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Marine and estuarine methylotrophs: Their abundance, activity and identity

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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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*For correspondence. (e-mail:
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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
Keywords: Adaptation, estuarine, methylotrophs, marine,
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
CURRENT SCIENCE, VOL. 90, NO. 7, 10 APRIL 2006 985
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
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
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
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
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
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
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
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
M. G. Thakkar* and Bhanu Goyal
Department of Geology, R. R. Lalan College, Bhuj, Kachchh 370 001,
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,
... A variety of methylotrophic microorganisms are found in almost all kinds of natural habitats Chistoserdova et al., 2009;Faria and Bharathi, 2006;Meena et al., 2015;Thulasi et al., 2018). Methylotrophs are part of different branches of the tree of life such as yeast, archaea and bacteria . ...
The objective of this project was the development of enhanced methylotrophic chassis strains capable of converting methanol as carbon and energy source into biomass and ultimately into commodity chemicals under industrial conditions. Methanol is an alternative to carbohydrates as feedstock in industrial biotechnology as its use does not interfere with food supply and its production can start from CO2.A prerequisite for an efficient and large scale industrial fermentation is stable growth of the methylotrophic producer strain on high methanol concentrations. For this purpose, two closely related methylotrophic strains, Methylobacterium extorquens AM1 and TK 0001, which both have a growth optimum at about 1% methanol, were adapted in continuous culture to proliferate stably in the presence of methanol of up to 10% (v/v). The adaptations were conducted using GM3 devices enabling automated long term cultivation of microorganisms.Growth curves recorded for isolates obtained from evolved populations showed enhanced proliferation in the presence of methanol at 5% as compared with wild type cells. The isolates showed comparable albeit not identical growth pointing to heterogeneity among the adapted cells in the population.Genomic sequencing of isolated clones at different steps of the adaptation revealed differences in their mutation profiles. The gene metY coding for O-acetyl-L-homoserine sulfhydrylase was found to be mutated in all isolates. This enzyme undergoes a side reaction with methanol leading to the production of the methionine analogue methoxinine known to be toxic through incorporation into proteins.Enzymatic tests conducted with these mutants showed an almost complete loss of activity even with their natural substrates, validating the involvement of MetY in methanol toxicity.Transcriptomic analysis was performed to study the gene expression response of an evolved derivative of M. extorquens TK 0001 to short and long term exposure to high methanol and compared with the response of the ancestor strain. Genes implicated in cell division, ribosomal and flagellar structures, protein stability and iron uptake showed differences in expression patterns between the strains.The M. extorquens TK 0001 cells adapted to high methanol produced more biomass from methanol than the wildtype cells. This suggests that a compound synthesized through a pathway branching from the central metabolism would be produced in higher yield from methanol by the adapted cells compared to the wildtype cells. The production of D-lactate was tested for wildtype and evolved cells both overexpressing native lactate dehydrogenase. The evolved cells produced more lactate than the control cells, confirming the interest of this methanol adaptation.
... The presence of Pseudomonas spp around the abattoir vicinity could be due to the presence of hydrocarbons within the abattoir. This observation supports the report by Faria and Bharathi[13] that Pseudomonas is ubiquitous in the environment and stated that they could contribute to the oxidation of hydrocarbons in the environment.In this study, a lot of the isolates were yielding readily to Cipro-floxacin. Most of the gram negative bacteria were resistant to some of the antibiotics used and a lot of the gram positive bacteria were 60 Microbiological Analysis of Abattoir Effluent Discharged into Eliozu River Citation: CJ Ugboma., et al. "Microbiological Analysis of Abattoir Effluent Discharged into Eliozu River". ...
... Some of the organisms encountered are majorly normal flora of a typical tropical soil whose growth must have been encouraged by the presence of animal droppings (Ayansina et al., 2014). Presence of Pseudomonas aeruginosa within the farm environment and its wide spread, supports the report by Faria and Bharathi (2006). ...
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Shiga toxin E.coli O157:H7 was isolated from chicken faecal matter, pen, feed, and water. All E.coli O157:H7 were 100% resistant to the selected antibiotics.
... The presence of Escherichia coli, Salmonella sp., Klebsiella sp., Proteus sp. and Serratia sp. in the soil and wastewater could be as result of faecal contamination and the run-off of wastes into the reservoirs [13,18]. The presence of Pseudomonas sp. in these ecologies may be the result of hydrocarbon production during burning of firewood, as this organism has been linked with hydrocarbon oxidation [19]. These bacteria from abattoir wastes may be discharged into water columns and can subsequently be absorbed to sediments, and when the bottom stream is disturbed, the sediment releases the bacteria back into the water columns presenting long term health hazards [20,21].The accumulation of the faecal materials act as a collection basin for pathogenic microorganisms which may spread between animals and man leading to zoonoses [22]. ...
