University of Southern California Dissertations and Theses

Unexplored microbial communities in marine sediment porewater

(USC Thesis Other)

Unexplored microbial communities in marine sediment porewater

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Content UNEXPLORED MICR OBIAL COMMUNITIES IN MARINE SEDIMENT POREW A TER
b y
Bingran Cheng
A Dissertation Presen ted to the
F A CUL TY OF THE USC GRADUA TE SCHOOL
UNIVERSITY OF SOUTHERN CALIF ORNIA
In P artial F ulfillmen t of the
Requiremen ts for the Degree
DOCTOR OF PHILOSOPHY
(MARINE BIOLOGY AND BIOLOGICAL OCEANOGRAPHY)
Ma y 2020
Cop yrigh t 2020 Bingran Cheng
T able of Con ten ts
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii
Chapter 1: In tro duction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Chapter 2: A Comparison of Microbial Div ersit y among the Sea w ater, the P orew ater and
the Sedimen t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
In tro duction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Metho ds and Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
A c kno wledgemen ts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Figures and T ables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Supplemen tary Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Chapter 3: Microbial A ctivities of Carb on T urno v er in a Comparison Bet w een F ree-living
and P article-attac hed Comm unities in Marine Sedimen ts . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
In tro duction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Metho ds and Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
A c kno wledgemen ts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Figures and T ables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Supplemen tary Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Chapter 4: Prop elling to Surviv al: Ho w Motilit y Structures Microbial Comm unities in Ma-
rine Sedimen t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
In tro duction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Metho ds and Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
A c kno wledgemen ts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Figures and T ables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Supplemen tary Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Chapter 5: Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
ii
Abstract
Coastal sedimen ts harb or div erse microbial comm unities in an abundance a v eraging
10
9
to 10
11
cells p er cm
3
, 3 to 4 orders of magnitude greater than the a v erage cell abun-
dance in the sea w ater (10
6
cells cm
3
). Ben thic microbial comm unities adopt t w o con-
trasting lifest yles: free-living in the p ore space or attac hed to sedimen t particles. These
t w o fractions are not distinguished in con v en tional b en thic microbial studies, whic h ana-
lyze the sedimen t in “bulk” . Therefore, the div ersit y , structure, and ecological functions
of the p orew ater comm unit y remains unkno wn. My thesis fo cused on c haracterizing this
unexplored p orew ater comm unit y for its abundance, div ersit y , and metab olic activities, in
comparison to the particle-attac hed comm unit y . In v estigations w ere carried out in an in-
tertidal m udflat system (Catalina Harb or, Catalina Island, CA). Results sho w ed that the
free-living comm unit y in the p orew ater w as not only abundan t (10
7
to 10
8
cells cm

3 ) but
also differed greatly from the particle-attac hed comm unit y and the microbial comm unit y
in the o v erlying sea w ater based on 16S rRNA sequencing. More imp ortan tly , radiotracer
studies rev ealed significan tly higher carb on turno v er rates (acetate and formate o xidation)
of the p orew ater comm unit y than its particle-attac hed neigh b ors. This finding w as further
supp orted b y a com bination of thermo dynamic calculations, metagenomic analysis and
nano-calorimetry . Results also sho w ed that a large fraction (up to 90%) of the free-living
comm unit y w as motile and displa y ed c hemosensory resp onse to added n utrien ts (e.g. ni-
trate), whic h could partially explain their elev ated metab olic activities. Although sulfate
reduction is regarded as the most imp ortan t pro cess for the anaerobic mineralization of
organic matter in marine sedimen ts, rep eated radiotracer measuremen ts using
35
S-SO
2
4
sho w ed that the p orew ater comm unit y , surprisingly , did not use sulfate as a terminal elec-
tron acceptor, in con trast to the particle-attac hed comm unit y . In conclusion, the p orew a-
ter comm unit y is a distinct comp onen t of the b en thic microbiome, ho w ev er its role in b en-
thic biogeo c hemical cycles has b een greatly o v erlo ok ed and deserv es further in v estigations
in differen t sedimen t settings, including the deep biosphere.
iii
Chapter 1: In tro duction
The v ast o cean co v ers ab out 71% of our planet’s surface. Belo w the w ater column (an a v-
erage depth of 4 km), marine sedimen ts co v er the Earth’s crust at v arious thic knesses, thin
(a few h undreds of meters) in the p elagic o ceans to o v er thousands of meters on con tinen-
tal margins (Whittak er et al., 2013). Within the la y ers of sedimen t dep osits, hidden is the
history of b oth the con tinen ts and the o ceans. In the mean time, ongoing diagenesis con tin-
ues to shap e the curren t o cean flo or.
Sedimen t particles are formed through v arious pro cesses and encompass four ma jor
categories in size: gra v el (> 2 mm), sand (2 - 0.0625 mm), silt (0.0625 - 0.0039 mm) and
cla y (< 0.0039 mm) (Sc h ulz & Zab el, 2006). The fine-grained, mainly non-biogenic sed-
imen ts whic h consist of a mixture of silt and cla y are what w e normally refer to as m ud.
Muddy sedimen ts mak e up the ma jorit y of the deep o cean flo or, while in con tinen tal shelf
and coastal regions, sand co v ers more than 50% of the seaflo or (Hall, 2002). The solid par-
ticles and the p ore space in b et w een constitute the sedimen t matrix. The v olume ratio of
p ore space to the bulk sedimen t, termed as p orosit y , v aries dep ending on sedimen t t yp e
and depth. Sandy sedimen ts mark the lo w er end of the p orosit y sp ectrum, ranging from
0.4 to 0.6, whereas the p orosit y of deep-sea sedimen ts can b e as high as 0.9 (Hamilton,
1971). P orosit y generally decreases with sedimen t depth, but ev en at a depth of 0.5 km, a
p orosit y of 0.5 can still b e observ ed (Hamilton, 1976).
The p ore space is filled with sea w ater, called the p orew ater, whic h connects the sed-
imen t with the o v erlying sea w ater b y allo wing the exc hange of solutes and small partic-
ulates b et w een the t w o compartmen ts. The p ermeabilit y (k) of the sedimen ts, whic h de-
notes the abilit y for the p orew ater to flo w through the sedimen t column, is dep enden t
on the grain size distribution (Burdine, 1953; Marshall, 1958). The lo w-p orosit y , coarse-
1
grained sandy sedimen ts generally exhibit higher p ermeabilit y than the high-p orosit y , fine-
grained m uddy sedimen ts (Huettel et al., 2014; Neumann et al., 2017). The p ermeabilit y
of the sedimen ts determines the relativ e imp ortance b et w een the t w o main transp ort pro-
cesses in marine sedimen ts: adv ectiv e flo w and molecular diffusion (Huettel et al., 2014).
In p ermeable sedimen ts (k > 10
12
m
2
), the main transp ort pro cess is through ad-
v ection (Huettel et al., 2014). A dv ectiv e flo ws, generated b y pressure gradien ts, can pump
w ater through the sedimen ts column rapidly . The v elo cit y and spatial scale of adv ectiv e
flo ws are dep enden t on the complex in terpla y b et w een surface top ograph y , b ottom cur-
ren ts, w a v es and the burro wing activities of sedimen tary animals (Huettel & R usc h, 2000;
Huettel et al., 2014; Kessler et al., 2012; Ziebis et al., 1996a,b). T o adapt to an en viron-
men t with constan t p orew ater flo w, micro organisms here migh t ha v e dev elop ed differen t
strategies suc h as a particle-attac hed living st yle (Musat et al., 2006).
In little- to non-p ermeable sedimen ts (k < 10
12
m
2
), the transp ort pro cess of so-
lutes and small particles are dominated b y molecular diffusion (Huettel et al., 2014). Dif-
fusiv e fluxes (J) can b e determined follo wing Fic k’s la w of diffusion:
J =D
eff
dC
dx
(1)
dC/dx is the c hange of solute concen trations o v er a giv en distance. The effectiv e diffusion
co efficien t ( D
eff
) is dep enden t on the diffusion co efficien t of the solute ( D
w
), as w ell as the
tortuosit y ( ) and the p orosit y ( ) of the sedimen ts. In sedimen ts with high p orosit y (
> 0.7), an empirical relationship is found b et w een the p orosit y and tortuosit y (
3
)
(Ullman & Aller, 1982). The effectiv e diffusion co efficien t is written accordingly:
D
eff
=D
w

2
(2)
The concen tration gradien ts are determined b y the rates of microbial consumption and
2
generation, and th us the diffusiv e transp ort of solutes, giv en the p orosit y ( ), are largely
mediated b y microbial activities (Berg et al., 1998; Jahnk e et al., 1982; Jørgensen, 1994;
Seidel et al., 2012). The o v erall microbial pro cesses in sedimen ts with lo w p ermeabilit y
differ from those in p ermeable sedimen ts (Boudreau et al., 2001; de Beer et al., 2005; Heise
& Gust, 1999; Huettel et al., 2014; Kessler et al., 2012; Probandt et al., 2017; Rao et al.,
2007; Rasheed et al., 2003). Our study fo cuses on sedimen ts with lo w to none p ermeabilit y
and should not b e mixed with a p ermeable system.
One eviden t result of the microbial regulation of solute transp orts in non-p ermeable
sedimen ts is the v ertical stratification of geo c hemical zones (Bö er et al., 2009; Canfield et
al., 1993; Jørgensen et al., 2012; Miy atak e et al., 2012; P olymenak ou et al., 2005). Oxy-
gen supplied from the sea w ater is preferen tially used b y aerobic micro organisms in the
surface sedimen t for organic carb on o xidation. In areas with high organic con ten t (0.5 to
3 %), aerobic respiration depletes o xygen in millimeters within the sedimen t (Jørgensen
& Des Marais, 1990). Nitrate reduction o ccurs near the o xic-ano xic transition zone as ni-
trate reducers are not completely inhibited b y the presence of o xygen (Chen et al., 2017;
Seitzinger, 1988; Usui et al., 1998). Manganese and iron o xides are strong o xidan ts and
highly reactiv e with reduced pro ducts suc h as H
2
S, solid-phase sulfides and iron sulfides
(Aller & R ude, 1988; Burdige & Nealson, 1986; Sc hipp ers & Jøgensen, 2001). Therefore
the biological reduction of manganese and iron o xides can only o ccur ab o v e the sulfidic
zone (Burdige, 1993). The con tribution of metal o xides reduction to remineralization is
further hindered b y their solid-phase ph ysical forms (Nealson & My ers, 1992). Sulfate is
considered the most imp ortan t terminal electron acceptor due to its high concen trations in
marine en vironmen ts. Sulfate reduction can o ccur throughout the sedimen t column, ev en
b elo w the sulfate-methane transition zone (Holmkvist et al., 2011; Jo c h um et al., 2017),
and accoun t for o v er half of the organic matter degradation in marine sedimen t (F ossing &
Jørgensen, 1989; Jørgensen, 1982; Sørensen et al., 1981). The reduced pro ducts from rem-
ineralization including NH

4
, Mn
2+
, F e
2+
and H
2
S diffuse up w ards and b ecome re-o xidized
3
in the surface la y ers, either biologically or c hemically , reinforcing the stabilit y of the redo x
cycles.
The coastal o cean represen ts one of the most pro ductiv e areas on Earth (Cap one &
Hutc hins, 2013; Hedges et al., 1988; Müller & Suess, 1979). A fraction of the organic ma-
terial generated in the photic zone through primary pro duction can ev ade the “amicro-
bial lo op” in the w ater column and ev en tually reac h the underlying seaflo or (Quijón et al.,
2008; Shanks, 2002). The high organic con ten t in coastal sedimen ts can supp ort high rates
of microbial activities (Hansen & Alongi, 1991; Kristensen, 2000; Kuehl et al., 1996) that
are estimated to b e resp onsible for 90% of the global b en thic organic matter remineraliza-
tion (Gattuso et al., 2009).
Cell abundances in surface coastal sedimen ts a v erage 10
9
10
10
cells cm
3
(P ark es et
al., 2014; Sc hmidt et al., 1998), that is 3 orders of magnitude higher than the t ypical cell
abundances found in 1 cubic cen timeter of sea w ater (F uhrman & Noble, 1995). It is gener-
ally b eliev ed that the ma jorit y (> 99%) of b en thic comm unities liv e attac hed to sedimen t
particles (R usc h et al., 2003), where they can access the organic material adsorb ed to the
particle surfaces (Keil & Geology , 1993), and form biofilms for sharing metab olic pro ducts
(Dang & Lo v ell, 2016; Giesek e et al., 2005; Sc hreib er et al., 2009). A particle-attac hed liv-
ing st yle migh t b e particularly b eneficial in p ermeable sedimen ts b ecause it prev en ts cells
from b eing w ashed a w a y b y the dynamic p orew ater flo w (Musat et al., 2006). In con trast,
in non-p ermeable sedimen ts, the h ydro dynamic stabilit y should fa v or a motile living st yle
(T a ylor & Sto c k er, 2012) and allo w the microbial cells to freely o ccup y the p ore space.
Motilit y can b e a great adv an tage for micro organisms as motile cells can mo v e a w a y
from less desirable en vironmen ts or comp etitors (Matz & Jürgens, 2005) and o v ercome
limitations of diffusiv e transp ort (Sto c k er, 2012). In addition, some swimming micro or-
ganisms can sense and trac k gradien ts, an activit y called taxis (A dler, 1966; Benc harit
& W ard, 2005; Dusen b ery , 1998; Jerome et al., 2018; Kennedy & La wless, 1985; Sto c k er
4
& Seymour, 2012). Protein-enco ding genes in v olv ed in motilit y and c hemotaxis are fre-
quen tly detected in metagenomic studies of b en thic en vironmen ts but exp erimen tal ev al-
uations of cell motilit y in marine sedimen ts are scarce. The ecological feasibilit y and im-
p ortance of microbial motilit y in marine sedimen ts, where small-scale and steady gradien ts
are commonly presen t, has previously b een h yp othesized (F enc hel 2002). In a later study ,
F enc hel (2008) found that at least 20% of cells in the sedimen t sw am, and motilit y p er-
sisted ev en at 35 cm b elo w the sedimen t-w ater in terface. He also disco v ered that dissolv ed
organic matter promoted the ratio of motile cells.
Con v en tionally , microbial comm unities in marine sedimen ts are studied in “bulk” and
not distinguished b et w een the attac hed and the free-living fractions. The p orew ater, es-
sen tially sea w ater, is exp ected to harb or micro organisms in an abundance at least similar
to, if not more than, that in the sea w ater (10
6
cells ml
1
). Considering the large v olume
of the p ore space in marine sedimen ts, the free-living comm unit y in the p orew ater consti-
tute a significan t fraction of the marine microbiome. With their metab olic activities p o-
ten tially enhanced b y activ e taxis, the previously unexplored p orew ater comm unit y migh t
con tribute greatly to the b en thic biogeo c hemical pro cesses. The goal of m y thesis is to
c haracterize the p orew ater microbial comm unit y in marine sedimen ts, sp ecifically an in-
tertidal m udflat, in regard to its abundance, div ersit y and metab olic activities. The three
c hapters approac hed this o v erall goal from differen t angles and eac h driv en b y differen t
questions:
• Chapter I I Ob jectiv e: T o c haracterize the microbial div ersit y in the p orew ater in
comparison to the sea w ater and on the particle surface:
1. Ho w abundan t are the free-living comm unit y in comparison to the particle-
attac hed comm unit y?
2. What microbial groups are free-living in the p orew ater?
5
3. Do the p orew ater comm unit y differ from the sea w ater comm unit y? Are there
exc hanges b et w een the t w o comm unities?
4. Do the free-living comm unit y differ from the particle-attac hed comm unit y?
5. Do the b en thic bacterial comm unities displa y nic he-partition?
• Chapter I I I Ob jectiv e: T o in v estigate the metab olic activities of the free-living com-
m unities:
1. Are the free-living comm unit y metab olically activ e?
2. Ho w m uc h do the free-living comm unit y con tribute to the b en thic carb on rem-
ineralization in comparison to the particle-attac hed comm unit y?
3. Do the free-living and particle-attac hed comm unities differ in metab olic flexibil-
it y (i.e. using differen t electron donors or acceptors)?
• Chapter IV Ob jectiv e: T o examine cell motilit y in marine sedimen ts and its resp ec-
tiv e effect on the free-living and particle-attac hed comm unities:
1. What p ercen tage of the free-living comm unit y exhibit motilit y?
2. Do the particle-attac hed cells exhibit p erio dic motilit y?
3. Is motilit y adv an tageous for the free-living comm unit y?
4. Do es motilit y pla y a role in the nic he-partition b et w een the free-living and
particle-attac hed comm unities
6
Chapter 2: A Comparison of Microbial Div ersit y among the
Sea w ater, the P orew ater and the Sedimen t
In collab oration with Wiebk e Ziebis
Abstract
Ben thic microbial comm unities adopt t w o con trasting lifest yles: free-living in the p ore
space or attac hed to sedimen t particles. The microbial groups o ccup ying the t w o differen t
nic hes p oten tially differ in comm unit y comp osition and ecological functions. Con v en tional
studies of b en thic microbial ecology do not differen tiate b et w een the t w o fractions. The
iden tities of the free-living and the particle-attac hed microbial comm unities remain un-
kno wn. Here w e used fluorescence microscop y and 16S rRNA tag sequencing to un v eil the
abundance and div ersit y of the p orew ater comm unit y in comparison to the bacterial com-
m unities in the o v erlying sea w ater and on the sedimen t particles in an in tertidal m udflat
(Catalina Harb or, Catalina Island, CA). The free-living comm unit y in the p orew ater, ev en
though only accoun ted for a minor fraction of the b en thic comm unities, constituted a high
n um b er in abundance (10
7
10
8
cells cm
3
), 1 to 2 orders of magnitude higher than that
in the sea w ater. The p orew ater comm unit y could b e clearly distinguished from the o v er-
lying sea w ater alb eit a connectivit y of the t w o compartmen ts. The sp ecies ric hness of the
p orew ater comm unit y w as comparable to that of the particle-attac hed comm unit y and the
t w o compartmen ts sho w ed little o v erlap at the amplicon sequence v arian ts (ASV) lev el.
In addition, 66.8% of the b en thic ASV s w ere found to b e either free-living or attac hed to
particles, suggesting a strong nic he-partition b et w een the t w o b en thic fractions. The nic he-
partitioning of the b en thic microbial comm unities is lik ely ph ylogenetically conserv ed and
do es not represen t a sto c hastic pro cess.
7
In tro duction
Marine sedimen ts harb or a great div ersit y of microbial comm unities who are critically
in v olv ed in v arious o ceanic biogeo c hemical pro cesses (Dekas et al., 2018; Jørgensen et
al., 2012; Jo y e et al., 2004; Kimes et al., 2013; Meziane et al., 1997; Orcutt et al., 2011;
Sapp et al., 2010; T esk e et al., 2000; W ang et al., 2014). In high-pro ductivit y area suc h as
coastal sedimen ts, microbial abundances can reac h up to 10
9
to 10
10
cells p er cm
3
(Jør-
gensen & Bo etius, 2007; Kallmey er et al., 2012), that is more than 3 orders of magnitude
higher than the a v erage cell abundance in the sea w ater (F uhrman & Noble, 1995). The
sedimen t matrix consists of the solid particles of v arious origins and the p ore space in b e-
t w een. This ph ysical structure defines the area that microbial cells can o ccup y: the p ore
space that is filled with p orew ater or the particle surface. Con v en tionally , the b en thic mi-
crobial comm unities are only analyzed in “bulk”, meaning that the p oten tial differen tiation
b et w een the cells that are free-living in the p orew ater and the cells that form biofilms on
particle surfaces has long b een o v erlo ok ed.
A partition b et w een the free-living and particle-attac hed lifest yles is commonly de-
tected in the sea w ater. The particle-asso ciated comm unities ha v e b een found to b e more
div erse than the free-living comm unities in v arious p elagic regions, suc h as the ano xic
Cariaco Basion (Suter et al., 2018), the Beaufort Sea in the Arctic (Ortega-Retuerta et al.,
2013), the Baltic Sea (Riec k et al., 2015) and the lo w-o xygen zone of the Blac k Sea (F uc hs-
man et al., 2011). In addition, a distinction in taxonomic comp osition b et w een the free-
living and the particle-attac hed assem blages has b een observ ed (DeLong et al., 1993; Riec k
et al., 2015; Smith et al., 2013). The rep eated disco v eries of non-random distributions
of sp ecific taxa in either the particle-asso ciated or free-living fraction of the planktonic
comm unities ha v e led to the conclusion that the preference for a free-living or particle-
attac hed lifest yle is a ph ylogenetically conserv ed trait (Salazar et al., 2015; Suter et al.,
2018).
8
In con trast to the sea w ater, marine sedimen ts of lo w p ermeabilit y are comparably
stagnan t. The absence of large-scale p orew ater-flo w mak es the p ore space a stable en vi-
ronmen t for microbial inhabitation. In lo w-p ermeable sedimen ts, molecular diffusion is
the dominan t transp ort pro cess (Iv ersen & Jørgensen, 1993; Jørgensen, 1994; Seidel et al.,
2012) whic h allo ws for the establishmen t of strong and steady v ertical gradien ts of dis-
solv ed gases, organic material and n utrien ts within the sedimen t (Blac kburn & F enc hel,
1999; Bo oij et al., 1994; Jørgensen & Des Marais, 1990; Jørgensen, 1994). The great spa-
tial heterogeneit y within cen timeter-depth in the sedimen t p ermits the co-existence of
div erse microbial comm unities with differen t ecological functions (Llob et-Brossa et al.,
2002, 1998; Probandt et al., 2017). The division b et w een the free-living and the particle-
attac hed comm unities in sedimen ts with lo w p ermeabilit y is lik ely (F ranco et al., 2007),
but has rarely b een studied. T o our kno wledge, only one study on fresh w ater lak e sedi-
men ts has compared the p orew ater to the sedimen t particles in terms of microbial div er-
sit y (Keshri et al., 2018). In their study , the authors observ ed a clear difference b et w een
the p orew ater and the particle-asso ciated comm unities.
This study is the first to compare the difference b et w een the free-living and particle-
attac hed microbial comm unities in marine sedimen ts. W e in v estigated the microbial com-
m unit y comp osition in sedimen ts of an in tertidal m udflat in Catalina Harb or, Catalina
Island, CA. Our goal w as to distinguish the microbial comm unities of the o v erlying sea-
w ater, the p orew ater and the particles using 16S rRNA tag sequencing. The sp ecific ob-
jectiv es of this study w ere (1) to c haracterize the comm unit y comp ositions of the three
compartmen ts, (2) to determine the similarit y and difference in comm unit y comp osition
b et w een the three compartmen ts (3) determine whether the b en thic comm unities displa y
nic he-partition b et w een the p orew ater and the particles and (4) if so, whic h groups sho w a
preference o v er whic h lifest yle and to what exten t.
9
Metho ds and Materials
Sampling site
San ta Catalina Island (33°23’N 118°25’W) is lo cated off the coast of southern California,
appro ximately 35 km south w est of the cit y of Los Angeles, CA. Sedimen t samples w ere
collected from an in tertidal m udflat in Catalina Harb or, a small harb or lo cated on the
south side of the isthm us of San ta Catalina Island. The o v erlying sea w ater, the p orew a-
ter and the sedimen t w ere collected for molecular analyses in Octob er 2014.
Sample collection
Sedimen t cores w ere collected using cylindrical p olycarb onate coreliners (10 cm diameter,
35 cm length). Eigh t parallel cores w ere collected during lo w tide with appro ximately 10 to
15 cm of w ater co v erage. Up on retriev al, PEEK stopp ers with o-ring fittings w ere used to
seal the sedimen t cores at the b ottom to prev en t p orew ater leakage. The o v erlying w ater
in the cores w ere k ept o xygenated using battery-p o w ered air pumps. The o v erlying sea w a-
ter directly ab o v e the sedimen t-w ater in terface w as collected in duplicates using a buc k et
and temp orarily stored in t w o sterile carb o y . The sedimen t cores and the sea w ater w ere
pro cessed immediately after transp ortation to the lab facilit y (less than 30 min).
Geo c hemical analyses
Oxygen profiles w ere measured using an amp erometric o xygen micro electro de (100 µm tip,
A/V Unisense, Denmark). The microsensor w as attac hed to a motorized micromanipula-
tor moun ted on a hea vy stand for lab oratory measuremen ts. The micromanipulator w as
computer-con trolled for precise v ertical mo v emen t in micrometer steps. One sedimen t core
with partial microbial mats co v erage at the sedimen t-w ater in terface w as profiled for o xy-
gen concen tration. T w o measuremen ts w ere conducted under indirect ligh t and one addi-
10
tional measuremen t in direct ligh t. The profiling started from 4 mm ab o v e the sedimen t-
w ater in terface (SWI) in 200 µm-incremen ts and stopp ed at 6mm b elo w the SWI. A t w o-
p oin t calibration w as p erformed b y measuring the electrical signals in sea w aters at 0% sat-
uration (de-gassed with N
2
for 5 min) and 100% saturation (o xygenated with an air pump
for 5 min). A standardized o xygen solubilit y table w as consulted to determine the o xygen
concen tration at 100% saturation based on the temp erature and the salinit y of the sea w a-
ter.
F our out of the eigh t parallel sedimen t cores w ere pro cessed for geo c hemical analy-
ses. F rom t w o of the cores, p orew ater w as extracted using rhizon samplers (2.5 mm di-
ameter, 0.15 µm p ore size, Rhizosphere Researc h Pro ducts, The Netherlands) from 0 to
10 cm depth in 1 cm-in terv als and analyzed for dissolv ed inorganic nitrogen, i.e. nitrate
(NO

