Estuarine, Coastal and Shelf Science 82 (2009) 29–34

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Estuarine, Coastal and Shelf Science

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Limno-tolerant bacteria govern nitrate concentration in Mandovi Estuary, India

Baby Divya, Sheryl O. Fernandes, G. Sheelu 1, Shanta Nair, P.A. Loka Bharathi*, D. Chandramohan
Biological Oceanography Division, National Institute of Oceanography, Dona Paula, Goa 403 004, India

a r t i c l e i n f o a b s t r a c t

Article history: The spatial and temporal abundances of limno-tolerant and halo-tolerant bacteria were investigated in
Received 23 May 2008 the tide-dominated Mandovi estuary along the west coast of India. These investigations were carried out
Accepted 13 November 2008 in relation to various environmental parameters on a monthly basis at three fixed stations for a year. On
Available online 3 December 2008
an annual basis, the estuary showed an average salinity of 28.2, 17.4, and 12.6 at the mouth, midstream
and upstream region. Halo-tolerant retrievable count (HTRC) and limno-tolerant retrievable count (LTRC)
Keywords:
of bacteria were in the order of 106 L1. Among the environmental parameters, a strong negative rela-
bacteria
tionship between salinity and nitrate (r ¼ 0.806; p < 0.001) suggested that 64% of the variation could be
limno-tolerant
halo-tolerant due to fresh water influence in the estuary. The limno-tolerant retrievable count (LTRC) brought about
salinity 23% variations in nitrate concentration. This influence was maximum during the monsoon (r ¼ 0.522;
nitrate p < 0.05) especially in the surface waters (r ¼ 0.624; p < 0.001) suggesting nitrate reduction by LTRC.
estuarine environment Measurements of nitrate reducing activity (NRA) in whole-water samples along the salinity gradient in
Mandovi the estuary also revealed higher reduction rates at lower salinity upstream. This was further confirmed
by culture experiments where the limno-tolerant bacteria showed higher NRA than halo-tolerant forms.
It is therefore suggested that LTRC is more actively involved in the variation of nitrate that enters the
Mandovi estuary particularly during the monsoon.
Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction The Mandovi is a tide-dominated tropical estuary, located

between 15 250 to 15 310 N and 73 450 to 73 590 E on the west coast
An estuary, as the meeting point of fresh and saline waters, is of India and is well mixed throughout the year. Due to its location in
a dynamic environment which is subjected to a wide variation in the south-eastern Arabian Sea, it is subjected to wind-forcing
physical and chemical parameters. In the estuary, the bacterial resulting from the annual reversal of monsoon winds (Wiggert
community may comprise physiologically versatile species et al., 2005). The estuary receives an annual rainfall of the order
belonging to the autochthonous estuarine bacterial population 250–300 cm yr1 during the southwest monsoon (June–
and/or the allochthonous fresh water or terrestrial populations. September) and less than 10 cm yr1 during the rest of the year
Although the fresh water bacteria are introduced with the nutrient (Shetye and Murthy, 1987). It shows a characteristic shift from an
rich waters, they could be adversely affected by an increase in autotrophic phase during the non-monsoon period to heterotro-
salinity in the estuary (Hyun et al., 1999) compared to the phic mode during the monsoon (Ram et al., 2003).
autochthonous estuarine bacteria. Estuarine microorganisms thus The nitrate concentration is high (Sardessai and Sundar, 2007) in
require the capacity for adaptation to the continuous mixing of this estuary and the sources of nitrate are from terrestrial influx, the
waters, the different residence times and transportation of mate- mangrove swamps (Qasim and Sengupta, 1981) and from the iron
rials from diverse sources (Igeno et al., 1995; Almeida et al., 2001). ore extraction-plant discharge (De Souza, 1983) located upstream.
The adaptive response of the bacteria may be either by changing Do bacteria from these estuarine waters govern the level of nitrate?
their abundance and/or their activity. To address this question, we examined both autochthonous halo-
tolerant and allochthonous limno-tolerant community along with
nitrate concentrations in the Mandovi estuary. We hypothesize that
the allochthonous limno-tolerant bacteria are responsible for
governing the concentration of nitrate in the Mandovi estuary. Our
* Corresponding author.
hypothesis is further developed by laboratory experiments with
E-mail address: loka@[Link] (P.A. Loka Bharathi).
1
Present address: Indian Institute of Chemical Technology, Hyderabad 500 007, whole-water sample and isolates of limno-tolerant and halo-
Andhra Pradesh, India. tolerant forms on nitrate reducing activity at different salinities.

