R E S EA RCH AR T I C L E

Differences between aerobic and anaerobic degradation ofmicrophytobenthic 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: bamcke@essex.ac.uk

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

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 microphy-

tobenthos 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 Desulfob-

acteraceae (relative sequence abundance increased from 1.9% to 12.2%) and

Desulfobulbaceae (increased from 1.5% to 4.3%) were the main sulphate reduc-

ers, 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 metha-

nogens) 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 sedi-

ments, significantly influencing carbon and nitrogen bud-

gets of coastal environments. The most labile carbon

inputs originate in photosynthetic microbial biofilms (mi-

crophytobenthos) that occur extensively in such habitats

and generate up to half of the total autochthonous car-

bon production (Heip et al., 1995; Underwood & Kromk-

amp, 1999). Microphytobenthos excrete large quantities

of extracellular polymeric substances (EPS), predomi-

nantly polysaccharides; and 14C- and 13C-tracer studies

have shown rapid transfer (within 3 h) of photo-assimi-

lated carbon into the extracellular dissolved pool and

then into heterotrophic microorganisms (Smith & Under-

wood, 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 carbo-

hydrates are rapidly degraded under oxic conditions, fol-

lowed 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 sedi-

ment 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

FEMS Microbiol Ecol 84 (2013) 495–509 ª 2013 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

MIC

ROBI

OLO

GY

EC

OLO

GY

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 commu-

nity (Hanlon et al., 2006).

Bacteria in the Bacteroidetes are known degraders of

complex biopolymers and, together with Actinobacteria,

Alpha-, Beta- and Gammaproteobacteria, have been identi-

fied 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). A13C-stable isotope probing study in subtidal sandy sedi-

ments found that Gammaproteobacteria, Bacteriodetes and

some Firmicutes could utilise planktonic algal dissolved

organic matter; 13C-enriched sequences for sulphate-

reducing 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 inter-

tidal sediments, with anaerobic conditions almost reach-

ing the sediment surface during darkness (Underwood &

Kromkamp, 1999; B€ottcher et al., 2000). Microphytoben-

thic biofilms are frequently buried due to sediment resus-

pension 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 degrada-

tion of EPS in anaerobic conditions remains unknown.

The structural complexity of algal EPS raises the possi-

bility that numerous bacterial groups are involved in its

complete degradation (Bellinger et al., 2009). There is evi-

dence of partitioning between rapidly turned-over EPS

carbohydrate components and more structurally complex

EPS-carbon that may be subjected to longer-term reten-

tion and diagenesis in sediments (Cook et al., 2007; Evr-

ard 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

zones (Kristensen et al., 1995; Amtoft Neubauer et al.,

2004; Lomstein et al., 2006), rather than a underlying char-

acteristic 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 polysaccha-

rides (Arnosti & Repeta, 1994; Arnosti et al., 1994), so

should be able to utilise freshly buried biofilm organic

matter. We hypothesised that anaerobic bacterial commu-

nities would degrade fresh biofilm EPS as rapidly as aero-

bic bacteria. We therefore compared changes in

concentrations of different fractions of DOC and carbohy-

drates, 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 diatom-

dominated microphytobenthic biofilm) was collected at

low tide from Pyefleet mudflat, Colne Estuary, East Mer-

sea (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 para-

film, and the same number of anoxic slurries were sealed

with gas-tight septa and flushed with oxygen-free N2.

Concentrations of sediment carbohydrates (total, dis-

solved, 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 slur-

ries (204 and 3.1 lg C mL�1 for total and dissolved car-

bohydrate, 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 inde-

pendent 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, carbohy-

drate fractions, methane, aminopeptidase and b-glucosi-dase activity (as a proxy for bacterial activity). The

significance of any differences in these parameters was

ª 2013 Federation of European Microbiological Societies FEMS Microbiol Ecol 84 (2013) 495–509Published by Blackwell Publishing Ltd. All rights reserved

496 B.A. McKew et al.

Table

1.Chan

ges

inconcentrationsan

dratiosoforgan

iccarbonan

dcarbohydrate

fractionswithin

thesedim

entslurries.