3
) + nitrite (NO

2
) and ammonium (NH
+
4
). NO
x
w as determined colorimetrically us-
ing a metho d adapted for small sample v olumes (Jones, 1984). NH
+
4
w as determined using
a fluorometric tec hnique (Holmes et al., 1999). The other t w o cores w ere sectioned from
0 - 10 cm in 1 cm-in terv als for bulk sedimen t analyses. P orosit y w as determined b y mea-
suring the w eigh t loss of re-saturated sedimen t samples after drying at 60° o v er nigh t. Or-
ganic con ten t w as determined as the w eigh t loss after the com bustion of the dry sedimen t
at 450

C for 8 hours (loss of ignition). Chloroph yll concen tration w as measured follo wing
a widely used proto col (IOC-Proto cols, 1994). T otal HCl extractable iron w as measured
colorimetrically follo wing proto cols (K ostka & Luther, 1994; Lo vley & Phillips, 1987).
Determination of microbial abundance
Microbial abundances w ere determined for the o v erlying sea w ater (ab o v e the sedimen t col-
lected using coreliners), the p orew ater and the bulk sedimen t from v arying depths (sec-
tioned in 1 cm in terv als from 0 - 20 cm) follo wing proto cols b y Epstein and Rossel (1995)
and P atel (2007) with mo dification. The detailed pro cedures are listed in the Supple-
11
men tary Material. In brief, sedimen t samples of 1 cm
3
v olume w ere fixed with 9 ml 4%
sea w ater-formaldeh yde solution (0: 02 µm filtered). The 1 : 10 diluted sedimen t slurries
w ere further diluted (1:10 v/v) with 4% sea w ater-formaldeh yde solution (0: 02 µm filtered)
and sonicated using a sonicator tip to dislo dge the cells off the particles. This solution w as
then again diluted (1: 10) in the same fashion. 2 ml subsamples of the p orew ater of eac h
depth w ere transferred in to resp ectiv ely lab elled 2 ml cry o vials and fixed with 0 : 02 µm fil-
tered formaldeh yde to a final concen tration of 2%. 100 ml of the o v erlying sea w ater w as
collected at the sampling site and fixed with 0 : 02 µm filtered formaldeh yde to a concen tra-
tion of 2%.
A defined v olume of the formalin-fixed subsamples from eac h nic he (200 µL for the
p orew ater subsamples, 2 ml of the sea w ater subsamples, or 1 ml of 1:1000 diluted sedi-
men t solution) w as filtered on to a 0 : 22 µm Nucleop ore p olycarb onate filter (Whatman, GE
Healthcare) using a hand pump. Sterile-filtered marine PBS w as used to w ash off the inner
w all of the filter funnel and the w ash fluid w as collected on the filter to catc h all cells that
w ere stuc k on the glass. The filter w as then air-dried and transferred on to a sterile glass
microscop e slide and stained with a SYBR green staining solution (1:15:1 1% Ascorbic
acid: Mo wiol moun ting solution: SYBR Green). Eac h prepared slide w as visualized and
en umerated under a fluorescence microscop e (Nik on Eclipse 80i, 100 oil immersion). F or
eac h sample, triplicate filters w ere prepared. Eac h filter w as coun ted for 10 fields of view.
DNA extraction, 16S rRNA sequencing and pro cessing
The remaining four sedimen t cores w ere pro cessed for molecular analyses. The parallel
sedimen t cores w ere sectioned in 2 cm-in terv als from 0 to 8 cm deep. The sections from
all cores of the same depth w ere com bined to ensure a sufficien t amoun t of cells in eac h
sample for DNA extraction. P orew ater samples w ere obtained using a p ore-w ater pressing
b enc h (25 cm
3
POM, K C Denmark A/S, Silk eb org, Denmark). Eac h subsample w as placed
12
and sealed in to individual pressing house. Ultrapure nitrogen w as applied through the top
of eac h pressing house and gen tly exp el the p orew ater from the sedimen t matrix through a
pre-installed 8 µm Nucleop ore mem brane filter (Whatman,GE Healthcare) in to resp ectiv e
collecting tub es. The remaining dry sedimen ts in the pressing house w ere considered to b e
p orew ater-free and con tain only the particle-attac hed comm unit y .
F or eac h of the four depth in terv als (0 - 2, 2 - 4, 4 - 6 and 6 - 8 cm), In tact cells w ere
extracted from a defined v olume (25 ml) of the p orew ater samples, as w ell as from the cor-
resp onding p orew ater-free sedimen t samples (200 cm
3
). A densit y cen trifugation metho d
using Nyco denz w as used to concen trate cells (Kallmey er et al., 2008). Cells obtained
from the p orew ater w ere regarded as the free-living cells and the cells retriev ed from the
p orew ater-dev oid sedimen t w ere considered particle-attac hed. 5 L of eac h sea w ater sample
w as filtered through an 8 µm Nucleop ore mem brane filter (Whatman,GE Healthcare) and
on to a 0.22 µm Nucleop ore filter (Whatman, GE Healthcare).
DNA w as extracted from the 0.22 µm filters with the sea w ater samples and the p ellets
of in tact cells using the NucleoSpin Kit (Mac herey-Nagel Inc., Bethlehem, P A). The 16S
rRNA gene w as partially amplified using general bacterial primer sets (Bacterial 27F 5’-
A GA GTTTGA TCMTGGCTCA G-3’, 806R 5’-GGA CT A CNNGGGT A TCT AA T-3’). DNA
w as sequenced on a MiSeq (Illumina) using the 2 300 bp paired-end c hemistry according
to man ufacturer’s instructions at the Biomedical Researc h F acilit y in UC San Diego.
Dem ultiplexed 16S rRNA sequences w ere trimmed and filtered using T rimmomatic
(Bolger et al., 2014; LEADING:28 TRAILING:28 MINLEN:100). The remaining sequences
w ere imp orted in to QI IME2 for further analyses (Boly en et al., 2019). Amplicon sequence
v arian ts (ASV s) w ere constructed using D AD A2 (Callahan et al., 2016). ASV s with less
than 10 sequences and observ ed in only one sample w ere remo v ed. T axonom y of eac h
remaining feature (ASV) w as assigned using q2-feature-classier (Bokulic h et al., 2018)
trained on the Silv a v.132 99% OTUs (Quast et al., 2013).
13
Div ersit y analyses
Sampling co v erage w as assessed b y calculating the rarefaction curv e for the samples origi-
nating from the three defined nic hes (sea w ater, p orew ater and sedimen t) using the Alpha
rarefaction function at step = 10000 in QI IME2. Comm unit y ric hness and div ersit y at the
ASV lev el w ere estimated using m ultiple calculators (SOBs, Shannon, Fisher’s alpha and
Pielou’s ev enness) in R using the “microbiome” pac kage (Lah ti, 2017).
The SOB w as further decomp osed follo wing Eq.1 of Crist et al. (2003) and Roth-
Sc h ulze et al. (2016):

=
1
+
m
X
i=1

i
(1)
The total div ersit y (
) w as partitioned in to the a v erage div ersit y within samples ( ), and
among samples ( ) at differen t hierarc hiacal (i) lev el Here the lo w est hierarc hical lev els (i
= 1) is the individual sample and the second lev el (i = 2) is the sp ecific nic he (sea w ater,
p orew ater and sedimen t). Div ersit y among eac h sample and among compartmen ts are de-
fined as -in tra and -in ter div ersit y .

average
=
X

ij
NumberofSeqsinj
TotalnumberofSeqs
(2)

intra
=
2

1
(3)

