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Microbial Processes of the Methane Cycle at the North-western Shelf of the Black Sea
Estuarine, Coastal and Shelf Science (2002) 54, 589–599 doi:10.1006/ecss.2000.0667, available online at http://www.idealibrary.com on
Microbial Processes of the Methane Cycle at the North-western Shelf of the Black Sea M. V. Ivanova,c, N. V. Pimenova, I. I. Rusanova and A. Yu. Leinb a
Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia Shirshov Institute of Oceanology, Russian Academy of Sciences, Moscow, Russia
b
Received 10 October 1999 and accepted in revised form 20 June 2000 During expeditions in August 1995 and May 1997, the distribution of methane and the rates of its production and oxidation were studied in water and bottom sediments of the north-western shelf of the Black Sea. Experiments that involved the addition of 14CH3COOH and 14CO2 to sediment samples showed the main part of methane to be formed from CO2. Maximum values of methane production (up to 560–680 mol m 2 day 1) were found in coastal sediments in summer-time. In spring, methane production in the same sediments did not exceed 3·6 mol m 2 day 1. The 13C values of methane ranged from 70·7 to 81·8‰, demonstrating its microbial origin and contradicting the concept of the migration of methane from the oilfields located on the Black Sea shelf. Experiments that involved the addition of 14 CH4 to water and sediment samples showed that a considerable part of methane is oxidized in the upper horizons of bottom sediments and in the water column. Nevertheless, it was found that, in summer, part of the methane (from 20 to 235 mol m 2 day 1) arrives at the atmosphere. 2002 Elsevier Science Ltd. All rights reserved. Keywords: methane oxidation and production; Black Sea Shelf; methane flux to the atmosphere
Introduction Under the thermodynamic conditions of the biosphere, the processes of methane production and oxidation are controlled by specialized groups of micro-organisms. Along with carbon dioxide and hydrogen sulphide, methane is one of the terminal products of anaerobic decomposition of organic matter. It is generated by obligatory anaerobic microbial associations that include both autotrophic and heterotrophic methanogens. The autotrophs reduce carbon dioxide at the expense of hydrogen, either formed during anaerobic fermentation of organic matter or coming from subsurface sources. The heterotrophic methanogens produce methane from methyl groups of low-molecular organic acids, alcohols and methyl sulphoxides, which are also formed during anaerobic decomposition of organic matter (Oremland, 1988). When studying the methane cycle in marine ecosystems, one should also take into account the possibility of arrival of subsurface methane in the form of so-called ‘ cold methane seeps ’. This methane can be of either biogenic or abiogenic origin, as shown by studies on the ratio of the 12C and 13C stable isotopes (Ivanov et al., 1993). The existence of numerous c
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methane seeps in the north-western part of the Black Sea was demonstrated in the late 1980s (Ivanov et al., 1989). The second stage of the cycle of methane, its oxidation, is driven by the activity of methanotrophic bacteria, which yields carbon dioxide, biomass and organic exometabolites. In some freshwater reservoirs, the process of methane oxidation significantly affects the oxygen regime of near-bottom water, since up to three molecules of oxygen are spent on the oxidation of one methane molecule (Kuznetsov, 1970). Unfortunately, the role of micro-organisms in the methane cycle in marine reservoirs, including the Black Sea, is poorly studied. The pioneering data on the role of micro-organisms in the methane cycle were obtained during the expedition on board the RV Professor Stockman in December 1980, when the distribution and geochemical activity of methanogens and methanotrophs in the Bulgarian sector of the Black Sea were studied (Ivanov et al., 1983). In 1989, we published a paper concerned with the role of micro-organisms in the oxidation of the methane of methane seeps (Ivanov et al., 1989). In 1991, a paper by American microbiologists was published, reporting the results of their studies of anaerobic oxidation of methane at one deep-sea and one shelf station in the southern part of the Black Sea (Reeburgh et al., 1991). 2002 Elsevier Science Ltd. All rights reserved.
590 M. V. Ivanov et al.
The aim of our work, which was a part of the European River–Ocean System 2000 (EROS 2000) multidisciplinary international project, was to obtain quantitative estimates of the role of methanogens in the anaerobic decomposition of organic matter and in methane production in the bottom sediments of the north-western Black Sea, and to study the processes of aerobic and anaerobic oxidation of methane in both bottom sediments and the water column. Comparison of the intensities of methane production and oxidation was to clarify the question on the contribution of marine methane to the atmospheric pool of this greenhouse gas. Studies of methane fluxes from the water column of the Baltic Sea to the atmosphere (Bange et al., 1994) showed that the largest amounts of methane are emitted from coastal sediments in the southern, most-polluted part of the Sea. In summer, methane emission was much greater than in winter, indicating the existence of seasonal dynamics in the activity of methanogenic bacteria of bottom sediments. According to our data for the Kara Sea, the highest rate of methane production and oxidation also occurred in the shallow-water shelf sediments and in the estuaries of the Ob and Yenisei rivers (Lein et al., 1996). Since the north-western shelf is the most polluted part of the Black Sea (Tolmazin, 1985; Mee, 1992; Zaitzev, 1992), we could anticipate high rates of methane cycle processes in this region. To monitor the possible seasonal dynamics of methane production and oxidation, investigations were carried out in August 1995 and May 1997. Materials and methods Samples of water and bottom sediments were taken at 22 stations in August 1995 during the 45th cruise of the RV Professor Vodyanitsky and, repeatedly, at 16 stations in May 1997 during the 49th cruise of the same vessel (Figure 1). Near-bottom water samples were taken by modified Niskin bottles and a rosette system with 10 Go-Flo bottles combined with the Mark-III CTD. Sediments were sampled by a Tripod sampler, box corer and multiple corer, which allowed sediments to be sampled to a depth of 30–50 cm, virtually without disturbing their structure. The detailed lithological–geochemical peculiarities of the sediment cores are presented by Lein et al. (2000) in this issue. Gas chromatography For methane analysis, a headspace technique was applied (Bolshakov & Egorov, 1987; Egorov &
Ivanov, 1998). A 5-cm3 section of wet sediments was placed into a glass vial, 30 cm3 in volume. The sediment was flooded with distilled water, leaving 4 cm3 of the bottle volume free and covered by rubber stopper. A 24-h waiting period was used with periodic shaking of the bottle to mix the water and gas phases and to establish phase equilibrium. For chromatographic analysis, 0·25 ml of the headspace gas was collected by a syringe running. Results are recalculated according to the volume of wet sediment using Henry’s law (Bolshakov & Egorov, 1987). The field chromatography HPM-2 with a flame ionization detector was used. The total relative error of the determinations was about 8% (by parallel definitions).
Radiotracer experiments The rates of methanogenesis and methane oxidation in sediments were determined by the radioisotopic method. Subsamples of 3·5 ml were taken in a 5-ml cut-off plastic syringe, and the end was sealed with a rubber bung. Then, 100 l of 14C-substrate was injected into the sediment through the rubber bung. The rate of methanogenesis was measured using H14CO3 (10 Ci per sample) and 14CH3COO (10 Ci per sample) solutions in sterile de-gassed seawater. The samples for determination of methane oxidation rate were supplemented with 0·2 ml of sterile de-gassed water containing dissolved CH4 with a total activity of 4 Ci. The syringes were incubated in the dark in a water bath at an in situ temperature (7·5–10 C) for 24–36 h. After incubation time, the samples were fixed with 1 ml of 2 M KOH. Water samples were taken in 30-ml glass bottles and stopped by rubber bung without a gas bubble. Then, 100 l of CH4 (4 Ci) was injected through the rubber bung. The bottles were incubated in the dark in a water bath at an in situ temperature for 24 h and, after incubation time, were fixed with 1 ml of 2% glutarealdehyde. In the case of methanogenesis, the CH4 formed was driven-off in a stream of air under boiling for 2 h and burnt at 800 C on a silica gel catalyst impregnated with cobalt salts. The carbon dioxide formed from the methane burnt was trapped in a scintillation liquid containing 2-phenylethylamine, and its radioactivity was measured in a scintillation counter. Water and sediment samples for the determination of methane oxidation were acidified, and CO2 was driven-off. Organic matter was then completely oxidized by the persulphate method (Rusanov et al.,
The methane cycle at the north-western shelf of the Black Sea 591
F 1. Distributions of the main lithological types of sediments on the shallow water NW shelf of the Black Sea (45th/49th) cruise of the RV Professor Vodyanitsky. (a) Carbonate-terrigenous estuarine sediments: (i) with polychaetes, (ii) without polychaetes; (b) sediments of the continental shelf: (i) clay muds with shells and shell sediments with terrigenous material on the outer terrace of the coastal part of the shelf, (ii) non-uniformly-grained carbonate–terrigenous sediments (with shells), exposed to the southern flow of sediment input by the Danube River; (c) sediments of the continental shelf; shell rock of the part of the shelf, poor with terrigenous sedimentary material; (d) sediment input sampling stations; (e) isobars.
