Growth and root oxygen release by Typha latifolia and its effects on sediment methanogenesis

Aquatic Botany 61 (1998) 165±180

Growth and root oxygen release by Typha latifolia and its effects on sediment methanogenesis Dorthe N. Jespersen, Brian K. Sorrell1, Hans Brix* Department of Plant Ecology, Institute of Biological Sciences, University of Aarhus, Nordlandsvej 68, DK-8240 Risskov, Denmark Received 7 August 1997; accepted 14 February 1998

Abstract Growth of Typha latifolia L. and its effects on sediment methanogenesis were examined in a natural organic sediment and a sediment enriched with acetate to a concentration of 25 mM in the interstitial water. The lower redox potential and higher oxygen demand of the acetate-enriched sediment did not significantly impede growth of T. latifolia despite some differences in growth pattern and root morphology. Plants grown in acetate-enriched sediment were ca. 15% shorter than plants grown in natural sediment, but the former produced more secondary shoots at earlier stages, which resulted in similar total biomasses after 7 weeks of growth in the two sediment types. Plants grown in acetate-enriched sediment had thicker and much shorter roots than plants grown in natural sediment. This difference did not significantly affect the release of oxygen from the roots when measured under laboratory conditions, which was 0.12±0.20 mmol O2 gÿ1 DW hÿ1. Enrichment with acetate resulted in much higher sediment methanogenesis rates (643 vs. 90 nmol CH4 gÿ1 sediment DW hÿ1). Growth of T. latifolia significantly reduced methanogenesis in both types of sediment, but the effect was twice as marked in the natural sediment (34%) as in the acetateenriched sediment (18%), although in absolute terms the reduction was higher in the enriched sediment. The data suggest that this effect of plant growth was via root oxygen release and its effect on redox conditions. In the natural sediment, oxygen release resulted in a significantly higher redox potential and lower sediment oxygen demand, whereas there were no significant changes in the acetate-enriched sediment. The very high oxygen demand of this sediment probably masked the effect of root oxygen release so that a significant reduction in methanogenesis occurred without any significant increase in the redox potential. This demonstrates how root oxygen release from plants like T. latifolia can significantly alter rates of biogeochemical processes such as methanogenesis,

* Corresponding author. Tel.: 45 8942 4714; fax: 45 8942 4747; e-mail: [email protected] 1 Present address: National Institute of Water and Atmospheric Research Ltd., PO Box 8602, Riccarton, Christchurch, New Zealand 0304-3770/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved PII S 0 3 0 4 - 3 7 7 0 ( 9 8 ) 0 0 0 7 1 - 0

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even in sediments with high oxygen demands where this is not evident from instantaneous parameters such as redox potential. # 1998 Elsevier Science B.V. All rights reserved Keywords: Cattail; Aeration; Oxygen stress; Root development; Redox potential; Sediment oxygen demand

1. Introduction Emergent aquatic macrophytes are intimately involved in the production, oxidation and release of methane from freshwater wetlands (Cicerone and Oremland, 1988; Chanton and Whiting, 1995). Most of the large amounts of organic matter produced by these plants is decomposed anaerobically in the sediment, resulting in high rates of methanogenesis and saturation of methane in the interstitial water (SchuÈtz et al., 1991; Chanton and Whiting, 1995). Rates of methane production and consumption in sediments are controlled by the relative availability of substrates for methanogenesis (acetate, carbon dioxide and hydrogen) and methane oxidation (methane and oxygen), which plants can affect through transpiration, organic carbon excretion from roots, and root oxygen release (Dacey and Howes, 1984; Lambers, 1987; Armstrong et al., 1991). Furthermore, sites colonized by emergent macrophytes generally have higher rates of methane release to the atmosphere than unvegetated sites, due to internal gas transport processes in plants, and therefore retain less interstitial methane (Chanton et al., 1993; Sorrell and Boon, 1994; Brix et al., 1996). Determining how, or indeed whether, macrophytes modify any particular aspect of methane cycling in wetlands is not straightforward because resolving the various, often conflicting effects they can have is fraught with difficulties. For example, the low methane concentrations frequently observed in rhizospheres could result from internal methane transport and release through the macrophytes, but also from effects of root oxygen release. This rhizosphere oxidation could inhibit methanogenesis by regenerating electron acceptors used by more energy-efficient bacteria (Conrad, 1989), or alternatively allow methane oxidation (King, 1996; Lombardi et al., 1997). These issues prompted us to investigate whether root oxygen released by Typha latifolia L., which has an effective aeration system based on internal convective through-flow of gases (Bendix et al., 1994), can reduce methanogenesis in the reducing, organic sediments in which it grows. This has involved growing T. latifolia under conditions in which roots were the only possible oxygen source for the sediment, and measuring methanogenesis in vitro on sediment samples from the rhizosphere under conditions excluding the possibility of methane oxidation. 2. Materials and methods 2.1. Sediment and plant material Sediment rich in organic material was collected from Lake Brabrand, a natural wetland near Aarhus, Denmark. After the sediment had been sealed in 10 l buckets for two weeks