inter
=
2
(4)
W e then remo v ed the ASV s from the o v erlying sea w ater and rep eated the div ersit y parti-
tion for the b en thic ASV s alone. Instead of grouping individual samples b y the nic hes they
b elong to, the b en thic samples w ere group ed b y depth. Therefore, the second lev el (i = 2)
represen ted the differen t depth in terv als sampled (i.e. 0 - 2 cm, 2 - 4 cm, 4 - 6 cm and 6 -
8 cm).
Differences in comm unit y structure b et w een the o v erlying sea w ater, the p ore w ater,
14
and the sedimen t w ere ev aluated b y calculating the Bra y-Curtis dissimilarities b et w een
eac h sample pair. T o comp ensate for v arying sample size, w e randomly subsampled from
1000 to 25000 sequences (n = 20) without replacemen t from eac h library (W eiss et al.,
2017).
T esting nic he-nartition of the b en thic microbial comm unities
Differen tial abundance testing w as p erformed for the ASV shared b et w een the p orew ater
and the sedimen t comm unit y using the DESeq function (Anders & Hub er, 2010; Lo v e et
al., 2014) implemen ted in ph yloseq (McMurdie & Holmes, 2013). Only ASV s with differ-
en tial abundance significan tly great than 4-fold (log2fold > 2, adjusted p-v alue < 0.05)
b et w een the p orew ater and the sedimen t w ere retained. T axonom y w as assigned to the
highest tax rank with confidence v alue > than 0.95.
A “particle-attac hmen t nic he index” (P AN index) (Salazar et al., 2015) w as also adopted
to n umerically c haracterize and compare the nic he-preference for eac h ASV presen t in the
b en thic samples. F or eac h ASV, its P AN index mean w as calculated as suc h:
PAN =
P
n
i=1
RAwt
P
n
i=1
RA
(5)
RA is the relativ e abundance of a giv en ASV in a giv en sample, wt is 0 for p orew ater sam-
ples or 1 for sedimen t samples, n is the n um b er of samples. An ASV o ccurring only in
p orew ater samples w ould ha v e a P AN-index v alue of 0 and an ASV o ccurring in only sed-
imen t samples w ould ha v e a P AN-index v alue of 1. A P AN-index v alue of 0.5 means an
equal distribution b et w een the t w o nic hes.
ASV s w ere then group ed b y ph ylum (class for Proteobacteria) and higher taxonomic
ranks (gen us or order). T o test whether ASV s b elonging to a giv en lineage sho w the same
degree of particle asso ciation the group ed P AN index v alues w ere tested for significance,
15
using the n ull h yp othesis that ASV s in the same taxonomic group are ev enly distributed
without displa ying a preference for either lifest yle (P AN index = 0.5). The significance w as
tested using one-sample Wilco xon signed rank tests (T aheri & Hesamian, 2013). Lineages
con taining less than 5 ASV s w ere not included.
Results
Geo c hemical parameters
The sedimen t in the in tertidal m udflat in Catalina harb or displa y ed t ypical c haracteristics
of a shallo w in tertidal area (Fig. 1). The p orosit y v aried b et w een 0.6 0.7 at the surface
and decreased with depth to 0.5 at 10 cm. The organic con ten t v aried b et w een 0.8 to
1.6 % in the upp er 10 cm and stabilized at 0.8% b elo w 10 cm (Fig. 1d). Oxygen p en-
etrated less than 3mm in to the sedimen t (Fig. 1a), resulting in a mostly ano xic sedimen t
column b elo w a thin surface la y er. Nitrate concen tration in the p orew ater ranged from
7.3 to 12: 7 µM at the sedimen t surface and decreased to close to 0 b elo w 4 cm with some
p eaks at depth (Fig. 1c). Oxidized iron (F e(I I I)) con ten t w as highest at the sedimen t sur-
face and generally decreased with depth. Ammonium concen trations w ere close to zero at
the sedimen t surface and increased with depth to close to 100 µM at 10 cm. Reduced iron
con ten t generally increased with depth but with minor fluctuations. Chloroph yll concen-
tration w as highest in the top sedimen t la y er and decreased with depth but could still b e
measured at 24 - 25 cm in the sedimen t.
Microbial abundance
Microbial abundance in the bulk sedimen t w as highest (7.62 10
9
cells cm
3
) at the sedimen t-
w ater in terface and decreased with depth (Fig. 2). The same trend w as observ ed for the
free-living comm unities in the p orew ater. A t all depth, free-living cells (1:0 3:8
16
10
7
cells ml
1
) accoun ted for around 0.5% of the total sedimen tary microbial abundance
and w ere 1 to 2 orders of magnitude higher in abundance than the sea w ater comm unities
(6:2 10
5
cells ml
1
).
Ov erview of bacterial div ersit y
A total of 1069165 sequences w ere retriev ed from the three differen t en vironmen tal nic hes
of the Catalina harb or ecosystem. 111054 sequences w ere eliminated b y the D AD A2 fil-
tering and denoising pro cedure. After c himera filtration, the remaining 645566 sequences
w ere represen ted b y 3467 unique bacterial amplicon sequence v arian ts (ASV s). 597 of these
ASV s that had at least 10 sequences and w ere presen t in more than one sample and w ere
k ept for further div ersit y analyses.
201 bacterial ASV s w ere presen t in the sea w ater samples, 273 ASV s in the p orew ater,
and 276 ASV s on the surface of the sedimen t particles (T able.1). The rarefaction curv es
suggest the sampling efforts of the sea w ater, the p orew ater and the particles surface reac hed
saturation despite unev en sequencing depth (Fig.S1). The free-living and the particle-
attac hed comm unities in surface sedimen ts sho w ed higher ric hness but lo w er ev enness than
the comm unities in the o v erlying sea w ater (T able.1). A decrease in microbial div ersit y
with depth w as observ ed for b oth the particle-attac hed and the free-living comm unities
(T able.1).
The Bra y-Curtis dissimilarit y b et w een the p orew ater and the sea w ater comm unities at
the ASV lev el w as in a v erage close to 1 (Fig. 3a), indicating the clear distinction b et w een
the t w o habitats. Bet w een the free-living and the particle-attac hed comm unities, the Bra y-
Curtis dissimilarit y w as 0.26 0.01 at 0 - 2cm and increased with depth to 0.69 0.02
(Fig. 3b) at 6 - 8 cm. The increasing difference with depth b et w een the t w o nic hes w as
further evidenced b y the decreasing n um b er of shared ASV s b et w een the p orew ater and
the sedimen t with depth. Ov erall, the dissimilarit y in comm unit y comp osition b et w een
17
the p orew ater and the sea w ater w as greater than the difference b et w een the t w o b en thic
nic hes.
The o v erall ASV div ersit y (Fisher’s alpha = 65.44) of all samples can b e decomp osed
in to -div ersit y (38.31%), -in tra-div ersit y (b et w een individual sample, 19.83%) and -
in ter-div ersit y (b et w een compartmen ts, 41.86%) (T able. 2). This suggests that a large
prop ortion (41.86%) of the o v erall div ersit y in this system could b e attributed to differ-
en tiation among the three nic hes. F or the b en thic samples, the o v erall taxonomic div ersit y
(Fisher’s alpha = 50.37) can b e decomp osed in to -div ersit y (50.01%), -in tra-div ersit y
(b et w een individual sample, 8.12%) and -in ter-div ersit y (b et w een depth, 41.87%) (T a-
ble.2), suggesting that depth hea vily affects the b en thic comm unit y comp osition. The
particle-attac hed comm unit y displa y ed a higher degree of microbiota div ergence than the
free-living comm unit y (Fig. S3), suggesting that depth exerted a stronger influence on the
particle-attac hed comm unit y .
The taxonomic comp osition of the three nic hes
The o v erlying sea w ater harb ored 201 bacterial ASV s encompassing 7 main ph yla: Pr o-
te ob acteria (80.10%), Bacter oidetes (14.91%), Cyanob acteria (2.54%), A ctinob acteria (2.35%),
V erruc omicr obia (0.05%), Planctomyc etes (0.02%) and F usob acteria (0.01%) (T able. 3).
A lphapr ote ob acteria (50.11%), mainly represen ted b y R ho dob acter ales , SAR 11 , Punic eispir-
il lales , Parvib aculales , R ickettsiales , R hizobiales , R ho dospiril lales and Sphino gomonadales
dominated the sea w ater comm unit y . Mem b ers of Bacter oidetes (mainly Flavob acteriac e ae ),
Gammapr ote ob acteria (includiing Cel lvibrionales , Oc e anospiriilales and SAR86 ) and Cyanob ac-
teria w ere also abundan t. The abundance of the ASV s in the sea w ater fit a log-normal dis-
tribution, with a few ASV s con taining large fraction of the sequences. The 14 most abun-
dan t ASV s (> 1% relativ e abundance) accoun ted for 65% of the total sequence abundance
(Fig. 4). 148 of the ASV s found in the sea w ater w ere unique to this nic he (Fig.1), accoun t-
18
ing for 45.9% of the total microbial abundance in the sea w ater.
Descending from the sea w ater to the p orew ater, the microbial comm unit y shifted
drastically . The 273 ASV s found in the p orew ater could b e classified in to 11 ph yla in-
cluding Pr ote ob acteria (90.98%), Epsilonb acter ae ota (8.67%), Dep endentiae (0.31%), Bac-
ter oidetes (0.17%), Planctomyc etes (0.04%), Cyanob acteria (0.02%), Firmicutes (0.02%)
and Patescib acteria (0.02%) (T able 3). Gammapr ote ob acteria w as the predominan t class
in the p orew ater and the most abundan t ASV s w ere classified in to the orders V ibrionales ,
A lter omonadales and Oc e anospiril lales (Fig. 4). Epsilonb acter ae ota w as the second most
abundan t ph ylum in the p orew ater with most sequences classified in to one gen us A r c o-
b ater .
The particle-attac hed comm unit y shared similarit y with the free-living comm unit y
to some exten t, esp ecially at the surface, as suggested b y the Bra y-Curtis dissimilarit y
(Fig. 3). The particle-attac hed comm unit y consisted of mainly Pr ote ob acteria (94.47%),
Epsilonb acter ae ota (3.99%), Bacter oidetes (0.31%), A cidob acteria (0.2%), Planctomyc etes
(0.19%), Gemmatimonadetes (0.15%), Dep endentiae (0.14%), Chlor oflexi (0.1%), Fir-
micutes (0.08%) and Patescib acteria (0.08%), Spir o chaetes (0.05%) and A ctinob acteria
(0.04%). The orders V ibrionales and A lter omo danles within the Gammapr ote ob acteria
dominated this b en thic fraction.
The microbial comm unit y in the o v erlying sea w ater could b e clearly distinguished
from those in the b en thic nic hes. The most abundan t ASV s (> 1% relativ e abundance) in
the sea w ater w ere in either in extremely lo w abundance or completely absen t in the b en-
thic system (Fig. 4). Bet w een the p orew ater and the sea w ater 14 ASV s w ere shared. That
is less than 5% of the total ASV s presen t within the t w o comm unities (Fig.1). The shared
ASV s b et w een the sea w ater and the p orew ater sho w ed higher relativ e abundance in the
sea w ater (Fig. S2) and w ere only found in the surface p orew ater (0 - 2 cm).
The free-living and particle-attac hed comm unities con tained ASV s unique to eac h
19
nic he (Fig. 5). With 158 unique ASV s (Fig.1), the free-living comm unit y con tributed greatly
to the o v erall b en thic microbial ric hness. The taxonomic comp osition of the p orew ater-
sp ecific microbial comm unit y differed from that of the sedimen t-sp ecific comm unit y . Most
apparen tly , mem b ers of the order Oc e anospiril lales (39.5%) and the gen us A r c ob acter (27.8%)
dominated the p orew ater-sp ecific comm unit y . These t w o groups w ere in lo w abundance in
the sedimen t-sp ecific comm unit y (4.7% and absen t, resp ectiv ely). Deltapr ote ob acteria ac-
coun ted for 14.9% of the sedimen t-sp ecific sequences but w ere virtually absen t in the p ore-
w ater.
The n um b er of ASV s shared b et w een the p orew ater and the sedimen t totaled 101 and
decreased with depth (Fig. 3). Among the ASV s shared b et w een the t w o compartmen ts,
27 sho w ed differen tial abundance of greater than 4-fold (Fig. 6). The ASV s with higher
relativ e abundance in the p orew ater b elonged to the gen us A r c ob acter , A mphrite a and Psy-
chr osphaer a . The ASV s with higher represen tation in the sedimen t w ere assigned to the
taxa Marinifilum , Sulfur ovum , Dehalo c o c c oidia , Desulfobulb ac e ae , Glacie c ola , Shewanel la ,
Thio alkalispir ac e ae and V ibrio . Neptuniib acter and Pseudo alter omonas con tained ASV s
with con trasting preference in habitat.
The nic he-partition b et w een the free-living and the particle-attac hed comm uni-
ties in the sedimen t
The P AN index describ ed the degree of particle-asso ciation for the b en thic ASV s presen t
in this study , from completely free-living (P AN index = 0) to alw a ys attac hed to particles
(P AN index =1) (Fig. 7). 66.8% (300 / 449 ASV s) of the ASV s had extreme P AN-index
v alues (0 or 1), among whic h 32.3% w ere free-living and 34.5% w ere attac hed to particles.
27 ASV s had P AN-index v alue b et w een 0.4 and 0.6 suggesting only a small fraction of the
b en thic ASV s w ere ev enly distributed b et w een the t w o nic hes.
A t the ph ylum lev el, A cidob acteria , A ctinob acteria , Bacter oidetes , Gemmatimon-
20
adetes , Patescib acteria , Planctomyc etes and Spir o chaetes sho w ed strong preference for a
particle-attac hed lifest yle (Fig. 8a). The genera A r c ob acter and Sulfur ovum within the
ph ylum Epsilonb acter ae ota exhibited con trasting patterns (Fig. 8b). Within A lphapr o-
te ob acteria , mem b ers of K or diimonadales and R ho dospiril lales w ere mainly free-living whereas
R ho dob acter ales con tained mem b ers that w ere either free-living or attac hed to particles.
In Gammapr ote ob acteria , cells b elonging to Diplorickettsiales w ere mainly free-living and
mem b ers of Ster oidob acter ales w ere all attac hed. Oc e anospiril lales sho w ed a significan t
preference for the free-living lifest yle as they w ere in m uc h higher relativ e abundance in
the p orew ater than on the sedimen t particles. The orders A lter omonadales and V ibrionales
con tained mem b ers distributed across the sp ectrum of particle-asso ciation.
Discussion
The con tin uum of b en thic-p elagic b oundary system
The b en thic-p elagic b oundary is a complex in terface where the fluid and dynamic w ater
column meets the rigid but p orous sedimen ts. These t w o seemingly separate en vironmen ts
are connected b y the p orew ater that fills the in terstitial space b et w een the sedimen t par-
ticles. In the transition zone from the sea w ater to non-p ermeable sedimen ts, suc h as the
system of in terest in this study , the geo c hemical conditions c hange drastically b elo w the
sedimen t-w ater in terface (SWI) o v er short distances (Jørgensen & Des Marais, 1990; Kris-
tensen, 2000; Reimers et al., 2001; Small et al., 2014; T urley , 2000). Oxygen is quic kly de-
pleted in surface sedimen ts due to aerobic respiration and re-o xidation of reduced com-
p ounds (Dolgonoso v, 2000; Hansen & Blac kburn, 1991; Jørgensen & Des Marais, 1990),
resulting in an ano xic en vironmen t b elo w the SWI. As the ano xic condition p ersists, other
electron acceptors (i.e. nitrate, metal o xides, sulfate and carb on dio xide) are sequen tially
used in the degradation of organic material (Edlund et al., 2008; Seidel et al., 2012) b y dif-
feren t groups of micro organisms (Bö er et al., 2009; Chen et al., 2017; Jo c h um et al., 2017;
21
P etro et al., 2017; Preisler et al., 2007; W ang et al., 2014).
The in tertidal m udflat in Catalina harb or receiv es input of organic matter from macroal-
gae, ph ytoplankton and b en thic photosyn thesis, evidenced b y the high c hloroph yll concen-
tration in the sedimen t (Fig. 1b), as w ell as b y the presence of fresh and deca ying macroal-
gae in the o v erlying w ater during sampling. Slo w b ottom curren ts allo ws the dep osition
of algae-deriv ed organic matter and lo w p ermeabilit y slo ws do wn the remo v al of decom-
p osition pro ducts (Huettel et al., 2014), resulting in carb on build-up in the system (Fig.
1d). The c hloroph yll concen tration within the sedimen ts reac hes v alues that are orders of
magnitude higher than in the o v erlying sea w ater and p ersists in to greater depth (Fig.1b).
The supply of organic material supp orts high rates of microbial activit y whic h quic kly de-
plete the o xygen (Arndt et al., 2013; Beulig et al., 2018; Jørgensen & Des Marais, 1990)
within millimeters of the sedimen t surface, ev en with activ e b en thic photosyn thesis o c-
curring concurren tly (Fig. 1a). Belo w the thin o xic la y er, high-energy electron acceptors
suc h as nitrate and iron o xides are a v ailable (Fig. 1c) for anaerobic respiration (Burdige,
1993; Laursen & Seitzinger, 2002; Seitzinger, 1988). Sulfate reduction has also b een found
to b e a ma jor terminal electron-accepting pro cess in this lago on en vironmen t (Bertics and
Ziebis, 2010). The sedimen ts in Catalina harb or displa y ed a general v ertical zonation of
electron acceptors with o ccasional p eaks of o xidan ts at greater depths (Fig. 1c). This is
caused b y the bioturbation activities of macrofauna suc h as the ghost shrimp (Neotrypaea
californiensis) (Bertics & Ziebis, 2010; Bertics & Ziebis, 2009). Ev en through area of hea vy
bioturbation w as a v oided in this study , effects of bioturbation could still b een seen.
Three connected but distinct ecological nic hes
Oxygen p enetrates only a few millimeters in the sedimen t in Catalina harb or (Fig. 1a) and
separates the t w o sides of the sedimen t-w ater in terface in to t w o regimes: the o xic and the
ano xic. The b en thic microbial comm unities w ere more div erse than the sea w ater comm u-
22
nities (T able 1) as has b een previously disco v ered (Lozup one & Knigh t, 2007; Probandt
et al., 2017; Zinger et al., 2011). This is in agreemen t with the concept that spatial hetero-
geneit y promotes sp ecies div ersification and co existence (Kassen, 2002; Zinger et al., 2011).
The main bacterial taxa found in the o v erlying sea w ater (T able 3) in Catalina harb or are
frequen tly encoun tered in the o xic surface o cean globally (F uhrman et al., 2006; Gilb ert et
al., 2012; Laura et al., 2015). In a study comparing microbial comm unities in the sea w ater
and the sedimen ts at a global scale, Zinger et al. (2011) found that the b en thic microbial
comm unities differed from those living in the p elagic en vironmen ts in taxonomic comp osi-
tion, consisten t with what w e observ ed in this system (Fig. 4,5).
In terestingly , Zinger et al. (2011) found that bacterial comm unities from ano xic w a-
ters o v erlapp ed with b oth p elagic and b en thic comm unities. If in fact the p orew ater ex-
c hanged its microbiome with the o v erlying sea w ater, the p orew ater, analogous to the ano xic
sea w ater in the aforemen tioned study , w ould lik ely harb or microbial taxa found b oth in
the sea w ater and on particle surfaces in the sedimen t. In con trast to this h yp othesis, only
20 ASV s (less than 5% of the total ASV s presen t within the t w o nic hes) w ere shared b e-
t w een the sea w ater and the p orew ater (Fig. 5). The ma jorit y of these shared ASV s sho w ed
a m uc h higher relativ e abundance in the sea w ater than in the p orew ater. A dditionally ,
these ASV s w ere only found in the p orew ater sample from the v ery surface (Fig. S2). It is
lik ely that these ASV s primarily dw ell in the sea w ater. They could remain viable in sur-
face p orew ater but failed to dev elop in to abundance. The drastic difference in comm unit y
comp osition b et w een the sea w ater and the p orew ater suggests that the free-living cells in
the p orew ater are not just temp orarily transp orted in to the b en thos from the sea w ater but
constitute a stable and unique comm unit y separate from the o v erlying sea w ater.
Within the sedimen t the bacterial ric hness in the p orew ater w as similar to what w as
observ ed for the particle-attac hed comm unit y (T able 1) ev en though the cell abundance
in the p orew ater w as in general 2 orders of magnitude lo w er than the particle-attac hed
comm unit y (Fig. 2). This confirms that microbial abundance is not directly related to mi-
23
crobial div ersit y (F ranco et al., 2007). T o our kno wledge only one study fo cused on fresh-
w ater lak es has compared the microbial div ersit y b et w een the p orew ater and the sedimen t
particles (Keshri et al., 2018) where they found, in con tradiction to our result, the former
to harb or m uc h lo w er div ersit y . This discrepancy lik ely stems from the difference in geo-
c hemical conditions b et w een the t w o study sites, particularly the salinit y , sedimen t t yp e,
organic material and n utrien ts whic h are kno wn factors con trolling microbial comm unit y
structure (Duyl et al., 1992; Hamdan & Jonas, 2006; K ondo et al., 2007; Mou et al., 2008).
In addition, Keshri et al. (2018) separated the p orew ater comm unit y from the bulk sedi-
men t via cen trifugation. This metho d lik ely precipitated a part of the p orew ater cells in to
the sedimen t fraction (Henry , 2004) and resulted in the underestimation of microbial div er-
sit y in the p orew ater.
The shared microbial comm unities b et w een the p orew ater and the sedimen t accoun ted
for 22.1% of the ric hness observ ed in the t w o compartmen ts com bined (Fig. 5) and sho w ed
highly differen tial distribution b et w een the t w o nic hes (Fig. 6). This finding corrob orates
with the study b y Keshri et al. (2018) where they found little o v erlap of microbial div er-
sit y at the 97% OTU lev el b et w een the p orew ater and the sedimen t. It is exp ected that
the div ersit y of the b en thic comm unities v aries with depth (Fig. 3b) due to the c hanging
a v ailabilit y of organic material and the v ertical stratification of electron acceptors (Bö er et
al., 2009; Jo c h um et al., 2017; Sapp et al., 2010). Unlik e the particle-attac hed comm unit y ,
the free-living microb es p oten tially enjo y a higher lev el of flexibilit y and v ertical mobilit y
(Grossart et al., 2001; Matz & Jürgens, 2005; Mitc hell & K ogure, 2006) and exhibited less
v ariance in comm unit y structure with depth than the particle-attac hed comm unit y (Fig.
S3). More imp ortan tly , the presence of 158 unique ASV s in the p orew ater suggests that a
large fraction of the p orew ater microbial div ersit y ma y ha v e its o wn origin separate from
the rest of the b en thic comm unit y .
24
The nic he-partition b et w een the free-living and particle-attac hed comm unities
Nic he partition b et w een the free-living and particle-attac hed lifest yle has b een rep eat-
edly observ ed in marine w aters (Ghiglione et al., 2007; Suter et al., 2018; Ortega-Retuerta
et al., 2013 ), ca v e w aters (Macalady et al., 2008), subsurface (Flynn et al., 2008; Long-
nec k er & Kuja winski, 2013) and fresh w ater lak e sedimen ts (Keshri et al., 2018). The sp e-
cific mec hanism underlying the differen tiation b et w een free-living and particle-attac hmen t
is not explicitly understo o d, but sev eral driving factors ha v e b een prop osed suc h as the
t yp e of organic carb on, energy a v ailabilit y , bacterial predation and microb e-microb e in ter-
action (Ganesh et al., 2014; Mou et al., 2008; Ortega-Retuerta et al., 2013. In addition,
it is suggested that particle-asso ciation is a ph ylogenetically conserv ed traits as microb es
b elonging to the same taxonomic rank seem to exhibit similar degree of particle asso cia-
tion (Salazar et al., 2015; Suter et al., 2018). Ev en though the particles o ccupied b y mi-
crob es in marine sedimen ts differ from those in the w ater column in particle comp osition
and the p orew ater differs from the sea w ater in terms of o xygen and n utrien t concen tration,
the pattern of bacterial b eha vior in relation to particle-attac hmen t still holds.
In Catalina harb or sedimen t, a strong preference for a particle-attac hed lifest yle w as
observ ed for sev eral ph yla (Fig. 8a). The groups Bacter oidetes and Planctomyc etes for ex-
ample are kno wn to b e asso ciated with particles in aquatic en vironmen ts (DeLong et al.,
1993; F on tanez et al., 2015; F uc hsman et al., 2011; Milici et al., 2016; Riec k et al., 2015;
Salazar et al., 2016; Suter et al., 2018). Most mem b ers of these groups are in v olv ed in
c hemo organotroph y and are able to degrade p olymers and particulate material (Beatriz
et al., 2013; Ganesh et al., 2014), suggesting that taxa with v aried metab olisms asso ciated
with organic material degradation can co-exist on particles.
Microbial groups with sp ecific metab olic activities sho w ed high lev els of nic he-sp ecificit y ,
for example, the sulfate-reducing Deltapr ote ob acteria and the sulfur-o xidizing Epsilonb ac-
ter ae ota . Epsilonb acter ae ota reside in v arious en vironmen ts particularly in habitats with
25
high sulfide concen trations (Ak erman et al., 2013; Campb ell et al., 2006; Headd & En-
gel, 2014; Hub er et al., 2010, 2007; Risto v a et al., 2015). The t w o main genera found in
Catalina harb or sedimen ts sho w ed opp osite trends of particle-asso ciation: the gen us A r-
c ob ater sho w ed strong and significan t preference for the free-living lifest yle whereas Sul-
fur ovum w as predominan tly attac hed to particles. Although the metab olic capabilities of
most arcobacters ha v e not b een studied in detail, man y cultured represen tativ es ha v e b een
sho wn to gro w on h ydrogen, h ydrogen sulfide, thiosulfate and elemen tal sulfur (Roalkv am
et al., 2015; Wirsen et al., 2002). The abilit y to use a v ariet y of electron acceptors suc h as
nitrate, o xygen, elemen tal sulfur, p erc hlorate and p oten tially manganese o xide (Carlström
et al., 2013; Roalkv am et al., 2015; V andiek en et al., 2012) is also c haracteristic for A r-
c ob acter . Sulfur ovum are commonly found in similar en vironmen ts as A r c ob ater (Campb ell
et al., 2006; Hub er et al., 2007; Inagaki et al., 2004; W ang et al., 2015) and are capable to
o xidize elemen tal sulfur for energy gain (Inagaki et al., 2004; Mori et al., 2018; P ark et al.,
2012).
Sulfate reduction has b een sho wn to b e an imp ortan t terminal o xidation pro cess in
Catalina harb or sedimen ts (Bertics and Ziebis 2009). The pro duced sulfide reacts quic kly
with the o xidized iron whic h is enric hed in this sedimen t. The concen tration of H
2
S is b e-
lo w detection limit in Catalina harb or (Bertics & Ziebis, 2009), but the strong signals of
the t w o p oten tial sulfur o xidizers suggest that an activ e sulfur cycle is at pla y in this sys-
tem (Canfield et al., 2010). A similar situation w as observ ed in the ano xic Cariaco Basin
where in w aters with no measurable sulfide, high abundance of putativ e sulfide o xidiz-
ers and abundance of elemen tal sulfur (pro duct of H
2
S o xidation) w ere found (Suter et
al., 2018). In terestingly , the putativ e sulfur o xidizers w ere presen t in b oth free-living and
particle-attac hed fractions in their study . The differen tial distribution of A r c ob acter spp.
and Sulfur ovum b et w een the p orew ater and the sedimen t is surprising and not fully un-
dersto o d. One lik ely driving factor of this partitioning is the differen t ph ysical forms of
the sulfur sp ecies they can utilize: the soluble sulfide for A r c ob acter and the solid-phase
26
S
0
for Sulfur ovum . The main source of h ydrogen sulfide in this system is sulfate reduc-
tion (Bertics & Ziebis, 2009; Zhang et al., 2017). Hydrogen sulfide can then react with
other o xidan ts biologically or c hemically to form v arious in termediate sulfur sp ecies in-
cluding elemen tal sulfur (W asm und et al., 2017). The sulfate reducing Deltapr ote ob acte-
ria in Catalina harb or sedimen ts w ere mostly attac hed to sedimen t particles (Fig. 7) and
th us the sulfide pro duced on the particles can b e o xidized to elemen tal sulfur and easily
accessed b y mem b ers of Sulfur ovum or diffuses in to the p orew ater fueling the free-living
arcobacters.
A lter omonadales and V ibrionales b elonging to the class Gammapr ote ob acteria w ere
ev enly distributed b et w een the t w o nic hes and w ere among the most abundan t taxa in
Catalina harb or sedimen ts. V ibrionales and A lter omonadales are commonly found in aquatic
en vironmen ts from coastal area to the deep o cean (T ak em ura et al., 2014; Thompson et
al., 2006). Both groups are c haracterized as high metab olic flexibilit y and genome plas-
ticit y (Amaral et al., 2014; Grimes et al., 2009; Lauro et al., 2009) whic h allo w them to
successfully o ccup y b oth nic hes. Mem b ers within the order V ibrionales and A lter omon-
adales can utilize a wide range of organic carb on comp ounds (Garrit y et al., 2015; Li et
al., 2019; Lin et al., 2018) and are often closely asso ciated with macro- (Aires et al., 2018;
Huggett et al., 2006; P atel et al., 2003) and microalgae (Buc han et al., 2014; Main et al.,
2015; T eeling et al., 2012, 2016). In p elagic en vironmen ts, V ibrio spp. are usually detected
in lo w abundance (Mansergh & Zehr, 2014; Sib oni et al., 2016; Thompson et al., 2004) but
their presence can b e stim ulated b y pulses of n utrien ts and organic matter (Baffone et al.,
2006; Balmon te et al., 2019; W estric h et al., 2016; Zhang et al., 2018). Similarly , sev eral
ph ylogenetic groups within A lter omonadales could b e stim ulated with the addition high-
molecular-w eigh t (HMW) dissolv ed organic matter in sea w ater incubations (Balmon te et
al., 2019; Ja y et al., 2010; T a ylor & Cunliffe, 2017). The V ibrionales and A lter omonadales
in this system ma y ha v e b een brough t in b y the sinking algae and are able to main tain
their high abundance with the con tin uous supply of algal organic material.
27
More than 120 V ibrio sp ecies ha v e b een describ ed to date, y et their distribution pat-
tern in marine sedimen ts ha v e rarely b een studied. A recen t study b y W ang et al. (2019)
analyzed sedimen ts in v arious lo cations in Chinese marginal seas and found the b en thic
V ibrio spp. to b e more abundan t than the p elagic V ibrio spp. in the same ecosystem.
Since sympatric sp eciation and the co-existence of free-living and particle-attac hed V ib-
rio spp. are commonly do cumen ted in the sea w ater (Hun t et al., 2008; Thompson et al.,
2004), a sp ecies-sp ecific preference o v er either lifest yle (free-living or particle-attac hmen t)
is lik ely to o ccur. In fact ASV s b elonging to the gen us V ibrio w ere found across the P AN
sp ectrum with o v er half of the ASV s at the extremes (i.e. only presen t in the p orew ater or
on the particle surface Fig. 8b). Differen tial usage of organic carb on at the sp ecies lev el
has b een found in Pseudo alter omonas spp. and V ibrio spp. (Mahmoudi et al., 2019) and is
p ossibly widespread within the ubiquitous Gammapr ote ob acteria lineages (Dyksma et al.,
2016; Spring & Riedel, 2013; T eira et al., 2008). The v arying lev els of particle-asso ciation
among A lter omonadales spp. and V ibrionales spp. could b e related to the differen tial dis-
tribution of organic comp ounds b et w een the t w o compartmen ts (Burdige, 2007; F orb es
et al., 1998; Ma y er, 1994). The in tra-gen us div ersit y within these taxa is difficult to cap-
ture using general 16S rRNA bacterial primers (Jensen et al., 2009; Jesser & Noble, 2018;
Zhang et al., 2018). F urther analyses resolving taxonomic div ersit y at resolution are needed
to illuminate the complete pattern of nic he-partitioning b et w een the free-living and particle-
attac hed comm unities in marine sedimen ts.
In summary , the p orew ater harb ors an abundan t free-living microbial comm unit y that
riv als the particle-attac hed comm unit y in div ersit y . The p orew ater comm unit y could b e
distinctly differen tiated from the comm unities in the o v erlying sea w ater and those attac hed
to sedimen t particles. Bet w een the t w o b en thic nic hes, i.e. the p orew ater and the parti-
cle surface, sev eral groups of generalists b elonging to the class Gammapr ote ob acteria w ere
shared, who accoun ted for 22.1% of the total b en thic ric hness. The ma jorit y of the b en thic
sp ecies sho w ed strong preference to w ards a particle-attac hed or a free-living lifest yle, and
28
this nic he-partition w as ph ylogenetically conserv ed. The nic he-sp ecificit y could b e a taxo-
nomic trait due to cell prop erties suc h as size, shap e, electrostatic state of the mem brane,
and esp ecially metab olic activities. The free-living comm unit y in the p orew ater p oten-
tially pla y a significan t role in b en thic biogeo c hemistry , differen t from the particle-attac hed
comm unit y . F urther studies addressing the differences b et w een the t w o b en thic nic hes in
regard to their resp ectiv e ecological functions are needed.
A c kno wledgemen ts
I w ould lik e to thank m y collab orators (Karsten Zengler, Mallory Em bree, Cameron Mar-
tino, Ric hard Szubin) at USCD for their supp ort in the molecular w ork that is in tegral
to this w ork. Dr. Ben T ully at C-DEBI has pro vided m uc h supp ort in the bioinformatic
analyses. The past mem b ers of the Ziebis Lab (Jane Den tinger, Abb y Lam bretti, Emily
W aggoner) ha v e con tributed greatly to sample collection and further pro cessing. The main
b o dy of w ork w as supp orted through the Ziebis lab researc h fundings. The W rigley In-
stitute of Marine Studies has also pro vided m uc h logistical and financial supp ort for this
w ork.
29
Figure 1: a. Oxygen concen tration at the sedimen t-w ater in terface with partial microbial
mat co v erage (t w o measuremen ts with indirect ligh t, one with direct ligh t). b. Chloroph yll
concen tration within the sedimen t in Catalina harb or (shaded bars represen t area with
microbial mats, empt y bars represen t area without microbial mats co v erage). c. concen-
trations of nitrate + nitrite and ammonium in the p orew ater and extractable F e(I I I) and
F e(I I) con ten t in the sedimen t. d. organic con ten t and p orosit y of the sedimen ts from 1 to
10 cm.
30
Figure 2: A v erage (n=3) microbial cell abundances in sea w ater, p orew ater and sedimen t.
31
Figure 3: a. Bra y-Curtis dissimilarit y b et w een the p orew ater at differen t depths and the sea w ater;
b et w een the p orew ater and the sedimen t at differen t depths. 20 subsamples of eac h sample w ere
tak en and used for the calculation. b. n um b er of amplicon sequence v arian ts (ASV s) shared b et w een
the sedimen t and the p orew ater samples or unique to either compartmen t at v arying depth.
32
Figure 4: The relativ e abundances of the abundan t amplicon sequence v arian ts (ASV s, >
1% of the relativ e abundance) in the three compartmen ts.
33
Figure 5: Bacterial amplicon sequence v arian ts (ASV s) in the sea w ater, the p orew ater and the sedimen t compart-
men ts. The n um b ers in the V enn diagram indicate the n um b er of ASV shared b y/ unique to the compartmen ts.
The piec harts represen t the taxonomic comp osition of the unique ASV s in eac h compartmen t.
34
Figure 6: T axonomic assignmen ts of the amplicon sequence v arian ts (ASV s) with differen tial abundance ( > 4
fold difference in abundance) b et w een the p orew ater and the sedimen t. Only ASV s shared b et w een the t w o com-
partmen ts w ere included in this analysis.
35
Figure 7: The distribution of the particle-asso ciation nic he (P AN)-index v alues for eac h
amplicon sequence v arian ts (ASV s) presen t in the b en thic comm unities.
36
Figure 8: The distribution of the particle-asso ciation nic he (P AN)-index v alues group ed b y (a) ph y-
lum or class and (b) gen us or order. Empt y circles represen t statistical outliers. Shaded b o xes indi-
cate statistical significance (p-v alue < 0.05) for a deviation from a P AN-index v alue of 0.5. In (b),
eac h jitter p oin ts represen ts an individual amplicon sequence v arian t (ASV).
37
Table 1. Amplicon S equence Varia nt (ASV) statistics and diversity estimates
Sample
Observed
ASVs
Depth
(cm)
Observed
ASVs
Fisher's
alpha
Shannon
Index
Pielou's
evenness
- 180 25.13 3.68 0.71
- 201 25.24 3.59 0.68
0 - 2 232 28.76 3.37 0.62
2 - 4 203 27.29 3.23 0.61
4 - 6 110 13.77 3.01 0.64
6 - 8 70 8.27 2.30 0.54
0 - 2 196 24.44 3.00 0.57
2 - 4 251 30.79 2.74 0.59
4 - 6 154 18.26 2.57 0.51
6 - 8 65 9.09 2.97 0.71
Porewater
Sediment
Seawater 201
273
276
38
Table 2. Diversity Partition of Fisher's alpha
Division beta_inter beta_intra alpha gamma
All ASVs niche 27.39 (41.86%) 12.97 (19.83%) 25.07 (38.31%) 65.44
Benthic ASVs depth 21.09 (41.87%) 4.09 (8.12%) 25.19 (50.01%) 50.37
39
Table.3 Relative abundance of main taxa (>0.1%) in the three niches
- - 0 - 2 cm 2 - 4 cm 4 - 6 cm 6 - 8 cm 0 - 2 cm 2 - 4 cm 4 - 6 cm 6 - 8 cm
Proteobacteria
Alphaproteobacteria
Rhodobacterales 40.40 46.83 0.54 0.33 0.29 - 0.82 0.34 0.28 0.25
SAR11 clade 3.29 1.95 - - - - - - - -
Parvibaculales 1.31 0.89 - - - - - - - -
Puniceispirillales 4.19 2.49 - - - - - - - -
Rhizobiales 0.23 0.45 - - - - - - - -
Rickettsiales 0.46 0.27 0.21 0.17 - - 0.22 - - -
Rhodospirillales - - 1.73 0.37 - - - - - -
Deltaproteobacteria
Bdellovibrionales 2.14 0.98 - - - - - - - -
PB19 0.28 0.11 - - - - - - - -
Desulfobacterales - - 0.34 0.50 - - 0.96 1.62 0.95 0.72
Syntrophobacterales - - - - 0.19 - - 0.12 - -
Gammaproteobacteria
Oceanospirillales 20.44 13.74 10.02 6.77 17.38 4.74 10.81 2.16 1.66 3.15
Vibrionales 0.50 0.26 61.45 71.52 65.51 87.05 68.39 83.49 90.59 64.96
Alteromonadales 0.50 0.80 16.37 7.24 1.89 1.62 13.60 6.29 3.34 15.09
Betaproteobacteriales 1.25 1.16 - - - - - - - -
Cellvibrionales 2.12 2.07 - - - - - - - -
Coxiellales 0.31 0.24 0.17 0.21 - - - - - -
Diplorickettsiales - - 0.19 0.17 - - - - - -
Legionellales - - 0.21 0.50 - - 0.20 - - -
MBAE14 - - 0.46 0.37 0.28 - - - - -
OM182 clade 0.29 - - - - - - - - -
SAR86 clade 2.02 1.13 - - - - - - - -
Steroidobacterales - - - - - - 0.34 0.42 0.15 0.38
Thiohalorhabdales - - - - - - - 0.13 - -
Bacteroidetes -
Overlying Seawater Porewater (Free-living) Sediment (Particle-attached)
40
Chitinophagales 0.23 0.31 - - - - - - - -
Flavobacteriales 14.15 18.66 0.21 0.37 - - 0.36 0.21 0.12 -
Ignavibacteriales - - - - - - - 0.13 - -
Sphingobacteriales 0.44 0.61 - - - - - - - -
Epsilonbacteraeota
Arcobacter - - 4.45 8.60 13.32 6.09 0.19 0.89 1.09 1.73
Sulfurimonas - - - - - - - 0.15 0.19 -
Sulfurovum - - - - - - - 0.30 0.35 2.04
Cyanobacteria 2.54 1.34 - - - - - - - -
Actinobacteria 2.35 4.82 - - - - 0.13 - - -
Dependentiae - - 0.21 0.59 0.41 - 0.13 0.17 0.11 0.14
Verrucomicrobia - - - - - - - - - -
Planctomycetes - - - - - - 0.57 0.17 - -
Fusobacteria - - - - - - - - - -
Acidobacteria - - - - - - 0.45 0.29 - -
Chloroflexi - - - - - - - - - 0.25
Firmicutes - - - - - - - - 0.18 -
Gemmatimonadetes - - - - - - - 0.16 - 0.27
Patescibacteria - - - - - - - 0.17 - -
Spirochaetes - - - - - - - 0.11 - -
41
Supplemen tary Materials
Figure S1: Rarefaction curv e sho wing the n um b er of amplicon sequence v arian ts (ASV s)
against the n um b er of reads (calculated at step = 1000).
42
Figure S2: Relativ e abundance of the shared amplicon sequence
v arian ts (ASV s) b et w een the sea w ater and the b en thic com-
partmen ts. Individual ASV s w ere group ed in to ph ylum for clar-
it y of the figure.
43
Figure S3: Microbiota div ergence with depth of the free-living
and the particle-attac hed comm unities.
44
Chapter 3: Microbial A ctivities of Carb on T urno v er in a Com-
parison Bet w een F ree-living and P article-attac hed Comm uni-
ties in Marine Sedimen ts
In collab oration with Alb erto Robador and Wiebk e Ziebis
Abstract
Coastal marine sedimen ts harb or div erse microbial comm unities in an abundance a v erag-
ing 10
9
10
10
cells p er cm
3
, 3 orders of magnitude greater than the a v erage cell abun-
dance in the sea w ater. A fraction of the b en thic microbial comm unities are free-living in
the p ore space and the rest are attac hed to sedimen t particles. The con v en tional micro-
bial studies of the bulk sedimen ts disregard the differences b et w een the t w o nic hes and fail
to recognize the p orew ater comm unit y as an in tegral comp onen t of b en thic biogeo c hem-
ical cycles. This study fo cused on c haracterizing the unexplored p orew ater comm unit y
in comparison to the particle-attac hed comm unities in metab olic activities, sp ecifically
carb on turno v er. Rate measuremen ts of acetate and formate o xidation using
14
C-lab eled
tracers sho w ed m uc h higher carb on turno v er rates of the free-living comm unit y than their
particle-attac hed neigh b ors. A com bination of thermo dynamic calculations, metagenomic
analysis and nano calorimetry rev ealed the nic he-partition b et w een the free-living and the
particle-attac hed comm unities in carb on degradation and the terminal o xidation pro cesses.
W e therefore conclude that the con tribution of the free-living comm unit y to b en thic bio-
geo c hemical pro cesses has b een m uc h underestimated.
45
In tro duction
Coastal marine en vironmen ts suc h as tidal m udflats host a great abundance of microbial
comm unities, in a v erage 10 - 10000 times the n um b er found in the same unit v olume of
sea w ater (Jørgensen & Bo etius, 2007). The high abundance of microbial comm unities
in these systems are supp orted mainly b y organic material dep osited from the w ater col-
umn, including the pro ducts of p elagic primary pro duction as w ell as detritus of seagrass
and algae (Berner, 1978; Duarte & Cebrián, 1996; F ranco et al., 2007; F reese et al., 2008;
Jahnk e, 1996; V olkman et al., 2000). During lo w tide when sunligh t p enetrates through
the o v erlying w ater, b en thic photosyn thesis at the sedimen t surface b y micro eukary otes,
macroph ytes and bacteria also pro vides large amoun t of fresh organic material (de Co ok
et al., 2004; de Beer et al., 2005; F reese et al., 2008; Graue et al., 2011). The frequen t in-
put of labile organic material can fuel high rates of microbial activities that quic kly de-
plete o xygen in the surface sedimen ts (Kristensen et al., 1995). These remaining organic
material are then degraded sequen tially b y div erse groups of anaerob es through pro cesses
including h ydrolysis of p olymers, fermen tation of monomers, and terminal o xidation of the
fermen tation pro ducts (Laan bro ek & V eldcamp, 1982).
V olatile fatt y acids (VF As), suc h as formate, acetate, lactate, propionate and bu-
t yrate are k ey in termediates during the anaerobic degradation of organic materials. They
are end-pro ducts of microbial fermen ters and widely-used sources for terminal respiration
(Christensen & Blac kburn, 1982; Glom bitza et al., 2015; Mic helson et al., 1989; Sha w et
al., 1984; Thauer & Zinkhan, 1989). T o simply put it, the organic carb ons flo w through
simple VF As and ev en tually are o xidized to carb on dio xide. The rates of VF A o xidation to
carb on dio xide can th us b e used as an index to estimate the o v erall rates of organic carb on
remineralization, and ha v e b een measured in v arious en vironmen ts suc h as a mangro v e,
coastal lago ons, in tertidal flats and arctic fjords (Ansbaek & Blac kburn, 1980; Christensen,
1984; Christensen & Blac kburn, 1982; Fink e et al., 2007; Glom bitza et al., 2015; Kris-
46
tensen et al., 1994; Sha w et al., 1984; Sørensen et al., 1981). In b oth fresh w ater and ma-
rine sedimen ts, acetate is found to b e the most imp ortan t electron donor for anaerobic res-
piration including iron and sulfate reduction, follo w ed b y lactate, propionate and but yrate
(Balba & Nedw ell, 1982; Fink e et al., 2007; Laan bro ek & Pfennig, 1981; Sansone, 1986;
Thauer & P ostgate, 1982). T o the b est of our kno wledge, in-situ rate of formate o xidation
has nev er b een rep orted. The con tribution of formate o xidation to anaerobic respiration is
therefore unclear.
With the dev elopmen t of high-throughput sequencing, metagenomic approac hes ha v e
b een used to study the complex microbial pro cesses in marine sedimen ts (Biddle et al.,
2008; Dyksma et al., 2016; P ern thaler et al., 2008). Protein-co ding genes can b e iden tified
and assessed in natural comm unities to elucidate the in tegral steps in the degradation of
organic carb on and to iden tify the p oten tial pla y ers (Kimes et al., 2013; Kirc hman et al.,
2014; Mason et al., 2014; Rasigraf et al., 2019). In addition, metagenomic studies ha v e di-
rected the disco v eries of no v el metab olisms of a wide range of carb on resources (Castelle et
al., 2018; Ev ans et al., 2015; W asm und et al., 2019; Y au et al., 2013). Up on understanding
the correlation b et w een the abundance of protein-co ding genes estimated using metage-
nomics and the actual rates of geo c hemical pro cesses they catalyze, the predictiv e abilit y
of metagenomic studies ma y further lend help to quan tify other geo c hemical pro cesses that
can not b e measured directly (Ro cca et al., 2015).
The main pro cesses of carb on mineralization at the terminal step are w ell c haracter-
ized through rate measuremen ts and mark er gene analyses. The upstream pro cesses in-
cluding the breakdo wn of complex organic matter and fermen tation remain elusiv e. As
metab olic activities all pro duce sp ecific heat signals (R ussell & Co ok, 1995), calorimetric
measuremen ts of heat pro duction can b e used to infer the rates of metab olic pro cesses.
Nano calorimetry is an extremely sensitiv e tec hnique whic h allo ws for real-time measure-
men t of heat flo w in the range of micro w atts (Larsson et al., 1991; Mask o w & P aufler,
2015). This no v el tec hnique has b een successfully used to iden tify the differen t phases of
47
fermen tation of the microbial comm unities in a tidal flat (Graue et al., 2012), to c haracter-
ize the cell activities in deep subsurface o ceanic crustal fluids (Robador et al., 2016), and
to ev aluate the calorespirometric ratio (the ratio of heat pro duction to CO
2
pro duction) as
an index of microbial metab olism and carb on use efficiency (Herrmann & Bölsc her, 2015).
Nano calorimetry offers a non-in trusiv e and direct estimate of the o v erall microbial activi-
ties in en vironmen tal samples whic h is difficult to capture otherwise.
Marine sedimen ts consist of sedimen t particles and the p ore space in b et w een. The
v olume ratio of the p ore space to the bulk sedimen t, termed as p orosit y , v aries from 0.5
- 0.9 dep ending on sedimen t t yp e and depth (Hamilton, 1976). If the p ore space is com-
pletely filled with p orew ater, the v olume of p orew ater in global sedimen ts is estimated to
b e 8:46 10
7
km
3
(+15.6%/–14.1%) (LaRo w e et al., 2017). The p orew ater, essen tially
sea w ater, is exp ected to harb or microbial cells at a concen tration at least similar to that
in the sea w ater (10
6
cells cm
3
). Giv en the large v olume of marine p orew ater, the free-
living microb es comprise an in tegral comp onen t of the b en thic microbial comm unities. In
addition, a previous study b y Hewson and F uhrman (2003) found higher viral pro duction
rates in the p orew ater than the p orew ater-free sedimen ts in Los Angeles Harb or and San ta
Catalina Island (Los Angeles, CA), suggesting that the free-living comm unit y in the p ore-
w ater ma y b e more pro ductiv e than the particle-attac hed bacteria.
Con v en tionally microbial comm unities in the sedimen ts are studied in “bulk”, mean-
ing that the free-living and particle-attac hed comm unities ha v e nev er b een in v estigated
separately . The goal of this study is to in v estigate the role of the unexplored free-living
comm unit y in b en thic biogeo c hemical pro cesses via a comparison of the microbial activ-
ities in carb on degradation b et w een the free-living and particle-attac hed comm unities.
In-situ rate measuremen ts using radiotracers w ere com bined with metagenomic analy-
ses of carb on metab olizing genes to assess the relativ e con tributions to b en thic carb on
mineralization b y the free-living and particle-attac hed comm unities. W e also used nano-
calorimetry to monitor the heat pro ductions of the free-living comm unit y o v er 24h. Based
48
on the heat flux profiles, thermo dynamic calculations w ere conducted to piece together the
differen t pro cesses in organic carb on degradation.
Metho ds and Materials
Study site
Sedimen t samples w ere collected from an in tertidal m udflat in Catalina Harb or, a small
harb or lo cated on the south side of the isthm us of San ta Catalina Island. San ta Catalina
Island (33°23’N 128°25’W) is lo cated off the coast of southern California, appro ximately
35 km south w est of the cit y of Los Angeles, CA. This site has b een previously c haracter-
ized for its macrofauna activities and biogeo c hemical pro cesses (Bertics and Ziebis, 2009;
Bertics and Ziebis, 2010; Bertics et al., 2010; Lam et al., 2018).
Sample collection and pro cessing
Sedimen t samples w ere collected using p olycarb onate coreliners (10 cm diameter) in dif-
feren t seasons (July and A ugust 2016, Marc h and No v em b er 2017, Ma y 2019, detailed in
eac h section). The samples w ere collected during lo w tide with appro ximately 15 cm of
w ater co v erage. The temp erature of the sedimen t-w ater in terface w as b et w een 12 - 15