1998), and again CO2 was driven-off. Both CO2 portions were trapped in a scintillation liquid containing 2-phenylethylamine. In all cases, radioactivity was measured on a RackBeta scintillation counter (LKB, Sweden). To calculate the rate of methanogenesis from carbonates and acetate, we determined the alkalinity and acetate concentration in porewaters of bottom sediments using a standard Merk kit (Germany) and ion chromatography, respectively. Porewaters were squeezed by N2 from freshly sectioned sediment samples through (0·45-m) membrane filters. The rates of methanogenesis and methane oxidation were calculated using the equation: I (rate)=
rC RT
(1)
where r is the radioactivity of the formed substrate, C is the natural concentration of the substrate, R is the total radioactivity of the added 14C-compounds (CH4, H14CO3 and 14CH3COO ) and T is the incubation time.
Mass spectrometry For the determination of the stable isotopic composition of methane carbon, gas samples were passed through a Porapak column to obtain methane free of CO2 and higher hydrocarbon homologues. Methane was then burnt to CO2 in an atmosphere of pure oxygen at 800 C in a tube filled with CuO. The products of methane combustion, CO2 and H2O, were separated by freezing. CO2 was collected in ampoules and used to determine the 13C/12C
592 M. V. Ivanov et al. T 1. Methane concentration and the rate of methane oxidation in the water column of the Black Sea shelf (August 1995)
Horizon (m) Station 1, depth 55 m 0 30 50 Near bottom
Methane concentration (mol l 1)
Rate of methane oxidation (nmol l 1 day 1)
Part of CH4 oxidizing to CO2 (%)
0·027 0·036 0·040 0·045
0·04 0·18 0·22 0·36
40 42 38 13
Totala (mol m 2 day 1) Station 4, depth 68 m 0 45 Near bottom
8·8 0·031 0·036 0·067
Total (mol m 2 day 1) Station 12, depth 53 m 0 30 Near bottom
0·022 0·027 0·031
0·022 0·134 0·245 0·156 0·246 0·246
70 70 70
0·36 0·36 1·12 0·98 1·34 1·70
70 80 70 40 60 100
34·9 0·031 0·027 0·022 0·045
Total (mol m 2 day 1) Station 28, depth 120 m 0 50 100 Near bottom
0·22 0·13 0·09 7·8
Total (mol m 2 day 1) Station 25, depth 55 m 0 30 50 Near bottom
40 44 26
3·8
Total (mol m 2 day 1) Station 14, depth 63 m 10 30 40 50 60 Near bottom
0·04 0·04 0·13
0·13 0·09 0·09 1·34
70 70 80 33
8·7 0·027 0·054 0·058 0·089
Total (mol m 2 day 1)
0·09 0·13 0·18 0·13
80 70 25 35
16·5
a Total rate of methane oxidation (MO) was calculated using the equation: C(total)=r1v1 +r2v2 + . . . rNvN, where r is the rate of MO in mol m 3 day 1 and v is the thickness of the water layer in m.
ratio on an MI-1201V two-collector isotopic mass spectrometer. Results Most of the detailed investigation into the distribution of methane and the intensity of the microbial pro-
cesses of its production and oxidation was carried out in August 1995. Gas-chromatographic analysis showed that methane occurred in all samples of water (Tables 1 and 2) and bottom sediments (Tables 3 and 4). In the water column of the open shelf, methane content did not exceed 100 nmol l 1, and was usually at a minimum in the upper horizons (Table 1).
The methane cycle at the north-western shelf of the Black Sea 593 T 2. Methane concentration and the rate of methane oxidation in the water column of the Black Sea coastal area
Horizon (m) Station 16, depth 16 m 0 10 12 Near bottom
Methane concentration (mol l 1)
Rate of methane oxidation (nmol l 1 day 1)
Part of CH4 oxidizing to CO2 (%)
0·313 0·335 0·201 0·223
1·78 2·19 0·36 1·20
50 57 100 33
Totala (mol m 2 day 1) Station 20, depth 26 m 0 8 Near bottom
25·5 0·223 0·134 0·201
Total (mol m 2 day 1) Station 22, depth 27 m 0 10 20 25 Near bottom
0·054 0·058 0·170 0·080 0·103
0·04 0·13 0·27 0·18 0·31
70 75 86 81 62
4·5 0·045 0·067 0·089 0·380
Total (mol m 2 day 1) Station 32, depth 50 m 0 20 Near bottom
40 33 71
47·4
Total (mol m 2 day 1) Station 30, depth 21 m 0 10 18 Near bottom
3·21 2·68 2·45
0·09 0·22 0·31 0·63
80 80 70 100
5·1 0·045 0·045 0·107
Total (mol m 2 day 1)
1·43 1·34 1·52
48 41 38
35·3
a Total rate of methane oxidation (MO) was calculated using the equation: C(total)=r1v1 +r2v2 + . . . rNvN, where r is the rate of MO in mol m 3 day 1 and v is the thickness of the water layer in m.
Somewhat higher methane concentrations (130– 250 nmol) were found only at station 14, located in the zone of cold methane seeps. Methane jets at station 14 were easily recorded by lateral locators, and large methane bubbles could be visually observed from on board the ship. Higher methane concentrations were found in the water column at coastal stations (Table 2), in particular at stations 16 and 20, situated in the zone influenced by Danube River discharge. At some of these stations (Table 2, stations 16 and 20), methane concentrations were at a maximum in the surface–water horizons, rather than in near-bottom water; this
phenomenon can be explained by the arrival of a part of methane with the fresh water of the Danube River. However, the main part of the methane occurring in water was formed in bottom sediments, as can be deduced from comparing methane concentrations in the water column and bottom sediments both at open-shelf stations (Tables 1 and 3) and coastal stations (Tables 2 and 4). In both ecosystems, the content of methane in the uppermost horizon of the sediments exceeded its content in the near-bottom water by at least one order of magnitude. At virtually all stations, the content of methane in bottom sediments noticeably increased downward along their
594 M. V. Ivanov et al. T 3. Methane concentration and the rates of methane generation and methane oxidation in bottom sediments of the Black Sea shelf (August 1995) Rate of methane generation (nmol dm 3 day 1) Horizon (cm)
Rate of methane oxidation (nmol dm 3 day 1)
Acetate Part of CH4 Methane Part of CH4 Alk content produced from CO2 oxidizing to CO2 content (%) Total (%) (mol dm 3) (mmol dm 3) (mol dm 3) Total
Station 1, depth 55 m 0–3 4–5 7–20 20–30
0·313 0·402 0·714 0·670
3·3 3·8 3·9 4·1
33 38 45 40
Totala (mol m 2 day 1) Station 4, depth 68 m 0–3 3–5 12–17 24–30
0·446 0·580 0·446 0·536
3·5 3·9 4·1 4·5
25 30 37 50
0·268 0·402 0·893
4·2 4·0 5·0
20 20 25
0·313 0·469 1·562 3·884
4·0 4·2 5·5 6·5
83 107 17 23
12·3 0·7 0·9
5·2 23·4 — 47·7
0·268 0·201 0·107
3·5 4·0 4·5
25 17 22
— 23 665
98 33 50
3·5 4·5 5·0
Total
25 23 25
— 98·0 241 40·0
1·3 0·6 0·5 0·7
100 100 100 100
1·1 1·2 1·4
100 100 100
0·37 100 100 — 97
210 14 6·8 4·8
23 47 100 100
5·25 — 97 95
85·8 0·379 0·670 9·375
66 100 100 100
0·20
4·70
Total Station 28, depth 120 m 0–2 9–13 20–30
— 64 79 94
0·70
Total Station 25, depth 55 m 0–1 6–10 25–30
0 2·5 10·1 20·1
7·2 2·1 0·9 1·7 0·60
2·19
Total Station 14, depth 63 m 0–1 2–5 9–15 22–30
— 100 95 100
4·12
Total Station 12, depth 53 m 2–5 7–12 15–30
0 11·5 20·5 10·2
7·5 2·5 5·7
50 75 100
1·33 — 95 97
24 1·3 17
30 22 50
3·75
a Integrated rate of methane generation (MG) and methane oxidation (MO) per m2 per day, calculated according to the equation: C(total)=r1v1 +r2v2 + . . . rNvN, where r is the rate of MO in mol dm 3 day 1 and v is the thickness of the sediment layer in mm.