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to ensure anoxic conditions, half of it was enriched with acetate to a concentration of 25 mM in the interstitial water in order to obtain substrate-saturating conditions for methanogenic bacteria. The natural organic sediment (without acetate enrichment) will henceforth be termed `low C sediment' whereas acetate-enriched sediment will be termed `high C sediment'. Seeds of T. latifolia were germinated in vermiculite flooded with a commercial nutrient solution (Pioneer, Lyngby, Denmark). When the seedlings were 10±15 cm high, they were transplanted into 20 cm height individual glass jars of 1565 ml volume filled with the two sediments. 2.2. Experimental design The interactive effects of sediment type (low C sediment vs. high C sediment) and vegetation (planted vs. unplanted) were examined in a two by two factorial experiment with five replicates per treatment. The planted jars contained one plant each. The jars were placed in a plastic box, which was subsequently filled with de-oxygenated water to a height of a few cm above the rim of the jars. Supplements of de-oxygenated water were added as required throughout the incubation period in order to keep the water surface well above the jars. To prevent light penetration, a piece of black plastic with holes for the shoots was placed ca. 2 cm above the water surface. These precautions prevented oxygen from entering the sediments by diffusing from the overlying water, by plant transpiration, or by algal photosynthesis so that root oxygen release was the only possible source of oxygen for the sediment. The boxes were placed in a growth chamber (Weiss Technik GMBH, Lindenstruth, Germany) for 11 weeks at 258C, 85% RH, and a light intensity of 300 mmol mÿ2 sÿ1 PAR at the base of the plants. The chamber provided a 16:8 h light:dark cycle. To maintain a high acetate concentration in the high C sediment, 30 ml of a 1 M sodium acetate solution was distributed into the sediments every week using a syringe and a long needle. Shoot sizes and methanogenesis rates were measured approximately once a week during the first seven weeks of plant growth. Sediment characteristics, including pH, redox potential, and content of organic acids in the pore water, were measured after seven weeks. To further test the influence of plant growth, the shoots were cut below the water surface just above the sediment surface after eight weeks of growth in order to block the gas transporting tissues of the plants. The experiment was continued for a further three weeks after which methanogenesis rates, pH and redox potentials were measured again. 2.3. Analyses 2.3.1. Plant morphology The length of the longest leaf was measured weekly and the number of leaves on each main shoot counted. Shoot dry weights of main and secondary shoots were determined in week 8 when the shoots had been cut. Root and rhizome dry weights were determined in

