RESEARCH ARTICLE
Differences between aerobic and anaerobic degradation of
microphytobenthic biofilm-derived organic matter within
intertidal sediments
Boyd A. McKew, Alex J. Dumbrell, Joe D. Taylor, Terry J. McGenity & Graham J.C. Underwood
School of Biological Sciences, University of Essex, Colchester, UK
Correspondence: Boyd A. McKew, School
of Biological Sciences, University of Essex,
Colchester CO4 3SQ, UK. Tel.: +44 (0) 1206
873345; fax: +44 (0) 1206 872592;
e-mail: [email protected]
Received 9 November 2012; revised 14
January 2013; accepted 20 January 2013.
Final version published online 19 February
2013.
DOI: 10.1111/1574-6941.12077
MICROBIOLOGY ECOLOGY
Editor: Tillmann Lueders
Keywords
DOC; EPS; microbial community; mudflat;
exopolymers; microphytobenthos.
Abstract
Within intertidal sediments, much of the dissolved organic carbon (DOC)
consists of carbohydrate-rich extracellular polymeric substances (EPS) produced
by microphytobenthic biofilms. EPS are an important source of carbon and
energy for aerobic and anaerobic microorganisms owing to burial of microphytobenthos and downward transport of their exudates. We established slurries of
estuarine biofilms to determine the fate of organic carbon and EPS fractions,
differing in size and complexity, under oxic and anoxic conditions. DOC and
hot-water-extracted organic matter (predominately diatom chrysolaminarin)
were utilised rapidly at similar rates in both conditions. Concentrations of
insoluble, high-molecular-weight EPS were unchanged in oxic microcosms, but
were significantly degraded under anoxic conditions (39% degradation by day
25). Methanogenesis and sulphate reduction were major anaerobic processes in
the anoxic slurries, and 16S rRNA gene pyrosequencing revealed that Desulfobacteraceae (relative sequence abundance increased from 1.9% to 12.2%) and
Desulfobulbaceae (increased from 1.5% to 4.3%) were the main sulphate reducers, whilst Clostridia and Bacteroidetes were likely responsible for anaerobic
hydrolysis and fermentation of EPS. We conclude that a diverse consortium of
anaerobic microorganisms (including coexisting sulphate reducers and methanogens) degrade both labile and refractory microphytobenthic-derived carbon
and that anaerobic degradation may be the primary fate of more structurally
complex components of microphytobenthic EPS.
Introduction
Substantial quantities of particulate organic carbon and
DOC, particularly carbohydrates (Burdige et al., 2000),
are transformed by microorganisms in intertidal sediments, significantly influencing carbon and nitrogen budgets of coastal environments. The most labile carbon
inputs originate in photosynthetic microbial biofilms (microphytobenthos) that occur extensively in such habitats
and generate up to half of the total autochthonous carbon production (Heip et al., 1995; Underwood & Kromkamp, 1999). Microphytobenthos excrete large quantities
of extracellular polymeric substances (EPS), predominantly polysaccharides; and 14C- and 13C-tracer studies
have shown rapid transfer (within 3 h) of photo-assimilated carbon into the extracellular dissolved pool and
FEMS Microbiol Ecol 84 (2013) 495–509
then into heterotrophic microorganisms (Smith & Underwood, 1998; Middleburg et al., 2000; Bellinger et al.,
2009), meiofauna and other deposit feeders (Middelburg
et al., 2002; Cook et al., 2007; Oakes et al., 2010).
Lower-molecular-weight (LMW) algal-derived carbohydrates are rapidly degraded under oxic conditions, followed by utilisation of larger colloidal, and then more
insoluble, EPS constituents (Goto et al., 2001; Giroldo
et al., 2003; Haynes et al., 2007; Hofmann et al., 2009).
Polysaccharides comprise a significant portion of sediment organic carbon (Arnosti, 2000), and the molecular
size spectrum and complexity of EPS requires bacteria
to produce extracellular enzymes that hydrolyse EPS
before uptake (Simon et al., 2002; Arnosti et al., 2009;
Arnosti, 2011). Therefore, correlations occur between
enzyme activity rates (e.g. b-glucosidase), decreases in
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B.A. McKew et al.
496
concentrations of relatively insoluble EPS, increases in
colloidal carbohydrate concentrations (Haynes et al.,
2007; Bhaskar & Bhosle, 2008; Hofmann et al., 2009;
McKew et al., 2011) and shifts in the bacterial community (Hanlon et al., 2006).
Bacteria in the Bacteroidetes are known degraders of
complex biopolymers and, together with Actinobacteria,
Alpha-, Beta- and Gammaproteobacteria, have been identified as key bacteria involved in dissolved organic matter
and EPS turnover in estuarine water (Elifantz et al., 2005)
and Gammaproteobacteria (especially Acinetobacter sp.) in
sediments (Haynes et al., 2007; Webster et al., 2010). A
13
C-stable isotope probing study in subtidal sandy sediments found that Gammaproteobacteria, Bacteriodetes and
some Firmicutes could utilise planktonic algal dissolved
organic matter; 13C-enriched sequences for sulphatereducing Desulfobacteraceae (Deltaproteobacteria) were
also detected (Chipman et al., 2010).
Microphytobenthic photosynthesis, coupled with high
rates of bacterial respiration and chemical oxidation,
results in steep and temporally dynamic (over diel and
tidal cycles) gradients in oxygen concentrations in intertidal sediments, with anaerobic conditions almost reaching the sediment surface during darkness (Underwood &
Kromkamp, 1999; B€
ottcher et al., 2000). Microphytobenthic biofilms are frequently buried due to sediment resuspension and deposition, or biotubation (Underwood &
Paterson, 1993; Christie et al., 1999; Lawler et al., 2001;
Mitchell et al., 2003; Chipman et al., 2010; Uncles &
Stephens, 2010). Such buried organic matter will be
exposed to anoxic conditions. However, the relative
importance of different bacterial groups in the degradation of EPS in anaerobic conditions remains unknown.
The structural complexity of algal EPS raises the possibility that numerous bacterial groups are involved in its
complete degradation (Bellinger et al., 2009). There is evidence of partitioning between rapidly turned-over EPS
carbohydrate components and more structurally complex
EPS-carbon that may be subjected to longer-term retention and diagenesis in sediments (Cook et al., 2007; Evrard et al., 2008). Such observations support the
conceptual model (Bellinger et al., 2009) that structurally
complex and refractory EPS may become available to
anaerobic bacteria located deeper in the sediment, due to
transportation via diffusion, porewater pumping by
infauna or burial (Aller & Aller, 1992; Chipman et al.,
2010; Jorgensen & Parkes, 2010), whereas labile EPS
should be rapidly utilised in the upper aerobic zones of
microphytobenthic biofilms.
