Limnol. Oceanogr., 57(6), 2012, 1846-1856
© 2012, by the Association for the Sciences of Limnology and Oceanography, Inc.
doi: 10.4319 /I0 .2 OI2.57.06.1846
T ransform ation and fate o f m icrophytobenthos carbon in subtropical shallow subtidal
sands: A 13C-labeling study
Joanne M. Oakes,3’* Bradley D. Eyre,3 and Jack J. Middelburg b’c
a Centre for Coastal Biogeochemistry, Southern Cross University, Lismore, New South Wales, Australia
b D epartm ent of Ecosystems, Royal Netherlands Institute for Sea Research, Yerseke, The N etherlands
e Faculty of Geosciences, U trecht University, U trecht, The N etherlands
A bstract
M icrophytobenthos (MPB) in photic sediments are highly productive but the fate of this production remains
uncertain. Over 33 d, tracing of 13C from added bicarbonate in subtropical shallow subtidal sand showed rapid
transfer of MPB-derived carbon to deeper sediment; below 2 cm (31% within 60 h) and 5 cm (18%). Despite their
high turnover (5.5 d) and only representing ~ 8% of sediment organic carbon, MPB represented up to 35% of the
13C retained in sediments, dem onstrating substantial carbon recycling. Carbon was rapidly transferred to
heterotrophs, but their contribution to sediment 13C was similar to their biomass contribution (< 0.1% to 3.8%),
with the exception of the foraminifera Cellanthus craticulatus, which accounted for up to 33% of the 13C within
sediment. There was little loss of MPB-derived carbon via dissolved organic carbon (DOC) effluxes (3%) or
resuspension (minimal). Respiration was the m ajor loss pathway (63%), reflecting the high microbial biomass
typical of lower latitudes. Given that MPB take up dissolved inorganic carbon (DIC) from overlying water, and
the carbon they fix is released as D IC, MPB in subtropical sands are unlikely to substantially alter the form of
carbon transported offshore (i.e., there is no conversion to DOC), but processing within the sediment may alter its
<513C value. Given that 31% of fixed carbon remained in sediments after 33 d, subtropical sands may act as a
carbon sink, thereby affecting the quantity of carbon transported offshore.
Sedim ents w ithin the photic zone m ay be colonized by
highly productive m icroscopic photosynthetic organism s
(C ahoo n 1999), collectively referred to as m icrophyto­
benthos (M PB), and m ay therefore play an im p o rtan t role
in intercepting and m odifying carb o n inputs from the coast
to the ocean.
T he functional and tro p h ic im portance o f M PB in
coastal systems is widely recognized. M PB can affect
m ovem ent o f n utrients, oxygen, and carb o n across the
sedim ent-w ater interface (C ah o o n 1999; Eyre and F erg u ­
son 2005). P articularly w here nutrien ts are lim ited (C ook et
al. 2007), m uch o f the carb o n fixed by M PB is secreted as
extracellular polysaccharides (EPS; u p to 73% o f p ro d u c ­
tivity; G o to et al. 2001), prim arily carb o h y d rates (U nder­
w ood et al. 2004; O akes et al. 20106). EPS reduces
resuspension by stabilizing sedim ents (C ahoon 1999) and,
in additio n to direct grazing on M PB , can be a significant
labile carb o n source for consum ers (M iddelburg et al.
2000 ).
T rophic tran sfer o f carb o n fixed by M PB has received
considerable atten tio n , p articularly in intertid al silty and
m uddy sedim ents (M iddelburg et al. 2000; M oens et al.
2002; O akes et al. 2010«). M PB are aiso an im p o rtan t
carb o n source in sands (H erm an et al. 2000; M iddelburg et
al. 2000; E vrard et al. 2010), b u t studies have largely been
in tem perate regions. C arb o n cycling in the subtropics m ay
be fundam entally different, due to the high bacterial
biom ass at low latitudes (A longi 1994). H ow ever, the role
o f M PB as a carb o n source for consum ers in subtropical
sands is poorly understood.
* Corresponding author: joanne.oakes@ scu.edu.au
A fu rther gap in current understanding o f coastal carbon
cycling is the fate o f M PB-derived carbon. Follow ing
tran sfo rm atio n an d /o r burial, M PB -derived carb o n m ay be
lost to the w ater colum n via resuspension, fluxes o f
dissolved inorganic carb o n (D IC ) follow ing m ineralization
or respiration (G oto et al. 2001; E vrard et al. 2012), and/or
fluxes o f dissolved organic carb o n (D O C) derived from
exudates (Sm ith and U nderw ood 2000) and, to a lesser
extent, cell lysis during grazing. These pathw ays m ay be an
im p o rta n t com p o n en t o f coastal c arb o n cycling. F o r
exam ple, sedim ent fluxes can contribute significantly to
the D O C in coastal w aters (M aher and Eyre 2011), and
M PB has been proposed as a m ajor source (M aher and
Eyre 2010). H ow ever, the pathw ays for loss o f M PBderived c a rb o n fro m sedim ents have n o t been wellcharacterized.
F o r tem perate, photic, subtidal, sandy sedim ents C ook
et al. (2007) and E v rard et al. (2008) reported negligible loss
o f M PB-derived carbon to overlying w ater over 6 d and 4 d,
respectively. C ook et al. (2007) recovered only 1-5% o f
fixed carbon as D O C . In co ntrast, Oakes et al. (20106)
reported loss from in situ surficial subtropical photic sands
o f 85% o f M PB-derived carbon over 33 d. The pathw ays
responsible for this loss were n o t determ ined, b u t this
highlights th at loss to the w ater colum n m ay be the m ain
fate o f c a rb o n fixed by M PB in th is en v iro n m en t.
A lternatively, carb o n m ay have been buried deeper w ithin
sedim ents (below 2 cm; O akes et al. 20106). The greater loss
o f carbon from subtropical sands com pared w ith tem perate
sands m ay relate solely to the longer period o f study or the
m ethods used. F o r exam ple, given th a t the sedim ent
studied by C ook et al. (2007) and E v rard et al. (2008)
was ex situ, and therefore w as n o t subject to natu ral
1846
M icrophytobenthos carbon fa te in sands
environm ental flows and the effects o f m obile grazers, loss
o f fixed carb o n m ay have been underestim ated. In support
o f this, M iddelburg et al. (2000) reported substantial loss of
fixed carb o n ( ~ 53%) over 4 d from in situ tem perate
sands, albeit in the in tertidal zone. This w as estim ated to be
prim arily due to resuspension ( ~ 60%; M iddelburg et al.
2000), b u t losses o f carb o n as D IC and D O C were not
m easured. The different loss rates for carb o n in subtropical
an d te m p e ra te san d s m ay also reflect c h ara cteristic
differences betw een sands at these latitudes. H igh bacterial
biom ass and productivity is typical o f low er latitudes
(A longi 1994; Eyre and Balls 1999) and this m ay account
for the greater loss o f fixed carb o n observed for subtropical
sands.
Stable isotopes at n a tu ra l ab undance are a p o p u lar tool
for determ ining carb o n flows in aquatic systems, but
in terp retatio n can be com plicated by fractionation and
variability an d /o r sim ilarity in source signatures (Peterson
1999). D eliberate application o f the rare stable isotope of
carb o n , 13C, can overcom e these problem s, allow ing
tro p h ic transfers o f carb o n to be traced and quantified
(M iddelburg et al. 2000; O akes et al. 2010«). M ore recently,
it has been possible to trace 13C into fluxes o f D O C and
D IC . This has been d em onstrated for n atu ral abundance
<513C (R aym ond and B auer 2001; O akes et al. 2010c; M aher
and E yre 2011), and in 13C-labeling experim ents (Andersson et al. 2008; O akes et al. 2011; van den M eersche et al.
2011). In the current study, we aim ed to use a com bination
o f 13C-labeling, com pound-specific isotope analysis o f
biom arkers, and isotope analysis o f D O C and D IC to
investigate the fate o f M PB -derived carb o n in subtropical
photic subtidal sandy sedim ents. Specifically, we aim ed to
d ete rm in e ra te s o f c a rb o n tra n s fe r w ith in sedim ent
com p artm en ts and rates o f c arb o n loss to the w ater
colum n. W e hypothesized th a t D IC fluxes w ould be a
m ajo r path w ay for loss o f M PB-derived carbon, due to the
high bacterial biom ass typical o f the subtropical sedim ents
studied, and the high affinity o f bacteria for the labile D O C
exuded by M PB. Because we were particularly interested in
pathw ays o f carbo n loss, the experim ent w as done in situ,
allow ing n atu ral loss processes to occur, and over an
ex ten d ed tim e p e rio d (33 d). G iv en th e p au city o f
info rm atio n on the tran sfo rm atio n and fate o f carbon
fixed by M PB , particularly in subtropical photic sands, this
is a valuable step to w ard understan d in g the role o f M PB in
coastal carb o n cycling.