profile, reaching 1000–1340 mol dm 3 of sediment at some coastal stations; when lifted on board the ship, such sediment samples exhibited swelling and gas evolution (Table 4, stations 19 and 20). The results of our determinations of methanogenesis intensity were in good agreement with data on
methane distribution. In the upper (0–3 cm) horizon of open-shelf sediments, the conditions were aerobic, i.e. unfavourable for methanogens. Therefore, methanogenesis either did not occur here (Table 3, stations 1 and 4) or was characterized by a very low rate and was evidently restricted to anaerobic
T 4. Methane concentration and the rates of methane generation and methane oxidation in coastal sediments of the Black Sea (August 1995) Rate of methane generation (nmol dm 3 day 1) Horizon (cm)
Acetate Methane Part of CH4 produced from CO2 Alk content content (%) (mol dm 3) (mmol dm 3) (mol dm 3) Total
Station 7, depth 8 m 0–1 2–5 6–12 12–30
1·518 3·304 4·732 12·950
4·0 6·0 9·0 19·0
30 28 26 17
Totala (mol m 2 day 1) Station 16, depth 16 m 0–1 1–3 3–5 9–12 15–20 20–30
2·589 2·679 2·813 2·991 24·550 44·640
4·0 4·8 5·0 14·0 18·0 25·0
162 90 67 135 180 147
4·2 5·5 16·0 30·0
250 300 333 335
Total 4·1 4·2 4·5 21·7 32·0
170 175 180 195 164
Total
43·8 652 512 3790
— 22·3 56·9 183 2110
0·112 0·223 0·446 0·848
3·5 4·0 5·5 25·0
75 75 75 32
— 41·9 198 906
34 5 14 2
4·0 4·3 5·0 6·0
17 13 20 20
Total
— 120 570 440
— 82 85 69 4
4·0 4·5 22·0 29·0
25 38 93 67
40·2 158 1680 6110 681
88·0 39·7 32·1 23·7 372 4020
58 76 87 100 100 100
290 4440 23 000 319
65 95 100 100
176 181 2830 179 000 133 900
55 66 100 100 100
26 140 — 30 50 90
2·32 3·75 3·75 4·46
50 15 15 0
1·1 — 100 95 90
113 4·464 8·929 267·900 446·400
91 97 100 100
2476
123 1·34 1·79 2·90 2·01
15·80 27·90 41·52 144·20
430
215
Total
Total
82 80 3 80 81 13
560
Station 20, depth 26 m 0–1 2·232 1–2 4·464 3–7 102·700 12–17 848·200 25–30 1339·000
Station 32, depth 50 m 0–2 3–5 15–20 25–30
12·5 37·7 15·0 131 115 756
Total
Part of CH4 oxidizing to CO2 (%)
30·0
93·1
Station 19, depth 20 m 0–2 23·210 3–5 66·960 11–15 817·900 20–30 1004·000
Station 30, depth 21 m 0–1 3–5 15–20 25–30
91 97 98 100
55·6
Total
Station 22, depth 27 m 0–1 2–5 9–13 25–30
12·3 32·8 89·5 268
Rate of methane oxidation (nmol dm 3 day 1)
127 17 57 154
40 80 100 100
22·5 92 99 100 93
920·10 163·20 1403·00 2274·00
20 90 100 100
410
a Integrated rate of methane generation (MG) and methane oxidation (MO) per m2 per day, calculated according to the equation: C(total)=r1v1 +r2v2 + . . . rNvN, where r is the rate of MO in mol dm 3 day 1 and v is the thickness of the sediment layer in mm.
596 M. V. Ivanov et al. T 5. Integrated methane production in the upper 30 cm of sediment at the north-western Black Sea shelf (mol m 2 day 1) August 1995 Area
May 1997
Station CH4 Station CH4 no. production no. production
Danube delta and prodelta (coastal area)
19 20 22
Open shelf
4 12
560 215 123 2·2 0·7
BS 3 BS 5 BS 8
1·4 3·6 3·3
BS 10 BS 16
2·3 2·0
microzones (Table 3, station 14). In deeper horizons of shelf sediments, the methanogenesis rate increased (Table 3, stations 1, 4 and 14). Generally speaking, a similar pattern was observed in coastal sediments; here, however, rather intense methanogenesis was found in the surface (0–1 cm) layer, immediately below a thin oxidized film (Table 4). In deeper horizons, the methanogenesis rate increased, reaching several mol per dm3 per day in the lowest horizons sampled (20–30 cm, see Table 4, stations 19, 20 and 32). In virtually all sediment samples taken at the open shelf, and in most samples of coastal sediments, the main part of methane was formed at the expense of carbon dioxide reduction (Tables 3 and 4). However, in some sediment horizons at the coastal stations 16 and 20, and also in all sediment samples taken at station 19, the major part of methane was formed from acetate, whose concentration in porewater at station 19 reached 335 mol dm 3. An apt illustration of the intensity of microbial methanogenesis in various sediments of the northwestern shelf in August 1995 is provided by Table 5, which contains values of methane production by the 0–30 cm layer of sediments per m2 per day. It can be seen that, in summer, methane production in coastal sediments is two orders of magnitude greater than in sediments of the open shelf (Table 5, stations 4 and 12). During the second expedition, in May 1997, we repeatedly determined methanogenesis intensity and methane content in the sediments of some stations (Table 5 and Figure 2). It can be seen that, in spring, the methanogenesis intensity in coastal sediments of the Danube delta and prodelta noticeably decreased (Table 5), which resulted in a considerable decrease in the methane content of the upper horizons of these sediments (Figure 2). Conversely, no significant
seasonal changes in methanogenesis intensity were found in sediments of the open shelf (Table 5, stations 4 and 12). Table 6 presents data of the analysis of the carbon stable isotopic composition of methane samples taken by us during three expeditions: in December 1980 (Ivanov et al., 1983), August 1995 and May 1997. These data unambiguously demonstrate the microbial origin (Ivanov & Lein, 1997) of methane of the bottom sediments of the western and north-western Black Sea. Samples of water and bottom sediments, for determination of the methane oxidation rate, were taken from the sediment and water column horizons in which methane concentration was determined. Data presented in Tables 1 and 2 show that in the water column, methane oxidation occurred at a very low rate, not exceeding several nmoles CH4 per litre per day, even in the shallow water of the coastal zone. Methanotrophic activity was most probably limited by low methane concentrations. Due to the same reason, the rate of methane oxidation was also low (0·5–24 nmol dm 3 day 1) in bottom sediments of the open shelf (Table 3, stations 1, 4, 12, 25 and 28). The only exception was station 14, where the rate of methane oxidation in the surface horizon of the sediments was essentially higher than at other stations of the open shelf. This can be explained by the occurrence of numerous methane seeps, near station 14, emitting methane into the water column, as a result of which the concentration of methane in the near-bottom water here was higher than in any other water sample analysed (Table 2). The rates of methane oxidation were considerably higher in shallow-water coastal sediments, especially at stations located in the area of the Danube delta (Table 4, stations 16, 19 and 20). The maximum rates of methane oxidation were found in the lower horizons of the sediment thickness, where the conditions were anaerobic. It should be stressed that methanogenesis and methane oxidation proceeded in the same horizons of the sediments, the rate of methane oxidation often being higher than the rate of methanogenesis in the same horizon (Table 4). Thus, methanogenesis seems to occur mainly in deeper sediment horizons, and from these horizons, methane enters the zone of its anaerobic and aerobic oxidation. Further convincing evidence of methane arrival from deeper horizons is provided by data on the distribution of methane along the sediment vertical profiles (Table 4).
