All the microoaerphilic isolates were purified by
streaking on to congored medium.
3. The cellulolytic ability of all the nitrogen-fixing isolates were then tested
by inoculating them in the nitrogen-free cellulose (NFC) medium. The
cellulase activity was indicated by the production o f reducing sugar in the
culture. Gradual increase in SCP output in the nitrogen free culture
medium also suggests availability o f nitrogen through nitrogen-fixation
and utilization o f cellulose as energy source by the isolates.
4. It was found that only 29 isolates out o f 73 isolates tested in NFC
medium could utilize cellulose. These include 11 microoaerphilic isolates
also. Cellulolytic ability o f these was ascertained by repeated test in NFC
medium. The purified isolates were maintained in cellulose medium. The
rate of utilization o f cellulose was however slow for a number o f days
because o f the lack o f initial nitrogen source in the medium. With time
the rate o f cellulose breakdown increased as the supply o f nitrogen
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increased with time. Gradual increase in the SCP output also support this
fact.
5. All the 29 isolates that showed cellulolytic ability were subjected to
ARA. It was confirmed that out" of the 29 isolates that showed cellulolytic
ability only 24 isolates could reduce acetylene in varying degree. The rate
o f AR may be affected by various factors such as the choice o f medium,
physical conditions etc.
6. All the isolates showing cellulolytic as well as nitrogenase activities were
than
identified using
standard
morphological,
physiological
and
biochemical tests. It was found that 29 isolates were actually belonged to
8 different species which could not be distinguished at the time of
isolation.
7. The eight identified species are as follows:
i)
Bacillus brevis
ii)
Bacillus firm us
iii)
Bacillus coagulans
iv)
Arachnia propionica
v)
Kurthia sp.
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vi)
Agrobacterium rhizogenes
vii)
Azospirillum lipoferum
viii) Azospirillum brasilense.
It is noteworthy to mention that out o f eight species o f bacteria
identified, three species namely Kurthia sp, Arachnia propionica and
Agrobacterium rhizogenes are aerobic. No aerobic species having both the
abilities except, the one reported by Waterbury in 1983 was not known. All
the above species are also not usual cellulose decomposers. Moreover,
Kurthia, Arachnia and Agrobacterium are not known nitrogen-fixers.
Repeated tests were carried out to confirm their cellulolytic and nitrogenase
activities. O f course, the amount o f nitrogen fixed and the cellulose
decomposed should not be considered as being very important at this stage.
The ability o f these bacteria to take part in both carbon cycle and nitrogen
cycle o f nature has been taken into consideration only as the quantity of
nitrogen produced and cellulose decomposed under laboratory condition
may not be considered as being taken place under most ideal condition.
The present work is a preliminary one. It may open up further line of
investigation for isolation and identification o f microorganisms having dual
function which was unknown till 1983 and has not received much attention
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since then. This has great practical importance in that biological nitrogen
fixation accounts for 60% of the total nitrogen fixation in the biosphere.
BNF has however certain limitations. A major part o f the energy released by
the heterotrophic cellulose decomposers is utilized by the cellulose
decomposers themselves leaving veiy
little for the nitrogen-fixing
organisms. Consequently, the nitrogen-fixing organisms suffer from lack o f
supply o f adequate level of energy. Nitrogen fixation in Indian agricultural
field depends upon availability o f left over straw and agricultural wastes. As
straw is removed as animal fodder, a serious energy crisis prevails in
agriculture
soil
which
considerably
hampers
nitrogen-fixation.
Microorganisms having both cellulolytic and nitrogen-fixing ability may
help to manage the crisis and hence search for such microorganisms may be
a positive as well as rewarding exercise. The present investigation is limited
to focusing the attention that there are some microorganisms which are selfsustaining with respect to their energy and nitrogen demand.
In future, efforts may be made to isolate highly efficient strains which
may be used for conversion o f nitrogen-poor lignocellulosic wastes into
useful products. Suitable strains may also be selected as inoculants for use in
soil having high cellulosic materials and also in energy poor agricultural soil
to reduce nitrogen depletion. Such findings may stimulate investigation into
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the mechanism o f dual activity acquired by such organisms in the long
course o f evolution.
This in future may help in development of high
efficient strains having such dual or multiple functions through genetic
engineering.
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