Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e57126, enero-diciembre 2024 (Publicado Abr. 16, 2024)
Methanogenesis in sediments of a tropical coastal wetland:
a culture-dependent method
María del Rocío Torres-Alvarado*1; https://orcid.org/0000-0002-1919-7400
Teresa Pérez-Moz1; https://orcid.org/0009-0001-2744-0677
Neivy Betsabet Maldonado-Vela1; https://orcid.org/0000-0003-3439-9526
1. Department of Hydrobiology, Universidad Autónoma Metropolitana-Iztapalapa, Mexico City, Mexico;
rta@xanum.uam.mx (*Correspondence), tepm@xanum.uam.mx, neivymaldonado14@gmail.com
Received 23-X-2023. Corrected 05-II-2024. Accepted 10-IV-2024.
Introduction: Methanogenic archaea (MA), participate in the anaerobic mineralization of organic matter in
mangrove sediments, their activity is related to atmospheric warming due to the production of methane; several
environmental variables can influence the presence of MA and methane production in these sediments.
Objective: To analyze, through culture-dependent techniques, viable methanogenic archaea (VMA) in the sedi-
ments, and the production of methane from acetate in different climatic periods in the mangrove El Morro-La
Mancha, Veracruz, Gulf of Mexico.
Methods: From May to November 2019, following a salinity transect, sediment samples from El Morro-La
Mancha mangrove were collected at three locations, in three different climatic seasons, dry (May), rainy
(October) and northern (November) (N = 9). VMA in the sediments was quantified using the Most Probable
Number (MPN) technique with acetate and methanol as substrates. The influence of sulfate on methane produc-
tion was analyzed from acetate in microcosm by gas chromatography and the chemical variables of salinity, pH,
Eh, carbohydrates, organic content, and carbonates in the sediments were evaluated.
Results: The abundance of VMA was 102 to 108 MPN/g of wet sediment, higher than that reported in other stud-
ies, this abundance was higher when methanol (104-108 MPN/g sediment) was used as substrate, compared to
acetate (102-105 MPN/g sediment); methane production in the microcosms increased in sulfate-free conditions
(29.78-929.75 nmol CH4/month) and in the sediments of the rainy season.
Conclusion: The influence of the chemical conditions of the mangrove sediments on the methanogenic dynam-
ics is highlighted, determining that in the rainy season, the decrease in salinity, more electronegative Eh, and the
increase in organic fractions favored the methanogenesis.
Key words: acetate; climatic season; mangrove; methanol; microcosms; viable methanogenic archaea; sulfate.
Metanogénesis en sedimentos de un humedal costero tropical: un método dependiente de cultivo
Introducción: Las arqueas metanogénicas (MA) participan en la mineralización anaerobia de la materia orgánica
en sedimentos de manglar, su actividad está relacionada con el calentamiento atmosférico por la producción de
metano; diversas variables ambientales pueden influir en la dinámica metanogénica y la producción de metano
en estos sedimentos.
Objetivo: Analizar, mediante técnicas dependientes de cultivo, la abundancia de las arqueas metanógenas viables
(AMV) y la producción de metano en diferentes épocas climáticas en los sedimentos del manglar El Morro-La
Mancha, Veracruz, Golfo de México.
2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e57126, enero-diciembre 2024 (Publicado Abr. 16, 2024)
Mangrove forests are coastal wetlands
located in tropical and subtropical latitudes,
adjacent to coastal lagoons and estuaries, which
occupy an area of approximately 1.8 km2 ×
105 km2 in tropical and subtropical latitudes
(Alongi, 2002; Zhang et al., 2021). They are
extremely complex ecosystems formed by
woody trees that experience tidal and freshwa-
ter influence, and with numerous interactions
between vegetation, animals, and microorgan-
isms, Mangrove forests are considered among
the most productive wetlands on Earth, with
high rates of recycling of organic matter and
nutrients. These forests harbor high biodiver-
sity and provide several ecosystem services
(Zhang et al., 2021).
The sediments of the mangroves are char-
acterized by a high content of salts (20.8-22.8
PSU), hydrogen sulfide (1-25 mM) (Bhattacha-
ryya et al., 2015), a redox values low (-150 to
-200 mV), a low or no oxygen content and a
high proportion of organic matter (8-30 %),
mainly coming from the accumulation of leaf
litter of the mangrove trees (Lyimo et al., 2002a;
MacFarlane et al., 2007; Zhou et al., 2010).
