Pressure sensitivity of ANME-3 predominant anaerobic methane oxidizing community from coastal

37 Pages·2017·1.55 MB·English

bioRxiv preprint first posted online Apr. 25, 2018; doi: http://dx.doi.org/10.1101/307082. The copyright holder for this preprint
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1 Pressure sensitivity of ANME-3 predominant anaerobic methane oxidizing
2 community from coastal marine Lake Grevelingen sediment
3 C. Cassarinia,b*, Y. Zhangc and P. N. Lensa,b
4 aUNESCO-IHE, Institute for Water Education, P.O.Box 3015, 2601 DA , Delft, The Netherlands
5 b National University of Ireland Galway, University Road, Galway H91 TK33, Ireland
6 cState Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology,
7 Shanghai Jiao Tong University, Shanghai, People's Republic of China
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13 *Corresponding author
14 Chiara Cassarini
15 National University of Ireland Galway
16 University Road, Galway H91 TK33, Ireland
17 Phone: [phone]
18 e-mail: [email protected]
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bioRxiv preprint first posted online Apr. 25, 2018; doi: http://dx.doi.org/10.1101/307082. The copyright holder for this preprint
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19 Abstract
20 Anaerobic oxidation of methane (AOM) coupled to sulfate reduction is mediated by,
21 respectively, anaerobic methanotrophic archaea (ANME) and sulfate reducing bacteria (SRB).
22 When a microbial community from coastal marine Lake Grevelingen sediment, containing
23 ANME-3 as the most abundant type of ANME, was incubated under a pressure gradient (0.1- 40
24 MPa) for 77 days, ANME-3 was more pressure sensitive than the SRB. ANME-3 activity was
25 higher at lower (0.1, 0.45 MPa) over higher (10, 20 and 40 MPa) CH total pressures. Moreover,
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26 the sulfur metabolism was shifted upon changing the incubation pressure: only at 0.1 MPa
27 elemental sulfur was detected in a considerable amount and SRB of the Desulfobacterales order
28 were more enriched at elevated pressures than the Desulfubulbaceae. This study provides
29 evidence that ANME-3 can be constrained at shallow environments, despite the scarce
30 bioavailable energy, because of its pressure sensitivity. Besides, the association between ANME-
31 3 and SRB can be steered by changing solely the incubation pressure.
32 Importance
33 Anaerobic oxidation of methane (AOM) coupled to sulfate reduction is a biological process
34 largely occurring in marine sediments, which contributes to the removal of almost 90% of
35 sedimentary methane, thereby controlling methane emission to the atmosphere. AOM is
36 mediated by slow growing archaea, anaerobic methanotrophs (ANME) and sulfate reducing
37 bacteria. The enrichment of these microorganisms has been challenging, especially considering
38 the low solubility of methane at ambient temperature and pressure. Previous studies showed
39 strong positive correlations between the growth of ANME and the methane pressure, since the
40 higher the pressure the more methane is dissolved. In this research, a shallow marine sediment
2
bioRxiv preprint first posted online Apr. 25, 2018; doi: http://dx.doi.org/10.1101/307082. The copyright holder for this preprint
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
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41 was incubated under methane pressure gradients. The investigated effect of pressure on the
42 AOM-SR activity, the formation sulfur intermediates and the microbial community structure is
43 important to understand the pressure influence on the processes and the activity of the
44 microorganisms involved to further understand their metabolism and physiology.
45 Introduction
46 Anaerobic oxidation of methane (AOM) coupled to sulfate reduction (SR) is a major sink in the
47 oceanic methane (CH ) budget. The net stoichiometry of this reaction is shown in Eq. 1 (1):
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48 CH +SO2-→ HCO-+HS-+H O ∆G° '= -16.6 kJ mol-1 CH (Eq. 1)
4 4 3 2 4
49 The thermodynamics of this reaction depend on the concentration of dissolved CH . CH is
4 4
50 poorly soluble: 1.3 mM is its concentration in sea water at ambient pressure and at 15°C (2).