C.
Up on retriev al, sedimen t cores w ere closed with rubb er stopp ers. The o v erlying w ater in
the cores w ere k ept o xygenated using air pumps. The sedimen t cores w ere either pro cessed
shortly after transp ortation to lab facilit y or k ept at 4

C un til pro cess.
Subsamples for measuring acetate turno v er rates, nano-calorimetry , determining mi-
crobial abundance and molecular analyses w ere collected b y either homogenizing the upp er
10 cm or sectioning in 1 - 2 cm in terv als (detailed in eac h section). These subsamples w ere
sub jected to p orew ater separation using a p orew ater pressing b enc h (25 cm
3
POM, K C
Denmark A/S, Silk eb org, Denmark). Eac h subsample w as placed and sealed in to individ-
49
ual pressing house. Ultrapure nitrogen w as applied through the top of eac h pressing house
and gen tly exp el the p orew ater from the sedimen t matrix through a pre-installed 8 µm Nu-
cleop ore mem brane filter (Whatman,GE Healthcare) in to resp ectiv e collecting tub es. The
remaining dry sedimen ts in the pressing house w ere considered to b e p orew ater-free and
con tain only the particle-attac hed comm unit y .
Determination of microbial abundance
Microbial abundances w ere determined for the o v erlying sea w ater (ab o v e the sedimen t col-
lected using coreliners), the p orew ater and the bulk sedimen t from v arying depths (sec-
tioned in 1 cm in terv als from 0 - 20 cm) follo wing proto cols b y Epstein and Rossel (1995)
and P atel (2007) with mo dification. The detailed pro cedures are listed in the Supple-
men tary Material. In brief, sedimen t samples of 1 cm
3
v olume w ere fixed with 9 ml 4%
sea w ater-formaldeh yde solution (0: 02 µm filtered). The 1 : 10 diluted sedimen t slurries
w ere further diluted (1:10 v/v) with 4% sea w ater-formaldeh yde solution (0: 02 µm filtered)
and sonicated using a sonicator tip to dislo dge the cells off the particles. This solution w as
then again diluted (1: 10) in the same fashion. 2 ml subsamples of the p orew ater of eac h
depth w ere transferred in to resp ectiv ely lab elled 2 ml cry o vials and fixed with 0 : 02 µm fil-
tered formaldeh yde to a final concen tration of 2%. 100 ml of the o v erlying sea w ater w as
collected at the sampling site and fixed with 0 : 02 µm filtered formaldeh yde to a concen tra-
tion of 2%.
A defined v olume of the formalin-fixed subsamples from eac h nic he (200 µL for the
p orew ater subsamples, 2 ml of the sea w ater subsamples, or 1 ml of 1:1000 diluted sedi-
men t solution) w as filtered on to a 0 : 22 µm Nucleop ore p olycarb onate filter (Whatman, GE
Healthcare) using a hand pump. Sterile-filtered marine PBS w as used to w ash off the in-
ner w all of the filter funnel and the w ash fluid w as collected on the filter to catc h all cells
that w ere stuc k on the glass. The filter w as then air-dried and transferred on to a sterile
50
glass microscop e slide and stained with a SYBR green staining solution (1:15:1 1%Ascor-
bic acid: Mo wiol moun ting solution: SYBR Green). Eac h prepared slide w as visualized
and en umerated under a fluorescence microscop e (Nik on Eclipse 80i, 100 oil immersion).
F or eac h sample, triplicate filters w ere prepared. Eac h filter w as coun ted for 10 fields of
view.
DNA extraction and sequencing
The p orew ater and the p orew ater-free sedimen ts w ere sampled for molecular analyses (Oc-
tob er 2014). The parallel sedimen t cores w ere sectioned in 2 cm-in terv als from 0 to 8 cm
deep. The sections from all cores of the same depth w ere com bined to ensure a sufficien t
amoun t of cells in eac h sample for DNA extraction. P orew ater samples w ere obtained as
describ e in the ab o v e section. The remaining dry sedimen ts in the pressing house w ere
considered to b e p orew ater-free and con tain only the particle-attac hed comm unit y . F or
eac h of the four depth in terv als (0-2, 2-4, 4-6 and 6-8 cm), In tact cells w ere extracted from
a defined v olume (25 ml) of the p orew ater samples, as w ell as from the corresp onding
p orew ater-free sedimen t samples (200 cm
3
). A densit y cen trifugation metho d using Ny-
co denz w as used to concen trate cells (Kallmey er et al., 2008). DNA w as extracted from
the p ellets of the in tact cells using the NucleoSpin Kit (Mac herey-Nagel Inc., Bethlehem,
P A). Metagenomic sequencing w as p erformed on an Illumina HiSeq platform using the 2
150 bp paired-end c hemistry according to man ufacturer’s instructions at the Biomedical
Researc h F acilit y in UC San Diego.
Metagenomic assem bly , binning, and annotation
Ra w reads w ere trimmed using T rimmomatic (Bolger et al., 2014) to remo v e adapters
and lo w qualit y sequences. Sequences that passed qualit y trimming w ere co-assem bled to
con tigs using MEGAHIT (Li et al., 2015) with the meta-sensitiv e preset (min-coun t=1,
51
kmers=21, 29, 39, 49, ... ,129, 141). Primary con tigs w ere clustered using CD-HIT-EST
(F u et al., 2012, sequence iden tit y = 0.95, w ord length = 10). The con tigs set w ere fur-
ther merged using minim us2 (T reangen et al., 2011, O VERLAP=50, MINID=95). Final
con tigs with length greater than 1000 w ere imp orted in to An vi’o (Eren et al., 2015) and
analyzed follo wing the An vi’o metagenomic w orkflo w. In short, eac h sample w as profiled
b y mapping reads originated from that sample to the con tigs with b o wtie (Langmead et
al., 2009). Protein-co ding genes w ere predicted using Pro digal (Hy att et al., 2010) and an-
notated using GhostKO ALA (Kanehisa et al., 2016). The relativ e abundance of genes of
in terest w ere calculated as co v erage of eac h gene divided b y the o v erall sample mean co v er-
age.
Measuremen t of VF A o xidation rate
The anaerobic acetate and formate o xidation activities of the free-living and particle-attac hed
microbial comm unities w ere ev aluated b y incubating resp ectiv e samples with
14
C-lab eled
tracers and measuring the radioactivit y of the pro duced
14
CO
2
(Balba & Nedw ell, 1982).
A depth-profile of the o v erall acetate o xidation rates o v er 20 h and m ultiple time series of
acetate/formate o xidation rates of the upp er 10 cm of the sedimen ts w ere determined in
separate incubations detailed b elo w.
VF A
Pro duct(s)
Measured
Depth (cm) Time P oin ts (h)
Sampling
Time
A cetate CO
2
0 - 1, 1 - 2, 2 - 4,
4 - 6, 6 - 8, 8 - 10
0, 0.5, 2, 20 July 2016
A cetate CO
2
, CH
4
0 - 10
0, 2, 4, 6, 8, 12, 16, 20, 24,
30, 48
A ug 2016
A cetate CO
2
, CH
4
0 - 10
0, 0.5, 2, 4, 6, 8, 10, 14, 18,
22, 26, 30, 40, 48
Mar 2017
F ormate CO
2
0 - 10
0, 2, 4, 6, 8, 12, 16, 20, 24,
30, 48
Ma y 2019
After p orew ater separation, parallel incubations with the p orew ater and the p orew ater-
52
free sedimen ts w ere prepared for eac h set of exp erimen t. 5 cm
3
prew ater-free sedimen t or
p orew ater w ere mixed with 5 cm
3
0: 02 µm filtered sea w ater in eac h incubation vial (25 ml
serum vial, anaerobically sealed with rubb er but yl stopp er and alumin um crimp ed cap).
100 µl
14
C-acetate (So dium acetate [2-
14
C], 2 µCi, 58 mCi/ mmol, American Radiolab eled
Chemicals, diluted to 10 µCi/ml) or
14
C -formate (So dium formate [1 µCi, 56 mCi/ mmol,
American Radiolab eled Chemicals, diluted to 10 µCi/ml) w as injected in to eac h incuba-
tion vial using a gas -imp ermeable 1 ml micro-syringe (SGE, A ustralia). A t eac h time
p oin t, duplicate vials for eac h set of parallel incubations w ere terminated b y adding 5 ml
5% NaOH and stored at 20

C. Radioactivit y of the pro duced lab eled carb on pro ducts,
in the form of
14
CO
2
or
14
CH
4
, w ere determined at the end of eac h incubation.
Radioactivit y of
14
CO
2
w as measured follo wing the proto col b y T reude et al. (2003)
with mo dification. In brief, the con ten t of the incubation vial, together with the w ash flu-
ids (2 x 5ml 5% NaOH solution) w as emptied in to a 250 ml Erlenmey er flask. The flask
w as immediately capp ed using a rubb er stopp er. 6ml 6M HCl w as added to the Erlen-
mey er flask using a 3 inc h h yp o dermic needle. The
14
CO
2
de-gassed after acidification of
the samples w ere trapp ed inside a 7 ml scin tillation vial attac hed to the rubb er stopp er
prefilled with 1 ml phen yleth ylamine and 1 ml 2.5% NaOH. 3 ml scin tillation co c ktail (Ul-
tima GoldTM XR, P erkinElmer) w as added to eac h scin tillation vial. Radioactivit y w as
measured using a scin tillation coun ter (LS 6500 Multipurp ose Scin tillation Coun ter, Bec k-
man Coulter). The reco v ery rate of
14
CO
2
w as 94% (Details in Supplemen tary Materials).
The measuremen ts of radioactivit y of
14
CH
4
in addition to
14
CO
2
w ere conducted
mo difying pro cedure b y de Graaf et al. (1996). Compressed air w as purged through the
sample vial, flushing
14
CH
4
sequen tially through a copp er furnace at 850

C, a safet y b ot-
tle with ethanol and in to t w o 20ml scin tillation vials, eac h with 1ml phen y eth ylamine
and 7 ml eth ylene glycol monometh yl ether (2-Metho xy ethanol) as the absorb en t. After
30 min purging,7 ml 6 M HCl w as injected in to the sample vial, the
14
CO
2
w as then car-
ried through the system and in to t w o new scin tillation vials con taining the same absorb en t
53
describ ed ab o v e. 10 ml of scin tillation co c ktail (Ultima GoldTM XR, P erkinElmer) w as
added to eac h scin tillation vial. Radioactivit y w as measured using a scin tillation coun ter
(LS 6500 Multipurp ose Scin tillation Coun ter, Bec kman Coulter). The reco v ery rates of
14
CO
2
and
14
CH
4
w ere 92% and 94% resp ectiv ely (Details in Supplemen tary Materials).
The rate of VF A o xidation w as determined as the rate of disapp earance of acetate or
formate o v er time, calculated as the p o ol size of acetate or formate the rate constan t (k)
(Ansbaek & Blac kburn, 1980). The VF A o xidation rate constan t k w as calculated from the
equation kt=ln[A(A–a)] (Sha w et al., 1984), where A is the initial radioactivit y of acetate
or formate, a is the radioactivit y of pro duced
14
CO
2
at time t. The acetate and formate
concen tration in our sample w ere b elo w detection limit, th us w as assumed 10 µM based on
literature rep orts from similar en vironmen ts. The VF A o xidation rates of the free-living
comm unities w ere adjusted as b elo w, to accoun t for the dilution of initial ino culum and
p orosit y .
Ratepercm
3
sediment
=Rate
porewater

CellAbundance
porewater
CellAbundance
inoculum
porosity (1)
The adjusted n um b er w as normalized to p er v olume of bulk sedimen t and th us repre-
sen ted the amoun t of VF A o xidized b y the free-living comm unities within p er unit v olume
sedimen t p er uin t time. By the same tok en, the rate of VF A o xidation b y the particle-
attac hed comm unities presen ted in the results represen ted the con tribution b y the particle-
attac hed comm unities to p er unit v olume bulk sedimen t.
Thermo dynamic calculation of microbial heat pro duction
Reactions using acetate and formate as electron donors coupled with differen t electron ac-
ceptors w ere written and c hemically balanced. W e then used Thermo dyn (Damgaard &
Hanselmann, 1999; Hanselmann, 1991) to calculate the en thalp y c hange of eac h reaction
54
under standard state. The heat pro duction, estimated as en thalp y c hange, within 24h in-
cubation w as calculated as:
q = substrateH
r
(2)
where q stands the heat pro duced (kJ), substrate is the amoun t of substrate con-
sumed (mol), H
r
is the en thalp y of the reaction (kJ/mol).
Measuring microbial heat pro duction with nano calorimetry
Calorimetric measuremen ts of heat pro duction w ere used to directly assess the metab olic
activit y of the free-living and particle-attac hed microbial comm unities in the sedimen ts.
Surface sedimen t (upp er 10 cm) of parallel cores (No v 2017) w ere subsampled for p orew a-
ter separation as describ ed ab o v e. Samples and references w ere prepared in 4 ml calorimet-
ric vials (cleaned and com busted at 450

C for 6 h), all crimp ed and sealed with but yl rub-
b er stopp ers. F or the free-living comm unities, samples consisted of 4 ml of extracted p ore-
w ater. The reference consisted of 4 ml of 0.02 µm filtered and UV-sterilized p orew ater.
F or the particle-attac hed comm unities, 2 cm
3
of the p orew ater-free sedimen ts (re-saturated
with 0: 02 µm filtered p orew ater, transferred using a sterile cut-off syringe) w as mixed with
2 ml 0: 02 µm filtered and UV-sterilized o v erlying sea w ater. The reference consisted of pre-
com busted sedimen ts (450

C, o v ernigh t) prepared as in the sample.
The heat flo w in a pair of calorimetric vials (sample and reference) w as measured
isothermally (20

C) for 24 hours. The heat flo w due to metab olic activit y w as tak en as
the difference b et w een the sample and the reference. Nano calorimetric exp erimen ts w ere
p erformed using a thermal activit y monitor mo del T AM I I I equipp ed with a nano calorime-
ter (T A Instrumen ts, Lindon, UT, USA). In addition, three replicate samples w ere pre-
pared the same w a y as calorimetric exp erimen ts and incubated outside the nano calorime-
55
ter at 20

