Western Indian ANAEROBIC MICROBIAL PROCESSES IN7180, 2002 MANGROVE SEDIMENTS Ocean J. Mar. Sci. Vol. 1, No. 1, pp.

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Methane Emission, Sulphide Concentration and Redox Potential Profiles in Mtoni Mangrove Sediment, Tanzania
Thomas J. Lyimo1, Arjan Pol2 and Huub J.M. Op den Camp2
1

Applied Microbiology Unit, Department of Botany, Faculty of Science, University of Dar es Salaam, P.O. Box 35060, Dar es Salaam, Tanzania; 2 Department of Microbiology, Faculty of Science, University of Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands

Key words: mangrove sediment, organic matter, methanogenesis, sulphate reduction, redox potential, bacteria biomass AbstractMangrove forest sediments were investigated with respect to organic matter content, bacterial numbers, methane emission, sulphide concentration and redox potential profiles. The sediments had characteristically high organic matter content (varying from 8 to 30 % of dry weight) and high bacterial numbers (varying from 5 to 20 x 108 cells/g fw). Both values decreased with depth (030 cm). Methane emission, sulphide and redox profiles on the other hand, showed remarkable differences between and among sampling sites. In areas with a high abundance of leaf litter and few trees very high sulphide concentrations were encountered: increasing from about 1 mM at the surface to 25 mM at 3040 cm depth. Redox values were consistently low; from -150 to -200 mV. Near trees, in between the roots, a remarkably lower sulphide concentration was observed in the top 530 cm of the sediment. Methane emission varied from 0 to 500 mol/m2/h. In vitro incubations showed that methanogenic activity extended down to at least 30 cm depth. In other areas where there was no visible leaf litter accumulation and with many aerial roots the sulphide concentration was much lower ( 1 M) in the top 30 cm layer with correspondingly higher redox values (+100 mV to -150 mV). Below 30 cm sulphide concentrations increased to a maximum of 2 mM. Methane emissions from these areas were very low or even negative (-1 to 8 mol/m2/h). In vitro measurements showed CH4 production in the top layer occurring at a rate much higher than estimated from the emissions. The results clearly demonstrate the importance of the anaerobic microbial activity in mangrove sediments and indicate that aerial roots are responsible for a major input of oxygen into the sediment, which speeds up oxidation.

INTRODUCTION
Mangrove forests are usually considered to be high productivity areas that support highly developed detritus-based food webs (Odum & Heald, 1975; Robertson, 1986). The high primary productivity of mangroves implies a high demand for nutrients essential to plant growth and this demand appears to be met by a highly efficient system of nutrient trapping, uptake and recycling (Kristensen et al., 1991; 1992; 1994; Alongi, 1992; 1994 a,b; Alongi
Corresponding author: TJL. E-mail: lyimo@amu.udsm.ac.tz

et al., 1992; 1993; Robertson et al., 1992; Morell & Corredor, 1993; Hemminga et al., 1994; Nedwell et al., 1994). The organisms within mangrove ecosystems, including microorganisms, plants and animals, show complex interactions. Microorganisms are intimately involved in biogeochemical cycling and in many instances are the only biological agents capable of regenerating forms of the elements used by other organisms, particularly plants. The