51 Theoretically, an elevated CH pressure favors the AOM coupled to SR (AOM-SR)
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52 bioconversion since i) the Gibbs free energy becomes more negative at higher CH partial
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53 pressures and ii) the dissolved CH concentration increases and is thus more bioavailable (Table
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54 1). Thus, the activity and the growth of the microorganisms mediating the process, namely
55 anaerobic methanotrophs (ANME) and sulfate reducing bacteria (SRB), is expected to be higher
56 at elevated pressures.
57 ANME are grouped into three distinct clades, i.e. ANME-1, ANME-2 and ANME-3 based on the
58 phylogenetic analysis of their 16S rRNA genes (3-5). In vitro incubations of ANME-1 and
59 ANME-2 dominated microbial communities from deep sea sediments in high-pressure reactors
60 showed indeed a strong positive relation of the activity of the microorganisms capable of the
61 AOM-SR process with the CH partial pressure, up to 12 MPa (6-10). In the ANME-2 dominated
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bioRxiv preprint first posted online Apr. 25, 2018; doi: http://dx.doi.org/10.1101/307082. The copyright holder for this preprint
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62 shallow marine sediment of Eckernförde Bay, the AOM-SR rate increased linearly with the CH
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63 pressure from 0.00 to 0.15 MPa when incubated in batch, determining an affinity constant (K )
m
64 for CH at least higher than 0.075 MPa (1.1 mM) (11). The K for CH of ANME-2 present in
4 m 4
65 Gulf of Cadiz sediment is about 37 mM (9), which is equivalent to 3 MPa CH partial pressure.
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66 This ANME-2 dominated sediment has its optimum pressure at the in situ pressure (S. Bhattarai,
67 Y. Zhang, and P.N.L Lens, submitted for publication). In contrast, the CH partial pressure
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68 influenced the growth of different subtypes of ANME-2 and SRB (i.e. at 10.1 MPa only the
69 ANME-2c and SEEP-SRB2 subtypes were enriched) from the Eckernförde Bay marine sediment
70 incubated for 240 days in a high-pressure membrane capsule bioreactor (12).
71 Studying the effect of pressure on ANME and SRB will thus help understand the growth of the
72 different ANME clades and their SRB partner. Finding ANME-SRB consortia that can grow fast
73 at ambient pressure is of great importance for the application of AOM-SR in the desulfurization
74 of industrial wastewater. Sulfate and other sulfur oxyanions, such as thiosulfate, sulfite or
75 dithionite, are contaminants discharged in fresh water by industrial activities such as food
76 processing, fermentation, coal mining, tannery and paper processing. Biological desulfurization
77 under anaerobic conditions is a well-known biological treatment, in which these sulfur oxyanions
78 are anaerobically reduced to sulfide (13-15). The produced sulfide precipitates with the metals,
79 thus enabling their recovery (16). In the process of groundwater, mining or inorganic wastewater
80 desulfurization, electron donor for sulfate reduction needs to be supplied externally. Electron
81 donors such as ethanol, hydrogen, methanol, acetate, lactate and propionate (13) are usually
82 supplied, but these increase the operational and investment costs (16). The use of easily
83 accessible and low-priced electron donors such as CH is therefore appealing for field-scale
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bioRxiv preprint first posted online Apr. 25, 2018; doi: http://dx.doi.org/10.1101/307082. The copyright holder for this preprint
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84 applications (17). Moreover, from a logistic, economical and safety view point, bioreactors
85 operating at ambient conditions are preferred over those operated at high pressures.