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The constituents of wastes generated from abattoir activities create conducive environment for microbial proliferation, most of which are pathogenic. Infections caused by these microorganisms could result to zoonoses. This study was to determine the distribution of bacterial isolates and their biomass from different abattoirs in Port Harcourt. Samples like waste blood, table swab, service water, faecal matter, soil and wastewater from abattoirs in Iwofe, Rumuodomaya and Trans-Amadi were collected from October 2017 to November, 2018 and analysed using standard microbiological procedures. Results obtained revealed that the total heterotrophic bacterial count of blood samples ranged from 8.33x101 to 3.33x102 cfu/ml for Trans-Amadi and Iwofe abattoirs, table swabs ranged from 6.74x104 to 4.88x106 cfu/ml, water samples ranged from 1.56x104 to 2.07x104 cfu/ml; faecal matter had THB counts ranging from 9.97x107 to 1.06x108 cfu/g; and soil samples ranged from 1.11x1010 to 1.17x1010 g, while wastewater counts ranged from 1.03x108 to 1.08x108 cfu/ml. The predominant Bacterial isolates were of the genera Micrococcus, Staphylococcus, Serratia, Pseudomonas, Proteus, Klebsiella, Escherichia and Chromobacterium, Serratia sp. only was isolated from Iwofe and Rumuodomaya abattoirs within April to October while Chromobacterium sp. was isolated in Trans-Amadi and Rumuodomaya abattoirs within the months of May to October. Among the isolates, Escherichia coli and Klebsiella species occurred more compared to others in all the three locations. A higher percentage of microorganisms were recorded in the month of May compared to other months. It is presumed that abattoir wastes harbour many microorganisms of public health importance. The occurrence of these microbes, most of which are enteric pathogens, poses a public health challenge as infections by them could result in illnesses such as gastroenteritis, septicaemia and pneumonia especially in the absence of good hygiene around abattoirs. Proper sanitation in abattoirs as well as management of abattoir wastes is important in reducing the spread of these microorganisms.
... The same reason is applicable to Achromobacter and Acinetobacter, which are among the hydrocarbon degraders. The incidence of Staphylococcus species in this study is also in agreement with the report that Staphylococcus species is naturally found in the hides of cattle and Flavobacterium which is said to be authochthonous to the environment [54]. ...
... within the abattoir environment is probably due to the presence of hydrocarbons within the abattoir. This observation supports the report by Faria and Bharathi [18] that Pseudomonas spp. is widespread in the environment and concluded that they could contribute to the oxidation of hydrocarbons in the environment. ...
The study investigated the bacteriological effluent qualities of abattoirs in Abakaliki, Southeast Nigeria between June and September 2015. Wastewater samples were collected from two abattoirs, from their point of discharge into surface water bodies with sterile sample bottles and transported to the Laboratory for bacteriological analyses. Bacteria species were isolated, characterized, and identified using standard microbiological and biochemical techniques. Antibiotic susceptibility study was carried out using Kirby-Bauer disc diffusion method. The result of the total heterotrophic bacteria count obtained from Nkwo–Ezzangbo abattoir ranges from 7.00 × 106 Colony Forming Unit (CFU)/ml to 7.90 × 106 CFU/ml, while that of Abakaliki abattoir ranges from 5.50 × 106 CFU/ml to 6.95 × 106 CFU/ml. Antibiotic studies showed that majority of the gram-negative isolates were sensitive to the antibiotics. Resistance was obtained against augmentin, nalidixic acid (NAL) for P. aeruginosa. E. coli had resistance against ceporex, septrin, and NAL, while the gram-positive streptococci had resistance against cetriaxone and ampicillin. The presence of these multi-drug resistant strains of the isolated organism in abattoir effluents could act as a vehicle to disseminate antibiotic resistance to other bacteria. This emphasizes the need for proper treatment and safe disposal of abattoir effluents in the study area.
... Methylotrophic bacteria are a group of organisms that can utilize single carbon compounds (C 1 ) such as methane and methanol as their source for carbon and energy and have been reported from different environments (Faria and LokaBharathi 2006;Jhala et al. 2014;Meena et al. 2015). Methylotrophs can also consume other C 1 compounds including methylated amines, glycines and sulfur species as well as halomethanes. ...
Methylotrophs present in the soil play an important role in the regulation of one carbon compounds in the environment, and thereby aid in mitigating global warming. The study envisages the isolation and characterization of methanol-degrading bacteria from Kuttanad wetland ecosystem, India. Three methylotrophs, viz. Achromobacter spanius KUT14, Acinetobacter sp. KUT26 and Methylobacterium radiotolerans KUT39 were isolated and their phylogenetic positions were determined by constructing a phylogenetic tree based on 16S rDNA sequences. In vitro activity of methanol dehydrogenase enzyme, responsible for methanol oxidation was evaluated and the genes involved in methanol metabolism, mxaF and xoxF were partially amplified and sequenced. The specific activity of methanol dehydrogenase (451.9 nmol min⁻¹ mg⁻¹) observed in KUT39 is the highest, reported ever to our knowledge from a soil bacterium. KUT14 recorded the least activity of 50.15 nmol min⁻¹ mg⁻¹ and is the first report on methylotrophy in A. spanius.