The development of anaerobic metabolisms
is favored in these sediments, highlighting
the sulfate reduction and methanogenesis in
the terminal phases of the mineralization of
organic matter (Taketani et al., 2010).
Methanogenesis is a process carried out
by the methanogenic archaea (MA), a group of
prokaryotes of anoxic habitats (Conrad, 2020),
due to the sensitivity of methyl-coenzime M
reductasa to oxygen, which catalyzes the last
reaction for the formation of methane (CH4);
however, methanogens that have genes against
oxidative stress have been reported in arid areas
(Lyu et al., 2018). Historically MA had been
classified as a phylogenetically diverse group
of the phylum Euryarchaeota (Methanococ-
cales, Methanopyrales and Methanobacteria-
les). However, new phyogenetic analyzes have
revealed greater diversity, which includes other
phyla, Halobacteriota (Methanomicrobiales,
Methanocellales, Methanonatronarchaeales,
and Methamosarcinales), Thermoplasmatota
(Methanomassiliicoccales), and potentially Cre-
narchaeota, and this continues to be reviewed.
In addition to methanogenesis carried out by
archaea, it has been proposed that other meta-
bolic pathways present in fungi, plants and ani-
mals can produce methane (Bueno de Mesquita
et al., 2023; Lyu et al., 2018; Zhang et al., 2021).
The energy metabolism of methanogenesis
involves the formation of CH4 from organic
compounds such as acetate, formate, or short-
chain methylated compounds like methanol;
Métodos: De mayo a noviembre 2019, siguiendo un transecto de salinidad, se recolectaron sedimentos del
manglar del Morro-La Mancha, en tres localidades, abarcando tres diferentes épocas climáticas, secas (mayo),
lluvias (octubre) y nortes (noviembre) (N = 9), cuantificándose la abundancia de las AMV mediante la técnica
del Número Más Probable (NMP) con acetato y metanol como substratos; la influencia de los sulfatos en la
producción de metano a partir de acetato en microcosmos, se analizó por cromatografía de gases y se evaluaron
las variables químicas de salinidad, pH, Eh, carbohidratos, contenido orgánico y carbonatos en los sedimentos.
Resultados: Se determinó una abundancia de AMV de 102 a 108 NMP/g de sedimento húmedo, superior a la
reportada en otros estudios, dicha abundancia se incrementó cuando se utilizó metanol (104-108 NMP/g sedi-
mento) como sustrato, en comparación con el acetato (102-105 NMP/g sedimento); la producción de metano en
los microcosmos aumentó en condiciones libres de sulfatos (29.78-929.75 nmol CH4/mes) y en los sedimentos
de la temporada de lluvias.
Conclusión: Se destaca la influencia de las condiciones químicas de los sedimentos del manglar sobre la dinámica
metanógena, determinándose que, en la temporada de lluvias, una disminución de la salinidad, Eh más electro-
negativos y el incremento de las fracciones orgánicas favorecieron la metanogénesis.
Palabras clave: acetato; arqueas metanógenas viables; estación climática; manglar; metanol; microcosmos;
Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e57126, enero-diciembre 2024 (Publicado Abr. 16, 2024)
or MA can also grow autotrophically, from
the reduction of CO2 by H2 (Kurth et al.,
2020; Thauer et al., 2008). In this sense, the
acetate and the H2-CO2 also represent impor-
tant substrates for the sulfate reducing bacteria
(SRB), so that a competition for such substrates
can start between those and the MA (Con-
rad, 2020). However, in the mangrove sedi-
ments, the coexistence of both microbial groups
has been demonstrated (Lyimo et al., 2002b;
Taketani et al., 2010). In the mangrove sedi-
ments, it has been established that the abun-
dance and richness of MA are related to several
environmental factors, especially temperature,
pH, oxide-reduction potential (Eh) and quan-
tity and quality of organic matter (Euler et al.,
2020; Yasawong et al., 2013).
The study of the methanogenesis process
can be carried out using specific culture media
that allow the growth and quantification of
viable microorganisms, as well as laboratory
experiments (microcosm) in which, under con-
trolled conditions, the aim is to analyze the
utilization of a substrate by MA or the effect of
an electron acceptor on methanogenic metabo-
lism (Bueno de Mesquita et al., 2023). One of
the traditional microbiological methods used
to study MA is MPN, which evaluates the size
of a microbial population in a liquid culture
medium and is useful for estimating the abun-
dance of VMA from the formation of their
product metabolic, CH4 (Wagner et al., 2012).