The methane cycle at the north-western shelf of the Black Sea 597
–3
(a) 1
Content (µmolCH4 dm ) 10 100 1000 10 000
–3
(b) 0.1 1
Content (µmolCH4 dm ) 1 10 100 1000 10 000
5
4 Sediment depth (cm)
Sediment depth (cm)
10 15 20 25 30
10 15 20 25 30 35 40
–3
(c) 0.1 0
–1
Rate (nmolCH4 dm day ) 1 10 100 1000 10 000
(d) 0.1 0
–3
–1
–3
–1
Rate (nmolCH4 dm day ) 1 10 100 1000 10 000
5 5 Sediment depth (cm)
Sediment depth (cm)
10 15 20
10 15 20 25
25
30
30
35 –3
(e) 0
Rate (µmolCH4 dm 5 10 15
–1
day ) 20
(f) 25
0
5
5
15
20
25
30
Sediment depth (cm)
Sediment depth (cm)
10
Rate (µmolCH4 dm day ) 50 100 150 200
10 15 20 25 30 35
F 2. (a, b) Methane content and (c, d) the rate of methane generation and (e, f) methane oxidation in the coastal sediments of stations BS 3/19 and BS 5/20. ( ), BS 3, May 1997; ( ), BS 5, May 1997; ( ), station 19, August 1995; ( ), station 20, August 1995.
598 M. V. Ivanov et al. T 6. Isotopic composition of methane from sediments of the Black Sea (13C, ‰) Station no.
Depth (m)
Sediment layer (cm)
13C (‰) Station no.
a
December 1980 590 59 545 1620
140–160 100–120
78·4 81·8
August 1995 32
50
20–30
75·1
May 1997 BS 3 BS 5 BS 13
20 26 11
30–40 30–40 20–35
70·7 77·7 71·7
a
T 7. Daily methane production (P) and consumption of methane in sediments (Cs) and water column (Cw); minimal fluxes of methane from sediments to water (S
Ivanov et al. (1983).
Discussion As follows from the data in Table 4, the highest methane concentrations in bottom sediments of the north-western shelf were found in shallow-water sediments opposite the Danube delta (stations 19 and 20) and in sediments of the Bulgarian shelf (station 32). Industrially exploited oilfields are situated in the sedimentary rocks of the near-Danube zone of the Black Sea. Therefore, methane occurring in the sediments of this region may, theoretically, be of both microbial and migrational origin. All our data testify to the microbial origin of this methane: (1) Methanogens proved to be widespread in the anaerobic sediments of the Black Sea shelf (Ivanov et al., 1983). (2) The experiments that involved short-term incubation of sediment samples with 14CH3COOH and 14CO2 showed production of 14CH4, which was maximum for sediments characterized by a high content of methane (see Tables 3 and 4). (3) Methanogenesis intensity in coastal sediments exhibited seasonal dynamics (Table 5): it was maximum in August and considerably lower in May. The content of methane in the upper horizons of the sediments also showed seasonal variations (Figure 1), which cannot be explained by the concept of the migrational origin of methane. (4) Data on the isotopic composition of the methane of the bottom sediments (Table 6) also unambiguously demonstrate its microbial origin. The high methanogenesis rates in coastal sediments (Table 4) and high daily production of methane in summer (Table 5) are due to the same reasons as the
22 25 28 30 32
Depth (m)
P
Cs
Cw
S
W
27 55 120 21 50
123 85·8 40·0 113 681
1·1 1·3 3·8 22·5 410
4·5 8·7 16·5 5·1 35·3
122 84·5 36·2 90·5 271
117·5 75·8 19·7 85·4 235·7
high sulphate reduction rate in these sediments (Lein et al., 1997; 2000). The main reason is the arrival of large amounts of allochthonous and autochthonous organic matter in shallow-water bottom sediments of the near-Danube zone. The considerable decrease in the methane production that we recorded during our spring expedition in 1997 confirms the above statement, since it can be easily explained by a decrease in the inflow of organic matter occurring in winter due to the freezing of rivers and because of a decrease in the production of autochthonous organic matter. Along with methanogenesis, relatively active methane oxidation also occurs in the sediments and water column of the region studied. Based on data on the rates of methane production and oxidation in bottom sediments (Tables 3 and 4) and in the water column (Tables 1 and 2), we calculated the daily production and consumption of methane and the flux of methane from sediments to the water column and from the water column to the atmosphere. Data in Table 7 show that, despite the rather intense consumption of methane by methanotrophic bacteria, a considerable part of the methane produced in sediments escapes to the atmosphere, contributing to the atmospheric pool of greenhouse gases. It should be noted that our estimates of the methane flux to the atmosphere (Table 7) are in good agreement with the estimates made by the Dr S. Rapsomanikis’ team, which worked in the same region in summer 1995 and calculated methane flux to the atmosphere based on the data on methane concentrations in the surface water layer and in the lower atmospheric layers above the sea surface (Amouroux et al., 2000). According to these data, the daily emission of methane on the north-western Black Sea shelf varied from 28·9 to 469·2 mol m 2 day 1; in our case, this diapason ranged from 19·7 to 235 mol m 2 day 1.
The methane cycle at the north-western shelf of the Black Sea 599
The sufficient agreement between the estimates of the methane flux to the atmosphere made by two independent methods shows that our method, based on the results of the experimental determination of methane production and consumption rates (Table 7), is suitable for the assessment of methane fluxes in summer. However, our data cannot be used for the evaluation of annual methane flux, since methane production and oxidation rates strongly vary throughout the year, decreasing in winter and spring (Table 5). Thus, our quantitative investigations of the methane cycle at the north-western shelf of the Black Sea demonstrate that part of the microbial methane formed in the anaerobic sediments of the shelf can arrive at the atmosphere. The observed seasonal dynamics of the microbial activity calls for new, more detailed investigations of the rate of microbial processes in different seasons. Acknowledgements This work was supported by the Russian governmental programme ‘ Global Changes of the Environment and Climate ’, the Russian Foundation for Basis Research (96-04-48823), PECO (CT94-0115), INTAS (93-01-45) and INCO COPERNICUS (IC20-CT96-0065). This is publication No. 193 of the EU-ELOISE initiative. We thank the officers and crew of the RV Professor Vodyanitsky for helping us fulfil the programme of cruises and obtain the unique material. Special thanks to Prof. Nikolay Panin and Dr Victor Egorov, who promoted organization of the cruises. References Amouroux, D., Roberts, G., Rapsomanikis, S. & Andreae, M. 2002 Biogenic gas (CH4, N2O, DMS) emission to the atmosphere from near-shore and shelf waters of the north-western Black Sea. Estuarine, Coastal and Shelf Science 54, 575–587. Bange, H. W., Bartell, U. H., Rapsomanikis, S. & Andreae, M. O. 1994 Methane in the Baltic and North Seas and a reassessment of the marine emission of methane. Global Biogeochemical Cycles 8, 465–480.
