Indian Journal of Geo-Marine Science
Vol. 44(3), March 2015, pp. 318-322
Assessment of nitrogen fixing bacterial community present in the
rhizosphere of Avicennia marina
B. Baskar1, 2, 3,* & P. Prabakaran3
1
Cooperative research, Lincoln University, Jefferson City, Missouri 65101, USA.
2
Marine Biotechnology, Andaman and Nicobar Centre for Ocean Science and Technology, National Institute of Ocean
Technology, PortBlair, Andaman and Nicobar Islands 744 103, India.
3
Department of Microbiology, PRIST University, Thanjavur, Tamil Nadu 614904, India.
*
[E-mail address: [email protected]; [email protected]]
Received 17 January 2014; revised 11 February 2014
Present study served to identify an indigenous diazotrophic bacterial community as a biofertilizer for the improvement
of nursery techniques in mangrove restoration. Plant species Avicennia marina was selected for investigation in three
geographically different regions of the Pichavaram mangrove forest, India. A total of 23 diazotrophic bacteria were isolated from
the rhizosphere of the plant. Nitrogen fixing ability of the bacteria was quantitatively assessed using the Acetylene Reduction
Assay. Highest nitrogenase activity was recorded from members of Azotobacter and Azospirillum. Nitrogenase activity of the
three rhizosphere soils was evaluated and positively correlated with the diazotrophic species diversity.
[Keywords: Mangrove restoration, Avicennia marina; nitrogen fixing bacteria; diazotroph; nitrogenase]
Introduction
Mangrove forests are a highly Nitrogen
(N2) limiting environment because of various
physiochemical factors involved in the implicit
ecosystem1, 2. It is thus important to find
indigenous N2-fixing bacterial communities for
the augmentation of N2 content in order to
encourage the development of microbe-based
conservation and restoration techniques of the
mangrove ecosystem. There are reports available
on the diversity of N2 fixing bacterial
communities present in the mangrove rhizosphere,
but they are not adequate for the mangrove
restoration program. Concerning this, the present
study designed to identify the different bacterial
members capable of fixing atmospheric N2 in the
rhizosphere of A. marina and the N2-fixing ability
was quantitatively identified in in vitro
conditions.
Materials and Methods
The study was conducted at Pichavaram
mangrove ecosystem (latitude 11°23′ –11°30′ N
and longitude 79°45′ – 79°50′ E) in which three
geographically different sites were selected for the
study. Site 1 (Dense mangrove forest region)
located at the landward end of the forest, Site 2
(Coleroon estuary region) is an area heavily
influenced by freshwater and Site 3 (Chinnavoikal
region) is an area influenced by neritic water
entering from the sea. From each site, three A.
marina plants were selected and collected the
rhizosphere (root adhered) soil sample in
triplicates. Equal proportion of plant samples
were pooled together for each site separately.
Collected samples were subjected to serial
dilution technique and plated in Nitrogen free
Burk’s medium (modified with 2% sodium
chloride and 2% glucose instead of sucrose).
Inoculated plates were incubated at ambient
temperature for 2 days.
The selected isolates were inoculated in
50 ml sterile saline bottles containing 25 ml liquid
medium and the final turbidity of the culture
maintained as 0.5 McFarland turbidity standard.
Then all the bottles were sealed using rubber
stoppers. From the bottles, 10% of gaseous space
was removed, and the same volume of acetylene
gas (0.1 atm) was injected. For each sample
triplicates were maintained and one set of control
was maintained without bacterial culture in broth
(Burk’s medium). Prepared bottles were incubated
for 12 h. After incubation, one ml of gaseous part
was collected and subjected to
Gas
chromatography (GC). By using GC (Shimadzu,
Japan) with Flame Ionising Detector, Poropac-T
column with 3 and 3.1 mm stainless steel (column
temperature 60°C), 80-100 mesh, maintained at
the injection port temperature of 50°C and
ignition of 150°C and the carrier gas hydrogen at
the flow rate of 25 ml/min and oxygen at 300
BASKAR et al.: NITROGEN FIXING RHIZOSPHERE BACTERIAL COMMUNITY
ml/min. Finally, the nitrogenase
expressed as nM of Ethylene ml-1.
319
activity
acetylene, and another one was only with sterile
mangrove water. Samples were incubated for 12
Five grams of rhizosphere soil samples from the
selected three Sites were transferred separately
into 65 ml of sterile saline bottle contains 25 ml
of sterile (sterilized by 0.2 µm membrane filter)
mangrove water collected from the same Site.
