AEM Accepts, published online ahead of print on 5 September 2014 Appl. Environ. Microbiol. doi:10.1128/AEM.02146-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Stratified Microbial Structure and Activity in Sulfide- and
2
Methane- Producing Anaerobic Sewer Biofilms
3 4
Jing Sun1, Shihu Hu1, Keshab Raj Sharma1,2, Bing-Jie Ni1, Zhiguo Yuan1,2*
5 6
1
7
Queensland, Australia;
8
2
Advanced Water Management Center, The University of Queensland, St. Lucia, 4072,
CRC for Water Sensitive Cities, PO Box 8000, Clayton, Victoria, 3800, Australia
9 10
*Corresponding author
11
Email: [email protected]
12
Phone: +61 (0)7 3365 4374
13
Fax: +61 (0)7 3365 4726
14 15 16
ABSTRACT
17
Simultaneous production of sulfide and methane by anaerobic sewer biofilms has
18
recently been observed, suggesting that sulfate-reducing bacteria (SRB) and
19
methanogenic archaea (MA), microorganisms known to compete for the same substrates,
20
can coexist in this environment. This study investigated the community structures and
21
activities of SRB and MA in anaerobic sewer biofilms (average thickness of 800 μm)
22
using a combination of microelectrode measurements, molecular techniques and
23
mathematical modelling. It was seen that sulfide was mainly produced in the outer layer
24
of the biofilm, between 0 - 300 μm, which is in good agreement with the distribution of
25
SRB population as revealed by cryosection - fluorescence in situ hybridization (FISH). 1
26
SRB have a higher relative abundance of 20% on the surface layer, which decreased
27
gradually to below 3% at a depth of 400 μm. In contrast, MA mainly inhabited the inner
28
layer of the biofilm. Their relative abundances increased from 10% to 75% at depths of
29
200 μm and 700 μm, respectively, from the biofilm surface layer. High throughput
30
pyrosequencing of 16S rRNA amplicons showed that SRB in the biofilm were mainly
31
affiliated
32
Desulfatiferula and Desulforegula, while about 90% of the MA population belonged to
33
the genus Methanosaeta. The spatial organization of SRB and MA revealed by
34
pryosequencing were consistent with the FISH results. A biofilm model was constructed
35
to simulate the SRB and MA distributions in the anaerobic sewer biofilm. The good fit
36
between model predictions and the experimental data indicates that the coexistence and
37
spatial structure of SRB and MA in the biofilm resulted from the microbial types, their
38
metabolic transformations and interactions with substrates.
with
five
genera:
Desulfobulbus,
Desulfomicrobium,
Desulfovibrio,
39 40
INTRODUCTION
41
Sewer biofilms comprise complex multi-species microflora with a typical thickness of
42
only about one millimeter (1). According to the electron donors and electron acceptors
43
present in the wastewater, different carbon transformation processes can occur in close
44
proximity in the sewer biofilms. Domestic wastewater normally contains a significant
45
concentration of sulfate (ca. 100–1000 μM) but negligible nitrite and nitrate (2, 3).
46
Therefore, under anaerobic conditions (normally occurs in pressure sewers fully filled
47
with wastewater), sulfate reduction carried out by the sulfate-reducing bacteria (SRB)
48
could be an important terminal electron accepting process in the sewer biofilms. The
49
sulfate reduction activity in anaerobic sewers is important as the production of sulfide
50
produced can be transferred to the gas phase of partially-filled gravity sewers and cause 2
51
extensive corrosion of concrete sewer pipes (4, 5). Also, the emission of sulfide from
52
sewers can cause odor problems to the surrounding area and pose health risks to sewer
53
workers (6, 7). Apart from sulfate reduction, methanogenesis by the respiration of
54
methanogenic archaea (MA) could also be a key terminal process in anaerobic sewer
55
biofilms (8, 9). Guisasola and colleagues found that methanogenesis accounted for more
56
than 70% of the chemical oxygen demand (COD) loss in laboratory anaerobic sewer
57
biofilm reactors (9). A recent report suggests that methane emissions from sewers
58
contribute significantly to the total greenhouse gas footprint of wastewater systems (10).
59
Under anaerobic conditions, both sulfate reduction and methanogenesis can
60
potentially occur in the same system while competing for the same electron donors,
61
primarily hydrogen and acetate. In the presence of adequate sulfate concentrations, SRB
62
will typically outcompete MA due to kinetic and thermodynamic advantages (11-13).
