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Biological nitrogen fixation associated with living and decomposing macroalgae
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Content
Biological nitrogen fixation associated with living and decomposing
macroalgae
By: Yubin Raut
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(OCEAN SCIENCES)
December 2021
Copyright (2021) Yubin Raut
ii
DEDICATION
I would like to dedicate the entirety of this Ph.D. dissertation to my wonderful and loving parents:
Yogendra Man Singh Raut and Ambika Sitoula Raut. While I finished my thesis in 2021, the
journey towards higher education began over 25 years ago when my dad emigrated from Nepal,
followed soon after by mom, and ultimately with my immigration to the United States of America
in 2001. The conferral of this Ph.D. is a culmination of many years of struggles in which countless
sacrifices were made by my parents and numerous obstacles, challenges, trials, and tribulations
were overcome as a family. From the bottom of my heart, I thank you two for providing me with
such a golden opportunity in life. I am honored to pay this forward to the vast number of people
around the world that could benefit from being presented a similar opportunity to use education as
a platform to better their lives.
iii
ACKNOWLEDGEMENTS
I would like to thank my advisor and dissertation committee chair, Dr. Douglas G. Capone, for a
wonderful Ph.D. experience, superb mentoring and guidance, and countless opportunities to grow
and develop as a researcher. I greatly appreciate the time, feedback, and guidance from the rest of
my dissertation committee members (Professors Sergio Sañudo-Wilhelmy, David A. Hutchins,
and John Heidelberg) who also served on my Ph.D. qualifying exam committee (in addition to Dr.
Carly Kenkel). I thank the USC Wrigley Institute for Environmental Studies for supporting much
of my dissertation work and hosting me at the Wrigley Marine Science Center (WMSC) on Santa
Catalina Island for numerous summers. This work would not be possible without the logistical
support from Lauren Oudin-Czarnecki, Kellie Spafford, and many WMSC staff and crew. I thank
the generous financial support from several Victor J. Bertics and Wrigley Graduate Summer
Fellowships spearheaded by Dr. Linda Duguay and the USC Research Enhancement Fellowship.
The numerous experiments conducted for my dissertation could not have been viable without the
help from wonderful undergraduate summer researchers (Shannon Matzke, Camille Vieira, Taylor
Dillon, Calyn Crawford, Sarah Ortiz) as part of the Research Experiences for Undergraduates
program led by Dr. Diane Kim and Dr. Karla Heidelberg. I would like to thank all members of the
Capone lab, especially Michael Morando, Troy Gunderson, Miguel Rincon, Suriya Tanjasiri, and
Weimin Deng, for all their help. I would also like to thank numerous labs (Fuhrman lab, Hutchins
lab, Ziebis lab, Nealson lab), countless colleagues (Yi-Chun Yeh, Nina Yang, Bingran Cheng,
Casey Barr, Elaina Graham, Jason Wang, Kenneth Bolster, Joshua Kling, Babak Hassanzadeh,
Gerid Ollison, Heidi Aronson, Maria Ruggeri) from the Marine Environmental Biology program
at USC, BISC administrative staff, and many Wrigley Fellows (Jack May, Kathryn Scafidi, Erika
Nava, Emily Ryznar, Melissa Dellatorre, Lauren Smith, George Jarvis) for all their help and
incredible company throughout this journey. Lastly, I would like to thank my family, friends, and
partner in crime (Amanda Godbold) for all their love and support.
iv
TABLE OF CONTENTS
Dedication…………...……………………………………………………………………………ii
Acknowledgements ...……………………………………………………………………………iii
List of Tables ……………………………………………………………………………………..v
List of Figures ...…......…………………………………………………………………………...vi
Abstract…………………………………………………………………………………………..vii
Chapter 1: Introduction …………………………………………………………………………...1
References …………………………………………………………………………...........8
Chapter 2: Diazotrophic Macroalgal Associations with Living and Decomposing Sargassum…13
Abstract…………………………………………………………………………………..13
Introduction………………………………………………………………………………14
Methods…………………………………………………………………………………..17
Results……………………………………………………………………………………23
Discussion………………………………………………………………………………..31
Conclusion……………………………………………………………………………….41
References………………………………………………………………………………..42
Supplemental Material…………………………………………………………………...49
Chapter 3: Macroalgal detrital systems: An overlooked ecological niche for heterotrophic
nitrogen fixation………………………………………………………………………………….57
Summary…………………………………………………………………………………57
Introduction………………………………………………………………………………57
Results and Discussion…………………………………………………………………..62
Materials and Methods…………………………………………………………………...79
References………………………………………………………………………………..84
Supplementary Information……………………………………………………………...90
Chapter 4: Conclusion and Future Directions…………………………………………………..110
References………………………………………………………………………………117
v
LIST OF TABLES
Chapter 1……………………………………………………………………………………………
Table 1…………………………………………………………………………………….5
Chapter 2……………………………………………………………………………………………
Supplementary Table 1, 2………………………………………………………………..49
Supplementary Table 3, 4………………………………………………………………..50
Supplementary Table 5…………………………………………………………………..51
Supplementary Table 6…………………………………………………………………..52
Supplementary Table 7…………………………………………………………………..53
Supplementary Table 8…………………………………………………………………..54
Chapter 3……………………………………………………………………………………………
Table 1……………………………………………………………………………...60 – 61
Supplementary Table 1…………………………………………………………………..97
Supplementary Table 2…………………………………………………………………..98
Supplementary Table 3……………………………………………………………99 – 101
Supplementary Table 4…………………………………………………………………102
Supplementary Table 5…………………………………………………………………103
Supplementary Table 6…………………………………………………………………104
Supplementary Table 7…………………………………………………………………105
Supplementary Table 8…………………………………………………………………106
Supplementary Table 9…………………………………………………………………107
Supplementary Table 10, 10.1………………………………………………………….108
Supplementary Table 11………………………………………………………………..109
vi
LIST OF FIGURES
Chapter 1……………………………………………………………………………………………
Figure 1……………………………………………………………………………………1
Figure 2……………………………………………………………………………………3
Figure 3……………………………………………………………………………………6
Chapter 2……………………………………………………………………………………………
Figure 1…………………………………………………………………………………..23
Figure 2…………………………………………………………………………………..25
Figure 3…………………………………………………………………………………..26
Figure 4…………………………………………………………………………………..27
Figure 5…………………………………………………………………………………..28
Supplementary Figure 1………………………………………………………………….55
Supplementary Figure 2………………………………………………………………….56
Chapter 3……………………………………………………………………………………………
Figure 1…………………………………………………………………………………..66
Figure 2…………………………………………………………………………………..68
Figure 3…………………………………………………………………………………..75
Figure 4…………………………………………………………………………………..76
Supplementary Figure 1………………………………………………………………….90
Supplementary Figure 2………………………………………………………………….91
Supplementary Figure 3………………………………………………………………….92
Supplementary Figure 4………………………………………………………………….93
Supplementary Figure 5………………………………………………………………….94
Supplementary Figure 6………………………………………………………………….95
Supplementary Figure 7………………………………………………………………….96
Chapter 4……………………………………………………………………………………………
Figure 1…………………………………………………………………………………110
Figure 2…………………………………………………………………………………114
vii
Abstract
Macroalgae, or seaweed, are cosmopolitan organisms that populate global coastlines
ranging from the poles to the tropics. They contribute significantly to photosynthetic primary
production and create diverse habitats, providing food and shelter for a wide array of micro- and
macro-organisms. Macroalgae require nitrogen (N) and typically obtain N (e.g., ammonium,
nitrate) via direct uptake from the surrounding water. Biological nitrogen fixation (BNF) is the
process in which specialized prokaryotes, termed diazotrophs, use the nitrogenase enzyme to
convert dinitrogen (N2) gas into a bioavailable form of N. The most striking advantage for
macroalgae to host diazotrophs would be direct access to a N source. While microbial macroalgal
interactions have been well documented and identified to play an important role in the life cycle
of macroalgae, the prevalence of diazotrophic macroalgal associations (DMAs) are not well
characterized, especially in coastal ecosystems.
BNF rates were measured, using the acetylene reduction assay (ARA), with an invasive
benthic brown seaweed, Sargassum horneri, around Santa Catalina Island, CA. BNF associated
with S. horneri was absent throughout most of its life cycle, but higher BNF rates, supporting ~3
– 36 % of the required N demand of juvenile S. horneri during the late summer suggests BNF may
be important during periods of N deficiency. A larger investigation consisting of macroalgae from
all 3 clades corroborates this hypothesis, as DMAs persisted during periods of low nitrate
availability in the waters surrounding Santa Catalina Island. While it is unlikely that these lower
BNF rates provide a significant contribution towards the host macroalgal N demand, these results
further solidify that DMAs with living macroalgae are also prevalent in coastal environments.
Seaweeds continue to serve a myriad of ecological functions after death as they enter the
detrital food web, support benthic coastal ecosystems, and contribute to carbon (C) sequestration.
viii
Interestingly, diazotrophic activity was more pronounced throughout the decomposition of S.
horneri, suggesting another role for N2 fixers during the microbial breakdown of organic rich
macroalgal detritus. Diazotrophic activity was measured using the ARA during several long-term
litter bag decomposition experiments with a diverse set of brown, green, and red macroalgae. The
results suggest that decomposing brown and green macroalgal systems support higher rates of BNF
than red macroalgae.
This may largely be linked to the availability of N throughout decomposition, as inferred
from the C:N ratio of the aging macroalgal detritus. The higher C:N content of brown macroalgae
may result in faster utilization of the available N by the microbial community degrading the
macroalgal tissue. This can potentially lead to N limitation throughout decomposition, promoting
diazotrophs, who supply their own N, to facilitate the breakdown of the N limited, organic
macroalgal detritus. Accordingly, the 2 green and 1 red macroalgae which had higher C:N content
also exhibited greater diazotrophic activity throughout decomposition. Lastly, a series of sodium
molybdate amendments resulted in severe inhibition of nitrogenase activity, suggesting the
importance of sulfate reducing diazotrophs in macroalgal detrital systems.
Taken together, the results from this dissertation greatly expand upon our previously
limited understanding of DMAs with both living and decomposing macroalgae, further identifying
macroalgal detrital systems as a novel, globally relevant niche for BNF. These data were also used
in combination with a literature review of diazotrophic activity during decomposition of other
macrophytes to propose a theoretical model that predicts nitrogenase activity throughout
macrophyte degradation in three phases. Finally, the results from this thesis raises questions about
the influence of macroalgal detritus on benthic diazotrophic communities found in coastal marine
sediments.
1
Chapter 1: Introduction
Marine macroalgae or seaweed are a diverse group of photoautotrophic organisms that are
divided into 3 major lineages: Chlorophyta or green seaweed, Phaeophyceae or brown seaweed,
and Rhodophyta or red seaweed. Macroalgae from all 3 clades are found ubiquitously throughout
the global oceans (Figure 1) where they contribute ~4 – 5% to global net primary production
(Duarte and Cebrián, 1996; Raven, 2018). They extend across polar, temperate, and tropical
latitudes in both coastal and open ocean settings where they are further involved in a wide variety
of ecological functions (Keith et al., 2014). In coastal benthic habitats, macroalgae only occupy a
small fraction of the ecosystem (~5%) but disproportionally contribute up to 50% of global coastal
gross primary production, 41% of global coastal respiration, and 74% of global coastal net
ecosystem production (Duarte et al., 2005).
Furthermore, macroalgae such as the giant kelp (Macrocystis) create structurally complex
habitats and serve as the base of the food-web structure with large fractions of macroalgal
productivity also entering the detrital food chain (Mann, 1973). These ecosystems can also
influence water motion, nutrients, and light concentrations in the surrounding water column and
underlying sediment (Graham et al., 2007). Aside from being the major primary producer,
Figure 1: Macroalgal distribution and genus richness in the global oceans for the 3 major
lineages: A) Rhodophyta, B) Chlorophyta, and C) Phaeophyceae. Figure adapted from Keith et
al. (2014).
2
macroalgae play an additional role in the carbon (C) cycle with significant contributions to global
C sequestration (~173 TgC yr
-1
) where about 90% of this C is exported to the deep ocean and the
remaining portion is sequestered in coastal marine sediments (Smith, 1981; Krause-Jensen and
Duarte, 2016). More recently, the role of macroalgal sequestration is gaining traction as a
potentially important means of blue C storage similar to other macrophytes, e.g. mangroves, salt
marshes, and seagrasses (Trevathan-Tackett et al., 2015; Raven, 2018).
Macroalgae, particularly two major species of brown seaweed – Sargassum fluitans and S.
natans, can also proliferate in open ocean settings (e.g. Sargasso Sea) primarily as large floating
drift communities of pelagic macroalgae (Butler and Stoner, 1984). Pelagic Sargassum
communities contribute to total primary production (TPP), providing up to ~60% of the TPP in the
upper 1m of the water column in the Sargasso Sea (Coston-Clements et al., 1991). They also host
a diverse assemblage of epiphytes and fungi while serving as habitats for turtles and more than a
100 species of invertebrates and fishes, providing these organisms foraging grounds and protection
from potential predators (Coston-Clements et al., 1991).
Alternatively, distinct rafts of benthic macroalgae (e.g. M. pyrifera) can often become
uprooted from the substratum via different processes (e.g. grazing, storms) and transported
offshore to pelagic waters (Hobday, 2000). Positively buoyant drift macroalgae with large
pneumatocysts (air bladders), such as Macrocystis, can act as a substrate for other negatively
buoyant species of macroalgae to attach to (Figure 2). Together, the entangled macroalgae provide
diverse habitats for a wide assemblage of marine invertebrates and vertebrates and can even
become potential modes of dispersal for associated organisms such as the white abalone, Haliotis
sorenseni (Thiel and Gutow, 2005; McCormick et al., 2008). Although found primarily in the
upper water column, these floating macroalgal communities can alter nutrient concentrations and
3
the depth of light penetration throughout the photic zone, changing the food web structure deeper
down in the water column. Ultimately, these floating macroalgal systems are not constrained to
the upper water column and in fact, downwelling clumps of Sargassum can even sink out to the
bottom of the aphotic zone and further act as a resource for the benthic ecosystem (Schoener and
Rowe, 1970; Kokubu et al., 2012).
Macroalgae are truly cosmopolitan organisms that are widespread throughout the global
oceans where they serve a wide array of ecological functions and influence ecosystems from the
surface ocean to the seafloor. The success of these macroalgae in all the aforementioned
environments depend largely on the availability of light and nutrients. With regards to nutrients,
Figure 2: Schematic representation of macroalgal export within macroalgal beds and to
offshore, pelagic waters. Figure adapted from Krause-Jensen and Duarte (2016).
4
nitrogen (N) is usually reported as a major controlling factor in the growth of cycle of macroalgae
and oftentimes, it is the dominant limiting factor for macroalgal productivity (Gagné et al., 1982;
Hanisak, 1983; Fujita et al., 1989; Pedersen and Borum, 1996; Larned, 1998). Macroalgae are
typically thought to obtain N from the dissolved fraction that accumulate in the water column via
different means: 1) seasonal and local upwelling events that can be episodic and can provide large
pulses of dissolved inorganic N, 2) winter mixing events, 3) eutrophication and N loading events,
4) regeneration of nutrients from the underlying sediments, and 5) nutrient excretions by associated
organisms (e.g. fishes) within macroalgal habitats (Chapman and Craigie, 1977; Gagné et al.,
1982; Fujita et al., 1989; Valiela et al., 1997; Lapointe et al., 2014).
Unlike other biologically relevant elements (e.g. C, P, S, Fe, etc.) which have a significant
geologic source, the largest reservoir of N, an essential component of many of the key building
blocks to life (e.g. DNA, RNA, proteins), is in the atmosphere where it exists largely as dinitrogen
gas (N2). Despite making up ~78% of the atmosphere, N is thought to be the prominent nutrient
limiting primary productivity (including macroalgal productivity) in much of the global oceans
today and also in the past (Falkowski, 1997; Capone, 2000; Gruber and Galloway, 2008; Canfield
et al., 2010). Biological nitrogen fixation (BNF) is the conversion of N2 gas into a biologically
available form of N (e.g. NH3) mediated by a specialized group of prokaryotic organisms known
as diazotrophs. Diazotrophs use the conventional Mo-dependent nitrogenase metalloenzyme (or
one of the alternative Fe-only or V-based nitrogenases) to catalyze this energetically expensive
reaction (Boyd et al., 2011).