For the cultivation of VMA, a medium that
contains minerals, trace metals, vitamins and
methanogenic substrates is necessary, and these
components are in Balchs culture medium.
The methanogenic activity is related to
the global climate change through production
CH4 and CO2, important gases involved in the
greenhouse effect, responsible for global warm-
ing (Conrad, 2020). Estuaries, coastal lagoons,
and mangroves are the main marine ecosystems
that emit methane into the atmosphere (Arai et
al., 2021; Purvaja et al., 2004).
Globally, Mexico has an area of 51 610
km2 of mangrove forests, occupying the fourth
place in coastal wetlands extension, after Indo-
nesia, Brazil, and Australia. The mangroves
in Mexico are under the category of special
protection according to the Official Mexican
Standard NOM-059-ECOL-2010 (2010), being
threatened by the deforestation for agricultural
activities and the urban development. It has
been estimated that the mangrove deforestation
generates about 10 % of the global carbon emis-
sions per year (SEMARNAT, 2016).
The mangrove El Morro-La Mancha is
located around the coastal lagoon of La Man-
cha, in the state of Veracruz (Gulf of Mexico).
In this ecosystem, as far as it is known, there is
no information available on the methanogenic
activity in sediments, although there is a study
on the dynamics of MA and SRB in the estua-
rine sediments of the La Mancha coastal lagoon
(Torres-Alvarado et al., 2016). Therefore, the
objective of the present study was to analyze,
using the cultivation dependent techniques,
viable methanogenic archaea (VMA) in the
sediments at different climatic periods, and the
influence of sulfates on methane production
from acetate in microcosms, in the mangrove El
Morro-La Mancha, associated with an intermit-
tent coastal lagoon and its possible relation to
chemical characteristics of the sediments.
Study site: The mangrove forest El Morro-
La Mancha borders the coastal lagoon of La
Mancha. It is located on the coastal plain of
the Gulf of Mexico in the state of Veracruz, at
19º51’-19°61’ N & 96º37’-96°44’ W. The climate
of the region is warm-subhumid, with three cli-
matic seasons: dry (March to May), rainy (June
to October) and northern (November to Feb-
ruary). The dry season has high temperatures
and low rainfall (44 ± 37 mm), while maximum
rainfall (224 ± 25 mm), as well as an increase
in river volume and land runoff characterize
the rainy season. In the northern season, cold
fronts cause a temperature decrease and occa-
sional rains.
The mangrove forest El Morro-La Mancha
is influenced by the freshwater inflow of Caño
Grande River, as well as the marine influ-
ence through the partial communication of the
lagoon with the ocean, which generally is inter-
rupted during the northern season. The process
4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e57126, enero-diciembre 2024 (Publicado Abr. 16, 2024)
of opening and closing of the inlet influences
the dynamics of physicochemical parameters.
The Morro-La Mancha is a low tree man-
grove, whose trees have an average height
of 8.1 m, formed mainly by the basin man-
grove and, to a lesser extent, by the fringe
and riverside mangroves. Its characteris-
tic species are Avicennia germinans L. (black
mangrove), Laguncularia racemosa (L.) C. F.
Gaertn (white mangrove), Rhizophora mangle
L. (red mangrove) and Conocarpus erectus L.
(button mangrove) (Moreno-Casasola, 2003).
The four species are under the category of
threatened in the Official Mexican Standard
NOM-059-SEMARNAT-2010. Nevertheless,
the mangrove in some areas is impacted by the
livestock activities (paddock transformation)
and the agricultural ones (sugar cane crop).
The anthropogenic activities have caused the
decrease of the mangrove forest extension from
430 ha in 1976 to 268 ha in 2010 (López-Porti-
llo et al., 2009).
The mangrove species of El Morro-La
Mancha are distributed in three hydrographic
regions related to the salinity gradient: a) Oli-
gohalin Region in the inflow of Caño Grande
river, where R. mangle predominates with 73-76
% of the total abundance, followed by A. germi-
nans; b) Oligohalin-Mesohalin Region, in the
impacted area, with 64.4 % of R. mangle, and
c) Mesohalin region, close to the communica-
tion with the sea, with a predominance of L.
racemosa, which can reach up to the 89.8 % of
the total of mangrove species.