Bolshakov, A. M. & Egorov, A. V. 1987 Using the phaseequilibrium degassing method for gasometric studies. Oceanology 27, 861–862. Egorov, A. V. & Ivanov, M. K. 1998 Hydrocarbon gases in sediments and mud breccia from the central and eastern part of the Mediterranean Ridge. Geo-Mar. Letters 18, 127–138. Ivanov, M. V. & Lein, A. Yu. 1997 Changes in the stable isotope composition of gases and minerals as a result of microbial activity. In Instruments, Methods and Mission for the Investigation of Extraterrestrial Microorganisms, Proceedings of the SPIE 3111, 395–4404. Ivanov, M. V., Vainstein, M. B., Galchenko, V. F., Gorlator, S. N. & Lein, A. Yu. 1983 Distribution and geochemical activity of bacteria in sediments of the western part of the Black Sea. In Geochemical Processes in the Western Part of the Black Sea. Bulgarian Academy of Science, Sofia, pp. 150–181 (in Russian). Ivanov, M. V., Policarpov, G. G., Lein A. Yu., Galchenko, V. F., Egorov, V. N., Gulin, S. B., Gulin, M. B., Rusanov, I. I., Miller, Yu. M. & Kupzov, V. I. 1989 Biogeochemistry of carbon cycle on the Black Sea region of CH4 gas seeps. Dokladi Academy Nauk USSR 320 (N5), 1235–1240 (in Russian). Kuznetzov, S. I. 1970 Microflora of Lakes and their Geochemical Activity. Nauka, Moscow, 440 pp. Lein, A. Yu., Pimenov, N. V., Rusanov, I. I., Miller, Yu. M. & Ivanov, M. V. 1997 Geochemical consequences of microbiological processes on the northwestern Black Sea shelf. Geochemistry International 10, 985–1004. Lein, A. Yu., Rusanov, I. I., Savvichev, A. S., Miller, Yu. M., Pimenov, N. V., Pavlova, G. A. & Ivanov, M. V. 1996 Biogeochemical processes of the sulfur and carbon cycle in the Kara Sea. Geochemistry International 11, 1027–1044. Lein, A. Yu., Pimenov, N. V., Martin, Y.-M., Lancelot, Ch., Rusanov, I. I., Yusupov, S. K., Miller, Yu. M. & Ivanov, M. V. 2000 Seasonal dynamics of sulphate reduction rate at the northwestern Black Sea shelf. Estuarine, Coastal and Shelf Science (this issue). Mee, L. D. 1992 The Black Sea in crisis: a need for concerted international action. Ambio 21, 278–286. Oremland, R. S. 1998 Biogeochemistry of methanogenic bacteria. In (Zehnder, A. J. B., ed.) Biology of Anaerobic Bacteria. John Wiley and Sons, New York, pp. 640–681. Reeburgh, W. S., Ward, B. B., Whalen, S. C., Sandbeck, K. A., Kilpatrick, K. A. & Kerkhof, L. J. 1991 Black Sea methane geochemistry. In (Murrey, J. W., ed.) Black Sea Oceanography, Results from the 1988 Black Sea Expedition. Deep-Sea Research 38 (Suppl. N2A), 1189–1210. Rusanov, I. I., Savvichev, S. K., Yusupov, N. V., Pimenov, N. V. & Ivanov, M. V. 1998 Production of exometabolites in the microbial oxidation of methane in marine ecosystems. Microbiology 67, 590–596. Tolmazin, D. 1985 Changing oceanography of the Black Sea. Progress in oceanography 15, 217–276. Zaitzev, Yu. P. 1991 Anthropogenic changes in the communities of biologically active zones of the Black Sea. In Variability of the Black Sea Ecosystem (Vinogradov, M., ed.). Nauka, Moscow, pp. 290–298 (in Russian).
Microbial Processes of the Methane Cycle at the North-western Shelf of the Black Sea M. V. Ivanova,c, N. V. Pimenova, I. I. Rusanova and A. Yu. Leinb a
Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia Shirshov Institute of Oceanology, Russian Academy of Sciences, Moscow, Russia
b
Received 10 October 1999 and accepted in revised form 20 June 2000 During expeditions in August 1995 and May 1997, the distribution of methane and the rates of its production and oxidation were studied in water and bottom sediments of the north-western shelf of the Black Sea. Experiments that involved the addition of 14CH3COOH and 14CO2 to sediment samples showed the main part of methane to be formed from CO2. Maximum values of methane production (up to 560–680 mol m 2 day 1) were found in coastal sediments in summer-time. In spring, methane production in the same sediments did not exceed 3·6 mol m 2 day 1. The 13C values of methane ranged from 70·7 to 81·8‰, demonstrating its microbial origin and contradicting the concept of the migration of methane from the oilfields located on the Black Sea shelf. Experiments that involved the addition of 14 CH4 to water and sediment samples showed that a considerable part of methane is oxidized in the upper horizons of bottom sediments and in the water column. Nevertheless, it was found that, in summer, part of the methane (from 20 to 235 mol m 2 day 1) arrives at the atmosphere. 2002 Elsevier Science Ltd. All rights reserved. Keywords: methane oxidation and production; Black Sea Shelf; methane flux to the atmosphere
Introduction Under the thermodynamic conditions of the biosphere, the processes of methane production and oxidation are controlled by specialized groups of micro-organisms. Along with carbon dioxide and hydrogen sulphide, methane is one of the terminal products of anaerobic decomposition of organic matter. It is generated by obligatory anaerobic microbial associations that include both autotrophic and heterotrophic methanogens. The autotrophs reduce carbon dioxide at the expense of hydrogen, either formed during anaerobic fermentation of organic matter or coming from subsurface sources. The heterotrophic methanogens produce methane from methyl groups of low-molecular organic acids, alcohols and methyl sulphoxides, which are also formed during anaerobic decomposition of organic matter (Oremland, 1988). When studying the methane cycle in marine ecosystems, one should also take into account the possibility of arrival of subsurface methane in the form of so-called ‘ cold methane seeps ’. This methane can be of either biogenic or abiogenic origin, as shown by studies on the ratio of the 12C and 13C stable isotopes (Ivanov et al., 1993). The existence of numerous c
Corresponding author. E-mail: [email protected]
0272–7714/02/030589+11 $35.00/0
methane seeps in the north-western part of the Black Sea was demonstrated in the late 1980s (Ivanov et al., 1989). The second stage of the cycle of methane, its oxidation, is driven by the activity of methanotrophic bacteria, which yields carbon dioxide, biomass and organic exometabolites. In some freshwater reservoirs, the process of methane oxidation significantly affects the oxygen regime of near-bottom water, since up to three molecules of oxygen are spent on the oxidation of one methane molecule (Kuznetsov, 1970). Unfortunately, the role of micro-organisms in the methane cycle in marine reservoirs, including the Black Sea, is poorly studied. The pioneering data on the role of micro-organisms in the methane cycle were obtained during the expedition on board the RV Professor Stockman in December 1980, when the distribution and geochemical activity of methanogens and methanotrophs in the Bulgarian sector of the Black Sea were studied (Ivanov et al., 1983). In 1989, we published a paper concerned with the role of micro-organisms in the oxidation of the methane of methane seeps (Ivanov et al., 1989). In 1991, a paper by American microbiologists was published, reporting the results of their studies of anaerobic oxidation of methane at one deep-sea and one shelf station in the southern part of the Black Sea (Reeburgh et al., 1991). 2002 Elsevier Science Ltd. All rights reserved.
590 M. V. Ivanov et al.
The aim of our work, which was a part of the European River–Ocean System 2000 (EROS 2000) multidisciplinary international project, was to obtain quantitative estimates of the role of methanogens in the anaerobic decomposition of organic matter and in methane production in the bottom sediments of the north-western Black Sea, and to study the processes of aerobic and anaerobic oxidation of methane in both bottom sediments and the water column. Comparison of the intensities of methane production and oxidation was to clarify the question on the contribution of marine methane to the atmospheric pool of this greenhouse gas. Studies of methane fluxes from the water column of the Baltic Sea to the atmosphere (Bange et al., 1994) showed that the largest amounts of methane are emitted from coastal sediments in the southern, most-polluted part of the Sea. In summer, methane emission was much greater than in winter, indicating the existence of seasonal dynamics in the activity of methanogenic bacteria of bottom sediments. According to our data for the Kara Sea, the highest rate of methane production and oxidation also occurred in the shallow-water shelf sediments and in the estuaries of the Ob and Yenisei rivers (Lein et al., 1996). Since the north-western shelf is the most polluted part of the Black Sea (Tolmazin, 1985; Mee, 1992; Zaitzev, 1992), we could anticipate high rates of methane cycle processes in this region. To monitor the possible seasonal dynamics of methane production and oxidation, investigations were carried out in August 1995 and May 1997. Materials and methods Samples of water and bottom sediments were taken at 22 stations in August 1995 during the 45th cruise of the RV Professor Vodyanitsky and, repeatedly, at 16 stations in May 1997 during the 49th cruise of the same vessel (Figure 1). Near-bottom water samples were taken by modified Niskin bottles and a rosette system with 10 Go-Flo bottles combined with the Mark-III CTD. Sediments were sampled by a Tripod sampler, box corer and multiple corer, which allowed sediments to be sampled to a depth of 30–50 cm, virtually without disturbing their structure. The detailed lithological–geochemical peculiarities of the sediment cores are presented by Lein et al. (2000) in this issue. Gas chromatography For methane analysis, a headspace technique was applied (Bolshakov & Egorov, 1987; Egorov &
Ivanov, 1998). A 5-cm3 section of wet sediments was placed into a glass vial, 30 cm3 in volume. The sediment was flooded with distilled water, leaving 4 cm3 of the bottle volume free and covered by rubber stopper. A 24-h waiting period was used with periodic shaking of the bottle to mix the water and gas phases and to establish phase equilibrium. For chromatographic analysis, 0·25 ml of the headspace gas was collected by a syringe running. Results are recalculated according to the volume of wet sediment using Henry’s law (Bolshakov & Egorov, 1987). The field chromatography HPM-2 with a flame ionization detector was used. The total relative error of the determinations was about 8% (by parallel definitions).