Traditionally, upwelling events bringing up large reservoirs of nitrate (NO3
-
) entrenched in
deeper waters was considered to be the primary external source of N, but more recently, BNF has
also come to be considered a potential source of new N and an important part of the N cycle that
5
could be of greater importance in supporting primary productivity and C sequestration in the
current and future global oceans (Capone, 2001). There are several groups of marine diazotrophs
that are thought to make significant global contributions to the marine N budget: 1) non-
heterocystous forming, filamentous cyanobacteria (e.g. Trichodesmium), 2) unicellular
cyanobacteria (e.g. Crocosphaera watsonii), and 3) heterocystous forming diatom symbionts such
as Richelia intracellularis (Sohm et al., 2011). Over the recent years, research has expanded the
presence and roles of these phototrophic diazotrophs outside the typical temperature constrained
sub-tropical to tropical latitudes while also seeing an increase in the reports and prevalence of
heterotrophic N2 fixation throughout the global oceans (Sohm et al., 2011; Bombar et al., 2016).
Interestingly, the use of stable isotope approach and the more common acetylene reduction
method have also routinely identified diazotrophic associations with a wide variety of living
macroalgae such as Microdictyon sp., Dictyota sp. (Capone et al., 1977), Acanthophora sp.
(Goldner, 1980; France et al., 1998), and Caulerpa sp. and Lobophora sp. (Tilstra et al., 2017).
Additionally, there have also been several reports of contributions of fixed N (~2 – 50%: Table 1)
to various macroalgae by their associated diazotrophic community. Despite these numerous
Table 1: Biological nitrogen fixation (BNF) contributions to the required nitrogen (N) demand
for different green (Chlorophyta), red (Rhodophyta), and brown (Phaeophyceae) macroalgae.
6
accounts of diazotrophic
macroalgal associations
(DMAs), BNF is often
overlooked as a source of N
for most macroalgal
species. Accumulating data
shows that prokaryotic
partners, consisting of a
diverse community of
microorganisms including N2 fixers, are an integral part of macroalgal microbiomes (Figure 3).
Surface microbes provide a number of beneficial services such as growth-benefitting compounds,
nutrient acquisition, and protection from pathogens (Egan et al., 2013; Singh and Reddy, 2014).
However, the focus of this dissertation will be limited to investigating the role of diazotrophs
associated with living macroalgae.
Not surprisingly, microbes continue to play an important role with the remineralization of
macroalgal biomass but the role of diazotrophs throughout macroalgal decomposition remains
poorly investigated. Paralleling the diagenesis of organic matter in marine sediments, often
referred to as the 3G model (Westrich and Berner, 1984; Boudreau and Ruddick, 1991; Hülse et
al., 2018), macroalgae undergo three distinct phases of decomposition which influence the
availability of labile C: 1) rapid mechanical decay and leaching of soluble compounds, 2) slower
phase of leaching dominated by microbial degradation of organic matter, and 3) slowest phase of
decay due to the refractory nature of remaining detritus (Valiela et al., 1985; Rieper-Kirchner,
1989; Krumhansl and Scheibling, 2012). The remaining macroalgal detritus can either be
Figure 3: Schematic diagram depicting various microbial
macroalgal interaction. Figure adapted from Singh and Reddy
(2014).
7
sequestered in the sediments or subjected to further degradation by the benthic microbial
community (Castaldelli et al., 2003). Consequently, N is usually not considered to be limiting to
the microbial community during remineralization of such organic-rich matter. However, several
studies have shown that N can become limiting to the microbial community throughout macroalgal
decomposition (Tenore et al., 1979; Robinson et al., 1982). As many macroalgae have greater C
to N content, it is conceivable that the available N is utilized faster than the labile C pool. In these
N limited conditions, diazotrophs would have an advantage.
In fact, BNF has been routinely observed in various marine macrophyte detrital systems
where it might make significant contributions to immobilization, the process of nutrient
enrichment of different organic detritus. For instance, BNF associated with decomposing
mangrove leaf litter is quite common, contributing 13 – 21% of the N enrichment observed with
decaying Ceriops tagal and Rhizophora mucronata leaf detritus (Fell et al., 1975; Gotto and
Taylor, 1976; Woitchik et al., 1997). There are also several reports of nitrogenase activity
associated with senescing seagrass systems, again suggesting a contribution from BNF to the
enrichment of organic detritus (Capone and Budin, 1982; Kenworthy et al., 1987; O’Donohue et
al., 1991). N enrichment throughout macroalgal decomposition is also a common observation but
it’s typically attributed to the increasing microbial biomass associated with aging macroalgal
detritus (Rieper-Kirchner, 1989). While macroalgae have a relatively greater spatial coverage than
mangrove and seagrass ecosystems, there are only a limited number of investigations reporting
increased BNF activity associated with macroalgal detritus (Hanson, 1977; Zuberer and Silver,
1978; Hamersley et al., 2015).
The potential for BNF associated with decomposing macroalgae highlights a major gap in
our understanding of the role diazotrophs play during organic matter remineralization and further
8
suggests that DMAs may be much more widespread and not just limited to living macroalgae.
Furthermore, these macroalgal detrital systems may also serve as a previously unexplored niche
for heterotrophic N2 fixers. Lastly, we hypothesize that different N2 fixers are responsible for BNF
associated with living and decomposing macroalgae with probable shifts in the diazotrophic
community linked to the different stages of the host macroalgal physiological status. In the face of
changing environmental conditions, particularly increasing eutrophication, coastal macroalgal
blooms are increasing in frequency (Teichberg et al., 2010), making it even more pertinent to
understand the role of BNF throughout the macroalgal life cycle.
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13
Chapter 2: Diazotrophic Macroalgal Associations with Living and
Decomposing Sargassum
Abstract
Despite several studies reporting diazotrophic macroalgal associations (DMAs), biological
nitrogen fixation (BNF) is still largely overlooked as a potential source of nitrogen (N) for
macroalgal productivity. We investigated the role of BNF, via the acetylene reduction method,
throughout different life stages of the invasive macroalga, Sargassum horneri, in its non-native
Southern California coastal ecosystem. Throughout most of its life cycle, BNF rates were not
detectable or yielded insignificant amounts of fixed N to support S. horneri productivity. However,
during late summer when nutrient concentrations are usually at their minimum, BNF associated
with juvenile S. horneri contributed ~3 – 36% of its required N, potentially providing it with a
competitive advantage. As DMAs remain poorly understood within macroalgal detrital systems,
long term (15 – 28 days) laboratory decomposition time series were carried out to investigate the
role of BNF throughout decomposition of the endemic macroalga, S. palmeri, and the invasive S.
horneri. Nitrogenase activity increased drastically during the second phase of decomposition,
when increasing microbial populations are typically thought to drive macroalgal degradation, with
BNF rates associated with S. palmeri and S. horneri reaching up to 65 and 247 nmol N × g
-1
(dw)
× h
-1
, respectively. Stimulation of BNF rates by glucose and mannitol additions, up to 42x higher
rates observed with S. palmeri, suggest that labile carbon may be limiting at varying degrees in
these detrital systems. Comparable, if not higher, dark BNF rates relative to light incubations
during S. horneri decomposition suggest an important contribution from heterotrophic N2 fixers.
Inhibition of nitrogenase activity, up to 98%, by sodium molybdate additions also suggest that
sulfate reducers may be an important constituent of the detrital diazotrophic community. As labile
N can become limiting to the microbial community during macroalgal decomposition, our results
14
suggest that BNF may provide a source of new N, alleviating this limitation. Additionally, while
BNF is rarely considered as a source for N enrichment with aging macroalgal detritus, we found
it to account for ~1 – 11% of N immobilized with decaying S. horneri. Our investigations suggest
that DMAs may be globally important with Sargassum and potentially occur within other
macroalgal detrital systems.
Introduction
Benthic and pelagic marine macroalgae, encompassing 3 divisions (Rhodophyta,
Chlorophyta, Phaeophyceae), are a diverse group of photoautotrophic organisms that extend across
polar, temperate, and tropical latitudes (Keith et al., 2014). Species of brown macroalgae from the
genus Sargassum truly highlight this cosmopolitan distribution as they occur in diverse benthic
habitats along coastal zones throughout the world, spanning from the Gulf of California (Pacheco-
Ruíz et al., 1998) to the rocky intertidal shores of the Japan Sea (Umezaki, 1984), occur in coral
reefs such as the Great Barrier Reef (McCook, 1997) and form pelagic communities throughout
the Sargasso Sea, Gulf of Mexico, and Gulf Stream (Butler and Stoner, 1984). Additionally,
substrate-attached benthic macroalgae are often uprooted via different processes, e.g. exposure to
waves and currents, storm events, or grazing, and transported offshore where they proliferate as
rafts of drifting seaweed (Komatsu et al., 2008).
Benthic macroalgae can make important seasonal contributions to primary productivity in
nearshore fringing reefs of the central Great Barrier Reef (Schaffelke and Klumpp, 1997) and
dominate productivity along coastal ecosystems, contributing up to 50% of global coastal gross
primary production (Duarte et al., 2005). Similarly, pelagic Sargassum communities can also make
important contributions to upper ocean primary production with an input of ~60% of total primary
production in the upper 1m of the water column in the Sargasso Sea (Coston-Clements et al.,
15
1991). Although primary production by macroalgae provides fixed carbon to other organisms
through leaching of dissolved organic carbon or direct grazing, the vast majority of macroalgal
production (estimates up to 90%; Mann, 1973) ultimately enters the detrital food chain or is
sequestered in marine sediments or the deep ocean (Krause-Jensen and Duarte, 2016).
Macroalgae also create diverse habitats ranging from small drifting clumps of seaweed to
structurally complex three dimensional communities such as giant kelp forests. These communities
can host a rich assemblage of micro- and macro-organisms (e.g. prokaryotic and fungal epiphytes,
a wide array of fishes, turtles and other vertebrates, gastropods, amphipods, nematodes,
hydrozoans, and other invertebrates) that vary from larvae to juveniles to adults (Ingólfsson, 2000;
Graham et al., 2007; Goecke et al., 2010; Abé et al., 2013). Furthermore, macroalgal habitats (e.g.
Sargassum beds) can also control nutrient concentrations, pH, dissolved oxygen (O2)
concentrations, and photon flux in the immediate ecosystem surrounding it (Komatsu, 1989;
Lapointe, 1997; Zhang et al., 2008). The myriad of ecological functions that benthic, pelagic, or
drifting macroalgae serve exemplifies their versatility and importance in marine ecosystems.
The success of benthic and pelagic macroalgae, such as the globally relevant Sargassum
species, are largely dependent on the availability of light and nutrients. With regard to nutrients,
nitrogen (N) is usually reported as a major controlling factor in the growth cycle of macroalgae
and oftentimes, it is the dominant limiting factor for macroalgal productivity (Gagné et al., 1982;
Hanisak, 1983; Fujita et al., 1989; Pedersen and Borum, 1996; Larned, 1998; Zhang et al., 2008).
Macroalgae obtain N, along with other nutrients, from the water column where N can accumulate
via different avenues including seasonal and local upwelling events (Gagné et al., 1982; Fujita et
al., 1989), winter mixing events (Chapman and Craigie, 1977), nutrient excretions by associated
16
organisms (Lapointe et al., 2014), nutrients from the underlying sediments, and eutrophication
from N loading events such as wastewater discharge (Valiela et al., 1997).
Biological N fixation (BNF) is the conversion of dinitrogen gas (N2) into a biologically
available form of N (e.g. NH3) mediated by a specialized group of prokaryotic organisms known
as diazotrophs. Diazotrophs use the nitrogenase metalloenzyme to tap into the vast dissolved
reservoir of N2 and provide a new source of N, making significant contributions to the global N
budget and primary production (Capone, 2001). BNF associated with pelagic Sargassum species
(i.e. S. fluitans, S. natans) has been examined by a number of investigators (e.g. Carpenter 1972;
Hanson 1977; Phlips, Willis, and Verchick 1986) with varying estimates of contributions of fixed
N to sustain macroalgal productivity. In contrast, studies on benthic Sargassum species are less
frequent (Phlips et al., 1986; Odintsov, 1992). Despite several additional accounts (as determined
via the acetylene reduction (AR) method or stable isotope approach) of diazotrophic macroalgal
associations (DMAs) observed with other benthic macroalgae 1) Codium decorticatum (Rosenberg
and Paerl, 1981), 2) C. fragile (Head and Carpenter, 1975; Gerard et al., 1990), 3) Microdictyon
sp., Dictyota sp. (Capone et al., 1977), 4) Acanthophora sp. (Goldner, 1980; France et al., 1998),
5) Laurencia sp. (Capone, 1977), and 6) Caulerpa sp. and Lobophora sp. (Tilstra et al., 2017),
BNF is still rarely considered as a potential source of N for macroalgal productivity.
While there are a limited number of studies investigating BNF associated with living
macroalgae, it remains virtually unexplored with decomposing macroalgal systems. In contrast,
BNF dynamics have been routinely investigated with other macrophyte litter such as mangrove
leaf detritus and senescing sea grass meadows which have repeatedly been shown to support
diazotrophic activity (Gotto and Taylor, 1976; Zuberer and Silver, 1978; Capone and Budin, 1982;
O’Donohue et al., 1991). The only prior publication (to our knowledge) that directly investigated
17
BNF activity associated with decomposing seaweed reported higher BNF rates associated with
Macrocystis pyrifera detritus compared to healthy M. pyrifera blades (Hamersley et al., 2015).
Hamersley et al. (2015) proposed that while BNF did not contribute significantly to the N quota
required during macroalgal production, it played an important role with M. pyrifera post-
senescence by potentially improving the nutritional quality of the degrading macroalgal substrate
as it entered the detrital food web.
Although Sargassum beds in native ecosystems serve important ecological functions and
environmental services, the roles of various introduced Sargassum species (e.g. S. muticum, S.
horneri) within non-native macroalgal systems suggest a potentially harmful ecological impact
(Stæhr et al., 2000; Cruz-Trejo et al., 2015; Caselle et al., 2018). The introduction of an invasive
macroalga, S. horneri, to the eastern Pacific (Miller et al., 2007) and its increasing range expansion
throughout the Southern California ecosystem (Cruz-Trejo et al., 2015; Marks et al., 2015;
Kaplanis et al., 2016) provided us with a unique opportunity to tackle many of the aforementioned
gaps in the literature regarding DMAs. Primarily, we sought to investigate the role of diazotrophs
throughout the life cycle of S. horneri to assess whether BNF provided an advantage to this
globally important macroalga in a non-native ecosystem, thereby also expanding upon the limited
literature regarding BNF associated with benthic Sargassum species in general. As BNF dynamics
associated with decomposing macroalgal systems remain poorly understood, we also thought it
pertinent to 1) investigate BNF associated with aging S. horneri detritus in comparison to other
endemic species in the Southern California coastal ecosystem (e.g. S. palmeri) and 2) explore if
diazotrophs may influence the nutritional quality of S. horneri detritus.
Methods
2.1 Sample collection and experimental set up
18
Substrate attached S. horneri at various life stages, i.e. juvenile, immature, mature, and
senescent, were collected throughout different seasons over two years (2016 and 2017) at various
locations surrounding Santa Catalina Island, California, USA (Table S1). Substrate attached S.
palmeri that was beginning to senesce was also collected during the summer of 2016 (Table S1).
Upon collection, most samples were transported back to the Wrigley Marine Science Center where
the bulk of the experiments and incubations took place. However, there were some instances when
fresh samples were transported back to the University of Southern California (USC) main campus
in a bucket of seawater that was aerated with a bubbling stone prior to experimental setup.
For long term laboratory decomposition experiments, freshly collected samples were
randomly separated into multiple 200 µm white mesh litter bags and allowed to degrade inside
continuous flow-thru seawater tanks. The 2016 experiments took place in outdoor tanks with shade
screening resulting in ~40 – 60% of ambient surface photosynthetically active radiation (PAR),
resembling conditions experienced by floating rafts of S. horneri. The 2017 experiments took place
in indoor tanks without any screening resulting in ~4 – 10% of ambient surface PAR, resembling
decomposition conditions more likely to exist in the benthos. A different litter bag was sub-
sampled every few days and the macroalgal contents were apportioned into homogenous groups
that were then incubated for ~24 – 48 hours in serum vials with different amendments for
quantification of BNF rates via the AR method (discussed below). In order to track loss of biomass,
the same litter bag was manually squeezed to remove excess water and the wet weight was
recorded every few days over a 20-day period. The initial weight is considered to be at full
saturation and every weight measured thereafter is reported as the percentage of biomass remaining
relative to the initial weight (Figure S1).