Field work: Sediment samples were col-
lected in three mangrove sites associated with
the salinity gradient in the months of dry
(May), rainy (October) and northern (Novem-
ber) seasons. Site one was in the influence of the
Caño Grande River (oligohalin region), site two
was located on La Pajarera island (oligohalin-
mesohalin region) and site three was located
near of communication to the ocean, where
marine influence increases (mesohalin region).
The sediment was collected with a 4.5 cm
diameter acrylic corer which was introduced
to a depth of 20 cm and the temperature was
evaluated only in the superficial layer with
a thermometer. Subsequently each core was
stored vertically in a container until their pro-
cessing in the laboratory.
Laboratory work: Vertical profiles of pH
and Eh of each sediment core were evaluated
with a Unisense brand Microprofiler (RD-N-
5409/picoammeter PA 2000/ Reference-5160);
for the concentration of hydronium ions, a
Unisense-3390 pH sensor was used, and for the
redox potential, an RD-N-5409 Unisense sen-
sor and a reference one (5160).
Sample preparation: Every core had a
total depth of approximately 14-15 cm and
was divided into two strata of sediment in an
atmosphere of nitrogen to carry out the micro-
biological and chemical analyses: the first layer
(surface) was 0 to 6 cm deep and the second
one (bottom) was 6 to 12 cm deep. The selec-
tion of the layer was related to the intervals of
quantified Eh, slightly reducing from 0-6 cm
(-10 to - 100 mV) and totally reducing from
6-12 cm (-100 to - 300 mV).
Quantification of viable methanogenic
archaea: The technique of the Most Probable
Number (MPN) was used, performing serial
dilutions of each sample (10-1 a 10-10), with
three tubes per dilution and a control one.
The culture medium of Balch et al. (1979),
with acetate and methanol as substrates, at a
final concentration of 20 mm, was used. Acetate
and methanol are important substrates for
methanogenesis in mangrove sediments, ace-
tate represent approximately 67 % of methane
production, while methanol is an important
non-competitive substrate in brackish envi-
ronments with high organic matter content
(Conrad, 2020). The pH and the salinity of the
medium were adjusted to match those evalu-
ated in the sediment samples. NaS-9H2O at
2.5 % was added to decrease the Eh.
The incubation was carried out for 30 days
with the temperature quantified in the surface
layer of sediment, at the end of the incuba-
tion, the tubes that produced methane were
Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e57126, enero-diciembre 2024 (Publicado Abr. 16, 2024)
considered positive. Methane was detected with
a GOW-MAC Series 580 GC Gas Chromato-
graph, equipped with a thermal conductivity
detector (Torres-Alvarado et al., 2016).
Methane production experiments: The
experiments consisted in inoculating 5 ml of
wet sediment in serum bottles, with 45 ml of
the basic culture medium of Balch et al. (1979),
adding sodium acetate, as a substrate, to a
final concentration of 20 mM. To evaluate the
effect of sulfates on the production of methane
from acetate, the experiments were carried out
by duplicate both in the presence and in the
absence of sodium sulfate at a final concentra-
tion of 20 mM. The bottles were incubated
in the darkness at 32 °C and the mineraliza-
tion was evaluated by measuring changes in
the percentage of methane for 30 days, using
a GOW-MAC gas chromatograph, equipped
with a thermal conductivity detector (Torres-
Alvarado et al., 2016).
Analysis of the chemical characteristics:
40 g of homogenized sediment were weighed
and centrifuged at 3 600 rpm at a room tem-
perature for 20 min, to separate the interstitial
water from the sediment. In the interstitial
water, the salinity was determined by a refrac-
tometer (Hanna Instruments) and the total dis-
solved carbohydrates were evaluated using the
Strickland and Parsons’ methodology (1972).
In the wet sediment samples, the volatile
solids (VS, organic fraction) and fixed solids
(FS, inorganic fraction) was quantified using
the American Public Health Association meth-
odology (APHA et al., 2005). The carbonate
content was determined from the ignition of
the sample at 990 °C.
Data analysis: The data matrix included
the abundances of VMA, and the chemical
variables. To meet the assumptions of normal-
ity, the data of the variables were logarithmi-
cally transformed (Zar, 1999). For the seasonal
analysis, the variables were grouped into three
seasons (dry, rainy, and northern); for spatial
analysis, the data were grouped into two depth
categories (surface area, from 0 to 6 cm, and
the bottom one, from 6 to 12 cm). An analysis
of variance (ANOVA) was performed to test
significant differences between stations, depth,
and the behavior in methane production in
the microcosmos. Subsequently, through the
Tukey test, a multiple comparison of means
was made (Zar, 1999). In addition, a principal
components analysis (PCA) and a redundancy
analysis (RDA) were made to investigate the
relationship between the microbial density, and
the environmental variables (Infante-Cangrejo
& Donato-Rondón, 2017; Lozano et al., 2019;
Van den Wollenberg, 1977). All the analyses
were performed using the statistical package R
(R Core Team, 2020).