Radiotracer experiments The rates of methanogenesis and methane oxidation in sediments were determined by the radioisotopic method. Subsamples of 3·5 ml were taken in a 5-ml cut-off plastic syringe, and the end was sealed with a rubber bung. Then, 100 l of 14C-substrate was injected into the sediment through the rubber bung. The rate of methanogenesis was measured using H14CO3 (10 Ci per sample) and 14CH3COO (10 Ci per sample) solutions in sterile de-gassed seawater. The samples for determination of methane oxidation rate were supplemented with 0·2 ml of sterile de-gassed water containing dissolved CH4 with a total activity of 4 Ci. The syringes were incubated in the dark in a water bath at an in situ temperature (7·5–10 C) for 24–36 h. After incubation time, the samples were fixed with 1 ml of 2 M KOH. Water samples were taken in 30-ml glass bottles and stopped by rubber bung without a gas bubble. Then, 100 l of CH4 (4 Ci) was injected through the rubber bung. The bottles were incubated in the dark in a water bath at an in situ temperature for 24 h and, after incubation time, were fixed with 1 ml of 2% glutarealdehyde. In the case of methanogenesis, the CH4 formed was driven-off in a stream of air under boiling for 2 h and burnt at 800 C on a silica gel catalyst impregnated with cobalt salts. The carbon dioxide formed from the methane burnt was trapped in a scintillation liquid containing 2-phenylethylamine, and its radioactivity was measured in a scintillation counter. Water and sediment samples for the determination of methane oxidation were acidified, and CO2 was driven-off. Organic matter was then completely oxidized by the persulphate method (Rusanov et al.,
The methane cycle at the north-western shelf of the Black Sea 591
F 1. Distributions of the main lithological types of sediments on the shallow water NW shelf of the Black Sea (45th/49th) cruise of the RV Professor Vodyanitsky. (a) Carbonate-terrigenous estuarine sediments: (i) with polychaetes, (ii) without polychaetes; (b) sediments of the continental shelf: (i) clay muds with shells and shell sediments with terrigenous material on the outer terrace of the coastal part of the shelf, (ii) non-uniformly-grained carbonate–terrigenous sediments (with shells), exposed to the southern flow of sediment input by the Danube River; (c) sediments of the continental shelf; shell rock of the part of the shelf, poor with terrigenous sedimentary material; (d) sediment input sampling stations; (e) isobars.
1998), and again CO2 was driven-off. Both CO2 portions were trapped in a scintillation liquid containing 2-phenylethylamine. In all cases, radioactivity was measured on a RackBeta scintillation counter (LKB, Sweden). To calculate the rate of methanogenesis from carbonates and acetate, we determined the alkalinity and acetate concentration in porewaters of bottom sediments using a standard Merk kit (Germany) and ion chromatography, respectively. Porewaters were squeezed by N2 from freshly sectioned sediment samples through (0·45-m) membrane filters. The rates of methanogenesis and methane oxidation were calculated using the equation: I (rate)=
rC RT
(1)
where r is the radioactivity of the formed substrate, C is the natural concentration of the substrate, R is the total radioactivity of the added 14C-compounds (CH4, H14CO3 and 14CH3COO ) and T is the incubation time.
Mass spectrometry For the determination of the stable isotopic composition of methane carbon, gas samples were passed through a Porapak column to obtain methane free of CO2 and higher hydrocarbon homologues. Methane was then burnt to CO2 in an atmosphere of pure oxygen at 800 C in a tube filled with CuO. The products of methane combustion, CO2 and H2O, were separated by freezing. CO2 was collected in ampoules and used to determine the 13C/12C
592 M. V. Ivanov et al. T 1. Methane concentration and the rate of methane oxidation in the water column of the Black Sea shelf (August 1995)
Horizon (m) Station 1, depth 55 m 0 30 50 Near bottom
Methane concentration (mol l 1)
Rate of methane oxidation (nmol l 1 day 1)
Part of CH4 oxidizing to CO2 (%)
0·027 0·036 0·040 0·045
0·04 0·18 0·22 0·36
40 42 38 13
Totala (mol m 2 day 1) Station 4, depth 68 m 0 45 Near bottom
8·8 0·031 0·036 0·067
Total (mol m 2 day 1) Station 12, depth 53 m 0 30 Near bottom
0·022 0·027 0·031
0·022 0·134 0·245 0·156 0·246 0·246
70 70 70
0·36 0·36 1·12 0·98 1·34 1·70
70 80 70 40 60 100
34·9 0·031 0·027 0·022 0·045
Total (mol m 2 day 1) Station 28, depth 120 m 0 50 100 Near bottom
0·22 0·13 0·09 7·8
Total (mol m 2 day 1) Station 25, depth 55 m 0 30 50 Near bottom
40 44 26
3·8
Total (mol m 2 day 1) Station 14, depth 63 m 10 30 40 50 60 Near bottom
0·04 0·04 0·13
0·13 0·09 0·09 1·34
70 70 80 33
8·7 0·027 0·054 0·058 0·089
Total (mol m 2 day 1)
0·09 0·13 0·18 0·13
80 70 25 35
16·5
a Total rate of methane oxidation (MO) was calculated using the equation: C(total)=r1v1 +r2v2 + . . . rNvN, where r is the rate of MO in mol m 3 day 1 and v is the thickness of the water layer in m.
ratio on an MI-1201V two-collector isotopic mass spectrometer. Results Most of the detailed investigation into the distribution of methane and the intensity of the microbial pro-
cesses of its production and oxidation was carried out in August 1995. Gas-chromatographic analysis showed that methane occurred in all samples of water (Tables 1 and 2) and bottom sediments (Tables 3 and 4). In the water column of the open shelf, methane content did not exceed 100 nmol l 1, and was usually at a minimum in the upper horizons (Table 1).
The methane cycle at the north-western shelf of the Black Sea 593 T 2. Methane concentration and the rate of methane oxidation in the water column of the Black Sea coastal area
Horizon (m) Station 16, depth 16 m 0 10 12 Near bottom
Methane concentration (mol l 1)
Rate of methane oxidation (nmol l 1 day 1)
Part of CH4 oxidizing to CO2 (%)
0·313 0·335 0·201 0·223
1·78 2·19 0·36 1·20
50 57 100 33
Totala (mol m 2 day 1) Station 20, depth 26 m 0 8 Near bottom
25·5 0·223 0·134 0·201
Total (mol m 2 day 1) Station 22, depth 27 m 0 10 20 25 Near bottom
0·054 0·058 0·170 0·080 0·103
0·04 0·13 0·27 0·18 0·31
70 75 86 81 62
4·5 0·045 0·067 0·089 0·380
Total (mol m 2 day 1) Station 32, depth 50 m 0 20 Near bottom
40 33 71
47·4
Total (mol m 2 day 1) Station 30, depth 21 m 0 10 18 Near bottom
3·21 2·68 2·45
0·09 0·22 0·31 0·63
80 80 70 100
5·1 0·045 0·045 0·107
Total (mol m 2 day 1)
1·43 1·34 1·52
48 41 38
35·3
a Total rate of methane oxidation (MO) was calculated using the equation: C(total)=r1v1 +r2v2 + . . . rNvN, where r is the rate of MO in mol m 3 day 1 and v is the thickness of the water layer in m.