19
In the 2016 decomposition experiment, S. horneri and S. palmeri were sub-sampled for AR
assays on days 0, 5, 11, 21, and 28. All serum vials used for AR assays were incubated in the same
outdoor tanks where macroalgal litter bags were undergoing long term decomposition. In the 2017
decomposition experiment, S. horneri was sub-sampled on days 0, 3, 8, 12, and 15 for AR assays.
These serum vials were incubated inside a temperature and light controlled incubator held at a
constant 18°C with 12-hour diel cycles at ~3 – 10% of ambient surface PAR. From seasonal field
sampling efforts, fresh macroalgal samples were placed immediately (~2 – 5 hours upon
collection) in serum vials and assayed for nitrogenase activity to quantify ambient BNF rates.
Similar to the decomposition experiment, these serum vials underwent short-term incubations for
~24 – 48 hours either in the flow-thru seawater tank inside the laboratory (~4 – 10% PAR) or in a
16°C temperature controlled incubator with 12-hour diel cycles at ~5% PAR.
2.2 Light vs. dark assays
Parallel light and dark assays took place for all fresh and long-term decomposition
experiments. All light assays were set up by placing macroalgal samples in clear 14 or 27 mL
serum vials with 0.2 µm filtered seawater without any further changes to the illuminance. Dark
assays took place in 14 or 27 mL serum vials wrapped in aluminum foil with 0.2 µm filtered
seawater. For the 2016 decomposition experiment of S. horneri and S. palmeri, dark vials were
also purged with N2 gas for ~1.5 minutes before beginning the AR assays. Dark vials for the 2017
S. horneri decomposition experiment were not purged with N2 gas and light inhibition remained
to be the only perturbation to these assays.
2.3 Carbon additions: glucose and mannitol amendments
Primary stocks of D-glucose (C6H12O6) and mannitol (C6H14O6) were made in the same
batch of 0.2 µm filtered seawater as what was used for incubating macroalgae in the serum vials
20
for the AR assays. For the 2016 S. horneri and S. palmeri decomposition experiments, glucose and
mannitol amendments at different concentrations were carried out in in both light and dark vials
in parallel to the control light and dark assays. On day 0, 100 uM of glucose and mannitol was
added to all assays. However, glucose and mannitol amendments were increased to 1mM additions
for all subsequent assays (i.e. days 5, 11, 21) throughout the decomposition experiment except on
day 28 when only control assays were carried out due to a shortage in remaining biomass to carry
out the carbon amendments.
2.4 Sodium molybdate assays
Primary stocks of sodium molybdate dihydrate (Na2MoO4·2H2O), a known inhibitor of
sulfate reducing bacteria (Oremland and Capone, 1988), were made in the same batch of 0.2 µm
filtered seawater as what was used for incubating macroalgae in the serum vials. For the duration
of the 2017 S. horneri decomposition experiment (i.e. days 0, 3, 8, 12, and 15), 20mM sodium
molybdate amendments were carried out in both light and dark vials concurrent to control light
and dark assays.
2.5 AR assay
The widely used AR assay was utilized to quantify ethylene (C2H4) production and assess
nitrogenase activity (Capone, 1993). For each amendment (e.g. control light/dark, glucose
light/dark, etc.), triplicate 14 or 27 mL serum vials were capped with gray butyl stoppers and
crimped with aluminum crimp caps before injecting with 1 or 2 mL of acetylene (C2H2) gas
(produced by reacting water with calcium carbide) using a disposable BD syringe with luer lock
tips. Regardless of the size of the serum vial (14 vs 27 mL), the ratio of aqueous to gas phase was
kept constant at ~67:33%, respectively, and the volume of C2H2 injected (~20% of the gas phase
or ~7% of total volume) was enough to saturate the nitrogenase enzyme (Flett et al., 1976). The
21
vials were gently shaken (~5 inversions) upon injection of C2H2 to equilibrate the vapor phase with
the aqueous phase and incubated for ~24 – 48 hours in the aforementioned temperature and light
conditions. Negative controls without any C2H2 introduced into the serum vials containing
macroalgae were also routinely tested in order to ensure there was no background C2H4 production
during these incubations.
Starting with a T0 time-point upon initial C2H2 injection, 100 µL subsamples were taken
from the headspace of all replicates using a Hamilton gas tight syringe and injected into a gas
chromatograph (Shimadzu Mini 2) equipped with a flame ionization detector at different time-
points (every ~3-8 hours) throughout the duration of the incubation. The PeakSimple
chromatography data system was used to quantify C2H4 peak heights at these different time-points
and subsequently converted to nmol of C2H4 produced (Breitbarth et al., 2004; Capone, 1993).
Instantaneous rates between time-points which contributed less than 30% to the overall rate were
used to identify occurrences of severe lags in the beginning of incubations and plateaus towards
the end of an incubation. Finally, rates of C2H4 production excluding lags and plateaus were
determined using linear regressions and subsequently, theoretical BNF rates were calculated using
a 3:1 C2H2 reduced: N2 reduced ratio (Capone, 1993) and multiplied by 2 to express as nmol N ×
g
-1
(dry weight, dw) × h
-1
.
2.6 C and N content and δ
15
N analysis
Throughout the 2017 S. horneri decomposition experiment, fresh detritus (FD) was
subsampled on days 0, 3, 8, 12, and 15 for CN and δ
15
N isotope analysis. Additionally, a portion
of the FD subsampled on the aforementioned days were incubated for ~48 hours for AR assays
and retrieved on days 2, 5, 10, 14, and 17 for CN analysis. These post-incubation detritus samples
will be referred to as PID. Dried macroalgal samples, stored in aluminum pockets were transported
22
back to the USC main campus where they were homogenized to a fine powder using a mortar and
pestle. All samples ranging between ~1-1.3 mg were then encapsulated in tin capsules and
pelletized for elemental (CN) and/or isotopic (δ
15
N) analysis on a Micromass IsoPrime continuous
flow isotope ratio mass spectrometer with CHN analyzer/sample front ends.
2.7 Light and O2 measurements
PAR for the surface ocean (< 1 ft.), outdoor tank, indoor tank, and incubator was measured
at the same time (within 10 minutes) using a handheld quantum PAR meter (Biospherical
Instruments Inc., San Diego, California, USA) over multiple days. The surface ocean readings
were assumed to be fully saturated and all other conditions are expressed as a percentage relative
to the ambient surface conditions from that day. Dissolved O2 concentrations inside an open 27
mL serum vial with 0.2 µm filtered seawater and senescent S. horneri or without any S. horneri
were measured using an O2 microelectrode (Ox-50, Unisense A/S, Aarhus, Denmark). Multiple
depth profiles, over ~30 mm, were taken at 1 mm intervals and several depth profiles at close
proximity to the seaweed were also taken at the µm scale using the micromanipulator (Unisense
A/S).
2.8 Statistical Analysis
Since not all assumptions (i.e. normality, homoscedasticity) were met for standard tests
such as ANOVA, non-parametric equivalents of the one way ANOVA (Wilcox, 2012b) and two
way ANOVA (Wilcox, 2012c) were utilized to analyze the data sets for BNF rates, %C, %N, and
C:N ratios. In conjunction with these analyses, a non-parametric equivalent to the classic Tukey’s
HSD post-hoc analysis which allows heteroscedasticity, lincon function on the R software
(Wilcox, 2012a), was utilized to conduct pairwise comparisons between various treatments for the
23
different data sets. Statistically significant differences for p-values less than 0.05, 0.01, and 0.001
are included in the various figures.
Results
3.1 Role of BNF during different life stages of S. horneri
Seasonal BNF rates associated with freshly collected S. horneri (Table S2) yielded
significantly lower rates than those associated with decomposing macroalgal detritus (Tables S3,
S4). BNF rates were not detectable with juvenile and immature S. horneri collected during the fall
and winter. However, BNF rates close to the limit of detection (~0.7 – 2 nmol N × g
-1
(dw) × h
-1
)
were observed with adult S. horneri towards the end of winter. Higher BNF rates (~12 – 24 nmol
N ×g
-1
(dw) × h
-1
) associated with freshly collected S. horneri were measured in the summer when
necrosis was already beginning to take place with the seaweed upon time of collection. The highest
BNF rates (~28 – 91 nmol N × g
-1
(dw) × h
-1
) with living S. horneri was associated with juveniles
in the late summer (Figure 1).
Figure 1: NF rates associated with juvenile S. horneri recruits on two separate trials. (Error
bars represent SE)
24
Relative growth rates expressed in terms of percent increases in blade weight or fresh
weight per day, ranging from 4 – 5.2 % (Choi et al., 2008; Gao and Hua, 1997), were used to
estimate increases in biomass of whole juveniles collected in this study as g (dw) per day. These
growth rates were found to be in the same range as growth rates of different S. horneri juveniles
reported in other studies (Umezaki, 1984; Yoshida et al., 2001), ranging from 0.003 to 0.01 g (dw)
per day (Table S5). In concert with the estimated and previously reported growth rates of S. horneri
juveniles from approximately the same time, the average %N (~1.01 ± 0.04%, 1.07 ± 0.07%) of S.
horneri juveniles collected in July and August, respectively, were used to calculate g of N required
to sustain growth of S. horneri juveniles. As both trials in July and August yielded higher BNF
rates in the light than the dark, the average dark and light BNF rate (~47 and 61 nmol N × g
-1
(dw)
× h
-1
, respectively) was assumed to represent the overall diazotrophic activity in a full day. This
was used to calculate potential contributions of fixed N from diazotrophic activity to meet N
demands for S. horneri juveniles during this period. The contribution of fixed N from diazotrophic
activity ranged from 3 – 36% with a mean contribution of ~9.6%. The results from this analysis
can be found in the Supplementary Material (Table S6).
3.2 Trends in diazotrophic activity throughout macroalgal decay
Two separate laboratory decomposition experiments carried out in subsequent summers
showed recurring shifts in diazotrophic activity associated with the degradation of the invasive
macroalga, S. horneri (Figures 2A, B). Initially, when seaweed samples were relatively fresh and
only beginning to senesce, lower BNF rates were observed (~12 – 24 nmol N × g
-1
(dw) × h
-1
).
Following just a few days (~3 – 5 days) of decomposition during which there was ~20% loss in
biomass (Figure S1), there was a distinct increase (up to 10x) in diazotrophic activity with BNF
rates reaching up to 247 nmol N × g
-1
(dw) × h
-1
(Figures 2A, B). This increase in nitrogenase
25
activity was sustained for a number of days (until day ~11 – 12) by which time there was an
additional ~40% loss in biomass (Figure S1). This trend in rapid biomass loss continued from day
12 to 14 during which time there was a further ~9% loss in biomass (Figure S1). The ensuing
plateau in macroalgal degradation from days 14 to 20, with only ~1% loss in biomass, coincided
with a substantial decrease in diazotrophic activity (~day 15), resulting in BNF rates comparable
to the initial rates or in some cases, lower than the initial rates (Figures 2A, B; S1). These same
trends in diazotrophic activity were also observed during decomposition of the endemic Sargassum
species, S. palmeri (Figure 1C). Lastly, while several instances of S. horneri dark assays yielded
higher BNF rates, dark assays of S. palmeri did not result in higher nitrogenase activity than light
assays (Figure 1). All calculated BNF rates for control treatments are reported in the
Supplementary Material (Tables S3, S4).
Figure 2: NF rates associated with decomposing S. horneri (a&b) and S. palmeri (c) under dark
and light incubations during two summers (2016 and 2017). (Error bars represent SE) * p <
0.05
26
3.3 Impact of carbon additions on BNF rates
For S. horneri, we observed that additions of 100 µM glucose and mannitol on day 0
stimulated nitrogenase activity in the dark assays by a factor of ~3 and 2.5, respectively, but no
stimulation of BNF rates were observed in the light assays (Figures 3A, B). In contrast, there were
low to non-detectable BNF rates associated with S. palmeri with the same carbon amendments
(Figures 3C, D). Higher concentrations of glucose (ranging from ~56 µM – 56 mM) have been
used in prior investigations looking at DMAs (Head and Carpenter, 1975) and thus, in order to
ensure an increase in measurable nitrogenase activity, the amendment concentration for both
glucose and mannitol were increased to 1 mM for all subsequent days which yielded mixed results.
In both light and dark assays for S. horneri, adding 10x more glucose and mannitol failed
to further stimulate the diazotrophic community associated with it until day 21. On day 21, glucose
and mannitol amendments in the dark and light assays stimulated BNF rates by 5.3 – 15, but even
Figure 3: Differences in NF rates associated with decomposing S. horneri and S. palmeri
between control conditions, glucose (100µM/1mM) and mannitol (100µM/1mM) additions
under dark and light incubations. (Error bars represent SE) * p < 0.05, ** p < 0.01, *** p <
0.001
27
these elevated rates were either comparable to or lower than the control rates observed earlier in
the decomposition period (Figure 3A). For S. palmeri, higher concentrations of glucose and
mannitol stimulated BNF rates (~3.7 and 3-fold, respectively) on day 5 in dark assays but failed
to further stimulate nitrogenase activity in the light assays (Figures 3C, D). However, for the
remainder of the decomposition period of S. palmeri, carbon amendments greatly stimulated BNF
rates (with up to ~42-fold stimulation) in both light and dark assays (Figure 3C, D). A complete
list of BNF rates for various amendments with both species of macroalgae are available in the
Supplementary Material (Table S3).
3.4 Impact of sodium molybdate amendments on nitrogenase activity
It is evident in the dark assays that the addition of 20 mM sodium molybdate routinely
suppressed nitrogenase activity throughout most of the decomposition period and with increasing
intensity, reaching the highest reduction (~98%) in BNF rates on day 12 (Figure 4A). The same
amendments produced mixed results in the light assays but apart from day 0 where there was
Figure 4: Differences in NF rates associated with decomposing S. horneri between control
conditions and sodium molybdate (20mM) additions under dark (left) and light (right)
incubations. (Error bars represent SE) * p < 0.05, ** p < 0.01
28
stimulation of nitrogenase activity (Figure 4A), it either severely inhibited (maximum reduction
of ~95% on day 12) or did not significantly impact nitrogenase activity (Figure 4B). The complete
list of BNF rates are available in the Supplementary Material (Table S4).
3.5 Changes in C and N content (%), C:N ratio and δ
15
N signature of aging S. horneri detritus
There is an overall decrease in C, by 2.73%, with fresh S. horneri detritus subsampled over
the first 12 days of decomposition (Figure 5A). In contrast, N increases by 40.8% with FD during
this time (Figure 5B). Between day 12 and 15, FD exhibited a sudden increase in C by 20% and N
continued to increase by 14% (Figures 5A, B). The resulting C:N ratio with FD decreased by 31%
Figure 5: Changes in %C (a), %N (b) and C:N ratio (c) associated with decomposing S. horneri
in control (blue) and molybdate amendments (green) in both dark and light incubations as well
as pre-incubation samples (red dashed line) (2017). Additional changes in δ
15
N (d) with fresh
detritus throughout decomposition. (Error bars represent SE) * p < 0.05, ** p < 0.01, *** p <
0.001
29
over the first 12 days of decomposition and despite increasing from day 12 to 15 by 5.2%, the final
C:N ratio exhibited an overall decrease of 27.3% (Figure 5C). The δ
15
N of FD continuously
decreased from 12.2‰ to 9.3‰ over the course of the decomposition, resulting in an overall
decrease of 24.1% by day 15 (Figure 5D).
There is also an overall decrease in C, between 6.1 to 16.4%, with PID recovered at the
end of ~48-hour dark and light incubations under control and sodium molybdate treatments over
the first 12 days of decomposition (Figure 5A). Similar to the increase observed with day 15 FD,
PID recovered at the end of this incubation also exhibited an increase in C, between 3.5 to 10%,
amongst all treatments (Figure 5A). All PID exhibited increases in N, between 52.2 to 74.7%, over
the first 8 days of decomposition (Figure 5B). Subsequently, there was an overall decrease in N,
between 8.4 to 29.4%, with all PID over the remainder of the decomposition experiment (Figure
5B). Despite this decrease at the end, all PID were still relatively enriched, by 18.5 to 39.4%, in N
compared to PID from the onset of decomposition (Figure 5B). The resulting C:N ratio for all PID
exhibit an inverse pattern to the %N trends, expressing an overall decline over the first 8 days of
decomposition followed by an increase until the end of the decomposition experiment (Figure 5C).
Despite this increase at the end, there is an overall decrease with the C:N ratio of PID at the end
of decomposition relative to the initial (day 2) PID (Figure 5C). The PID recovered from sodium
molybdate treatments resulted in higher C:N ratio compared to the control treatments throughout
the duration of the experiment (Figure 5C).