Environmental characteristics: The
chemical characteristics of the mangrove
sediments in El Morro-La Mancha, presented
changes related to the climatic season and the
sediment depth (Table 1). In the dry season
there was a greater salinity (P < 0.05) than in
northern and rainfall due to a decrease in the
freshwater inflow, while, the pH values were
in the basic range, being higher in rainfall.
The greatest variations were determined in the
Eh and the VS concentration (P < 0.05). The
Eh was less electronegative in the dry season,
compared to the rainy and northern ones. A
similar behavior was recorded in the concen-
tration of the VS, with minimums in the dry
climate and maximum values in rainfalls (P
< 0.05). Likewise, a significant increase in the
amount of carbohydrates was determined in
the rainy season compared to the northern and
dry ones (P < 0.05). There were no significant
changes neither in the concentration of the FS
nor the carbonates (P < 0.05). Spatially, no sig-
nificant changes in the chemical characteristics
were determined, an exception was the salinity
(P < 0.05).
Structure of the abundance and the dis-
tribution of VMA: The abundance of two
6Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e57126, enero-diciembre 2024 (Publicado Abr. 16, 2024)
nutritional groups of VMA was quantified:
those that grow from the acetate, or acetoclas-
tic (VMA-A) and those that use methanol, or
methylotrophic (VMA-M). The global den-
sity of VMA was in an interval from 102 to
108 MPN/g of wet sediment. By physiological
group, the abundance of VMA-A, had an inter-
val from 102 to 105 MPN/g of wet sediment and,
although their density was higher in rains (Fig.
1), with lower salinity conditions, no differ-
ences were registered between climatic seasons
(P > 0.05). On the contrary, with respect to
the vertical distribution, the highest abun-
dance was determined by increasing the depth
of the sediment, mainly in the rainy season
(P < 0.05, Fig. 1).
In the case of VMA-M, their density had
an interval from 104 to 108 MPN/g of wet sedi-
ment, higher than that of VMA-A and, contrary
to these, there were important changes in their
abundance between the climatic seasons, reach-
ing higher levels in rainy and northern ones
than in the dry one (P < 0.05). However, its dis-
tribution by sediment layer was homogeneous
(P > 0.05. Fig. 1). This study has revealed that
apparently the effect of the depth is important
in the changes of the density of the VMA-A,
while for the group that grows from methanol
their distribution pattern is more related to the
weather season, regardless of the depth.
Methane production experiments: In all
experiments, with sulfates or sulfate-free condi-
tions, CH4 production was quantified; however,
in the case of microcosms with sediments from
the rainy season, production began from day 6
while in the dry and northern samples, the for-
mation of CH4 was recorded until days 12-24.
Tabl e 1
Environmental variables in the sediments of the mangrove La Mancha, Veracruz.
Season Northern Dry Rainy
Layer (cm) 612612612
Salinity 20 ± 0 22 ± 3.46 22.67 ± 2.08 25 ± 1.0 18.67 ± 0.58 20.33 ± 0.58
pH 7.59 ± 06 7.50 ± 0.09 7.69 ± 0.15 7.57 ± 0.09 7.89 ± 0.39 8.43 ± 0.44
Eh (mV) -137.65 ± 104.64 -153.61 ± 120.24 -13.94 ± 8.18 -27.25 ± 23.54 -125.61 ± 78.5 -170.83 ± 109.1
FS (g/l) 345.5 ± 286.4 340.2 ± 299.2 170.8 ± 192.6 147.8 ± 128.8 179.4 ± 74.9 229.8 ± 113.3
VS (g/l) 188.61 ± 93.1 216.34 ± 186.6 7.16 ± 5.49 6.94 ± 4.02 270.78 ± 65.39 347.91 ± 49.6
Carbonates 6.53 ± 2.84 7.80 ± 4.41 4.83 ± 4.57 5.41 ± 4.96 12.23 ± 10.87 12.25 ± 11.46
Carbohydrates (g/l) 0.08 ± 0.03 0.09 ± 0.03 0.07 ± 0.08 0.04 ± 0.05 0.14 ± 0.01 0.13 ± 0.02
Mean ± Standard deviation.