Although measured organic carbon mineralisation rates
are often lower in anoxic compared with oxic sediments,
this is probably due to the refractory nature of the organic
carbon content generally available in deeper anaerobic
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zones (Kristensen et al., 1995; Amtoft Neubauer et al.,
2004; Lomstein et al., 2006), rather than a underlying characteristic of anaerobic degradation. A key aim of our study
was to determine whether rates of loss of organic carbon in
different biofilm EPS fractions would be slower under
anoxic conditions. Anaerobic sediment bacteria can rapidly
hydrolyse a wide range of oligosaccharides and polysaccharides (Arnosti & Repeta, 1994; Arnosti et al., 1994), so
should be able to utilise freshly buried biofilm organic
matter. We hypothesised that anaerobic bacterial communities would degrade fresh biofilm EPS as rapidly as aerobic bacteria. We therefore compared changes in
concentrations of different fractions of DOC and carbohydrates, and extracellular enzyme activity, and changes in
the microbial assemblages in fresh microphytobenthic
biofilm-rich sediments within both oxic and anoxic slurries.
Materials and methods
Experimental overview
Surface sediment (top 2–3 mm containing a diatomdominated microphytobenthic biofilm) was collected at
low tide from Pyefleet mudflat, Colne Estuary, East Mersea (51° 48′N, 0° 22′E) in August 2009. The sediment was
loosened with sea water and sieved (0.5 mm) to remove
macrofauna before gently homogenising. Fifteen grams of
wet sediment (equivalent to 4.59 g dry sediment) was
added to each of 70 glass serum bottles (125 mL) with
50 mL of filtered (0.2 lm) seawater from the same site
(salinity 35). Oxic slurries (35) were capped with parafilm, and the same number of anoxic slurries were sealed
with gas-tight septa and flushed with oxygen-free N2.
Concentrations of sediment carbohydrates (total, dissolved, hot water (HW) and hot bicarbonate (HB)
extracted – see below) and DOC in the original sediment
(Table 1) were within the range of values typical for
mudflats in the Colne Estuary (Hanlon et al., 2006;
Haynes et al., 2007; Bellinger et al., 2009; McKew et al.,
2011). Measurements of sediment chl a at this site in
August range from 39 to 79 lg (g dry wt. sediment)1
(mean 59 2; n = 18). Initial concentrations in the slurries (204 and 3.1 lg C mL1 for total and dissolved carbohydrate, respectively) were similar to previous slurry
experiments (Haynes et al., 2007; Hofmann et al., 2009).
The slurries were incubated in darkness (to prevent
primary production) at 100 r.p.m. and 20 °C. Five independent replicates for each treatment were sampled at 0,
1, 3, 7, 10, 15 and 25 days. The slurries were analysed for
sulphate, sulphide, acetate, ammonium, DOC, carbohydrate fractions, methane, aminopeptidase and b-glucosidase activity (as a proxy for bacterial activity). The
significance of any differences in these parameters was
FEMS Microbiol Ecol 84 (2013) 495–509
FEMS Microbiol Ecol 84 (2013) 495–509
Total
carbohydrate
(TCHO)
TCHO:TOC
Total organic
carbon (TOC)
Hot-bicarbonateextracted organic
carbon (HB-OC)
Hot-bicarbonateextracted
carbohydrate
(HB-CHO)
HB-CHO:HB-OC
HW-CHO:HW-OC
Hot-waterextracted organic
carbon (HW-OC)
Hot-waterextracted
carbohydrate
(HW-CHO)
DCHO:DOC
All organic carbon, POC and DOC
fractions, including amino acids,
lipids
All dissolved and particulate carbon,
structural polysaccharides, detritus
Insoluble heteropolymers, gels rich in
deoxy sugars, uronic acids
Insoluble and HMW material, EPS
stalks, gels, cell contents
Heat-solublised extra- and
intracelluar constituents
(e.g. carbon, protein)
Chrysolaminarin (glucose), LMW and
EPS carbohydrate, pentose/hexose
rich
Carbon (40%), amino acids (12%),
other LMW organic carbon, for
example, acetate
25% colloidal EPS,75%
nonpolymeric carbo., including
LMW (glucose-rich)
Dissolved organic
carbon (DOC)
Dissolved
carbohydrate (DCHO)
Major constituents (where known)
Fraction
De Brouwer et al. (2003),
Hanlon et al. (2006)
Lomstein et al. (1998)
Abdullahi et al. (2006),
Bellinger et al., 2005; De Brouwer et al.
(2003), Hanlon et al. (2006)
Hanlon et al. (2006)
Abdullahi et al. (2006),
Bellinger et al. (2005),
Chiovitti et al. (2004),
Hanlon et al. (2006),
Staats et al. (1999)
Chiovitti et al. (2004), Staats
et al. (1999)
Bellinger et al. (2005), Burdige &
Gardner (1998), Lomstein
et al. (1998), Repeta et al. (2002)
Bellinger et al. (2005), De Brouwer
et al. (2003), Hanlon et al. (2006),
Staats et al. (1999)
Reference
1899 172
0.24
0.18
2894 139
611 78
2529 80
1893 93
0.21
425 45
2001 95
0.45
269 11
328 14
0.41
601 17
0.36
73 1
202 18
Anaerobic
799 14
488 121
2708 59
0.64
729 36
1141 66
0.16
19 1
44 15
0.08
117 18
Aerobic
Day10
565 31
Day 0
Initial
lg C (g dry wt. sediment)1
0.11
2046 299
18 788 1498
0.21
478 74
2318 54
0.53
248 8
472 8
0.58
117 6
203 29
Aerobic
Day 25
0.11
1948 55
18 480 343
0.27
444 14
1659 26
0.58
245 5
421 10
0.56
175 10
314 40
Anaerobic
Table 1. Changes in concentrations and ratios of organic carbon and carbohydrate fractions within the sediment slurries. Values expressed as lg C g1 dry wt. of original sediment; 4.59 g of
dry weight sediment was used in 65 mL final volume of slurry. Major constituents (based on published sources) of the four different organic carbon and carbohydrate fractions used in this study
are indicated
Anaerobic degradation of biofilm organic matter
497
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B.A. McKew et al.
498
determined by two-way analysis of variance, with treatment (oxic or anoxic) and time (day 0, 1, 3, 7, 10, 15
and 25) as factors. P-values are provided for treatment
(Ptrt), time (Ptime) and the interaction between these
factors (Ptrt9time). Pairwise comparisons were determined
using Tukey’s honestly significant difference (HSD)
method. On days 0, 3 and 10, a detailed analysis of the
bacterial (and chloroplast-containing eukaryote) community composition was undertaken.