86 Coastal marine sediment from Lake Grevelingen (the Netherlands) hosts both ANME and SRB
87 (18). Among the ANME types, ANME-3 is predominant, which makes this sediment a beneficial
88 inoculum to investigate the effects of pressure on ANME-3. ANME-3 is often found in cold seep
89 areas and mud volcanoes with high CH partial pressures and relatively low temperatures (10,
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90 19, 20). Therefore, the shallow marine sediment from Lake Grevelingen was incubated at
91 different pressures (0.1, 0.45, 10, 20, and 40 MPa) to study the influence of pressure on the
92 AOM-SR activity, but also on the methanogenic activity and the potential formation of carbon,
93 e.g. acetate or methanethiol, (21) and sulfur, e.g. elemental sulfur or polysulfides (22),
94 intermediates. Moreover, phylogenetic analysis and visualization of microorganisms by
95 fluorescence in-situ hybridization (FISH) were used to study the activity and the shifts in cell
96 morphology, community composition and aggregation upon incubation of marine Lake
97 Grevelingen sediment at different pressures in batch for 77 days.
98 Results
99 Conversion rates of sulfur compounds
100 The highest sulfide production rates of the coastal marine Lake Grevelingen sediment was in the
101 incubations at the in situ pressure (0.45 MPa) and 10 MPa: 270 and 258 µmol g -1 d-1,
VSS
102 respectively (Figure 1a). The sulfide production rate at 40 MPa was 109 µmol g -1 d-1,
VSS
103 comparable to the rate with no CH in the headspace, 99 µmol g -1 d-1 (Figure 1a). Similarly,
4 VSS
104 high SR rates were recorded for the incubations at 0.45 MPa and 10 MPa (Figure 1b): 297 and
5
bioRxiv preprint first posted online Apr. 25, 2018; doi: http://dx.doi.org/10.1101/307082. The copyright holder for this preprint
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105 278 µmol g -1 d-1, respectively. In contrast, the SR rate at 0.1 MPa was 257 µmol g -1 d-1,
VSS VSS
106 while the sulfide production was only 157 µmol g -1 d-1 (Figures 1b and 1a).
VSS
107 Sulfide was produced in almost all the incubations (Figure 2), with the exception of the
108 incubation without biomass (Figure 2g). The sulfate concentration profiles varied with initial
109 incubation pressure: at 0.45 MPa sulfate was reduced to sulfide in a 1:1 ratio (Figure 2b),
110 whereas sulfate was not reduced anymore after 40 days of incubation at 40 MPa (Figure 2e). At
111 0.45 MPa, 0.98 mmol of sulfate was consumed and exactly 0.98 mmol of total dissolved sulfide
112 was produced, closing the sulfur balance. In the incubation at 0.1 MPa, 0.37 mmol of elemental
113 sulfur was produced along with 0.54 mmol of sulfide (Figure 2a). In the other incubations at
114 different pressures, hardly any elemental sulfur was formed (Figures 2c-2g). Instead, long chain
115 polysulfides were formed along the incubation depending on the pressure (Figure 3), but in small
116 amounts (≤ 2 µmol per vessel): 2 µmol S 2- per vessel was determined at 0.45 MPa CH pressure
6 4
117 (Figure 3b) and 1.2 and 1.4 µmol S 2- per vessel at 10 MPa and 20 MPa, respectively (Figures 3c
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118 and 3d).
119 AOM rates
120 The AOM rates were calculated from the DIC produced from 13CH , from which the K for CH
4 m 4
121 of the marine Lake Grevelingen sediment was determined to be around 1.7 mM. The DIC
122 production rates followed a similar trend as the sulfide production rates: the highest rate was
123 found at 0.45 MPa and the lowest rate at 40 MPa: 320 and 38 µmol g -1 d-1, respectively
VSS
124 (Figure 1c). In the incubation at 0.45 MPa (in situ pressure), the total DIC produced from CH
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125 was similar to the sulfide produced: ~0.9 mmol per vessel (Supporting information, Figure S1b
126 and Figure 2b). However, sulfide was produced from the start for all the incubations, while the
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127 total DIC from CH was mainly produced only after 40 days of incubation (Figure 2 and
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128 Supporting information, Figure S1). Similar trends were found for all the other incubations at
129 different pressure, except for the vessel without CH , where only sulfide production (0.3 mmol/
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130 vessel) was recorded.