Fig. 1. Seasonal and spatial variation in the abundance of VMA in the sediment of the mangrove El Morro-La Mancha,
Veracruz, Gulf of Mexico.
Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e57126, enero-diciembre 2024 (Publicado Abr. 16, 2024)
Significant differences (P < 0.05) were
determined in methane production between
treatments, under sulfate-free conditions the
production of methane was higher than in
the experiments with sulfates, quantifying an
interval from 29.78-929.75 nmol CH4/month,
on the contrary, an interval of 7.88-347.38 nmol
CH4/month was determined in the experi-
ments with sulfates. Likewise, the differences in
methane production were significant (P < 0.05)
between the samples from the different climatic
periods, an increase in the methane produc-
tion can be observed in the absence of sulfates
in rainfall, compared to northern and the dry
seasons (Fig. 2).
Environmental variables and VMA: The
diagram obtained by the PCA allowed to quan-
tify the effect of the environmental variables
regarding the seasons and the structure of
the VMA. For the analysis of the abundances,
two components were obtained: the first one
explained the 44.80 % of the total variance
and the second one the 21 %, which, together,
explain an accumulated variance of 65.80 %
(Fig. 3). The variables with the highest partici-
pation in the definition of the first component
were the salinity, pH, Eh, VS and carbohy-
drates; they contributed to the presence of the
VMA of both nutritional groups. The second
component was defined by the FS and the car-
bonate content, with little effect on the VMA. It
was also observed that the chemical conditions
in the rainy season contribute to increasing the
density of the VMA (Fig. 3).
Considering the redundancy analysis
(RDA), the component 1 showed that the envi-
ronmental variable which had a greater direct
influence on the VMA-A was the Eh, contrary
to the VS and the carbohydrates. In addition,
considering component 2, the environmental
Fig. 2. Methane production from acetate, with sulfates and sulfate-free conditions.
Fig. 3. PCA showing the relationship between VMA and
environmental variables in the sediment of the El Morro-La
Mancha mangrove, Veracruz, Gulf of Mexico.
8Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e57126, enero-diciembre 2024 (Publicado Abr. 16, 2024)
variable that had the greatest inverse influence
on the VMA-M was the salinity (Fig. 4). Like-
wise, Eh and the salinity contribute to charac-
terize the dry season, the FS characterizes the
northern, while the VS, carbohydrates and pH
characterize the rainy season (Fig. 4).
Environmental characterization: The
changes observed in chemical characteristics of
the mangrove sediments along the climatic sea-
son and the depth, were similar those reported
in other studies (Bhattacharyya et al., 2015;
Dias et al., 2011; Taketani et al., 2010). The
changes in the interstitial water salinity were
caused mainly by the influence of the freshwa-
ter contributions. It has been established that,
on a seasonal scale, in the rainy season, when
the volume of the river discharge and precipita-
tion increases, there is a decrease in the salin-
ity, while during the months of northern the
salinity is the result of the dilution effect caused
by the rains of the previous season (Díaz et
al., 2017). On the contrary, the increase in the
salinity during the dry season is caused by a
lower inflow of freshwater, zero or low precipi-
tation and an increase in the evaporation rate
(Lara-Domínguez et al., 2006).
In the coastal ecosystems the variations of
pH depend on the marine influence, precipita-
tion, the amount of runoff, the origin of the
soils, the removal of the sediments by currents
and the biological activity of the organisms.
Biological processes, such as photosynthesis,
respiration, and mineralization of the organic
matter, are the ones that most influence pH,
due to the changes they cause in the concentra-
tion of the carbon dioxide (De La Lanza Espino,
1994; Torres-Alvarado et al., 2016). In the
lagoon of La Mancha to which the mangrove
forest studied is associated, the changes in the
pH have been related to the cycle of high and
low tides: the first ones cause an increase of pH
due to the adding of the alkaline seawater, while
the latter cause the opposite process.
The sediments presented reducing charac-
teristics (Eh < 0). The vertical changes observed
could be related to a lower diffusion of the oxy-
gen in the sediments, which causes a decrease
of the Eh with the depth, forming a strong
gradient of the redox potential that influences
the sequence of metabolic reactions that occur
during the degradation of the organic matter
(Stolzy et al., 1981). It has been established that
the sediment reducing characteristics favor the
production of methane (Li, 2000).