Methods
Two o f the experim ental plots used in the current study,
and the cores collected from these plots, were also used by
Oakes et al. (20106). H ow ever, the current study utilized an
additional 13C-labeled plot (i.e., three plots in total).
Study site— The subtidal study site ( ~ 1.5 m below
average sea level) in the Brunsw ick R iver, subtropical
A ustralia, w as described by O akes et al. (20106). The site
w as net au to tro p h ic (production : respiration ~ 1.2 over
24 h; J. M . O akes unpubl.). G ross productivity based on
C 0 2 fluxes averaged ~ 105 m m ol C m -2 d -1 (J. M . Oakes
1847
unpubl.). T he organic carbon (OC) content o f sedim ent to
10 cm depth w as 18.6 m ol C m - 2 . Surface sedim ents (0 2 cm) had a higher OC content (0.18%) and a lower m olar
C : N ratio (6.9 ± 0.7) th a n deeper sedim ents (0.16%, 9.7 ±
0.8 at 2-5 cm; 0.15% , 9.8 ± 0.6 at 5-10 cm). The sands were
n o t perm eable. Sedim ent at all depths was com posed
prim arily o f fine (125-250 /im; 72-79% ) and m edium (250500 /im; 17-24% ) qu artz sand grains.
13C-labeling— 13C-labeling o f three experim ental plots
(1.2 m 2) w as achieved by 13C-labeling the w ater colum n
D IC pool (23% 13C) in benthic cham bers using N a H 13C 0 3,
as described by O akes et al. (20106). Pum ps m aintained
lam inar flow o f w ater across the sedim ent w ithin the
cham bers for 24 h. C ham bers were then rem oved.
Sam ple collection— Im m ediately after cham ber rem oval
and at 1, 3, 10, 20, and 30 d thereafter, tw o sedim ent cores
(9 cm diam eter X 20 cm deep, w ith 30 cm overlying w ater)
from each labeled experim ental plot and tw o control cores
from 5 m to 8 m outside o f each plot (for background
isotope values) were collected. In the lab o rato ry , control
and labeled cores w ere placed in sep a ra te ta n k s o f
unlabeled site w ater at in situ tem perature (± 1°C) and
light levels (± 5%, ~ 400 /im ol p h otons m -2 s-1 at
sedim ent surface, 12 h) and were preincubated (12 h dark,
then 12 h light), then sealed and incubated (12 h d ark , then
12 h light) as described by O akes et al. (20106). M agnetic
bars ~ 10 cm above the sedim ent gently stirred w ater
w ithin cores. P reincubation was intended to allow sedim ent
m icrohabitats to re-establish, thus m inim izing the effects on
fluxes due to sedim ent disturbance, but use o f cham bers
and preincubation prevented m onitoring o f 13C losses over
the first 48 h, and sedim ent transfers over the first 60 h,
following labeling. The focus o f the study w as therefore on
longer term transfer and loss processes.
A t the beginning and end o f the d ark incubation period
and at the end o f the light incubation period, ~ 50 m L o f
w ater w as rem oved from each core to determ ine concen­
tratio n s and carbon stable isotope ratios (<513C) o f D O C
and D IC . Sam ples were syringe-filtered (precom busted G F /
F), leaving no headspace, into precom busted 40 m L glass
vials w ith T eflon-coated septa and were killed (200 /tL
50: 50 w : V ZnC L) and refrigerated until analysis. Sample
w ater was replaced, as it w as w ithdraw n, w ith site w ater
from a collapsible reservoir. A dditional w ater sam ples, not
included in this study, were also collected.
Sedim ent w as extruded and sectioned (0-2 cm, 2 -5 cm,
and 5-10 cm depths) for one core from each plot and for
one o f each p air o f control cores at the end o f the dark
incubation, and from rem aining cores at the end o f the light
incubation. A know n p o rtio n o f each sedim ent sam ple was
freeze-dried fo r <513C analysis o f sedim ent O C and
phospholipid-derived fatty acid (PL FA ) biom arkers for
bacteria and M PB. The rem ainder was stored frozen
(-2 0 ° C ) for separation o f fauna.
Low a b u n d a n c e o f m a c ro fa u n a at the stu d y site
prevented assessm ent o f their use o f M PB. W e therefore
focused on th e m eio b en th o s, w hich, at th e tim e o f
sam pling, w as dom inated by three species o f foram inifera
1848
Oakes et al.
(Cellanthus craticulatus, A m m onia beccarii, and Elphidium
advenum; 95-98% o f to tal m eiobenthos biom ass). F o ram i­
nifera were picked, by h an d , from sedim ent sam ples to
ob tain sufficient m aterial for isotope analysis.
Sam ple analysis— D O C and D IC co ncentrations and
<513C were m easured, as described by O akes et al. (2010c,
2011), via co ntinuous-flow w et-o x id atio n iso to pe-ratio
m ass spectrom etry using an A u ro ra 1030W to ta l organic
carb o n analyzer coupled to a T herm o D elta V Plus Isotope
R atio M ass Spectrom eter (IR M S). Sodium bicarbonate
(D IC ) and glucose (D O C ) o f know n isotope com position
dissolved in helium -purged milli-Q were used for drift
correction and to verify co ncentrations and S 13C values.
R eproducibility for D O C and D IC , respectively, was ±
0.3 m g L -1 and ± 0.4 m g L -1 (concentrations) and ±
0.08%o and ± 0.10%o (<513C).
P L F A biom arkers to determ ine 13C u p tak e into bacteria
and M PB were extracted from lyophilized sedim ents using a
m odified Bligh and D yer m ethod, as described by O akes et
al. (20106). A n internal stan d ard (tridecanoic acid, C i3) was
added at the beginning o f the extraction procedure. Extracts
were analyzed via gas ch ro m ato g rap h y -iso to p e ratio m ass
spectrom etry, as described by O akes et al. (20106).
F oram inifera and sedim ent sam ples were dried in silver
cups (60°C to co n stan t weight), acidified (10% HC1),
redried (60°C), then analyzed for S 13C and % C (% o f OC
in the unacidified sam ple) using a T herm o F innigan Flash
E A 112 interfaced via a T herm o C onfio III w ith a Therm o
D elta V Plus IR M S . H elium dilution o f the carrier stream
was tu rn ed o ff for foram inifera sam ples to reduce the
required mass.
Calculations— P L F A S 13C values were corrected for the
additio n o f a carb o n ato m during m éthylation, as described
by Jones et al. (2003). N a tu ra l abundance <513C values for
bacteria and M PB were calculated using 6 13C values o f
P L F A s specific to bacteria and diato m s from control cores,
as described by O akes et al. (20106).
T otal u p tak e (in corporation) o f 13C into sedim ent OC,
foram inifera, bacteria, and M PB (/anoi 13C m -2 ) was
calculated as the p ro d u ct o f excess 13C (fraction 13C in
labeled sam ple - fraction 13C in control) and the m ass o f
OC in each com partm ent. F o r sedim ent and foram inifera,
OC m ass was the p ro d u ct o f % C and to ta l dry m ass per
unit area. F o r foram inifera, counts o f individuals w ithin
sedim ent subsam ples were scaled u p to provide an estim ate
o f the to tal num ber o f individuals per m 2 at each depth.
T o tal m ass w as determ ined by m ultiplying this estim ate by
the average m ass o f an individual, w hich w as determ ined by
w eighing a know n num ber o f individuals.
C oncentrations o f P L F A biom arkers for bacteria and
M PB were calculated based on their peak areas relative to
th a t o f the C 13 internal stan d ard . T o tal biom ass o f bacteria
and M PB w as calculated as described by O akes et al.
(20106), based on the bacterial biom arkers i l 5:0 and al5:0,
and the algal biom arkers 16:l(n-7) and 20:5(n-3), which
gave distinct chrom ato g rap h ic peaks. T he average fraction
o f M PB P L F A s th a t is typically accounted for by the
biom arkers considered is 0.37 (V olkm an et al. 1989).