0272-7714/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/[Link].2008.11.020
30 B. Divya et al. / Estuarine, Coastal and Shelf Science 82 (2009) 29–34

2. Materials and methods along salinity gradients in the Mandovi estuary i.e. towards the
upstream (salinity ¼ 5), midstream (salinity ¼ 17) and mouth
Surface and bottom waters were collected from the mouth (Lat (salinity ¼ 34). Water samples were transferred to sterile conical
15 290 35.8100 N; Long 73 480 35.3300 E), midstream (Lat 15 300 13.1900 N; flasks. Allylthiourea (ATU) at a final concentration of 86 mM was
Long 73 540 00.8300 E) and upstream (Lat 15 320 35.2100 N; Long added to inhibit additional nitrate input through ammonium
73 570 24.4300 E) of the estuary at monthly intervals for a period of one oxidation (Ginestet et al., 1998). Triplicates were maintained
year using Zobell water samplers (Fig. 1). throughout the experiment and the flasks were incubated in the
dark at room temperature in a static condition. The fall in nitrate
2.1. Physico-chemical parameters content from each test flask was estimated at 0–1.5 and 3 h as
described above. Cell counts were also done using a cell counting
Water temperature was measured immediately after collection chamber under a bright field microscope (Nikon Eclipse 50i).
of sample onboard using a field thermometer. All the other Nitrate reducing activity was estimated with representative
physico-chemical variables were estimated in triplicate in the halo-tolerant and limno-tolerant isolates. These isolates were
laboratory after the transport of water samples in refrigerated grown on respective media. Pure cells harvested from culture plates
conditions. pH was determined using pH meter (Elico LI 614 pH were used. Cell suspension was vortexed and centrifuged at
Analyser). Salinity, nitrate, and nitrite were estimated as described 8000 rpm for 10 min using a REMI R-24 centrifuge. The supernatant
by Strickland and Parsons (1968). For oxygen estimation, the water was discarded and washing procedure was repeated. The cell
samples that were fixed immediately on board and were then suspension with 0.1 OD600 was then added to 500 ml of test
analyzed using Winkler’s titrimetric method (Carpenter, 1965). medium amended with ATU. Nitrate reducing ability in low (5) and
Chlorophyll was estimated based on the fluorimetry method of high (34) saline conditions was determined as described above and
Yentsch and Menzel (1963). Suspended load was determined are expressed as aM cell1 h1. Data of only representative cultures
gravimetrically on pre-weighed GF/F (Whatman) filters as are presented.
described by Krey (1964).
3. Results
2.2. Microbiological parameters
3.1. Variation in physico-chemical parameters
Total bacterial numbers were enumerated by the acridine
The temperature variation throughout the year was negligible
orange direct count (AODC) method (Hobbie et al., 1977). Samples
with slight drop during cold seasons and rainy months. Annual
were fixed immediately with formaldehyde (2% final concentra-
average temperature of the estuary was 27.2 (2.8)  C. The annual
tion). A fixed amount was filtered through 0.2 mm pore size black
average salinity was 20.3 (12.3) for the whole estuary. The average
polycarbonate membrane filters (Nucleopore), stained with 0.01%
salinity varied from 12.6 (11.2) in the upstream through 17.4 (12)
acridine orange and enumerated using an epifluorescence
in the midstream to 28.2 (9.25) in the mouth. During the
microscope (Olympus BH). Total viable counts were estimated
monsoon period the salinity dropped to a low of 0.11, 0.11, and 12.7
(Kogure et al., 1979) by incubating the sample with yeast extract
at upstream, midstream and mouth respectively. The average
for 6 h with subsequent filtration, staining, and enumeration as
concentration of nitrate during the monsoon was 6.2 (3.1) mM
above. Abundance of limno-tolerant and halo-tolerant bacteria
NO3–N whereas the value decreased drastically to 1.7 (1.4) mM
was estimated from the CFU (Colony Forming Units) retrieved on
NO3–N in the non-monsoon. Salinity and nitrate showed opposite
nutrient agar prepared in distilled water (salinity ¼ 0) and aged
trend (Fig. 2). In general, variation was more pronounced when
estuarine water (salinity ¼ 30) respectively (Krumbein, 1971).
data was analyzed seasonally (Table 1). In the surface waters
Enumeration was carried out after 48 h of incubation at 28  2  C
salinity varied from 0.11 to 18.7 in the monsoon and from 0.9 to 34.7
and counts were expressed in numbers per liter. Interrelation-
in the non-monsoon. However, in the bottom waters the salinity
ships between bacterial and environmental parameters were
ranged from 0.09 to 31.7 and from 13.6 to 34.9 during the monsoon
statistically examined using Statistica and Analysis Tool Pack in
Microsoft Excel. The microbial variables were log transformed
before analyses. 12
Nitrate (µ M)