Control and sodium molybdate incubations for ~48 hours in the dark and light repeatedly
resulted in PID that was more depleted, between 3.9 to 32.1%, in C relative to the FD (Figure 5A).
For the most part, sodium molybdate incubations in the dark and light also resulted in PID that
were more depleted, between 5.1 to 17.4%, in C compared to control PID from parallel incubations
30
(Figure 5A). While control PID in the light repeatedly resulted in higher %C than the dark
incubations, the opposite trend was observed with sodium molybdate treatments which
consistently resulted in slightly higher %C with substrate recovered from dark incubations (Figure
5A).
Control incubations in the dark and light resulted in PID that were depleted, between 4.8
to 29.7%, in N compared to FD at the beginning (day 0) and end (day 15) of decomposition (Figure
5B). However, PID retrieved at the end of day 3 and day 8 from dark and light control incubations
exhibited higher N, between 4.6 to 32.4%, relative to the FD collected on those corresponding
days (Figure 5B). While PID recovered at the end of day 12 light incubations resulted in slightly
higher N, by 1.26%, PID from dark incubations were 17.1% more depleted in N compared to the
FD collected initially on day 12 (Figure 5B). Apart from day 8 when PID in the light were more
enriched in N, by 10.1%, PID recovered at the end of both light and dark sodium molybdate
incubations were more depleted in N, between 2.4 to 37.6%, compared to corresponding FD
subsampled on days 0, 3, 8, 12 and 15 (Figure 5B). For both dark and light incubations, PID from
sodium molybdate treatments always resulted in lower N, between 9.8 to 35.7%, compared to
control PID from parallel incubations (Figure 5B). The %C, %N, C:N ratio, and δ
15
N values for
the aforementioned samples can be found in the Supplementary Material (Table S7).
From all treatments throughout the decomposition experiment, there were several
instances, on days 3 and 8, when PID exhibited higher mean %N relative to FD collected at the
beginning of the incubation (Figure 5B). Assuming a dry weight of 1 g of S. horneri detritus, the
theoretical mass of N (mg) required to result in the observed increase in %N at the end of the
incubation was calculated. The average BNF rates associated with the corresponding treatments,
expressed as nmol N × g
-1
(dw) × h
-1
, were transformed into nmol of N fixed by multiplying the
31
BNF rates by the assumed dry weight of the S. horneri detritus (1g) and length of the incubation
(~48 hours) and subsequently converted to mg of N. Lastly, the percent contribution of BNF to N
immobilization was calculated by dividing the theoretical mass of N (mg) required by the
theoretical mass of N (mg) fixed during the incubation. The percent contribution to N
immobilization by BNF ranged from 0.94 to 10.6% with an average contribution of 4.5%. The
complete results can be found in the Supplementary Material (Table S8).
Discussion
4.1 Role of BNF in N acquisition by living S. horneri
Throughout much of the life cycle of S. horneri, nitrogenase activity was not detectable or
exhibited relatively low BNF rates that yielded insignificant amounts of fixed N to support
macroalgal productivity. However, on two separate occasions, active BNF was observed with
living S. horneri juveniles (5-8 cm long) collected during late July and early August of 2017
(Figure 1). Ambient nutrient concentrations are usually at their minimum during late summer in
the Southern California Bight and one study conducted at Santa Catalina Island (same site as this
study) found the low nutrient concentrations in the surrounding waters during this time to be
insufficient to support maximal growth of the endemic macroalgae, M. pyrifera (Zimmerman and
Kremer, 1984). Similarly, it may also be possible that the low nutrient conditions are not ideal to
support growth of S. horneri juveniles. Thus, appreciable amounts of fixed N, ~3 – 36% of required
N with an average contribution of ~9.6%, derived from diazotrophic activity during this time might
potentially supplement their N requirement and thereby provide a competitive edge. These values
are comparable to previous estimates of BNF contributions to sustain macroalgal productivity of
various species such as C. decorticatum (~5%; Rosenberg and Paerl 1981), Laurencia sp. (14 –
18%; Capone 1977), and pelagic Sargassum community (~40%; Hanson 1977). Subsequent
32
sampling efforts of juveniles in mid-September, October and late-November resulted in
undetectable rates of BNF. Substantial increases in nutrient concentrations during the fall and
winter, with maximum nitrate concentrations observed during this time (Zimmerman and Kremer,
1984) may explain the absence of BNF activity associated with S. horneri and potential shift back
to nutrient uptake from the surrounding water column to sustain its growth and productivity.
4.2 Significance of BNF activity associated with decomposing Sargassum detritus
It is well established that various marine macrophytes such as salt marsh grasses (Valiela
et al., 1985), mangrove leaves (Cundell et al., 1979) and sea grasses (Godshalk and Wetzel, 1978)
undergo an initial phase of rapid biomass loss due to autolysis and leaching of soluble compounds.
This is followed by a longer phase of biomass loss driven by microbial degradation of organic
matter before entering the slowest phase of decay due to the refractory nature of the remaining
detritus. Similarly, numerous studies have also observed macroalgal decay to follow this three-
phase decomposition model (Hunter, 1976; Rieper-Kirchner, 1989; Robinson et al., 1982; Smith
and Foreman, 1984; Williams, 1984). Furthermore, the trend of biomass loss observed in this study
(Figure S1) also adheres to this three-phase decomposition model.
Observations of microbial densities throughout these three phases have generally found
there to be relatively low microbial biomass during the initial phase of decomposition. This is
followed by a rapid increase in microbial colonization, indicative of a transition to microbial
degradation of macroalgal detritus, with maximal microbial biomass being sustained for several
days before microbial populations decrease, marking the final shift towards a more recalcitrant
macroalgal detritus (Rice and Hanson, 1984; Rieper-Kirchner, 1989; Robinson et al., 1982; Sathe-
Pathak et al., 1993). Recurring shifts in nitrogenase activity during decomposition of S. horneri
and S. palmeri resemble this characteristic shift in microbial biomass, though with a much steeper
33
decrease in BNF rates towards the end of the decomposition period (Figure 2). If the diazotrophic
population is assumed to be changing in a similar fashion to the whole microbial community, the
lower BNF activity observed at the beginning and end of decomposition could be attributed to
potentially lower diazotrophic biomass present during the initial and terminal phases of
decomposition. The BNF hot spot observed in the middle of decomposition may be a result of
greater diazotrophic recruitment concomitant with increasing microbial populations that generally
transpires during the second phase of macroalgal decomposition.
As most macroalgae have a relatively high C:N ratio (>20:1 as observed with S. horneri
samples in this study), increased microbial colonization during macroalgal degradation and rapid
utilization of resources would lead to depletion in N before C. This may result in N limitation of
the organic rich macroalgal substrate, thereby creating a niche for N fixing organisms. Several
studies have shown additions of N to support greater microbial populations associated with
macroalgal detritus. Consequently, increased microbial biomass were observed to utilize more of
the available detrital carbon, expediting macroalgal degradation and highlighting the potential for
N limitation to retard microbial degradation and impact the overall rate of macroalgal
decomposition (Rice and Hanson, 1984; Robinson et al., 1982; Tenore et al., 1979). Additionally,
it is also well established that N enrichment observed with aging macroalgal detritus does not
necessarily indicate an increasing pool of labile N (e.g. proteins, amino acids) but rather, it might
be an accumulation of recalcitrant N which also results in N limitation (Rice, 1982). BNF may
provide new, bioavailable N for the associated microbial community, possibly alleviating N
limitation and stimulating microbial degradation of macroalgae.
Interestingly, BNF rates associated with freshly collected S. horneri, which has been noted
to begin senescing in its non-native eastern Pacific habitat during the spring (Cruz-Trejo et al.,
34
2015; Miller et al., 2007), expressed a seasonal increase towards the beginning of summer when it
was physically observed to be senescent. Similarly senescing S. horneri in the western Pacific
(Yoshida et al., 1998, 2001) might also potentially support diazotrophic activity as they form
drifting rafts during spring to early summer (Komatsu et al., 2007, 2008; Mizuno et al., 2014;
Yatsuya, 2008). Additionally, despite having a relatively longer floating period due to its lower
density (Yatsuya, 2008), S. horneri debris have also been found on the offshore deep sea floor
during the summer (Kokubu et al., 2012). Therefore, it may also be possible that sinking S. horneri
debris may further serve as a niche for diazotrophs in the deep sea benthos.
4.3 Presence of heterotrophic N fixers
In different marine ecosystems such as the anoxic waters (200m) of the Baltic Sea (Farnelid
et al., 2013), aphotic zone (500 and 885 m) in the San Pedro Basin (Hamersley et al., 2011), and a
wide assortment of benthic systems (Howarth et al., 1988), dark incubations have been utilized to
estimate rates of heterotrophic BNF. While dark BNF rates could also include contributions from
chemoautotrophic and methanotrophic diazotrophs, we are assuming BNF in the absence of light
to be primarily a result of heterotrophic BNF. In this study, dark vials were purged with N2 gas in
the 2016 S. horneri and S. palmeri decomposition experiment to promote an anaerobic
environment and to protect the nitrogenase enzyme from oxygen toxicity and ensure measurable
rates of BNF. While it could be argued that purging dark vials with N2 gas and leaving the light
vials unperturbed might have resulted in elevated dark BNF rates in the first experiment, the 2017
S. horneri decomposition experiment, which utilized unpurged dark and light vials, also
consistently exhibited higher rates of BNF associated with aging S. horneri detritus in the dark
than in the light (Figures 2A, B). The second experiment, despite using unpurged vials, also
expressed higher overall dark BNF rates compared to the 2016 experiment. Furthermore, similarly
35
prepared dark, anaerobic assays routinely yielded lower BNF rates throughout the decomposition
of S. palmeri (Figure 2C). These findings suggest that laboratory perturbations of dark vials in the
first S. horneri experiment most likely did not provide any significant advantage to specific
physiological groups of N fixers in the dark (Figure 2).
O2 depth profiles in an uncapped, illuminated serum vial containing senescent S. horneri
samples in 0.2 µm filtered seawater showed that O2 concentrations drop well below hypoxic levels,
< 63 µM as defined for coastal ecosystems (Middelburg and Levin, 2009), when approaching the
thicket of S. horneri but remain constant when only exposed to seawater (Figures S2A, B).
Furthermore, depth profiles measured using an O2 microelectrode reveal that O2 concentrations
can drop close to 0 µM when in close proximity to senescent S. horneri particles (Figures S2C,
D). As observed with colonies of Trichodesmium (Paerl et al., 1989), localized areas of O2
consumption probably lead to formation of microaerobic niches with aging macroalgal detritus
and provide protection from oxygen toxicity to diazotrophs. Additionally, heterotrophic respiration
and inhibition of overall photosynthetic activity in the dark assays could have also prevented
accumulation of high concentrations of O2 in the vials and further alleviated the nitrogenase
enzyme from oxygen toxicity, resulting in higher rates of BNF in dark assays throughout the
decomposition of S. horneri. As the exact nature of the diazotrophic population is currently
unknown, N fixers in these detrital systems are most likely employing an array of methods to
protect the nitrogenase enzyme from oxygen toxicity (Fay, 1992).
Similar to S. palmeri, dark BNF rates associated with living S. horneri juveniles were also
consistently lower than in the light, suggesting a lesser contribution from heterotrophic N fixers in
these systems (Figures 1, 2C). One possible explanation for higher dark BNF rates observed with
decomposing S. horneri detritus compared to aging S. palmeri detritus might be differences in the
36
diversity, abundance, and activity of the heterotrophic diazotrophs associated with the different
macroalgae. Similarly, the difference in dark BNF rates associated with S. horneri at different life
stages, i.e. juvenile vs. decomposing, leads us to hypothesize that there are most likely changes in
the diazotrophic community composition associated with the host macroalga throughout its life
cycle. This has been suggested with changes in the general microbial community associated with
different macroalgae (Goecke et al., 2010) and future molecular work specifically targeting the
nifH gene would better elucidate this hypothesis.
In many marine systems such as the oligotrophic open ocean and with previously reported
DMAs (e.g. pelagic Sargassum species), phototrophic diazotrophs (e.g. Trichodesmium,
Crocosphera, Calothrix, Dichothrix fucicola) are often regarded as the most important contributors
to BNF in the upper ocean (Capone et al., 1997; Carpenter, 1972; Montoya et al., 2004; Phlips et
al., 1986). However, it seems heterotrophic diazotrophs may have an important association with
decomposing Sargassum and Macrocystis (Hamersley et al., 2015), but with varying degrees of
contribution to BNF. The possibility for DMAs to occur within numerous macroalgal detrital
systems, as discussed previously, may provide an unexplored niche for heterotrophic N fixers,
requiring additional investigation.
4.4 Impact of labile carbon on nitrogenase activity
The concentrations of various organic molecules (e.g. alginates, phenols, total
carbohydrates, proteins, reducing sugars, etc.) generally decline throughout macrophyte
decomposition, resembling trends in loss of biomass throughout the three stages of decomposition
(Buchsbaum et al., 1991; Cundell et al., 1979; Sathe-Pathak et al., 1993). Generally, rapid build-
up in microbial biomass, observed after leaching of organic compounds during the first phase of
decomposition, begins to decline as the detritus enters a more refractory phase, suggesting that the
37
utilization of labile carbon is an important controlling factor for sustaining microbial populations
during macrophyte decomposition (Cundell et al., 1979; Sathe-Pathak et al., 1993). Considering
the high energetic cost of BNF and assuming the diazotrophic population is following similar
changes in biomass to the whole microbial community, it is likely that the availability of various
labile organic compounds may also have influenced nitrogenase activity associated with aging S.
horneri and S. palmeri detritus.
During the first phase of S. horneri decomposition (~days 0 – 5), the availability of labile
carbon may have been limiting BNF in the dark, and thus, even relatively lower (100 µM)
concentrations of glucose and mannitol were able to stimulate BNF on day 0 (Figure 3A).
However, 10x higher (1 mM) glucose and mannitol additions did not significantly stimulate BNF
throughout the second phase of decomposition (~days 5 – 14), suggesting that availability of labile
organic compounds during this phase were sufficient to support diazotrophic activity (Figures 3A,
B). Interestingly, these same amendments in both the light and dark were able to stimulate
diazotrophic activity, by ~5 – 15 fold, associated with the more recalcitrant S. horneri detritus
remaining on day 21 (Figures 3A, B). While this suggests that labile carbon may also be limiting
BNF at the end of decomposition, these elevated BNF rates stimulated by carbon additions were
relatively lower than control BNF rates observed throughout the second phase of decomposition
(i.e. days 5, 11). This indicates a potential decrease in diazotrophic population and activity by the
end of decomposition. In contrast, carbon additions routinely stimulated BNF rates, particularly in
the dark, throughout the different stages of S. palmeri decomposition (Figures 3C, D). This
suggests that the release of organic compounds throughout S. palmeri decomposition was not as
accessible to the diazotrophic community, resulting in labile carbon limitation for N fixers
associated with the aging S. palmeri detritus.
38
4.5 Sulfate reducing diazotrophs
While there have been several studies reporting sulfate reduction associated with sediments
in different macrophyte systems such as salt marshes (Hines et al., 1989), seagrasses (Blaabjerg
and Finster, 1998; Capone, 1982) and mangroves (Kristensen et al., 1994), there are not many
investigations looking directly at sulfate reduction associated with degrading macroalgae.
Interestingly, a few studies have reported increased sulfate reduction associated with the
decomposition of the green macroalga, Ulva lactuca (Lomstein et al., 2006; Nedergaard et al.,
2002). Nedergaard et al. (2002) even found sulfate reduction to be more prominent with
macroalgal-associated sulfate reducers than sediment associated and free living sulfate reducers
during the degradation of U. lactuca, suggesting that the presence of sulfate reducers in macroalgal
detrital systems may not be that uncommon. In this study, higher rates of BNF in the dark and
possible microaerobic niches associated with S. horneri detritus suggested a potential role for
sulfate reducers, which generally are thought to be active in anoxic zones (Canfield et al., 2010)
but have also been observed in ostensibly oxic environments (Canfield and Marais, 1991).
Fulweiler et al., 2015 have demonstrated that the active microbial community can change
by the end of an AR incubation and more specifically, the presence of C2H2, which is known to
inhibit methanogens (Oremland and Capone, 1988), can result in up or down regulation of
sulfur/sulfate reducers. However, despite potential changes induced by the presence C2H2, the use
of sodium molybdate additions, a known inhibitor of sulfate reducers (Oremland and Capone,
1988), in the 2017 S. horneri decomposition experiment yielded reproducible instances of
significantly reduced nitrogenase activity (~53 – 98% inhibition). This suggests that sulfate
reducers can contribute to BNF and at times, dominate the diazotrophic community in these detrital
systems. Surprisingly, this translated into noticeable changes with the C:N ratio associated with S.