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decomposition involves various trophic groups of microorganisms acting in a multi-step process. The first step is an enzymatic hydrolysis of polymeric material to soluble monomeric and oligomeric compounds. Under oxic conditions the soluble compounds are directly mineralised to carbon dioxide and water. Under anoxic conditions various physiological groups are involved in degradation after the initial depolymerisation. Fermentative bacteria convert the products of hydrolysis to a variety of products, mainly short chain fatty acids, carbon dioxide and hydrogen. Further conversion through the action of secondary fermenters, sulphate-reducers, acetogens and methanogens produces the end products CO2, CH4 and H2S, which may escape into the atmosphere. All three are important greenhouse gases. The extent of the fluxes depends on the penetration of oxygen and the activity of aerobic bacteria in the surface layer, which can oxidise sulphide and methane. Hydrogen sulphide can be oxidised to elemental sulphur, thiosulphate or sulphate. Hydrogen sulphide also precipitates easily with metal ions to the corresponding metal sulphide, for instance FeS, which gives many anoxic sediments their black coloration. Methanotrophic bacteria can oxidise CH4 to carbon dioxide. The literature shows that mangrove soils are sulphidic and variable, since their chemistry is regulated by a variety of factors such as texture, tidal range and elevation, redox state, bioturbation intensity, forest type, temperature and rainfall (Lugo & Snedaker, 1974; Alongi et al., 1992; 1993). Different mangrove forest differ in their sulphide concentration because the roots (pneumatophores) of some species of mangrove can oxidise the surrounding soil while others do not have this ability (Nickerson & Thibodeau, 1985; Lacerda et al., 1993). The role of anaerobic processes in mangrove sediment, however, is largely unresolved. Only few studies have dealt with anaerobic decomposition (Benner et al., 1986) sulphate reduction (Kristensen et al., 1991; 1994) and methanogenesis (Ramamurthy et al., 1990; Mohanraju & Natarajan, 1992; Mohanraju et al., 1997) in this ecosystem. There are only a few studies pertaining to depth profile for sulphide concentration and redox potential. Measurements of sulphide and redox

potential with electrodes in undisturbed sediments give better understanding of microbial interactions and sulphur cycling under natural conditions (Visscher et al., 1991). Moreover, most of the available information on anaerobic microbial activities in mangrove sediment is mainly based on studies from North America, Australia and Southeast Asia. In this paper we focus on in situ sulphide and redox profiles as well as methane emissions in sediments from mangroves at Mtoni, Tanzania.

MATERIALS AND METHODS

Sampling stations
The sampling stations were located in Mzinga creek, in Mtoni mangrove forest, along the Dar es Salaam coastline, at approximately 6o 5' S latitude and 39o 41' E longitude (Fig. 1). Four stations were chosen and designated A, B, AC and BC. At low tide all stations were exposed while at high tide the tidal water depth ranged from 0.5 to 2 m depending on lunar cycle and location. Sediment sampling and field measurements were conducted during low tide when sampling stations were accessible by foot. A large proportion of the studied area was muddy. The mangrove trees are found on both sides of the creek, occupying approximately 4 km2. Station A was located more inland and was characterised by open spaces without pneumatophores, clearly as a result of tree-cutting. The top layer of the sediment surface contained abundant mangrove litter. The sediment was more sandy below 20 cm. High sulphide concentrations were evidenced by the characteristic smell and white streams of elemental sulphur, which appear on the surface during low tide. The sediment was blacker than at the other stations, indicating the presence of high amount of metal sulphides. The main mangrove trees at this sampling station were Rhizophora mucronata mixed with some trees of Avicennia marina and Sonneratia alba. Station B was located at the seaside where there was no visual leaf litter accumulation. The area was rich in pneumatophores of S. alba trees that formed a pure stand. Crab holes were scattered all over the area.

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Fig. 1. Location of sampling stations for mangrove sediments near Dar es Salaam city

Station AC resembled station A but had less deep mud and was much closer to land. The dominant mangrove trees were R. mucronata. Station BC was intermediate between stations A and B, closer to land. The dominant trees here were S. alba but they were more scattered. Many small crab holes were observed in this station.

Physical-chemical parameters and total bacteria

The surface water and sediment temperatures and pH were measured with a portable field pH meter (Sentron pH system, Netherlands). The salinity of the drained seawater was measured using a hand refractometer (ATAGO, S-10, SALI. 0~10%, Japan). Sediments from station A were further characterised in terms of organic matter content, concentration of some inorganic elements and total bacteria numbers. Duplicate sediment cores were collected as described by Lyimo et al. (2000). The samples were transported to the laboratory (Nijmegen University, the Netherlands). Upon arrival (~24 h