T otal 13C transfer into w ater colum n D O C and D IC was
calculated for the beginning and end o f the d a rk incubation
period and for the end o f the light period as the p ro duct o f
excess 13C in D O C or D IC (fraction 13C in labeled sam ple
- fraction 13C in equivalent control), core volum e, and the
concentration o f D O C or D IC . T he to tal flux o f excess 13C
in D O C or D IC during d a rk or light incubation w as then
calculated as follows:
Excess13C flux = (Excess13C start —Excess13C end ) /S A /r
(1)
where excess 13C start and excess 13Cend represent excess 13C at
the beginning and end o f the dark or light incubation period,
SA is the sediment surface area w ithin a core, and t represents
hours o f dark or light incubation. N et fluxes (excess 13C
m -2 d -1 ) o f D O C or D IC were calculated as follows:
N et flux = (dark flu x /d ark hours)
+ (light flux/light hours) x 24 hours
W e interpolated between m easured net flux values and
estim ated the total quantity o f 13C lost via fluxes o f D O C and
D IC from the end o f labeling up until each sam pling period
by determ ining the area under the curve.
D ata analysis— Sedim ent OC, foram inifera, bacteria,
and M PB excess 13C values w ere determ ined for the end
o f b o th d ark and light periods. W e therefore used threeway analyses o f variance (A N O V A s) to determ ine w hether
these d a ta should be treated separately for each depth or
for each time. F actors o f depth, day, and light exposure
(dark or light) were used to test for an effect o f light
exposure alone or in co m b in atio n w ith o th er factors
(interaction w ith depth and/or day). Levene’s test was
significant in each case (variances were heterogeneous), so
alpha w as set at 0.01 to reduce the possibility o f falsely
rejecting the null hypothesis. Significant three-w ay in terac­
tions were explored using tw o-w ay A N O V A s, and signif­
icant tw o-w ay interactions were explored using one-way
A N O V A s. F o r exam ple, w here there was a depth X day
interaction, one-w ay A N O V A s were used to determ ine
w hether there w as a difference am ong depths on each
individual day. W here A N O V A s indicated a significant
m ain effect, post hoc Tukey tests showed w hich groups
were statistically different.
A 2-G m odel (W estrich and Berner 1984) w as used to
determ ine the rate o f 13C loss from to ta l sedim ent OC (0 10 cm) from each experim ental plot using the average o f
light and d a rk sedim ent <513C values (because these were not
significantly different) at each tim e, as follows:
G t( 0 = G\ [exp( —k \t)\ + G2[exp( —k 2t)\ + G n r
(3)
where GT is the in co rp o ratio n o f 13C w ithin sedim ent OC, t
is tim e, Gi, G2, and GNR represent the in co rp o ratio n o f 13C
into highly reactive, less reactive, and nonreactive (over the
tim escale o f the experim ent) fractions o f sedim ent OC, and
l<i and k 2 represent first-order decay constants specific to
Gi and G2, respectively.
T he isotope m ixing p ro g ram IsoSource (Phillips and
G regg 2003) calculates all feasible com binations o f m ultiple
1849
M icrophytobenthos carbon fa te in sands
possible sources to a m ixture, based on isotope ratios, and
w as therefore used to estim ate the likely co n trib u tio n of
M PB , p h y to p la n k to n , an d te rre stria l p lan ts to D O C
effluxes from the sedim ent.
Results
Three-w ay A N O V A s showed th a t there w as no signifi­
cant effect o f light exposure during lab o ra to ry incubation
on excess 13C in sedim ent O C, bacteria, foram inifera, or
M PB. Light and d a rk excess 13C values were therefore
com bined for subsequent calculations.
Sedim ent organic carbon composition and depth distribu­
tion — Sedim ent O C c o n ten t w as g reater in shallow er
sedim ents, w ith 223.56 ± 12.34 h mol C m L -1 in 0-2 cm
sedim ent com pared w ith 185.88 ± 8.71 /¡mol C m L -1 in 2 5 cm sedim ent and 172.12 ± 7.39 /¡mol C m L -1 in 5-10 cm
sedim ents. M PB , b acteria, an d fo ram in ifera to g eth er
accounted for 16.7%, 15.7%, and 18.0% o f the sediment
O C, respectively, in these sedim ent depths (Table 1). The
co n trib u tio n o f M PB and foram inifera to sedim ent OC was
greatest in surface sedim ents (Table 1), w hereas bacteria
m ade the greatest co n trib u tio n to OC in deeper sedim ents
(Table 1).
>> «2
-I
SÁ
S "g
U
<->
PD
-I
<
u ^O
r-v-tí
U £
o
° ë
N—
✓<L>
g s
’S
I
tí ití
°o tí
S
'S x>
§ 2
'O
r-i O O
2
1
V1
c- ^,10
08 ^ o Çp'Ç
'p
tíf
\ / 0^
co \c i—
io o V
—• 08 '■OOO'"t Cd CO
"o
OO k'OOb' i—i Cd
cc a 80
O
80 08Cd tí 80
'O V
O
ca oc lh rH
a
u
t—
h
OO O
O,
Vi 'C N Cd co 08 co
08 tri tri oc oc ö
777777?
£ 'S
1tí
«
i 'D
o **> a
js tí
u
o
tí r ,
au
CO
o
cd
tri
8¿
V 00
co co ;
co
q ^ q sw
tít-cöO
i—i Ö Ö Ö cd
'tí] cd Cd —<Cd xb
.2 "o
tri tri cd cd Ö '—i
ca a C- O
O1—
I 'tí co o
a
u
S ÖX)
OÖ
^ & 2
Ti
CO tí
tri
Crf
tri tri cd O co 'tí;
o o o V ¿ d
tí ootri en tí tí en
oi tí tí ÛO 80 o O
T TT T T i i
rn «3 ^
2O? M
co "to
(U
0 8 T t OO 8 0 H
O 8¿ h o O O tí
C
coO Ü
C
u
Incorporation o f 13 C and transfer am ong sedim ent
com partm ents — By the tim e the first sedim ent w as collected
(60 h after label addition), 1492 /onoi 13C m -2 was in
sedim ent OC 0-10 cm deep. A llow ing for m easured loss of
13C as D O C and D IC during the d ark in cubation period
preceding sedim ent sam ple collection, 1809 ± 1 1 0 a mol 13C
m -2 w as in 0-10 cm sedim ents 48 h after label addition.
This equated to an in co rp o ratio n o f 75 /onoi 13C m -2 light
h -1 until this point, w ith — 0.01% o f sedim ent OC replaced
w ith 13C. T rue rates o f 13C in co rp o ratio n are likely to have
been som ew hat higher, how ever, because we were unable to
account for losses o f 13C from the sedim ent during the
preincubation period. Based on 13C in co rp o ration, and
allow ing for the p artial labeling o f available D IC (23%
13C), to tal c arb o n fixation was estim ated to be — 328 /onoi
C m -2 light h -1 . This is far low er th a n the gross prim ary
productivity for the study site based on 0 2 and C 0 2 fluxes
(— 4000 /m o l m -2 h - 1 ; J. M . O akes unpubl.).
Fixed 13C w as detected in deeper sedim ents (2—5 cm and
5-10 cm deep) from the first sam pling tim e (Fig. 1). W ithin
60 h o f label addition, 31.5% o f the in co rp o rated 13C in
sedim ents was below 2 cm and 18.6% w as betw een 5 cm
an d 10 cm. A ssum ing fix atio n o f c a rb o n in deeper
sedim ents w as negligible, this gives tra n sp o rt rates of
8o co tn 8o i— < ■
O
t í (N L-
u ^ 2
N atural abundance stable isotope ratios — Sedim ent OC
w as m ore enriched in 13C in 0 -2 cm sedim ent th a n in deeper
sedim ents ( —17.9%o v s .
19.5%0; T able 1). A t all depths,
m ean d 13C values o f bacteria were depleted by up to 0.9%o
com pared w ith M PB (Table 1). A cross depths, sediment
OC w as depleted in 13C by 1.6%o to 5.6%o com pared w ith
M PB and bacteria.