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week 11 at the end of the incubation period. Dry weights were measured after drying for 24 h at 1058C. 2.3.2. Sediment characteristics Loss on ignition of dried sediment was determined by combustion at 5508C for at least 9 h. The dried sediment's content of organic carbon was determined titrimetrically after wet combustion in H2SO4/K2Cr2O7. Redox capacity was measured on fresh sediment by titration with FeCl3 under anoxic conditions, using platinum electrodes and calomel reference electrodes. All methods followed Schierup and Jensen (1981). The contents of dissolved organic acids in the sediment interstitial waters were determined on pore water samples obtained by pressing 1 ml sediment samples through a 0.2 mm filter. The pore water samples were then analyzed by HPLC using a UV-VIS detector and a Supelcogel C610-H column (LC-6A, Shimadzu, Kyoto, Japan). Since measurements of pH and redox potential caused major disturbance of the sediment in the jars, they were only performed twice: after seven weeks of plant growth and at the end of the experiment (week 11). Measurements of pH were taken with a Radiometer pH electrode at a depth of approximately 4 cm in each jar. A platinum electrode, referenced against a saturated calomel electrode, was used to measure redox potentials at 0, 6 and 13 cm depth in each jar (max. depthˆ15 cm). The electrodes were allowed to stabilize for 10 min before each reading. Quinhydrone solutions with known Eh were used for calibration of the electrodes (Sùndergaard and Riemann, 1979). All measurements were converted to E7 to allow comparisons between the sediments. 2.3.3. Methanogenesis Three sediment subsamples (ca. 5 ml) were taken from various depths in each jar by inserting a 7 mm diameter plastic tube connected to a syringe into the sediment. The sampling was carried out carefully in order to minimize disturbance of the sediment and avoid damaging the roots. The subsamples were transferred to 30 ml incubation flasks and moved as quickly as possible to an anaerobic chamber (COY Laboratory Products, Michigan, USA) where 2 ml of de-oxygenated cysteine solution (0.03%) were added. The flasks were then flushed for 1 min with gaseous N2 in order to remove pre-existing methane and ensure anoxic conditions. They were sealed with plastic caps fitted with teflon-lined septa and incubated in darkness in a shaking water-bath at 258C for about 24 h. Headspace samples (1.5 ml) were withdrawn by syringe and needle over time to monitor the development of methane in the headspace. One ml of these samples were injected into and analyzed on a gas chromatograph equipped with a flame ionization detector (GC-8A, Shimadzu, Kyoto, Japan). Before sampling, the incubation flasks were shaken thoroughly to ensure equilibrium between headspace and sediment methane concentration. After each sampling, 1.5 ml N2 gas were injected into the incubation flasks to compensate for the amount of headspace removed. Following a lag phase of about 5±7 h, the methane concentration in the flasks increased linearly with time (r2 generally >0.98), and rates were estimated from the slope of methane concentration against time during this linear period. On completion of the incubations, headspace volumes were determined and sediment dry weights measured after drying for 24 h at 1058C. Methanogenesis rates were

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expressed on dry weight bases. The methanogenesis rate for each single jar was an average of the three sediment subsamples taken from each. 2.3.4. Root oxygen release To confirm that oxygen was released from the roots and examine whether this release was influenced by sediment type, additional plants were grown in separate jars in low C and high C sediments for four weeks. Plants of similar size were selected from the two sediment types. Oxygen release from the roots was examined using a titanium(III) citrate buffer, which allows root oxygen release measurements in a reducing, oxygen-scavenging solution with a low redox potential (Kludze et al., 1993; Sorrell and Armstrong, 1994; Kludze and DeLaune, 1996). After the plant roots had been carefully washed clean of sediment, they were immersed in 300 ml nutrient solution (Pioneer, Lyngby, Denmark) in a conical flask. The solution was sparged with N2 gas for 45 min to remove any oxygen dissolved in the solution, and a 10 mm thick layer of paraffin oil was placed on top of the solution to prevent re-aeration from the atmosphere. The basal part of the shoot was wrapped with Parafilm to prevent the oil from infiltrating the aerenchyma. Sparging with N2 gas continued while 15 ml of a titanium(III) citrate stock solution (89.9 mM, pH 5.9) were injected with a syringe, the stirring from the sparging being necessary for complete mixing. Blank flasks without plants were prepared similarly. The flasks, which were covered with foil to prevent light penetration, were incubated for 12 h in a growth chamber at 208C, 85% RH, and 300 mmol mÿ2 sÿ1 PAR. As the blue titanium(III) citrate solution gradually became clear during oxidation, small samples of the solutions in the incubation flasks were taken regularly with a small syringe and the absorbance at 527 nm measured immediately on a spectrophotometer (UV-1201, Shimadzu, Kyoto, Japan). The flasks were gently shaken immediately before sampling to even out any colour gradients around the roots. The absorbances of the samples were compared to those of solutions with a known concentration of Ti3‡. After an equilibration phase of 4 h, the blanks were stable for the following 4±5 h during which the concentration of Ti3‡ in the flasks with plants decreased linearly (r2 on average 0.90). Because the oxidation of Ti3‡ is stoichiometric, rates of root oxygen release could be calculated from the rate of decrease in the concentration of Ti3‡ in the incubation flasks. After the incubation, length and mean diameter of each root were measured and the surface area of the main roots of the root systems calculated. The percentage of root length that was covered with laterals was also measured, but the surface area of laterals was not estimated. Root dry weights were determined after drying for 24 h at 1058C. 2.4. Statistical analyses Plant characteristics and data on root oxygen release were tested in two-tailed t-tests. Homogeneity of variance was tested with Cochran's C tests, which showed that heterogeneity of variance was significant in the data on methanogenesis, redox potential and organic acids. In these cases, a two-way ANOVA was applied to logarithmically transformed data. For the remaining data, ANOVA was applied directly. Significant differences between individual means were distinguished by Tukey tests. Overall effects of time, sediment type and presence/absence of plant (plants) on methanogenesis were tested by a three-way ANOVA on logarithmically transformed data.