Kilwa

Vijibweni

A AC BC

Mtoni Kijichi
Mangroves Sampling sites
0 500 1000 m

after sampling), the cores were sectioned at 05, 510, 1015, 1520, 2025 and 2530 cm intervals. The organic matter content of the sediment core subsections was calculated as differences in weight between dry weight (80 oC until a constant weight) and ash weight (550 oC for 4 h). Concentrations of calcium, magnesium, manganese, iron, silicon, zinc, phosphorus, sulphur (as sulphate), aluminium, sodium, potassium and chloride in the (sediment subsections) interstitial water were measured with an inductive coupled plasma (ICP)-Atomic emission spectrometer and a flame photometer, FLM3, Radiometer, Copenhagen, as described by Roelofs (1991). The total numbers of bacteria in the sediment subsections were determined using the methods described by Kepner & Pratt (1994).

Measurements of sulphide and redox potential profiles

Sulphide and redox potential profiles were measured using electrodes built in stainless steel

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needles of 1 mm tip, 60 cm long (microscale measurements, Netherlands). Calibrations of the electrodes were done as described in the protocol of the manufacturer. Field measurements were done by gently lowering the needle electrodes and reference electrode (Ag/AgCl) into the sediment as described by Vischer et al. (1991).

Methane emission measurements

Methane emission was estimated using the static chamber technique. The chambers were round brown glass bottles cut at the bottom and tightly closed with butyl rubber stoppers on top. The chamber covered an area of 0.0123 m2 and had an internal volume of 1.4 to 1.8 l. The bottles were gently pushed 12 cm into the sediment surface to ensure a seal against the ambient atmosphere. About 10 to 20 chambers were kept at a time at a sampling site to obtain representative data. Gas samples were taken with a gas-tight syringe (1 ml) at an interval of 0.51 h for 24 h. The samples were analysed in the laboratory on the same day by a HP 5890 II gas chromatograph (GC) equipped with a flame ionisation detector (FID) and a column packed with HayeSep Q, 6080 mesh. The carrier gas was nitrogen at 40 ml/min; oven, detection and injection port temperatures were 100, 200 and 170 oC, respectively.

done by adding the amount produced by each section divided by the surface area covered by the sampler. The most-probable number (MPN) method (3 tube series) was used to compare the numbers of methanogenic archaea in station A and B. The preparation of medium and handling of samples were done as described by Lyimo et al. (2000). A combination of methanogenic substrates; acetate (5 mM), formate (5 mM), methanol (5 mM), trimethylamine (2 mM) and dimethylsulphide (0.6 mM) was used.

RESULTS
Physical-chemical parameters and total bacteria
The salinity of the seawater ranged from 19 to 35 0 /00, the low salinity was caused by intrusion of fresh water especially during the rainy season. Temperature varied from 25 to 32 oC. However, the temperature of the top layer of the sediment during low tide on sunny days ranged from 35 to 39 o C. The pH of the sediment showed no significant differences with depth, varying from 5.5 to 7.1 with an average of 6.6 0.4. The variation of organic matter content with depth in the sediment is indicated in Table 1. The surface sediment samples (010 cm) had significantly higher (P < 0.05, student t-test) organic matter content than the deeper samples (1030 cm). The average water content in the sediment core subsections from 030 cm was 62 12% of the fresh weight (fw).

In vitro methane measurements

Out of a few randomly selected static chambers, sediment cores were taken with a sampler (plastic cylinder, 5.5 cm inside diameter and 45 cm long) immediately after field measurements. The cores were taken from exactly where the static chamber was kept. The samples were processed within 1 2 h after sampling. The sediment core was opened and cut into sections of 2 or 5 cm under a stream of N 2 gas. These sections were immediately transferred into 570 ml serum bottles which were closed with black butyl rubber stoppers. The bottles were evacuated and the atmosphere was replaced with N 2 (0.5 atm overpressure) to maintain anaerobic conditions. Incubations were done at 30 oC without shaking. Methane production was measured on a GC as described above. Calculations of the amounts produced per time and area were

Table 1. Variations of organic matter and total number of bacteria with depth at station A Number of bacteria (x108) (g fresh weight)-1 n Mean 20 16 14 10 6 Range n 3 3 3 3 3 2