131 Methanogenesis
132 CH was produced in all the incubations, with the exception of the batches without biomass
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133 (Supporting information, Figure S2). The highest amount of CH formed was recorded in the
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134 vessel at 0.1 MPa (Supporting Information, Figure S2b). The highest methanogenic rate was
135 determined in the control vessel without CH (N in the headspace) and at 0.1 MPa: 44 and 31
4 2
136 µmol g -1 d-1, respectively, while it was below 5 µmol g -1 d-1 in all the other batch
VSS VSS
137 incubations (Supporting information, Figure S2a). Assuming that all the total 12C-DIC was
138 produced from the oxidation of other carbon sources than CH , its production rate was low in
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139 almost all the incubations: lower than 3 µmol g -1 d-1, except for the incubation without CH
VSS 4
140 (64 µmol g -1 d-1, data not shown).
VSS
141 Community shifts as a function of incubation pressure: total cell numbers
142 The total bacterial and archaeal cellular numbers were accessed from Q-PCR data performed on
143 samples after 77 days of incubation (Figure 4). The highest increase in active cells, from 6 to
144 8×107 cells ml-1, was found in the incubation at the in situ pressure of 0.45 MPa. In the
145 incubation at 40 MPa, the amount of active total bacteria and archaea cells decreased from 6.5 to
146 5.8×107 cells ml-1 (Figure 4a). Based on Q-PCR results, archaea grew in all the incubations,
147 while copy numbers of bacteria decreased in the incubation without CH and at 20 MPa. The
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148 total number of archaea increased the most in the incubation at 20 MPa (Figure 4b).
7
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149 Based on the 16s rRNA gene analysis, both archaeal and bacterial communities had shifted along
150 the 77 days incubation (Figure 5). The most abundant OTUs (operational taxonomic unit) with
151 archaeal signature are shown in Figure 5a. Specifically, the abundance of ANME-3 among all the
152 archaea increased the most in incubations at 0.45 and 0.1 MPa, i.e. respectively three and two
153 times more than at the start of the incubation (Figure 5a). ANME-2a/b reads increased the most
154 at 20 MPa: 27 times more than at the start of the incubation (Figure 5a). Sequences of
155 methanogens, specifically belonging to the Methanomicrobiales, were more abundant after the
156 incubation at 0.1 MPa, rather than at higher partial pressures, where Thaumarchaeota and
157 Woesearchaeaota were more abundant in incubations at 10, 20 and even 40 MPa (Figure 5a).
158 The bacterial communities were very diverse in all the incubations, the ones with the highest
159 percentage are shown in Figure 5b. The absolute abundance of the Desulfobulbaceae (DBB) as
160 calculated from their 16s rRNA gene according to Q-PCR and Miseq results increased or
161 remained similar at the lower pressure incubations (0.1 and 0.45 MPa), but the percentage of
162 DBB in the total bacterial community decreased at more elevated pressures (10, 20 and 40 MPa).
163 Differently, the absolute abundance of Desulfobacteraceae, as DSS, increased in all the
164 incubations at different pressures, with the highest percentage of reads retrieved in the incubation
165 at 20 MPa (Figure 5b). The percentage of OTUs as assigned to Desulfovibrio, Desulfuromonas,
166 Halomonas and Sulfurovum genes decreased in all the batch incubations (Figure 5b).
167 Community shifts as a function of incubation pressure: FISH analysis
168 ANME-3 and DBB were visualized in all the batch incubations (Figures 6a-6c). At the start of
169 the incubation (t = 0 days), ANME-3 cells were preferentially visualized in aggregates with other
170 cells (data not shown). FISH images after 77 days of incubation showed variations in the
8
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171 aggregate morphology depending on the incubation pressure (Figure 6). At 0.1 and 0.45 MPa,
172 ANME-3 was more abundant than at the start, while the DBB cells were not found concomitant
173 to the ANME-3 cells (Figure 6a) and, even if present, the ANME-3 cells outnumbered the DBB
174 cells (Figure 6b). In the 10 MPa incubation, ANME-3 was visualized more scattered and not in
175 clusters as at the lower pressures, whereas DBB cells were even more rarely pictured (Data not
176 shown). At 20 MPa, ANME-3 and DBB cells were rare, however, the stained cells formed tight
177 ANME-3/DBB aggregates (Figure 6c). At 40 MPa, ANME-3 and DBB were the least abundant
178 and scattered and no aggregates could be found (data not shown).