Carbohydrates constitute a fundamental
fraction of the organic carbon, they contribute
between 14 % and 21 % of it to the coastal
ecosystems (Børsheim et al., 1999; Preston
& Prodduturu, 1992). In the mangrove of El
Morro-La Mancha, the highest concentration
of carbohydrates was determined in the rainy
Fig. 4. Redundancy analysis of the relationship between environmental variables and MA in the sediment of the mangrove
El Morro-La Mancha, Veracruz, Gulf of Mexico.
Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e57126, enero-diciembre 2024 (Publicado Abr. 16, 2024)
season, when the freshwater inflow increase.
Carbohydrates can be added by rivers and by
vegetable matter, roots, and leaves of the man-
groves; although they can be also originated by
the agricultural activities of the cane industry
and the discharge of the wastewater (Paez-
Osuna et al., 1998).
Methanogenic dynamics: The VMA were
present in an interval higher than that reported
by Lyimo et al. (2009), who quantified an abun-
dance of 105-106 MPN/g cells in the sediments
of the mangrove of Mtoni, Dar-ES-Salaam,
Tanzania. The seasonal and spatial variations
observed in the abundance of the MA of the
nutritional groups analyzed were related to
the changes in the chemical characteristics
of the sediments. The relative abundance of
different types of methanogens in mangrove
sediments may be regulated by the availability
of substrates (quantity and quality of organic
compounds), as well as by various environmen-
tal variables, including temperature, salinity,
pH, and oxide-reduction potential of sediment
(Bueno de Mesquita et al., 2023; Liu & Whit-
man, 2008).
In the present study, the abundance of
the VMA in the rainy season was favored by
the existing environmental conditions, mainly
salinity (18 to 21), alkaline pH conditions (7.8
to 8.4) and Eh reducers (-125 to -250 mV), like
those reported in other studies in mangrove
sediments (Lyimo et al., 2009). The presence
of MA in mangrove sediments has been associ-
ated with pH values of 6.6 to 7.2 (Mohanraju &
Natarajan, 1992). Also, in the rainy season, the
increase of VS and carbohydrates was impor-
tant for the development of the MA. Taketani
et al. (2010) mention that the presence of
the methanogenic community in mangrove
sediments is favored by organic matter, since its
abundance and variety increase with a greater
concentration of organic components.
Although the presence of the MA was
determined in the two layers of the sediment, as
it has been reported in other studies, in which
an active methanogenic community and the
production of methane have been detected at
depths of the sediment ranging from 0 to 30
cm (Lyimo et al., 2002b), it is evident that there
were spatial changes, where the greatest abun-
dance of the MA was found at greater depth.
In several studies, the greatest abundance and
methanogenic activity have been recorded in
deep layers of the sediment, where the redox
potential is more electronegative and where
the concentration of sulfates decreases and the
content of organic matter increases (Jing et al.,
2016; Wilms et al., 2007). In the sediments of
mangrove forests, with a high contribution of
organic matter, methanogenesis can become
the predominant terminal process in the anaer-
obic food chain, once the sulfate concentration
decreases (Parkes et al., 2007).
Regarding the dynamics of nutritional
groups, the lower density of VMA-A, compared
to the VMA-M, may be a result of acetate also
being used by the SRB, although in this study
the presence of the SRB was not analyzed,
Torres-Alvarado et al. (2016) demonstrated the
presence of the SRB and the MA, which used
acetate in the sediments of La Mancha lagoon.
The acetate is a product that is released during
the fermentation processes of organic matter
and, being a common source of carbon for SRB
and MA, there may be competition between
both microbial groups (Holmer & Kristensen,
1994). The sulfate reduction process from ace-
tate is favored for thermodynamic reasons,
since it produces more energy per mole of
acetate (∆G° of -43.8 kJ/mol), while the forma-
tion of methane produces a ∆G° of -19.9 kJ/mol
(Canfield et al., 2005; Howarth, 1993).
The influence of the sulfate in the produc-
tion of methane from acetate was observed
in microcosm experiments, where methane
production decreased in culture media where
sulfates were added. In laboratory experiments
it has been shown that methane production
decreases when more energetically favorable
electron donors are added, such as sulfates
(Achtnich et al., 1995) and although with
the presence of sulfates it dominates sulfate
reduction, methanogenesis can continue at the
expense of other substrates, such as differ-
ent methylated compounds. In addition, it
10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e57126, enero-diciembre 2024 (Publicado Abr. 16, 2024)
has been demonstrated in experiments with
mixed pure cultures, that the MA and the SRB
can coexist in syntrophic relationships due
to the release and consumption of hydrogen
(Ozuolmez et al., 2015).