C to serv e as a con trol.
A cetate o xidation rates w ere determined in all incubated samples (inside and outside
the nano calorimeter). Before eac h incubation 100 µl 10 µCi/ ml
14
C -lab eled so dium acetate
w as added to eac h vial. A t the end incubations w ere terminated b y transferring incubated
samples in to 50 ml cen trifuge tub es con taining 5 ml 5% NaOH. In addition, eac h calori-
metric vial w as w ashed with 2 x 4 ml 5% NaOH. The w ash fluid w as added to the resp ec-
tiv e cen trifuge tub es. The radioactivit y of pro duced
14
CO
2
w as subsequen tly measured as
describ ed ab o v e.
Normalization of initial heat flo w
Before and after the metab olic heat flo w w as measured, baseline v alues w ere recorded for
30 min without a vial inside the calorimeter in order to ensure that no offset due to an y
thermal activit y w as recorded. Before actual calorimetric measuremen ts w ere initiated,
sample and reference vials w ere lo w ered in to equilibration p osition inside the nano calorime-
ter to allo w for the thermal equilibration of the samples to the incubation temp erature of
20 °C. Once equilibration w as reac hed, t ypically after 15 min, vials w ere lo w ered in to the
measuring p osition. During this pro cess, disturbances due to the residual transfer of heat
from the thermal media to the sample pro duced an artifact of negativ e heat-flo w. There-
fore, another equilibration of 3 hours w as necessary to reac h a stable signal. The initial
heat effect is prop ortional to the temp erature difference b et w een the thermal media and
the sample appro ximating a logarithmic deca y . T o remo v e this initial effect, an exp onen-
tial function w as fitted to the heat curv es in the initial 3 hours and subtracted from the
en tire heat curv e of the resp ectiv e exp erimen ts. This allo w ed heat curv es to start at
0 µW with little noise or initial drift.
56
Results
Microbial abundance
Microbial abundance in the bulk sedimen t w as highest (7.83 10
9
cells cm
3
) at the sedimen t-
w ater in terface and decreased with depth (Fig. 1). The same trend w as observ ed for the
free-living comm unities in the p orew ater. A t all depth, free-living cells (1:0 4:0
10
7
cells ml
1
) accoun ted for around 0.5% of the total sedimen tary microbial abundance
and w ere 1 to 2 orders of magnitude higher in abundance than the sea w ater comm unities
(5:8 10
5
cells ml
1
).
High VF A o xidation rates of the free-living comm unities
A cetate o xidation rates of the b en thic microbial comm unities generally decreased with
depth (Fig. 2). Ranging from 16.3 to 5.9 nmol cm
3
sediment
d
1
, acetate o xidation rates of
the free-living comm unities w ere consisten tly higher than those of the particle-attac hed
comm unities (5.8 - 1.3 nmol cm
3
sediment
d
1
) at all depths.
T w o separate exp erimen ts w ere conducted in A ugust 2016 and Marc h 2017 to mea-
sure the acetate o xidation rates in the surface sedimen t (upp er 10 cm). During the first 24
hours of incubation, the free-living and the particle-attac hed comm unities sho w ed differ-
en tial patterns in o xidizing acetate (Fig. 3). The particle-attac hed comm unities started
o xidizing acetate at the highest rate (2.43 0.43, 2.83 0.41 nmol cm
3
sediment
d
1
) at
the b eginning of the incubations. As the incubation progressed the rate of acetate o xi-
dation decreased. In con trast the free-living microb es sho w ed increasing rates of acetate
o xidation with a slo w start and exceeded their particle-attac hed coun terpart after t w o
hours. The acetate o xidation rate of the free-living comm unities p eak ed (12.08 0.07
nmol cm
3
sediment
d
1
) around 12h of the incubation in A ugust 2016. During the incubation
in Marc h 2017, a relativ ely smaller p eak (7.05 0.35 nmol cm
3
sediment
d
1
) w as observ ed
57
around 8 - 10h. After 24h, minimal amoun t of acetate w as o xidized in b oth free-living and
particle-attac hed comm unities (Fig. S1). During the course of incubation, CO
2
w as the
main pro duct as minimal amoun t of CH
4
w as pro duced (Fig. S2).
Similarly , the formate o xidation rates of the particle-attac hed comm unities decreased
from 42.21 6.53 to 0.19 0.19 nmol cm
3
sediment
d
1
during the 24h incubation (Fig. 4).
In con trast, the formate o xidation rates of the free-living comm unities increased to 36.03
and 29.68 nmol cm
3
sediment
d
1
in duplicate exp erimen ts at 12h and 16h resp ectiv ely and
decreased afterw ards. By 24h, the formate o xidation rates of b oth free-living and particle-
attac hed comm unities dropp ed to appro ximately 0.
A v eraged o v er 24h, the particle-attac hed comm unities o xidized acetate at a rate of
0.98 and 1.49 nmol cm
3
sediment
d
1
in t w o separate exp erimen ts, while the acetate o xidation
rates of the free-living comm unities to w ered at 7.96 and 4.45 nmol cm
3
sediment
d
1
(Fig. 5).
In a v erage, the acetate o xidation rate of the free-living comm unities w as appro ximately 5
times that of the particle-attac hed comm unities (Fig. 5). In essence, the free-living com-
m unities, accoun ting for only 1% of the b en thic microbial comm unities in abundance, w ere
resp onsible for o v er 80% of the acetate turno v er in the sedimen t. The disparit y of formate
o xidation rates b et w een the free-living and the particle-attac hed comm unities w as less ap-
paren t. In a v erage, the formate o xidation rates of the particle-attac hed and the free-living
comm unities w ere 11.23 and 14.11 nmol cm
3
sediment
d
1
(Fig. 5). Ov erall, for ev ery mole of
formate o xidized, the free-living comm unities con tributed appro ximately 55.8%.
Mark er genes for carb on metab olisms
Genes enco ding for three acetate metab olizing proteins: (ADP-forming) acetate-CoA ligase
( A c d ), acetate kinase ( A ck ), and acet yl-CoA syn thetase ( A cs ) w ere detected in b oth free-
living and particle-attac hed comm unities (Fig. 6). In b oth nic hes, A cs and A ck w ere more
prev alen t than A c d . Genes enco ding for formate deh ydrogenase ( Fdh/Fdo ), the mark er
58
gene for formate o xidation, w as found in high abundance in b oth free-living and particle-
attac hed comm unities (Fig. 6). Genes enco ding for p yruv ate formate ly ase ( Pfl ), whic h
catalyzes formate pro duction from p yruv ate, w as detected in b oth nic hes but in lo w abun-
dance. Cyto c hrome-dep enden t ( Lldh ) and NAD(P)-dep enden t ( L dh ) lactate deh ydrogenase
enco ding genes, in v olv ed in lactate metab olism, w ere b oth presen t. Genes enco ding for
aldeh yde ( A ldh ) and alcohol ( A dh ) deh ydrogenase, essen tial for ethanol metab olism, w ere
also detected.
Among the genes in v olv ed in organic carb on metab olism, genes enco ding for alde-
h yde and alcohol deh ydrogenase sho w ed highest abundance, follo w ed b y genes enco ding
for formate deh ydrogenase, acetate metab olizing proteins, and lactate deh ydrogenases.
Except for formate deh ydrogenase-enco ding genes, whic h sho w ed similar abundance in
the free-living and particle-attac hed comm unities, all other genes asso ciated with car-
b on metab olism assessed w ere more prev alen t in the free-living comm unities, esp ecially
at greater depth (4 - 6cm).
In addition, capabilit y of CO
2
fixation of the t w o comm unities w ere assessed b y the
presence of genes enco ding for R uBisCO ( Cbb ), phosphoribulokinase ( Prk ), A TP-citrate
ly ase ( A cl ), and carb on mono xide deh ydrogenease (Cdh), mark er genes for CO
2
fixation
using the Calvin-Benson-Bassham cycle, reductiv e citric acid cycle, and the W o o d-Ljungdahl
path w a y , resp ectiv ely . The former three sho w ed lo w relativ e abundance whereas Cdh w as
not detected in either nic he.
Microbial heat pro duction: measured and calculated
Nano calorimetry w as used to measure the heat flux due to biological activities. The heat
flo w of the free-living comm unities sho w ed a gradual increase in the first quarter of the 24-
hour incubation (Fig. 7). The rate of heat pro duction increased and p eak ed around hour
10, follo w ed b y a rapid decrease. Heat pro duction fell b elo w detection limit after 14 15
59
hours. The same trend w as observ ed in t w o separate exp erimen ts.
In tegrating heat flo w (µW) o v er time, w e w ere able to calculate the total heat pro duc-
tion in the 24h incubation (Fig.7). In the t w o separate exp erimen ts, microbial activities
generated 16.30% and 22.71% of the total heat in the first 8 hours. The ma jorit y of bio-
logical activities (83.70% and 77.29%) w ere observ ed in the follo wing 5 hours (S.T able 1).
Ov erall, microbial activities generated heat in the amoun t of 0.140J and 0.102J in t w o re-
p eat exp erimen ts.
The acetate o xidation rates measured using
14
C-tracer for samples in and outside of
the nano calorimeter w ere similar to the a v erage rates measured in previous exp erimen ts.
Based on the en thalp y c hange of acetate and formate o xidation coupled to differen t elec-
tron acceptors (T able 1), anaerobic acetate o xidation w as predicted to generate heat in
the amoun t of 0.0114 0.0032J, 0.0134 0.0038J, and 0.00149 0.00030J using nitrate,
amorphous iron o xyh ydro xide, and sulfate as the sole electron acceptor, resp ectiv ely . The
rate of formate o xidation, although not measured here, is exp ected to b e similar to what
w as measured in separate exp erimen ts. F ormate o xidation w as predicted to ha v e yielded
0.00840 0.000596J, 0.00964 0.000683J, and 0.00316 0.000224J of heat. The calcu-
lated heat from the terminal o xidation of acetate and formate p oten tially accoun ted for up
to 20% of the total heat generated.
Discussion
High VF A turno v er rates of the free-living comm unit y
In diffusion-dominated m uddy sedimen ts, suc h as the in tertidal m udflat in Catalina Har-
b or, microbial activities generally decrease within depth due to the decreasing a v ailabil-
it y of labile organic material and the decreasing redo x energy for microbial respiration
(Bec k et al., 2009; Miy atak e et al., 2012; O’Sulliv an et al., 2013; Sansone, 1986; Seidel et
60
al., 2012). A similar trend w as observ ed in this study as the cell abundance, as w ell as the
acetate o xidation rates of the free-living and particle-attac hed comm unities b oth decreased
with depth (Fig. 1, Fig. 2).
No prior studies ha v e directly compared the metab olic rates of the free-living and
particle-attac hed bacterial comm unities in marine sedimen ts. Our results, for the first
time, sho w ed that the free-living comm unit y could o xidize VF A m uc h faster than the particle-
attac hed comm unit y (Fig. 5). If w e assume equal cell activit y within eac h nic he, a free-
living cell in the p orew ater could outcomp ete a particle-attac hed cell in the race of carb on
turno v er b y a h uge margin (Fig. S4). In a study in v estigating viriob en thos pro duction,
Hewson and F uhrman 2003 found that the ma jorit y (1:710
8
and 4:610
8
VLP cm
3
h
1
;
61 and 92% of total b en thic virus pro duction for L.A. Harb or and San ta Catalina Island
resp ectiv ely) of viruses pro duced in sedimen ts w ere from infected bacteria that w ere free-
living in the p orew ater. This hin ts that the free-living comm unit y in the p orew ater ma y b e
more pro ductiv e than the particle-attac hed comm unit y , in agreemen t with our results.
It is not clear at this p oin t wh y the free-living comm unit y could outcomp ete the particle-
attac hed comm unit y in accessing the VF As. One p oten tial explanation lies in one in trinsic
c haracteristic differen tiating the free-living and particle-attac hed comm unities: motilit y .
In surface sedimen t, ph ytoplankton excretion and lo cal photosyn thesis can frequen tly gen-
erate patc h y distributions of organic matter (F ranco et al., 2007; Graue et al., 2011; P an
et al., 2013; Smriga et al., 2016; Usui et al., 1998). The free-living comm unit y could swim
to a p oin t-source of organic carb on (A dler, 1966; A dler & T empleton, 1967) b efore it dif-
fuses to the particle-attac hed cells (Blac kburn & F enc hel, 1999; Bo oij et al., 1994; Iv ersen
& Jørgensen, 1993).
The “bulk” rates of acetate o xidation ha v e b een measured in other marine sedimen ts
using t w o main tec hniques: (1) direct measuremen ts of the decrease in lab eled substrate
and the increase of lab eled pro duct (Ansbaek & Blac kburn, 1980; Christensen & Blac k-
61
burn, 1982; Sansone, 1986; Sha w et al., 1984; Sha w & McIn tosh, 1990; W ellsbury & P ark es,
1995); (2) Measuremen ts of VF A accum ulations with molyb date inhibition (Christensen,
1984; F ukui et al., 1997; Sha w & McIn tosh, 1990; Sørensen et al., 1981). The tracer metho d
in v olv es measuring the size of the acetate p o ol and calculating the acetate o xidation rate
constan t. In marine sedimen t, particle adsorption and c hemical complexion renders part
of the acetate p o ol (9-23%, Sansone, 1986, 10-40%, Sha w et al., 1984) una v ailable to mi-
cro organisms. The added tracer, in comparison, is more accessible to micro organisms. In
addition, a fraction of the dissolv ed acetate can b e c hemically complexed and the c hemical
analysis of acetate generally o v erestimates the freely a v ailable acetate. These t w o factors
seem to lead to an o v erestimation of the acetate o xidation rate (Fink e et al., 2007; Glom b-
itza et al., 2015). The second metho d assumes that all terminal VF A o xidation is coupled
to sulfate reduction, whic h disregards other electron acceptors in marine sedimen ts suc h as
metal o xides and underestimates the rates of VF A o xidation.
The acetate o xidation rates of the bulk sedimen t, calculated b y summation of the
rates measured for the free-living and particle-attac hed comm unities, ranged from 5.3 to
18.1 nmol cm
3
sediment
d
1
. The v alues of rate constan t for acetate o xidation in this study
(0.008 - 0.078 h
1
) w ere lo w er than those measured using the tracer metho d for sedimen ts
in Cap e Lo ok out Bigh t (Sansone, 1986), a Mangro v e in Thailand (Kristensen et al., 1994),
Danish Coastal w aters (Christensen & Blac kburn, 1982), and Limfjorden, Denmark (Ans-
baek & Blac kburn, 1980), and higher than those measured with molyb date inhibition for
sedimen ts in a Scottish Estuary (P ark es et al., 1989), and a Danish coastal lago on (Chris-
tensen, 1984), and similar to the v alue measured b y Fink e et al. (2007) in a study care-
fully a v oiding the issues men tioned b efore (details b elo w).
Measuring the rates of acetate o xidation separately for the free-living and particle-
attac hed comm unities yielded reliable n um b ers compared to the rates measured in other
similar systems. Ho w ev er, the absence of particles in the incubations with p orew ater elim-
inated the effect of adsorption whic h migh t lead to an o v erestimation for the free-living
62
comm unit y . This effect w as minimal in our study . When calculating the VF A o xidation
rates, w e assumed a constan t VF A concen tration whic h has b een observ ed in natural en-
vironmen ts (Glom bitza et al., 2015, 2014). A dditionally , the pro cess of adsorption should
not affect the rate constan t of the VF A o xidation (Mic helson et al., 1989). Therefore, the
size of the free VF A p o ol w ould not c hange the shap e of the curv es or the relativ e rates
b et w een the free-living and particle-attac hed comm unities.
Differen tial usage of electron donor and acceptor b y the free-living and particle-
attac hed comm unities
Sulfate reduction has b een recognized as the most imp ortan t terminal pro cess for the o x-
idation of VF As in marine sedimen ts (Jørgensen, 1982). By measuring the accum ulation
of VF As with sulfate reduction inhibited, previous studies compared the con tribution of
differen t VF As to the organic carb on mineralization supp orted b y sulfate reduction. In a
sulfide-ric h sedimen t from a shallo w coastal lago on, 10% of the sulfate reduction w as cou-
pled to the o xidation of h ydrogen, 40–50% to acetate, 10–20% to propionate and 10–20%
to but yrate (Sørensen et al., 1981). In a similar exp erimen t, Christensen (1984) found a
con tribution of 65% to sulfate reduction from acetate, follo w ed b y 14% propionate, 8%
but yrate, and 6% isobut yrate. Ho w ev er, molyb date could complex VF A and p oten tially
in terfered with the determination of VF A concen trations in these studies. One the other
hand, studies that measured VF A o xidation rates using the tracer metho d found acetate
o xidation rates m uc h higher than what could p ossibly b e supp orted b y sulfate reduction
(Ansbaek & Blac kburn, 1980; Christensen & Blac kburn, 1982). This could b e due to an
o v erestimation of the free acetate p o ol as the complexed acetate could not b e differen ti-
ated from the bio-a v ailable acetate in c hemical analyses. In addition, the added tracers
w ere not c hemically complexed and w ould b e o xidized faster than the total dissolv ed VF As
resulting in an o v erestimated rate constan t as w ell.
63
T o accoun t for this effect, Fink e et al. (2007) equilibrated the tracers in sterilized
p orew ater b efore the exp erimen t so that the tracers could b e c hemically complexed the
same w a y as the p orew ater acetate th us represen ting the in-situ rate of VF A o xidation
more accurately . In the same study , the authors also calculated VF A o xidation rates b y
measuring the accum ulation of VF As while inhibiting sulfate reduction with selenate. The
turno v er rates determined through the t w o metho ds w ere similar. 40% sulfate reduction
w as attributed to acetate, 8% to propionate, 1.3% but yrate, and 3% to lactate. T aking
these n um b ers, Glom bitza et al. (2015) estimated acetate o xidation rates to b e 0.2 - 5
nmol cm
3
d
1
, a n um b er similar to this study , based on measured sulfate reduction rates
in Go dthåbsfjord sedimen ts.
If w e add up the ratios rep orted, eviden tly acetate, propionate, but yrate, and lactate
com bined did not accoun t for all the sulfate reduced in their study (Fink e et al.,2007). In
fact, these VF A only explained 21% and 52% of sulfate-fueled mineralization at depth of
0 - 2 cm and 5 - 9 cm resp ectiv ely . Similarly in our study acetate turno v er rates in bulk
sedimen t (8.93/5.93 nmol cm
3
sediment
d
1
) accoun ted for 40% and 34% of sulfate reduction
(22.59/17.50 nmol cm
3
sediment
d
1
, Fig. S2, detailed data sho wn in c hapter 4) in separate
exp erimen ts. This suggests that other electron donors are at pla y .
The energy yield of anaerobic respiration using formate as the electron donor is com-
parable to that of acetate (T able.1) and it has b een sho wn that formate as the sole elec-
tron donor can supp ort the gro wth of iron and sulfate reducers (Hausmann et al., 2016;
Kane et al., 2016; Lo vley et al., 1989; Sp eers & Reguera, 2012). In sedimen ts where mea-
suremen ts w ere a v ailable, formate sho w ed similar concen tration lev el as acetate (Glom bitza
et al., 2015). In our study , the formate o xidation rates of the bulk sedimen t w ere in a v-
erage 25.34 nmol cm
3
sediment
d
1
, higher than the a v erage rate of acetate o xidation. If w e
assume formate o xidation w as coupled to sulfate reduction alone, it could accoun t for 28%
36% of the total sulfate reduced.
64
The sulfate reduction rate (SRR) calculated for the bulk sedimen ts in our study w as
comparable to other similar en vironmen ts, but surprisingly , SRR w as b elo w detection limit
for the free-living comm unit y alone (Fig. S4). F or the particle-attac hed comm unit y , for-
mate app eared to b e the fa v ored electron donor rather than acetate. F ormate and ac-
etate accoun ted for in a v erage 14% and 6% of the sulfate reduction, suggesting other elec-
tron donors are used b y the particle-attac hed comm unit y . The biofilm-forming consortia
are kno wn to k eep extracellular enzymes within the extracellular p olymeric substances
(EPS, Flemming & Wingender, 2010). The particle-attac hed comm unit y could p oten-
tially pro duce div erse simple organic carb ons from high-molecular organic material and
shared within the EPS matrix. F or the free-living comm unit y , since sulfate reduction w as
absen t, the high rates of VF A o xidation m ust ha v e b een supp orted b y other terminal elec-
tron acceptors suc h as nitrate, iron and manganese o xides, whic h ha v e b een found to sup-
p ort large prop ortions of carb on mineralization in marine sedimen ts (Canfield et al., 1993;
Fink e et al., 2007; Hyun et al., 2017; V andiek en & Nic k el, 2006).
Protein-co ding gene abundance and VF A turno v er rates
Genes enco ding for anaerobic formate (formate deh ydrogenase), acetate (acet yl-CoA syn-
thetase, acetate kinase and ADP-forming acetate-CoA ligase, (note these enzymes can
catalyze reactions in b oth directions dep ending on en vironmen tal conditions) and lactate
metab olisms (lactate deh ydrogenase) w ere presen t in b oth the free-living and the particle-
attac hed comm unities (Fig. 6), suggesting microbial groups with the abilit y to turno v er
these three v olatile fatt y acids inhabited b oth nic hes.
Higher abundances of genes enco ding for acetate metab olizing enzymes w ere observ ed
for the free-living comm unities than the particle-attac hed comm unit y at all depths ( A c d :
p.v alue = 0.042; A cs : p-v alue = 0.08; A ck : p-v alue = 0.002; Fig. 6). This w as consisten t
with the higher rates of acetate o xidation measured for the free-living comm unit y . No dis-
65
cernible difference in formate deh ydrogenase-enco ding gene abundance w as observ ed b e-
t w een the free-living and particle-attac hed comm unities (p-v alue = 0.75). This could p o-
ten tially explain the similar formate o xidation rates measured b et w een the t w o comm uni-
ties. F or genes in v olv ed in lactate metab olism, only cyto c hrome-dep enden t lactate deh y-
drogenase sho w ed significan tly higher abundance in the free-living comm unities than the
particle-attac hed comm unities (p-v alue = 0.0085).
Mark er genes resp onsible for formate o xidation w as more prev alen t than those for ac-
etate o xidation (Fig. 6). Concomitan tly , the formate o xidation rates measured w ere higher
than the acetate o xidation rates for the free-living comm unities as w ell as the particle-
attac hed comm unities. Genes enco ding for lactate deh ydrogenases w ere in similar abun-
dance compared to the genes enco ding for acetate metab olisms, in agreemen t with the
comparable rates of acetate and lactate o xidations, whic h ha v e b een rep orted in other
marine sedimen ts (Fink e et al., 2007; Glom bitza et al., 2015). Measured formate and ac-
etate o xidation only accoun ted for 20% of the sulfate reduction for the particle-attac hed
comm unit y , suggesting that lactate migh t b e of greater imp ortance as an electron donor
than acetate and formate. High abundance of ethanol and aldeh yde deh ydrogenase genes
suggested that ethanol fermen tation migh t b e an imp ortan t carb on-metab olizing pro cess,
whic h has b een observ ed in other ano xic sedimen ts from b oth metagenomic and geo c hemi-
cal analyses (Rasigraf et al., 2019; Zh uang et al., 2014; Zink e et al., 2019)
Genes are prerequisites for enzyme-catalyzed reactions. The “sequence h yp othesis”
predicts that the abundance of genes should correlate with rates of biogeo c hemical pro-
cesses they are in v olv ed in (Cric k, 1958). P ositiv e relationships b et w een the abundance of
protein-enco ding genes and corresp onding pro cesses ha v e b een observ ed in natural en vi-
ronmen ts (Fierer et al., 2011; P etersen et al., 2012). Ho w ev er through a meta-analysis of
studies that quan tified gene abundance/enzyme transcripts and measured biogeo c hemical
rates, Ro cca et al. (2015) found significan t but w eak relationship b et w een the t w o. This
suggests that ecological factors suc h as substrate lev el migh t also pla y an imp ortan t role
66
in determining the rates of microbially mediated pro cesses. In our study the substrates
of in terest (acetate and formate) w ere b oth pro duced through fermen tation and generally
main tained at lo w concen trations in marine sedimen ts (Cselo vszky et al., 1992; Fink e &
Jørgensen, 2008; V aldemarsen & Kristensen, 2010) and th us the effect of substrate lev el
w as p oten tially minimal. As a result, a p ositiv e correlation b et w een gene abundance and
activit y rate w as eviden tly observ ed.
Can microbial heat pro duction reflect metab olic pro cesses?
Nano calorimetry w as used to monitor the heat generated o v er time b y biological activities
for the free-living comm unit y . Thermo dynamic calculations based on the rates of acetate
and formate o xidation estimated that up to 20% of the heat pro duced could b e attributed
to the terminal o xidation of these t w o simple organic carb ons. Other heat-generating pro-
cesses could b e terminal o xidation of other electron donor, microbial anab olism, and more
imp ortan tly fermen tation (Graue et al., 2012). Lo w VF A concen trations in marine sed-
imen ts indicate a close coupling b et w een fermen tation and terminal o xidation pro cesses
in these systems (Christensen & Blac kburn, 1982; Fink e & Jørgensen, 2008; W ellsbury &
P ark es, 1995; W u & Scran ton, 1994). The VF As pro duced through fermen tation w ere im-
mediately consumed, resulting in a steady and lo w standing sto c k of these in termediates.
The profile of heat flux mirrored that of the VF A o xidation rates during the incuba-
tions (Fig. 3, Fig. 4, Fig. 7). The t w o lines of evidence sho w ed that the metab olic activi-
ties of the free-living comm unit y w ere in fact lo w at the b eginning. Immediately follo wing
the p orew ater separation, the free-living cells w ere remo v ed from their original en viron-
men t and encoun tered geo c hemical conditions differen t from b efore. Only after the genes
enco ding for enzymes required in the new en vironmen t w ere turned on, did the free-living
cells start metab olizing the VF As presen t, corresp onding to the increasing rate of VF A
o xidation. In the first half of the incubation, the pro duction of VF As from fermen tation
67
p oten tially surpassed the consumption. This resulted in an accum ulation of VF As whic h
supp orted the increasing rates of acetate and formate o xidation. The p eak of the curv e
represen ted the balanced state b et w een fermen tation and terminal o xidations. After that,
the a v ailabilit y of the carb on monomers could not supp ort high rates of fermen tation. The
VF A concen trations decreased and so did the rates of VF A o xidation.
In con trast, the particle-attac hed comm unit y sho w ed high rates of the VF A o xidation
at the b eginning of the exp erimen t (Fig. 3, Fig. 4). Micro organisms living in biofilm are
kno wn to k eep and share enzymes within the extracellular p olymeric matrix (Flemming &
Wingender, 2010). It is lik ely that the enzymes necessary for VF A o xidation w ere readily
a v ailable for the particle-attac hed comm unit y at the b eginning of the incubation. Ho w ev er,
the rate of VF A remained lo w for the particle-attac hed cells and decreased o v ertime p o-
ten tially due to the exhaustion of substrates or energy . Calorimetric measuremen ts for the
particle-attac hed comm unities w ere attempted but did not yield meaningful heat profiles
due to the nature of the samples. The preparation of the measuremen ts included lo w er-
ing the sample vials in to the measuring c ham b er (see Metho ds and Material for details)
and this pro cess stirred the sedimen t particles within the sample vials. The mo v emen ts
of the particles generated heat and caused to o m uc h noise at the b eginning of the incuba-
tion where most of the VF A o xidation activities w ere exp ected to o ccur (Fig. 3). The heat
profiles for the particle-attac hed comm unities w ere therefore disregarded.
Ov erall the heat flux measured b y nano calorimetry matc hed with the measuremen ts
of VF A o xidation rates. Calorimetric measuremen ts pro v ed to b e useful in determining the
differen t phases of carb on mineralization and accurate in estimating the o v erall microbial
activities in samples of this nature.
In summary , the free-living microbial comm unit y in coastal marine sedimen ts, for
the first time, w ere studied separately from the particle-attac hed comm unit y . Radiotrac-
ers, metagenomic analyses and nano calorimetry w ere com bined to ev aluate the role of the
68
free-living comm unit y in b en thic carb on turno v er and their o v erall metab olic activities,
in comparison to the particle-attac hed comm unit y . The free-living cells, ev en though in
m uc h lo w er abundance than their particle-attac hed neigh b ors, sho w ed m uc h higher rates of
acetate and formate o xidation. Differen tial patterns in carb on metab olisms w ere also ob-
serv ed b et w een the t w o comm unities. The free-living comm unit y used acetate and formate
as the ma jor electron donors, supp orted b y high energy-yielding electron acceptors other
than sulfate. The particle-attac hed cells lik ely relied on electron donors other than acetate
and formate. This system therefore pro vides a great study mo del for understanding mi-
crobial nic he-partition. The previously o v erlo ok ed free-living comm unit y can con tribute
greatly to the b en thic biogeo c hemical pro cesses.
A c kno wledgemen ts
I w ould lik e to thank m y collab orators (Karsten Zengler, Mallory Em bree, Cameron Mar-
tino, Ric hard Szubin) at USCD for their supp ort in the molecular w ork. Dr. Ben T ully
and Elaina Graham ha v e pro vided m uc h help in the metagenomic assem bly and subse-
quen t analyses. I also thank mem b ers of the Ziebis Lab (Jane Den tinger, Abb y Lam bretti,
Emily W aggoner) for their help in sample collection and pro cessing. The main b o dy of
w ork w as supp orted through the Ziebis lab researc h fundings. The W rigley Institute of
Marine Studies has pro vided logistical supp ort for this w ork as w ell as partial financial
supp ort.
69
Figure 1: A v erage (n=3) microbial cell abundances in sea w ater, p orew ater and sedimen t.
70
Figure 2: A cetate o xidation rates of the free-living comm unities and the particle-attac hed
comm unities normalized to p er cm
3
of bulk sedimen ts at v arying depth. Duplicates are
b oth represen ted.
71
Figure 3: A cetate o xidation rate of the free-living comm unities and the particle-attac hed
comm unities in ev ery cm
3
of bulk sedimen ts c hanges during the course of 24 h incubation.
Samples w ere p o oled from the upp er 10 cm of the sedimen ts. Results from t w o indep en-
den t incubations are presen ted. The trend lines indicated the a v erage of the duplicates in
b oth incubations.
72
Figure 4: F ormate o xidation rate of the free-living comm unities and the particle-attac hed
comm unities normalized to ev ery cm
3
of bulk sedimen ts c hanges during the course of 24 h
incubation. Samples w ere p o oled from the upp er 10 cm of the sedimen ts. Results from du-
plicate incubations are presen ted. The trend lines indicated the a v erage of the duplicates
in b oth incubations.
73
Figure 5: A cetate and formate o xidation rates of the free-living comm unities and the
particle-attac hed comm unities (normalized to ev ery cm
3
of bulk sedimen ts) a v eraged o v er
24 h incubation. Samples w ere p o oled from the upp er 10 cm of the sedimen ts. T w o sepa-
rate measuremen ts w ere conducted for acetate o xidation and one measuremen t w as con-
ducted for formate o xidation.
74
Figure 6: Abundance (sum of gene co v erage / a v erage sample co v erage) of mark er genes
for carb on metab olisms.
75
Figure 7: Real-time heat flo w (µW) measured at 7.56s in terv al (solid blac k line) and the
accum ulativ e heat (J) pro duced (gra y line) of the free-living comm unities. Results from
t w o indep enden t incubations are presen ted.
76
77
Supplemen tary Materials
Determination of cell abundances in the sea w ater, the p orew ater and the sedimen t
F or the sedimen t samples, subsamples of 1 cm
3
w ere collected using cut-off 5 ml sterile sy-
ringes and immediately fixed with 9 ml 4% sea w ater-formaldeh yde solution (0 : 02 µm fil-
tered) in 15 ml cen trifuge tub es. 2 ml subsamples of the p orew ater w ere transferred in to
resp ectiv ely lab elled 2 ml cry o vials and fixed with 0 : 02 µm filtered formaldeh yde to a fi-
nal concen tration of 2%. 100 ml subsamples of the o v erlying sea w ater w ere collected at the
sampling site and fixed with 0 : 02 µm filtered formaldeh yde to a concen tration of 2%. The
p orew ater and the o v erlying sea w ater w ere fixed o v ernigh t at 4