179 Differently than ANME-3, more ANME-2 cells were visualized in the 77 days incubations at
180 higher (10, 20 and 40 MPa) than at lower (0.1 and 0.45 MPa) incubation pressures (Figures 6d-
181 6f). DSS, the most common SRB bacterial partner of ANME-2, were most abundant at 0.1 MPa.
182 At lower pressure they were mainly visualized together with ANME-2 (Figure 6d). At 20 MPa
183 only clusters of ANME-2 cells were visualized without DSS (Figure 6e).
184 Discussion
185 Pressure effect on AOM in marine Lake Grevelingen sediment
186 This study showed that AOM and SR processes in Lake Grevelingen sediment depend on the
187 CH total pressure. According to Eq. 1, the reaction rate is expected to be stimulated by the
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188 elevated CH partial pressure when the other parameters remain the same (Table 1). This
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189 expectation has been commonly accepted and has been shown in communities dominated by
190 ANME-1, i.e. hydrocarbon seep in the Monterey canyon sediment (23) and ANME-2, i.e.
191 Eckernförde Bay (24, 25) and Gulf of Cadiz sediment (S. Bhattarai, Y. Zhang, and P.N.L. Lens,
192 submitted for publication). In contrast, the AOM-SR process by the ANME-3 dominated marine
9
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193 Lake Grevelingen sediment has an optimal pressure at 0.45 MPa among all tested conditions
194 (Figure 1). This is in accordance with their natural habitat, i.e. the in situ pressure of marine Lake
195 Grevelingen is 0.45 MPa, but contrasts the theoretical thermodynamic calculation (Table 1), that
196 predicts a higher CH solubility and thus a higher activity based on reported Km values, i.e. 37
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197 mM as calculated from an ANME-2 predominant enrichment originated from the Gulf of Cadiz
198 (9). The calculated K value on CH based on our ANME-3 dominated inoculum is much lower
m 4
199 than previously reported: around 1.7 mM. Thus, the ANME cells from Grevelingen marine
200 sediment have a higher affinity for CH than the ANME-2 from the Gulf of Cadiz, explaining
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201 why a higher pressure, i.e. higher dissolved CH concentration, did not result in a higher AOM
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202 rate (Figu 1c). As a matter of fact, higher pressure resulted in a lower number of ANME-3 cells
203 and ANME-3 proliferated only at lower pressures incubation (0.1 and 0.45 MPa). This indicates
204 ANME-3 cells of marine Lake Grevelingen are non-piezophilic, which are easily damaged upon
205 pressure elevation and require extra energy to cope with the damage (26). ANME-3 are found in
206 cold seep areas and mud volcanoes with high CH partial pressures (~10 MPa) and relatively low
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207 temperatures (10, 19, 20). However, Lake Grevelingen is a shallow sediment with high
208 abundance of ANME-3 (18) and likely contains different subtypes than the ones found in deep
209 sea sediments, which cannot cope with a high pressure.
210 Pressure effect on ANME types
211 The ANME-3 type is usually visualized in association with DBB as sulfate reducing partner (10,
212 19). FISH analysis showed that the DBB cells were not as high in number as the ANME-3 cells
213 in any of the incubations (Figures 6a-6c), but they increased the most at the 0.1 MPa incubation
214 (data not shown). In a recent study describing the microbial ecology of Lake Grevelingen
215 sediment (incubation pressure = 0.1 MPa), the two species (ANME-3 and DBB) could not be
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