Methylotrophic methanogenesis is com-
mon in saline environments, such as coastal
wetlands, where AM convert so-called non-
competitive substrates such as methanol, meth-
yl and trimethylamines, and methyl sulfides to
CH4 and CO2. Methanol is a fraction of plant
organic matter, it is released as a product of
the degradation of pectin and lignin, being
important in mangrove areas and favors the
growth and development of the MA in the pres-
ence of the SRB (Lyimo et al., 2002b), which
would explain the dominance of the VMA-M.
In the sediments associated with mangroves,
the MA that use methanol, methylaminas and
trimethylamines are an important methanogen
component (Bueno de Mesquita et al., 2023;
Mohanraju et al., 1997).
Although in the present study a higher
MPN of VMA-M was quantified compared to
VMA-A, it is important to consider that some
studies report that their contribution to meth-
ane formation is reduced, between 1-10 % of
the total, since it can be used as a carbon source
in other anaerobic metabolisms such as denitri-
fication (Conrad & Claus, 2005). In this sense,
it is mentioned that, in marine environments
and coastal wetland sediments, methylamine
could represent a more important methyl-
ated substrate than methanol. Methylamine is
formed from the production of glycine beta-
ine, an excretion product of marine organisms
and is an easily degradable substrate through
methanogenesis and, like methanol, is a “non-
competitive” substrate (Conrad, 2020; Bueno
de Mesquita et al., 2023); therefore, it would be
of interest to consider the use of this substrate
in subsequent studies of methanogenesis in
mangrove sediments. The use of specific inhibi-
tors for methanogenesis (2-Bromoethanesul-
fonic acid or chloroform) or sulfate reduction
(sodium molybdate), could also provide more
detailed information on the importance of
acetate in metabolic processes.
The objective of this manuscript was to
analyze the presence of viable methanogenic
archaea in mangrove sediments in different
climatic periods, as well as the influence of
sulfates on the production of methane from
acetate in laboratory experiments. However,
although methanogenesis can be analyzed using
culture-based methods, the use of molecular
analyzes could provide important information
related to the composition of MA, the genes
and biochemical pathways involved in methane
production with different environmental con-
ditions. In this regard, studies based on inde-
pendent cultivation techniques have reported
a number of archaeal 16S rRNA genes ranges
from 107 to 1010 copies/g dry sediment (Zhou
et al., 2017), and a diversity of acetoclastic MA
that includes members of the families Metha-
nosarcinaceae and Methanotrichaceae, while
methylotrophic methanogens have a phyloge-
netic affiliation with the genera Methanococcoi-
des and Methanometylovorans (Conrad, 2020;
Kurth et al., 2020; Lyimo et al., 2009; Yasawong
et al., 2013).
It was identified that salinity, pH, redox
potential, volatile solids, and carbohydrates
were the main variables that were influenced
by the seasonality in the area, and the seasonal
variation of the chemical characteristics of the
sediments of the mangrove of El Morro-La
Mancha, influence the presence and distri-
bution of the VMA-A and VMA-M. In the
rainy season, the viable methanogenic archaea
increased when lower salinity, more electro-
negative oxide-reduction potentials and a high-
er concentration of organic components were
recorded. These characteristics would deter-
mine that rains are an important factor related
to the climate change. The production of meth-
ane from acetate in the presence of sulfates and
the quantification of VMA indicates that the
processes of sulfate reduction and methano-
genesis can coexist in mangrove sediments of El
Morro-La Mancha. However, due to the lack of
molecular analysis, diversity of methanogenic
communities cannot be draw, further research
is necessary at respect.
Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e57126, enero-diciembre 2024 (Publicado Abr. 16, 2024)
Ethical statement: the authors declare that
they all agree with this publication and made
significant contributions; that there is no con-
flict of interest of any kind; and that we fol-
lowed all pertinent ethical and legal procedures
and requirements. All financial sources are fully
and clearly stated in the acknowledgments sec-
tion. A signed document has been filed in the
journal archives.
This study was supported by the project
Caracterización Ecológica de los Ambientes
Costeros Mexicanos. 2019-2022”, approved by
the División de Ciencias Biológicas y de la
Salud, Universidad Autónoma Metropolitana-
Iztapalapa, Mexico City, Mexico.
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