The effect of the physicochemical parameters of water and soil on the distribution of nitrogen-fixing bacteria and their nitrogen-fixing capacity was studied. Four species of nitrogen-fixing bacteria, e. g. Azotobacter chroococcum, A. vinelandii, A. beijerinckii and A. armeniacus, were recorded from water and soil samples of Mumbai coast. A higher number of bacterial populations were observed in sediment than in water samples. A positive correlation was observed between the dissolved organic matter and nitrogen fixing bacterial populations of water as well as between available phosphorus and the nitrogen-fixing bacteria of sediment. The nitrogen-fixing capacity of A. chroococcum was found to be 1.076 nmol C sub(2) H sub(4)/l/d and that of A. vinelandii was 0.965 nmol C sub(2) H sub(4)/l/d. Station 1 showed higher level of nitrogenase activity in comparison to other four stations

Pandey, P.K.

Sreelu, N.S.

Aquatic Commons

J. Indian Fish. Assoc., 33: 95-102, 2006 95 
NITROGEN-FIXING AEROBIC BACTERIA AND THEIR 
NITROGENASE ACTIVITY IN NORTH MUJ.\;IBAI COASTAL \VATERS 
P. K. Pandey and N. S. Sreelu 
Central Institute of Fisheries Education, Versova, Mumbai- 400 061, India 
ABSTRACT 
The effect of the physicochemical parameters of water and soH on the 
distribution of nitrogen-fixing bacteria and their nitrogen-fixing capacity was 
studied. Four species of nitrogen-fixing bacteria, e. g., Azotobacter 
chroococcunz, A. vinelandii, A. beijerinckii and A. armeniacu . .,· were recorded 
from water and soil samples of Mumbai coast. A higher number of bacterial 
populations were observed in sediment than in water samples. A positive 
correlation was observed between the dissolved organic matter and nitrogen-
fixing bacterial populations of water as well as between available phosphorus 
and the nitrogen-fixing bacteria of sediment. The nitrogen-fixing capacity of 
A. chroococcum was found to be 1.076 nmol C2H./l/d and that of A. vinelandii 
was 0.965 nmol C2H/l/d. Station 1 showed higher level of nitrogenase activity 
in comparison to other four stations. 
Keywords: Nitrogen-fixing bacteria,AzotobacteJ~ nitrogenase activity 
INTRODUCTION 
Nitrogen availability is a key factor 
regulating primary productivity in 
pelagic, coastal, marine and estuarine 
waters. The quantitative role of nitrogen 
fixation in the ocean's nutrient budget is 
of considerable scientific interest (Karl 
et ar, 1997). Chronic nitrogc11 
deficiencies in such systems would 
appear to be of selective advantage to 
nitrogen-fixing microorganisms to 
convert nitrogen to ammonia, a 
biologically utilizable fonn of nitrogen. 
The mechanism of nitrogen fixation has 
been studied in several free-living 
aerobic and anaerobic bacteria 
(Quispel, 1974), in the root nodules of 
leguminous plants (Bergersen, 1971, 
1977; Mortenson, 1978), in Azolla 
containing nitrogen-fixing blue-green 
algal endophyte (Peters and Calvert, 
1982), in free-living photoautotrophic 
blue-green algae (Venketaraman, 1993) 
and nitrogen-fixing phototrophic 
bacteria (Sprent and Sprent, 1990). In 
recent years, research on nitrogen 
fixation in marine as well as freshwater 
habitats has expanded due to its 
recognition as an impo1iant process 
from a budgetary point of view. 
The present investigation was 
undertaken to enumerate, isolate and 
96 P. K. Pandey and N. S. Srcclu 
characterize nitrogen-fixing bacteria in 
coastal waters and soils, and to study the 
influence of different physicochemical 
parameters on their nitrogen-fixing 
capacity. 
MATERIAL AND METHODS 
Five stations (S 1 to S5) were 
selected in and around the creek 
discharging drainage water near Juhu 
beach along the coast of Mumbai for a 
period of three and a half months ( 105 
days) from 01 October 2002 to 15 
January 2003. S 1 was a creek connected 
to sea in which all the drainage water 
from the nearby locality was drained 
and got mixed with seawater. The other 
stations were selected on either side of 
the creek at a distance of 1 km from each 
other. S2 was at Seven Bungalows 
coast and S3 was at Versova coast; these 
two stations were located on the right-
hand side of the creek. S4 was at 
Chaupatty and S5 at Juhu on the left-
hand side of the creek. Water and soil 
samples were collected at fortnightly 
intervals during the study period. For 
microbial studies, water and sediment 
samples were collected aseptically 
using sterile test tubes and Petri plates. 