Organic matter (% of dry weight) Depth (cm) 0 5 5 10 10 15 15 20 20 25 25 30 Mean 22 6 21 4 18 2 18 3 17 4 14 4 Range 16 30 5 17 25 5 15 21 5 13 21 5 13 22 5 8 18 4

6 14 27 9 6 23 9 6 26 4 6 13 3 3 8 5.5 5 6

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The concentration of various elements in the interstitial water of the sediments did not show correlation with depth. The mean concentrations for all sub-samples in mM ( SD, n = 13) were as follows: Ca, 7.0 1.2; Mg, 35 6; Mn, 0.003 0.004; Fe, 0.02 0.01; Si, 0.6 0.3; Zn, 0.20 0.05; P, 0.2 0.1; S as SO42-, 21 9; Al, 0.2 0.1; -K, 13 1; Na, 502 38; and Cl, 492 37. The total number of bacteria estimated at different depths intervals is also shown in Table 1. The total counts of bacteria decreased with depth and were significantly higher (P < 0.05) at 05 cm as compared to 1030 cm depth. The numbers of bacteria (g fw)-1 ranged from 2.7 x 109 at the surface to 3 x 108 at 2025 cm depth.

Sulphide and redox profiles

Station A, which was characterised by a high abundance of leaf litter, showed remarkable differences between non-rooted and rooted areas. In the non-rooted areas very high sulphide concentrations were measured which increased from about 1 mM at the surface to 25 mM at 30 40 cm depth (Table 2). Redox values were consistently low (-150 mV to -300 mV). This suggests an active sulphate reduction along the entire sediment column which was examined (0 60 cm depth). Between the pneumatophores of S. alba or A. marina, a remarkably lower sulphide

concentration was observed in the top 030 cm of the sediment. A typical example of sulphide and redox potential profiles in the rooted and nonrooted sediments is shown in Fig. 2. At station B, which had no visible leaf litter but had many pneumatophores, the sulphide concentrations were much lower ( 1 mM) in the top 30 cm layer and correspondingly redox values were much higher (+250 mV to -150 mV). Below 30 cm, sulphide concentrations increased to a maximum of 2 mM and redox values decreased to -250 mV. A typical example of sulphide and redox potential profiles from station B is shown in Fig. 3. Generally the low sulphide zone extended deeper in the sediments at points that were adjacent to trees. Despite the very low sulphide concentrations at the surface, oxygen could not be detected in the upper layer (< 2% air saturation). Besides roots, crab holes were abundant and could contribute to the oxygenation of the sediment. Preliminary results using aerated sediment of station B showed that the capacity to oxidise sulphide by far (1000 fold) exceeded the sulphide production rate under anaerobic conditions.

Methane emission
Methane emission was estimated from static chamber experiments. Placing the bottles at random in sulphide-rich areas (Station A) a broad

Table 2. In situ methane emission and sulphide profiles of mangrove sediments from Mtoni, Dar es Salaam Sulphide profiles CH4 emission Station Characteristics A High sulphide smell, many leaves, deep mud, near Avicennia trees Resembles A, less deep mud, closer to land Intermediate of A & B, closer to land, less roots, many small crab holes No sulphide smell, no leaf deposition, many large crab holes, abundant roots mol/m /h
2

Maximum (mM) 2 8 2 25 15 6

Gradient at (cm) 1 20 25 40 <5 5 25 30 45 2550 Remark Non-rooted Rooted Non-rooted Non-rooted Rooted Rooted

Range - 0.6 520

79 133

AC BC

51 66 26 91

0.6 186 - 3.0 380

1 5 0.5 2 0.02 2

1.3

2.0

- 0.72

8.4

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Sulphide (mM) 0 0 5 10 15 I 20 0 5 10 15 20 II

10

20

Depth (cm)

30

40

50 Non-rooted 60 -250 -150 -50 50 -250 -150 Redox potential (mV) -50 Rooted 50

Fig. 2. Typical sulfide and redox profiles of sediments in non-rooted (I) and rooted (II) areas at station A. Redox profile ( ), sulphide profile ( )