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3. Results 3.1. Plant morphology There were significant differences between plants grown in the two types of sediment. Leaves of high C plants were generally narrower, thinner, and darker green than leaves of low C plants. Maximum leaf lengths of low C and high C main shoots were similar after two and three weeks of incubation, but thereafter leaves of low C main shoots were significantly longer than leaves of high C main shoots (P<0.01; Table 1). The numbers of live leaves were similar on low C and high C plants throughout the seven weeks of plant growth. After the third week of incubation, the plants started producing secondary shoots, which were more frequent in the high C sediment at earlier stages (Table 1). The biomass of low C main shoots was significantly higher than that of high C main shoots, but biomasses of secondary shoots, total aboveground biomass, and biomass of roots and rhizomes did not differ significantly between low C and high C plants (Fig. 1). Observations made on plants from the root oxygen release experiment showed that the highly reducing high C sediment affected root morphology (Table 2). The surface area of the main roots did not differ significantly between low C and high C plants and neither Table 1 Characteristics of Typha latifolia main shoots grown for 7 weeks in a natural organic sediment (low C plants) and in a sediment enriched with acetate (high C plants) Time (week)

Max. leaf length (cm) Low C plants

2 3 4 5 7

54.6 67.4 74.8 77.6 79.0

a

(15.0) (11.3) a (6.9) b (4.4) b (3.2) b

# of leaves per shoot

High C plants 48.0 55.2 59.8 65.6 68.8

a

(8.8) (9.7) a (6.5) a (1.5) a (2.8) a

Low C plants 7.6 8.4 7.8 7.6 8.0

a

(1.8) (1.3) a (1.1) a (0.5) a (0.7) a

Proportion of plants with secondary shoots

High C plants 8.8 9.2 7.4 7.6 7.6

a

(0.8) (0.8) a (0.9) a (1.1) a (0.5) a

Low C plants

High C plants

0/5 1/5 1/5 1/5 4/5

0/5 2/5 3/5 4/5 4/5

Numbers in brackets are 1 SD; nˆ5; for each parameter, means within row with different letter superscripts are significantly different (P<0.05).

Table 2 Root morphology of Typha latifolia grown for 4 weeks in a natural organic sediment (low C plants; nˆ4) and in a sediment enriched with acetate (high C plants; nˆ5) Parameter

Low C plants ÿ1

Number of roots (# plant ) Mean root length (cm) Mean root diameter (mm) Surface area of main roots (cm2 plantÿ1) Lateral root coverage (% of root length) Root biomass (mg DW plantÿ1)

a

49 (18) 16.2 (7.0) b 0.87 (0.21) a 223 (107) a 70 (30) b 131 (77) a

High C plants 58 (16) a 4.4 (3.0) a 1.16 (0.43) b 97 (31) a 34 (36) a 153 (48) a

Numbers in brackets are 1 SD; means within row with different letter superscripts are significantly different (P<0.05).

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Fig. 1. Biomass of main and secondary shoots, total aboveground biomass, and biomass of roots and rhizomes of Typha latifolia grown for 7 weeks in a natural organic sediment (low C plants; open bars) and in a sediment enriched with acetate (high C plants; solid bars). Error bars are 1 SD; nˆ5. Significant differences within plant part are indicated by $ (P<0.05).

did the number of roots per plant, but there were significant differences in mean root length and mean root diameter of plants grown in the two types of sediment. The main roots of low C plants were longer and thinner than the roots of high C plants (P<0.001), and roots of low C plants bore laterals on twice as high a proportion of each root as high C plants (P<0.001). However, the laterals on high C plants tended to be longer and more densely packed than laterals on low C plants. 3.2. Root oxygen release Roots from both low C and high C plants released oxygen into the medium, as the Ti3‡ was oxidized only in the flasks that contained plants. The root weight specific oxygen release rates tended to be higher for the low C plants than the high C plants, whereas the opposite was observed for rates expressed on a root surface area basis (Table 3). These differences were, however, not statistically significant. It should be noted that the root surface area specific release rates were overestimated, as the surface areas of laterals were not included in the calculations. Oxygen release from the roots was also visible as oxygenated rhizospheres in the planted sediments. After four weeks of growth, the black high C sediment was slightly grey in the areas just around the roots, but the oxidation was more noticeable in the greyish-black low C sediment where the oxygenated zones were up to four to six times as wide, with conspicuous reddish-brown areas in the otherwise light grey rhizospheres.