The heterotrophic and nitrogen-fixing 
bacterial populations were enumerated 
using standard pour plate method. For 
the total aerobic heterotrophic bacterial 
count, nutrient agar in seawater was 
used as the medium. For the total 
nitrogen-fixing bacterial count, 
Jensen's medium in seawater was used. 
Fifty bacterial isolates were taken on to 
agar slants from Jensen's medium and 
maintained at room temperature. Based 
on the different morpho logical 
observations and biochemical tests such 
as Gram's staining, starch hydrolysis, 
hydrogen sulphide production, 
peroxidase reaction, nitrate reduction 
and antibiotic sensitivity, the bacterial 
isolates were identified following Krieg 
(1984). 
Water quality parameters such as 
temperature, salinity, dissolved oxygen, 
total alkalinity, dissolved organic 
matter, ammonia-nitrogen, nitrite-
nitrogen, nitrate-nitrogen, phosphate-
phosphorus, etc. were estimated 
following APHA (1998). Moisture 
content, pH and organic carbon of 
sediment were analysed following 
Walkley and Black (1934). Available 
nitrogen (Jhingran et al., 1969) and 
phosphorus (Olsen et al., 1954) m 
sediment were also estimated. 
Nitrogenase activity was 
determined following the acetylene 
reduction assay (ARA) in a gas 
chromatograph (Shimadzu Class-
GClO) using Porapak-N column and 
flame ionization detector (FID). The 
temperature regime was: injection 
temperature - 100°C, column 
temperature- 80°C and FID temperature 
150°C. The carrier gas (nitrogen) flow 
rate of 40 ml/min was maintained. A 
standard chromatogram was obtained 
by injecting 1 111 of standard ethylene. 
Further, 1 111 of gaseous phase in the 
headspace from different samples was 
injected, and the ARA value was 
calculated and expressed as nmol 
C2Hilld. 
NITROGEN-FIXING AEROBIC BACTERIA AND THEIR 
NITROGENASE ACTIVITY IN NORTH MUMBAI COASTAL WATERS 
97 
RESULTS AND DISCUSSION 
Physicochemical Parameters of 
Water 
The water temperature at the study 
sites remained below 30°C. Since . all 
known heterotrophic. nitrogen-fixing 
bacteria belong to the mesophilic group 
(Jensen, 1981 ), the temperature 
observed was ideal for the growth and 
natural occurrence of the nitrogen-
fixing bacteria. 
Salinity values during the study 
period were more or less 35°/00 • During 
the first half of November, the salinity 
values of S 1, S2 and S3 stations were 
low due to mixing of seawater with land 
drainage. Kawai and Sugahara (1971a) 
reported nitrogen~ fixing bacteria at 
salinity of34°/00; however, the nitrogen-
fixing bacterial count was independent 
of the salinity value. The present study 
also showed the same result as the 
nitrogen-fixing bacterial count was 
independent of salinity. During the 
present study, the value of dissolved 
oxygen ranged from nil to 6.4 mg/1. 
However, the nitrogen-fixing bacterial 
count showed no correlation with the 
dissolved oxygen content in the present 
study as reported by Kawai et a!. 
(1971). The total alkalinity values 
recorded during the course of study 
were. in the range of 85-142 mg 
CaC0/1. A significant difference in the 
levels of total alkalinity was recorded 
both between different stations (F 4, 
24 2.9; p<0.05) as well as between 
periods (F6 24= 14.39;p<0.05). 
According to Paerl et a!. (1987), 
deficiencies in organic matter may play 
key roles in limiting and regulating 
marine nitrogen fixation. The dissolved 
organic matter values recorded during 
the course of study were in the ranges of 
3.6-24.8, 0.4-13.6, 2.0-16.0, 2.8-18.0 
and 1.6-14.8 mg/1 at Sl, S2, S3, S4 and 
S5, respectively (Fig. 1). A significant 
Oct111t Oct1Sth Nov1st Nov151h Dtoc15th Jon1st 
Sampling porfods 
I 
-0-- 1 -§- 2 -+-- 3 --+-- 4 -<>-- 5 1 
Fig. 1: Variations in dissolved organic matter 
(ppm) of water 
difference in the levels of dissolved 
organic matter was observed between 
periods (F6, 24=5.44;p<0.05). A positive 
correlation was observed between the 
dissolved organic matter and the 
nitrogen-fixing bacteria of water 
(r=0.45;p<0.05). The dissolved organic 
matter contents in different waters 
indicated the levels of degradation of 
organic matter taking place. 