... They also represent promising organisms in biotechnology for the conversion of onecarbon (C1) substrates to value-added products such as single cell proteins, vitamins, aminoacids, biopolymers, enzymes, products of biotransformation and also for their role in carbon-cycling, bioremediation and in replacing petrochemical-based chemical processing in future (Chistoserdova et al., 2003;Schradar et al., 2009).Members of this group possess great metabolic versatility with the ability to scavenge trace amounts of single carbon, nitrogen and resistance to a certain degree of desiccation which contributes their survival in the hostile environments (Anthony and Williams, 2003). Faria and Lokabharathi (2006) reported the marine and estuarine methylotrophs abundance in the samples of Dona Paula beach, Goa, India. Active methylotrophs present in the sediments of Lonar Lake, India, was reported earlier (Antony et al., 2010). ...
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Though the single carbon utilizing bacterial system, Methylotrophs are rich source of biological compounds and good source of bio-remeidation, their diversity in river system have not paid much attention. In this study, the population of methylotrophs in two main water ways, river Coovum and river Adyar of Chennai, fourth cosmopolitan city, of India was carried out. The samples were collected for 12 months from four different sites every fortnight and grouped into four seasons. The sampling sites are either ecologically sensitive or severely affected by contamination. The methylotrophic densities were found high in river Cooum 5.9 to 6.7 Log CFU mL-1 than Adyar river 5.5 to 5.9 Log CFU mL-1 and also fluctuated significantly over time which showed a clear seasonal variation. The bacterial count also varied depending upon the nature of site of sample collection with gradual decrease from upstream to downstream. Cluster analysis (similarity) based on the average values of methylotrophic counts explained the site-wise and month�wise relationship individually.
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Criteria for the selection of sampling sites were based on the probability of the occurrence of methylotrophs and pink pigmented facultative methylotrophs (PPFMs), especially in the ecologically fragile and environmentally polluted coastal and terrestrial areas. Nearly 10% of total methylotrophs were identified as PPFMs, exhibiting varying colony morphology and pigmentation in the methanol mineral salts medium, methanol served as a carbon source. The occurrence of methylotrophs and PPFMs in all marine water samples during four seasons in the period of investigation was in the range of 8-160 and 1-19 CFU/ml x 103 respectively. Methylotrophs and PPFMs were enumerated and the populations were in the range of 7-96 and 1-7 CFU/g x 103 respectively in marine soil samples. Methylotrophs and PPFMs were in the range of 25-116 and 1-9 CFU/g x 104 in terrestrial samples. This is study given the detailed information about the methanol utilizing bacterial communities are continuously affected by seasonal variations and shown the ability to survive with the available resources.
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The pattern of methane (CH4) oxidation and population size of methane oxidizing bacteria (MOB) were studied in three different soils (rhizosphere, bulk and bare) of a dryland rice (Oryza sativa L. cultivar Narendra-118) field. The rhizosphere soil exhibited the strongest CH4 oxidation activity and bare soil the weakest. MOB population size was significantly higher in the rhizosphere (671.0 × 105 cells g-1 soil) than in the bulk (569.0 × 105 cells g-1 soil) or the bare soil (49.2 × 105 cells g-1 soil), and NH4+-N concentration was highest in the bare soil (6.74 μg g-1 soil) followed by the bulk (5.58 μg g-1 soil) and rhizosphere soil (4.02 μg g-1 soil). Half saturation constant (Km) and maximum oxidation rate (Vmax) decreased significantly from the rhizosphere to bulk to bare soil and ranged from 84.01 to 5.81 μg g-1 dry soil and 0.62 to 0.05 μg h-1 g-1 dry soil, respectively. The rice rhizosphere not only supports a larger population of MOB but also contributes substantially to the capacity of soil for CH4 uptake, leading to a predictable spatial pattern in CH4 sink strength within the dryland rice ecosystem.
Scientific interest in methane dates back to 1776, when Alessandro Volta reported on the “aria infiammabile” which he had collected in a wetland (Paolini, 1976). This early observation already showed two things: The flux of CH4 from the biosphere to the atmosphere, and the possible role of wetlands as a source of atmospheric CH4. The first methane-oxidizing bacteria (MOB) were isolated at the beginning of the 20th century (Söhngen, 1906). The role of wetland plants was addressed for the first time in the 1970s (de Bont et al, 1978; Yoshida, 1978). Many of the following studies were stimulated by the potential impact of CH4 on our climate (e. g. Holzapfel-Pschorn et al, 1985, 1986; Holzapfel-Pschorn and Seiler, 1986; Schütz et al, 1989a). Current research becomes more focused on the interaction between MOB and wetland plants, and the spectrum of plants is widening from rice to emergent plants of natural wetlands.