Somewhat higher methane concentrations (130– 250 nmol) were found only at station 14, located in the zone of cold methane seeps. Methane jets at station 14 were easily recorded by lateral locators, and large methane bubbles could be visually observed from on board the ship. Higher methane concentrations were found in the water column at coastal stations (Table 2), in particular at stations 16 and 20, situated in the zone influenced by Danube River discharge. At some of these stations (Table 2, stations 16 and 20), methane concentrations were at a maximum in the surface–water horizons, rather than in near-bottom water; this
phenomenon can be explained by the arrival of a part of methane with the fresh water of the Danube River. However, the main part of the methane occurring in water was formed in bottom sediments, as can be deduced from comparing methane concentrations in the water column and bottom sediments both at open-shelf stations (Tables 1 and 3) and coastal stations (Tables 2 and 4). In both ecosystems, the content of methane in the uppermost horizon of the sediments exceeded its content in the near-bottom water by at least one order of magnitude. At virtually all stations, the content of methane in bottom sediments noticeably increased downward along their
594 M. V. Ivanov et al. T 3. Methane concentration and the rates of methane generation and methane oxidation in bottom sediments of the Black Sea shelf (August 1995) Rate of methane generation (nmol dm 3 day 1) Horizon (cm)
Rate of methane oxidation (nmol dm 3 day 1)
Acetate Part of CH4 Methane Part of CH4 Alk content produced from CO2 oxidizing to CO2 content (%) Total (%) (mol dm 3) (mmol dm 3) (mol dm 3) Total
Station 1, depth 55 m 0–3 4–5 7–20 20–30
0·313 0·402 0·714 0·670
3·3 3·8 3·9 4·1
33 38 45 40
Totala (mol m 2 day 1) Station 4, depth 68 m 0–3 3–5 12–17 24–30
0·446 0·580 0·446 0·536
3·5 3·9 4·1 4·5
25 30 37 50
0·268 0·402 0·893
4·2 4·0 5·0
20 20 25
0·313 0·469 1·562 3·884
4·0 4·2 5·5 6·5
83 107 17 23
12·3 0·7 0·9
5·2 23·4 — 47·7
0·268 0·201 0·107
3·5 4·0 4·5
25 17 22
— 23 665
98 33 50
3·5 4·5 5·0
Total
25 23 25
— 98·0 241 40·0
1·3 0·6 0·5 0·7
100 100 100 100
1·1 1·2 1·4
100 100 100
0·37 100 100 — 97
210 14 6·8 4·8
23 47 100 100
5·25 — 97 95
85·8 0·379 0·670 9·375
66 100 100 100
0·20
4·70
Total Station 28, depth 120 m 0–2 9–13 20–30
— 64 79 94
0·70
Total Station 25, depth 55 m 0–1 6–10 25–30
0 2·5 10·1 20·1
7·2 2·1 0·9 1·7 0·60
2·19
Total Station 14, depth 63 m 0–1 2–5 9–15 22–30
— 100 95 100
4·12
Total Station 12, depth 53 m 2–5 7–12 15–30
0 11·5 20·5 10·2
7·5 2·5 5·7
50 75 100
1·33 — 95 97
24 1·3 17
30 22 50
3·75
a Integrated rate of methane generation (MG) and methane oxidation (MO) per m2 per day, calculated according to the equation: C(total)=r1v1 +r2v2 + . . . rNvN, where r is the rate of MO in mol dm 3 day 1 and v is the thickness of the sediment layer in mm.
profile, reaching 1000–1340 mol dm 3 of sediment at some coastal stations; when lifted on board the ship, such sediment samples exhibited swelling and gas evolution (Table 4, stations 19 and 20). The results of our determinations of methanogenesis intensity were in good agreement with data on
methane distribution. In the upper (0–3 cm) horizon of open-shelf sediments, the conditions were aerobic, i.e. unfavourable for methanogens. Therefore, methanogenesis either did not occur here (Table 3, stations 1 and 4) or was characterized by a very low rate and was evidently restricted to anaerobic
T 4. Methane concentration and the rates of methane generation and methane oxidation in coastal sediments of the Black Sea (August 1995) Rate of methane generation (nmol dm 3 day 1) Horizon (cm)
Acetate Methane Part of CH4 produced from CO2 Alk content content (%) (mol dm 3) (mmol dm 3) (mol dm 3) Total
Station 7, depth 8 m 0–1 2–5 6–12 12–30
1·518 3·304 4·732 12·950
4·0 6·0 9·0 19·0
30 28 26 17
Totala (mol m 2 day 1) Station 16, depth 16 m 0–1 1–3 3–5 9–12 15–20 20–30
2·589 2·679 2·813 2·991 24·550 44·640
4·0 4·8 5·0 14·0 18·0 25·0
162 90 67 135 180 147
4·2 5·5 16·0 30·0
250 300 333 335
Total 4·1 4·2 4·5 21·7 32·0
170 175 180 195 164
Total
43·8 652 512 3790
— 22·3 56·9 183 2110
0·112 0·223 0·446 0·848
3·5 4·0 5·5 25·0
75 75 75 32
— 41·9 198 906
34 5 14 2
4·0 4·3 5·0 6·0
17 13 20 20
Total
— 120 570 440
— 82 85 69 4
4·0 4·5 22·0 29·0
25 38 93 67
40·2 158 1680 6110 681
88·0 39·7 32·1 23·7 372 4020
58 76 87 100 100 100
290 4440 23 000 319
65 95 100 100
176 181 2830 179 000 133 900
55 66 100 100 100
26 140 — 30 50 90
2·32 3·75 3·75 4·46
50 15 15 0
1·1 — 100 95 90
113 4·464 8·929 267·900 446·400
91 97 100 100
2476
123 1·34 1·79 2·90 2·01
15·80 27·90 41·52 144·20
430
215
Total
Total
82 80 3 80 81 13
560
Station 20, depth 26 m 0–1 2·232 1–2 4·464 3–7 102·700 12–17 848·200 25–30 1339·000
Station 32, depth 50 m 0–2 3–5 15–20 25–30
12·5 37·7 15·0 131 115 756
Total
Part of CH4 oxidizing to CO2 (%)
30·0
93·1
Station 19, depth 20 m 0–2 23·210 3–5 66·960 11–15 817·900 20–30 1004·000
Station 30, depth 21 m 0–1 3–5 15–20 25–30
91 97 98 100
55·6
Total
Station 22, depth 27 m 0–1 2–5 9–13 25–30
12·3 32·8 89·5 268
Rate of methane oxidation (nmol dm 3 day 1)
127 17 57 154
40 80 100 100
22·5 92 99 100 93
920·10 163·20 1403·00 2274·00
20 90 100 100
410
a Integrated rate of methane generation (MG) and methane oxidation (MO) per m2 per day, calculated according to the equation: C(total)=r1v1 +r2v2 + . . . rNvN, where r is the rate of MO in mol dm 3 day 1 and v is the thickness of the sediment layer in mm.
596 M. V. Ivanov et al. T 5. Integrated methane production in the upper 30 cm of sediment at the north-western Black Sea shelf (mol m 2 day 1) August 1995 Area
May 1997
Station CH4 Station CH4 no. production no. production
Danube delta and prodelta (coastal area)
19 20 22
Open shelf
4 12
560 215 123 2·2 0·7
BS 3 BS 5 BS 8
1·4 3·6 3·3
BS 10 BS 16
2·3 2·0
microzones (Table 3, station 14). In deeper horizons of shelf sediments, the methanogenesis rate increased (Table 3, stations 1, 4 and 14). Generally speaking, a similar pattern was observed in coastal sediments; here, however, rather intense methanogenesis was found in the surface (0–1 cm) layer, immediately below a thin oxidized film (Table 4). In deeper horizons, the methanogenesis rate increased, reaching several mol per dm3 per day in the lowest horizons sampled (20–30 cm, see Table 4, stations 19, 20 and 32). In virtually all sediment samples taken at the open shelf, and in most samples of coastal sediments, the main part of methane was formed at the expense of carbon dioxide reduction (Tables 3 and 4). However, in some sediment horizons at the coastal stations 16 and 20, and also in all sediment samples taken at station 19, the major part of methane was formed from acetate, whose concentration in porewater at station 19 reached 335 mol dm 3. An apt illustration of the intensity of microbial methanogenesis in various sediments of the northwestern shelf in August 1995 is provided by Table 5, which contains values of methane production by the 0–30 cm layer of sediments per m2 per day. It can be seen that, in summer, methane production in coastal sediments is two orders of magnitude greater than in sediments of the open shelf (Table 5, stations 4 and 12). During the second expedition, in May 1997, we repeatedly determined methanogenesis intensity and methane content in the sediments of some stations (Table 5 and Figure 2). It can be seen that, in spring, the methanogenesis intensity in coastal sediments of the Danube delta and prodelta noticeably decreased (Table 5), which resulted in a considerable decrease in the methane content of the upper horizons of these sediments (Figure 2). Conversely, no significant
seasonal changes in methanogenesis intensity were found in sediments of the open shelf (Table 5, stations 4 and 12). Table 6 presents data of the analysis of the carbon stable isotopic composition of methane samples taken by us during three expeditions: in December 1980 (Ivanov et al., 1983), August 1995 and May 1997. These data unambiguously demonstrate the microbial origin (Ivanov & Lein, 1997) of methane of the bottom sediments of the western and north-western Black Sea. Samples of water and bottom sediments, for determination of the methane oxidation rate, were taken from the sediment and water column horizons in which methane concentration was determined. Data presented in Tables 1 and 2 show that in the water column, methane oxidation occurred at a very low rate, not exceeding several nmoles CH4 per litre per day, even in the shallow water of the coastal zone. Methanotrophic activity was most probably limited by low methane concentrations. Due to the same reason, the rate of methane oxidation was also low (0·5–24 nmol dm 3 day 1) in bottom sediments of the open shelf (Table 3, stations 1, 4, 12, 25 and 28). The only exception was station 14, where the rate of methane oxidation in the surface horizon of the sediments was essentially higher than at other stations of the open shelf. This can be explained by the occurrence of numerous methane seeps, near station 14, emitting methane into the water column, as a result of which the concentration of methane in the near-bottom water here was higher than in any other water sample analysed (Table 2). The rates of methane oxidation were considerably higher in shallow-water coastal sediments, especially at stations located in the area of the Danube delta (Table 4, stations 16, 19 and 20). The maximum rates of methane oxidation were found in the lower horizons of the sediment thickness, where the conditions were anaerobic. It should be stressed that methanogenesis and methane oxidation proceeded in the same horizons of the sediments, the rate of methane oxidation often being higher than the rate of methanogenesis in the same horizon (Table 4). Thus, methanogenesis seems to occur mainly in deeper sediment horizons, and from these horizons, methane enters the zone of its anaerobic and aerobic oxidation. Further convincing evidence of methane arrival from deeper horizons is provided by data on the distribution of methane along the sediment vertical profiles (Table 4).