Methane was measured by extracting 100 lL of headspace
gas with a Gas Tight Analytical Syringe (SGE Analytical
Science) and injecting into a Phillips PU4500 gas chromatograph with a 2-m Restek Molecular Sieve packed 5A
column (100/120 mesh) with flame ionisation detector.
N2 carrier gas was set at 40 mL min1, oven temperature
at 100 °C and the injector and detector at 160 °C.
overall total organic carbon (TOC) and total carbohydrate concentrations during degradation. Subsamples of
slurry (5 mL) were centrifuged (4000 g, 15 min), and the
supernatant was removed and diluted 20-fold with MilliQ
water to measure DOC on a Shimadzu TOC-VCSH Analyzer using the nonpurgeable organic carbon method with
1% acid ratio and 3-min sparge. The sediment pellets
(retained after removing the supernatant from the centrifuged slurries for the DOC analysis) were subject to
sequential extractions of hot-water-extracted (95 °C)
organic carbon (HW-OC, in 2.5% NaCl) and hot bicarbonate-extracted (95 °C) organic carbon (HB-OC, in
0.5 M NaHCO3), using methods developed to extract
intracellular and more tightly bound polymers from
diatoms and sediments (Bellinger et al., 2005; Hanlon
et al., 2006). On day 25, 5 mL of slurry (including suspended sediment and biomass) was dried, acidified with
2 M HCl (to remove inorganic carbon) and measured for
TOC using a Shimadzu SSM5000 analyzer.
Sulphate and acetate
Carbohydrate
5 mL of slurry was filtered (0.2 lm) and diluted 100-fold
or 10-fold (for sulphate and acetate, respectively) with MilliQ water. The sulphate and acetate ion concentrations
were then quantified against Na2SO4 and C2H3NaO2
analytical standards on a Dionex Ion Chromatogram
ICS3000 with an AS18 column and KOH and MilliQ water
eluents (increasing from 0 to 50 mM KOH over 90 min).
Carbohydrate in the four different fractions was measured
spectrophotometrically (485 nm) after the phenol–sulphuric acid reaction (Dubois et al., 1956) as described by
Hanlon et al. (2006) and quantified as glucose equivalents
(lg g1 dry weight sediment). These fractions included
total carbohydrates (TCHO; measured on complete slurry
including suspended sediment and biomass), dissolved
and colloidal carbohydrate (DCHO; measured in the DOC
fraction detailed above) and the HW and HB extracts
[termed hot-water-extracted carbohydrate (HW-CHO)
and hot bicarbonate-extracted carbohydrate (HB-CHO)].
Methane
Sulphide
Dissolved and precipitated sulphide was measured spectrophotometrically as a colloidal solution of copper
sulphide as previously described (Cord-Ruwisch, 1985).
No dissolved sulphide was detected, so reported values
represent sulphide precipitated as FeS, measured after
acidification with 0.5 vol of 4 M HCl.
Organic carbon
Organic carbon and carbohydrates were determined in
four inter-related fractions extracted sequentially from the
sediment slurry. The most soluble fractions of organic
material (DOC, dissolved or colloidal carbohydrates, see
Table 1) are obtained from saline extractions of slurry,
followed by a hot-water extraction that solubilises intraand extracellular constituents, including the diatom
storage compound chrysolaminarin, and finally a hot
bicarbonate extraction, which dissolves less soluble EPS
structures (Table 1). This approach permits the characterisation of changes in various organic fractions identified on the basis of solubility, as well as decreases in the
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Extracellular b-glucosidase and aminopeptidase
activity
Maximum potential rates of extracellular b-glucosidase
and aminopeptidase activity were determined using saturating concentrations (1 mM) of fluorescently labelled
substrates MUF-b-D-glucopyranoside or leucine-7-aminomethylcoumarin (Sigma-Aldrich, Dorset, UK) (Hoppe, 1983)
after adding 0.2% w/v sodium azide (metabolic inhibitor
to prevent de novo enzyme production). Incubation conditions, controls, spectrophotometry and calculation of relative rates (based on the rate of MUF or coumarin cleavage
over a 30-min incubation) were performed as described
previously (McKew et al., 2011).
Bacterial community analysis
DNA was extracted from slurry samples, and the V3
region of the 16S rRNA gene was PCR-amplified and
FEMS Microbiol Ecol 84 (2013) 495–509
499
Anaerobic degradation of biofilm organic matter
used to analyse the bacterial community composition by
454 pyrosequencing of amplicon libraries. DNA was
extracted from pellets after centrifuging (16 100 g,
15 min) 2 mL of slurry using a bead beating and phenol–
chloroform–isoamyl alcohol method as previously
described (McKew et al., 2011).
The V3 region of the 16S rRNA gene was PCR-amplified using separate 454 fusion primers for each sample,
each of which contained a unique 10-base barcode on the
forward primer to distinguish each sample (Parameswaran et al., 2007), and the V3 target sequence (Muyzer
et al., 1993) (forward 5′ - CC TAC GGG AGG CAG CAG
- 3′; reverse 5′ - ATT ACC GCG GCT GCT GG - 3′).
DNA extract (2 lL) was amplified in 50-lL reactions
containing 0.4 mM of the primers, 0.1 mM dNTPs,
2.5 U of Taq DNA polymerase (Qiagen) and 5 lL of
reaction buffer (Qiagen). The amplification programme
was 95 °C for 30 s, 30 cycles of 94 °C for 30 s, 55 °C for
30 s and 72 °C for 30 s, with a final elongation of 72 °C
for 10 min, and was performed in a Gene Amp PCR
system 9700 Thermocycler (Applied Biosystems). The
PCR products were cleaned using a QIAquick PCR purification kit (Qiagen) and quantified with a Nanodrop
ND-1000 spectrophotometer, replicates were pooled in
equimolar concentrations and approximately 200 ng of
DNA per sample was analysed using 454 pyrosequencing
at the NERC Molecular Genetics Facility (University of
Liverpool). The 5 samples (day 0, day 3 and day 10, aerobic and anaerobic) were multiplexed on 1/8 of a 454 GSFLX LR70 pyrosequencing plate.