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Table 3 Oxygen release rates from roots of Typha latifolia previously grown for 4 weeks in a natural organic sediment (low C plants; nˆ4) and in a sediment enriched with acetate (high C plants; nˆ5) Sediment type

Low C High C

O2 release rate Root weight specific (mmol O2 gÿ1DW hÿ1)

Root surface area specific (mmol O2 mÿ2 hÿ1)

0.20 (0.16) a 0.12 (0.03) a

1.26 (1.08) a 1.79 (0.30) a

Numbers in brackets are 1 SD; means within column with different letter superscripts are significantly different (P<0.05).

3.3. Sediment characteristics Sediment characteristics and the results of the analysis of variance performed on the data are presented in Tables 4 and 5, respectively. Loss on ignition did not differ between treatments, and the content of organic carbon was not influenced by the type of sediment but was significantly increased by plant growth. Redox capacity was about 14% higher in the high C than in the low C sediment (Table 4). Overall, plant growth did not affect redox capacity significantly, but the Tukey test showed that there was a significant difference between low C sediment with and without plants, the capacity being lower in sediment with plants. Redox potentials differed markedly between the two sediment types. The high acetate concentration in the high C sediment led via increased microbial activity to a highly reducing sediment with an E7 of ÿ170 to ÿ220 mV (Table 4). The redox potentials in high C sediments were significantly lower than those in low C sediments in both week 7 and 11. After 7 weeks of plant growth, there was significant interaction between the effects of plants in the two types of sediment. Plant growth significantly increased the redox potentials at 6 and 13 cm depth, on average from ÿ7 to ‡160 mV in low C sediment and from ÿ190 to ÿ160 mV in high C sediment, but the increase in the high C sediment was not significant in the Tukey test. In week 11, three weeks after the shoots had been cut, the effect of the former plant growth was no longer significant and neither was the interaction. After seven weeks of plant growth, pH differed significantly between treatments, and the interaction between sediment type and plants was significant (Tables 4 and 5). Generally, pH was higher in the high C sediment, and in both sediments plant growth led to a pH decrease, probably because of root respiration and proton release connected with uptake of cations and/or microbial nitrification in the rhizosphere. Three weeks after the shoots had been cut, in week 11, the difference in pH between the two sediment types still prevailed, as did the effect of the earlier plant growth, but now the pH decrease in the low C sediment was not significant in the Tukey test. The interaction between sediment type and plants was still significant showing that the effect of the earlier growth of plants on pH was dependent on the type of sediment. The total concentration of organic acids in the interstitial water was significantly higher in the high C sediment probably mainly because of the addition of acetate and the

209 (48) b ÿ55 (63) a, ÿ42 (42) b 6.82 (0.11) a 7.01 (0.05) a

208 (15) b ÿ9 (8) b ÿ34 (15) b 7.20 (0.05) b 7.20 (0.05) a 131 (73) b 98 (58) a 25 (37) a ND ND

0 cm depth 6 cm depth 13 cm depth

Redox potential, E7 (mV), week 11

pH, week 7 pH, week 11

Lactic acid (mmol lÿ1) Formic acid (mmol lÿ1) Acetic acid (mmol lÿ1) Propionic acid (mmol lÿ1) Butyric acid (mmol lÿ1)

27 (28) a 446 (676) a 88400 (38700) b 474 (61) a 163 (41) a

8.46 (0.06) d 8.72 (0.15) c

ÿ74 (55) a ÿ188 (26) a ÿ217 (11) a

ÿ42 (23) a ÿ171 (11) a ÿ206 (14) a

20.8 (0.7) 8.2 (1.2) a 5.66 (0.25) c

b

22 (32) a 109 (33) a 60800 (17300) b 416 (171) a 83 (57) a

7.89 (0.07) c 7.89 (0.17) b

23 (45) a, b ÿ132 (22) a, ÿ173 (13) a

29 (118) b ÿ146 (12) a ÿ174 (8) a

20.4 (0.7) a 10.3 (0.5) b 5.66 (0.08) c

Planted

Parameters were generally measured after seven weeks of growth of Typha latifolia (week 7). Sediment pH and redox potential were also measured three weeks after harvest of the shoots (week 11). Numbers in brackets are 1 SD; nˆ5; NDˆnot detectable. Means within row with different letter superscripts are significantly different (P<0.05).