Values

expressed

aslgC

g�1

dry

wt.oforiginal

sedim

ent;4.59gof

dry

weightsedim

entwas

usedin

65mLfinal

volumeofslurry.

Majorconstituen

ts(based

onpublished

sources)ofthefourdifferentorgan

iccarbonan

dcarbohydrate

fractionsusedin

thisstudy

areindicated

Fraction

Majorconstituen

ts(w

herekn

own)

Referen

ce

lgC

(gdry

wt.sedim

ent)�1

Day

0

Initial

Day10

Day

25

Aerobic

Anaerobic

Aerobic

Anaerobic

Dissolved

organ

ic

carbon(DOC)

Carbon(40%),am

inoacids(12%),

other

LMW

organ

iccarbon,for

exam

ple,acetate

Bellinger

etal.(2005),Burdige&

Gardner

(1998),Lomstein

etal.(1998),Rep

etaet

al.(2002)

565�

31

117�

18

202�

18

203�

29

314�

40

Dissolved

carbohydrate

(DCHO)

25%

colloidal

EPS,75%

nonpolymeric

carbo.,including

LMW

(glucose-rich)

Bellinger

etal.(2005),DeBrouwer

etal.(2003),Han

lonet

al.(2006),

Staats

etal.(1999)

44�

15

19�

173�

1117�

6175�

10

DCHO:DOC

0.08

0.16

0.36

0.58

0.56

Hot-water-

extractedorgan

ic

carbon(HW-O

C)

Heat-solublised

extra-

and

intracelluar

constituen

ts

(e.g.carbon,protein)

Chiovittiet

al.(2004),Staats

etal.(1999)

1141�

66

799�

14

601�

17

472�

8421�

10

Hot-water-

extracted

carbohydrate

(HW-CHO)

Chrysolaminarin

(glucose),LM

Wan

d

EPScarbohydrate,pen

tose/hexose

rich

Abdullahiet

al.(2006),

Bellinger

etal.(2005),

Chiovittiet

al.(2004),

Han

lonet

al.(2006),

Staats

etal.(1999)

729�

36

328�

14

269�

11

248�

8245�

5

HW-CHO:HW-O

C0.64

0.41

0.45

0.53

0.58

Hot-bicarbonate-

extractedorgan

ic

carbon(HB-O

C)

Insoluble

andHMW

material,EPS

stalks,gels,

cellcontents

Han

lonet

al.(2006)

2708�

59

2529�

80

2001�

95

2318�

54

1659�

26

Hot-bicarbonate-

extracted

carbohydrate

(HB-CHO)

Insoluble

heteropolymers,

gelsrich

in

deo

xysugars,

uronic

acids

Abdullahiet

al.(2006),

Bellinger

etal.,2005;DeBrouwer

etal.

(2003),Han

lonet

al.(2006)

488�

121

611�

78

425�

45

478�

74

444�

14

HB-CHO:HB-O

C0.18

0.24

0.21

0.21

0.27

Totalorgan

ic

carbon(TOC)

Allorgan

iccarbon,PO

Can

dDOC

fractions,

includingam

inoacids,

lipids

Lomsteinet

al.(1998)

18788�

1498

18480�

343

Total

carbohydrate

(TCHO)

Alldissolved

andparticulate

carbon,

structuralpolysaccharides,detritus

DeBrouwer

etal.(2003),

Han

lonet

al.(2006)

2894�

139

1899�

172

1893�

93

2046�

299

1948�

55

TCHO:TOC

0.11

0.11

FEMS Microbiol Ecol 84 (2013) 495–509 ª 2013 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Anaerobic degradation of biofilm organic matter 497

determined by two-way analysis of variance, with treat-

ment (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) commu-

nity composition was undertaken.

Methane

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 chro-

matograph 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 min�1, oven temperature

at 100 °C and the injector and detector at 160 °C.

Sulphate and acetate

5 mL of slurry was filtered (0.2 lm) and diluted 100-fold

or 10-fold (for sulphate and acetate, respectively) with Mil-

liQ 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).