The pyrosequence reads were analysed using the
QIIME pipeline and associated modules (Caporaso et al.,
2010). All sequences were checked for the presence of
correct pyrosequencing adaptors, 10-base barcodes and
the 16S rRNA gene-specific primers, and any sequences
containing errors in these regions were removed from the
analysis. Any sequences < 150 bp and > 200 bp in read
length, containing homopolymer inserts, with low quality
scores (< 20) or chimaeras, were also removed from further analysis. The remaining reads were clustered into
operational taxonomic units (OTUs) using the UClust
algorithm (Edgar, 2010) at the 5% level. Representative
sequences from each OTU were identified using RDP
classifier, which assigns taxonomic identities against the
RDP database using a na€ıve Bayesian classifier (Wang
et al., 2007). All singletons were removed before further
analysis. The microbial communities across all slurries
were analysed by nonmetric multidimensional scaling
(NMDS) ordination of distance matrices calculated from
the OTU pyrosequence-read matrix using the Jaccard’s
index. Differences in bacterial composition and diversity
were compared between treatments (aerobic or anaerobic)
and time (day 0, day 3 and day 10). Species diversity
FEMS Microbiol Ecol 84 (2013) 495–509
(number and relative abundance of OTUs) was calculated
using Shannon–Wiener’s index, and differences in composition were calculated using Jaccard’s index and compared between treatments and time points using a simple
randomisation test, based on 10 000 randomisations
(Solow, 1993). This randomisation test treats the entire
community as a single data set and is an absolute statistical measure that does not require replication to produce
probabilities (Macek et al., 2011). All analyses were conducted in the R statistical language version 2.7.2 using
the R standard libraries and the community ecology analysis-specific package ‘vegan’ (R Development Core Team,
2011).
Results
Development of anaerobic conditions
There was evidence of anaerobic conditions in the sediment slurries within 3 days. Sulphate concentrations
reduced significantly from day 3 (Fig. 1a), declining from
31 to 15 mM between days 3 to 15, coupled with a significant increase in sulphide concentrations from 0 to 6 mM
over the first 15 days (Fig. 1b). In the aerobic slurries,
sulphate concentrations remained constant at approximately 32 mM and sulphide remained undetectable
throughout the experiment. Concentrations of acetate
significantly increased only in the anoxic slurries, from
undetectable starting concentrations to a maximum concentration of 191 lM on day 3, before reducing sharply
to the initial levels by day 10 (Fig. 1c). Methane concentrations in the anoxic microcosms increased significantly
from day 3 and steadily throughout the experiment to
2964 ppm (Fig. 1d).
In both treatments, there were significant spikes in
ammonium concentrations (from 4 to 65 lM) (Fig. 1e)
followed by rapid declines. In the anoxic microcosms, this
increase occurred after day 1, peaking at day 3, after
which ammonium concentrations decreased (by day 10)
to below 1 lM for the remainder of the experiment. In
the oxic treatments, the increase occurred after day 3,
with the highest concentration on day 7.
Changes in organic carbon and sediment
carbohydrate concentrations
Biofilm and initial slurry
The microphytobenthic biofilm-rich sediment used in the
experimental slurries contained high concentrations of
TCHO [7.24 mg (gluc. equiv. g dry wt. sediment)1] and
DOC (as DOC and in extractable fractions, HW-OC and
HB-OC; Table 1). The slurries contained high initial
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Published by Blackwell Publishing Ltd. All rights reserved
B.A. McKew et al.
500
Sulphate (mM)
Aerobic degradation
(a)
40
30
P trt <0.0001
P time <0.0001
P trt x time <0.0001
20
10
Oxic
Anoxic
0
(b)
Sulphide (mM)
8
6
P trt < 0.0001
P time <0.0001
P trt x time <0.0001
4
2
0
(c)
Acetate (μM)
250
200
P trt < 0.001
P time <0.0001
P trt x time <0.0001
150
100
50
0
(d)
Methane (ppm)
4000
3000
2000
P trt < 0.0001
P time <0.0001
P trt x time <0.0001
1000
Ammonium (μM)
0
(e)
80
60
P trt = 0.023
P time <0.0001
P trt x time <0.0001
40
20
0
0
5
10
15
20
25
Day
Fig. 1. Changes in concentrations of sulphate (a), sulphide (b),
acetate (c), methane (d) and ammonium (e) in the oxic and anoxic
slurries. Shown are the means SE (n = 5). P values given show
significant effects of both treatment (Ptrt), time (Ptime) and the
interaction between the two factors (Ptrt 9 time) (two-way ANOVA), and
star symbols (*) indicate a significant difference between the
measured values in oxic and anoxic slurries on the given day (Post hoc
Tukey’s HSD test).
concentrations of extractable fractions of carbohydrates
[DCHO, HW-CHO and HB-CHO fractions totalled
7.42 mmol C L1, equivalent to 1261 lg C (g dry wt. sediment)1], DOC [DOC, HW-OC and HB-OC fractions
totalled 25.97 mmol C L1, equivalent to 4414 lg C (g
dry wt. sediment)1] and TCHO [17.03 mmol C L1,
equivalent to 2894 lg C (g dry wt. sediment)1]. Carbohydrate-carbon represented between 8% (dissolved extracts)
and 64% (HW extracts) of the TOC in these fractions
(Table 1).
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Concentrations of DOC decreased rapidly and significantly in the oxic slurries, with 75% being consumed
within 3 days (Fig. 2a). Similar declines were seen in
DCHO concentrations (65% reduction between day 0
and day 3, Fig. 2b). After 10 days, there were no further
reductions in DOC with concentrations increasing slightly
over the remaining 15 days. DCHO concentrations rose
significantly between days 10 and 25, resulting in a significant increase in the DCHO/DOC ratio in the slurry
supernatant (from 0.08 to 0.58 on day 25, Table 1).
There was no change in the HW-OC and HW-CHO
concentrations in the first 24 h, after which concentrations decreased significantly over 24 days (Fig. 2c and d).
By day 10, HW-OC had decreased by 30%, and at day
25, by 59%. Identical changes occurred in the HW-CHO
concentrations (correlation between HW-CHO and
HW-OC, r = 0.927, P < 0.001). The HW-OC fraction
contained a high percentage (64%) of dissolved carbohydrate at the beginning of the experiment, declining to
53% after 25 days (Table 1). In contrast, HB-OC concentrations [a higher-molecular-weight (HMW), more insoluble fraction of the organic carbon pool] remained
relatively constant in the oxic microcosms, with no significant changes over 25 days (Fig. 2e). TCHO concentrations (Fig. 2f) increased initially in the first 24 h, but
then reduced significantly by approximately 33% over the
first 10 days (Fig 2f).
Maximum potential rates of extracellular enzyme activity (aminopeptidase and b-glucosidase) declined by 40%
and 92%, respectively, in the oxic slurries during the
experiment. Enzyme activity rates were significantly positively correlated with TCHO, HW-CHO and HW-OC
concentrations (r > 0.695, P < 0.001 in all cases). Highest
partial correlation coefficients were between b-glucosidase
and HW-OC (r = 0.976, P < 0.001) and aminopeptidase
and HW-OC (r = 0.483, P < 0.01). Activities of both
extracellular enzymes were negatively correlated with
ammonium concentration (P < 0.05), and aminopeptidase activity with DCHO concentrations (r = 0.582,
P < 0.001). The rate of reduction in DOC
(mmol DOC L1 day1), calculated between each time
point, was positively correlated with the rates of b-glucosidase (r = 0.84, P < 0.05) and aminopeptidase (r = 0.93,
P < 0.01) activity.