150 (31) b 93 (35) a ND ND ND

348 (47) c 188 (78) c 127 (94) c

323 (76) c 5 (11) b ÿ20 (11) b

0 cm depth 6 cm depth 13 cm depth

Redox potential, E7 (mV), week 7

b

20.7 (1.7) 10.1 (0.9) b 4.78 (0.30) a

22.0 (1.0) 9.8 (0.8) a, b 5.15 (0.06) b

Loss on ignition (% DW) Total organic carbon (% DW) Redox capacity (meqFe3‡ gÿ1DW)

a

Unplanted a

Unplanted

Planted

High C sediment

Low C sediment

a

Parameter

Table 4 Sediment characteristics and concentrations of organic acids in the interstitial water of a natural organic sediment (low C sediment) and of a sediment enriched with acetate (high C sediment)

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Table 5 Results of two-way ANOVAs for sediment characteristics measured in two types of sediment (low C and high C sediment) with and without growth of Typha latifolia Parameter

Source of variance Main effects

Interaction

Sediment

Plants

Sediment  Plants

0.1601 0.1053 0.0000

0.0959 0.0107 0.0566

0.3630 0.0368 0.0608

0.0000 0.0000 0.0000 0.0000 0.0021 0.0000

0.0548 0.0000 0.0000 0.0849 0.8264 0.4972

0.0637 0.0000 0.0000 0.0817 0.5182 0.9081

pH, week 7 pH, week 11

0.0000 0.0000

0.0000 0.0000

0.0000 0.0000

Total organic acids

0.0000

0.5604

0.2770

Loss on ignition Total organic carbon Redox capacity Redox potential, week 7 Redox potential, week 11

0 cm depth 6 cm depth 13 cm depth 0 cm depth 6 cm depth 13 cm depth

All measurements were taken after seven weeks of growth except for pH and redox potential, which were also measured three weeks after harvest of the shoots (week 11). P-values are the probability of a greater F-value. Bold indicates P<0.05.

associated microbial processes. Concentrations of formic acid were similar in the two types of sediment, whereas lactic acid concentrations were higher in low C than in high C sediment. The concentrations of all other analyzed acids were significantly higher in the high C sediment, with acetate accounting for far the greatest part. Plant growth had no effect on the concentrations of any of the organic acids analyzed (Table 5). 3.4. Methanogenesis Methanogenesis rates were significantly influenced by incubation time, type of sediment, and presence/absence of plants, and all interaction terms were highly significant (Table 6). Methanogenesis was seven-fold higher in the acetate-enriched sediment than in the low C sediment (643 vs. 90 nmol CH4 gÿ1DW hÿ1) when averaged over the entire incubation period (Fig. 2). Generally, methanogenesis rates in planted sediments were lower than in unplanted sediments (Fig. 2). After two weeks of incubation, no effect of plants could be detected in either of the sediments, but in weeks 3 and 4, plant growth significantly reduced the rate of methanogenesis in the low C sediment but not in the high C sediment, as also indicated by the significant interaction terms (Table 6). After 5 weeks of incubation, plant growth also reduced the rate of methanogenesis in the high C sediment, and this effect prevailed in both sediments until the plants were harvested. In the periods when plant growth influenced methanogenesis significantly, rates were on average reduced by 34% (from 93 to 61 nmol CH4 gÿ1DW hÿ1) in the low C sediment (weeks 3 to 7) and by 18% (from 818 to

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Table 6 Three-way analysis of variance (ANOVA) for methanogenesis rates measured during an incubation period of seven weeks in two types of sediment (low C and high C sediment) with and without growth of Typha latifolia (plants) Source of variance

d.f.

Sum of squares

F-ratio

P

Main effects

Time Sediment plant

4 1 1

3.518 26.616 0.754

48.1 1454.7 41.2

0.0000 0.0000 0.0000

Interactions

Time  sediment Time  plant Sediment  plant Time  sediment  plant

4 4 1 4

3.282 0.679 0.582 0.974

44.8 9.3 31.8 13.3

0.0000 0.0000 0.0000 0.0000

80

1.464

Residual

Fig. 2. Methanogenesis rates in a natural organic sediment (low C sediment) and a sediment enriched with acetate (high C sediment) with and without growth of Typha latifolia. Rates were measured during seven weeks of growth and three weeks after harvest of the shoots (week 11). Low C sediment without plants (*), low C sediment with plants (*), high C sediment without plants (&), high C sediment with plant (&). Means1 SD; nˆ5.