Sulphide

Dissolved and precipitated sulphide was measured spec-

trophotometrically 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 intra-

and 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 charac-

terisation of changes in various organic fractions identi-

fied on the basis of solubility, as well as decreases in the

overall total organic carbon (TOC) and total carbohy-

drate 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 Ana-

lyzer using the nonpurgeable organic carbon method with

1% acid ratio and 3-min sparge. The sediment pellets

(retained after removing the supernatant from the centri-

fuged 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 bicar-

bonate-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 sus-

pended sediment and biomass) was dried, acidified with

2 M HCl (to remove inorganic carbon) and measured for

TOC using a Shimadzu SSM5000 analyzer.

Carbohydrate

Carbohydrate in the four different fractions was measured

spectrophotometrically (485 nm) after the phenol–sulphu-ric acid reaction (Dubois et al., 1956) as described by

Hanlon et al. (2006) and quantified as glucose equivalents

(lg g�1 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)].

Extracellular b-glucosidase and aminopeptidase

activity

Maximum potential rates of extracellular b-glucosidaseand aminopeptidase activity were determined using satu-

rating concentrations (1 mM) of fluorescently labelled

substrates MUF-b-D-glucopyranoside or leucine-7-amino-

methylcoumarin (Sigma-Aldrich, Dorset, UK) (Hoppe, 1983)

after adding 0.2% w/v sodium azide (metabolic inhibitor

to prevent de novo enzyme production). Incubation condi-

tions, controls, spectrophotometry and calculation of rela-

tive 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

ª 2013 Federation of European Microbiological Societies FEMS Microbiol Ecol 84 (2013) 495–509Published by Blackwell Publishing Ltd. All rights reserved

498 B.A. McKew et al.

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-ampli-

fied 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 (Parameswa-

ran 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 °Cfor 10 min, and was performed in a Gene Amp� PCR

system 9700 Thermocycler (Applied Biosystems). The

PCR products were cleaned using a QIAquick PCR purifi-

cation 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, aero-

bic and anaerobic) were multiplexed on 1/8 of a 454 GS-

FLX 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 fur-

ther 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

(number and relative abundance of OTUs) was calculated

using Shannon–Wiener’s index, and differences in com-

position were calculated using Jaccard’s index and com-

pared 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 statisti-

cal measure that does not require replication to produce

probabilities (Ma�cek et al., 2011). All analyses were con-

ducted in the R statistical language version 2.7.2 using

the R standard libraries and the community ecology anal-

ysis-specific package ‘vegan’ (R Development Core Team,

2011).

Results

Development of anaerobic conditions

There was evidence of anaerobic conditions in the sedi-

ment 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 signif-

icant 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 approxi-

mately 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 con-

centration of 191 lM on day 3, before reducing sharply

to the initial levels by day 10 (Fig. 1c). Methane concen-

trations 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

FEMS Microbiol Ecol 84 (2013) 495–509 ª 2013 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Anaerobic degradation of biofilm organic matter 499

concentrations of extractable fractions of carbohydrates

[DCHO, HW-CHO and HB-CHO fractions totalled

7.42 mmol C L�1, equivalent to 1261 lg C (g dry wt. sed-

iment)�1], DOC [DOC, HW-OC and HB-OC fractions

totalled 25.97 mmol C L�1, equivalent to 4414 lg C (g

dry wt. sediment)�1] and TCHO [17.03 mmol C L�1,

equivalent to 2894 lg C (g dry wt. sediment)�1]. Carbohy-

drate-carbon represented between 8% (dissolved extracts)

and 64% (HW extracts) of the TOC in these fractions

(Table 1).