The methane cycle at the north-western shelf of the Black Sea 597
–3
(a) 1
Content (µmolCH4 dm ) 10 100 1000 10 000
–3
(b) 0.1 1
Content (µmolCH4 dm ) 1 10 100 1000 10 000
5
4 Sediment depth (cm)
Sediment depth (cm)
10 15 20 25 30
10 15 20 25 30 35 40
–3
(c) 0.1 0
–1
Rate (nmolCH4 dm day ) 1 10 100 1000 10 000
(d) 0.1 0
–3
–1
–3
–1
Rate (nmolCH4 dm day ) 1 10 100 1000 10 000
5 5 Sediment depth (cm)
Sediment depth (cm)
10 15 20
10 15 20 25
25
30
30
35 –3
(e) 0
Rate (µmolCH4 dm 5 10 15
–1
day ) 20
(f) 25
0
5
5
15
20
25
30
Sediment depth (cm)
Sediment depth (cm)
10
Rate (µmolCH4 dm day ) 50 100 150 200
10 15 20 25 30 35
F 2. (a, b) Methane content and (c, d) the rate of methane generation and (e, f) methane oxidation in the coastal sediments of stations BS 3/19 and BS 5/20. ( ), BS 3, May 1997; ( ), BS 5, May 1997; ( ), station 19, August 1995; ( ), station 20, August 1995.
598 M. V. Ivanov et al. T 6. Isotopic composition of methane from sediments of the Black Sea (13C, ‰) Station no.
Depth (m)
Sediment layer (cm)
13C (‰) Station no.
a
December 1980 590 59 545 1620
140–160 100–120
78·4 81·8
August 1995 32
50
20–30
75·1
May 1997 BS 3 BS 5 BS 13
20 26 11
30–40 30–40 20–35
70·7 77·7 71·7
a
T 7. Daily methane production (P) and consumption of methane in sediments (Cs) and water column (Cw); minimal fluxes of methane from sediments to water (S
Ivanov et al. (1983).
Discussion As follows from the data in Table 4, the highest methane concentrations in bottom sediments of the north-western shelf were found in shallow-water sediments opposite the Danube delta (stations 19 and 20) and in sediments of the Bulgarian shelf (station 32). Industrially exploited oilfields are situated in the sedimentary rocks of the near-Danube zone of the Black Sea. Therefore, methane occurring in the sediments of this region may, theoretically, be of both microbial and migrational origin. All our data testify to the microbial origin of this methane: (1) Methanogens proved to be widespread in the anaerobic sediments of the Black Sea shelf (Ivanov et al., 1983). (2) The experiments that involved short-term incubation of sediment samples with 14CH3COOH and 14CO2 showed production of 14CH4, which was maximum for sediments characterized by a high content of methane (see Tables 3 and 4). (3) Methanogenesis intensity in coastal sediments exhibited seasonal dynamics (Table 5): it was maximum in August and considerably lower in May. The content of methane in the upper horizons of the sediments also showed seasonal variations (Figure 1), which cannot be explained by the concept of the migrational origin of methane. (4) Data on the isotopic composition of the methane of the bottom sediments (Table 6) also unambiguously demonstrate its microbial origin. The high methanogenesis rates in coastal sediments (Table 4) and high daily production of methane in summer (Table 5) are due to the same reasons as the
22 25 28 30 32
Depth (m)
P
Cs
Cw
S
W
27 55 120 21 50
123 85·8 40·0 113 681
1·1 1·3 3·8 22·5 410
4·5 8·7 16·5 5·1 35·3
122 84·5 36·2 90·5 271
117·5 75·8 19·7 85·4 235·7
high sulphate reduction rate in these sediments (Lein et al., 1997; 2000). The main reason is the arrival of large amounts of allochthonous and autochthonous organic matter in shallow-water bottom sediments of the near-Danube zone. The considerable decrease in the methane production that we recorded during our spring expedition in 1997 confirms the above statement, since it can be easily explained by a decrease in the inflow of organic matter occurring in winter due to the freezing of rivers and because of a decrease in the production of autochthonous organic matter. Along with methanogenesis, relatively active methane oxidation also occurs in the sediments and water column of the region studied. Based on data on the rates of methane production and oxidation in bottom sediments (Tables 3 and 4) and in the water column (Tables 1 and 2), we calculated the daily production and consumption of methane and the flux of methane from sediments to the water column and from the water column to the atmosphere. Data in Table 7 show that, despite the rather intense consumption of methane by methanotrophic bacteria, a considerable part of the methane produced in sediments escapes to the atmosphere, contributing to the atmospheric pool of greenhouse gases. It should be noted that our estimates of the methane flux to the atmosphere (Table 7) are in good agreement with the estimates made by the Dr S. Rapsomanikis’ team, which worked in the same region in summer 1995 and calculated methane flux to the atmosphere based on the data on methane concentrations in the surface water layer and in the lower atmospheric layers above the sea surface (Amouroux et al., 2000). According to these data, the daily emission of methane on the north-western Black Sea shelf varied from 28·9 to 469·2 mol m 2 day 1; in our case, this diapason ranged from 19·7 to 235 mol m 2 day 1.
The methane cycle at the north-western shelf of the Black Sea 599
The sufficient agreement between the estimates of the methane flux to the atmosphere made by two independent methods shows that our method, based on the results of the experimental determination of methane production and consumption rates (Table 7), is suitable for the assessment of methane fluxes in summer. However, our data cannot be used for the evaluation of annual methane flux, since methane production and oxidation rates strongly vary throughout the year, decreasing in winter and spring (Table 5). Thus, our quantitative investigations of the methane cycle at the north-western shelf of the Black Sea demonstrate that part of the microbial methane formed in the anaerobic sediments of the shelf can arrive at the atmosphere. The observed seasonal dynamics of the microbial activity calls for new, more detailed investigations of the rate of microbial processes in different seasons. Acknowledgements This work was supported by the Russian governmental programme ‘ Global Changes of the Environment and Climate ’, the Russian Foundation for Basis Research (96-04-48823), PECO (CT94-0115), INTAS (93-01-45) and INCO COPERNICUS (IC20-CT96-0065). This is publication No. 193 of the EU-ELOISE initiative. We thank the officers and crew of the RV Professor Vodyanitsky for helping us fulfil the programme of cruises and obtain the unique material. Special thanks to Prof. Nikolay Panin and Dr Victor Egorov, who promoted organization of the cruises. References Amouroux, D., Roberts, G., Rapsomanikis, S. & Andreae, M. 2002 Biogenic gas (CH4, N2O, DMS) emission to the atmosphere from near-shore and shelf waters of the north-western Black Sea. Estuarine, Coastal and Shelf Science 54, 575–587. Bange, H. W., Bartell, U. H., Rapsomanikis, S. & Andreae, M. O. 1994 Methane in the Baltic and North Seas and a reassessment of the marine emission of methane. Global Biogeochemical Cycles 8, 465–480.
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