63
However, the coexistence of SRB and MA has been observed in anaerobic sewer
64
biofilms in the presence of sulfate. Guisasola et al. (9) hypothesized the coexistence of
65
SRB and MA in sewer biofilms was due to the penetration limitation of sulfate into the
66
biofilms, resulting in a stratified biofilm structure, with SRB being predominant in the
67
outer zone, nearer to the wastewater, while MA inhabit the inner zone, nearer the sewer
68
pipe. However, to date this hypothesis has not been verified. A few studies have
69
investigated the veritical distribution of SRB in oxic-anoxic sewer biofilms (biofilms
70
attached on gravity sewer pipe with the presence of oxygen or nitrate in wastewater), but
71
studies on the SRB distribution in the anerobic sewer bioflims is scarce (14). In addition,
72
the distubtion of MA in sewer biofilms and their interaction with SRB have not been
73
explored yet. Similarly, the phylogenitic diversity of SRB and MA in the anaerobic
74
sewer biofilms is rarelly reported. These fundamental information could provide a better
75
understanding of the sulfate reduction and methanogenesis processes in sewer systems,
3
76
which would be useful for sewer management. Therefore, the aims of this study are to
77
investigate the community structures of both SRB and MA and to determine their spatial
78
arrangement in anaerobic sewer biofilms.
79
Both experimental investigations and modeling analyses were conducted to achieve
80
the aims of this study. The experiments were carried out in an annular biofilm reactor
81
mimicking anaerobic sewer conditions, which was fed with real domestic wastewater.
82
Firstly, microelectrodes were applied to determine the spatial distribution of in situ
83
sulfide production activity within the biofilms. Although it would have been ideal to
84
determine the distribution of methane production activity using the same method, this
85
was difficult to perform due to the lack of suitable microelectrodes (15). Secondly, the
86
spatial distributions of the SRB and MA in the biofilms and their abundance at different
87
depths were determined by fluorescence in situ hybridization (FISH) after
88
cryosectioning the biofilm samples. This method has been used frequently to determine
89
the spatial distributions of microbial communities in biofilms or granules. However,
90
phylogenetic information is hardly revealed due to the limitation of oligonucleotide
91
probes used in FISH (16). Therefore, 16S rRNA gene amplicon pyrosequencing was
92
applied to further investigate the phylogenetic diversity. In previous studies of sewer
93
biofilms, the phylogenetic analysis is performed on the entire biofilm, and information
94
on the different genera at different biofilm depths is rarely reported (2, 14). In this study,
95
we determined the phylogenetic diversity in different layers of the sewer biofilms by
96
innovatively using pyrosequencing combined with cryosectioning. To our knowledge, to
97
date, this method has not been applied in any other studies related to biofilms and
98
granules. Finally, a mathematical model focusing on the interaction between SRB and
99
MA in the sewer biofilm was developed to evaluate and interpret the experimental
100
results.
4
101
MATERIALS AND METHODS
102
Reactor configuration, operation and monitoring. An annular biofilm reactor made
103
of acrylonitrile butadiene styrene (ABS), one of the typical materials used for sewer
104
pipes, was set up to mimic an anaerobic sewer pipe section (Fig. 1). The reactor
105
consisted of an inner cylinder (of height 295 mm, and diameter of 130 mm) enclosed in
106
an outer cylinder (of height 345 mm and inner diameter of 160 mm). Wastewater was
107
filled in the gap between the two cylinders, with a volume of 3 L. Biofilms were grown
108
on the walls of both cylinders in contact with the wastewater, resulting in a biofilm area
109
to reactor volume (A/V) ratio of 119 m-1. Mixing was established by the rotation of the
110
inner cylinder driven by a motor at a speed of 200 rpm. The mixing is expected to create
111
a uniform shear stress on the reactor walls so that biofilms grow relatively evenly on the
112
wall. The average shear stress provided by the mixing was 2.11 N/m2, which is typical in
113
sewer systems (17). Eight removable ABS slides of width and length at 5 mm by 200
114
mm were mounted in recessed slots on the inside of the outer cylinder. The slides were
115
removable via ports on the top of the reactor for biofilm sampling. The reservoir on the
116
top of the reactor was used to ensure the reactor was full of wastewater during sampling.