Aerobic degradation

Concentrations of DOC decreased rapidly and signifi-

cantly 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 signif-

icant 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 concentra-

tions 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 carbohy-

drate at the beginning of the experiment, declining to

53% after 25 days (Table 1). In contrast, HB-OC concen-

trations [a higher-molecular-weight (HMW), more insol-

uble fraction of the organic carbon pool] remained

relatively constant in the oxic microcosms, with no signif-

icant changes over 25 days (Fig. 2e). TCHO concentra-

tions (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 activ-

ity (aminopeptidase and b-glucosidase) declined by 40%

and 92%, respectively, in the oxic slurries during the

experiment. Enzyme activity rates were significantly posi-

tively 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-glucosidaseand 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 aminopepti-

dase activity with DCHO concentrations (r = �0.582,

P < 0.001). The rate of reduction in DOC

(mmol DOC L�1 day�1), calculated between each time

point, was positively correlated with the rates of b-gluco-sidase (r = 0.84, P < 0.05) and aminopeptidase (r = 0.93,

P < 0.01) activity.

Anaerobic degradation

The development of anoxic conditions caused major dif-

ferences in the patterns of concentrations of organic car-

bon and carbohydrate and enzyme activities compared

with the oxic treatments. There was a significant 42%

0

20

40

60

80

0 5 10 15 20 25

0

1000

2000

3000

40000

50

100

150

200

2500

2

4

6

80

10

20

30

40

OxicAnoxic

Am

mon

ium

(μM

)M

etha

ne (p

pm)

Ace

tate

(μM

)S

ulph

ide

(mM

)S

ulph

ate

(mM

)

Day

P trt < 0.0001P time <0.0001P trt x time <0.0001

P trt <0.0001P time <0.0001P trt x time <0.0001

P trt < 0.001P time <0.0001P trt x time <0.0001

P trt < 0.0001P time <0.0001P trt x time <0.0001

P trt = 0.023P time <0.0001P trt x time <0.0001

(a)

(b)

(c)

(d)

(e)

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).

ª 2013 Federation of European Microbiological Societies FEMS Microbiol Ecol 84 (2013) 495–509Published by Blackwell Publishing Ltd. All rights reserved

500 B.A. McKew et al.

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 signifi-

cantly 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),

which continued until day 25 (39% decrease). This reduc-

tion in HB-OC did not consistently match changes in

HB-CHO concentrations, which declined by only approxi-

mately 10% at day 25 (Table 1). TCHO declined in a simi-

lar 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 poten-

tial rate) was reduced significantly in the anoxic micro-

cosms compared with oxic treatments (Fig. 2g). The

highest rates were significantly positively correlated with

TCHO, HW-CHO, DOC, HW-OC and HB-OC concen-

trations (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-glucosidaserates over time was identical in anoxic and oxic slurries

(Fig. 2h). However, in the anoxic treatment, there were

0255075

100125150 Oxic

Anoxic

0

25

50

75

100

125

0

25

50

75

100

125

0

25

50

75

100

125β-

gluc

osid

ase

TCH

OH

W-C

HO

DC

HO

Am

inop

eptid

ase

HB

-OC

HW

-OC

DO

C(r

elat

ive

rate

)(%

rem

aini

ng)

(% re

mai

ning

)(%

rem

aini

ng)

(rel

ativ

e ra

te)

(% re

mai

ning

)(%

rem

aini

ng)

(% re

mai

ning

)

P trt <0.0001P time <0.0001P trt x time <0.0001

P trt =0.002P time <0.0001P trt x time <0.0001

P trt <0.0001P time <0.0001P trt x time =0.003

P trt <0.0001P time <0.0001P trt x time <0.0001

0

100

200

300

400

0

25

50

75

100

125

0

25

50

75

100

125

0

25

50

75

100

125

0 5 10 15 20 25 0 5 10 15 20 25

P trt <0.0001P time <0.0001P trt x time <0.0001

P trt = nsP time <0.0001P trt x time <0.0001

P trt <0.009P time <0.0001P trt x time =0.034

P trt = nsP time <0.0001P trt x time = ns

Day Day

(a) (b)

(c) (d)

(e) (f)

(g) (h)

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).