Irrigated rice fields account for 10–30% of global methane emissions. Rice plants ventilate the soil and enlarge the oxic–anoxic interface by their root system, thus supplying the necessary O2 to aerobic CH4 oxidizing bacteria (MOB). Rice plants (Oryza sativa type japonica var. Roma) were grown in microcosms in a greenhouse. The roots were sandwiched between two blocks of flooded rice field soil separated by a nylon gauze bag. A root mat developed which mimicked the dense root texture in the upper layer of a natural rice field. Flux measurements under oxic and anoxic conditions showed that CH4 was oxidized with a constant rate of 19% of the anoxically emitted CH4, suggesting that CH4 oxidation in the rhizosphere was at least sometimes limited by CH4 availability. Washed rice roots could both produce and oxidize CH4, depending upon incubation conditions. CH4 production by washed rice roots accounted for at most 10% of the CH4 emitted under anoxic conditions. Initial CH4 oxidation rates of washed roots equaled oxidation rates calculated from the difference between oxic and anoxic fluxes in situ. Oxidation rates became twice as high after an induction period of 20h, indicating a limitation by O2 or CH4in situ. The micro-environmental conditions near to the root mat were measured using microelectrodes for O2, redox potential and NH4+ and diffusion probes for CH4. Up to 42μM O2 was detected in the root mat and concentrations were >2.5μM in 45% of all measurements. In the bulk soil, no O2 was detected below 2mm depth, but the root mat significantly increased the redox potential. Plant roots and associated bacteria decreased porewater CH4 and NH4+ concentrations. In the root mat, concentrations of dissolved CH4 were below the detection limit of our probes (
Methanotrophs, the inhabitants of irrigated rice soils, were monitored using molecular tools. Methanol dehydrogenase coded by mxaF locus has been used as the genus-specific locus. The presence of the locus has been demonstrated in DNA extracted from soil samples as well as in methanol utilizing isolates derived from those samples. Further, ARDRA pattern and cluster analysis revealed the following closely related genera - type-I methanotrophs Methylomonas and/or Methylocaldum in rhizosphere soil and coexistence of both type-I (Methylomonas and/or Methylocaldum) and type-II methanotrophs Methylobacterium and/or Methylocella in non-rhizosphere soil.
The low-temperature, methane-oxidizing activities and species composition of methanotrophic communities in various tundra bog soils were investigated by radioisotopic and immunofluorescent methods. Methanotrophic bacteria carried out the methane oxidation process through all horizons of seasonally thawed layers down to permafrost. The highest activity of the process has been observed in the water surface layer of overmoistured soils and in water-logged moss covers. Up to 40% of(14)CH4 added was converted into(14)CO2, bacterial biomass, and organic exometabolites. By immunofluoresecent analysis it was demonstrated that the representatives of I+X (Methylomonas, Methylobacter, andMethylococcus) and II (Methylosinus, Methylocystis) methanotrophic groups occurred simultaneously in all samples at 61.6% and 38.4%, respectively. The number of methane-oxidizing bacteria in the ecosystems studied was 0.1-22.9×10(6) cells per gram of soil. Methanotrophic organisms ranged from 1% to 23% of the total bacterial number.
The obligate methylotroph Methylosinus trichosporium OB3 b was capable of growth on methanol as sole carbon source at concentrations as high as 4% (v/v), and viability was maintained over many successive transfers. Methane mono-oxygenase, detected by epoxidizing and hydroxylating activity, was retained. The gross morphology of the organism on this substrate was dependent on culture conditions. It varied from organisms containing extensive peripheral membrane systems to those with extensive inclusions, the latter representing a poorly developed membrane system which predominated under most growth conditions.
trichloroethylene (TCE), an Environmental Protection Agency (EPA) priority pollutant, with the highest rate among biological systems (510 nmol/(min mg protein) in the presence of 20 mM formate as a reducing equivalent (source of NADH)33) and has a wide spectrum of sub- strates including benzene: chlor0form,3~ hydrochlo- rofluorocarb~ns,~ and vinyl chloride.l2 This enzyme also has potential applications for converting waste methane into methanol, synthesizing homochiral epoxides, and probing the biochemical fundamentals of the methane oxidation reaction. The sMMO expression in the wild- type strain is known to be regulated by the concentration of copper ions (sMMO is expressed at copper concentra- tions lower than 0.86 pmol/g dry cell weight; and partic-