39
horneri detritus, discussed below. Although sulfate reduction rates were not directly measured in
this study, increasing inhibition of nitrogenase activity (up to 98% by day 12) throughout
decomposition suggests that increasing sulfate reduction may also be associated with aging S.
horneri detritus as observed by Lomstein et al. (2006) and Nedergaard et al. (2002) with U. lactuca
decomposition. This suggests that sulfate reducers might be playing an important role in the
breakdown of the more recalcitrant macroalgal detritus left towards the latter phases of macroalgal
decomposition.
4.6 Imprint of BNF on S. horneri detritus
A common observation with aging macrophyte detritus is the decrease in C:N ratio
throughout decomposition which has been linked, with varying extent, to the associated
diazotrophic activity (Cundell et al., 1979; Kenworthy et al., 1987; Kenworthy and Thayer, 1984).
Additionally, there have also been several studies that directly link BNF observed in these marine
detrital systems to N immobilization of the aging detritus, highlighting the importance of
diazotrophs in marine detrital food chains (Pelegri and Twilley, 1998; Van der Valk and Attiwill,
1984; Woitchik et al., 1997). Not surprisingly, this trend of decreasing C:N ratio (observed in this
study with the 2017 S. horneri decomposition experiment, Figure 5C) has also been reported on
several accounts with aging macroalgal detritus but not linked directly to BNF and instead,
generally attributed to the buildup of microbial biomass (Banta et al., 2004; Duggins and Eckman,
1997; Rieper-Kirchner, 1989; Smith and Foreman, 1984). Alternatively, Rice (1982) argue that
increases in detrital N cannot be fully accounted for by microbial protein but rather, depend more
on the accumulation of non-labile N, which has also been suggested by others (Robinson et al.,
1982; Smith and Foreman, 1984). In this study, changes in the %C, %N, C:N ratio, and δ
15
N
40
alongside BNF rates from the second S. horneri decomposition experiment (Figures 5A-D, 4A, B)
highlight the potential influence that diazotrophs may have on aging macroalgal detritus.
For instance, the largest decrease in C:N ratio, taking place over the first few days of
decomposition, is accompanied by relatively little change in %C and is most likely driven by the
steep increase in %N (Figures 5A-C). The BNF rates associated with dark and light control
incubations on day 3 can account for ~5 and 11%, respectively, of the observed N immobilization.
On the contrary, parallel dark and light sodium molybdate incubations, where 66 and 88% of the
nitrogenase activity was inhibited (Figures 4A, B), resulted in PID that were ~24 and 28% more
depleted in N, respectively, than the control treatments (Figures 5B, C). The sharpest decrease in
δ
15
N of FD from 12.2‰ to 10.7‰ also occurred during this period (Figure 5D), further supporting
that BNF may be playing a direct role in influencing the changes associated with S. horneri detrital
N. On day 8, BNF can account for ~2 and 1% of the observed increase in N with PID from dark
and light control incubations. Interestingly, sodium molybdate amendments did not inhibit
nitrogenase activity in the light (Figure 4B) and the corresponding BNF accounted for 4% of the
N immobilization. These relatively low contributions suggest that BNF was not playing a
significant role in N immobilization during this period.
Instead, potential increases in microbial biomass, characteristic of the second phase of
decomposition, or retention of refractory N in aging S. horneri detritus might account for much of
the N immobilization. Nonetheless, the lack of nitrogenase inhibition by sodium molybdate on day
8 resulted in a ~11% more N enriched PID in the light than when BNF was strongly inhibited,
69%, in the dark (Figure 5B). Similarly, strong inhibition of nitrogenase activity on day 12, 95-
98%, by sodium molybdate treatments (Figures 4A, B) resulted in PID that was ~11-33% more N
depleted (Figure 5B). This, alongside a smaller difference in C content between light and dark
41
molybdate incubations, results in a significantly higher C:N ratio for PID when BNF is inhibited,
suggesting that BNF could still indirectly be influencing the nutritional quality of the macroalgal
detritus. While a few studies have suggested BNF as a potential source of N enrichment
(Hamersley et al., 2015; Hill and McQuaid, 2009), our results provide direct evidence for how
BNF, or therein lack of, can influence the nutritional quality of the aging S. horneri detritus.
Conclusion
Investigating the role of diazotrophs with S. horneri throughout its life cycle has provided
insight into the relevance of BNF with regard to the success of this globally relevant macroalga in
a non-native ecosystem. Our findings suggest, at least during periods of N deficiency, that BNF
can potentially provide significant amounts of fixed N to macroalgae and heterotrophic BNF
associated with living macroalgae may be more significant than what is currently thought. Higher
BNF rates observed with decomposing Sargassum in this study, which has also been reported in
the past with pelagic Sargassum (Hanson, 1977; Phlips et al., 1986), suggests that DMAs with
decomposing Sargassum species may be a global occurrence. Moreover, several studies reporting
higher BNF activity associated with other senescent brown (Hamersley et al., 2015) and green
(Zuberer and Silver, 1978) macroalgae further suggest that DMAs with aging macroalgal detritus
may not be a rare phenomenon. Macroalgae such as S. horneri may provide a niche for BNF and
these DMAs may ultimately allow for a more efficient decomposition of a large reservoir of
organic material. Currently, the macroalgal detrital food web remains a vastly unexplored niche
for N fixers. Our findings suggest that heterotrophic N fixers and sulfate reducing diazotrophs may
be important members of macroalgal detrital diazotrophic communities. As most macroalgal
production ultimately enters the detrital food web, future studies investigating DMAs with
decomposing macroalgae are necessary to elucidate the role of BNF in these detrital systems.
42
Acknowledgements
We would like to thank the USC Wrigley Marine Science Center (WMSC) and summer
Wrigley/Victoria J. Bertics Fellowships (2016-17) for supporting this research and the WMSC
staff, especially Lauren Czarnecki Oudin and Kellie Spafford, for all their accommodations. We
want to thank the participants from past Research Experience for Undergraduates programs (NSF-
1263356), Amanda DeLiberto, Shannon Matzke, Camille Vieira, and Taylor Dillon, for all their
contribution. We also want to thank all members of the Capone lab, especially Weimin Deng,
Miguel Rincon, Suryia Tanjasiri, Chrystal Li, Jill Sohm, and Troy Gunderson, for their help. We
thank the Ziebis lab, especially Bingran Cheng, for help with O2 measurements.
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49
Supplementary Table 1: Details of sample collection.
Date Location Latitude Longitude Species Life Stage
02/02/2016 Isthmus Reef 33.449 °N 118.489 °W S. horneri Mature Adult
03/25/2016 Isthmus Reef 33.449 °N 118.489 °W S. horneri Mature Adult
07/08/2016 Rippers Cove 33.428 °N 118.433 °W S. horneri Senescent Adult
07/08/2016 Rippers Cove 33.428 °N 118.433 °W S. palmeri Senescent Adult
06/18/2017 Isthmus Reef 33.449 °N 118.489 °W S. horneri Senescent Adult
07/27/2017 Isthmus Reef 33.449 °N 118.489 °W S. horneri Juvenile (5 cm)
08/02/2017 Isthmus Reef 33.449 °N 118.489 °W S. horneri Juvenile (8 cm)
09/13/2017 Isthmus Reef 33.449 °N 118.489 °W S. horneri Juvenile (5-11 cm)
09/13/2017 Isthmus Reef 33.449 °N 118.489 °W S. horneri Immature
10/18/2017 Isthmus Reef 33.449 °N 118.489 °W S. horneri Juvenile (8-17 cm)
10/18/2017 Isthmus Reef 33.449 °N 118.489 °W S. horneri Immature
11/30/2017 Isthmus Reef 33.449 °N 118.489 °W S. horneri Juvenile (5-8 cm)
11/30/2017 Isthmus Reef 33.449 °N 118.489 °W S. horneri Immature
12/8/2017 Isthmus Reef 33.449 °N 118.489 °W S. horneri Juvenile (6 cm)
Supplementary Table 2: NF rates of control incubations (~48 hours) under dark (D) and light (L)
treatments from different collections of S. horneri at various life stages. Rates are expressed as
nmol N × g
-1
(dw) × h
-1
± SE. Not Detectable (ND).
Day Season Life Stage Light
Treatment
Control
09/13/2017 Fall Juvenile (5-11 cm) D & L ND
09/13/2017 Fall Immature D & L ND
10/18/2017 Fall Juvenile (8-17 cm) D & L ND
10/18/2017 Fall Immature D & L ND
11/30/2017 Fall Juvenile (5-8 cm) D & L ND
11/30/2017 Fall Immature D & L ND
12/8/2017 Winter Juvenile (6 cm) D & L ND
02/02/2016 Winter Mature Adult D 2.01 ± 0.99
02/02/2016 Winter Mature Adult L 0.73 ± 0.43
03/25/2016 Spring Mature Adult D & L ND
06/18/2017 Summer Senescent Adult D 11.5 ± 8.27
06/18/2017 Summer Senescent Adult L 11.7 ± 8.64
07/08/2016 Summer Senescent Adult D 22.9 ± 10.2
07/08/2016 Summer Senescent Adult L 23.7 ± 5.04
07/27/2017 Summer Juvenile (5 cm) D
27.8 ± 4.12
07/27/2017 Summer Juvenile (5 cm) L
66.2 ± 11.7
08/02/2017 Summer Juvenile (8 cm) D
30.9 ± 3.28
08/02/2017 Summer Juvenile (8 cm) L
91.4 ± 21.1
50
Supplementary Table 3: NF rates with glucose and mannitol amendments and their impact on NF
stimulation (Gx, Mx) under dark/light treatments for 2016 S. horneri & S. palmeri
decomposition experiments. Rates are expressed as nmol N × g
-1
(dw) × h
-1
± SE. No Stimulation
(NS), Not Detectable (ND) and Not Applicable (NA). Due to insufficient biomass, G/M
amendments were not carried out on day 28 with S. horneri & S. palmeri.
Day Species Light Control Glucose (G) Mannitol (M) Gx Mx
0 S. horneri Dark
22.9 ± 10.2 66 ± 8.59 57 ± 8.3
3x 2.5x
5 S. horneri Dark
77.8 ± 8.69 68.2 ± 3.19 41 ± 7.81
NS NS
11 S. horneri Dark
167 ± 20 113 ± 12.2 86.6 ± 13.8
NS NS
21 S. horneri Dark
1.26 ± 0.03 6.62 ± 2.77 6.89 ± 1.91
5.3x 5.5x
28 S. horneri Dark
ND NA NA
NA NA
0 S. horneri Light
23.7 ± 5.04 19 ± 5.81 14.8 ± 11.4
NS NS
5 S. horneri Light
38.3 ± 8.24 68.2 ± 3.6 29.8 ± 0.09
1.8x NS
11 S. horneri Light
60.9 ± 5.94 98.5 ± 19.2 98.8 ± 8.89
1.6x 2x
21 S. horneri Light
2.08 ± 0.57 12.8 ± 4.11 31.7 ± 16.6
6.2x 15x
28 S. horneri Light
ND NA NA
NA NA
0 S. palmeri Dark
ND ND ND
NA NA
5 S. palmeri Dark
17.2 ± 7.79 64 ± 10.9 50.8 ± 14
3.7x 3x
11 S. palmeri Dark
35 ± 16.4 126 ± 20.8 107 ± 36
3.6x 3.1x
21 S. palmeri Dark
3.69 ± 0.69 154 ± 39.8 139 ± 12.2
42x 38x
28 S. palmeri Dark
2.3 ± 1.07 NA NA
NA NA
0 S. palmeri Light
1.31 ± 1.07 1.77 ± 0.44 2.69 ± 2.12
1.4x 2.1x
5 S. palmeri Light
44.9 ± 13.2 57.8 ± 12.7 47.9 ± 6.32
1.3x NS
11 S. palmeri Light
65.3 ± 16.9 124 ± 55.6 109 ± 37.9
1.9x 1.6x
21 S. palmeri Light
6.52 ± 0.98 237 ± 19.7 161 ± 2.12
36x 25x
28 S. palmeri Light
8.32 ± 3.94 NA NA
NA NA
Supplementary Table 4: NF rates for sodium molybdate additions and its inhibition on
nitrogenase activity under dark/light treatment during the 2017 S. horneri decomposition
experiment. Rates are expressed as nmol N × g
-1
(dw) × h
-1
± SE.
Day
Light
Treatment Control
Molybdate
% Inhibition
of NF Rates
0 Dark 11.5 ± 8.27 5.39 ± 3.46 53
3 Dark 247 ± 27.2 83.8 ± 10.6 66
8 Dark 154 ± 9.78 47.6 ± 1.38 69
12 Dark 167 ± 34.2 3.44 ± 1.26 98
15 Dark 12.2 ± 4.91 17.1 ± 2.67 No Inhibition
0 Light 11.7 ± 8.64 89.7 ± 48.1 No Inhibition
3 Light 141 ± 59 17.5 ± 8.01 88
8 Light 80.4 ± 13.8 106 ± 10.4 No Inhibition
12 Light 171 ± 7.48 8.73 ± 1.83 95
15 Light 13.3 ± 4.67 13.6 ± 1.87 No Inhibition
51
Supplementary Table 5: Compilation of reported growth rates as g (dw) × day
-1
(Umezaki 1984
1
,
Yoshida, Yoshikawa, and Terawaki 2001
2a, b
)
and estimations of growth rates as g (dw) × day
-1
for samples collected for this study on 07/27/2017 * and 08/02/2017 ** using previously
reported relative growth rate (RGR) expressed as % increase in fresh weight per day (Gao and
Hua 1997
3
) or % increase in blade weights per day (Choi et al. 2008
4a, b
).
Daily biomass increase (DBI): whole sample dry weight (0.384 g *, 0.566 g **) × RGR (%) = g (dw) × day
-1
Duration
Location DBI
g (dw) × day
-1
RGR (%)
1
08/03/1981-
10/31/1981
Obama Bay, Japan 0.003 NA
2a
08/10/1995-
09/07/1995
Hiroshima Bay, Japan 0.01 NA
2b
08/06/1996-
09/09/1996
Hiroshima Bay, Japan 0.01 NA
3
08/17/1987-
08/25/1987
Maizuru Bay, Japan 0.0200 *
0.0294 **
5.2
4a
Laboratory Jeonchonri, Wolsung,
Kyungbuk,
Korea
0.0154 *
0.0226**
4
4b
Laboratory Jeonchonri, Wolsung,
Kyungbuk,
Korea
0.0192 *
0.0283 **
5
52
Supplementary Table 6: Contributions of fixed N from diazotrophic activity to meet N demands
for juvenile S. horneri collected on 07/27/2017 * and 08/02/2017 **.
N Requirement: average %N of S. horneri (1.014 ± 0.0381 % *, 1.066 ± 0.0691 % **) × growth rates listed in Supp.
Table 3 as g (dw) × day
-1
= g N × day
-1
NF Production
A
: Average light and dark NF rates (nmol N × g
-1
(dw) × h
-1
) calculated using a 3 C2H2:1 N2
ratio
(47.0 *, 61.1
**) × whole sample dry weight (0.384 g *, 0.566 g **) × molecular weight of N (g/nmol) × 24 hours =
g of N fixed per day
NF Production
B
: Same calculation as NF Production
A
except using NF rates using a 4 C2H2: 1 N2 ratio yielding
(nmol N × g
-1
(dw) × h
-1
) of 35.3 * and 45.8 **.
% N Supplied by NF
A, B
: (NF Production
A, B
/N Requirement) × 100 = % contribution of N by NF.
Date of
Collection
N
Requirement
(g N × day
-1
)
NF Production
(g N × day
-1
)
A
NF Production
(g N × day
-1
)
B
% N
Supplied
by NF
A
% N
Supplied
by NF
B
07/27/2017 3.041 E-05
1
6.072 E-06 4.55 E-06 20.0 15.0
07/27/2017 1.014 E-04
2
6.072 E-06 4.55 E-06 5.99 4.49
07/27/2017 2.025 E-04
3
6.072 E-06 4.55 E-06 3.00 2.25
07/27/2017 1.56 E-04
4a
6.072 E-06 4.55 E-06 3.90 2.92
07/27/2017 1.95 E-04
4b
6.072 E-06 4.55 E-06 3.12 2.34
08/02/2017 3.20 E-05
1
1.16 E-05 8.72 E-06 36.3 27.3
08/02/2017 1.066 E-04
2
1.16 E-05 8.72 E-06 10.9 8.17
08/02/2017 3.14 E-04
3
1.16 E-05 8.72 E-06 3.70 2.78
08/02/2017 2.41 E-04
4a
1.16 E-05 8.72 E-06 4.82 3.61
08/02/2017 3.017 E-04
4b
1.16 E-05 8.72 E-06 3.85 2.89
Average 1.68 E-04 8.85 E-06 6.64 E-06 9.56 7.17
53
Supplementary Table 7: %C, %N, C:N ratio and δ
15
N values for samples (FD) before the start of
dark (D) and light (L) incubations for ~48 hours under control (Con) and sodium molybdate
(Mol) treatments and samples at the end of the incubation (PID) throughout the 2017 S. horneri
decomposition experiment.