W ater-colum n D IC and D O C in con tro l cores had m ean
<513C values o f 2.6%o and —23.3%o, respectively.
u
o
q
OC Cdt—<
O 08 co CO Ö O '
2
V1
o 2
I i Bcd
•Ü
»-i r—
OO
sCO ,
'o o
^
i—iö V
VV
V cd
oo
t í cd en
t í OÍ OÍ ^ t í
M 5^ N
Cd Ö
cd t í
ci
o
u
ö tí
o tí
CO t í T 80 T O
s s 'S
L" O 8080
■tí- 'tí i—i '
"tí-
ts U 3
'S
S oö %
o
^tí 'S
u
a 2
&
a xi <D
co tí
q 'tí; co q cd q 80
tri 80 o 80 tri oc
T T T T T TT
a
o
Ö B
B S
tí O
O h tí
sp «
«
i tí ^
Î .§ &
'2<u <
ÜD
co tí
C
c-d
co
tí
Oh
ao
O
^ O
o *tí
2Cu a<u
a 5
s ■S s3 O
utí
<U tí-: ! 'S
tí o : i 'S
y¡
S ’■o2tí
tí
fe] p
o 2
tí x
.
Oakes et al.
1850
0-2 cm
□
MPB
■
Bacteria
□
C. craticulatus
20- n
15-
A. beccarii
10-
E. advenum
□
5-
0
B i wl i i\
¿i i Ll \\è
10H 2-5 cm
uc/5
+1
§
U
6-
T3
D
tí
ë
i*
o
tí
4-
4" p II
: .I L Ü U
/
IL
5-10 cm
1 -
i
i
4
i
i
6
13
Days after labeling
r
23
33
Fig. 1. Excess 13C incorporation in m icrophytobenthos,
bacteria, and foraminifera (Cellanthus craticulatus, Ammonia
beccarii, and Elphidium advenum) at sediment depths of 0-2 cm,
2-5 cm, and 5-10 cm throughout the study period as a percentage
of the 13C initially incorporated into sediment organic carbon
(mean ± SE). N ote some bars and error bars are too small to be
seen. Scale on y-axis varies with sediment depth. Elphidium
advenum incorporation is too low to be seen.
10 /(mol 13C m -2 h _1 and 6 /anoi 13C m -2 h _1 to these
d epths, respectively. C onsidering th a t the added 13C
accounted for 23% o f the to tal w ater-colum n D IC available
to M PB, fixed carb o n was tran sp o rted to sedim ent below
2 cm at a rate o f 41 /onoi C m -2 h _1, and to sedim ent
below 5 cm at a rate o f 24 /onoi m -2 h _1.
Significantly m ore excess 13C w as m easured in sedim ent
OC at 0-2 cm th an at 2 -5 cm and 5-10 cm early in the
study (at 3 d and 4 d after label application, tw o-w ay
A N O V A : T2,5 = 12.515, p = 0.001 and Fx 5 = 8.005, p =
0.005, respectively; Fig. 1). H ow ever, by 13 d after label
application tran sp o rt o f 13C to deeper sedim ent, com bined
w ith 13C loss from surface sedim ents to the w ater colum n,
led to sim ilar excess 13C in sedim ent OC across depths (p >
0.01; depth X sam pling day interaction, three-w ay A N ­
OVA: ^0,35 = 3.836, p = 0.001; Fig. 1).
T he sedim ent com partm ents considered in the current
study (M PB, bacteria, and the three species o f foram inif­
era) accounted for approxim ately h a lf (51.6% ± 5.8%) o f
the 13C w ithin sedim ent OC across all sam pling times.
M PB in surface sedim ents (0-2 cm) initially accounted
for a far greater p o rtio n o f the 13C fixed into sedim ent OC
th an did M PB in deeper sedim ents (Fig. 1). W ithin each
sedim ent depth layer, the co n trib u tio n o f M PB to the 13C in
sedim ent OC far outw eighed the co n tribution o f M PB
biom ass to sedim ent OC. W hereas M PB biom ass repre­
sented only 6.6% to 9.0% o f the OC w ithin 0 -2 cm, 2 -5 cm,
and 5-10 cm sedim ent (Table 1), the average co n trib u tio n
o f M PB to 13C w ithin O C at each sedim ent d e p th
th ro u g h o u t the study period w as 34.8% ± 3.9%, 27.3%
± 3.7%, and 15.8% ± 3.5%, respectively. T h ro u g h o u t the
study, there was a m arked decrease in the 13C content o f
M PB in 0-2 cm sedim ent. By 33 d after label addition, only
2.9% o f the 13C initially fixed into sedim ent OC rem ained
in this com partm ent (Fig. 1). The in co rp o ratio n o f 13C into
M PB in 2 -5 cm and 5-10 cm sedim ents varied relatively
little th ro u g h o u t the study (Fig. 1) and w as significantly
low er th a n in 0 -2 cm sedim ents (three-w ay A N O V A : F->35
= 14.068, p < 0.001).
T he initial in co rp o ratio n o f fixed 13C into bacteria in 0 2 cm and 2 -5 cm sedim ent w as sim ilar (but note the
different volum es encom passed by the given depth ranges;
Fig. 1). A sm aller p o rtio n o f fixed 13C was in 5-10 cm
sedim ent at th e first sam p lin g p e rio d (Fig. 1). T he
co n trib u tio n o f bacteria to the 13C in OC w ithin each
sedim ent depth layer w as 6.7% ± 1.3%, 11.9% ± 2.2% , and
10.7% ± 3.5%, respectively, for sedim ent at depths o f 0 2 cm, 2-5 cm, and 5-10 cm. This was sim ilar to the
co n trib u tio n o f bacterial biom ass to OC w ithin each
sedim ent layer (Table 1). The 13C content o f bacteria was
sim ilar across sedim ent depths (p > 0.01) but varied
significantly am ong days (three-w ay A N O V A : F5tî5 =
3.559, p = 0.007; Fig. 1). This w as dom inated by a general
decrease in the 13C content o f bacteria in 0 -2 cm sedim ents
th ro u g h o u t the study period (Fig. 1).
T here w as evidence o f label u p tak e into all three
foram inifera species by the tim e the first sedim ent sam ple
was taken (Fig. 1). T he greatest in co rp o ratio n o f 13C, per
u n it b iom ass, w as by Cellanthus craticulatus, w hich
dom inated the foram inifera (Table 1). The greatest uptake
1851
M icrophytobenthos carbon fa te in sands
100✓
—\
w
on
+1
I
u
cn
80-
60-
T3
<U
1
I* 40o
5
20
-
0
5
10
15
20
Days after labeling
25
30
Fig. 2. Excess 13C incorporation in total sediment organic carbon (0-10 cm sediment depth) throughout the study period as a
percentage of the 13C initially incorporated into sediment OC (mean ± SE). Line represents the 2-G model predicting 13C loss from total
sediment organic carbon.
o f 13C by C. craticulatus was generally in sediment at 2-5 cm
(Fig. 1), where C. craticulatus accounted for u p to ~ 5% of
the to tal 13C initially fixed into sedim ents (Fig. 1). Despite
representing only 0.8% to 3.8% o f the biom ass w ithin each
sediment depth layer (Table 1), C. craticulatus accounted for,
on average, 9.6% ± 2.1%, 31.2% ± 5.9%, and 9.7% ± 2.9%
o f the 13C w ithin sediment O C at 0 -2 cm, 2 -5 cm, and 5 10 cm, respectively. In contrast, the contribution o f Am m onia
beccarii and Elphidium advenum to the 13C in sediment OC
was similar to their contribution to biom ass (Fig. 1; Table 1).
The incorporation o f 13C into A. beccarii was greater in 5 10 cm sediment th an in shallower sediments. A t any one time,
E. advenum accounted for < 0.01% o f the total 13C initially
incorporated into sediment OC (Fig. 1).
Loss o f 13C fro m sediments— The decline in 13C content
o f 0-10 cm sedim ents th ro u g h o u t the study period was
substantial and could be fitted w ith a 2-G m odel (R 2 =
0.98; Fig. 2). A highly reactive fraction o f sedim ent OC ( Gj :
60.67% ± 5.95%) was lost at a rate o f 1.51 ± 0.20 d -1 (iki),
and a less reactive fraction (G2; 17.18% ± 5.39%) degraded
at a rate o f 0.02 ± < 0.01 d _1 (k2). T he rem aining sediment
OC w as nonreactive over the tim escale o f this experim ent
(GNR; 24.09% ± 3.65%).