Anaerobic degradation
The development of anoxic conditions caused major differences in the patterns of concentrations of organic carbon and carbohydrate and enzyme activities compared
with the oxic treatments. There was a significant 42%
FEMS Microbiol Ecol 84 (2013) 495–509
501
150
125
Oxic
Anoxic
100
P trt <0.0001
P time <0.0001
P trt x time <0.0001
75
50
25
(b)
DCHO
(% remaining)
(a)
DOC
(% remaining)
Anaerobic degradation of biofilm organic matter
400
(d)
125
100
P trt =0.002
P time <0.0001
P trt x time <0.0001
50
25
(f)
125
50
25
75
50
25
TCHO
(% remaining)
0
P trt <0.0001
P time <0.0001
P trt x time =0.003
100
(h)
125
100
P trt <0.009
P time <0.0001
P trt x time =0.034
75
50
25
0
0
(g) 125
Aminopeptidase
(relative rate)
P trt = ns
P time <0.0001
P trt x time <0.0001
75
β-glucosidase
(relative rate)
HB-OC
(% remaining)
100
100
100
0
125
200
0
75
(e)
300
HW-CHO
(% remaining)
(c)
125
HW-OC
(% remaining)
0
P trt <0.0001
P time <0.0001
P trt x time <0.0001
P trt <0.0001
P time <0.0001
P trt x time <0.0001
100
75
50
25
P trt = ns
P time <0.0001
P trt x time = ns
75
50
25
0
0
0
5
10
15
20
25
Day
0
5
10
15
20
25
Day
Fig. 2. Relative changes (%) in concentrations of carbon and carbohydrate fractions of DOC (a), DCHO (b), HW-OC (c) HW-CHO (d), HB-OC (e),
TCHO (f) and relative rates of aminopeptidase (g) and b-glucosidase (h) extracellular enzyme activities in the oxic and anoxic slurries. Shown are
the means SE (n = 5). P values given show significant effects of both treatment (Ptrt), time (Ptime) and the interaction between the two factors
(Ptrt 9 time) (two-way ANOVA), and star symbols (*) indicate a significant difference between the measured values in oxic and anoxic slurries on the
given day (Post hoc Tukey’s HSD test).
increase in DOC in the anoxic microcosms over the first
3 days before concentrations reduced significantly (to
those measured in the oxic treatments) by day 10
(Fig. 2a). After 10 days, there were no further reductions
in the DOC pool and no significant difference between
the treatments. The release of DOC at the onset of anoxia
was not primarily carbohydrate; changes in DOC were
not mirrored in the DCHO concentrations, which
remained unchanged for the first 8 days, before increasing
significantly by the end of the experiment (Fig. 2b). The
carbohydrate content of the DOC pool increased significantly from day 0 to 10, with the DCHO/DOC ratio
changing from 0.08 to 0.56 (Table 1).
Both HW-OC and HW-CHO concentrations did not
alter for the first 24 h, but decreased significantly and faster
than in the oxic treatments over the first 10 days (Fig. 2c
and d). By 25 days, concentrations had decreased by 63%
to levels observed in oxic treatments. There was significant
loss of HB-OC by day 7 (24% decrease in concentrations),
FEMS Microbiol Ecol 84 (2013) 495–509
which continued until day 25 (39% decrease). This reduction in HB-OC did not consistently match changes in
HB-CHO concentrations, which declined by only approximately 10% at day 25 (Table 1). TCHO declined in a similar fashion to the oxic slurries (Fig. 2f). By 25 days, TOC
concentrations were very similar (P = not significant) in
both the anoxic [18.5 mg C (g dry wt. sediment)–1] and
oxic slurries [18.8 mg C (g dry wt. sediment)–1] (Table 1).
Extracellular aminopeptidase activity (maximum potential rate) was reduced significantly in the anoxic microcosms compared with oxic treatments (Fig. 2g). The
highest rates were significantly positively correlated with
TCHO, HW-CHO, DOC, HW-OC and HB-OC concentrations (r > 0.826, P < 0.001 in all cases) and negatively
with DCHO concentrations. Highest partial correlation
coefficients were between aminopeptidase and HW-OC
(r = 0.927, P < 0.001). The decrease in b-glucosidase
rates over time was identical in anoxic and oxic slurries
(Fig. 2h). However, in the anoxic treatment, there were
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Published by Blackwell Publishing Ltd. All rights reserved
B.A. McKew et al.
502
significant positive correlations between b-glucosidase
activity and the rates of loss (mmol L1 day1, calculated
between each time point) of HB-OC (r = 0.947,
P < 0.01) and DCHO (r = 0.848, P < 0.05), and between
b-glucosidase activity and increase in DOC concentrations (r = 0.834, P < 0.05).
Changes in bacterial community
Differences in the bacterial community composition were
determined by 454 pyrosequencing of DNA amplicon
libraries of the 16S rRNA gene. Amplified DNA from replicates was pooled and used to construct libraries from
the initial sediment (day 0) and from both the oxic and
anoxic microcosms on days 3 and 10. Across samples,
pairwise comparisons of the diversity of the libraries
revealed that bacterial assemblages were significantly more
diverse, as measured by the Shannon–Wiener index, in
anoxic microcosms compared with oxic microcosms
(DH0 > 0.47, P < 0.01 in all cases). However, in both
treatments, the diversity of bacterial assemblages did not
change with incubation time between day 3 and day 10
(DH0 < 0.14, P > 0.05 in all cases). NMDS ordination
(based on Jaccard’s index) showed compositionally distinct assemblages of bacteria between oxic and anoxic
microcosms that become increasingly dissimilar through
time (Fig. 3). To test this observation, pairwise randomisation tests comparing Jaccard’s index across samples
were conducted. Comparisons of Jaccard’s index (bacterial species turnover) confirmed significant differences in
bacterial composition between oxic and anoxic microcosms (Jd > 0.8, P < 0.001 in all cases). By day 10, the
bacterial composition from both aerobic and anaerobic
microcosms was significantly different from day 0
(Jd > 0.8, P < 0.01 in both cases).