Day Sample Light Treatment %C %N C:N Ratio δ
15
N
0 FD NA NA 29.0 ± 0.1 1.45 ± 0.02 19.9 ± 0.25 12.2 ± 0.87
3 FD NA NA 28.5 ± 0.06 1.72 ± 0.03 16.6 ± 0.32 10.7 ± 0.11
8 FD NA NA 28.5 ± 0.2 1.75 ± 0.03 16.3 ± 0.34 10.6 ± 0.79
12 FD NA NA 28.2 ± 0.13 2.05 ± 0.01 13.8 ± 0.05 9.83 ± 0.19
15 FD NA NA 33.8 ± 0.16 2.33 ± 0.03 14.5 ± 0.15 9.25 ± 0.23
2 PID D Con 25.8 ± 0.44 1.29 ± 0.02 20.0 ± 0.53 NA
5 PID D Con 25.8 ± 0.23 2.02 ± 0.02 12.8 ± 0.06 NA
10 PID D Con 25.0 ± 0.92 2.25 ± 0.09 11.1 ± 0.07 NA
14 PID D Con 21.5 ± 0.74 1.70 ± 0.09 12.7 ± 0.48 NA
17 PID D Con 23.7 ± 0.42 1.74 ± 0.05 13.6 ± 0.20 NA
2 PID D Mol 23.7 ± 0.92 1.13 ± 0.06 21.1 ± 0.61 NA
5 PID D Mol 23.2 ± 0.08 1.30 ± 0.10 18.1 ± 1.47 NA
10 PID D Mol 23.7 ± 0.87 1.71 ± 0.09 13.9 ± 0.27 NA
14 PID D Mol 22.3 ± 0.32 1.51 ± 0.06 14.8 ± 0.41 NA
17 PID D Mol 23.9 ± 0.89 1.57 ± 0.06 15.3 ± 1.08 NA
2 PID L Con 27.9 ± 0.79 1.38 ± 0.02 20.1 ± 0.48 NA
5 PID L Con 26.9 ± 0.97 1.8 ± 0.06 15.0 ± 0.21 NA
10 PID L Con 26.1 ± 1.32 2.32 ± 0.17 11.4 ± 0.41 NA
14 PID L Con 24.3 ± 0.8 2.10 ± 0.07 11.8 ± 0.18 NA
17 PID L Con 25.2 ± 0.61 1.64 ± 0.04 15.4 ± 0.23 NA
2 PID L Mol 23.4 ± 0.29 1.19 ± 0.06 19.7 ± 1.03 NA
5 PID L Mol 22.2 ± 0.41 1.25 ± 0.14 18.3 ± 2.42 NA
10 PID L Mol 23.1 ± 0.75 1.93 ± 0.15 12.1 ± 0.53 NA
14 PID L Mol 21.2 ± 1.19 1.38 ± 0.08 15.3 ± 0.50 NA
17 PID L Mol 23.0 ± 0.92 1.46 ± 0.08 15.9 ± 1.14 NA
54
Supplementary Table 8: Theoretical percent contribution to N immobilization (Imm.) by NF in
the various treatments during the 2017 S. horneri decomposition experiment: dark/light control
(DC/LC) and dark/light molybdate (DM/LM).
All calculations are based on 1 g (dry weight, dw) of S. horneri detritus
Δ g N: ((%N of TF/100) × 1 g) - ((%N of T0/100) × 1 g) = Δ g N for the various treatments
% N Imm. : (Average NF rates corresponding to the various treatments, from Supp. Table 6) × 1 g (dw) × 48 hours
× (14.001 g of N/1,000,000,000 nmol of N) × (1/ Δ g N) × 100 = % N Imm. for the various treatments
Day Δ g N
(DC)
Δ g N
(DM)
Δ g N
(LC)
Δ g N
(LM)
% N
Imm.
(DC)
% N
Imm.
(DM)
% N
Imm.
(LC)
% N
Imm.
(LM)
0 -1.65 E-03 -3.29 E-03 -7.03 E-04 -2.62 E-03 NA NA NA NA
3 3.02 E-03 -4.19 E-03 7.84 E-04 -4.72 E-03 5.49 NA 12.1 NA
8 4.98 E-03 -4.16 E-04 5.66 E-03 1.77 E-03 2.07 NA 0.954 4.02
12 -3.51 E-03 -5.41 E-03 2.58 E-04 -6.63 E-03 NA NA 44.6 NA
15 -5.95 E-03 -7.65 E-03 -6.94 E-03 -8.77 E-03 NA NA NA NA
55
Supplementary Figure 1: Percent loss in biomass of S. horneri throughout decomposition.
56
Supplementary Figure 2: Variations in oxygen (O2) concentrations surrounding senescent S.
horneri in an open serum vial in the light (A&B, blue profile representing O2 concentrations
of just the seawater without any senescent seaweed present). Finer (µm) resolution snapshots
of O2 concentrations approaching senescent S. horneri detritus (C&D).
57
Chapter 3: Macroalgal detrital systems: an overlooked ecological
niche for heterotrophic nitrogen fixation
SUMMARY
Diazotrophic macroalgal associations (DMAs) can contribute fixed nitrogen (N) to the host
macroalgae. Biological nitrogen fixation (BNF) rates investigated using acetylene reduction assays
with living macroalgae surrounding Santa Catalina Island were low (maximum: 36 nmol N × g
-1
(dw) × h
-1
) and probably insufficient towards helping meet macroalgal N demand. However,
DMAs were observed during periods of low nitrate availability in Southern California coastal
waters, highlighting the potential importance of diazotrophs during N depleted conditions. Eleven
long-term (16 – 32 days) litter bag decomposition experiments with various macroalgae, especially
those with high (> 10) C:N ratios, resulted in much higher BNF rates (maximum: 693 nmol N × g
-
1
(dw) × h
-1
) than observed with living macroalgae. BNF rates were lower at the beginning of
macroalgal decomposition but rapidly increased during the second phase before declining towards
the end of decomposition. Labile carbon availability is likely influencing BNF rates throughout
macroalgal degradation and limits BNF in the final decomposition stage. Comparable dark and
light BNF rates with most macroalgae surveyed suggest macroalgal detrital systems are an
overlooked, potentially global, niche for heterotrophic N2 fixation. Lastly, suppressed BNF rates
with sodium molybdate additions highlight the prevalence of sulfate reducing diazotrophs.
INTRODUCTION
Macroalgae have long been considered an important food source to coastal food webs,
either through detrital pathways or direct grazing (Graham et al., 2007). The first phase in
macroalgal decomposition is typically initiated by physical processes (e.g. storms) or autolysis,
resulting in fragmentation of the macroalgal tissue which is then subject to microbial degradation
during the second phase (Rieper-Kirchner, 1989; Krumhansl and Scheibling, 2012). The third and
58
final phase of decomposition is marked by the more recalcitrant nature of the remaining macroalgal
detritus which can either be sequestered in the sediments or subjected to further degradation by
the benthic microbial community (Castaldelli et al., 2003). More recently, the role of macroalgal
sequestration is gaining traction as a potentially important process of removing atmospheric carbon
dioxide, resulting in another means of blue carbon (C) storage similar to other macrophytes, e.g.
mangroves, salt marshes, and seagrasses (Trevathan-Tackett et al., 2015; Raven, 2018).
Although macroalgal detrital systems provide numerous beneficial ecological services, the
byproducts of macroalgal decay have also been reported to have negative impacts on littoral
organisms (Eklund et al., 2005). Detrimental ecological effects, e.g. hypoxia or anoxia leading to
the death of benthic organisms (Valiela et al., 1997), are especially likely to occur when macroalgal
productivity results in excessive standing biomass. In the face of changing environmental
conditions, particularly increasing eutrophication, coastal macroalgal blooms are increasing in
frequency (Teichberg et al., 2010), making it pertinent to understand the different factors
influencing macroalgal decomposition.
Microbial interactions are one such factor which influence the rate of macroalgal
decomposition and play an important role in determining the fate of macroalgal detritus
(Krumhansl and Scheibling, 2012). Nitrogen (N) enrichment throughout macroalgal
decomposition is a common observation, typically attributed to the increasing microbial biomass
associated with aging macroalgal detritus (Rieper-Kirchner, 1989). Consequently, N, an important
macronutrient, is usually not considered to be limiting to the microbial community during
remineralization of such organic-rich matter. However, several studies have shown that N can
become limiting to the microbial community throughout decomposition (Tenore et al., 1979; Rice,
1982; Robinson et al., 1982). As many macroalgae have relatively high C to N content, it is
59
conceivable that available N is utilized faster than the labile C pool. Diazotrophs, specialized
prokaryotic organisms possessing the nitrogenase enzyme, can supply their own N via biological
nitrogen fixation (BNF), possibly providing them with an advantage in these N-limited conditions.
Not surprisingly, diazotrophic activities have been observed many times in similarly organic-rich,
reducing environments, e.g. seagrass beds (Capone and Budin, 1982), mangrove litter (Gotto and
Taylor, 1976; Woitchik et al., 1997), and sediments (Capone, 1982).
In other nutrient depleted ecosystems (e.g. coral reefs, Sargasso Sea), diazotrophic activity
has also been widely reported with living macroalgae from all three major clades of macroalgae
including Phaeophyceae (Carpenter, 1972; Hanson, 1977), Rhodophyta (Capone, 1977), and
Chlorophyta (Head and Carpenter, 1975; Capone et al., 1977). To our knowledge, observations of
diazotrophic macroalgal associations (DMAs) with degrading tissue date as far back as the 1970s
(Hanson, 1977; Zuberer and Silver, 1978) but investigations focusing primarily on BNF associated
with macroalgal detritus are sparse. Two recent studies (Hamersley et al., 2015; Raut et al., 2018)
looked at BNF associated with macroalgae but only within the Phaeophyceae clade. Thus, the
primary goal of this study is to investigate the prevalence of DMAs with living and decomposing
macroalgae across all three clades. This includes 2 macroalgae within Chlorophyta (Codium
fragile and Ulva sp.), 6 macroalgae within Phaeophyceae (Colpomenia sinuosa, Cystoseria sp.,
Dictyota sp., Dictyopteris sp., Sargassum horneri, and Zonaria farlowii), and 3 macroalgae within
Rhodophyta (Asparagopsis taxiformis, Laurencia sp., and Plocamium sp.). These macroalgae can
be found across a wide range of habitats (Table 1). Notably, several macroalgae (e.g. A. taxiformis,
C. sinuosa, Dictyota sp., Dictyopteris sp., Laurencia sp., Plocamium sp., Ulva sp.) investigated in
this study have a cosmopolitan distribution (Table 1) which warrants future studies to confirm
whether the localized findings of this study persist globally.
60
Macroalgae Clade Habitat Regions References BNF Measured
Codium
fragile
Chlorophyta
(Green)
N. Pacific, N. Atlantic,
Mediterranean, S. Pacific
Provan et al., 2005
Tyberghein et al., 2012
Living (†, HI): (L/D)
18-days (‡, HI): (L/D)
32-days (‡, HI): CON
(L/D), SM (L/D)
Ulva sp. Chlorophyta
(Green)
Australia (Indian,
Pacific), N. America
(Atlantic, Pacific),
Europe (Atlantic), Japan
(Pacific), S. Korea
(Pacific), China (Yellow
Sea)
Kirkendale et al., 2013
Cao et al., 2019
32-days (‡, HI): CON
(L/D), SM (L/D)
Colpomenia
sinuosa
Phaeophyceae
(Brown)
E. Asia, S.E. Asia, S.W.
Asia, S. Africa, Europe,
Australasia, Central
Pacific, N. America, S.
America
Lee et al., 2013
Living (†, HI): (L/D)
Degrading (†, HI):
(L/D)
Degrading (†, DT):
(L/D)
Cystoseira sp. Phaeophyceae
(Brown)
N.E. Atlantic,
Mediterranean, Indian,
N. Pacific
Draisma et al., 2010
18-days (‡, HI): (L/D)
16-days (‡, HI): CON
(L/D), SM (L/D)
Degrading (†, DT):
(L/D)
Dictyota sp. Phaeophyceae
(Brown)
N. Atlantic, S. Atlantic,
N. Pacific, S. Pacific,
Indian, Mediterranean,
Australasia, Central
Pacific
Bogaert et al., 2020
18-days (‡, HI): (L/D)
16-days (‡, HI): CON
(L/D), SM (L/D)
Dictyopteris
sp.
Phaeophyceae
(Brown)
N. Atlantic, S. Atlantic,
Mediterranean, Indian,
N. Pacific, Australia (S.
Pacific), Hawaii
Zatelli et al., 2018
16-days (‡, DT): CON
(L/D), SM (L/D)
Table 1. Summary of the species, clade, geographical distribution, and type of biological
nitrogen fixation (BNF) measurements conducted with all macroalgae surveyed in this study.
BNF was measured in short-term single experiments (†) with living or degrading macroalgae
that were either holdfast-intact (HI) or drifting (DT) at the time of collection. HI or DT
macroalgae were also used in long-term decomposition experiments (‡) and subsampled
separately for BNF rate measurements on different days throughout the decomposition period.
BNF rates were measured in light and dark (L/D) incubations constituting of only the
macroalgae without any further amendments and separate 20 mM sodium molybdate (SM)
amendments used to investigate the prevalence of sulfate reducing diazotrophs in parallel with
control (CON) L/D incubations for a subset of macroalgae surveyed in this study.
61
Table 1. Continued
Macroalgae Clade Habitat Regions References BNF Measured
Sargassum
horneri
Phaeophyceae
(Brown)
Pacific coast N. America
(S. California, USA and
Baja California, Mexico),
W. Pacific (Yellow Sea,
E. China Sea)
Marks et al., 2015
Zhuang et al., 2021
Degrading (†, DT):
(L/D)
Zonaria
farlowii
Phaeophyceae
(Brown)
S. California (Pacific) Haupt, 1932
16-days (‡, DT): CON
(L/D), SM (L/D)
Asparagopsis
taxiformis
Rhodophyta
(Red)
Mediterranean, S.
California (Pacific), N.
America (Atlantic),
Central America
(Atlantic), Australasia,
Hawaii, S.W. Asia
(Pacific), Indian
Andreakis et al., 2007,
2009
18-days (‡, HI): (L/D)
Laurenica sp. Rhodophyta
(Red)
W. Atlantic, E. Atlantic,
Mediterranean, Indian,
Central Pacific, E.
Pacific, W. Pacific
Sentíes et al., 2019
Living (†, HI): (L/D)
Degrading (†, DT):
(L/D)
Plocamium sp. Rhodophyta
(Red)
S. Africa (Atlantic,
Indian), Australasia
(Indian, Pacific), N.
America (Pacific,
Atlantic), S. America
(Pacific, Atlantic),
Subantarctic islands
Wynne, 2002
18-days (‡, HI): (L/D)
Whereas DMAs with living macroalgae can provide the host with a direct N supply and
support macroalgal production (Egan et al., 2013; Singh and Reddy, 2014), the role of BNF in
macroalgal detrital systems is poorly understood. Diazotrophic associations with other
macrophytes, e.g. mangrove litter (Woitchik et al., 1997; Pelegri and Twilley, 1998), contribute to
N immobilization, a process often observed during decomposition of N poor organic matter where
microbes assimilate inorganic N. Similarly, BNF has also been suggested to influence the
nutritional quality of the macroalgal detritus (Hamersley et al., 2015).
This study aims to better elucidate the role of BNF throughout macroalgal decomposition.