C, and the sedimen t sam-
ples w ere fixed for 1 w eek b efore further pro cessing.
Before slide preparation, 1 ml of the 1:10 (v/v) diluted sedimen t slurries w ere trans-
ferred in to 9 ml 4% sea w ater-formaldeh yde solution (0: 02 µm filtered) in a 15 ml cen trifuge
tub e using a cut-off pip et tip for a second 1:10 dilution. A sonicating tip w as used to dis-
lo dge cells off the sedimen t particles (Branson Sonifier 250, output = 3, amplitude = 135
microns, 30 s on/off in terv al for a total of 6 min utes). This solution w as then again diluted
1:10 in the same fashion.
Eac h subsample of a defined v olume (200 µL p orew ater, 2 ml sea w ater, or 1 ml of 1:1000
diluted sedimen t, optimized for cell densit y on the filter, 100 200 cells p er field) w as added
to 5 ml of sterile-filtered marine PBS solution and gen tly v acuum-filtered on to a 0 : 22 µm
Nucleop ore p olycarb onate filter (Whatman, GE Healthcare) using a hand pump. Sterile-
filtered marine PBS w as used to w ash off the inner w all of the filter funnel and the w ash
fluid w as collected on the filter to catc h all cells that w ere stuc k on the glass. The filter
w as then air-dried and transferred on to a sterile glass microscop e slide. 20 µL of the SYBR
green staining solution (1:15:1 1%Ascorbic acid: Mo wiol moun ting solution: SYBR Green)
w as dropp ed on top of the filter and gen tly pressed with a sterile co v er slip. The prepared
78
slide w as visualized and en umerated under a fluorescence microscop e (Nik on Eclipse 80i,
100 oil immersion). F or eac h sample, triplicate filters w ere prepared. Eac h filter w as
coun ted for 10 fields of view.
79
Determination of the reco v ery rate for the measuremen ts of VF A o xidation rates
CO
2
tr apping
14
C-so dium bicarb onate of differen t radioactivit y (0 µCi, 0 : 5 µCi, 1 µCi, 2 µCi) w as
added in to four incubation vials con taining 10 mL filtered sea w ater. 5 mL 5% NaOH w as
injected in to eac h b ottle. The subsequen t measuremen ts w ere conducted the same w a y as
the Erlenmey er flask metho d (detailed in Metho ds and Material).
80
CH
4
and CO
2
14
C-methane (0: 4 µCi) w as added to the same incubation vials con taining 10 mL fil-
tered sea w ater. 5 mL 5% NaOH w as injected in to the b ottle. The vial w as connected in to
the com bustion setup the same w a y as the sample. The radioactivit y of reco v ered CH
4
w as
determined. T riplicate measuremen ts w ere conducted.
A mixture of
14
C-methane (0: 4 µCi) and
14
C-bicarb onate (0: 2 µCi) w ere added to the
same incubation vials con taining 10 mL filtered sea w ater. 5 mL 5% NaOH w as injected
in to the b ottle. The vial w as connected in to the com bustion setup the same w a y as the
sample. The radioactivit y of reco v ered CH
4
and CO
2
w as determined. T riplicate measure-
men ts w ere conducted.
81
Figure S1: Amoun t of acetate o xidized b y free-living comm unities and the particle-
attac hed comm unities in ev ery cm
3
of bulk sedimen ts o v er the course of 48 h incubation.
Samples w ere p o oled from the upp er 10 cm of the sedimen ts. Results from t w o indep en-
den t incubations are presen ted.
82
Figure S2: Pro duction rates of CH
4
(shaded) and CO
2
during the acetate o xidation assa ys
for the free-living and particle-attac hed comm unities)
83
Figure S3: A cetate o xidation rates of the free-living comm unities and the particle-attac hed
comm unities normalized to p er cell at v arying depth. Duplicates are b oth represen ted.
84
Figure S4: Sulfate reduction rates of the free-living and the particle-attac hed comm unities
a v eraged o v er 24h incubation in t w o separate exp erimen ts.
85
86
Chapter 4: Prop elling to Surviv al: Ho w Motilit y Structures
Microbial Comm unities in Marine Sedimen t
In collab oration with Wiebk e Ziebis
Abstract
Marine sedimen ts represen t a heterogeneous en vironmen t c haracterized b y the spatial v ari-
ations and temp oral fluctuations of biogeo c hemical parameters. A microb e with the abilit y
to mo v e can theoretically tak e full adv an tage of the patc h y distribution of substrates and
steep gradien ts of o xidan ts that are t ypical in marine sedimen ts. The imp ortance of mi-
crobial motilit y in marine sedimen ts has b een recognized but rarely in v estigated. In this
study , w e sampled an in tertidal m udflat (Catalina Harb or, Catalina Island, CA) to exam-
ine the imp ortance of microbial motilit y in marine sedimen ts and the p oten tial differen-
tial usage of the acceptors b et w een the free-living and the particle-attac hed comm unities.
Our results sho w ed that up to 90% of the p orew ater comm unit y w ere motile, a fraction
of whic h only exhibited p erio dical motilit y . The ma jorit y of the particle-attac hed cells
did not sho w motilit y . The motile cells in the p orew ater could outcomp ete the particle-
attac hed cells in accessing fa v orable electron acceptors including the nitrate through c hemo-
taxis and the solid-phase manganese o xide. Surprisingly , the p orew ater comm unit y did not
use sulfate as an electron acceptor, whereas sulfate reduction w as clearly an imp ortan t ter-
minal carb on o xidation pro cess for the attac hed cells. These findings suggest that micro-
bial motilit y is a ubiquitous c haracteristic within the p orew ater comm unit y in marine sedi-
men ts. Moreo v er, the free-living comm unit y within the p orew ater differs from the attac hed
comm unit y in its c hemosensory resp onse to a v ailable electron acceptors and its metab olic
flexibilit y .
87
In tro duction
The abilit y to mo v e is a living organism’s most fundamen tal w a y to in teract with its en-
vironmen t, ev en for a single-celled bacterium (Mitc hell & K ogure, 2006; Shapiro, 2007).
This ancien t and ubiquitous c haracteristic of bacteria enables the microb es to explore and
exploit its surroundings for n utrien ts that are almost alw a ys unev enly distributed in the
en vironmen t (F enc hel, 2002; Grün baum, 2012; Sto c k er, 2012; W ei et al., 2011). The abil-
it y for a cell to prop el itself to a more fa v orable condition through sensing and resp onding
to certain en vironmen tal cues is called taxis (A dler, 1966; Alon et al., 1999; Bi & Sourjik,
2018; Micali & Endres, 2016). Microbial taxis is usually coupled with motilit y in a wide
range of microbial groups and can b e induced b y v arious attractan ts suc h as organic car-
b ons (Bell & Mitc hel, 1972; Smriga et al., 2016; Y ang et al., 2015), soluble and insoluble
electron acceptors (Benc harit & W ard, 2005; Childers et al., 2002; Kennedy & La wless,
1985; Nealson & Moser, 1995), and most p eculiarly , magnetic field (Blak emore, 1975; De-
Long & F rank el, 1993).
When a swimming bacterium, while tracing a gradien t of an attractan t, encoun ters a
surface, suc h as a ph ytoplankton (K ogure et al., 1981; T amplin et al., 1990; V aque et al.,
1989), marine sno w (Grossart et al., 2006; Kiørb o e et al., 2003), or solid particles (W eise
& Ecology , 1977), it has the option to initiate attac hmen t and ev en tually b ecome immobi-
lized in a biofilm or remain free-living (Vigean t et al., 2002). Surface can pro vide ph ysical
protection against grazing (Kiørb o e et al., 2003), concen trate n utrien ts and organic mate-
rial in oligotrophic en vironmen ts (Zob ell, 1943) and enhance cell-cell in teraction through
biofilm formation (Flemming & Wingender, 2010). Corresp ondingly , whether the cell b e-
comes attac hed is affected b y v arious factors, suc h as the t yp e of surface (Bolster et al.,
2001; Sherer et al., 1992), en vironmen tal conditions (Zhang et al., 2016), the cells that
ha v e already o ccupied the surface (Dang & Lo v ell, 2016; Elifan tz et al., 2013) and the
cell’s o wn metab olic prop erties (Das et al., 2010; Suter et al., 2018). If the cell remains
88
free-living, its swimming b eha vior can b e excited b y in tensified gradien ts of substrates or
temp orarily inhibited when the energy in v ested in motilit y is not comp ensated b y the b en-
efit of taxis (Grossart et al., 2001; Mitc hell et al., 1995).
The sympatric presence of b oth particle-attac hed and free-living micro organisms, ev en
within the same sp ecies, are common in natural en vironmen ts (Baffone et al., 2006; De-
Long et al., 1993; Riec k et al., 2015; Suter et al., 2018; Xu et al., 2018). The partitioning
b et w een the free-living and the particle-attac hed lifest yles is lik ely flexible and sub jected
to c hange. This could mean an o v erlap b et w een the particle-attac hed and free-living com-
m unities at a giv en momen t suggesting a constan t exc hange b et w een the t w o comm unities
(Hollibaugh et al., 2000), or a shifting ratio of particle-attac hed:free-living with c hanging
en vironmen tal conditions (Bengtsson, 1989; Ghiglione et al., 2007).
Microbial motilit y and c hemotaxis ha v e b een recognized to b e imp ortan t in marine
sedimen ts (F enc hel, 2002), esp ecially in non-p ermeable systems where steep gradien ts of
substrate and o xidan ts/reductan ts are presen t (Iv ersen & Jørgensen, 1993; Jørgensen &
Des Marais, 1990; V anderb orgh t & Billen, 1975; W ebster et al., 2011). Previous studies on
microbial mobilit y in marine sedimen ts ha v e mainly fo cused on the sulfide-o xidizing bac-
teria (Larkin & Henk, 1996; Nelson et al., 1986; Preisler et al., 2007). They use gliding
motilit y to na vigate themselv es along the sulfide - o xygen gradien t for energy gain. These
sulfur bacteria ha v e b een sho wn to b e c hemotactic in o xygen gradien ts (Thar & F enc hel,
2001). The c hemosensory motile b eha vior is not limited to sulfur bacteria in marine sedi-
men ts, as genes in v olv ed in cell motilit y and c hemotaxis are abundan t and widespread in
the b en thic comm unities (Dinsdale et al., 2008; Harrington et al., 2007). Ho w ev er, only a
few studies directly in v estigated the o v erall motile b eha viors of the b en thic microbial com-
m unities (F enc hel, 2008; Thar & F enc hel, 2005). If the b en thic microb es are motile and
c hemotactic, they are faced with the same c hoice b et w een attac hmen t and free-living. Our
previous findings sho w ed that differen t bacterial groups exhibited strong preference for ei-
ther a free-living or particle-attac hed lifest yle. This suggested that the division b et w een
89
free-living and particle-attac hmen t is unlik ely a sto c hastic pro cess. It is of great in terest
to understand whether, and if so to what exten t, motilit y and c hemotaxis affect the nic he-
partition of the b en thic microbial comm unities.
It is tec hnically extremely difficult to visualize the motile b eha vior of the b en thic mi-
crobial comm unities within bulk sedimen ts. In this study a metho d successfully applied
b y F enc hel (2008) as adapted to estimate the fraction of motile cells in marine sedimen ts.
This metho d is based on quan tifying the n um b er of microb es that are able to migrate in to
h yp o dermic needles that are inserted in to in tact sedimen t columns. Needles are either
filled with sterile sea w ater to test for cell motilit y , or with sea w ater amended with n utri-
en ts to in v estigate the c hemosensory resp onse of cells. In this study , in addition to quan ti-
fying the p ercen tages of motile cells in the p orew ater, w e tested the resp onse of the motile
cells to nitrate additions. In addition, w e examined the abilit y to reduce MnO
2
and SO
2
4
for gro wth in a comparison of the free-living and the particle-attac hed comm unities using
enric hmen t cultures (manganese reducers) or direct rate measuremen ts (sulfate reduction)
using radiotracers. The ob jectiv es of this study w ere to determine the motile abilit y and
c hemotaxis of the b en thic microbial comm unities, and to explore a nic he-partitioning b e-
t w een the free-living and particle-attac hed comm unities in regard to metab olic activities.
Metho ds and Materials
Sampling site
Sedimen t samples w ere collected from an in tertidal m udflat in Catalina Harb or, a small
harb or lo cated on the south side of the isthm us of San ta Catalina Island. San ta Catalina
Island (33°23’N 128°25’W) is lo cated off the coast of southern California, appro ximately
35 km south w est of the cit y of Los Angeles, CA. This site has b een previously c haracter-
ized for its macrofauna activities and biogeo c hemical pro cesses (Bertics and Ziebis, 2009;
90
Bertics and Ziebis, 2010; Bertics et al., 2010; Lam et al., 2018).
Sample collection
Sedimen t cores w ere collected using cylindrical p olycarb onate coreliners (10 cm diame-
ter, 35 cm length). F or eac h sampling campaign (timeline detailed b elo w), parallel cores
w ere collected during lo w tide with appro ximately 10 to 15 cm of w ater co v erage. Up on
retriev al, stopp ers with o-ring fittings w ere used to seal the sedimen t cores at the b ottom
to prev en t p orew ater leakage. The o v erlying w ater in the cores w ere k ept o xygenated us-
ing air pumps. The sedimen t cores w ere pro cessed immediately after transp ortation to lab
facilit y (within 30 min).
F or quan tifying the p ercen tage of cells with flagellum in the p orew ater, the enric h-
men ts of manganese reducers and the sulfate reduction rate measuremen ts, the p orew a-
ter samples w ere collected using a p orew ater pressing b enc h (25 cm
3
POM, K C Denmark
A/S, Silk eb org, Denmark). Eac h subsample w as placed and sealed in to individual pressing
house. Ultrapure nitrogen w as applied through the top of eac h pressing house and gen tly
exp el the p orew ater from the sedimen t matrix through a pre-installed 8 µm Nucleop ore
mem brane filter (Whatman,GE Healthcare) in to resp ectiv e collecting tub es. The remain-
ing dry sedimen ts in the pressing house w ere considered to b e p orew ater-free and con tain
only the particle-attac hed comm unit y .
Geo c hemical parameters
F or eac h sampling, additional sedimen t cores w ere pro cessed for geo c hemical analyses.
F rom t w o of the cores, p orew ater w as extracted using rhizon samplers (2.5 mm diame-
ter, 0.15µm p ore size, Rhizosphere Researc h Pro ducts, The Netherlands) from 0 to 10 cm
depth in 1cm-in terv als and analyzed for dissolv ed inorganic nitrogen, i.e. nitrate (NO

3
)
+ nitrite (NO

2
) and ammonium (NH
+
4
), and dissolv ed Mn
2+
(dissolv ed Mn
2+
w as only
91
measured in 2018 together the manganese reducer enric hmen t). NO
x
w as determined col-
orimetrically using a metho d adapted for small sample v olumes (Jones, 1984). NH
+
4
w as
determined using a fluorometric tec hnique (Holmes et al., 1999). F or dissolv ed manganese,
filtered p orew ater from 0 - 5 cm w ere acidified with trace-metal free HCl to pH=2 and
measured using ICP-MS. F rom the bulk sedimen t slices, w e determined the p orosit y and
HCl-extractable iron con ten t. P orosit y w as determined through measuring the w eigh t loss
of re-saturated samples b y drying at 60ºC till w eigh t stabilizes. T otal HCl extractable
iron w as measured colorimetrically follo wing proto cols (K ostka & Luther, 1994; Lo vley
& Phillips, 1987).
T esting cell motilit y of the p orew ater comm unit y
Flagellum is a strong indication of motilit y for microbial cells. W e quan tified the cells
with flagellum in the p orew ater to estimate the p ercen tage of motile cells. The sedimen t
core w as sectioned from 1 to 10 cm in 1 cm in terv als. The p orew ater w as extracted as
describ ed ab o v e. F or eac h depth in terv al, the p orew ater samples w ere fixed with 0.2 µm
sterile-filtered formaldeh yde solution (4%) o v ernigh t. 200 µl of eac h fixed p orew ater sam-
ple w as filtered on a 0 : 22 µm Nucleop ore p olycarb onate filter (Whatman, GE Healthcare)
using a hand pump. The filters w ere then stained with NanoOrange, mo difying a staining
proto col b y Grossart et al. (2000). The NanoOrange dy e (500x, Life T ec hnologies. Carls-
bad, CA) w as mixed with Mo wiol moun ting solution (2.4 g Mo wiol + 6 ml glycerol + 6
ml MQ + 14 ml 1 PBS) and Ascorbic acid solution (1% v ol/v ol 1M ascorbic acid in 1x
PBS) at a ratio of 1:15:1 to mak e up the NanoOrange Mo wiol moun ting solution. F or eac h
sample filter 25 µl of this solution w as added.
W e then determined the p ercen tage of activ ely mo ving cells in a separate exp erimen t.
A lab oratory flo w b o x (Fig. 1S) made of acrylic glass w as used for exp erimen tal manipu-
lation. The flo w b o x w as filled with surface sedimen ts (top 10 cm) collected at the study
92
site. The flo w b o x has an inflo w and outflo w p ort at opp osite ends to pro vide flo w across
the sedimen t surface. F resh sea w ater w as supplied through the inflo w p ort. The w ater w as
k ept saturated with o xygen b y bubbling the w ater in the inflo w compartmen t with air us-
ing an aquarium pump. Before starting the exp erimen t, the flo w b o x w as allo w ed to stabi-
lize for 3 da ys with con tin uous sea w ater flushing. The fron t w all of the flo w b o x w as p erfo-
rated with silicone-filled holes in a 1 - cm grid (25 cm wide and 12 cm high) that serv ed as
sampling p orts.
Cell motilit y w ere tested with a metho d adapted from F enc hel (2008). F or quan tifying
cell motilit y , a 26 1/2 G needle w as filled with 0.2 µm sterile-filtered ano xic sea w ater and
attac hed to a 1 ml sterile syringe. Needles w ere inserted in to the in tact through the sam-
pling p orts , and k ept in the sedimen t at predetermined time in terv als b efore b eing pulled
out. Immediately after retriev al, the cells that had migrated in to the needle w ere fixed b y
dra wing up 1 ml 4% formaldeh yde-sea w ater solution (0.2 µm sterile-filtered) through the
needle and in to the syringe. The con ten t w as then transferred in to a 2 ml cry o-vial for
subsequen t cell coun ts. This exp erimen t w as conducted at 6 depths (1, 2, 4, 6, 8, 10 cm)
o v er 6 time-in terv als (15min, 30min, 1h, 2h, 3h and 4h).
The cells within the needles w ere collected and fixed in 4% formaldeh yde-sea w ater
solution (1 ml of v olume) o v ernigh t. This solution con taining the cells w as filtered on a
0: 22 µm Nucleop ore p olycarb onate filter (Whatman, GE Healthcare) using a hand pump.
Sterile-filtered marine PBS w as used to w ash off the inner w all of the filter funnel and the
w ash fluid w as collected on the filter to catc h all cells that w ere stuc k on the glass. The
filter w as then air-dried and transferred on to a sterile glass microscop e slide and stained
with a SYBR green staining solution (1:15:1 1% Ascorbic acid: Mo wiol moun ting solution:
SYBR Green). Eac h prepared slide w as visualized and en umerated with a fluorescence mi-
croscop e (Nik on Eclipse 80i, 100 oil immersion). Eac h filter w as coun ted for 10 fields of
view.
93
T esting c hemotactic resp onse to w ards nitrate of the p orew ater comm unit y
The c hemotactic resp onse of the p orew ater comm unit y to w ards nitrate w as tested in July
2019. A lab oratory flo w b o x (Fig. 1S) w as set up as describ ed ab o v e. F or testing c hemo-
taxis, the needles w ere filled with amended sea w ater (50 or 100 µM p otassium nitrate).
T w o depths w ere selected, 3 cm and 10 cm, and based on the motilit y tests 2 time in ter-
v als w ere c hosen,15 min utes and 4 hours. F or eac h exp erimen tal condition, triplicate sam-
ples w ere tak en. The cells within the needles w ere collected and en umerated follo wing the
proto col used in the motilit y test.
Comparing the abilit y of the b en thic comm unities to use manganese o xide as an
electron donor
T o test for the manganese reducing p oten tial of the microbial comm unities in Catalina
harb or sedimen ts, enric hmen t cultures for manganese reducing bacteria w ere set up. A
sea w ater base medium (recip e in supplemen tary material) con taining minimal sulfate
(50 µM) w as prepared with either lactate (20 mM) or acetate (20 mM) as the sole electron
donor. Amorphous -MnO
2
w as prepared b y the o xidation of Mn
2+
b y p ermanganate in
the presence of so dium ion according to Balistrieri and Murra y (1982). The MnO
2
w as
then w ashed, freeze-dried and k ept in a desiccator. 18 ml base media, 6 ml molten agar
(3%) and -MnO
2
(final concen tration = 10 mM w ere mixed anaerobically in to pre-com busted
balc h tub es (18 x 150 mm). The tub es w ere capp ed with auto cla v ed blue rubb er stopp ers
and gen tly in v erted un til the MnO
2
particles w ere mixed ev enly .
Sedimen t sections of 0 - 0.5 cm, 1 - 4 cm and 9 - 11 cm w ere collected from three
sedimen t cores. The p orew ater and the sedimen t particles w ere separated for eac h se-
lected la y er using the p orew ater press as describ ed ab o v e. The gained p orew ater (0: 5 ml
or p orew ater-free sedimen t 0: 5 cm
3
w ere used as ino cula and mixed in to the surface of the
solidified agar under nitrogen atmosphere. After ino culation, the culture tub es w ere sealed
94
with rubb er stopp ers and crimp-capp ed. F or eac h sample, triplicates and a con trol w ere
prepared. Enric hmen ts w ere k ept at ro om temp erature for o v er 60 da ys. The disapp ear-
ance of manganese o xide particles o v er time w ere used to estimate the p oten tial of man-
ganese reduction.
Selected enric hmen t cultures sho wing manganese reduction w ere pro cessed for molecu-
lar analyses. Appro ximately 0: 5 cm
3
of the agar medium from zones with manganese o xide
disapp earance w ere used for DNA isolation using the P o w erSoil DNA Isolation Kit (MoBio
Lab oratories, Inc. Carlsbad, CA) follo wing the man ufacturer’s man ual. 16S V3-V4 rDNA
libraries w ere prepared and sequenced on Illumina MiSeq platform with a v3 reagen t kit
(600 cycles) at Zymo Researc h (Irvine, CA). 16S sequences w ere analyzed using the Dada2
pip eline (Callahan et al., 2016). T axonom y w as assigned using Uclust from Qiime v.1.9.1
against the Silv a 138 database. Comm unit y comp osition of the enric hmen ts w as compared
to that of the en vironmen tal samples from our previous studies.
T esting the usage of sulfate as an electron acceptor b y the b en thic comm unities
Sulfate reduction rates (SRR) in the p orew ater and the p orew ater-free sedimen ts w ere
measured for the upp er 10 cm of the Catalina harb or sedimen ts in No v em b er 2017 and
A ugust 2018 to accoun t for p oten tial seasonal v ariations. Sedimen t la y ers (0 to 10 cm)
w ere p o oled from four parallel sedimen t cores. F rom the p o oled sedimen t samples, the
p orew ater and the sedimen t w ere separated using the p orew ater press.
P arallel incubations w ere prepared. 5 cm
3
p orew ater-free sedimen t or p orew ater, re-
sp ectiv ely w ere mixed with 5 cm
3
0: 02 µm filtered sea w ater in eac h incubation vial (25 ml
serum vial, anaerobically sealed with a but yl rubb er stopp er and crimp ed with an alu-
min um cap). 50 µl
35
S-SO
2
4
) (10 mCi ml
1
, American Radiolab eled Chemicals, diluted
to 0: 1 µCi µl
1
) w as injected in to eac h incubation vial (final radioactivit y in vial = 5 µCi).
A t eac h time p oin t (0, 0.5, 2, 4, 6, 8, 10, 12, 14, 18, 22, 26, 30, 40, 48h), duplicate samples
95
w ere killed b y adding 5 mL 20% ZnA c solution and stored at 20