Ammonia-nitrogen levels 
recorded during the study were in the 
range of 0.03-3.72 mg/1. A significant 
difference in the levels of ammonia-
nitrogen was observed between 
different stations (F4 24=5.07; p<0.05). 
According to Dash (1993), the 
ammonia-nitrogen values obtained in . 
freshwater ponds were in the range of 
0.16-14.82 g-at N/1. But in the present 
98 P. K. Pandey and N. S. Srcc\u 
study, high values of ammonia-nitrogen 
(0.03-3.72 mg/1) were obtained. This 
may be due to the domestic drainage 
coming to the coastal waters at the site 
of study. The nitrite-nitrogen values 
recorded during the study period were 
in the ranges of 0.04-0.10, 0.03-0.17, 
0.05-0.50, 0.036-0.18 and 0.02-0.17 g-
atN/1 at stations Sl, S2, S3, S4 and S5, 
respectively. The nitrate-nitrogen of 
water was noted in the ranges of 1. 69-
20.70, 3.50-21.39, 4.50-20.35, 12.87-
26.46 and 14.81-26.36 g-at N/1 at 
stations S 1, S2, S3, S4 and S5, 
respectively. A significant difference in 
the levels of nitrate-nitrogen was 
recorded both between different 
stations (F4. 24=3.14; p<0.05) as well as 
between periods (F6• 24=4.19; p<0.05). 
Nitrite-nitrogen level was 
comparatively less than the value 
obtained by Kawai and Sugahara 
(1971 a) at Kumihama Bay. The nitrate-
nitrogen value was very high compared 
to the value obtained by Kawai and 
Sugahara ( 1971 a). This indicates that 
active oxidation of am1nonia is taking 
place converting it to nitrate which is 
less toxic and more useful as a nutrient 
to the aquatic ecosystem. 
Ecologically, phosphorus is often 
considered as the most critical single 
element in the maintenance of aquatic 
productivity (Banerjea, 1967). In the 
present study, phosphate-phosphorus 
was found in the range 12.5-93.0 g-at 
P /l. This is attributed to the domestic 
sewage supply from the adjacent 
locality. 
Sediment Characteristics 
Highly alkaline sediment in the pH 
range 8.6-9.34 was observed at the 
sampling stations. Carbon is an 
important constituent of all organic 
matter. According to Banerjea (1967), 
1.5 to 2.5% organic carbon appears to 
be optimal for a productive freshwater 
body. The present study showed a 
much l_ower value of organic carbon 
(0.0 1 to 0.52%). 
Nitrogen is of prime importance 
for energetic and synthetic processes, 
and sediments are the main source of 
nitrogen in an aquatic ecosystem. The 
present study showed comparatively 
low content of available nitrogen in 
sediment, values being in the range of 
10.08 to 23.52 mg N/1 00 g of soil. 
Available phosphorus was recorded in 
the ranges of 1.69-2.59, 1.53-2.43, 
1.70-2.68, 2.03-2.73 and 1.88-2.75 
mg/1 00 g of sediment at S 1, S2, S3, S4 
2.5 
0 
~ 2 
0, 
E 
; 1.5 
:J 
... 
0 
.1: 
~ 1 
0 
.l: 
a. 
0.5 
0~------~--~~~~~~ 
Oct 1st Oct 15th Nov 1st Nov 15th Dec 1st Dec 15th Jan 1st 
sampling periods _ 
1--2 ¢ 3-+-4---*:--5 
Fig. 2: Variations in available phosphorus 
(mg/lOOg) of soil 
NITROGEN-FIXING AEROBIC BACTERIA AND THEIR 
NITROGENASE ACTIVITY IN NORTH MUMBAI COASTAL WATERS 
99 
and S5, respectively (Fig. 2). The 
· present study showed a positive 
correlation between the available 
nitrogen content and available 
phosphorus. 
Total Heterotrophic Bacterial 
Population 
The bacterial coenoses serve as a 
link between the abiotic environment 
and the biotic communities in an aquatic 
ecosystem. Kawai and Sugahara 
(1971a) reported that the number of 
total heterotrophic bacteria in Maizuru 
Bay was 1 02 to 1 03 cells/ml, and in 
Surugama and Sagami Bay, were about 
1 03 to 1 05 cells/1. In the present study, the 
total heterotrophic bacterial count at 
sampling sites was comparatively 
higher than that reported by them 
(0.004-2.34x 106 cfu/ml), whichniaybe 
attributed to the higher levels of nutrient 
availability. Kawai and Sugahara 
(1971b) reported 104 to 105 cells/g mud 
in Surugama Bay and Sagami Bay. Tne 
present study also showed a 
comparatively high value of total 
heterotrophic bacterial count of 0.03 to 
32.22 x 106 cfu/g dry weight of soil. The 
observation that bacterial count in 
sediment is higher than that in water is 
consistent with this finding. 