117
The reactor was operated in a temperature-controlled room (20 ± 2 °C).Domestic
118
sewage, collected on a weekly basis from a local wet well (Brisbane, Queensland), was
119
used as the feed for the reactor. The sewage compositions varied to a certain extent in
120
terms of sulfate, Volatile fatty acid (VFA), and COD concentrations. The sewage
121
typically contained sulfate at concentrations of 10-25 mgS/L, sulfide at < 3 mgS/L,
122
Soluble COD (sCOD) at 200-300 mg/L, 50-120 mgCOD/L of VFAs and approximately
123
50 mgN/L of ammonium. Negligible amounts of sulfite, thiosulfate (
1
Stratified Microbial Structure and Activity in Sulfide- and
2
Methane- Producing Anaerobic Sewer Biofilms
3 4
Jing Sun1, Shihu Hu1, Keshab Raj Sharma1,2, Bing-Jie Ni1, Zhiguo Yuan1,2*
5 6
1
7
Queensland, Australia;
8
2
Advanced Water Management Center, The University of Queensland, St. Lucia, 4072,
CRC for Water Sensitive Cities, PO Box 8000, Clayton, Victoria, 3800, Australia
9 10
*Corresponding author
11
Email: [email protected]
12
Phone: +61 (0)7 3365 4374
13
Fax: +61 (0)7 3365 4726
14 15 16
ABSTRACT
17
Simultaneous production of sulfide and methane by anaerobic sewer biofilms has
18
recently been observed, suggesting that sulfate-reducing bacteria (SRB) and
19
methanogenic archaea (MA), microorganisms known to compete for the same substrates,
20
can coexist in this environment. This study investigated the community structures and
21
activities of SRB and MA in anaerobic sewer biofilms (average thickness of 800 μm)
22
using a combination of microelectrode measurements, molecular techniques and
23
mathematical modelling. It was seen that sulfide was mainly produced in the outer layer
24
of the biofilm, between 0 - 300 μm, which is in good agreement with the distribution of
25
SRB population as revealed by cryosection - fluorescence in situ hybridization (FISH). 1
26
SRB have a higher relative abundance of 20% on the surface layer, which decreased
27
gradually to below 3% at a depth of 400 μm. In contrast, MA mainly inhabited the inner
28
layer of the biofilm. Their relative abundances increased from 10% to 75% at depths of
29
200 μm and 700 μm, respectively, from the biofilm surface layer. High throughput
30
pyrosequencing of 16S rRNA amplicons showed that SRB in the biofilm were mainly
31
affiliated
32
Desulfatiferula and Desulforegula, while about 90% of the MA population belonged to
33
the genus Methanosaeta. The spatial organization of SRB and MA revealed by
34
pryosequencing were consistent with the FISH results. A biofilm model was constructed
35
to simulate the SRB and MA distributions in the anaerobic sewer biofilm. The good fit
36
between model predictions and the experimental data indicates that the coexistence and
37
spatial structure of SRB and MA in the biofilm resulted from the microbial types, their
38
metabolic transformations and interactions with substrates.
with
five
genera:
Desulfobulbus,
Desulfomicrobium,
Desulfovibrio,
39 40
INTRODUCTION
41
Sewer biofilms comprise complex multi-species microflora with a typical thickness of
42
only about one millimeter (1). According to the electron donors and electron acceptors
43
present in the wastewater, different carbon transformation processes can occur in close
44
proximity in the sewer biofilms. Domestic wastewater normally contains a significant
45
concentration of sulfate (ca. 100–1000 μM) but negligible nitrite and nitrate (2, 3).
46
Therefore, under anaerobic conditions (normally occurs in pressure sewers fully filled
47
with wastewater), sulfate reduction carried out by the sulfate-reducing bacteria (SRB)
48
could be an important terminal electron accepting process in the sewer biofilms. The
49
sulfate reduction activity in anaerobic sewers is important as the production of sulfide
50
produced can be transferred to the gas phase of partially-filled gravity sewers and cause 2
51
extensive corrosion of concrete sewer pipes (4, 5). Also, the emission of sulfide from
52
sewers can cause odor problems to the surrounding area and pose health risks to sewer
53
workers (6, 7). Apart from sulfate reduction, methanogenesis by the respiration of
54
methanogenic archaea (MA) could also be a key terminal process in anaerobic sewer
55
biofilms (8, 9). Guisasola and colleagues found that methanogenesis accounted for more
56
than 70% of the chemical oxygen demand (COD) loss in laboratory anaerobic sewer
57
biofilm reactors (9). A recent report suggests that methane emissions from sewers
58
contribute significantly to the total greenhouse gas footprint of wastewater systems (10).
59
Under anaerobic conditions, both sulfate reduction and methanogenesis can
60
potentially occur in the same system while competing for the same electron donors,
61
primarily hydrogen and acetate. In the presence of adequate sulfate concentrations, SRB
62
will typically outcompete MA due to kinetic and thermodynamic advantages (11-13).