Triplicates were maintained for each sample, and
two sets of control were maintained one without
h in ambient temperature and finally nitrogenase
activity was measured using ARA as described
previously. Nitrogenase activity of the soil
sample was expressed by the amount of ethylene
produced per gram of soil.
Table 1. Nitrogenase activity of the N2 fixing bacteria.
Site
Site 1
Site 2
Site 3
Isolate name
Azotobacter chroococcum BPRIST062
Nitrogenase activity (nM ethylene/ml)
210±4.1
Azotobacter vinelandii BPRIST064
192±1.6
Azospirillum brasilense BPRIST025
209±7.2
Bacillus thuringiensis BPRIST010
31±1.8
Enterobacter aerogenes BPRIST026
12±0.2
Pseudomonas aeruginosa BPRIST029
21±0.8
Vibrio natriegens BPRIST034
40±0.8
Vibrio parahaemolyticus BPRIST002
94±1.8
Vibrio proteolyticus BPRIST003
88±8.1
Azotobacter chroococcum BPRIST063
211±2.4
Azospirillum brasilense BPRIST040
251±5.9
Enterobacter aerogenes BPRIST042
34±0.4
Pseudomonas aeruginosa BPRIST046
28±1.3
Vibrio natriegens BPRIST057
57±1.8
Vibrio proteolyticus BPRIST030
89±5.1
Azotobacter sp. BPRIST061
183±0.8
Azospirillum brasilense BPRIST041
234±6.1
Bacillus cereus BPRIST015
17±1.0
Enterobacter aerogenes BPRIST043
18±1.6
Paenibacillus pabuli BPRIST071
56±2.0
Pseudomonas aeruginosa BPRIST047
20±0.3
Vibrio natriegens BPRIST058
45±4.3
Vibrio proteolyticus BPRIST048
88±2.8
INDIAN J MAR SCI VOL 44, No. 3 MARCH 2015
320
Results
Based on biochemical and 16S rDNA
sequence analysis (results not given) from the
isolated N2 fixing bacteria, 23 bacteria were
selected for further study, within this 11
different species from 7 genera are included.
Members
of
genus
Azotobacter
and
Azospirillum were showed highest amount of
nitrogenase activity than other species, in which
A. brasilense BPRIST040 (Isolated from Site 2)
showed the highest activity of 251±5.9 nM of
ethylene ml-1 of culture. Lowest
activities were recorded from the genus
Bacillus, Enterobacter and Pseudomonas and
moderate activity were observed from
Paenibacillus and Vibrio. The results of
Nitrogen fixing ability are summarized in Table
1.
All the three soils showed varied
nitrogenase activity. In which highest activity
was recorded for rhizosphere sample collected
from Site 1, next to that Site 3 showed good
nitrogenase activity and Site 2 showed less
nitrogenase activity. Amount of nitrogenase
activity of rhizosphere soils are given in Table
2.
Table 2. Total diazotrophs and nitrogenase activity of the
rhizosphere soils.
Rhizosphere
soil
Site 1
Site 2
Site 3
Total diazotrophs
8.1 × 105 CFU g-1
Nitrogenase activity
(nM ethylene/g of
soil)
62±12.76
6.3 × 105 CFU g-1
38±9.24
5
54±5.76
-1
7.2 × 10 CFU g
Discussion
The present study identified the
different bacterial members which fix
atmospheric N2 with the association of
Avicennia marina. Nitrogen fixing ability was
quantitatively identified in, in-vitro conditions
by ARA method. The nitrogen fixation in the
rhizosphere soils of three Sites significantly
correlated with total bacterial count, and the
number of nitrogen fixing species present in
each Site, this phenomenon is supported by
many works even from terrestrial one3, 4.
Though, there is affirmative relationship
between diazotrophic bacterial counts and
nitrogen fixation, it is not a direct proof of basis.
Variations in nitrogen fixation rates observed
among different species of bacteria associated
with nitrogen fixation is also inhibited by
several physical factors like light, temperature
and seasonal variations4, 5. Subsequently, apart
from diazotrophic bacteria, other rhizosphere
bacterial community also contributes in nitrogen
fixation process indirectly by providing energy,
electron and other sources to nitrogen fixers to
improve nitrogen fixing ability6, 7. Variation
between the sites may be due to the nature of
site, as Site 1 represents dense mangrove forest
where the possibilities are more to have stable
microbial population rather than other two sites.
Site 2 highly influenced by domestic and shrimp
farming activities and Site 3 vigorously
undergoes in their chemical and physical nature
by means of tidal variations. These kinds of
environmental factors always influences the
presence of stable microbial community in the
rhizosphere8, 9.