Sulfide (mM) 0.0 0 0.5 1.0 1.5 2.0

10

20 Depth (cm)

30

40

50

60 -200

-100 0 100 Redox potential (mV)

200

Fig. 3. A typical example of sulphide and redox profiles of sediments at station B. Redox profile ( ), sulphide profile ( )

range of CH4 emission values varying from -0.6 to 520 mol/m2/h were found (Table 2). Emission of methane by ebullition sometimes impaired accurate rate measurements (Fig. 4). No correlation between the nature (with or without litter, root or non-rooted) of the place (spot) and CH4 production was observed. In the station where the surface sediment was very poor in sulphide (Station B), CH4 emission was much lower or tended to be negative. This may indicate oxidation of CH4 at the oxycline, as a consequence CH4 does not reach the atmosphere. To verify this, anaerobic in vitro incubation was done with samples taken immediately after in vivo measurements. The results of this sediment showed that methane production extended down to at least 30 cm (Fig. 5) at any station. For station A, the in vitro production rate (82.2 53.4 mol CH4/m2/h) was almost the same as in vivo emission rate (113.4 94.2 mol CH4/m2/h). The in vivo rate values that have been considered are only those corresponding with the in vitro measurements. At station B, the in vitro production rate (14.4 2.4 mol CH4/m2/h) was about 10-fold the in vivo emission rate (1.2 0.4 mol CH4/m2/h). This

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50 45
Methane emission (nmol/cm )

40 35 30 25 20 15 10 5 0

50

100 150 Time (min)

200

250

Fig. 4. A typical example of time-dependent in situ methane emissions from mangrove sediment. Incubation bottles at station A showing constant emission ( ) and with ebullition ( )
Methane production (nmol/g.fw/h) 0.0 02 0.1 0.2 0.3 0.4 0.5 0.6 0.7

46

810

1215

Depth (cm)

2025 Station A 03 38 813 1318 1823 2328 2833 Station B

Fig. 5. A typical example of in vitro methane production rates from sediment core sectioned at various depths; sediment samples from station A and station B. Values are means of two independent incubation bottles

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could indicate oxidation of CH4 at station B due to the presence of many aerial roots. The number of methanogens in the top layer of the sediment were also estimated and compared between stations A and B. The numbers for both sites were of the same order of magnitude, 4.3 x 105 and 2.4 x 105 cells/g fw for stations A and B, respectively.

DISCUSSION
Mtoni mangrove sediment samples were characterised by a high organic matter content (8 30 %). Other authors (Kristensen et al., 1991; Sotomayor et al., 1994) have reported values ranging from 4 to 16 % in comparable mangrove ecosystems. The major input of organic material in the system is plant litter (e.g. leaves, stems) and roots exudates. During the time of conducting this research there were many recently cut mangrove trees that may have contributed to the observed high organic matter contents. The results for other physical-chemical parameters were within the expected ranges. For example concentration of sulphate was high (21 9 mM) as reported in other marine seawaters and does not appear to be limiting factor for sulphide production. High amounts of organic material support high densities of bacteria. In this study values for total bacterial counts are slightly lower than those reported from other mangrove sediments (Alongi, 1992; Boto et al., 1989) but the general tendency of decreasing with depth is consistent with other findings (Alongi, 1992). The rapid consumption of oxygen by aerobic microorganisms, driven by the high organic matter content, results into anaerobic conditions in the entire sediment column except at the surface. Anaerobic conditions and a high concentration of sulphate in the interstitial water support the growth of sulphate-reducing bacteria (SRB). The activity of SRB was obvious from the presence of up to 25 mM of sulphide. Oxidation of sulphide at the surface was evidenced by elemental sulphur deposition. The importance of sulphate reduction in the mineralisation of the organic matter has also been clearly demonstrated in laboratory incubations that the sulphide production rate was up to 200 times higher than the CH4 production rate. It was also shown that