There was generally an efflux o f D IC from sedim ent in
the d a rk (1677 ± 252 /anoi C m -2 h _1) and an uptake in
the light ( —2319 ± 278 /anoi C m -2 h _1), resulting in net
D IC u ptake. F o r D O C , there w ere effluxes in b o th the dark
and the light (596 ± 235 /onoi C m -2 h _1 and 482 ±
211 /anoi C m -2 h _1, respectively).
Loss o f 13C from sedim ent to the w ater colum n was
dom inated by fluxes o f D IC , w ith very little 13C lost as
D O C (Fig. 3). M ost o f the loss o f 13C as D IC occurred over
the first few days o f the study, with nearly 50% o f the 13C
initially incorporated into sediments having been lost to the
w ater colum n as D IC within 6 days o f label addition (Fig. 3).
By the conclusion o f the study ~ 63% (59-67% ) o f
incorporated 13C had been lost to the w ater colum n as D IC ,
and ~ 3% (1.4—4.0%) as DO C. A pproxim ately 31% o f the 13C
th at was initially incorporated by M PB remained within the
sediment OC pool. O f this, ~ 12% (4.0-33.1%) was in
sediment at 0-2 cm, ~ 10% (4.6% to 17.7%) was at 2-5 cm,
and ~ 9% (3.0% to 13.6%) was at 5-10 cm (Fig. 3). Based on
excess 13C per volume, this was dom inated by OC within
surface sediments. A t the conclusion o f the study , only ~ 3%
of the initially incorporated 13C could not be accounted for by
the pathw ays and com partm ents considered.
Discussion
The 13C-labeling experim ent described in the current
study was prim arily done in situ, w ith cores rem oved to the
lab o rato ry shortly before incubation for sedim ent fluxes.
F o r m ost o f the study period, the sedim ent studied was
therefore subject to n atu ral loss and transfer processes,
including grazing by m obile consum ers, b io tu rb atio n , and
physical mixing and resuspension by w ater m ovem ent.
These processes are unable to be adequately replicated in a
lab o rato ry environm ent; therefore, in situ experim entation
w as essential for our focus on the longer term fate o f M PBderived carbon. A lthough the fate and tran sfo rm atio n o f
M PB-derived carb o n has previously been investigated in
situ (M iddelburg et al. 2000; Bellinger et al. 2009), this was
done in tem perate, intertidal sedim ents, w hich are subject
Oakes et al.
1852
100
□
/•"■s
80
w
t/5
I DIC
□ 0-2 cm sediment
[jíj 2-5 cm sediment
□
+1
U
DOC
5-10 cm sediment
1 60
'w '
nU
’O
I 40
i*
O
.5
20
0
3
4
6
13
Days after labeling
23
33
Fig. 3. Carbon budget showing excess 13C within sediment organic carbon at 0-2 cm, 2 5 cm, and 5-10 cm at each sampling time, and the cumulative excess 13C lost via fluxes of D IC
and D O C from the end of 13C-labeling until each sampling time, as a percentage of the 13C
initially incorporated into sediment organic carbon (mean ± SE).
to fundam entally different environm ental conditions com ­
pared w ith the subtropical subtidal sedim ent we studied. In
addition, the study by M iddelburg et al. (2000) used a
m ass-balance ap p ro ach to estim ate losses to respiration
and resuspension and did n o t estim ate D O C losses, and
Bellinger et al. (2009) did n o t m easure D IC and D O C
fluxes. In com parison, the current study directly quantified
losses to b o th D O C and D IC . It is im p o rtan t to n ote th a t
our experim ental procedure m ay have affected shorter term
processes, because the first sam ples collected h ad been
w ithin cham bers and cores (i.e., isolated from in situ
conditions) for 60 h. Sim ilarly, cores collected at other early
tim e points h ad a p ro p o rtio n ally small period during which
they were exposed to in situ conditions. This m ay have
affected o u r observations o f carb o n cycling in the short
term , particularly carb o n loss. H ow ever, for cores collected
tow ard the end o f the study, w hich were prim arily exposed
to in situ conditions, the q u an tity o f 13C rem aining w ithin
sedim ent OC m atched th a t expected based on earlier
observations. Sim ilarly, o u r budget accounted for ~ 97%
o f the added 13C over 33 d. A ny effect o f our experim ental
treatm en t on short-term carb o n cycling processes therefore
appears to be negligible.
N atural abundance isotope ratios— We previously deter­
m ined th a t OC in surface sedim ents (0-2 cm) at o ur study
site is derived prim arily from M PB ( ~ 56%) and pelagic
algae ( ~ 32%), w ith only a small co n trib u tio n o f terrestrial
plan t m aterial ( ~ 12%; O akes et al. 20106). In the cu r­
rent study, S 13C o f O C in deeper sedim ents was depleted
(~ —19.5%0; Table 1) com pared w ith surface sedim ents
( —17.9%o ± 1.3%o), indicating an increased co n trib u tio n o f
13C -depleted O C, possibly derived from terrestrial plant
m aterial ( —23%o to -3 0 % o; M ichener and Schell 1994). This
reflects the greater biom ass o f relatively 13C-enriched M PB
(—15.4%o ± 0.4%o) in surface sedim ents com bined w ith
rapid processing o f relatively 13C-enriched labile M PB
(Oakes et al. 20106) and pelagic algae-derived carbon in
surface sedim ents, com pared w ith m ore refractory 13Cdepleted terrestrial organic m atter, prio r to OC burial.
N a tu ra l abundance <513C o f D IC at the study site (2.56%o
± 0.59%o) was w ithin the range o f coastal D IC (Schidlowski 2000); however, n a tu ra l abundance S 13C o f D O C
(—23.31%o ± 1.03%o) is m ore difficult to put in context and
to interpret. A ssum ing th a t D O C faithfully reflects the 6 13C
value o f the prim ary producer from w hich the carb o n is
ultim ately derived, isotope mixing calculations (IsoSource;
Phillips and G regg 2003) based on n atu ral abundance <513C
values for M PB ( —15.4%o, this study), pelagic algae ( —18%o
to —24%o, M ichener and Schell 1994), and terrestrial plants
(—23%o to —30%o, M ichener and Schell 1994) suggest th at
the carbon w ithin D O C in the lower Brunswick estuary is
ultim ately derived prim arily from carbon fixed by p h y to ­
p lan k to n (45% ± 37%, SD) and terrestrial plants (38% ±
37%). M PB appear to have a sm aller co n trib u tio n (18% ±
27%) b u t m ay contribute anyw here from 0% to 46% o f the
D O C . This m ay represent direct release from prim ary
producers, or release o f D O C by bacteria th a t have
co n su m ed c a rb o n th a t w as fixed by these p rim a ry
producers. The usefulness o f this analysis, however, is
M icrophytobenthos carbon fa te in sands
questionable because, ra th e r th a n faithfully reflecting its
source, preferential use o f m ore labile D O C com ponents
w ithin sedim ents (C hipm an et al. 2010) can result in fluxes
o f D O C b ein g co m p o sed o f a very en rich ed (e.g.,
carbohydrates) or depleted (e.g., lipids) fraction o f the
source organic m a tte r (M aher and Eyre 2011).
13C incorporation and transfer within sediments— W e
were unable to account for loss o f 13C th a t occurred before
th e first w a te r sam ple w as ta k e n (48 h a fter label
application). G iven th a t M PB -derived c a rb o n can be
rapidly processed (M iddelburg et al. 2000), it is therefore
likely th a t we underestim ated 13C incorp o ratio n. How ever,
the rates o f tran sfer and loss calculated from our starting
poin t (48 h) are independent o f the accuracy o f this
in co rp o ratio n rate.
C om pared w ith previous studies in tem perate intertidal
sands (M iddelburg et al. 2000) and tem perate subtidal
san d s (E v rard et al. 2008, 2010), th ere w as greater
dow nw ard tra n sp o rt o f 13C in the current study. In the
current study, 12.9% o f fixed 13C was in sedim ent OC at a
dep th o f 2 -5 cm 60 h after label application, and 18.6% of
fixed 13C w as transferred to sedim ents 5-10 cm deep. In
com parison, it to o k longer (3 d) for a sim ilar fraction o f 13C
to enter 2 -5 cm sedim ent in the studies o f tem perate sands
(M iddelburg et al. 2000; E vrard et al. 2008), and there was
negligible transfer o f 13C below 5 cm. This m ay reflect
differences in m ethodology an d /o r location (tem perate vs.
subtropical, intertid al vs. subtidal, differences in sediment
com position), and associated variation in rates o f b io tu r­
b atio n , active m igration o f M PB and hetero trophs, and
physical m ixing o f sedim ents by w ater m ovem ent. A further
explanation for differences in 13C in co rp o ratio n in deeper
sedim ents is differences in rates o f ch em o autotrophy.