C un til further pro cess.
The radioactivit y of the reduced sulfide in eac h vial w as measured follo wing a proto col b y
Kallmey er et al. (2004) for a cold c hromium distillation for radiolab eled sulfide, follo w ed
b y a scin tillation coun ting. The rate of sulfate reduction w as calculated using the follo wing
equation (Kallmey er et al., 2004):
SRR = [SO
2
4
]
a
TRIS
a
TOT

1
t
1:06 (1)
[SO
2
4
] is the sulfate concen tration, is the p orosit y , a
TRIS
is the radioactivit y of the re-
duced sulfur, a
TOT
is the total sample radioactivit y , t is the incubation time in da y , 1.06 is
the correction factor for the estimated microbial isotopic fractionation of sulfur. All rates
w ere presen ted p er v olume of undisturb ed sedimen t after con v ersion with the dilution fac-
tor and p orosit y .
Ratepercm
3
sediment
=Rate
porewater

CellAbundance
porewater
CellAbundance
inoculum
porosity (2)
T o examine the prop ortion of SRR-coupled acetate o xidation of the free-living and
particle-attac hed comm unities, the acetate o xidation rates w ere measured with and with-
out an addition of so dium molyb date (20 mM) to inhibit sulfate reduction. After p ore-
w ater separation, triplicate incubations with the p orew ater and the p orew ater-free sedi-
men ts w ere prepared for eac h set of exp erimen t. 5 cm
3
prew ater-free sedimen t or p orew ater
w ere mixed with 5 cm
3
0: 02 µm filtered sea w ater in eac h incubation vial (25 ml serum vial,
anaerobically sealed with rubb er but yl stopp er and alumin um crimp ed cap). 100 µl
14
C-
acetate (So dium acetate [2-
14
C], 2 µCi, 58 mCi/ mmol, American Radiolab eled Chemicals,
diluted to 10 µCi/ml) w as injected in to eac h incubation vial using a gas -imp ermeable 1ml
micro-syringe (SGE, A ustralia). Incubations w ere k ept for 24h, after whic h incubation vial
w as used for measuring the radioactivit y of
14
C-CO
2
.
96
Radioactivit y of
14
CO
2
w as measured follo wing the proto col b y T reude et al. (2003)
with mo dification. In brief, the con ten t of the incubation vial, together with the w ash flu-
ids (2 x 5 ml 5% NaOH solution) w as emptied in to a 250 ml Erlenmey er flask. The flask
w as immediately capp ed using a rubb er stopp er. 6 ml 6 M HCl w as added to the Erlen-
mey er flask using a 3 inc h h yp o dermic needle. The
14
CO
2
de-gassed after acidification of
the samples w ere trapp ed inside a 7 ml scin tillation vial attac hed to the rubb er stopp er
prefilled with 1 ml phen yleth ylamine and 1 ml 2.5% NaOH. 3 ml scin tillation co c ktail (Ul-
tima GoldTM XR, P erkinElmer) w as added to eac h scin tillation vial. Radioactivit y w as
measured using a scin tillation coun ter (LS 6500 Multipurp ose Scin tillation Coun ter, Bec k-
man Coulter).
The rate of acetate o xidation w as determined as the rate of disapp earance of acetate
o v er time, calculated as the p o ol size of acetate the rate constan t (k) (Ansbaek & Blac k-
burn, 1980). The VF A o xidation rate constan t k w as calculated from the equation (Sha w
et al., 1984):
kt =ln[A(A –a)] (3)
where A is the initial radioactivit y of acetate, a is the radioactivit y of pro duced
14
CO
2
at
time t. The acetate o xidation rates of the free-living comm unities w ere adjusted as b elo w,
to accoun t for the dilution of initial ino culum and p orosit y .
Ratepercm
3
sediment
=Rate
porewater

CellAbundance
porewater
CellAbundance
inoculum
porosity (4)
The adjusted n um b er w as normalized to p er v olume of bulk sedimen t and th us represen ted
the amoun t of acetate o xidized b y the free-living comm unities within p er unit v olume sed-
imen t p er uin t time. Studen t’s t-test w as used to test the statistical difference of acetate
o xidation rates b et w een the Mo-treated and un treated conditions.
97
Results
Geo c hemical parameters of the sedimen t
A v ertical zonation of geo c hemical parameters w as observ ed for the in tertidal m udflat in
Catalina harb or (Fig. 1). Nitrate w as a v ailable as an electron acceptor in the top 5 cm of
the sedimen t core (Fig. 1a). A quic k decrease in nitrate concen tration and a concomitan t
increase in ammonium with depth (Fig. 1b) suggested that nitrate w as used an electron
acceptor coupled to the degradation of organic material. Oxidized iron w as highest at the
surface follo w ed b y a sharp decrease around 2 cm and then p ersisted with depth. Sim ul-
taneously there w as a sligh t increase in reduced iron with depth. Dissolv ed F e
2+
w as not
detected in the p orew ater. A p eak of dissolv ed manganese in the p orew ater w as observ ed
at 2 cm suggestiv e of manganese reduction, either c hemically or biologically .
Cell motilit y
Cell motilit y w as in v estigated in the upp er 10 cm of the sedimen t (Fig. 3, Fig. 4). The
concen trations of the motile cells at a giv en depth w ere defined as the n um b er of motile
cells that had migrated in to the needle that w as inserted at a sp ecific depth. 15 min utes
w as c hosen as the cutoff time for the initial migration of p ermanen tly motile cells in to the
needle. Bet w een 15 min and 1 h there w as still an increase in cell concen trations, after 1
h a plateau indicated that a saturation (Fig. 3, Fig. S3) has b een reac hed. The additional
increase in cell concen tration after 15 min (and up to 4 h) w ere defined as the fraction of
p erio dically motile cells. Cells that w ere initially attac hed but b ecame motile later in the
exp erimen t.
The p ercen tage of motile cells in the whole p orew ater comm unit y w as determined as
the motile cell concen trations divided b y the concen tration of the total cell coun ts in the
p orew ater. Up to 90 % of the p orew ater microb es w ere motile in the upp er 10 cm of the
98
sedimen ts (Fig. 4), supp orted also b y the observ ation that 50 to 90 % of the microbial
cells p ossessed flagellum (Fig. 2). There w as no ob vious trend observ ed in the n um b er of
motile cells (p ermanen tly and p erio dically) with depth. A higher p ercen tage (92.3% and
71.7%) of motile cells in the p orew ater w as observ ed at 8 - 10 cm compared to the surface
la y ers (50.4 5.7%, 0 to 6 cm). The p ercen tage of the p erio dically motile cells w as fairly
consisten t with depth (19.5 2.1%) except for 8 cm depth (Fig S2).
Chemotactic resp onse of the free-living comm unit y to w ards nitrate
Chemotactic resp onse to w ards nitrate at a mo derate concen tration (50 µM) w as observ ed
for the free-living cells. The motile cells (p ermanen t and p erio dic, defined in the same
w a y as in the motilit y test ab o v e), at t w o distinct depths, one (3 cm) in the o xidized la y er
(with nitrate presen t) and the other (10 cm) in the reduced zone (no o xidan ts presen t),
w ere tested for their c hemotactic resp onse to nitrate of differen t concen trations (50 and
100 µM). With the addition of 50 µM nitrate, the motile cells in the o xidized zone exhib-
ited a p ositiv e c hemotactic resp onse, sho wn as a significan t increase of cell concen tration
in the needle compared to the con trol (no nitrate addition) (Fig .5). The motile cells in
the reduced zone also sho w ed a p ositiv e ho w ev er w eak er resp onse. In the o xidized la y er,
the p erio dically motile and the attac hed cells w ere not excited b y the nitrate as no in-
crease in the cell concen tration inside the needle w as observ ed after 15 min (Fig. S4).
Ho w ev er, the addition of nitrate at 100 µM resulted an adv erse resp onse from the p er-
manen tly motile cells in b oth o xidized and reduced zone as the cell concen tration in the
needle decreased in comparison to the con trol. The p erio dically and attac hed cells in the
o xidized zone w ere rep elled b y this high concen tration of nitrate as w ell. Ho w ev er, motilit y
and c hemotaxis w ere induced b y the addition of nitrate in the reduced zone where nitrate
w as not normally presen t.
99
Manganese o xide as an electron acceptor for the free-living and the particle-attac hed
comm unities
Disapp earance of manganese o xide w as observ ed in enric hmen t cultures with p orew ater
collected from v arious depths (0 - 0.5 cm, 1 - 4 cm, 9 - 11 cm) as the initial ino culum (Fig.
6). In enric hmen ts with lactate as the sole electron donor, the first sign of manganese re-
duction w as observ ed at da y 6 in cultures ino culated with p orew ater from the depth in-
terv al 1 - 4 cm. A t da y 7, enric hmen ts of the p orew ater from 0 - 0.5 cm and 9 - 11 cm
started reducing manganese o xides. Within 9 da ys of incubation, the greatest amoun t of
manganese reduction w as observ ed in enric hmen ts of p orew ater from the depth in terv al of
1 - 4 cm, and the least amoun t of manganese clearance w as found in enric hmen ts of p ore-
w ater from 9 - 11 cm. When acetate w as used as the electron donor, manganese reduction
w as observ ed after 30 da ys in enric hmen ts ino culated with p orew ater from the depth in ter-
v al of 1 - 4 cm (data not sho wn).
Enric hmen ts with the p orew ater-free particles from three distinct depths (0 - 0.5 cm,
1 - 4 cm, 9 - 11 cm) started sho wing disapp earance of manganese o xides at da y 6 with ei-
ther lactate or acetate as the sole electron donor. In enric hmen ts with acetate as the sole
electron donor, the highest amoun t of manganese clearance w as observ ed in enric hmen ts
of p orew ater-free particles from 1 - 4 cm, follo w ed b y ino culum from 0 - 0.5 cm and then 9
- 11 cm. In enric hmen ts with lactate as the sole electron donor, ino culum from 9 - 11 cm
cleared the manganese o xide the fastest, follo w ed b y ino culum from 0 - 0.5 cm. Ho w ev er,
large v ariance existed for the triplicate enric hmen ts for eac h depth group. Ov erall, enric h-
men ts with lactate as the electron donor sho w ed higher rates of manganese clearance than
enric hmen ts using acetate as the electron donor.
The comm unit y comp osition w as determined for selected enric hmen ts using 16S rRNA
sequencing. The only enric hed taxa with culture represen tativ es sho wing manganese re-
ducing abilit y w as the class Deltapr ote ob acteria (Fig. 7a). In the enric hmen ts with p orew a-
100
ter as the ino culum, Desulfovibrionales had a final relativ e abundance of 10.5% to 28.4%.
The relativ e abundance of Desulfovibrionales in the p orew ater sample w as b elo w detection
lev el. F or enric hmen ts with p orew ater-free particles, Desulfob acter ales , Desulfur omonadales
and Desulfovibrionales sligh tly increased in relativ e abundance in comparison to their rela-
tiv e abundances in the en vironmen ts.
The op erational taxonomic units (OTUs) asso ciated with the enric hed Deltapr ote ob ac-
teria sho w ed nic he-partitioning b et w een the free-living and particle-attac hed comm uni-
ties (Fig. 7b). The OTUs mainly found in the p orew ater w ere more closely related to eac h
other whereas the particle-attac hed OTUs group ed together on the ph ylogenetic tree. The
most closely related culture represen tativ es of the enric hed free-living Deltapr ote ob acte-
ria , mainly Desulfovibrionales , w ere capable of gro wing on manganese o xide (Fig. S5).
The closely related sp ecies to the enric hed particle-attac hed Deltapr ote ob acteria w ere not
kno wn to able to gro w b y manganese reduction. The rapid disapp earance of manganese
o xides in the enric hmen ts with the p orew ater-dev oid particles w as lik ely reduced b y the
sulfide pro duced b y sulfate reducers supp orted b y the lo w amoun t of sulfate presen t in the
enric hmen ts.
Lo w sulfate reduction rates of the free-living comm unit y
In t w o separate exp erimen ts in v estigating sulfate reduction rates in a comparison of the
free-living comm unit y and the particle-attac hed comm unit y , the most surprising result w as
that sulfate reduction for the p orew ater comm unities remained b elo w the detection limit
throughout the 24 h incubation p erio d (Fig. 8). In stark con trast, sulfate reduction rates
of the particle-attac hed comm unities increased with time starting from the b eginning and
p eak ed at 12 and 16 hours in to the exp erimen t in the t w o separate incubations, resp ec-
tiv ely . The a v erage sulfate reduction rates measured for the particle-attac hed comm unities
w ere 22.59 and 17.50 nmol cm
3
sediment
d
1
.
101
T o decipher to what exten t the acetate o xidation w as supp orted b y sulfate reduction,
the acetate o xidation rates of the free-living and particle-attac hed comm unities w ere mea-
sured while sulfate reduction w as inhibited using molyb date (20 mM). The goal w as to
in v estigate further what role sulfate reduction coupled to the o xidation of organic car-
b on pla ys in the t w o differen t compartmen ts. F or the particle-attac hed comm unities, ac-
etate o xidation rates reduced from 5.39 0.53 nmol cm
3
sediment
d
1
to 3.87 0.32 nmol
cm
3
sediment
d
1
(Fig. 8). F or the free-living comm unities, molyb date inhibition did not affect
the rate of acetate o xidation, supp orting the observ ation that the p orew ater comm unit y
do es not couple sulfate reduction to carb on o xidation but m ust use other electron accep-
tors.
Discussion
The ph ysical in teraction b et w een a microb e and surfaces
Microbial cells in marine sedimen ts liv e in extremely tigh t space. Cell concen trations in
coastal sedimen ts can reac h 10
9
10
10
cells cm
3
(Sc hmidt et al., 1998). If w e assume 10
9
microbial cells are ev enly distributed in 1 cm
3
(a cub e with 1 cm sides) of sedimen ts, the
distance b et w een t w o neigh b oring bacteria w ould b e 10 µm. This n um b er is a considerable
o v erestimation since only a fraction of the sedimen t v olume (i.e the p ore space and the
surface of sedimen t particles) is a v ailable for microbial o ccupancy . A t suc h small scale, cell
mo v emen ts due to Bro wnian motion alone allo w microb es to encoun ter particle surface
frequen tly (Kim, 1996; Sto c k & Jenkins, 1978).
When a bacterium approac hes a surface, it m ust first cross the h ydro dynamic b ound-
ary la y er ( 10 µm) of the solid particle and then o v ercome the electrostatic repulsion
as it mo v es closer to appro ximately 10 - 20 nm from the surface (Liao et al., 2015; Mar-
shall et al., 1971; Vigean t et al., 2002). Within 10 nm from the surface, the bacterium is
102
sub jected to v an der W aal attraction (Nir, 1977) and b oth electrostatic repulsion and at-
traction due to the heterogeneous distribution of negativ e and p ositiv e c harges on surfaces
(Huang et al., 2012; Song et al., 1994; W alk er et al., 2005). The strengths of these ph ysical
forces exerted on the bacterium are dep enden t on cell prop erties suc h as size, shap e, c hem-
ical comp osition and gro wth stage, the surface prop erties including comp osition, surface
top ograph y and coating, as w ell as en vironmen tal conditions: pH, ionic strength, temp er-
ature and macromolecule con ten t (Camesano & Logan, 1998; Dang & Lo v ell, 2016; Das et
al., 2010; F opp en & Sc hijv en, 2005; Gannon et al., 1991; Johnson & Logan, 1996; Keller &
A uset, 2007; Levy et al., 2007; Liao et al., 2015; Morisaki & T abuc hi, 2009).
The fate of a non-motile bacterium b et w een attac hmen t and free-living is solely de-
p enden t on its passiv e in teraction with the particles, whereas a swimming bacteria is able
to c ho ose, to some degree, b et w een the t w o lifest yles. Studies on mo del organisms suc h
as Escherichia c oli , Pseudomonas spp. and Bacil lus spp. suggest that, though coun terin-
tuitiv ely , motilit y b y the use of flagella is generally required for cell attac hmen t (Dang &
Lo v ell, 2016; George & K olter, 1998; Hölsc her et al., 2015; T urn bull et al., 2001). Motil-
it y b y the use of flagella increases the rate of surface encoun ter for a bacterium and helps
the cell to o v ercome repulsiv e forces at the particle surface (Gutman et al., 2013). One
the other hand, for a cell to a v oid or rev erse attac hmen t, motilit y is also required (Berg &
T urner, 1990; F rymier et al., 1995; La wrence et al., 1987) to escap e the attractiv e forces
from the particles. The dep endence on motilit y of b oth free-living and particle-attac hed
cells explains the high abundance of motilit y genes in marine sedimen ts (Dinsdale et al.,
2008; Harrington et al., 2007; Zink e et al., 2017)
A motilit y driv en nic he-partition in marine sedimen ts
Non-motile cells in the p orew ater can b e considered colloidal susp ensions, whic h are dis-
p ersions of small particles, ranging in size from 1 nm to 1 µm, in a solv en t (Kim, 1996).
103
The filtration theory (Nelson & Ginn, 2005) predicts that in lo w-v elo cit y fluid, esp ecially
at high cell concen trations, cell attac hmen t is enhanced (McDo w ell-Bo y er, 1992; Rijnaarts
et al., 1996). Ho w ev er, this rule do es not necessarily apply to motile cells as motilit y can
efficien tly help cells a v oid attac hmen t (Camesano & Logan, 1998). The lo w-p ermeable
sedimen ts in Catalina harb or represen ts a relativ ely stagnan t en vironmen t since they are
minimally impacted b y adv ectiv e transp ort (Rasheed et al., 2003; Sc h ulz & Zab el, 2006).
W e found that a large fraction (up to 90%) of the p orew ater comm unit y in Catalina har-
b or sedimen ts w as motile (Fig. 4), whic h confirms that motilit y is tigh tly coupled to the
free-living lifest yle (F rymier et al., 1995).
Our results sho w that a p ortion of the motile cells in Catalina harb or sedimen t ex-
hibit p erio dical motilit y (Fig. 4). These could b e the cells that sho w rev ersible attac h-
men t. Our assumption is that these are cells that w ere initially attac hed but the attac h-
men t w as rev ersible. During rev ersible attac hmen t, a bacterium remains in the pro ximit y
b y tethered spinning (Bennett et al., 2016) or mo ving laterally along a surface for a p erio d
of time (sev eral min utes to hours) (Marshall et al., 1971; Vigean t et al., 2002) and the cells
can ev en tually lea v e the surface (Berg & T urner, 1990; F rymier et al., 1995).
The p erio dically motile cells in this study are defined as cells that sw am in to the nee-
dles after 15 min. The cut-off of 15 min w as previously used b y F enc hel (2008) in a study
in v estigating bacterial motilit y in marine sedimen ts. This w as supp orted b y our results
that the c hange of bacterial concen trations o v er time in the needles w ere significan tly dif-
feren t (p-v alue < 0.001) b efore and after 15 min utes (Fig. S3), suggesting this is an appro-
priate cut-off v alue. In addition, it tak es appro ximately 15 min. for a non-motile cell with
a diffusivit y of 10
9
cm
2
/s (t ypical for non-motile cells, (Kim, 1996) to tra v erse 10 µm,
therefore non-motile cells are m uc h less lik ely to end up in the needles than the activ ely
swimming cells.
Detac hmen t and regained motilit y b y the particle-attac hed cells w as not observ ed in
104
our exp erimen t lasting 4 hours (Fig. 3, Fig. S3), confirming that the p o ol of motile cells
in the p orew ater is not replenished b y the attac hed cells. Motilit y of cells b y flagella is
essen tial for the initial surface attac hmen t as they facilitate the bacterium’s initial in terac-
tion with the surface (O’T o ole et al., 2000). Ho w ev er, flagellum is not required for further
biofilm dev elopmen t (Pratt & K olter, 1998). Once the con tact b et w een a bacterium and a
surface is ac hiev ed, lip op olysacc harides (LPS, sugar units attac hed to the cell surface) and
extracellular p olymeric substances (EPS, high-molecular-mass comp ounds secreted b y mi-
crob es) substan tiate the adhesion of the microb es (Flemming & Wingender, 2010; W alk er
et al., 2005). The immobilized cells b ecome em b edded, resistan t to simple w ashing (Zob ell,
1943) and ev en tually adapt to a life in biofilms (de la F uen te-Núñez et al., 2013; Lóp ez et
al., 2010; Shemesh et al., 2010).
Motilit y and metab olisms
Motilit y allo ws a microbial cell to o v ercome the ph ysical constrain ts on its in teraction with
sedimen t particles (Bolster et al., 2006; Camesano & Logan, 1998; Hölsc her et al., 2015;
Liao et al., 2015). The c hoice of a motile cell b et w een particle-attac hmen t and remain-
ing free-living is unlik ely random, evidenced b y the clear nic he-partitioning b et w een the
free-living and particle-attac hed comm unities in natural en vironmen ts (Ghiglione et al.,
2007; Ortega-Retuerta et al., 2013; Suter et al., 2018). The motile b eha vior of micro organ-
isms is inevitably related to cell metab olism as a strategy to surviv al (Jerome et al., 2018;
Sto c k er, 2012; Sto c k er & Seymour, 2012).
P article-attac hmen t can protect cells from predators and the subsequen t formation of
biofilm enhance microb e-microb e in teractions suc h as sharing the extracellular enzymes
and secondary metab olites (Dang & Lo v ell, 2016; Giesek e et al., 2005; Sc hreib er et al.,
2009). One apparen t trade-off is that attac hed cells ha v e reduced surface area for sub-
strate uptak e (Jeffrey & Microbiol., 1986). It has b een observ ed that cells gro wn in the
105
attac hed-states sho w higher expression of transp orters on the cell surface (T rémoulet et
al., 2002) p oten tially as a comp ensation mec hanism. In non-p ermeable sedimen ts, the
particle-attac hed cells rely solely on the slo w diffusion of substrates and n utrien ts that are
dissolv ed in the p orew ater (Blac kburn & F enc hel, 1999; Bo oij et al., 1994; Iv ersen & Jør-
gensen, 1993). The free-living cells, on the other hand, are able to prop el themselv es to
a more fa v orable condition through c hemo- and redo x- taxis (A dler, 1966; Jerome et al.,
2018). Motilit y also allo ws a microb e to access to a wider range of electron acceptors, suc h
as insoluble iron and manganese o xides (Nealson & Moser, 1995; Smriga et al., 2016; Y ang
et al., 2015). The selecting effect of a substrate on microbial motilit y has b een rep eatedly
sho wn in lab cultures (A dler & T empleton, 1967; Ames & Bergman, 1981; Childers et al.,
2002; Harris et al., 2010) and here w e sho w that the energy a v ailabilit y and the ph ysical
form of the electron acceptors exert an impact on a microbial cells’ c hoice b et w een the t w o
lifest yles in natural en vironmen ts.
Nitrate is the most fa v orable electron acceptor under anaerobic conditions due to its
high redo x p oten tial (Bonin et al., 1999; Straub et al., 1996). The limited source and the
fast consumption b y microbial respiration constrain the a v ailabilit y of nitrate to a thin and
shallo w la y er within the sedimen ts (Henriksen et al., 1981; Usui et al., 1998; V anderb orgh t
& Billen, 1975). The free-living and c hemotactic comm unities are exp ected to exploit this
high-energy electron acceptor b etter than the particle-attac hed cells (Kennedy & La wless,
1985). In the la y er where nitrate w as presen t, the motile cells exhibited acute c hemotaxis
to w ards a mo derate amoun t of nitrate but the p erio dically motile and attac hed cells w ere
unaffected (Fig. 5, Fig. S4). This c hemotactic b eha vior can help the free-living cells to
outcomp ete their particle-attac hed neigh b ors for this highly co v eted n utrien t(Mitc hell &
K ogure, 2006; Sto c k er & Seymour, 2012).
With increasing depth, the imp ortance of nitrate as an electron acceptor decreases
(Fig. 1). The relativ e abundance of activ e nitrate reducers should decrease accordingly
(P apasp yrou et al., 2014). In the p orew ater, a decrease in the relativ e abundance of genes
106
in v olv ed in nitrate reduction w as observ ed with increasing depth (Fig. S5), suggesting that
the distribution of the free-living nitrate reducers w ere tigh tly coupled to the a v ailabil-
it y of nitrate. A t greater depth (10 cm) where nitrate w as absen t, the motile cells in the
p orew ater did not exhibit immediate c hemotaxis to w ards nitrate (Fig. 5, Fig. S4). In ter-
estingly , for the particle-attac hed comm unit y , the relativ e abundance of nitrate-reducing
genes increased with depth (Fig. S5). The abilit y to reduce nitrate is wide-spread in bac-
terial ph yla (Courtney et al., 2013; Smith et al., 2007; Thorup et al., 2017) but unlik ely to
b e in use with the absence of nitrate (P apasp yrou et al., 2014). Ho w ev er, the expression of
nitrate reductase can b e induced b y nitrate (Marietou et al., 2009), sho wn in the dela y ed
but activ e c hemotactic resp onse of the microbial comm unities in the reduced la y er (Fig. 5,
Fig. S4).
The o xidizing p o w er of manganese o xide is highly comparable to nitrate (Burdige,
1993) but it is m uc h less accessible due to its ph ysical form, as extracellular electron trans-
p ort (EET) is needed for a microb e to harness the energy from the sold-phase manganese
o xide (Harris et al., 2018; Murra y , 1974; Nealson & Fink el, 2011). Sev eral mec hanisms for
EET ha v e b een un v eiled: (1) outer mem brane cyto c hromes (m trC / omcA) that catalyze
the direct electron transfer to solid-phase electron acceptors (Beliaev et al., 2001; Deng et
al., 2018), (2) electron sh uttles suc h as quinones and rib ofla vins that can carry electrons
from cells to solid substrates (Canstein et al., 2008; Newman & K olter, 2000), and (3) con-
ductiv e nano wires and extracellular p olymeric substances (EPS) (Borole et al., 2011).
It is generally b eliev ed that manganese reduction via direct electron transfer or con-
ductiv e nano wires and EPS usually require cells to main tain close con tact with the sub-
strates through attac hmen t, either rev ersibly or in biofilm (Nealson & My ers, 1992). Al-
though manganese reducers gro wing in biofilm can b e activ e for mon ths in lab cultures
(Nealson & Fink el, 2011), in natural en vironmen ts, they are p oten tially less stable when
faced with shortages of substrates. F or example, the mo del organism for manganese reduc-
tion, Shewanel la oneidensis MR-1 has b een sho wn to rapidly detac h from biofilm induced
107
b y a decrease of energy a v ailabilit y (Sa ville et al., 2011; Thormann et al., 2005). The re-
duction of manganese o xide b y S. oneidensis MR-1 without attac hmen t has also b een
observ ed through a unique b eha vior called electrokinesis, sho wn as “increased swimming
sp eeds and prolonged runs when cells are in close pro ximit y to a redo x activ e surface suc h
as MnO
2
particles or the w orking electro de of an electro c hemical cell” (Harris et al., 2010;
Oram & Jeuk en, 2019). MnO
2
particles w ere reduced without cells initiating attac hmen t
but only transien tly touc hing the particle surface.
A group of putativ e manganese-reducing Desulfovibrio using amorphous MnO
2
as the
electron acceptor, acetate or lactate as the electron donor, w as rep eatedly enric hed only
from the p orew ater (Fig. 7). The slo w reduction of MnO
2
in the enric hmen t, esp ecially
when acetate w as the sole electron donor, (Fig. 6) fits the description of other p oten tial
manganese reducing bacteria in coastal sedimen t (V andiek en et al., 2012). The rapid dis-
app earance of MnO
2
in enric hmen ts of the p orew ater-free particles w ere lik ely due to the
presence of H
2
S (My ers & Nealson, 1988) pro duced b y the lo w abundance Deltapr ote ob ac-
teria in the enric hmen ts. In the p orew ater enric hmen ts, sulfate reduction w as p ossibly
minimal esp ecially in enric hmen ts with acetate as the sole electron donor, as during the
first 40 da ys of the exp erimen t, no detectable disapp earance of manganese o xide w as ob-
serv ed (Fig. 6). This statemen t is ho w ev er offered ten tativ ely as no direct measuremen t of
sulfate reduction w as made.
Sev eral op erational taxonomic units (OTUs) within the group of p oten tial manganese
reducers w ere also presen t in the particle-attac hed comm unities but remained in extremely
lo w abundance in the enric hmen ts (Fig .7b). One p ossible explanation is that these particle-
attac hed Desulfo vibrios w ere unable to detac h themselv es and mo v e to w ards the MnO
2
particles, whic h tactic resp onse w as used b y the free-living Desulfovibrio cells. Another ex-
planation is that the c hemical reduction b y H
2
S b y the particle-attac hed Deltapr ote ob acteri
precluded the biological reduction of MnO
2
(Sc hipp ers & Jøgensen, 2001; Thamdrup et
al., 1994). The reason as to wh y the free-living Desulfovibrio did not reduce sulfate is not
108
clear but this un usual b eha vior w as consisten tly observ ed in our exp erimen ts.
In marine sedimen ts, sulfate is presen t at high concen tration ( 28 mM) therefore
sulfate reducers generally dominate the microbial comm unities in marine sedimen ts and
can accoun t for up to 80% of the degradation of organic carb on (Andreote et al., 2012;
Durbin & T esk e, 2011; Hoshino & Inagaki, 2013; Jørgensen, 1982). The sulfate reduction
rates (SRR) measured for the particle-attac hed comm unities in our study w ere compara-
ble to previous bulk measuremen ts at the same site (Bertics & Ziebis, 2010) and in other
shallo w sedimen ts (Christensen, 1984; Kristensen et al., 1994; Sha w et al., 1984; W ellsbury
& P ark es, 1995), but surprisingly , SRR w as b elo w detection limit for the free-living com-
m unities in separate exp erimen ts (Fig. 8). Corresp ondingly , the putativ e sulfate reducers
( Deltapr ote ob acteria ) w ere in lo w abundance in the free-living comm unit y (Fig. S7).
Sulfate reduction has b een recognized to con tribute greatly to the turno v er of organic
carb on in marine sedimen ts, fueling a large fraction of the terminal carb on o xidation pro-
cesses in differen t en vironmen ts (Christensen, 1984; Christensen & Blac kburn, 1982; Fink e
et al., 2007; Glom bitza et al., 2015). A cetate is an imp ortan t electron donor for sulfate re-
ducers (Sha w & McIn tosh, 1990; Thauer & P ostgate, 1982; W ellsbury & P ark es, 1995) and
is activ ely o xidized for energy acquisition b y the microbial comm unities in Catalina harb or
sedimen ts (Chapter I I I). Inhibiting sulfate reduction decreased the rate of acetate o xida-
tion for the particle-attac hed comm unities (Fig. 9) suggesting that sulfate reducers w ere
activ e mem b ers of the particle-attac hed comm unit y . In con trast, inhibiting sulfate reduc-
tion did not affect the acetate o xidation rate of the free-living comm unit y leading to the
conclusion that other electron acceptors are used b y the free-living comm unities. Motilit y
and c hemotaxis ma y allo w the free-living comm unities to access electron acceptors that
are more energetically fa v orable, suc h as nitrate and manganese o xide. In con trast, sul-
fate pro vides less energy but is supplied consisten tly in abundance to the particle-attac hed
comm unit y and therefore preferred.
109
In summary , this study sho ws that a large fraction (50 to 90%) of the free-living com-
m unit y in the p orew ater are motile and c hemotactic. The particle-attac hed comm unit y
do es not frequen tly detac h and en ter the free-living p o ol. Increasing n utrien t (NO