We utilize acetylene reduction assays comprised of dark and light incubations (24 – 48 hours) to
assess diazotrophic activity associated with macroalgae in different physiological states and during
62
longer decomposition experiments (16 – 32 days). In the absence of light, we assume
chemoheterotrophs, rather than photo- or chemo- autotrophs, are responsible for the observed
diazotrophic activity in dark incubations. However, future studies should utilize nifH amplicon
sequencing to better characterize the diazotrophic community and assess the prevalence of
heterotrophic diazotrophs. We also employ sodium molybdate (20 mM) amendments, an inhibitor
of sulfate reducers (Oremland and Capone, 1988), to evaluate whether inhibiting sulfate reducing
bacteria impacts BNF rates. Lastly, we assess the occurrence of DMAs with living macroalgae
surrounding Santa Catalina Island, a mesotrophic coastal ecosystem subject to periods of low
nitrate concentrations (Hewson and Fuhrman, 2003) and increased nitrate availability from
episodic and seasonal upwelling events (Zimmerman and Kremer, 1984), which hosts a varied
assemblage of macroalgae (Murray and Littler, 1989).
RESULTS & DISCUSSION
DMAs observed in all 3 macroalgal clades: Phaeophyceae, Rhodophyta, and Chlorophyta
In this study, the highest BNF rate, ~36 nmol N × g
-1
(dw) × h
-1
(averaged between dark
and light incubations), measured with living, substrate bound macroalgae was associated with
Laurencia sp. (hereafter referred to as Laurencia). However, this is only ~5 % of the average
nitrogenase activity (~768 nmol N × g
-1
(dw) × h
-1
)
previously reported with a Laurencia
diazotrophic association dominated by the heterocystous cyanobacteria, Calothrix, in nutrient poor
coral reef communities of the Grand Bahamas Island (Capone, 1977; Penhale and Capone, 1981).
Whereas BNF contributed up to 19 % of the N demand for Laurencia in the coral reef environment,
the average BNF rate measured in this study with living Laurencia would only supply ~1.1 % of
the N demand (47.2 µg N × g
-1
(dw) × h
-1
) previously estimated for Laurencia (Penhale and
Capone, 1981). In a more recent study, transcriptomic analysis of Laurencia from different coastal
63
sites along Brazil also revealed members of the Nostocales order, which includes Calothrix, as
well as diazotrophs of the Oscillatoriales and Chroococcales order to be dominant amongst the
cyanobacterial epiphytes associated with L. dendroidea (Oliveira et al., 2012). As light incubations
resulted in significantly higher BNF rates associated with substrate bound Laurencia
(Supplementary Table 1), it is likely that similar DMAs consisting of cyanobacterial epiphytes,
e.g. Calothrix, may be prevalent around Santa Catalina Island.
There were multiple instances where BNF rates, ranging from 2 – 14 nmol N × g
-1
(dw) ×
h
-1
, were associated with both adult and juvenile, holdfast-intact Codium fragile (Supplementary
Table 1). These were measured almost exclusively in the dark incubations while minimal
nitrogenase activity was observed during parallel light incubations, perhaps due to nitrogenase
inhibition by oxygen (Gallon, 1981) produced by macroalgal photosynthesis in the light
incubation. In contrast, both Dromgoole et al. (1978) and Gerard et al. (1990) mainly observed
epiphytic cyanobacterial (e.g. Calothrix, Microcoleus lyngbyaceus, Scytonema hofmannii)
associations with C. fragile. Head and Carpenter (1975) postulated that release of photosynthetic
metabolites by C. fragile supported diazotrophic activity by the heterotrophic bacterium,
Azotobacter, isolated from Codium and observed in dense populations on the macroalgal surface.
Despite fewer photosynthetic metabolites (e.g. glucose) being released by C. fragile in the dark
(Head and Carpenter, 1975), higher dark BNF rates were measured in this study. This may indicate
that a more prominent heterotrophic diazotrophic community, perhaps one resembling the
Azotobacter – C. fragile association (Head and Carpenter, 1975), may be prevalent with C. fragile
surrounding Santa Catalina Island.
The BNF rates measured with living C. fragile in this study fall towards the lower end of
previously reported BNF rates, 2 – 1042 nmol N × g
-1
(dw) × h
-1
, with C. fragile (Head and
64
Carpenter, 1975; Dromgoole et al., 1978; Gerard et al., 1990). Assuming the N requirement for C.
fragile used in this study is similar to previous reports of 1.25 mg N × g
-1
(dw) × d
-1
(Head and
Carpenter, 1975) or 96 µmol N × g
-1
(dw) × d
-1
(Pedersen and Borum, 1997), the observed
diazotrophic activity would only contribute 0.05 – 0.38 % towards the required macroalgal N
quota. Thus, diazotrophs, whether they be heterotrophic or photosynthetic, appear to be making
only a small contribution towards the N requirements of the living host macroalgae (i.e. Laurencia,
C. fragile) at our sites, further supporting previous observations reported by Hamersley et al.
(2015) and Raut et al. (2018).
Unlike what was observed with the dominant brown macroalgae, e.g. Macrocystis pyrifera
(Hamersley et al., 2015) and Sargassum horneri (Raut et al., 2018), surrounding Santa Catalina
Island, nitrogenase activity was not detected with healthy, substrate bound Dictyota sp. (hereafter
referred to as Dictyota) sampled in this study. However, diazotrophic activity associated with
Dictyota has been reported in the past, albeit in the nutrient depleted waters surrounding coral reef
communities (Capone et al., 1977). Interestingly, very low BNF rates (< 3 nmol N × g
-1
(dw) × h
-
1
) were measured in dark incubations with Cystoseira sp. (hereafter referred to as Cystoseira)
(Figure 1C). We also report for the first time (to our knowledge), diazotrophic activity (~15 nmol
N × g
-1
(dw) × h
-1
) associated with another cosmopolitan species of brown macroalga, Colpomenia
sinuosa (Supplementary Table 1).
While the factors influencing DMAs are not well understood, it is possible that
diazotrophic associations might be specific to certain macroalgae. All instances of DMAs observed
with living macroalgae at our sites around Santa Catalina Island overlapped with periods of low
nitrate concentrations (< 0.1 µM), derived using sea surface temperatures, in Southern California
coastal waters (Supplementary Figure 1B). This supports another hypothesis suggested by Raut et
65
al. (2018) that variability of N stress experienced by the host macroalgae at the time of
investigation might be an important factor influencing the presence or absence of diazotrophic
activity. While it is unlikely that these low BNF rates measured with different red, green, and
brown macroalgae provide a significant contribution towards the host macroalgal N demand, these
results further solidify that DMAs with living macroalgae are widespread and also exist along
typically under surveyed mesotrophic coastal zones.
Higher BNF rates associated with decomposing macroalgae
Apart from the brief mention of relatively high BNF rates observed with degrading brown
(Hanson, 1977) and green (Zuberer and Silver, 1978) macroalgae, there have been two direct
investigations of diazotrophic associations with aging brown macroalgal detritus. Both of these
studies also report significantly higher BNF rates with degrading macroalgal tissue (Hamersley et
al., 2015; Raut et al., 2018). In the current study, the BNF rates associated with holdfast-intact
brown macroalgae, Cystoseira and Dictyota, on the day of collection were very low or below the
detection limit in dark and light incubations during both 18-days and 16-days decomposition
experiments (Figure 1).
After 3 – 5 days of decomposition, there was a pronounced increase in nitrogenase activity
associated with both seaweeds in dark and light incubations (Figure 1). Similar to aging M. pyrifera
(Hamersley et al., 2015) and Sargassum spp. (Raut et al., 2018) detritus, this increased nitrogenase
activity persisted, although with some fluctuation, for several days during Cystoseira and Dictyota
decomposition (Figure 1). Contingent on the interval of sampling frequency, length of experiment,
and rate of decomposition, this intermediate period of increased diazotrophic activity is followed
by a gradual decrease or steep reduction in BNF rates (Figure 1). Organic C (e.g. glucose,
mannitol) amendments with aging Sargassum detritus (Raut et al., 2018) and mangrove leaf-litter
66
(Pelegri and Twilley, 1998) have shown significant stimulus of BNF rates towards the latter stages
of decomposition, suggesting that this decline in heterotrophic diazotrophic activity may be due to
labile C limitation.
Figure 1. Biological nitrogen fixation (BNF) rates derived from acetylene reduction assays
conducted with decomposing brown macroalgae: Cystoseira and Dictyota. The first
decomposition experiment with Cystoseira (A, n = 3) and Dictyota (B, n = 3) is comprised of
treatments constituting of macroalgal tissue subsampled on days 0, 5, 12, 15, and 18 and
incubated in dark and light serum vials without any further amendments. The second
experiment with Cystoseira (C, n = 3) and Dictyota (D, n = 3) includes control treatments in
dark and light incubations with macroalgae subsampled on days 0, 3, 6, 10, 14, and 16. The
control BNF rates (mean + SE) are represented by the dark or light brown bar graphs (A-D).
Sodium molybdate (20 mM) amendments, commonly used to inhibit sulfate reducers, were
carried out in conjunction with control treatments during the second experiment in separate
dark and light incubations throughout the 16-days decomposition period to assess the presence
of sulfate reducing diazotrophs. Sodium molybdate amendments resulting in reduced BNF rates
(mean ± SE) are represented by dotted lines overlaying the corresponding dark and light
incubations (C, D). BNF rates that were below the detection limit are labeled as BD. The mean
± SE for all BNF rates from the first (A, B) and second (C, D) experiments are presented in
Supplementary Table 5 and 6, respectively. Significance stars between bar graphs highlight the
difference in BNF rates between dark and light incubations. Significance stars inside the bar
graphs highlight the difference in BNF rates in the corresponding dark or light incubations
between control and sodium molybdate treatments. P < 0.001 (***), P < 0.01 (**), P < 0.05 (*)
67
The trends in nitrogenase activity throughout decomposition of brown macroalgae are
similar to those reported with mangrove leaf-litter (Pelegri et al., 1997; Woitchik et al., 1997;
Pelegri and Twilley, 1998) and also adhere strikingly well to the changes in microbial abundances
throughout macrophyte decomposition. Lower microbial abundances during the first phase of
decomposition generally precede a considerable increase in microbial abundances with various
macrophytes, e.g. seagrasses (Blum and Mills, 1991; Anesio et al., 2003), mangrove leaves
(Cundell et al., 1979; Raghukumar et al., 1995), and macroalgae (Rice and Hanson, 1984; Rieper-
Kirchner, 1989; Sathe-Pathak et al., 1993). This increase in microbial population typically
coincides with the second phase of macrophyte decomposition, during which loss of labile C, N,
and other organic molecules are thought to be mediated largely by microbial degradation (Cundell
et al., 1979; Buchsbaum et al., 1991; Sathe-Pathak et al., 1993; Raghukumar et al., 1995).
Consequently, the resulting scarcity of labile C and N can influence decreases in microbial
abundances, marking the final transition towards a refractory phase of macrophyte detritus
(Robinson et al., 1982; Buchsbaum et al., 1991; Sathe-Pathak et al., 1993). As previously
suggested, increased diazotrophic activity during the intermediate or second phase of
decomposition could potentially help alleviate N limitation to the microbial and detritovore
community (Hamersley et al., 2015; Raut et al., 2018).
Strikingly, the 32-days decomposition of substrate bound green macroalgae, C. fragile and
Ulva sp. (hereafter referred to as Ulva), exhibited a similar trend in diazotrophic activity where
lower initial nitrogenase activity was succeeded by a large increase in BNF rates which declined
by the latter stages of decomposition (Figure 2). The 18-days decomposition of C. fragile in 2017
also resembled similar trends in diazotrophic activity (Supplementary Figure 2). Furthermore, the
intermediate period of increased BNF rates also show a similar pattern of fluctuations (Figure 2)
68
as observed with decomposing brown macroalgae (Figure 1) and mangrove leaf-litter (Pelegri et
al., 1997; Pelegri and Twilley, 1998). During the second phase of macrophyte decomposition,
microbial abundances also exhibit episodic increases and decreases, which might be explained by
protistan grazing (Blum and Mills, 1991; Anesio et al., 2003).
Since N2 fixers are a subset of the overall microbial population, it is possible that the
fluctuation in nitrogenase activity may be a function of the variability in diazotrophic abundance
during this intermediate phase of decomposition. Despite these fluctuations, BNF rates, maximum
Figure 2. Biological nitrogen fixation (BNF) rates measured using acetylene reduction assays
with green macroalgae throughout the 32-days decomposition period: C. fragile (A, n = 4) and
Ulva (B, n = 3). Control treatments constituting of C. fragile subsampled on days 0, 2, 4, 7, 10,
13, 17, 22, 26, and 32 (A) and Ulva subsampled on days 0, 2, 4, 7, 10, 13, 17, 22, and 32 (B)
were incubated in dark and light serum vials without any further amendments. The control BNF
rates (mean + SE) are represented by the dark or light green bar graphs (A, B). Sodium
molybdate (20 mM) amendments, used to assess the presence of sulfate reducing diazotrophs,
resulting in reduced BNF rates (mean ± SE) are represented by dotted lines overlaying the
corresponding dark and light incubations (A, B). BNF rates that were below the detection limit
are labeled as BD. The mean ± SE for all BNF rates from the C. fragile and Ulva experiments
are presented in Supplementary Table 7 and 8, respectively. Significance stars between bar
graphs highlight the difference in BNF rates between dark and light incubations for control
treatments. Significance stars inside the bar graphs highlight the difference in BNF rates in the
corresponding dark or light incubations between control and sodium molybdate treatments. P
< 0.001 (***), P < 0.01 (**), P < 0.05 (*)
69
of 226 nmol N × g
-1
(dw) × h
-1
(Figure 2A), observed throughout C. fragile degradation were
consistently higher than BNF rates associated with healthy C. fragile in this study (Supplementary
Table 1). Interestingly, greater diazotrophic activity during C. fragile decomposition results in
BNF rates that are more comparable to previously reported BNF rates for healthy C. fragile (Head
and Carpenter, 1975; Dromgoole et al., 1978; Gerard et al., 1990). The highest BNF rates, 44 nmol
N × g
-1
(dw) × h
-1
(Figure 2B), observed with Ulva detritus is also comparable with previously
reported nitrogenase activity with drifting Ulva litter, ~43 nmol N × g
-1
(dw) × h
-1
(Zuberer and
Silver, 1978).
Since a substantial portion of global macroalgal net primary production is remineralized
(~37 %: Duarte and Cebrián, 1996), it is important to consider the different environmental settings
where particulate macroalgal remineralization occurs when extrapolating the results from litter bag
decomposition experiments carried out in this study. The majority (89 %) of the macroalgal
particulate organic C flux is thought to persist in coastal zones and the remaining 11 % is exported
to the deep sea (Krause-Jensen and Duarte, 2016). Thus, it is likely that DMAs similar to what was
observed throughout macroalgal decomposition in this study are most prevalent in coastal zones.
Indeed, DMAs have been detected via molecular efforts during green tides consisting of
Ulva prolifera blooms in coastal surface waters in the Yellow Sea (Zhang et al., 2015). The
sustained diazotrophic activity throughout Ulva decomposition (Figure 2B) identified in this study
suggests BNF might be a novel source of N to fuel additional ephemeral macroalgal growth,
resulting in prolonged green tides. Furthermore, high BNF rates measured with various drifting
macroalgal litter (Supplementary Figure 3) suggest that periods of increased diazotrophic activity
observed during litter bag decomposition can and does occur in situ coastal zones. For example,
BNF rates observed with noticeably degrading, drifting S. horneri (Supplementary Table 1) are
70
comparable to higher BNF rates previously reported with S. horneri during the second phase of
decomposition (Raut et al., 2018).
In order to investigate how diazotrophic activity might change throughout decomposition
of drifting macroalgal biomass, another 16-days decomposition experiment was conducted with
drifting Dictyopteris sp. (hereafter referred to as Dictyopteris) and Zonaria farlowii macroalgal
tissue that was markedly degraded upon the time of collection. Not surprisingly, the initial BNF
rates (88 – 154 nmol N × g
-1
(dw) × h
-1
) were substantially higher than any other BNF rates
measured with healthy, living macroalgae at the beginning of other long-term decomposition
experiments (Supplementary Figure 3). This is most likely due to the fact that the drifting
Dictyopteris and Z. farlowii biomass collected for the experiment had already undergone multiple
days of decomposition and were beyond the first phase of decomposition.
Despite this uncertainty, remarkably similar patterns in diazotrophic activity (as previously
discussed) were observed throughout the decomposition of drifting Dictyopteris and Z. farlowii
(Supplementary Figure 3). In contrast to incubation with other macroalgae, there was an
unexpected resurgence in nitrogenase activity on day 16 of the decomposition period
(Supplementary Figure 3). Since there was no way to ensure homogeneity in the length of
decomposition with drifting macroalgae prior to collection, it is possible that younger macroalgal
detritus, having undergone fewer cumulative days of decomposition, were subsampled on day 16.