667 nmol CH4 gÿ1DW hÿ1) in the high C sediment (weeks 5 to 7). There was no significant relationship between the methanogenesis rates from week 7 and root biomass for low C plants (Pˆ0.52), but for high C plants there was a highly significant decrease in methanogenesis rate of ca. 170 nmol CH4 gÿ1DW hÿ1 per g increase of root DW (P<0.001; data not shown).

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Three weeks after the shoots had been cut, in week 11, the methanogenesis rates in high C sediment remained significantly higher than the rates in low C sediment (P<0.001; Fig. 2). The prior reduction of the methanogenesis rates by plant growth had disappeared (Pˆ0.10), and there was no interaction between sediment and former plant growth (Pˆ0.10). 4. Discussion Although aquatic macrophytes survive in flooded soils by virtue of their internal gas transport and the ability of their rhizomes to survive prolonged hypoxia and anoxia (Armstrong et al., 1991; Crawford, 1992), these strategies do not provide unlimited tolerance of highly reducing sediments. The growth of many aquatic plants is inhibited as sediments become increasingly reducing (Kludze and DeLaune, 1996; Pezeshki et al., 1996), and even where total biomass accumulation is unaffected, changes in morphology provide clear evidence of the stress suffered. The shorter, smaller shoots and increased production of secondary shoots in the high C plants in this study almost certainly result from effects of the more reducing sediment. They were accompanied by differences in root morphology known to be caused by oxygen stress in highly reducing sediments, such as shorter, thicker roots and reduced lateral root formation (Kludze et al., 1993; Armstrong et al., 1996). Short, thick roots are favoured when plants are under oxygen stress because they have low axial resistances to oxygen diffusion, whereas lateral roots are more difficult to support, having limited oxygen transport capacities due to their narrow diameter and low porosity (Armstrong et al., 1990; Sorrell, 1994). Hence, although T. latifolia produced similar total biomass in the low C and high C sediments, the smaller individual shoots and roots are a similar negative response to sediment oxygen demand to that seen in other emergent macrophytes (e.g. Kludze and DeLaune, 1996; Pezeshki et al., 1996). Whilst inhibition of growth is a general response of most aquatic macrophytes to increasing sedimentary decomposition rates, some taxa are far more sensitive than others, due to poorer internal oxygen transport. Relative to many genera of wetland plants, Typha species have well-developed internal gas transport pathways with low internal resistances, and effective gas transport physiology (Brix et al., 1992; Bendix et al., 1994), and they tolerate deep water and reducing sediments well (Grace, 1988; Callaway and King, 1996). In particular, high rates of convective gas flow in the shoot systems of emergent macrophytes are associated with the ability to colonize deep water and reducing sediments, and all Typha species have efficient convective gas flow (Brix et al., 1992; Chanton et al., 1993; Bendix et al., 1994). This also indirectly benefits root aeration (Armstrong et al., 1992), which may partly explain the ability of T. latifolia here to maintain its root biomass in the high C sediment. Our data therefore agree with earlier studies suggesting a high ability to avoid sediment oxygen stress by internal gas transport in Typha species. However, T. latifolia does appear to have less efficient convective gas flow and root aeration than the more narrow-leaved Typha species (T. angustifolia L. and T. domingensis Pers.), which is one of the factors thought to restrict it to shallower water when competing with them (Grace, 1988; Tornbjerg et al., 1994).