2.3. Nitrate reduction along salinity gradients 9

6
Surface water samples were collected in sterile polypropylene
bottles for the determination of nitrate reducing activity (NRA) 3

74°45 50' 55' 74°00'E 0

40
N
Stn.3 30
Malim
Salinity

15°30' Stn.1 15°30'

.
Stn.2 20
N iR Panaji N
c anal

0 5 km
v
do
Man 10
Dona Paula
rj u a

Arabian Sea
ba

0
m

15°25' INDIA Zuari R. Cu 15°25' JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC
N N
Study Goa
area
Non monsoon Monsoon Non monsoon
Fig. 1. Sampling locations along the Mandovi estuary (Stn 1: mouth; Stn 2: midstream; Fig. 2. Annual variation of salinity and nitrate in the estuary. Mouth (6), midstream
Stn 3: upstream). (B) and upstream (,).
 B. Divya et al. / Estuarine, Coastal and Shelf Science 82 (2009) 29–34 31

Table 1
Physico-chemical variables in surface and bottom waters during monsoon and non-monsoon periods. Values in parenthesis – mean  SD.

Variables Monsoon Non-monsoon

Surface Bottom Surface Bottom

Temperature ( C) 25–31a (28  2.05) 24–30.5 (27  2.1) 20.5–32.5 (26.5  3) 19.5–32 (25  3.5)
pH 6.8–7.8 (7  0.31) 6.7–7.8 (7.3  0.3) 7.2–8.2 (7.7  0.26) 7.4–8.3 (7.87  0.2)
Salinity 0.11–18.7 (9.4  9.9) 0.09–31.7 (15  12) 0.9–34.7 (17.8  10.3) 13.6–34.9 (24  6.2)
Dissolved oxygen saturation (%) 94–73 (84  7.8) 104–53.7 (78.9  15.4) 106–61 (83  10) 111–62 (87  13.3)
Nitrate (mM) 3.6–14.1 (8.8  2.9) 1.17–13.3 (7.2  3.4) 0.05–6.44 (3.2  1.6) 0.09–3.6 (1.8  1.1)
Nitrite (mM) 0.6–6.84 (3.7  2.3) 0.72–8.9 (4.8  2.8) 0.001–2.3 (1.1  0.6) 0.001–2.2 (1.14  0.6)
Suspended load (g L1) 0.01–0.1 (0.06  0.04) 0.008–0.17 (0.09  0.06) 0.008–0.29 (0.14  0.08) 0.05–0.32 (0.18  0.07)
Chlorophyll (mg L1) 2.6–0.04 (1.3  0.86) 0.04–3.7 (1.8  1.1) 0.04–10.59 (5.3  2.8) 0.1–5.1 (2.6  1.32)
a
Range.