the most probable number (MPN) of SRB was 10 times higher compared to MPN of methanogens (Lyimo, unpublished data). The observed sulphide concentrations and redox potentials observed were within the range reported in literature (Nickerson & Thibodeau, 1985; Lacerda et al., 1993; Giani et al., 1996). At station B the steep part of the sulphide gradient was located below 30 cm, which suggests transport upward and a rapid oxidation of sulphide in the surface sediment layer. Station A exhibited much higher sulphide concentrations as compared to station B and other reports on mangrove sediments (Lacerda et al. 1993). This could obviously be due to the high deposition of organic matter in this area, although the station had fewer trees compared to other stations. The sulphide concentration was higher in the top 10 cm of the sediment indicating high production at the surface. The fact that near the trees and in between their roots the sulphide concentration was much lower and increased again below about 30 cm suggests increased oxidation of sulphide by roots. This observation indicates that there was significant contribution from roots in aerating the sediment. Methane emissions were comparable with literature values for mangrove sediments (Barber et al., 1988; Harris et al., 1988; Bartlett et al., 1989; Sotomayor, 1994). At station B methane emission was very low and in some cases emission could not be detected. However, slurry incubations with samples from sediment cores taken from this station produced 10-fold more methane as compared to the field measurements. The results correspond with those of Giani et al. (1996) who studied the Balandra mangrove sediment. The authors could not detect any in vivo emissions but in the laboratory incubation rates of 1 to 23 nmol/ ml sediment/day were estimated. As for sulphide this also suggests a balance between production and oxidation of methane in the rooted area of the forest. Methane emission rates at station A were much higher (up to a hundred-fold) than at station B, and of the same order of magnitude as found for the in vitro production rates. The reason may be due to lack of oxygen input in the sediment at this largely unrooted station, rather than the higher supply of organic material. Further support for this view

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comes from the fact that the in vitro CH4 production rates for station A were only 6-fold higher than for station B and the MPN of methylotrophic methanogens in the top layer of the sediment were of the same order of magnitude (105 cells/g fw). When data of various stations were combined (Table 2), a correlation between sulphide concentration and methane emission becomes obvious. This may be a result of the simultaneous oxidation of both products in the top layer, rather than differences in organic matter input. It is well known that plants growing in waterlogged soils, including mangrove trees (Scholander et al., 1955; Thibodeau & Nickerson, 1986; Lacerda et al., 1993) frequently evolve gases at the root hair region creating an oxidised rhizosphere within the anaerobic soil environment. This creates a microenvironment whose biogeochemistry differs from the surrounding sediment. This study has shown in the soil around Sonneratia spp. sulphide is virtually absent in the top sediment layer, most likely due to introduction of oxygen by pneumatophores of these trees. Similarly the rhizosphere of A. marina was highly oxidised, to the extent that sulphide was virtually absent. Nickerson & Thibodeau (1985) and Scholander et al. (1955) have demonstrated that A. germinans is able to bring air to roots growing in anaerobic substrate through its root aerenchyma. The findings from the present study imply that anthropogenic disturbance will endanger this ecosystem. The cutting of trees, which evidently had taken place at station A, leaves the area without roots. The lack of roots will result into less aeration of the sediment, and toxic sulphide will start to accumulate to very high concentrations, making the area inhospitable to both animals and plants. This may explain why crabs were absent in station A, which in turn will impair oxygen input via crab holes and diminution and burial of litter. Apart from crabs there was a striking absence of mangrove seedlings at station A. The increase of human pressure in utilisation of Tanzanian mangrove forest (Semesi, 1992; 1998) necessitates further research on the mangrove ecosystem. We recommended that the users of mangrove resources be educated on the negative impacts of clear-cutting and encouraged in more sustainable utilisation.

AcknowledgementsWe are indebted to the late Professor A.K. Semesi who contributed significantly to this study, but passed away before the work could be submitted for publication. We would like to dedicate this manuscript to her. We are grateful for the technical support received from technicians in the Microbiology Laboratory of the University of Dar es Salaam, Tanzania as well as the staff of the Microbiology Laboratory of the University of Nijmegen, the Netherlands. The project was financed by the Directorate General for International Cooperation (DGIS), Ministry of Foreign Affairs of the Netherlands.

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