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Differences in the extent to which aquatic macrophytes affect sediment biogeochemistry are related primarily to the extent of their root development, with only those species with extensive root systems affecting sediments to any degree (Chanton and Whiting, 1995; Wigand et al., 1997). Large emergent macrophytes such as T. latifolia, with their prolific root development, often dramatically alter nutrient concentrations and biogeochemical parameters through a range of physiological processes (Dacey and Howes, 1984; Boon and Sorrell, 1991). Their effects on redox-dependent processes are complicated by the fact that oxygen can enter sediments via mechanisms other than root oxygen release (Dacey and Howes, 1984). By excluding other possible factors in this study, we have demonstrated the ability of T. latifolia's root oxygen release to affect biogeochemical processes, supporting claims that root oxygen release can be a significant oxygen input into sediments colonized by emergent plants (Boon and Sorrell, 1991; Armstrong et al., 1990; 1992). Our measurements of root oxygen release by T. latifolia were at the upper end of comparable data on whole plants from earlier studies (Kludze et al., 1993; Sorrell and Armstrong, 1994; Kludze and DeLaune, 1996). These interspecific differences in rates of root oxygen release can result from differences in root development, morphology and porosity (Smits et al., 1990; Kludze and DeLaune, 1996; Wigand et al., 1997). Environmental control of root morphology can also cause considerable intraspecific variation in root oxygen release, as in this study, with the high C plants having shorter roots that released less oxygen per unit dry weight than the low C plants (Table 3), although this difference was not statistically significant. Laterals, with their high surface area:volume ratios, are important sites for root oxygen release (Armstrong et al., 1991; Sorrell, 1994). The greater similarity of root oxygen release rates between the low C and high C plants when expressed on a surface area basis therefore provides further evidence that the amount of permeable surface area of the root system is the major morphological factor limiting root oxygen release, although the surface area of laterals were not included in our calculations. Some plants, however, show noticeably higher root oxygen release rates when grown in more reducing sediments, as their root porosity increases in response to the higher external oxygen demand (Kludze and DeLaune, 1996). In contrast, root porosity is little affected by external oxygen demand in many wetland species that produce roots with high porosities under all conditions (Justin and Armstrong, 1987), and our data suggest that T. latifolia may be one of these. It is a common observation that root oxygen release by aquatic macrophytes can effect large changes in Eh and concentrations of oxidized compounds in oligotrophic sediments, whereas these effects are rarely as evident in fertile, reducing sediments (Barko et al., 1991). Rather than suggesting that roots do not release oxygen into more reducing sediments, which is highly unlikely given that the oxygen demands of root and sediment are competitive (Armstrong et al., 1991), this is normally interpreted as rapid consumption of the oxygen released by the external oxygen demand (Sorrell and Armstrong, 1994). Our data strongly support this, given that our high C plants released as much oxygen as our low C plants, but had relatively little apparent effect on Eh. Measurements of Eh and other indicators of soil aeration status, which provide instantaneous measurements of relative concentrations of redox-active substances, reveal

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little about fluxes of materials and rates of processes in the rhizosphere. High rates of oxygen flux from roots into reducing media can provide a narrow but active zone favourable for aerobic processes (Roden and Wetzel, 1996; Lombardi et al., 1997), and the reduction in methanogenesis in the vegetated sediment here is clear evidence of this. These reductions were unlikely to have resulted from other effects of roots: the methanogenesis rates in this sediment were apparently little affected by pH differences in the jars, whilst the plants had no apparent effect on sediment organic acid contents. The importance of root oxygen release in highly reducing sediments is evident from the degree of reduction in methanogenesis in our data: although the reduction in the high C sediment only averaged 18% whereas it was 34% in the low C sediment, this corresponds to a total loss of 151 nmol CH4 gÿ1DW hÿ1 in the high C sediment but only 32 nmol CH4 gÿ1DW hÿ1 in the low C sediment. Given that the organic carbon and acetate concentrations in our high C sediment were the same in both vegetated and unvegetated treatments, this represents a substantial flux of organic carbon passing through other, less anaerobic microbial pathways ± fluxes that are otherwise cryptic, because of the high turnover rates and their physical restriction to the narrow, highly active rhizosphere. The high root to sediment ratios that develop in greenhouse studies of this type can favour the oxidizing effect of roots in sediments more than may occur in the field (Schipper and Reddy, 1996; Lombardi et al., 1997). This, together with the decrease in oxygen release from roots as they age (Armstrong et al., 1990; Gilbert and Frenzel, 1995), suggests that our results are maximum estimates of the degree of methanogenic inhibition effected by T. latifolia roots. This ought, however, to be viewed in the context of the growth of this species as a rhizomatous perennial, where new rhizomes are forming with dense, young roots in some areas of the sediment, whilst elsewhere older material is decomposing and stimulating anaerobic processes (Sorrell et al., 1997). Sediments under these plants are therefore likely to be extremely heterogeneous, and our understanding of their nutrient cycling may be improved by better recognition of the mosaic of aerobic and anaerobic sites they generate. Acknowledgements This study was funded by the Environment and Climate Programme of the European Commission, contract no. ENV4-CT95-0147: ``EUREED: Dynamics and stability of reed-dominated ecosystems in relation to major environmental factors that are subject to global and regional anthropogenically-induced changes''. References Armstrong, J., Armstrong, W., Beckett, P.M., 1992. Phragmites australis: Venturi- and humidity-induced pressure flows enhance rhizome aeration and rhizosphere oxidation. New Phytol. 120, 197±207. Armstrong, J., Armstrong, W., van der Putten, W.H., 1996. Phragmites die-back: bud and root death, blockages within the aeration and vascular systems and the possible role of phytotoxins. New Phytol. 133, 399±414.

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