Relatively little is know n o f the im portance o f chem oau­
to tro p h y in coastal sands, b u t E vrard et al. (2008) identified
ch em o au to tro p h y as being responsible for a relatively small
p o rtio n o f 13C u p tak e at d ep th in tem perate sands. In the
current study the delay betw een label ap plication and
sam ple collection p rev en ted us fro m identifying any
c o n trib u tio n o f c h e m o a u to tro p h y to 13C u p tak e into
deeper sedim ents. H ow ever, there was no evidence o f 13C
up tak e by bacteria in the absence of, or in excess of, 13C
up tak e by M PB.
C onsiderable loss via resuspension (60%) in the study by
M iddelburg et al. (2000) m ay have reduced the carbon pool
available for dow nw ard tra n sp o rt, and the ex situ natu re of
the study by E vrard et al. (2008) isolated sedim ents from
n atu ra l w ater m ovem ent and b io tu rb atio n , w hich m ay have
lim ited carb o n burial. T he different d ep th distribution of
13C in the study by M iddelburg et al. (2000) com pared w ith
the current study m ay reflect differences in w ater flow and
sedim ent m ixing in in tertidal sands, com pared w ith the
subtidal sedim ents we studied. H ow ever, the 13C depth
distrib u tio n observed by M iddelburg et al. (2000) was
sim ilar to th a t observed for subtidal tem perate sands by
E v ra rd et al. (2008). T h e d ifferen ces in 13C d e p th
distrib u tio n betw een studies o f tem perate sands, and th at
seen in the current study, hints at the possibility th at
different processes influence carb o n burial in tem perate and
1853
subtropical sands. H ow ever, fu rth er replicated studies
across clim ate zones are required to determ ine w hether
this is the case.
In the current study, initial burial o f 13C occurred w ithin
cham bers and cores, and therefore occurred in the absence
o f n a tu ra l w ater flow. G iven th a t the sands were not
perm eable, advective flow w ould n o t be im p o rtan t in
carbon burial in this environm ent. T he m inim al loss o f
carbon via resuspension observed in the current study also
suggests th a t w ater m ovem ent (i.e., physical m ixing) had
little effect on sedim ents and their carbo n distribution.
G iven th a t b io tu rb atio n w ould be lim ited by the low
abundance o f fauna at the site, it is likely th a t high rates o f
M PB and h e te ro tro p h m ig ra tio n (i.e., m ig ra tio n by
bacteria and/or m eiobenthos) were responsible for the high
rate o f dow nw ard tra n sp o rt for 13C in the current study.
This m ay reflect the high heterotrophic activity typical o f
low er latitudes (Böer et al. 2009). Active m igration by M PB
can occur to considerable depths w ithin sands (U nderw ood
2002; Saburova and P olikarpov 2003) and this m ay account
for the considerable 13C content o f M PB in 2-5 cm and, to
a lesser extent, 5-10 cm sedim ents. This m ay be assisted by
the greater light availability at low latitudes, w hich m ay
m ake it possible for M PB in subtropical sands to persist at
greater sedim ent depths. M PB m ay also m ove to deeper,
aphotic layers o f sandy sedim ents to access nutrients
(S ab u ro v a an d P o lik a rp o v 2003). R eg ard less o f the
m echanism involved, the rapid dow nw ard tra n sp o rt o f
carbon observed in the current study has im plications for
the ultim ate fate o f carb o n fixed by M PB. Labile OC will
be respired, b u t the depth at w hich this occurs, and the
pathw ays leading to its respiration, will depend on mixing
regimes. T he efficient burial o f M PB -derived carbon is
likely to c o n trib u te to its long-term reten tio n w ithin
subtropical sandy sediments.
M PB-derived carbon appears to have been recycled by
M PB, bacteria, and foram inifera, w ith considerable 13C
detected in all o f these com partm ents th ro u g h o u t the
experim ental period. P articularly for Cellanthus craticulatus
in sedim ent at 0 -5 cm, and for bacteria at all depths, there
w as no ap p aren t decline in 13C content th ro u g h o u t the
experim ent. W hereas the fraction o f fixed 13C th at was
w ith in b acte ria w as sim ilar to th e ir c o n trib u tio n to
sedim ent O C, the fraction o f 13C w ithin M PB and C.
craticulatus far outw eighed their co n tribution to sediment
OC. This suggests th at M PB and C. craticulatus have a
greater role th an bacteria in the long-term retention o f fixed
carbon. The low biom ass : productivity ratio for M PB at
the study site ( ~ 5.5 d; O akes et al. 20106), possibly due to
fast tu rnover in response to high grazing pressure (H erm an
et al. 2000), m ay m ean th a t M PB need to intercept D IC
produced th ro u g h respiration, in addition to w ater-colum n
D IC , to satisfy their carbon dem and. F o r foram inifera, the
carbon source can vary w ith species (O akes et al. 2010«).
F oram inifera can obtain carb o n from M PB and phytode­
tritus (M oodley et al. 2002, O akes et al. 2010«), as well as
from bacteria (M ojtahid et al. 2011), m acrophyte detritus
(O akes et al. 2010«), and dissolved organic m atter (D eLaca
1982). L arger foram inifera can contain sym biotic algae,
allowing them to directly access inorganic carb o n as a
1854
Oakes et al.
source (Lee 1995). L arger foram inifera were n o t observed
in the cu rren t study, b u t fo ram in ifera in th e fam ily
Elphidiidae (which includes the genera Cellanthus and
Elphidium ) can con tain chloroplasts extracted from d ia­
tom s, w hich can rem ain photosynthetically active, fixing
carb o n for their hosts, for weeks or m o n th s (Lee 1995). The
high accum ulation o f 13C in C. craticulatus com pared w ith
A m m onia beccarii and Elphidium advenum , relative to their
biom ass, m ay therefore indicate the presence o f functional
chloroplasts in C. craticulatus. H ow ever, given th a t the 13C
content o f C. craticulatus in surface sedim ents did not
noticeably decline w ith time, in contrast to M PB , it appears
th a t C. craticulatus retain fixed carb o n for a longer period
th a n M PB an d /o r access M PB -derived organic carbon in
additio n to D IC . G iven th a t rates o f active m igration for
benthic foram inifera can be high (0.4-184.3 m m d _1;
B ornm alm et al. 1997), p articu larly in shallow -w ater
sedim ents and at higher tem peratures, C. craticulatus m ay
play an im p o rtan t role in the transfer o f fixed carbon to
deeper sediments.
T he 13C w ithin sedim ent OC th a t we w ere u nable to
account for m ay have been in co rp o rated by m eiobenthos
th a t we did n o t consider; occasional m acro fauna; or
senescent M PB , bacteria, or fauna. E xtracellular com ­
pou n d s (e.g., EPS; G o to et al. 2001) can also be a m ajor
reservoir fo r M PB -derived carbon. F o r exam ple, intraand extracellular carb o h y d rates to g eth er co ntained 1530% o f the 13C in co rp o rated in to 0 -2 cm sedim ent O C at
o u r site (O akes et al. 20106). D ep e n d in g o n th e ir
co n trib u tio n to to ta l carb o h y d rates, extracellular c a rb o ­
h y drates m ay th erefore co n trib u te significantly to the
uncharacterized p o rtio n o f the 13C w ithin sedim ent OC in
the curren t study.
Loss o f 13C fr o m sediments— W e previously observed
th a t 85% o f the carb on fixed by M PB w as lost from surface
sedim ents (0-2 cm) over 33 d (O akes et al. 20106).
H ow ever, the loss pathw ays were n o t determ ined and were
therefore the focus o f the current study.
Given th a t only ~ 3% o f the carb o n fixed by M PB was
n o t found w ithin sedim ents or accounted for by fluxes o f
D O C and D IC by the end o f the study, resuspension
apparen tly played only a m in o r role in the loss o f M PBderived c a rb o n from the subtropical subtidal sands studied.
T his m ay be due to low w ater m ovem ent over the sedim ent
surface, the observed rapid transfer o f carb o n to deeper
sedim ents, high p ro d u ctio n by M PB o f EPS (O akes et al.