In the present study glucose was used as
a sole carbon source, since this is the carbon
source utilized by all the bacterial isolates
isolated in this study. Generally malate is used
as a sole carbon source in the nitrogen free
medium, here it is not used because some of the
isolates selected in the study are not able to
utilize malate as a sole carbon source (Data not
given), and in the same way not used multiple
carbon sources also6.
Free living bacteria Azotobacter and
Azospirillum were widely reported for its
excellent nitrogen fixing characters both in, in
vitro and in vivo studies able to improve the
growth of the mangrove seedlings, isolated from
sediments, root surfaces and rhizosphere of the
mangroves3, 4, 10. As Ravikumar et al., 4 reported
Azotobacters such as A. chroococcum and A.
vinelandii from various mangrove plant
rhizospheres of the Pichavaram mangrove plants
including A. marine. Present study also reports
the presence of those two Azotobacter in the
rhizosphere of A. marina in comparison with in
vitro nitrogen fixation capability. In the same
way, the present study reported the maximum of
nitrogen fixation by Azospirillum brasilense
isolated from all the three Sites and it
corroborated with the reports of Ravikumar et
al.,3 where the nitrogen fixing Azospirillum
BASKAR et al.: NITROGEN FIXING RHIZOSPHERE BACTERIAL COMMUNITY
present in the rhizosphere of the Pichavaram
mangroves.
Several members of the genera Vibrio
are widely reported for its presence in the
rhizosphere soil of many terrestrial plants, but
only few were reported from mangrove related
environments for their ability to fix atmospheric
N2 namely V. campbelli, V. aestuarianus, V.
anguilarum, V. parahaemolyticus, V. natriegens,
V. mangrove, V. rhizosphaerae, V. proteresiae
and V. diazotrophicus6, 11, 12, 13. Vibrios are
generally pathogenic bacteria for marine
animals and a major cause of diseases with huge
economic losses in the aquaculture industry.
The study shows that Vibrios are the highest
nitrogen fixers next to Azotobacters and
Azospirillums with a large number of species
composition, which appear to be beneficial to
the mangroves and it is still unclear whether the
isolates are pathogenic to mangrove associated
fishes and other aquatic animals or not. This has
an insight to study on the criteria since
Pichavaram mangrove area is highly influenced
by shrimp farming discharges.
Though, Vibrios are moderate nitrogen
fixers in the rhizosphere having the ability to
dominate in their total number and more
diversity in species level, which leads the
importance of the community to be used as plant
growth promoting bacteria for mangrove
restoration. However, the presence of nitrogen
fixing V. parahaemolyticus and V. natriegens
were previously reported from mangrove
ecosystem11, the results of the present study has
added possible reports for the first time from the
Pichavaram mangroves. In the same way, V.
proteolyticus reported as a phosphate
solubilizing
bacteria
from
mangrove
rhizosphere12, 14 and it may be the first report on
the nitrogen fixing ability of the bacterium
present in this kind of ecological niche.
Paenibacillus pabuli showed moderate
nitrogen fixing aptitude, it is comparable with
some reported nitrogen fixers of the genus viz.,
P. polymyxa, P. macerans, P. odorifer, P.
graminis, P. forsythia, P. azotofixans and P.
durus; nevertheless these all were reported from
terrestrial ecosystems15, 16. This report may be a
novel one on the presence of N2 fixing
Paenibacillus associated with a mangrove plant.
321
Bacillus thuringiensis was widely
reported for its mosquitocidal activity and B.
cereus for its asparaginase production from
mangrove ecosystems17, 18, 19, 20. However,
according to current knowledge there were no
reports available on nitrogen fixing ability of
those two bacteria. In the same way, P.
aeruginosa were not reported as diazotroph
from the mangrove ecosystem but were reported
for other potentials like phosphate solubilizing
nature, heavy metal resistance etc., 21, 22, 23, 24. In
the case of E. aerogenes rarely reported as
nitrogen fixer in association with mangrove25.
These isolates showing different potential
characteristics apart that they were well adapted
to the respective niche, which may be new
findings, although a detailed study has to be
carry out.
Conclusion
Three studied sites were rich in
heterotrophic and diazotrophic bacterial
diversity.
Study also revealed the potent
bacterial members such as Azotobacter and
Azospirillum, which possess high nitrogenase
activity. This allows for the possibility of using
these
bacteria
to
improve
mangrove
conservation strategies by enhancing N2 fixation
in the extremely N2-limited ecosystem. Besides,
these results can be used as a starting point for
further more extensive analysis of diazotrophic
bacterial diversity, not only for aerobic groups
of bacteria, but also for microaerophilic and
anaerobic groups.
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