and non-monsoon respectively. The percentage of mean dissolved 3.3. Nitrate reducing activity
oxygen saturation during monsoon and non-monsoon were
(84  7.8) and (83  10) respectively in the surface waters. At the The initial concentrations of nitrate were 4.31 (0.2) mM NO3–N
surface, nitrate concentration ranged from 3.6 to 14.1 mM with an at 5, 3.5 (0.02) mM NO3–N at 17, and 3.0 (0.06) mM NO3–N at 34
average value of 8.8 (2.9) mM in the monsoon. The distribution in salinities (Fig. 4). Nitrification inhibited incubations with water
nitrite concentration followed similar trend. The suspended load collected along salinity gradients in the estuary revealed higher
was more during the monsoon compared to the non-monsoon NRA in low saline waters upstream as compared to the higher
period. Chlorophyll a concentration was higher during the salinity waters towards the mouth. Though increasing salinity
monsoon period both at the surface and bottom. generally inhibits the NRA activity, our observations show highest
NRA of up to 8.72 (0.1) aM cell1 h1 in midstream (salinity ¼ 17)
where the ambient nitrate concentration was 3.5 (0.02) mM. At
3.2. Bacterial parameters
salinity 5 specific NRA of limno-tolerant isolate was
55 aM cell1 h1 whereas for halo-tolerant it was 9.87 aM NO3–
The variations in total count (TC), total viable count (TVC),
N cell1 h1. Specific NRA in the estuarine isolate showed 11–92%
limno-tolerant retrievable count (LTRC), and halo-tolerant retriev-
repression of nitrate reduction at higher salinity (Table 5).
able count (HTRC) are shown in Table 2. The total count of bacteria
ranged from 108 to 109 L1. The mean annual value of TC showed
the maximum in the bottom waters during the non-monsoon 4. Discussion
season. The direct viable counts comprised 25% of the total bacterial
abundance. These varied from the lowest 19.5% at the bottom in the 4.1. Physico-chemical parameters
non-monsoon season to the highest 51.7% at the surface in the
monsoon season. The densities of LTRC and HTRC formed 0.008% Mandovi is a fresh water dominated estuary, considerably
and 0.014% respectively of the viable bacterial population in the influenced by tides. Even during the pre-monsoon period there is
estuary. On an average, the retrievability of halo-tolerant bacteria enough fresh water influx to keep salinity close to zero upstream to
was double that of limno-tolerant bacteria at 106 CFU L1 about 40 km which prevents high salinity waters from intruding
throughout the year (Fig. 3). The retrievability of LTRC during upstream (Shetye et al., 1995). In the present study, the average
monsoon was of the order 106 CFU L1, while during the non- salinity of the estuary was 12.6 (11.2) and 28.2 (9.25) in the head
monsoon their abundance decreased by an order. The highest and mouth respectively. The upstream and mid stations close to
abundance of HTRC and LTRC was detected in the non-monsoon lower region of the estuary (Fig. 1) were selected to evaluate the
and monsoon season respectively .The LTRC/HTRC ratios were high effect of salinity on nitrate concentration and also to enumerate
in midstream. The highest value of 14.2 (66.9) was recorded LTRC and HTRC.
during the monsoon season (Table 3). The halo-tolerant forms did The low salinity observed during the monsoon season in all the
not show any significant relation to the variation in physico- 3 stations was due to a considerable amount of run off from a larger
chemical parameters. The variation in the limno-tolerant counts tributary system and also because of its versatile topography
related significantly to the variation in nitrate by 23% (p < 0.001) on (Qasim and Sengupta, 1981). During the season, the estuary is
an annual basis. During monsoon, nitrate related negatively to known to be stratified 2–3 m below surface depending on the
salinity (r ¼ 0.536; p < 0.05). Variation in the LTRC was found to be depth. Two salt wedges are formed which extends 10 km from the
responsible for 27% (p < 0.05) of the variation in nitrate during this mouth of the estuary (Qasim and Sengupta, 1981) though it
season particularly in the surface waters (r ¼ 0.624; p < 0.001). remains well mixed during the non-monsoon (Varma et al., 1975).
During the non-monsoon, LTRC caused 27% variation in nitrite Therefore, the presence of lighter fresher water above and the
(Table 4). denser saline water below leads to a lesser extent of mixing. The

Table 2
Total count (TC), total viable count (TVC), halo-tolerant retrievable count (HTRC) and limno-tolerant retrievable count (LTRC) of bacteria in surface and bottom waters during
monsoon and non-monsoon periods. Values in parenthesis – mean  SD.

Variables (L1) Monsoon Non-monsoon

Surface Bottom Surface Bottom

TC (109) 4.05–0.9a (2.4  0.9) 12–0.6 (6.3  3.3) 13.2–0.7 (7  3) 41.4–0.74 (21  13.7)
TVC (109) 1.8–0.2 (1  0.5) 2.2–0.19 (1.2  0.4) 2.8–0.3 (1.6  0.6) 6.7–0.4 (3.6  1.8)
HTRC (106) 9.2–0.3 (4.7  2.5) 5.1–0.06 (2.6  1.5) 13–0.06 (6.6  3.2) 13.2–0.01 (6.6  3.5)
LTRC (106) 3.3–0.1 (1.7  1.05) 20–0.12 (10  5.5) 1.5–0.06 (0.75  0.35) 4.08–0.03 (2  1.1)
a
Range.
32 B. Divya et al. / Estuarine, Coastal and Shelf Science 82 (2009) 29–34

35 A B C
30
Log HTRC, LTRC

25

20

15

10

0
J F M A M J J A S O N D J F M A M J J A S O N D J F M A M J J A S O N D

Surface LTRC Surface HTRC Bottom LTRC Bottom HTRC

Fig. 3. Annual variation of halo-tolerant retrievable count (HTRC) and limno-tolerant retrievable count (LTRC) at the surface and bottom waters. A-mouth, B-midstream, C-
upstream.

present study also indicates that nitrate concentrations decreased non-monsoon as observed in the present study suggests nitrate
from the head towards the mouth of the estuary as has been removal from the estuary could be largely due to phytoplankton
reported elsewhere (Dong et al., 2000, 2002). Nitrate and nitrite uptake. Earlier studies by Dham et al. (2002) have also demon-
concentrations in the estuary were higher during the monsoon strated that the nitrate uptake by phytoplankton was more during
compared to the non-monsoon period with surface water recording the non-monsoon than the monsoon. In addition, chlorophyll
higher concentration than the bottom water. This is because the concentration was low during monsoon as compared to non-
estuary is also a receptacle for continuous nutrient and terrigenous monsoon season suggesting reduced primary production due to
organic inputs from riverine run off and mixing of fresh water and increased turbidity during the monsoon as reported for Schelde
seawater leading to chemical precipitation (Eckert and Sholkovitz, estuary (Soetaert and Herman, 1995). However, the nitrate and
1976; Karbassi et al., 2008) particularly during monsoons. The nitrite concentrations in these waters are generally high due to
higher level of suspended load in the monsoon (0.14 gL1) enrichment caused by the discharge from extraneous sources like
compared to non-monsoon (0.068 gL1) would have contributed to the iron ore screening plants throughout the year (De Souza, 1983).
the high level of nitrate. A similar observation was made in the
Zuari estuary (De Souza, 1977) where nitrate levels were high in the 4.2. Microbiological parameters
surface waters and was correlated to the influence of fresh water
influx following precipitation. The negative relationship between The bacterial abundance in this estuary is comparable to that of
salinity and nitrate in the surface waters observed in the present the earlier reports by De Souza et al. (2003) and Ram et al. (2003).
study could be attributed to extraneous input of nitrate into the About 25% of the total bacteria were viable in the whole of the
system by fresh water run off. The influx of fresh water in the head estuary. This study also finds that the culturable forms of HTRC and
region throughout the year, especially during the monsoon would LTRC are also high. The high amount of suspended load could
bring relatively high concentration of inorganic nitrogenous species provide the required microenvironments (Fletcher, 1991; Kirch-
into the estuary (Bhunia and Choudhary, 1982; Chandran and man, 1993). Previous reports show that LTRC is known to be
Ramamoorthy, 1984; Vijayakumar et al., 2000). The percent oxygen abundant when fresh water inflow is high (Nair and Bharathi,
saturation in the estuary ranged from 53 to 111% annually with the 1982). Interestingly, the present study reveals that the HTRC and
least values during the monsoon which suggests a predominance of LTRC were of similar magnitude. The culturability is different with
heterotrophic respiration. Increased allochthonous inputs have the halo-tolerant forms being marginally higher than and limno-
been shown to result in enhanced heterotrophic respiration and tolerant. This was in contrast to what has been reported in Gironde
reduced primary production in the estuary (Ram et al., 2003). estuary where halo-tolerant far out-numbered limno-tolerant
Intrinsic nitrification could also have a role in augmenting the bacteria (Prieur et al., 1987). Thus, the limno-tolerant forms in the
levels of this parameter. In the non-monsoon season the variation lower estuary seem to have adapted themselves to salinity fluctu-
in chlorophyll accounted for about 15% (p < 0.001) variation in ations as the estuary maintained a salinity close 20 throughout the
nitrate concentration, while this relationship did not prevail during year except during monsoon thereby deriving the benefit of
the monsoon. As there was no relationship between chlorophyll nutrients. Seasonal precipitation and salinity-induced stratification
and nitrate it is suggested that excess of nitrate in the water did not
limit the primary production during monsoon. Further, the signif- Table 4
Pearson’s correlation of variables with r-values in the estuary.
icant relationship between chlorophyll and nitrate during the
Variables Correlation r
Estuary Whole Nitrate LTRC 0.479b
Table 3
Estuary surface Nitrate Salinity 0.806b
Spatial and seasonal variation in LTRC/HTRC ratio.
Nitrate LTRC 0.624b
Location Estuary Season Monsoon Nitrate Salinity 0.536a
Nitrate LTRC 0.522a
Monsoon Non-monsoon Non-monsoon Nitrate Chlorophyll 0.383b
Mouth 0.62 (0.54) 0.8 (0.4) 0.53 (0.57) Nitrite LTRC 0.524b
Midstream 14.2 (66.9) 41.8 (115.8) 0.49 (0.4) a
p < 0.05.
Upstream 0.61 (0.4) 0.6 (0.31) 0.58 (0.55) b
p < 0.001.
 B. Divya et al. / Estuarine, Coastal and Shelf Science 82 (2009) 29–34 33

6 10 a 100% increase in salinity from 17 to 35 there was a decrease in

NRA by 50%. Recently, Miranda et al. (2008) have also shown that
9
nitrification rates are higher at intermediate salinities than in either
5
8 fresh water or seawater. Moreover, the ratio of the distribution of
LTRC/HTRC clearly shows that estuary is dominated by the limno-
7 tolerant forms especially at the midstream and during the
4
monsoon. Thus the higher NRA at the midstream is attributed to

aM cell-1h-1
6
the higher ratio. Further, the tighter coupling of LTRC to nitrate
µM