20106) th a t binds and stabilizes sedim ent, or the absence o f
m acrofau n a, w hich can destabilize sedim ent (H erm an et al.
2000). R egardless, the ap parently low resuspension rate
suggests th a t losses o f carb o n from sedim ent in the current
study reflect processing o f fixed carb o n by the sedim ent
com m unity.
M PB are a p otential source o f w ater-colum n D O C
(Porubsky et al. 2008). H ow ever, D O C derived from M PB
represented only a sm all fraction o f the to tal D O C efflux
from sedim ents in the current study (0.4% during the first
d a rk incubation, w hen excess 13C flux as D O C was highest)
and w as a m in o r pathw ay for loss o f 13C fixed by M PB
(2.64% over 33 d). T he D O C released from sedim ents at the
study site m ay be prim arily derived from carbon fixed by
M PB before label addition, b u t is m ore likely to be derived
from alternative sources, including p h y to p lan k to n and
terrestrial organic m atter, as suggested by n atu ral a b u n ­
dance carbon stable isotope ratios o f w ater-colum n D O C .
Given th a t alm ost all o f the substantial D O C pool exuded
by M PB at the study site (O akes et al. 20106) was
consum ed by heterotrophs, and is therefore apparently
highly labile, the D O C th a t is released from sedim ents is
presum ably dom inated by m ore refractory com ponents.
This is reflected in the relatively depleted n a tu ra l a b u n ­
dance <513C value o f D O C . G reater loss o f M PB-derived
carbon via D O C fluxes m ay be expected where M PB
productivity is high, b u t heterotrophic activity is low.
It is clear th a t n o t all o f the D IC p ro duced via
respiration o f M PB-derived carbon was released from
sediments. D IC w as the m ajor pathw ay for 13C recycling
into M PB, im plying th at D IC produced th ro u g h respira­
tion in the sedim ent is used by M PB in addition to D IC
from the overlying w ater colum n. T his is supported by the
observation th a t gross prim ary production, based on 0 2
and C 0 2 fluxes, w as far greater th an w as estim ated based
on M PB u p tak e o f 13C fro m overlying w ater. D IC
produced th ro u g h respiration m ay be particularly im p o r­
ta n t for M PB in deeper sedim ents, where D IC diffusion
from overlying w ater w ould be lim ited. This is evident in
the m aintenance o f 13C in co rp o ratio n in M PB w ithin the 5 10 cm sedim ent layer (Fig. 1).
Release o f D IC from sedim ents w as the m ajor pathw ay
for loss o f M PB-derived carbon from the sands studied.
This m ay reflect, in p a rt, the high bacterial productivity
typical o f subtropical sedim ents (Alongi 1994), although
this w ould be offset by M PB productivity. W here M PB
productivity is lower, there m ay be even greater loss o f
M PB-derived carbon via respiration.
Implications— This study dem onstrates the key role o f
M PB as a resource for benthic heterotrophs in shallow
photic subtidal sandy sedim ents in the subtropics. G iven
th a t M PB take up D IC from the overlying w ater and the
carbon they fix is released as D IC , M PB is unlikely to
substantially alter the form o f carbon tran sp o rted offshore
(i.e., there is no conversion to D O C ), although processing
w ithin the sedim ent m ay alter its S 13C value. H ow ever,
given th a t 35% o f fixed carb o n was retained over 33 d, the
sedim ents m ay act as a carbon sink, affecting the quantity
o f carb o n th at is tran sp o rted offshore.
Acknowledgments
We thank Pete Squire, Melissa Bautista, Terry Lewins, Michael
Brickhill, and Simon Turner for field assistance, Kym Haskins
and M ax Johnston for extraction of biomarkers, Melissa Gibbes
for fauna extraction, and M atheus Carvalho for isotope analysis.
This study was funded by A ustralian Research Council (ARC)
Discovery grants to B.D.E. and J.J.M . (DP0663159) and B.D.E.,
J.M .O., and J.J.M . (DP0878568), an A RC Linkage grant to
B.D.E. (LP0667449), and A RC Linkage Infrastructure, Equip­
ment and Facilities grants to B.D.E. and J.M .O. (LE0989952,
LE0668495), and an ARC Discovery Early Career Researcher
Award to J.M .O. (DE120101290). We thank two anonymous
reviewers for their constructive feedback.
M icrophytobenthos carbon fa te in sands
References
D. M . 1994. The role of bacteria in nutrient recycling in
tropical mangrove and other coastal benthic ecosystems.
Hydrobiologia 285 : 19-32, doi:10.1007/BF00005650
A n d e r s s o n , J. H., C . W o u l d s , M . S c h w a r t z , G. L . C o w i e , L . A .
L e v i n , K. S o e t a e r t , a n d J. J. M i d d e l b u r g . 2008. Short-term
fate of phytodetritus in sediments across the A rabian Sea
oxygen minimum zone. Biogeosciences 5: 43-53, doi:10.5194/
bg-5-43-2008
B e l l i n g e r , B. J., G. J. C. U n d e r w o o d , S. E. Z i e g l e r , a n d M. R.
G r e t z . 2009. Significant of diatom-derived polymers in
carbon flow dynamics within estuarine biofilms determined
through isotopic enrichment. A quat. M icrob. Ecol. 55 :
169-187, doi:10.3354/ame01287
B ó e r , S. I., C. A r n o s t t , J. E. E. v a n B e u s e k o m , a n d A. B o e t i u s . 2009.
Temporal variations in microbial activities and carbon turnover
in subtidal sandy sediments. Biogeosciences 6: 1149-1165.
B o r n m a l m , L., B. H. C o r l i s s , a n d K. T e d e s c o . 1997. Laboratory
observations of rates and patterns of movement of continental
m argin benthic foram inifera. M ar. M icropalaentol. 29 :
175-184, doi:10.1016/S0377-8398(96)00013-8
C a h o o n , L. B. 1999. The role of benthic microalgae in neritic
ecosystems. Oceanogr. Mar. Biol. 37 : 47-86.
C h i p m a n , L . , D. P o d g o r s k i , S. G r e e n , J. K o s t k a , W. C o o p e r , a n d
M. H u e t t e l . 2010. Decomposition of plankton-derived dis­
solved organic m atter in permeable coastal sediments. Limnol.
Oceanogn 55 : 857-871, doi: 10.4319/lo.2009.55.2.0857
C o o k , P . L . M., B . V e l i g e r , S. B o e r , a n d J. J. M i d d e l b u r g . 2007.
Effect o f nutrient availability on carbon and nitrogen
incorporation and flows through benthic algae and bacteria
in near-shore sandy sediment. Aquat. Microb. Biol. 49 :
165-180, doi:10.3354/ame01142
D e L a c a , T. E. 1982. Use of dissolved amino acids by the
foraminifer Notodendrodes antarctikos. Am. Zool. 22: 683-690.
E v r a r d , V . , P . L . M . C o o k , B . V e l i g e r , M . H u e t t e l , a n d J. J.
M i d d e l b u r g . 2008. Tracing carbon and nitrogen incorpora­
tion pathways in the microbial community of a photic
subtidal sand. Aquat. Microb. Ecol. 53 : 257-269, doi:10.
3354/ame01248
, M . H u e t t e l , P . L . M . C o o k , K. S o e t a e r t , C . H . R . H e i p ,
a n d J. J. M i d d e l b u r g . 2012. Relative importance of phytode­
tritus deposition and microphytobenthos for the heterotrophic
community of a shallow subtidal sandy sediment. Mar. Ecol.
Prog. Ser. 455 : 13-21, doi:10.3354/meps09676
, K. S o e t a e r t , C . H . R . H e i p , M . H u e t t e l , M . A .
X e n o p o u l o s , a n d J. J. M i d d e l b u r g . 2010. Carbon and
nitrogen flows through the benthic food web of a photic
subtidal sandy sediment. M ar. Ecol. Prog. Ser. 416 : 1-16,
doi:10.3354/meps08770
E y r e , B . , a n d P. B a l l s . 1999. A com parative study of nutrient
behavior along the salinity gradient of tropical and temperate
estuaries. Estuaries 22: 313-326, doi:10.2307/1352987
, a n d A . J. P . F e r g u s o n . 2005. Benthic metabolism and
nitrogen cycling in a subtropical east Australian Estuary
(Brunswick): Tem poral variability and controlling factors.
