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-Muñoz1; 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.

ABSTRACT

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.

RESUMEN

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.

https://doi.org/10.15517/rev.biol.trop..v72i1.57126

ECOLOGÍA ACUÁTICA

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e57126, enero-diciembre 2024 (Publicado Abr. 16, 2024)

INTRODUCTION

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;

sulfatos.

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 Balch’s 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.

MATERIALS AND METHODS

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).

RESULTS

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).

DISCUSSION

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.

ACKNOWLEDGMENTS

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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