63
However, the coexistence of SRB and MA has been observed in anaerobic sewer
64
biofilms in the presence of sulfate. Guisasola et al. (9) hypothesized the coexistence of
65
SRB and MA in sewer biofilms was due to the penetration limitation of sulfate into the
66
biofilms, resulting in a stratified biofilm structure, with SRB being predominant in the
67
outer zone, nearer to the wastewater, while MA inhabit the inner zone, nearer the sewer
68
pipe. However, to date this hypothesis has not been verified. A few studies have
69
investigated the veritical distribution of SRB in oxic-anoxic sewer biofilms (biofilms
70
attached on gravity sewer pipe with the presence of oxygen or nitrate in wastewater), but
71
studies on the SRB distribution in the anerobic sewer bioflims is scarce (14). In addition,
72
the distubtion of MA in sewer biofilms and their interaction with SRB have not been
73
explored yet. Similarly, the phylogenitic diversity of SRB and MA in the anaerobic
74
sewer biofilms is rarelly reported. These fundamental information could provide a better
75
understanding of the sulfate reduction and methanogenesis processes in sewer systems,
3
76
which would be useful for sewer management. Therefore, the aims of this study are to
77
investigate the community structures of both SRB and MA and to determine their spatial
78
arrangement in anaerobic sewer biofilms.
79
Both experimental investigations and modeling analyses were conducted to achieve
80
the aims of this study. The experiments were carried out in an annular biofilm reactor
81
mimicking anaerobic sewer conditions, which was fed with real domestic wastewater.
82
Firstly, microelectrodes were applied to determine the spatial distribution of in situ
83
sulfide production activity within the biofilms. Although it would have been ideal to
84
determine the distribution of methane production activity using the same method, this
85
was difficult to perform due to the lack of suitable microelectrodes (15). Secondly, the
86
spatial distributions of the SRB and MA in the biofilms and their abundance at different
87
depths were determined by fluorescence in situ hybridization (FISH) after
88
cryosectioning the biofilm samples. This method has been used frequently to determine
89
the spatial distributions of microbial communities in biofilms or granules. However,
90
phylogenetic information is hardly revealed due to the limitation of oligonucleotide
91
probes used in FISH (16). Therefore, 16S rRNA gene amplicon pyrosequencing was
92
applied to further investigate the phylogenetic diversity. In previous studies of sewer
93
biofilms, the phylogenetic analysis is performed on the entire biofilm, and information
94
on the different genera at different biofilm depths is rarely reported (2, 14). In this study,
95
we determined the phylogenetic diversity in different layers of the sewer biofilms by
96
innovatively using pyrosequencing combined with cryosectioning. To our knowledge, to
97
date, this method has not been applied in any other studies related to biofilms and
98
granules. Finally, a mathematical model focusing on the interaction between SRB and
99
MA in the sewer biofilm was developed to evaluate and interpret the experimental
100
results.
4
101
MATERIALS AND METHODS
102
Reactor configuration, operation and monitoring. An annular biofilm reactor made
103
of acrylonitrile butadiene styrene (ABS), one of the typical materials used for sewer
104
pipes, was set up to mimic an anaerobic sewer pipe section (Fig. 1). The reactor
105
consisted of an inner cylinder (of height 295 mm, and diameter of 130 mm) enclosed in
106
an outer cylinder (of height 345 mm and inner diameter of 160 mm). Wastewater was
107
filled in the gap between the two cylinders, with a volume of 3 L. Biofilms were grown
108
on the walls of both cylinders in contact with the wastewater, resulting in a biofilm area
109
to reactor volume (A/V) ratio of 119 m-1. Mixing was established by the rotation of the
110
inner cylinder driven by a motor at a speed of 200 rpm. The mixing is expected to create
111
a uniform shear stress on the reactor walls so that biofilms grow relatively evenly on the
112
wall. The average shear stress provided by the mixing was 2.11 N/m2, which is typical in
113
sewer systems (17). Eight removable ABS slides of width and length at 5 mm by 200
114
mm were mounted in recessed slots on the inside of the outer cylinder. The slides were
115
removable via ports on the top of the reactor for biofilm sampling. The reservoir on the
116
top of the reactor was used to ensure the reactor was full of wastewater during sampling.
117
The reactor was operated in a temperature-controlled room (20 ± 2 °C).Domestic
118
sewage, collected on a weekly basis from a local wet well (Brisbane, Queensland), was
119
used as the feed for the reactor. The sewage compositions varied to a certain extent in
120
terms of sulfate, Volatile fatty acid (VFA), and COD concentrations. The sewage
121
typically contained sulfate at concentrations of 10-25 mgS/L, sulfide at < 3 mgS/L,
122
Soluble COD (sCOD) at 200-300 mg/L, 50-120 mgCOD/L of VFAs and approximately
123
50 mgN/L of ammonium. Negligible amounts of sulfite, thiosulfate (