Aerobic Nitrogen-fixing Bacteria 
The heterotrophic free-living 
aerobic nitrogen-fixing bacteria have 
been considered to be significantly 
contributing to the productivity of 
natural and agricultural systems. 
Guerinot and ColwelL(1985) reported 
0.4 to 1.0 x 103/1 nitrogen-fixing 
bacteria in the water samples collected 
off the coast of Puerto Rico and 2.0 to 
5.5 x 102/1 in the Chesapeake Bay, which 
were quite comparable with the number 
of nitrogen-fixing bacteria recorded in 
water, i.e., 0.03 to 4.65 x 103 cfu!ml 
(Fig. 3) and was higher than the value 
reported for nitrogen-fixing bacteria in 
/ml 
' cfu 10 
O~t1l'!f Oct151h Nov1nt Nov15th Doc1:~t Dnc:151h J11n1\'lt 
Sampling periods 
1--1 --2 --3 --4 -.:.~5 
Fig. 3: Variations in total nitrogen-fixing bacteria 
(cfu103/ml) ofwater 
Surugama Bay and Sagami Bay by 
Kawai and Sugahara (1971b). The 
present study showed nitrogen-fixing 
bacterial count in the range of0.3-12.7 x 
103 cfu/g dry weight of soil (Fig. 4). 
Nitrogen-fixing bacterial population 
/g 
' du10 
Oe<:15th 
Sampling periods 
Fig. 4: Variations in total nitrogen-f"Irlng bacteria 
( cful 03 /ml) dry weight of soil 
100 P. K. Pandey and N. S. Srcclu 
was higher in sediments than those in 
overlying waters as recorded by J ana 
andRoy(1986). 
According to Guerinot and 
Colwell (1985), significant correlation 
was not observed between numbers of 
the nitrogen-fixing bacteria present at 
any given locality with the physical and 
chemical parameters. However, a 
positive correlation was observed 
between the dissolved organic matter 
and nitrogen-fixing bacteria of water at 
the present study sites. Dash (1993) 
attributed higher aerobic nitrogen-
fixing bacterial population in water to 
higher dissolved organic matter. A 
positive correlation between the 
available phosphorus and nitrogen-
fixing bacteria in soil was also observed 
in the present study. 
Four nitrogen-fixing bacterial 
species occurred in the water and soil 
samples. These were Azotobacter 
chroococcum, A. vinelandii, A. 
beijerinckii and A. armeniacus. Pandey 
(2002) reported A. chroococcum, A. 
vinelandii, A. beijerinckii, A. 
armeniacus and A. nigricans in 
freshwater fish ponds. Dash ( 1993) also 
reported A. chroococum, A. vinelandii, 
A. beijerinckii, A. armeniacus and A. 
nigricans in freshwater ponds. 
Nitrogenase Activity Studies by 
Acetylene Reduction Assay 
ARA value was 1.076 nmol 
C2H/lld when each millilitre contained 
48 colony-forming units of A. 
chroococcum and 0.965 nmol C2H4/l/d 
for A. vinelandii when each millilitre 
contained 35 colony-forming units. A. 
chroococcum showed higher level of 
nitrogenase activity thanA. vinelandii. 
S 1 (creek) was found to have high 
number of A. chroococcwn. The 
maximum ARA value was observed at 
S 1 followed by S3, S4, S5 and S2. 
Higher level of nitrogenase activity at 
S 1 is attributed to the higher load of 
nutrients through domestic sewage, 
mainly dissolved organic matter and 
available phosphorus, which showed 
positive correlation with aerobic 
heterotrophic nitrogen-fixing bacteria. 
The influx of nutrient-rich sewage 
water favours greater number of 
nitrogen-fixing bacteria enhancing the 
productivity ofthe seawater through the 
higher level of nitrogen. 
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Nitrogen-fixing aerobic bacteria and their nitrogenase activity in north Mumbai coastal waters

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Nitrogen-fixing aerobic bacteria and their nitrogenase activity in north Mumbai coastal waters

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