3
) could
induce motilit y from either the non-motile cells in the p orew ater or the rev ersibly attac hed
cells. The three main terminal electron acceptors tested here pro v e to ha v e a selecting
impact on the nic he-partitioning b et w een the free-living and particle-attac hed cells. The
solid-phase manganese o xide could b e accessed and utilized as an electron acceptor b y
the motile cells in the p orew ater, but most lik ely not b y the particle-attac hed cells. F or
the soluble electron acceptors, the lo w-concen tration and high-energy yielding nitrate is
more quic kly used b y the c hemotactic cells in the p orew ater. Sulfate, whic h is abundan t
but not as energetically-fa v orable, is not a preferred electron acceptor for the p orew ater
comm unit y , but serv es as the primary energy source for the particle-attac hed comm unit y .
The presence of a distinct, motile and metab olically activ e p orew ater comm unit y has b een
o v erlo ok ed and underestimated. Our study highligh ts the uniqueness and imp ortance of
this comm unit y whic h deserv ed further and more detailed understandings.
A c kno wledgemen ts
I w ould lik e to thank the Nealson Lab esp ecially Casey Barr for their help with the prepa-
ration of the enric hmen t cultures for the manganese reducers and the 16S rRNA sequenc-
ing of the enric hmen ts. I w ould also lik e to thank the Dr. James Moffett and Kenn y Bol-
ster for their help with the dissolv ed manganese analyses. I w ould lik e to thank m y collab-
orators (Karsten Zengler, Mallory Em bree, Cameron Martino, Ric hard Szubin) at USCD
for their supp ort in the molecular w ork. I also thank mem b ers of the Ziebis Lab (Jane
Den tinger, Abb y Lam bretti, Emily W aggoner) for their help in sample collection and pro-
cessing. The W rigley Institute of Marine Studies has pro vided logistical and financial sup-
p ort for this w ork.
110
Figure 1: a. Concen trations of NO

3
+NO

2
in the p orew ater and HCl extractable F e (I I I)
in the bulk sedimen t. b. Concen trations of NH
+
4
and Mn
2+
in the p orew ater and HCl ex-
tractable F e (I I) in the bulk sedimen t.
111
Figure 2: The a v erage (n = 3) p ercen tage of cells with flagel-
lum in the p orew ater
112
Figure 3: Concen trations of motile cells that migrated in to the needles at differen t time p oin ts. Exp erimen ts
w ere carried out for 6 differen t depths.
113
Figure 4: A v erage (n=3) p ercen tages of motile cells at v arying depths in the p ore-
w ater. In blue the p ermanen t motile cells, that w ere defined as the cells that
mo v ed in to the needles within the first 15 min utes. of the exp erimen t. In orange,
the p erio dically motile cells, that w ere defined as the cells that mo v ed in to the
needles after the 15-min time p oin t
114
Figure 5: Chemotactic resp onse (c hange in cell concen tration within the needle in
comparison to the con trol) of the p ermanen tly and the p erio dically motile cells,
using concen trations of 50 µM and 100 µM nitrate and samples from t w o differen t
depths. In the original sedimen t column, nitrate w as still presen t at 3 cm but w as
close to 0 at 10 cm.
115
Figure 6: A v erage (n=3) amoun t of the manganese o xide reduced o v er time in enric hmen t
cultures using acetate or lactate as the electron donor and with either the p orew ater or the
p orew ater-free sedimen t from three depths as the initial ino culum.
116
Figure 7: a. Relativ e abundances of p oten tial sulfate reducers in the original sedimen t vs. in the enric hmen ts.
b. The partitioning of Deltaproteobacterial op erational taxonomic units (OTUs) b et w een the free-living and the
particle-attac hed comm unities. The relativ e abundance of eac h OTU within the enric hmen t w as color-co ded.
117
Figure 8: Sulfate reduction rates of the free-living and particle-attac hed comm unities
incubated o v er a time p erio d of 24 hours. T w o separate exp erimen ts w ere conducted in
No v em b er 2017 (circles) and June 2018 (triangles)
118
Figure 9: A cetate o xidation rates of the free-living and particle-attac hed
comm unities with and without the addition of molyb date (20 mM
so dium molyb date) to inhibit sulfate reduction.
119
Supplemen tary Materials
Metagenomic assem bly and annotation
The samples for molecular surv ey of the Catalina harb or sedimen t w ere conducted in Oc-
tob er 2014. The upp er 8 cm of four parallel sedimen t cores w ere sectioned at 2cm eac h.
Sedimen t la y er of the same depth from all four cores w ere p o oled. The p orew ater and the
sedimen t w ere separated for eac h depth la y er. In tact cells w ere extracted from the p ore-
w ater (25 mL) and the bulk sedimen t (200 cm
3
) of four differen t depths b y densit y cetaxo-
nomicn trifugation through a cushion of Nyco denz (Kallmey er et al., 2008). Genomic DNA
w as extracted from the 0: 22 µm filter and in tact cells using the NucleoSpin Kit (Mac herey-
Nagel Inc., Bethlehem, P A). Metagenomic sequencing w as p erformed on Illumina HiSeq
platform using the 2 150 bp paired-end c hemistry according to man ufacturer’s instruc-
tions at the Biomedical Researc h F acilit y in UC San Diego.
Ra w reads w ere trimmed using T rimmomatic (Bolger et al., 2014) to remo v e adapters
and lo w qualit y sequences. Sequences that passed qualit y trimming w ere co-assem bled to
con tigs using MEGAHIT (Li et al., 2015) with the meta-sensitiv e preset (min-coun t=1,
kmers=21,29,39,49,...,129,141). Primary con tigs w ere clustered using CD-HIT-EST (F u
et al., 2012, sequence iden tit y = 0.95, w ord length = 10). The con tigs set w ere further
merged using minim us2 (T reangen et al., 2011, O VERLAP=50, MINID=95).
Final con tigs with length greater than 1000 w ere imp orted in to An vi’o (Eren et al.,
2015) and analyzed follo wing the An vi’o metagenomic w orkflo w. In short, eac h sample w as
profiled b y mapping reads originated from that sample to the con tigs with b o wtie (Lang-
mead et al., 2009). Protein-co ding genes w ere predicted using Pro digal (Hy att et al., 2010)
and annotated using GhostKO ALA (Kanehisa et al., 2016) and blastp against the Ref-
Seq database (O’Leary et al., 2016). Genes of in terest w ere aligned to closely related genes
using Clustalo (Siev ers et al., 2011). Ph ylogenetic trees w ere built using F astT ree. Abun-
120
dance of genes w ere calculated as the co v erage of the gene divided b y the a v erage co v erage
of one single cop y gene r e cA in resp ectiv e samples.
121
Sea w ater medium recip e
Sea w ater base medium
Comp onen t Final Concen tration (mM)
NaCl 342.2
MgCl
2
6H
2
O 14.8
CaCl
2
2H
2
O 1
K Cl 6.71
NH
4
Cl 10
Na
2
SO
4
1
KH
2
PO
4
/K
2
HPO
4
1
HCl-Dissolv ed T race Elemen ts
Comp onen t Final Concen tration (µM)
HCl 20
FeSO
4
7H
2
O 7.5
H
3
BO
3
2H
2
O 0.48
MnCl
2
4H
2
O 0.5
CoCl
2
6H
2
O 6.8
NiCl
2
6H
2
O 1
CuCl
2
2H
2
O 12 nM
ZnSO
4
7H
2
O 0.5
Na
2
MoO
4
2H
2
O 0.15
NaVO
3
2
Na
2
WO
4
1H
2
O 75 nM
Na
2
SeO
3
5H
2
O 23 nM
13-Vitamin solution
Comp onen t Final Concen tration (µg/ml)
10 MOPS pH 7.2 10 µM
Rib ofla vin 0.1
Biotin 0.03
Thiamine HCl 0.1
L-Ascorbic acid 0.1
d-Ca-pan tothenate 0.1
F olic acid 0.1
Nicotinic acid 0.1
4-aminob enzoic acid 0.1
p yrido xine HCl 0.1
Lip oic acid 0.1
NAD 0.1
Thiamine p yrophosphate 0.1
Cy ano cobalamin 0.01
122
Figure S1: Diagram of the flo w b o x used in the exp erimen ts to test for cell motilit y and c hemotaxis.
123
Figure S2: Concen trations of motile cells that migrated in to the needles at differen t time p oin ts. Ex-
p erimen ts w ere rep eated for samples from differen t depths.
124
Figure S3: Change of motile cell concen trations that migrated in to the needles b et w een
eac h time-in terv als. A v erages and standard deviations w ere tak en from the 6 differen t
depth that w ere analyzed.
125
Figure S4: P ercen tage of motile cells at t w o differen t depths in the con trol, and with additions of 50 µM and
100 µM nitrate to the ano xic sea w ater in the needle. The p ermanen t motile cells w ere defined as the cells that
mo v ed in to the needles within the first 15 min. The p erio dically motile cells w ere the cells that mo v ed in to the
needles after the 15min. time p oin t
126
Figure S5: Relativ e abundances (normalized to the cop y n um b ers of r e cA) of the essen tial genes in-
v olv ed in nitrate reduction sho wn for the p orew ater (pa, pb, p c, p d) and the sedimen t (sa, sb, sc,
sd). a - d represen ts the depth of the sample, corresp ondingly 0 - 2 cm, 2 - 4 cm, 4 - 6 cm, and 6 - 8
cm
127
Figure S6: Ph ylogenetic tree (Neigh b or-joining, b o otstrap = 100) of the op erational tax-
onomic units (OTUs) b elonging to the class Deltapr ote ob acteria from the manganese re-
ducer enric hmen ts and their closest relativ es.
128
Figure S7: Relativ e abundances of p oten tial sulfate reducers in the sea w ater, the p orew ater and the
sedimen t at differen t depths. T axonom y w as assigned based on 16 rRNA gene sequences similarit y
(97%) to the Silv a v132 database.
129
Chapter 5: Conclusion
Ben thic microbial comm unities ha v e b een w ell recognized for their essen tial roles in the
marine biogeo c hemical pro cesses. In marine sedimen t, micro organisms can either liv e at-
tac hed to sedimen t particles or liv e singly in the p ore space. The con v en tional approac h
examines the b en thic microbial comm unities in the “bulk” . Therefore, the p oten tial dif-
ferences b et w een the free-living and particle-attac hed comm unities in marine sedimen ts
remain unkno wn. Here I presen t, for the first time, a study fo cusing on the free-living bac-
terial comm unit y in marine sedimen ts and its ecological imp ortance.
The p orew ater in the in tertidal m udflat (Catalina Harb or, Catalina Island, CA) har-
b ors an abundan t microbial comm unit y with an abundance of 10
7
10
8
cells cm
3
, 1 to
2 orders of magnitude higher than the microbial abundance in the sea w ater. The p orew a-
ter comm unit y differed greatly with the sea w ater comm unit y suggesting little exc hange
b et w een the t w o habitats. The free-living comm unit y w ere equally div erse as the particle-
attac hed comm unit y and only 22% of the bacterial ric hness w ere shared b et w een the t w o
fractions. The b en thic bacterial cells exhibited strong nic he-partition b et w een the p orew a-
ter and particle surfaces as 67% of the amplicon sequence v arian ts (ASV s) w ere found in
only one of t w o nic hes. The preference for the free-living or the particle-attac hed lifest yle
app eared to b e ph ylogenetically conserv ed.
The free-living comm unit y w ere highly activ e, ev en more so than the particle-attac hed
comm unit y . Based on the measured rates of anaerobic acetate o xidation and anaerobic
formate o xidation, the free-living comm unit y is estimated to b e resp onsible for o v er half
of the organic carb on remineralization in surface sedimen ts. Up to 90% of the free-living
comm unit y exhibited swimming motilit y and o v er half w ere c hemotactic to w ards nitrate.
W e h yp othesize that the high metab olic rates of the free-living comm unit y are p o w ered b y
130
motilit y and c hemotaxis.
Differen tial usages of electron acceptors w ere observ ed for the free-living comm unit y
and the particle-attac hed comm unit y . Sulfate reduction, arguably the most imp ortan t ter-
minal o xidation pro cess in marine sedimen ts, supp orted a large fraction of the terminal
carb on o xidation for the particle-attac hed comm unit y but w as not detected in the p orew a-
ter. The disregard for sulfate b y the free-living comm unit y could p oten tially b e an adap-
tiv e c hoice: the c hemotactic cells could preferen tially use high-energy electron acceptors
suc h as nitrate or metal o xides when a v ailable. The particle-attac hed cells, relying solely
on the diffusion of substrates for surviv al, could main tain a slo w but steady gro wth on the
consisten t supply of sulfate.
The p orew ater comm unit y constitute a unique comp onen t of the b en thic microbiome.
They are abundan t, div erse and con tribute tremendously to the b en thic biogeo c hemical
pro cesses. My study is just b eginning to un v eil the differen tial b eha viors b et w een the free-
living and the particle-attac hed comm unities that ha v e b een o v erlo ok ed b y the con v en-
tional ”bulk” studies of marine sedimen ts. F urther studies are m uc h needed to shed ligh t
up on the long-neglected free-living comm unit y .
131
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Abstract (if available)

Abstract Coastal sediments harbor diverse microbial communities in an abundance averaging 10⁹ to 10¹¹ cells per cm³, 3 to 4 orders of magnitude greater than the average cell abundance in the seawater (10⁶ cells cm⁻³). Benthic microbial communities adopt two contrasting lifestyles: free-living in the pore space or attached to sediment particles. These two fractions are not distinguished in conventional benthic microbial studies, which analyze the sediment in “bulk”. Therefore, the diversity, structure, and ecological functions of the porewater community remains unknown. My thesis focused on characterizing this unexplored porewater community for its abundance, diversity, and metabolic activities, in comparison to the particle-attached community. Investigations were carried out in an intertidal mudflat system (Catalina Harbor, Catalina Island, CA). Results showed that the free-living community in the porewater was not only abundant (10⁷ to 10⁸ cells cm⁻³) but also differed greatly from the particle-attached community and the microbial community in the overlying seawater based on 16S rRNA sequencing. More importantly, radiotracer studies revealed significantly higher carbon turnover rates (acetate and formate oxidation) of the porewater community than its particle-attached neighbors. This finding was further supported by a combination of thermodynamic calculations, metagenomic analysis and nano-calorimetry. Results also showed that a large fraction (up to 90%) of the free-living community was motile and displayed chemosensory response to added nutrients (e.g. nitrate), which could partially explain their elevated metabolic activities. Although sulfate reduction is regarded as the most important process for the anaerobic mineralization of organic matter in marine sediments, repeated radiotracer measurements using ³⁵S-sulfate showed that the porewater community, surprisingly, did not use sulfate as a terminal electron acceptor, in contrast to the particle-attached community. In conclusion, the porewater community is a distinct component of the benthic microbiome, however its role in benthic biogeochemical cycles has been greatly overlooked and deserves further investigations in different sediment settings, including the deep biosphere.

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Asset Metadata

Creator Cheng, Bingran (author)

Core Title Unexplored microbial communities in marine sediment porewater

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Biology (Marine Biology and Biological Oceanography)

Publication Date 04/23/2021

Defense Date 12/04/2019

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag electron acceptor,marine sediment,microbial ecology,microbial motility,niche-partition,OAI-PMH Harvest,organic carbon turnover,porewater,terminal oxidation

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Ziebis, Wiebke (committee chair), Heidelberg, John (committee member), LaRowe, Douglas Edward (committee member), Moffett, James Wylie (committee member)

Creator Email bingran.cheng@gmail.com,bingranc@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-289661

Unique identifier UC11663600

Identifier etd-ChengBingr-8327.pdf (filename),usctheses-c89-289661 (legacy record id)

Legacy Identifier etd-ChengBingr-8327.pdf

Dmrecord 289661

Document Type Dissertation

Rights Cheng, Bingran

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA