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Marine and estuarine methylotrophs: Their abundance, activity and identity

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  • National Institute of Oceanography.

Abstract and Figures

Methanotrophs were up to 1 and 0.65% of the total counts in estuarine waters and offshore sediments respectively. Experimental tests on methanol utilization showed that the estuarine isolates grew best at 4% methanol whereas offshore ones grew at 5% at an optimum pH of 6 or 7. Methanol, when used as an additional carbon source, in the presence of nutrient broth concentration ranging from 0.08 to 0.4%, enhanced growth by 129% and respiration by 177% in estuarine isolates. Biochemical and physiological characteristics showed that estuarine methylotrophs exhibited taxonomic affinities to Pseudomonas I or II sp. The offshore genera were more varied and belonged to Flavobacterium and Pseudomonas I or II sp. The abundance, activity and identity suggest that these physiological groups could be widespread and therefore could perhaps contribute significantly to the changes in C1 compounds and even their derivatives in marine and estuarine environments.
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CURRENT SCIENCE, VOL. 90, NO. 7, 10 APRIL 2006
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*For correspondence. (e-mail: loka@nio.org)
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ACKNOWLEDGEMENTS. We thank Dr Harsh K. Gupta, Former
Secretary, DOD for his personal interest in launching the pilot expedi-
tion to the Southern Ocean. We also thank Officials at the Department
of Ocean Development, New Delhi and the National Centre for Antarc-
tic and Ocean Research, Goa for their untiring efforts in making this
long awaited programme a reality. Special thanks are also due to all
participating organizations and their respective scientists who have
very kindly provided inputs for the present paper. Captain, officers and
crew of PESO are acknowledged for their constant support during the
collection of samples.
Received 1 April 2005; revised accepted 3 December 2005
Marine and estuarine methylotrophs:
their abundance, activity and identity
Daphne Faria and P. A. Loka Bharathi*
Biological Oceanography Division, National Institute of Oceanography,
Dona Paula, Goa 403 004, India
Methanotrophs were up to 1 and 0.65% of the total
counts in estuarine waters and offshore sediments res-
pectively. Experimental tests on methanol utilization
showed that the estuarine isolates grew best at 4%
methanol whereas offshore ones grew at 5% at an opti-
mum pH of 6 or 7. Methanol, when used as an addi-
tional carbon source, in the presence of nutrient broth
concentration ranging from 0.08 to 0.4%, enhanced
growth by 129% and respiration by 177% in estuarine
isolates. Biochemical and physiological characteristics
showed that estuarine methylotrophs exhibited taxo-
nomic affinities to Pseudomonas I or II sp. The offshore
genera were more varied and belonged to Flavobacte-
rium and Pseudomonas I or II sp. The abundance, acti-
vity and identity suggest that these physiological groups
could be widespread and therefore could perhaps con-
tribute significantly to the changes in C1 compounds
and even their derivatives in marine and estuarine
environments.
Keywords: Adaptation, estuarine, methylotrophs, marine,
methanol.
METHYLOTROPHIC bacteria (MTB) are obligate aerobic
microorganisms recognized by their ability to grow on
carbon compounds more reduced than CO2, without any
CC bonds. They are even able to assimilate compounds
such as HCHO or a mixture of HCHO and CO2. MTB capa-
ble of oxidizing methane are methanotrophs (MOB). They
play an important role in the geochemical cycling of
methane and its derivatives. The oxidation of methane
can have major implications on the structure of food webs
and climate, especially in the current global scenario.
Hence, a study on their ecology would be pertinent to under-
stand the dynamics of methane and methane-derived
compounds, especially in marine and estuarine systems.
Though much work has been carried out on the mole-
cular1–3 and taxonomic aspects4 of methanotrophs, the study
is either restricted to lacustrine environment5,6 or terres-
trial regions7–9. Work on the marine environment is limited10.
Hence, the present study assesses the retrievable abun-
dance and distribution of methylotrophs and methano-
trophs. It also examines the activity of methylotrophs
from estuarine beach and offshore regions.
Sampling was carried out during low tide in Septem-
ber, representing the end of the southwest monsoon season
at Dona Paula beach (15°27N, 73°48E), a sheltered
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beach in North Goa situated at the confluence of the
Mandovi and Zuari estuaries. Three adjacent sediment
cores were collected using hand-corers. The cores were
sectioned into 05, 510 and 10–13 cm. Water and core
samples were transported to the laboratory in iceboxes
and analysed within 2–3 h of collection.
Water samples were collected into sterile polypropylene
bottles from which 5 ml was transferred into vials and
fixed with 250 µl buffered formalin. Total bacterial popu-
lations were estimated using acridine orange direct counts
(AODC) method11. For sediment samples, approximately
1 g was transferred to a 15 ml centrifuge tube filled with
9 ml autoclaved filtered sea water, vortexed for 5 min and
allowed to settle for 1 min. Five millilitre of supernatant
was transferred into vials and fixed with 250 µl buffered
formalin. One millilitre of the fixed sample was mixed
with acridine orange (final concentration 0.01% w/v) for
5 min. The contents were then filtered onto 0.22 µm pore-
size black-stained Nuclepore filter paper. Bacterial counts
were made at 100X with an epifluoresence microscope
(BH) using 515-barrier filter.
Isolation of methylotrophs and methanotrophs was carried
out onto ATCC #1306 medium12. The medium contained
[g l–1 sea water (50%) at pH 6.8]: MgSO4.7H2O, 1.0;
CaCl2.6H2O, 0.2; FeNH4EDTA, 0.004; KNO3, 1.0;
KH2PO4, 0.272; Na2HPO4.12 H2O, 0.717 and 0.5 ml trace
element solution. Plates were solidified with 1.8% of puri-
fied bacto agar. Approximately 5 g of sediment sample
was suspended in a conical flask containing 45 ml of sterile
sea water, vigorously shaken for 1 min and diluted up to
106. Suitable aliquots (50–100 µl) from 102 to106 dilutions
were surface-plated onto ATCC #1306 medium. The plates
were incubated with methane in Gas Pak jars in the dark
for 34 weeks and checked for bacterial growth against a
control. For the isolation of methylotrophs the procedure
was the same, except that methanol was supplied in the
vapour phase13 from a petri plate placed at the bottom of
Gas Pak jar. Growth was recorded for 7 to 10 days period
against a control. The counts were expressed as CFU g–1
dry sediment or CFU ml–1. Plates were divided into sectors
and a sector was randomly chosen and all CFU were iso-
lated, checked for purity and used for experiments and
characterization. Colonies of similar morphotypes were
isolated (in replicates of 4 to 5). Thus six methylotrophic
isolates each ES6, ES7, ES31, OF401, OF504 and OF507
from estuarine and offshore regions represent about 30
original isolates which were identified according to
Gerhardt14.
Adaptation to aqueous methanol was achieved by se-
quential transfer of cultures into media with progressively
higher concentration of methanol (0.1, 0.5, 1.0, 2.0, 3.0,
4.0, 5.0% v/v). Subsequently, experiments were carried
out with 4% methanol for estuarine and 5% for offshore
isolates. Experiments were carried out to measure growth
and respiration under varying methanol, organic carbon
concentrations, and pH. Growth was estimated at definite
intervals by direct cell counts on a haemocytometer. Simul-
taneously, respiration was monitored in terms of forma-
zan production from TTC15,16. Cultures were incubated at
(30 ± 2°C) for a period of 8 days.
To test the effect of organic carbon on growth and res-
piration, isolates were grown in mineral media containing
methanol supplemented with various concentrations (0.08,
0.16 and 0.4%) of nutrient broth.
To test the effect of pH on growth and respiration, iso-
lates were grown in mineral media containing methanol
adjusted to various pH values, viz. 69. Control tubes without
methanol were included.
Microbial Adhesion to Hydrocarbons (MATH) Assay
was carried out to assess the ability of the isolates to util-
ize hydrocarbons, as outlined by Rossenberg17. The assay
was carried out as follows: To a thick suspension of a day
old culture, 4 ml of phosphate buffer (pH 8) was added
and the same was vortexed and its initial OD was adjusted
to 0.2 at 550 nm. To this, 0.5 ml of n-undecane was
added and vortexed for 2 min. This was allowed to stand
at room temperature for 20 min for phase separation.
Turbidity of the lower aqueous phase was measured again
and fraction of adherence calculated.
Fraction of cell adherence is defined as the ratio of the
difference between the initial and final turbidity over the
initial value.
Fraction of adherence = (A C)/A,
where A is the initial turbidity and C the final turbidity.
Both water and sediment from the estuarine and off-
shore regions were tested for the presence of methylotrophs
and methanotrophs. Culturable methanotrophs ranged be-
tween 0.13 and 6.12 × 103 CFU g–1 estuarine sediment
and between 0.17 and 0.85 × 103 CFU g–1 offshore sedi-
ment. However, methanotrophs from water were two or-
ders more at 807.0 × 103 CFU ml–1 in the estuary than in the
sea, where they ranged between 101 and 103 ml–1. How-
ever, Faria et al.18 reported that the MOB could range
higher from 104 to 106 g–1 much later in the year (post-
monsoon) in offshore sediments of the Arabian Sea and
these were over three orders higher than the present val-
ues. Yet another comparison showed that abundance of
methanotrophs in this study is one order less than the
values reported by Takeuchi et al.19 from sediment using
MPN technique. The difference in abundance could be
partly due to differences in ecosystems and also differ-
ences in the techniques employed. Like methanotrophs,
methylotrophs retrieved on methanol-containing media
varied from 0.63 to 66.9 × 103 CFU g–1 estuarine sedi-
ment, but were below detection limits offshore. Methylo-
trophic abundance was higher in the estuarine waters at
49.60 × 103 CFU ml–1 than offshore (Table 1). Ross et al.20
reported higher values for methylotrophic bacterioplankton
that varied between 0.6 and 1.2 × 106 ml–1 in the winter of
1994, and 0.8 and 5.5 × 106 ml–1 in the summer of 1994–95
in the floodplain lake in northeastern Victoria, Australia
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Table 1. Comparison of abundance of methylotrophic and methanotrophic bacteria with other studies
Source Number of MTB Number of MOB Method Reference
Marine sediment (g–1)
Arabian Sea sedimentJanuary to February 2002 BDL 0.170.85 × 103 Surface plating Present study
Arabian Sea sedimentNovember 2002 ND 104106 Faria et al.18
Water (ml1) 0.084.00 × 103 0.087.3 × 103 Present study
Estuarine sediment 0.6366.9 × 103 0.136.12 × 103
Water 49.60 × 103 807.00 × 103
Others
Aquifer sediment 104 MPN Takeuchi et al.20
Floodplain lake water
1994 Winter 0.61.2 × 106 16S rRNA probes Ross et al.21
199495 summer 0.85.5 × 106
BDL, Below detection limit; ND, Not done; MTB, Methylotrophic bacteria; MOB, Methane-oxidizing bacteria; MPN, Most probable number technique.
(Table 1). Thus, our study shows that methylotrophs are
two orders less than the estimates made by Ross et al.20
using 16S rRNA probes. Irrespective of the region, water
recorded higher abundance than sediment in the present
study. The present study shows that the methylotrophs and
methanotrophs were generally more abundant in estuaries
with water showing a higher concentration than sediment,
suggesting that the substrates or substrate producers for
these organisms could be more abundant in estuarine waters.
Particles in estuarine waters could harbour higher number
of fermentative and methanogenic bacteria which could
provide these substrates.
The total bacterial counts in water were in the order of
107 ml–1. However, in the estuarine beach and offshore
sediments, they were in the order of 105 and 108 g–1 res-
pectively (Table 2). Takeuchi et al.19 reported total counts
in the order of 104 g–1 dry sediment, which is lower than
that observed in the present study. Methanotrophs formed
up to 1% of total counts in estuarine water and 0.005% in
sediment. The contribution of these forms amounted to
0.65 and 0.03% of the total counts in offshore sediments
and water respectively. These values are congruent with
those reported by other authors. Gilbert and Frenzel21 re-
ported that MOB estimated in a planted paddy soil accounted
for 1% of total counts (direct counts of total bacteria vs
MPN of MOB). MOB are also known to account for the
same number as aerobic heterotrophic bacteria22. However,
Vecherskaya et al.23 reported that MOB estimated by
immunofluorescence formed 123% of total counts in
peat soils from Siberian tundra.
Takeuchi et al.19 reported high percentages of 15.71,
4.78 and 5.08 for three aquifers from TCE-contaminated
site in Chikura, Chiba, Japan. The higher percentages of
MOB reported by these authors could perhaps be attributed
not only to the different ecosystems, but also to the lower
total bacterial counts obtained from that system. MPN
methods are known to yield higher numbers. Dubey et
al.24 reported that most quantification of MOB population
size relies on MPN methods. Frenzel22 highlights some of
the limitations of this method. These include microcolonies
that could be counted instead of single cells, the medium
could be selective for certain strains, and cells may be in
an unculturable state.
Despite the widely reported toxicity of methanol to ob-
ligate methylotrophs25–27, growth on methanol at high
concentrations is clearly possible for Methylocystis parvus
OBBP up to 4% w/v28, Methylococcus NCIB 11083 up to
0.2%, v/v28 and Methylosinus trichosporium OB3b up to
4% v/v29. In the present study, some of the estuarine iso-
lates grew and respired at 4 or 5% v/v concentration of
methanol. The mechanism by which these organisms
adapt to growth on methanol at high concentrations is un-
known, but may reflect physiological adaptations of the
population to the substrate or the selection of a mutant to
either methanol or formaldehyde29. Loss of this ability of
methanol-grown organisms on methane or substrate ana-
logues has been reported for bacterium B6 (ref. 30) and
for three other methylotrophs31. In contrast, other workers
have demonstrated the retention of methane-oxidizing ac-
tivity in type I methylotrophs25,28. Results of Best and
Table 2. Total counts of bacteria from estuarine and offshore waters
Estuarine Offshore
Total counts Water Sediment Water Sediment
(ml1/g–1) × 107 × 105 × 107 × 108
1.81.9 2.41.3 1.158.19 0.091.18
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CURRENT SCIENCE, VOL. 90, NO. 7, 10 APRIL 2006 987
Higgins13 were in contrast with those of Hou et al.31 for
the same strain of M. trichosporium. In the present study
the six isolates also displayed retention of such methanol-
oxidizing activity, suggesting that the enzyme is either
constitutive or induced by methanol.
Many methylotrophs are facultative or restricted facul-
tative, that grow on sugars, fatty acids, amino acids, inorganic
substrates, and complex media as well as one-carbon
compounds. However, in the present study, methylotro-
phs could utilize either methane or methanol effectively
for their growth in the presence of low concentration of
nutrient broth (0.1%). It has been reported that some cul-
tures utilizing methanol grow better on heterotrophic sub-
strates30,32, and it is suggested that methanotrophs with
heterotrophic tendencies would be more widespread than
strict autotrophic ones. According to Griffiths et al.33, there
is extensive documentation that methane-utilizing bacte-
ria can utilize a large number of organic compounds that
include not only simple hydrocarbons, but also more
complex organic molecules33–36.
Experiments with isolates showed that maximum
growth and respiration were recorded after a period of 8
days for all methylotrophs with the exception of OF507,
which showed maximum growth and respiration after 6
days. With increasing organic carbon from 0.08 to 0.4%,
enhanced growth (129%) and respiration (177%) were re-
corded for estuarine isolates. However, there was a 17%
decrease in growth and 396% increase in respiration for
their marine counterparts.
The optimum pH recorded for the estuarine and off-
shore isolates was 6 or 7. On an average, growth of off-
shore isolates was 199% of the control at pH 6 or 7, and
was better than its estuarine counterparts, which was only
Table 3. Molar growth yield constant (k) of estuarine and offshore MTB
Methanol
4% 5%
Estuarine methylotrophs Offshore methylotrophs
Ps. sp (n = 3) Ps. sp (n = 2) Fl sp (n = 1)
0.041 0.024 0.018
Avg. 0.04 0.02
Table 4. MATH assay
Initial Final Fraction of adherence
Culture turbidity (A) turbidity (C) (AC)
ES6 0.200 0.246 0.230
ES7 0.200 0.170 0.178
ES31 0.206 0.211 0.055
OF401 0.203 0.133 0.350
OF504 0.200 0.111 0.445
OF507 0.203 0.201 0.0098
166%. Respiration was stimulated by 80 and 70% of the
control for offshore and estuarine methylotrophs respec-
tively. At 4 and 5% methanol concentration for estuarine
and offshore respectively, their ability to grow at differ-
ent pH values, and organic carbon concentration (0.08
0.4%) showed that these isolates have an optimal pH at 6
or 7. Borne et al.37 reported methane oxidation from pH
3.5 to 8.0. However, Dedysh et al.38 reported isolation of
methanotrophs from Sphagnum peat bogs incapable of
growth at pH values below 5.0. Calhoun and King39 report
isolation of methanotrophs from freshwater macrophytes
at pH values between 6 and 7. Irrespective of the region,
isolates could grow in a broad range of pH values with an
optimum at 6 or 7 in this study.
Molar growth yield on methanol was 2 × 1010 cells
(± 3.4) and 4 × 1010 cells (± 1.2) for offshore and estua-
rine methylotrophs respectively. The molar growth yield
constant on methanol for offshore methylotrophs was 0.02
and for estuarine methylotrophs 0.04. Offshore forms are
lower in yield, but higher in activity (Tables 3 and 4). Faria
et al.18 also reported that offshore MOB are significantly
higher in abundance than near-shore ones (P > 0.001).
Thus it is suggested that offshore MOB are not only more
abundant in certain seasons, but also could be higher in
activity.
MATH assay, a cell-surface hydrophobicity test indicates
that growth of four of the six methylotrophs under the
conditions described displayed a hydrophobic character,
as evidenced by their association with the immiscible n-
undecane phase (Table 4). The fraction of adherence for
culture OF504 was high, due to its hydrocarbonoclastic
property. Raiker et al.40 observed the fraction of adher-
ence for thraustochytrids to be high and ranging between
0.2 and 0.42. The fraction of adherence was however less
for OF507 (0.0098), ES7 (0.178) and closer to that of
OF401 (0.35).
The high fraction of adherence for OF401 (0.445)
could be due to its hydrocarbonoclastic property. De
Souza et al.41 reported a high fraction of adherence (0.40.5)
for culture P43, a phosphate-solubilizing bacteria. In the
present study cultures ES6 and ES31 were hydrophilic,
exhibiting an adherence of 0.23 and 0.055 respectively.
The hydrophilic nature apparently reflects the presence of
vegetative cells42 and the probable presence of poly β hydr-
oxybutyrate granules43. Hydrophobic property exhibited by
methylotrophic bacteria makes them ideal candidates for
the bio-remediation of hazardous environmental wastes
like tricholoroethylene44 and other xenobiotics.
Phenotypic characterization based on key biochemical
(catalase, oxidase and modified oxidative fermentative
tests, ability to use methane, methanol and n-undecane)
and morphological traits (colony morphology, Gram stain,
motility, size, etc.) showed that 33% of the offshore iso-
lates and all the estuarine methylotrophs showed taxo-
nomic affinities to Pseudomonas I and II sp. The offshore
genera were more varied and belonged to Flavobacterium
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988
and Pseudomonas I and II sp. As these taxonomical
groups are widespread in the marine environment and as
the abundance of methylotrophs was as high as 1%, it is
speculated that they could contribute significantly to the
oxidation of methanol and its derivatives in nature. These
groups could perhaps also participate in the oxidation of
methane in the marine and estuarine environments. Experi-
ments are underway to examine their methane-oxidizing
activity in estuarine and marine sediments under simulated
in situ conditions.
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ACKNOWLEDGEMENTS. We thank Dr Chandramohan, Former
Deputy Director, HOD, BOD and Dr E. Desa, Ex Director NIO, Goa
for providing the opportunity to carry out the work. D.F. also thanks
Prof. G. N. Nayak, HOD Marine Sciences, and Goa University for pro-
viding the necessary facilities to carry out the dissertation. An anony-
mous referee helped improve the contents of the text. This is NIO
contribution no. 4080.
Received 2 July 2005; revised accepted 5 December 2005
Historic submergence and tsunami
destruction of Nancowrie, Kamorta,
Katchall and Trinket Islands of
Nicobar district: Consequences of
26 December 2004 SumatraAndaman
earthquake
M. G. Thakkar* and Bhanu Goyal
Department of Geology, R. R. Lalan College, Bhuj, Kachchh 370 001,
India
The 26 December 2004 SumatraAndaman earthquake
is one of the largest plate-boundary earthquakes in
the recent seismic history of the world. This earth-
quake has also generated the greatest tsunami run-up
and coastal devastation ever recorded. Our field study
at four major islands of Nancowrie group of Nicobar
District in Andaman and Nicobar archipelago has re-
vealed that the islands are vertically subsided by 1.0
1.75 m with the submergence of coastal land area by
thousands of square kilometers. It is also suggested
based on our field observations that societal and socio-
economic rejuvenation of the islands will need resur-
vey of entire topography of the islands, coastal
bathymetry mapping and identification of newly de-
veloped ecological regimes. We have also prepared
maps of the coastal submergence for these islands us-
ing field observations and remote sensing in this paper.
Based on the present field study and geodetic studies
by other workers, differential tilting of the Andaman
micro-plate is also inferred.
Keywords: Coastal submergence, Nancowire group,
Nicobar Islands, tsumani.
THE SumatraAndaman earthquake of 26 December 2004
occurred on 6:29 IST (0.58 UTC) at the subduction plate
boundary where the Indian and Australian plates converge
and plunge below the Sunda plate. The Mw 9.3 (revised
magnitude) plate boundary earthquake is located at 3.7°N
and 95°E off the Sumatra coast near the island of Simue-
lue with a focal depth1 of ~15 km. The earthquake is con-
sidered as the second largest ever recorded on the globe,
and it caused wobbling of the earth’s axis2. Distribution
of aftershocks reveals that the rupture plane is about
1200 km long extending to the north, up to the Andaman
and Nicobar Islands3,4. Immediate observation by the sat-
ellite imageries confirms large-scale subsidence around
the epicentral zone and many kilometers north of it, in the
Andaman and Nicobar Islands. The present study on pre-
liminary documentation of ground deformation and tsu-
nami effects on the Nancowrie group of islands of
Nicobar district, was carried out based on satellite image-
ries. These imageries provide information on inundation
of the islands and site-specific details of subsidence and
tsunami run-ups at each location. An attempt is also made
to prepare preliminary maps that show the coastal area of
subsidence on four islands Nancowrie, Kamorta, Katchall
and Trinket of the Nancowrie group. These inundation
maps could be used in future planning of developmental
activities in these islands. The coastal villages mentioned
later and farm fields on all four islands can be identified as
areas likely to be submerged in the future. A similar study
on the other islands of Andaman and Nicobar groups could
reveal the tectonic behaviour of the Andaman micro-plate.
The Andaman and Nicobar groups of 349 islands, situated
in the Bay of Bengal, are separated by the ten-degree
channel (Figure 1). The rocks of these islands are belie-
ved to have been formed from the sediments scraped off
the descending Indian plate interleaved with ophiolites
from the ocean floor beneath the Bengal Fan. Detailed
geology of the Andamans has been described by Oldham5
and Tipper6. The earliest rocks found in Andamans are
Upper Cretaceous clastics with ultramafic and mafic intru-
sives. A complete succession of Tertiary rocks is found in
the Andaman group of islands. Pleistocene sand beds,
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