Limnol. Oceanogr. 50 : 81-96, doi:10.4319/lo.2005.50.1.0081
G o t o , N ., O. M i t a m u r a , a n d H . T e r a i . 2001. Biodégradation of
photosynthetically produced extracellular organic carbon
from intertidal benthic algae. J. Exp. M ar. Biol. Ecol. 257 :
73-86, doi: 10.1016/S0022-0981 (00)00329-4
H e r m a n , P. M. J., J. J. M i d d e l b u r g , J. W i d d o w s , C. H . L u c a s ,
a n d C. H . R . H e i p . 2000. Stable isotopes as trophic tracers:
Combining field sampling and manipulative labeling of food
resources for m acrobenthos. Mar. Ecol. Prog. Ser. 204 : 79-92,
doi:10.3354/meps204079
A lo n g i,
1855
B., L. A. C i f u e n t e s , a n d J. E. K a l d y . 2003. Stable
carbon isotope evidence for coupling between sedimentary
bacteria and seagrasses in a sub-tropical lagoon. M ar. Ecol.
Prog. Ser. 255: 15-25, doi:10.3354/meps255015
L e e , J. J. 1995. Living sands: The symbiosis of protists and algae
can provide good models for the study of host/symbiont
interactions. BioScience 45 : 252-261, doi: 10.2307/1312418
M a h e r , D., a n d B. D. E y r e . 2011. Insights into estuarine benthic
dissolved organic carbon (DOC) dynamics using <513C-DOC
values, phospholipid fatty acids and dissolved organic
nutrient fluxes. Geochim. Cosmochim. Acta 75 : 1889-1902,
doi:10.1016/j.gca.2011.01.007
M a h e r , D. T., a n d B. D. E y r e . 2010. Benthic fluxes of dissolved
organic carbon in three tem perate A ustralian estuaries:
Implications for global estimates of benthic D O C fluxes.
J. Geophys. Res. 115: G04039, doi:10.1029/2010JG001433
M i c h e n e r , R. H., a n d D. M. S c h e l l . 1994. Stable isotope ratios
as tracers in marine aquatic food webs, p. 138-157. In K.
Lajtha and R. H. Michener [eds.], Stable isotopes in ecology
and environmental science. Blackwell Scientific.
M i d d e l b u r g , J. J., C. B a r r a n g u e t , H. T. S. B o s c h k e r , P. M. J.
H e r m a n , T. M o e n s , a n d C. H. R. H e i p . 2000. The fate of
intertidal m icrophytobenthos carbon: An in situ 13C-labeling
study. 45: 1224-1234.
M o e n s , T., C. L u y t e n , J. J. M i d d e l b u r g , P. M. J. H e r m a n , a n d
M. V i n c x . 2002. Tracing organic m atter sources of estuarine
tidal flat nematodes with stable carbon isotopes. M ar. Ecol.
Prog. Ser. 234: 127-137, doi:10.3354/meps234127
M o j t a h i d , M., M. V . Z u b k o v , M. H a r t m a n , a n d A. J. G o o d a y .
2011. Grazing of intertidal benthic foraminifera on bacteria:
Assessment using pulse-chase radiotracing. J. Exp. M ar. Biol.
Ecol. 399 : 25-34^ doi:10.1016/j.jembe.20fl.01.011
M o o d l e y , L., J. J. M i d d e l b u r g , H. T. S. B o s c h k e r , G. C. A.
D u i n e v e l d , R. P e l , P. M. J. H e r m a n , a n d C. H. R. H e i p .
2002. Bacteria and foraminifera: key players in a short-term
deep-sea benthic response to phytodetritus. M ar. Ecol. Prog.
Ser. 236 : 23-29, doi:10.3354/meps236023
O a k e s , J. M., M. D. B a u t i s t a , D. M a h e r , W . B. J o n e s , a n d B. D.
E y r e . 2011. C arbon self-utilization may assist Caulerpa
taxifolia invasion. Lim nol. O ceanogr. 56 : 1824-1831,
doi:10.4319/lo.2011.56.5.1824
, R. M. C o n n o l l y , a n d A. T. R e v i l l . 2010«. Isotope
enrichment in mangrove forests separates m icrophytobenthos
and detritus as carbon sources for animals. Limnol. Ocean­
ogr. 55 : 393^102, doi:10.4319/lo.2010.55.1.0393
, B. D. E y r e , J. J. M i d d e l b u r g , a n d H. T. S. B o s c h k e r .
2010/l Composition, production, and loss of carbohydrates in
subtropical shallow subtidal sandy sediments: R apid process­
ing and long-term retention revealed by 13C-labeling. Limnol.
Oceanogr. 55 : 2126-2138, doi:10.4319/lo.2010.55.5.2126
, --------- , D. J. Ross, a n d S. D. T u r n e r . 2010/l Stable
isotopes trace estuarine transform ations of carbon and
nitrogen from primary- and secondary-treated paper and
pulp mill effluent. Environ. Sei. Technol. 44 : 7411-7417,
doi:10.1021/esl01789v
P e t e r s o n , B. J. 1999. Stable isotopes as tracers of organic m atter
input and transfer in benthic food webs: A review. Acta
Oecol. 20 : 479-487, doi:10.1016/S1146-609X(99)00120-4
P h i l l i p s , D. L., a n d J. W . G r e g g . 2003. Source partitioning with
stable isotopes: Coping with too many sources. Oecologia
136: 261-269, doi:10.1007/s00442-003-1218-3
P o r u b s k y , W . P., L. E. V e l a s q u e z , a n d S. B. J o y e . 2008.
N utrient-replete benthic microalgae as a source of dissolved
organic carbon to coastal waters. Estuaries and Coasts 31 :
860-876, doi:10.1007/sl2237-008-9077-0
Jones, W .
1856
Oakes et al.
A., a n d J . E. B a u e r . 2001. Use of 14C and 13C
natural abundances for evaluating riverine, estuarine, and
coastal DOC and POC sources and cycling: A review and
synthesis. Org. Geochem. 32 : 469-485, doi:10.1016/S01466380(00)00190-X
S a b u r o v a , M. A., a n d I. G. P o l i k a r p o v . 2003. D iatom activity
within soft sediments: Behavioural and physiological processes.
Mar. Ecol. Prog. Ser. 251: 115-216, doi:10.3354/meps251115
S c h i d l o w s k i , M. 2000. C arbon isotopes and microbial sediments,
p. 84—104. In R. E. Riding and S. M. Awramik [eds.],
Microbial sediments. Springer-Verlag.
S m i t h , D. J., a n d G. J. C. U n d e r w o o d . 2000. The production of
extracellular carbohydrates by estuarine benthic diatoms: The
effects of growth phase and light and dark treatment. J.
Phycol. 36:^321-333, doi:10.1046/j,1529-8817.2000.99148.x
U n d e r w o o d , G. J. C. 2002. A daptations of tropical marine
m icrophytobenthic assemblages along a gradient of light and
nutrient availability in Suva Lagoon, Fiji. Eur. J. Phycol. 37 :
449-462, doi:10.1017/S0967026202003785
, M. B o u l c o t t , a n d C. A. R a i n e s . 2004. Environmental effects
on exopolymer production by marine benthic diatoms: Dynamics,
changes in composition, and pathways of production. J. Phycol.
40: 293-304, doi:10.1111/j.l529-8817.2004.03076.x
R aym ond, P.
K ., K. S O E TA ER T, A N D J . J . M ID D E L B U R G . 2011.
Plankton dynamics in an estuarine plume: A mesocosm 13C
and 15N tracer study. M ar. Ecol. Prog. Ser. 429 : 29^-3,
doi:10.3354/meps09097
V o l k m a n , J . K., S . W . J e f f r e y , P. D. N i c h o l s , G . I. R o g e r s , a n d
C.
D. G a r l a n d . 1989. F atty acid and lipid composition of 10
species of microalgae used in mariculture. J . Exp. M ar. Biol.
Ecol. 128: 219-240, doi:10.1016/0022-0981(89)90029-4
W e s t r i c h , J . T., a n d R. A. B e r n e r . 1984. The role of sedimentary
organic m atter in bacterial sulfate reduction: The G model
tested. Limnol. Oceanogr. 29 : 236-249, doi:10.4319/lo,1984.
29.2.0236
VANDEN M eE R S C H E ,
Associate editor: Bo Thamdrup
Received: 10 M a y 2012
Accepted: 04 Septem ber 2012
Amended: 05 Septem ber 2012