3 5 concentration as revealed by lowered NRA in estuarine isolates

under halo-tolerant conditions could be ascribed to repression of
4
nitrate reductase at higher salinity. Igeno et al. (1995) have
2
3 demonstrated that increase in salinity led to inhibition of nitrate
uptake resulting in a drop in intracellular nitrate, thus repressing
2
1 the nitrate reductase gene. Studies on the activity of nitrate
1 reductase enzyme in a halo-tolerant cyanobacterium Aphanothece
halophytica by Thaivanich and Incharoensakdi (2007), have also
0 0
shown decline in NRA when sodium chloride concentrations were
5 17 34
300 mM. Though it is generally known that salinity can have
Salinity
a significant effect on inorganic nitrogen cycling in estuarine
Fig. 4. Variation in nitrate concentration and specific nitrate reducing activity (NRA) ecosystems, the differential inhibition in the reducing potential of
along salinity gradients in the Mandovi estuary. Ambient nitrate: vertical bars; Specific estuarine bacteria observed in the present study is suggestive of
nitrate reducing activity: smooth line. optimal salinity favoring nitrate reduction. Although the nitrate
reductase gene is known to be constitutive and widespread, the
extent of influence of nitrate concentration on LTRC demonstrates
apparently control the availability of the major nutrients in the
that the environment could govern the degree of phenotypic
water column of the estuary as elsewhere (Vareethiah et al., 1998).
expression in the LTRC community. Moreover, the nitrate reducing
Nitrate levels may be influenced by microbially driven processes
activity was found to be more widely expressed in LTRC especially
(Smith et al., 2007) mediated by a diverse polyphyletic group of
during the monsoon during which the nitrate flux is pre-dominant.
bacteria (Zumft, 1997). Nitrate in the Mandovi estuary could be
Thus, nitrogen cycling in estuarine waters could be maximal at
removed through the reductive cycle by both halo-tolerant and
optimal salinity levels. LTRC could therefore play a significant role
limno-tolerant bacterial communities. LTRC was found to be
in the variation of nitrate particularly in the surface waters where
responsible for the variation in nitrate during the monsoon season
its concentration is greater.
particularly in the surface waters. Even during the non-monsoon
season, LTRC caused variation in nitrite. The significant relationship
between LTRC and nitrite concentration observed in the present 5. Conclusion
study emphasizes that the limno-tolerant bacteria could also be
responsible for nitrite reduction in the estuary. Yoshie et al. (2004) Limno-tolerant bacteria, though less in abundance, are actively
have shown that salinity decreases the nitrite reductase gene involved in the variation of nitrate that enters the Mandovi estuary
diversity in denitrifiers. Quantification of nitrate/nitrite reducing particularly during the monsoon. Nitrate reduction in the estuary is
phylotypes has shown that the gene copy numbers were detected enhanced at optimal salinity levels. Though the NRA activity is
the highest at the head of estuary where denitrification dominated generally inhibited by increasing salinity, our observations show
(Smith et al., 2007). Consequently, this high nitrate input could that different microbes exhibit different levels of inhibition varying
trigger higher population of nitrate reducers and therefore nitrate between 11 and 95%. The Mandovi estuary could therefore act as
reductase activity. a major sink of nitrogen throughout the year especially during the
Experiments to determine nitrate reducing activity in ambient monsoon.
water samples have shown higher NRA to occur in low saline
waters where nitrate concentrations were relatively high. Studies Acknowledgements
by Rysgaard et al. (1999) in estuarine sediments have shown that in
situ nitrification and denitrification decreased with increasing We are thankful to Dr. S.R. Shetye, Director, NIO for the facilities.
salinities, with the most pronounced reduction of approximately Divya and Sheryl thank the Council of Scientific and Industrial
50% occurring when the salinity was raised from 0 to 10. Our study Research (Govt. of India) for the award of Senior Research Fellow-
demonstrated that an increase in salinity up to 10 showed an ship. We are indebted to two anonymous reviewers for their
increase of NRA by about 33% and this trend was observed up to 17. valuable suggestions which improved the quality of the manu-
Nevertheless our experiments also indicated that when there was script. The authors are thankful to by Ms. Shiney Babu Koshy,
Vellore Institute of Technology University, Tamil Nadu, and Ms.
Lavanya C., Alagappa University, Karaikudi, Tamil Nadu, for help
Table 5
rendered during field sampling and lab analyses. This is NIO
Specific nitrate reducing activity (NRA) of limno-tolerant and halo-tolerant bacteria contribution no: 4472.
at salinity 5 and 34.

Isolate NRA (aM cell1 h1) Percentage reduction in NRA References

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Bhunia, A.B., Choudhary, A., 1982. Primary production of the estuarine water around
Halo-tolerant 9.87 (0.08) 8.72 (0.91) 11.65
Sagar Island, Sunderbans. Indian Journal of Marine Sciences 11, 87–89.
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