FEMS Microbiol Ecol 84 (2013) 495–509 ª 2013 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Anaerobic degradation of biofilm organic matter 501

significant positive correlations between b-glucosidaseactivity and the rates of loss (mmol L�1 day�1, 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 concentra-

tions (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 rep-

licates 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 dis-

tinct assemblages of bacteria between oxic and anoxic

microcosms that become increasingly dissimilar through

time (Fig. 3). To test this observation, pairwise randomi-

sation tests comparing Jaccard’s index across samples

were conducted. Comparisons of Jaccard’s index (bacte-

rial species turnover) confirmed significant differences in

bacterial composition between oxic and anoxic micro-

cosms (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). Clostri-

dia (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 con-

sensus sequences from the clusters within this group

revealed that the majority were most closely related to

Clostridia sequences (92–93% similarity). Deltaproteobac-

teria 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 num-

bers of Gammaproteobacteria sequences were approxi-

mately 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 rela-

tive abundance from 2.2% to 0.1% by day 3.

Survival of diatoms

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 rela-

tive abundance of both dead and live diatoms was deter-

mined by phase-contrast microscopy. Empty frustules

(dead cells) represented 80% (� 5.4%) of the total valve

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 FEMS Microbiol Ecol 84 (2013) 495–509Published by Blackwell Publishing Ltd. All rights reserved

502 B.A. McKew et al.

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 Navi-

cula 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 con-

trast, oxic slurries contained 22 morphologically identified

species that were alive, including the aforementioned taxa,

but also Gyrosigma attenuatum, G. limosum, Navicula gre-

garia, 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 abun-

dance 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 bacte-

rial degradation following leakage from live cells or

release from dying or dead cells (especially in the anaero-

bic treatments). Whilst some organic material may be

degraded faster under oxic conditions (e.g. chlorophyll

(Sun et al., 1993), and refractory material from aged dia-

toms (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 degrada-

tion rates of diatom EPS compared with glucose have

been measured in sediments (Goto et al., 2001), suggest-

ing some specificity for chemically complex carbohydrate-

rich 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

being attributed to the initial hydrolytic attack (Kristen-

sen 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 pro-

portion 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 (particu-

larly 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; Kristen-

sen, 1993; Volkman et al., 2000). Our findings of faster

rates of degradation of the more insoluble HB-OC frac-

tion, 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 bio-

film 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 commu-

nity (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-glucosi-dase is the rate-limiting step (Meyer-Reil, 1990). b-Gluco-sidase 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 heterotro-

phy 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 degrada-

tion of organic carbon were observed under both condi-

tions, 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).

FEMS Microbiol Ecol 84 (2013) 495–509 ª 2013 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Anaerobic degradation of biofilm organic matter 503

A multifunctional anaerobic community

Compositionally distinct bacterial communities devel-

oped 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 meta-

bolic diversity within the Clostridia, all highlight their

major role in anaerobic degradation in intertidal sedi-

ments. 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 sub-

strates, releasing a range of fermentation products, such

as acetate (which was shown to increase to highest lev-

els 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, particu-

larly sulphate-reducing bacteria in marine sediments

(Purdy et al., 2002).

Whilst methanogenic Archaea are not detected by

our Bacteria-specific primers, their presence is con-

firmed by the continuous production of methane in

the anoxic slurries (Fig. 1d). Some methanogens are

outcompeted by sulphate-reducing bacteria for sub-

strates 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 (McGe-

nity, 2010) from glycine betaine and dimethylsulpho-

niopropanate, 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 methanogen-

ic community in intertidal sediments (Purdy et al.,

2003) and that intertidal sediments can act as a signifi-

cant source of methane to the atmosphere (Middelburg

et al., 2002). Methanogens able to utilise competitive

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 contrib-

ute 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. Delta-

proteobacteria 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 Desulfo-

bulbaceae. Members of these families have diverse meta-

bolic 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 fermen-

tation by Clostridia and other fermenters. Rates of sul-

phide 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

ª 2013 Federation of European Microbiological Societies FEMS Microbiol Ecol 84 (2013) 495–509Published by Blackwell Publishing Ltd. All rights reserved

504 B.A. McKew et al.

with 13C-labelled Spirulina biomass in an intertidal sedi-

ment slurry (Graue et al., 2012). Graue et al. (2012)

showed that members of the phylum Fusobacteria were

also implicated in fermentative degradation of cyanobac-

terial biomass; however, whilst they increased in abun-

dance 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 sin-

gle-cell genomics to identify Verrumicrobia phylotypes as

the main degraders of the storage polysaccharides, lami-

narin and xylan, in coastal surface waters.