Ribosomal Database Project classification of the OTUs
revealed that the most notable differences in the anoxic
microcosms were large increases in relative abundance of
the Firmicutes and Deltaproteobacteria (Table 1). Clostridia (in the Firmicutes) constituted 0.3% to 0.5% of the
day 0 library and aerobic day 3 and day 10 libraries, but
increased to 6.3% and 6.5% in the anoxic libraries at day
3 and day 10, respectively. This was due mainly to
increases in relative abundance of sequences assigned to
Lachnospiraceae, Peptostreptococcaceae, Ruminococcaceae
and other unclassified Clostridiales. By day 3, unclassified
bacterial sequences had increased 2-fold in the anoxic
microcosms from 9% to 18%. Examination of the consensus sequences from the clusters within this group
revealed that the majority were most closely related to
Clostridia sequences (92–93% similarity). Deltaproteobacteria increased by approximately 2-fold (Table 1) in the
anoxic libraries (compared with the day 0 and day 3 and
day 10 oxic libraries), which was attributed primarily to
increases in sequences from families that consist mainly
of sulphate-reducing bacteria: Desulfobacteraceae (0.7%
and 0.8% in the oxic slurries increasing to 8.4% and
12.2% in the anoxic slurries) and Desulfobulbaceae from
the order Desulfobacterales (< 1% in the oxic slurries
increasing to 4.7% and 4.3% in the anoxic slurries).
Relative abundance of Alphaproteobacteria (particularly
Rhodobacteraceae) and Gammaproteobacteria sequences
decreased in the anoxic slurries (Table 1). Relative numbers of Gammaproteobacteria sequences were approximately 50% lower in the anoxic slurries by day 3.
Sequences assigned to Epsilonproteobacteria decreased in
both treatments over time. Cyanobacterial sequences
declined in both the oxic and anoxic microcosms and
were undetected at day 10, although the decrease was
more rapid in the anoxic microcosms, decreasing in relative abundance from 2.2% to 0.1% by day 3.
Survival of diatoms
Fig. 3. NMDS ordination of distance matrices calculated from the
OTU pyrosequence-read matrix using Jaccard’s index. 16S rRNA
bacterial and diatom community structure associated with the initial
sediment at day 0 (circle) and with the oxic (white) and anoxic (black)
slurries at day 3 (squares) and day 10 (triangles).
ª 2013 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
At the beginning of the experiment, 25.4% of sequences
detected came from diatom chloroplasts, decreasing to
18.2% and 18.3% at days 3 and 10, respectively, in the
oxic microcosms (Table 1). In the anoxic microcosms,
the relative abundance of diatom sequences also decreased
by day 3 (16.7%), but then decreased further to 9.2% by
day 10.
After 25 days in the dark-incubated slurries, the relative abundance of both dead and live diatoms was determined by phase-contrast microscopy. Empty frustules
(dead cells) represented 80% ( 5.4%) of the total valve
FEMS Microbiol Ecol 84 (2013) 495–509
Anaerobic degradation of biofilm organic matter
counts in anoxic slurries, compared with 22.6% ( 2.2%)
in oxic slurries. The live diatom assemblages in both
treatments were distinctly different, with < 35% similarity
(compared with 65% similarity within oxic and anoxic
replicates). Anoxic slurries were dominated by live Navicula phyllepta (> 75% of the live cells; but numbers of
live N. phyllepta were half that of the oxic slurries) and
also included Amphora spp. and Fallacia pygmeae. In contrast, oxic slurries contained 22 morphologically identified
species that were alive, including the aforementioned taxa,
but also Gyrosigma attenuatum, G. limosum, Navicula gregaria, Nitzschia sigma, Cylindrotheca closterium and small
species of Nitzschia (representing up to 40% of the total
live cell counts). These taxa either declined significantly
or died out completely under anoxic conditions.
Discussion
Degradation of organic carbon
The initial increases in ammonium and DOC in the
anoxic and oxic slurries (day 3) were probably due to
physical stirring, increased microbial activity and the
death of some organisms (e.g. declines in relative abundance of diatoms and Deltaproteobacteria). The DOC
released from stressed and dead cells contained many
labile LMW components that were utilised rapidly under
both oxic and anoxic conditions with similar rates and to
a similar extent. Loss rates of DCHO (containing both
colloidal EPS and LMW carbohydrate, Bellinger et al.,
2005) and the glucose-rich HW-OC and HW-CHO were
equally high in both treatments. The decrease in this
labile fraction, containing mainly glucans (Hanlon et al.,
2006), may be attributed to a combination of diatoms
using chrysolaminarin as a storage reserve in the dark
(Underwood et al., 2004; Hanlon et al., 2006) and bacterial degradation following leakage from live cells or
release from dying or dead cells (especially in the anaerobic treatments). Whilst some organic material may be
degraded faster under oxic conditions (e.g. chlorophyll
(Sun et al., 1993), and refractory material from aged diatoms (Kristensen et al., 1995)), our results indicate that
labile biofilm-derived organic carbon can be readily
degraded in both anoxic and oxic conditions (Lee, 1992;
Arnosti et al., 1994; Kristensen et al., 1995; Andersen,
1996; Arnosti, 2000; Dauwe et al., 2001). Higher degradation rates of diatom EPS compared with glucose have
been measured in sediments (Goto et al., 2001), suggesting some specificity for chemically complex carbohydraterich algal-derived organic matter by the heterotrophic
community (both aerobic and anaerobic) (Arnosti, 2000).
Old refractory material (by definition) resists anaerobic
degradation, with the limiting step of anaerobic decay
FEMS Microbiol Ecol 84 (2013) 495–509
503
being attributed to the initial hydrolytic attack (Kristensen et al., 1995; Arnosti, 2011). The HB-OC (i.e. the
high-molecular-weight pool of insoluble EPS) was the
least degraded fraction (Fig. 2e), but greater degradation
was seen under anoxic conditions. It is known that a proportion of microphytobenthic biofilm carbon remains
longer and penetrates to greater depths in sediments
(Evrard et al., 2008; B€
oer et al., 2009; Oakes et al., 2010).
Given the high rates of mineralisation activity (particularly of LMW labile material) in surface aerobic layers
(Goto et al., 2001; Hanlon et al., 2006; Oakes et al.,
2010), it is unlikely that the most labile carbon fractions
penetrate to greater depths (Middleburg, 1989; Kristensen, 1993; Volkman et al., 2000). Our findings of faster
rates of degradation of the more insoluble HB-OC fraction, and correlations between HB-OC loss rates and
b-glucosidase activity (with a concomitant liberation of
soluble DOC from the process) under anoxic conditions,
support the idea that anaerobic taxa may be pre-adapted
to breaking down the more refractory components of biofilm organic matter. There were significant increases in
DCHO in both treatments, which may represent the
accumulation of some soluble but refractory carbohydrate
moieties produced during degradation of complex EPS
(Burdige & Gardner, 1998), as well as nonlabile EPS–
polysaccharides produced by the active bacterial community (Eichinger et al., 2009).