Survival/growth of oxygenic phototrophs

Many diatoms in the anoxic slurries died during the experi-

ment, evidenced microscopically at day 25 and also by the

reduction in diatom chloroplast sequences at day 10. Dia-

toms 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

Relative abundance (%)

Acidobacteria; Acidobacteriaceae 0.0 0.2 0.5 0.1 0.1

Actinobacteria 1.5 0.2 1.0 0.5 0.4

Bacteroidetes; Bacteroidales 0.0 0.5 0.0 0.1 0.0

Bacteroidetes; Flavobacteriales 7.6 8.8 9.0 5.7 6.8

Bacteroidetes; Sphingobacteriales 0.5 0.9 1.5 0.9 0.2

Bacteroidetes; unclassified 4.1 5.5 5.7 9.9 11.0

Firmicutes; Clostridia 0.3 0.5 0.3 6.3 6.5

Firmicutes; other 0.0 0.2 0.3 0.6 0.9

Fusobacteria; Fusobacteriales 0.5 0.2 0.0 1.2 0.7

Alphaproteobacteria; Rhizobiales 1.8 0.2 0.0 0.5 0.0

Alphaproteobacteria; Rhodobacteraceae 7.5 7.4 3.6 2.5 1.7

Alphaproteobacteria; Sphingomonadaceae 0.3 0.2 0.5 0.0 0.2

Alphaproteobacteria; unclassified 1.5 1.4 0.3 0.9 0.9

Deltaproteobacteria; Bacteriovorax 0.1 0.5 0.3 0.2 0.0

Deltaproteobacteria; Desulfobacteraceae 1.9 0.7 0.8 8.4 12.2

Deltaproteobacterial; Desulfobulbaceae 1.5 0.2 0.8 4.7 4.3

Deltaproteobacteria; unclassified 0.0 0.0 0.0 0.1 0.1

Deltaproteobacteria; Desulfovibrio 0.0 0.0 0.0 0.2 0.0

Deltaproteobacteria; Desulfuromonales 0.0 0.0 0.0 0.2 0.0

Deltaproteobacteria; Myxococcales 0.0 0.5 0.3 0.1 0.2

Deltaproteobacteria; unclassified 4.4 9.7 7.2 6.9 4.9

Epsilonproteobacteria 3.6 1.2 0.8 0.7 0.5

Gammaproteobacteria; Alteromonadales 0.1 0.0 0.0 0.0 0.1

Gammaproteobacteria; Chromatiales 0.0 0.2 0.0 0.0 0.1

Gammaproteobacteria; Vibrionaceae 0.0 0.2 0.0 0.0 0.1

Gammaproteobacteria; unclassified 18.3 19.9 17.3 9.5 9.9

Proteobacteria; unclassified 5.4 5.5 7.0 2.5 5.0

Spirochaetes; Spirochaetaceae 0.0 0.0 0.0 0.8 2.1

Verrucomicrobia; Verrucomicrobiales 1.2 3.9 7.0 1.5 0.7

BRC1 genera incertae sedis 0.3 0.0 0.0 0.0 0.0

Unclassified bacteria 8.9 9.7 17.5 18.2 21.1

Cyanobacteria 2.2 2.5 0.0 0.1 0.0

Bacillariophyta (diatoms) 25.4 18.2 18.3 16.7 9.2

Unclassified 1.2 0.7 0.3 0.2 0.1

FEMS Microbiol Ecol 84 (2013) 495–509 ª 2013 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Anaerobic degradation of biofilm organic matter 505

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 dissimi-

latory 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 insol-

uble 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 refrac-

tory 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 deg-

radation of EPS consisted of diverse taxa with hydrolytic,

fermentative, methanogenic and particularly sulphate-

reducing capabilities. Some biofilm diatom taxa also sur-

vived the anoxic conditions, raising the question of het-

erotrophic 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).

References

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