Microbial extracellular enzymes play an important role
in carbon cycling (Someville & Billen, 1983; Danovaro
et al., 2001; Arnosti, 2011). Complex polysaccharides in
diatom EPS require hydrolysis into smaller molecules by
extracellular enzymes prior to uptake by bacteria, and it
has been proposed that bacterial extracellular b-glucosidase is the rate-limiting step (Meyer-Reil, 1990). b-Glucosidase activity declined equally in both treatments and
was correlated with the rates of loss of DOC and
HW-CHO. In anoxic treatments, loss rates of HB-OC
were correlated with b-glucosidase activity, suggesting
increased breakdown of more complex organic matter
(Haynes et al., 2007; Hofmann et al., 2009), with the
labile fractions produced being subsequently utilised by
the heterotrophic community under anoxic conditions.
Aminopeptidase activity is also an indicator of heterotrophy as these enzymes hydrolyse proteins and peptides
into oligopeptides and amino acids, which are key sources
of energy, carbon and nitrogen (Wheeler & Kirchman,
1986). Interestingly, although similar patterns of degradation of organic carbon were observed under both conditions, the maximum potential rates of aminopeptidase
were significantly lower in the anoxic slurries (Fig. 2g),
raising the question of whether alternative enzymes for
protein hydrolysis exist under anoxic conditions (Arnosti,
2011).
ª 2013 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
504
A multifunctional anaerobic community
Compositionally distinct bacterial communities developed between the oxic and anoxic slurries. The change
in relative abundance of Gammaproteobacteria that
dominated under oxic conditions to Firmicutes and
Deltaproteobacteria dominating in anoxic conditions
reflects the shifts seen in Bacterial phyla from surface
to deep tidal-flat sediments (K€
opke et al., 2005; Wilms
et al., 2006; Webster et al., 2010). The significant
increase in Clostridia sequences in anoxic slurries, the
wide diversity of phylotypes (15 distinct clusters, mostly
absent in aerobic libraries) and the recognised metabolic diversity within the Clostridia, all highlight their
major role in anaerobic degradation in intertidal sediments. Clostridia can degrade both refractory HMW
and labile LMW substrates (see Xing et al., 2011) and
have been shown to be the dominant bacteria during
degradation of freshwater Microcystis biomass (Xing
et al., 2011). Clostridia hydrolyse diverse organic substrates, releasing a range of fermentation products, such
as acetate (which was shown to increase to highest levels in the early stages of the experiment at day 3) and
ethanol, which serve as substrates for microorganisms
using a range of terminal electron acceptors, particularly sulphate-reducing bacteria in marine sediments
(Purdy et al., 2002).
Whilst methanogenic Archaea are not detected by
our Bacteria-specific primers, their presence is confirmed by the continuous production of methane in
the anoxic slurries (Fig. 1d). Some methanogens are
outcompeted by sulphate-reducing bacteria for substrates such as hydrogen and acetate, whilst others can
coexist by utilising noncompetitive substrates, such as
methylamines and dimethylsulphide (van der Maarel &
Hansen, 1997; Wilms et al., 2006; McGenity, 2010) that
are likely to have been fermentatively converted (McGenity, 2010) from glycine betaine and dimethylsulphoniopropanate, respectively, and released into the anoxic
slurries from dying obligate aerobes and phototrophs,
especially diatoms. In tidal-flat sediments stimulated
with Spirulina biomass, methanogenesis only occurred
after depletion of sulphate (Graue et al., 2012), and
often, methanogenesis is a relatively minor process until
deep in the sediment where sulphate concentrations are
much lower (Jorgensen & Parkes, 2010; Webster et al.,
2010). However, this study confirms the coexistence of
methanogenesis and sulphate reduction and supports
the previous claims that there is a primed methanogenic community in intertidal sediments (Purdy et al.,
2003) and that intertidal sediments can act as a significant source of methane to the atmosphere (Middelburg
et al., 2002). Methanogens able to utilise competitive
ª 2013 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
B.A. McKew et al.
and noncompetitive substrates have been detected in
Colne estuary sediments (Munson et al., 1997; Purdy
et al., 2003), and Purdy et al. (2003) showed that
Methanosarcinales increased to represent 16.5% of the
prokaryote community after addition of trimethylamine
to anaerobic slurries. Sediment-dwelling methanogens,
therefore, contribute to anaerobic degradation of buried
organic matter, using noncompetitive substrates from
the buried biomass, in the same way that they contribute to degradation in the guts of phytoplankton grazers
(de Angelis & Lee, 1994).
Sulphate reduction was a major anaerobic process
confirmed by the decrease in sulphate and increase in
sulphide concentrations (Fig. 1a and b), but also from
the 16S rRNA gene libraries that showed large increases
in the relative abundance of Deltaproteobacteria. Deltaproteobacteria were in fact already abundant in the initial
community (7.8%) and remained so in the oxic slurries,
but increased overall in relative abundance under anoxic
conditions, particularly Desulfobacteraceae and Desulfobulbaceae. Members of these families have diverse metabolic capabilities, but given the increases seen, they are
the taxa most likely to be responsible for the observed
reduction in sulphate (Fig. 1a and 1b), utilising a range
of LMW compounds as electron donors derived from
fermentative organisms. After the large increase in
acetate concentrations by day 3, the subsequent decrease
in acetate coincides with the loss of sulphate and
increase in sulphide from day 3 to day 15, suggesting
that the sulphate reducers are in fact utilising this acetate
and other (not measured) fermentation products (e.g.
propionate, butyrate), produced from the earlier fermentation by Clostridia and other fermenters. Rates of sulphide production decelerated after day 10, probably
linked to organic carbon and/or nutrient limitation as
both DOC and ammonium concentrations had reduced
to their lowest levels.
The Bacteroidetes represented 12.2% of the starting
community, and by day 10, constituted 16.2% and
18.2% of the oxic and anoxic slurries, respectively. There
was a phylogenetically diverse array of Bacteroidetes, with
104 OTUs, 21 of which were detected in both aerobic
and anaerobic slurries at day 10, suggesting that many
of the Bacteroidetes in mudflats can switch from aerobic
to fermentative degradation of DOC. This is supported
by the isolation of facultative anaerobes from Wadden
Sea tidal flats (K€
opke et al., 2005). Bacteroidetes are
implicated as being among the primary degraders of
algal-derived polymers (Bruckner et al., 2002; Grossart
et al., 2005), which was confirmed by the detection of
Bacteroidetes 16S rRNA genes from 13C-labelled nucleic
acids, after incubation with 13C-labelled diatom lysate in
an intertidal sand column (Chipman et al., 2010) and
FEMS Microbiol Ecol 84 (2013) 495–509
505
Anaerobic degradation of biofilm organic matter
with 13C-labelled Spirulina biomass in an intertidal sediment slurry (Graue et al., 2012). Graue et al. (2012)
showed that members of the phylum Fusobacteria were
also implicated in fermentative degradation of cyanobacterial biomass; however, whilst they increased in abundance in our anoxic slurries, they were in comparatively
low abundance. The function of difficult-to-culture
members of the phylum Verrucomicrobia is largely
unknown; however, the large increase in their relative
abundance in the oxic slurries (Table 2) is consistent
with their ability to degrade HMW polysaccharides, as
shown by Martinez-Garcia et al. (2012), who used single-cell genomics to identify Verrumicrobia phylotypes as
the main degraders of the storage polysaccharides, laminarin and xylan, in coastal surface waters.
Survival/growth of oxygenic phototrophs
Many diatoms in the anoxic slurries died during the experiment, evidenced microscopically at day 25 and also by the
reduction in diatom chloroplast sequences at day 10. Diatoms may be frequently buried in dark, anoxic sediments
(Kamp et al., 2011). In a natural burial event, some diatoms
could escape anoxia by migrating vertically (McKew et al.,
2011), but this was not a possibility in this experiment.
Numbers of sequences appeared unchanged by day 3, but
Table 2. Relative abundance (%) of 16S rRNA gene OTUs assigned to different microbial groups in 454 pyrosequencing libraries from the day 0,
day 3 and 10 oxic and anoxic slurries. Reads were clustered into OTUs using the UCLUST algorithm at the 5% level. Shaded groups are discussed
within the main text.
Number of OTUs (using rarefaction)
Day 0
Day 3
Oxic
Day 10
Oxic
Day 3
Anoxic
Day 10
Anoxic
153
165
151
190
198
0.5
1.0
0.0
9.0
1.5
5.7
0.3
0.3
0.0
0.0
3.6
0.5
0.3
0.3
0.8
0.8
0.0
0.0
0.0
0.3
7.2
0.8
0.0
0.0
0.0
17.3
7.0
0.0
7.0
0.0
17.5
0.0
18.3
0.3
0.1
0.5
0.1
5.7
0.9
9.9
6.3
0.6
1.2
0.5
2.5
0.0
0.9
0.2
8.4
4.7
0.1
0.2
0.2
0.1
6.9
0.7
0.0
0.0
0.0
9.5
2.5
0.8
1.5
0.0
18.2
0.1
16.7
0.2
0.1
0.4
0.0
6.8
0.2
11.0
6.5
0.9
0.7
0.0
1.7
0.2
0.9
0.0
12.2
4.3
0.1
0.0
0.0
0.2
4.9
0.5
0.1
0.1
0.1
9.9
5.0
2.1
0.7
0.0
21.1
0.0
9.2
0.1
Relative abundance (%)
Acidobacteria; Acidobacteriaceae
Actinobacteria
Bacteroidetes; Bacteroidales
Bacteroidetes; Flavobacteriales
Bacteroidetes; Sphingobacteriales
Bacteroidetes; unclassified
Firmicutes; Clostridia
Firmicutes; other
Fusobacteria; Fusobacteriales
Alphaproteobacteria; Rhizobiales
Alphaproteobacteria; Rhodobacteraceae
Alphaproteobacteria; Sphingomonadaceae
Alphaproteobacteria; unclassified
Deltaproteobacteria; Bacteriovorax
Deltaproteobacteria; Desulfobacteraceae
Deltaproteobacterial; Desulfobulbaceae
Deltaproteobacteria; unclassified
Deltaproteobacteria; Desulfovibrio
Deltaproteobacteria; Desulfuromonales
Deltaproteobacteria; Myxococcales
Deltaproteobacteria; unclassified
Epsilonproteobacteria
Gammaproteobacteria; Alteromonadales
Gammaproteobacteria; Chromatiales
Gammaproteobacteria; Vibrionaceae
Gammaproteobacteria; unclassified
Proteobacteria; unclassified
Spirochaetes; Spirochaetaceae
Verrucomicrobia; Verrucomicrobiales
BRC1 genera incertae sedis
Unclassified bacteria
Cyanobacteria
Bacillariophyta (diatoms)
Unclassified
FEMS Microbiol Ecol 84 (2013) 495–509
0.0
1.5
0.0
7.6
0.5
4.1
0.3
0.0
0.5
1.8
7.5
0.3
1.5
0.1
1.9
1.5
0.0
0.0
0.0
0.0
4.4
3.6
0.1
0.0
0.0
18.3
5.4
0.0
1.2
0.3
8.9
2.2
25.4
1.2
0.2
0.2
0.5
8.8
0.9
5.5
0.5
0.2
0.2
0.2
7.4
0.2
1.4
0.5
0.7
0.2
0.0
0.0
0.0
0.5
9.7
1.2
0.0
0.2
0.2
19.9
5.5
0.0
3.9
0.0
9.7
2.5
18.2
0.7
ª 2013 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
506
the build-up of toxic H2S may have contributed to the later
decline. Some diatoms survived, particularly H2S- and
ammonia-tolerant Fallacia pygmeae (Peletier, 1996) and
Amphora sp. Utilisation of b-glucan storage reserves would
be a survival strategy in the absence of photosynthesis
(Underwood et al., 2004), and the HW-CHO fraction,
which in sediment extracts contains significant proportions
of intracellular chrysolaminarin (Chiovitti et al., 2004;
Hanlon et al., 2006; Bellinger et al., 2009), decreased in a
similar fashion under both conditions. One survival strategy
may be respiration of stored nitrate when light and oxygen
are absent, resulting in ammonium production by dissimilatory nitrate reduction, a process recently confirmed in
Amphora aoffeaeformis (Kamp et al., 2011). This may also
account for some of the ammonium produced in the slurries.
Conclusion
We have shown that in estuarine sediments, the anaerobic
community can rapidly utilise labile biofilm DOC and
EPS (at the same rates as the aerobic community), but
has a greater potential for the degradation of more insoluble EPS components that remain refractory in aerobic
layers. This suggests anaerobic sediment communities
consist of many ‘opportuni-trophs’ (Polz et al., 2006)
that are adapted to exploit the arrival of labile and refractory biofilm-derived material upon its burial, which is a
resource that varies on both spatial and temporal scales.
The microbial community involved in the anaerobic degradation of EPS consisted of diverse taxa with hydrolytic,
fermentative, methanogenic and particularly sulphatereducing capabilities. Some biofilm diatom taxa also survived the anoxic conditions, raising the question of heterotrophic nutrition or nitrate respiration in these
species. Sediments play an important role in carbon
cycling, and our results reveal that multiple and complex
microbial interactions are involved in the degradation of
different elements of the sediment organic carbon pool.
Acknowledgements
We thank Tania Cresswell-Maynard and John Green for
their technical support. This work was funded by a grant
to GJCU and TJM from the UK Natural Environment
Research Council (NE/D003598/1).
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