This is corroborated by the highest BNF rates, up to 693 nmol N × g
-1
(dw) × h
-1
,
measured in this
study and C:N ratios which resemble values from the intermediate phase of decomposition (days
4 – 10) (Supplementary Figures 3, 4). While mesotrophic coastal areas may not foster particularly
high rates of BNF associated with living macroalgae, it appears that macroalgal degradation can
71
ubiquitously stimulate much higher diazotrophic activity and this may potentially be a novel source
of bioavailable N to coastal macroalgal ecosystems.
Nature of the diazotrophic community prevalent in macroalgal detrital systems
Dark BNF rates with living macroalgae can be ~5.5 – 52-fold lower than light BNF rates
(Capone, 1977; Capone et al., 1977). In contrast, dark and light BNF rates are much more
comparable throughout macroalgal decomposition across all three clades (Figures 1, 2;
Supplementary Figures 2, 3, 5). Compared to dark and light BNF rates (14 and 77 nmol N × g
-1
(dw) × h
-1
, respectively) reported with living Dictyota in a N limited coral reef ecosystem (Capone
et al., 1977), the decomposition of Dictyota in this study seems to promote greater diazotrophic
activity, up to 339 and 396 nmol N × g
-1
(dw) × h
-1
, respectively. This resulted in higher levels of
BNF rates in the dark (24-fold increase) than the light (5-fold increase). Taken together with
similar reports of increased diazotrophic activity throughout decomposition, particularly dark BNF
rates (Hamersley et al., 2015; Raut et al., 2018), this indicates that macroalgal decomposition
greatly stimulates heterotrophic diazotrophic activity. This also aligns well with the reports of
heterotrophic diazotrophic activity associated with degrading seagrass rhizomes (Capone and
Budin, 1982; Kenworthy et al., 1987), mangrove leaf-litter (Pelegri and Twilley, 1998), and
freshwater macrophytes (Šantrůčková et al., 2010). This suggests that the decomposition of
macrophytes might provide a global niche for heterotrophic N2 fixers.
In this study, there was minimal nitrogenase activity associated with living macroalgae.
The few instances of measurable BNF rates were typically higher in light incubations, which can
likely be attributed to various cyanobacterial epiphytes previously reported with other DMAs
(Capone, 1977; Capone et al., 1977; Dromgoole et al., 1978; Gerard et al., 1990). It is possible
that as macroalgae degrade, this cyanobacterial community tends to be replaced by a more
72
prominent heterotrophic diazotrophic community. Due to limited nifH targeted molecular
investigations of macroalgal systems, the identity of N2 fixers associated with living and degrading
macroalgae and the potential for this shift in the diazotrophic community remains largely
unknown. The results from this study provide a few examples that support a substantial shift in the
physiology and intensity of diazotrophic populations associated with macroalgae during the
transition from living to decomposing macroalgal biomass.
For instance, BNF rates were observed exclusively in the light incubations with healthy C.
sinuosa tissue whereas degrading C. sinuosa tissue (collected adjacent to the healthy macroalga)
resulted in similar BNF rates but strictly in the dark incubations (Supplementary Table 1). This
suggests that there might be differences in the diazotrophic community based on the physiological
status of the host macroalga. It is possible that greater availability of labile C, in the form of
dissolved organic C (García-Robledo et al., 2008) and other organic compounds (e.g.
carbohydrates, phenols, alginate, and mannitol) (Sathe-Pathak et al., 1993), with degrading
macroalgal tissue could fuel heterotrophic N2 fixation. Additionally, increased microbial and
protist colonization associated with degrading macroalgal tissue (Sathe-Pathak et al., 1993) could
result in increased community respiration that could potentially draw down oxygen concentrations
within the macroalgal detritus. This coupled with the lack of oxygen production via photosynthesis
by the degrading macroalgae could result in more favorable conditions to sustain heterotrophic
diazotrophic activity throughout macroalgal decomposition.
In another instance, light BNF rates associated with living Laurencia were significantly
greater than dark incubations (Supplementary Table 1) whereas dark BNF rates associated with
drifting Laurencia, having undergone an unknown period of decomposition, yielded comparable
results to parallel light incubations. Similarly, greater BNF rates were observed in light incubations
73
with healthy S. horneri juveniles while dark incubations resulted in comparable, if not higher, BNF
rates throughout S. horneri decomposition (Raut et al., 2018). Another study investigating
epiphytic bacterial communities associated with drifting S. horneri at different physiological
stages found that the host macroalgal physiological status greatly influences the associated
bacterial communities (Mei et al., 2019). Taken together, this supports an emerging perspective
that DMAs are similarly influenced by the host macroalgal physiology and most likely shift
towards a dominant heterotrophic N2 fixing community during decomposition.
A recent study analyzing nifH genes from surface waters populated by a macroalgal (U.
prolifera) bloom found a much higher abundance of nifH relative to non-covered surface waters,
suggesting that macroalgal decomposition does indeed support greater BNF (Zhang et al., 2015).
Notably, they found heterotrophic diazotrophs to dominate the diazotrophic community (Zhang et
al., 2015). The higher dark BNF rates associated with decomposing macroalgae and drifting
macroalgal samples, consisting of degrading macroalgal tissue, may also be dominated by similar
heterotrophic diazotrophs (Figures 1, 2; Supplementary Figures 2, 3, 5). Furthermore, severe
inhibition of nitrogenase activity, up to 97 %, by sodium molybdate amendments with aging Ulva
detritus, particularly in the dark incubations (Figure 2B), strongly indicates that sulfate reducing
diazotrophs are a constituent of the heterotrophic N2 fixing community.
Likewise, several instances of nitrogenase inhibition throughout macroalgal decomposition
by sodium molybdate amendments (Figures 1C, D, 2; Supplementary Figure 3; Raut et al., 2018)
suggest the presence of sulfate reducing diazotrophs. The presence of Desulfovibrio-related
phylotypes found exclusively in the surface waters dominated by Ulva blooms (Zhang et al., 2015)
corroborates the potential importance of heterotrophic sulfate reducing diazotrophs during
macroalgal decomposition. Moreover, direct measurements of sulfate-reduction during Ulva
74
(Nedergaard et al., 2002; Lomstein et al., 2006) decomposition exhibits a very similar progression
to the three-phases of diazotrophic activity (Figure 2B), suggesting sulfate reduction and BNF to
be very tightly coupled throughout Ulva decomposition.
The phenomenon of nifH phylotypes dominated by sulfate-reducing bacteria (e.g.
Desulfobulbaceae, Desulfobacteraceae) in the presence of macrophytes was also observed with
surface sediments populated by the seagrass, Zostera marina (Sun et al., 2015). Additionally,
direct measurements of sulfate reduction (Blaabjerg and Finster, 1998) and suppressed nitrogenase
activity in the rhizosphere sediments of Z. marina via sodium molybdate amendments (Capone,
1982) corroborate the importance of sulfate reducing diazotrophs in seagrass ecosystems (Nielsen
et al., 2001). Sulfate reducers are also prevalent in salt marshes (Hines et al., 1989) and mangroves
(Kristensen et al., 1994). Thus, it is entirely possible that the anaerobic conditions and high
bioavailability of organic matter found in various macrophyte detrital systems favor heterotrophic
diazotrophic communities dominated by sulfate-reducing N2 fixers. Interestingly, sodium
molybdate additions simultaneously inhibited nitrogenase activity while stimulating
methanogenesis (Capone, 1982), suggesting that when conditions no longer support sulfate
reduction, e.g. when macroalgal detritus becomes too refractory, methanogenic N2 fixers might be
favored.
C and N content of macrophyte detritus and its influence on diazotrophy
The C:N ratios of aging macroalgal detritus exhibit an inverse trend to BNF rates, having
initially higher values that decline throughout decomposition (Figure 3; Supplementary Figures 4,
6). Decreasing C:N ratios have routinely been observed throughout macrophyte decomposition
and are often linked to increasing N content due to microbial biomass buildup (Robinson et al.,
1982; Rieper-Kirchner, 1989), incorporation of microbial exudation products into the detrital
75
matrix (Rice and Hanson, 1984), or accumulation of recalcitrant N (Rice, 1982) within the detritus.
Not surprisingly, this trend was also observed in this study and principal component analysis
suggests that decreasing C:N ratios may similarly correspond with increasing N content of
macroalgal detritus (Figure 4).
A generalized additive model (R
2
= 0.842; p < 0.001; n = 299; deviance explained = 84.6%)
further supports % N to be a strong predictor of C:N ratios, but not necessarily in a linear fashion
(Supplementary Figure 7). Interestingly, BNF has also been proposed as a mechanism for N
immobilization with decomposing macrophytes such as mangrove leaf-litter (Woitchik et al.,
1997; Pelegri and Twilley, 1998) and macroalgae (Hill and McQuaid, 2009; Raut et al., 2018).
Although acetylene is a competitive inhibitor of BNF (Schöllhorn and Burris, 1967), theoretical
calculations utilizing BNF rates derived from the acetylene reduction assays conducted in this
study suggest BNF is probably a minor contributor, < 0.1 – 5.5 %, to N immobilization throughout
Figure 3. Carbon (C) to nitrogen (N) ratios of post-incubation macroalgal detritus from dark
and light acetylene reduction assays throuhgout the 18-days decomposition experiment with
red (A. taxiformis – A, Plocamium – B), green (C. fragile – C), and brown (Cystoseira – A,
Dictyota – B) macroalgae. The data is presented in a Tukey’s box and whisker plot. The
complete dataset consisting of % C, % N, and C:N ratios are listed in Supplementary Table 3.
76
macroalgal decomposition (Supplementary Table 2). Since bulk macrophyte detritus is comprised
of refractory and digestible fractions with different C:N ratios (Robinson et al., 1982), BNF might
potentially be contributing more to the labile N pool. However, determining the fate of fixed N
from diazotrophic activity is difficult to ascertain without undertaking stable isotope labeling.
Interestingly, C:N ratios of macroalgal detritus and differences between the refractory and
labile fractions may explain the differences in diazotrophic activity observed throughout
decomposition of different macroalgae. For instance, the C:N ratios of A. taxiformis (6.5: this
study) and Plocamium sp., hereafter referred to as Plocamium, (7.8: this study; 7.5: Weykam et
al., 1996), are relatively low compared to the brown and green macroalgae which had initially
Figure 4. Principal component analysis (PCA) based on the first 2 principal components of
different variables (% carbon (C), % nitrogen (N), C:N ratios, and biological nitrogen fixation
(BNF) rates). The variables were measured with macroalgal samples from dark and light
acetylene reduction assays throughout 3 long-term decomposition experiments with several
macroalgae (A. taxiformis, C. fragile, Cystoseira, Dictyota, and Plocamium) and 2 short-term
experiments with drifting and substrate bound Laurencia. The different ellipses represent 80 %
confidence intervals around data points representing the various macroalgae, broken down into
different clusters of green (C. fragile), brown (Cystoseira, Dictyota), and red (A. taxiformis,
Laurencia, Plocamium) macroalgae, colored correspondingly. Laurencia is represented by the
square points and clusters differently from the other red macroalgae.
77
higher C:N ratios (Figure 3; Supplementary Table 3). Notably, BNF rates associated with these
decomposing red macroalgae (Supplementary Figure 5) were orders of magnitude lower than that
measured with decomposing brown and green macroalgae. It is possible that the enriched N
content of the detritus inhibited higher BNF rates (Figure 4). The striking resemblance in
diazotrophic activity throughout decomposition (Supplementary Figure 5) to trends in diazotrophic
activity observed with decomposing brown and green macroalgae suggest other factors, e.g. the
lower availability of labile C, might have similarly influenced the N2 fixing community.
Indeed, red macroalgae such as Plocamium have been characterized to be relatively more
recalcitrant, consisting of low soluble (~28 %) and high crude fiber (~18 %) content (Smith and
Foreman, 1984) and other refractory compounds such as sulfated polysaccharides and alginates
(Trevathan-Tackett et al., 2015). The degradation of this more recalcitrant macroalgal tissue may
result in lower microbial abundances overall and reduced BNF rates compared to the
decomposition of more labile macroalgal detritus. Thus, labile organic C additions (e.g. glucose,
mannitol) may stimulate BNF rates throughout macroalgal decomposition of A. taxiformis and
Plocamium as previously observed with the brown macroalga S. palmeri (Raut et al., 2018).
Alternatively, the production of quorum sensing inhibitory substances and antimicrobial
compounds by red macroalgae like A. taxiformis (Genovese et al., 2012; Jha et al., 2013) may
possibly deter biofilm formation and microbial colonization during decomposition, also resulting
in lower diazotrophic abundance.
The various brown macroalgae investigated here and in other studies (e.g. Hamersley et
al., 2015; Raut et al., 2018) suggest that higher initial C:N ratios, indicating N depleted conditions,
may support greater diazotrophic activity throughout decomposition. The two green macroalgae
used in this study, C. fragile (C:N – 12: this study) and Ulva (C:N – 21: Hu et al., 2017), are also
78
characterized with C:N ratios greater than 10 and sustained higher BNF rates throughout
decomposition (Figures 2, 3). Several of these brown (e.g. Sargassum, Macrocystis, Colpomenia,
Dictyota) and green (e.g. Codium, Ulva) macroalgae have a cosmopolitan distribution and future
investigations may confirm whether or not these DMAs are globally ubiquitous.
In contrast, various red macroalgae seem to have relatively lower C:N ratios, ranging from
5.6 – 10.6 (Weykam et al., 1996), and thus, it is likely that lower BNF rates might be associated
with their decomposition as observed with A. taxiformis and Plocamium (Supplementary Figure
5). Strikingly, both healthy and degrading tissue of another red macroalga used in this study,
Laurencia, had a C:N ratio greater than 10 and correspondingly, supported much higher BNF rates.
Taken together, it is likely that ecosystems comprised of macroalgae with high (> 10) C:N ratios
might provide an overlooked niche for BNF throughout their decomposition.
However, this is not to say there is a linear relationship between C:N ratios of macrophytes
and BNF rates associated with their decomposition. In fact, higher C:N ratios observed with other
macrophytes, e.g. mangrove leaves (56 – 290: Woitchik et al., 1997; Pelegri and Twilley, 1998),
support comparable BNF rates to those observed throughout decomposition of brown and green
macroalgae with much lower C:N ratios (12 – 36). This is likely due to the fact that mangrove
leaves, despite having significantly higher C:N ratios, are much more recalcitrant than macroalgae
with a higher degree of structural macromolecules including cellulose, lignin, and other
polyphenols (Trevathan-Tackett et al., 2015). Thus, the availability of labile C could limit the
dominant heterotrophic N2 fixing community associated with decomposing mangrove leaf detritus
resulting in comparable BNF rates to that observed with decomposing macroalgae in this study.
The positive correlation (Pearson’s correlation coefficient: r = 0.37; p-value < 0.00001) between
% C and BNF rates observed with macroalgae surveyed in this study (Figure 4) further highlights
79
the potential influence of labile C availability on BNF rates. The host macrophyte’s cell wall
composition and structure might have the most influence on the associated diazotrophic activity
throughout decomposition and warrants future investigations.
Additionally, differences between refractory and labile fractions of different macrophyte
detritus also add to the complexity of interpreting nutritive value from bulk C:N ratios. Although
the C:N ratios of macroalgae are much lower at the end of decomposition (Figure 3; Supplementary
Figures 4, 6), much of the remaining detritus may be refractory (Rice, 1982; Robinson et al., 1982).
Thus, lower C:N ratios may not necessarily reflect greater nutritional value. Ultimately, BNF
during the decomposition of macroalgal detritus from several clades follow similar temporal
dynamics with ingrowth of diazotrophic heterotrophic microbes during intermediate stages of
decay. The overall C:N ratio of the residual material generally decreases as a result although the
effect on the nutritional value of the detritus remains to be determined.
MATERIALS AND METHODS
Collection of macroalgae and experimental setup
Macroalgal samples were collected between 2017 – 2019 at various locations surrounding
Santa Catalina Island, CA (Supplemental Table 4) via freediving (depth < 10 m for substrate bound
samples pulled off at the holdfast) or at the surface (for drift macroalgae). These were transported
back to the Wrigley Marine Science Center (WMSC), typically within 1 hour, in a bucket with
ambient seawater bubbled with aquarium air stone diffusers for downstream experimental
manipulations. Upon arrival at WMSC, macroalgal tissue were immediately placed in 14- or 27-
mL serum vials with 0.2 µm filtered seawater to conduct the acetylene reduction assay as described
below. For long term decomposition experiments, macroalgal biomass of similar physiological
status were gauged by physical observation and separated into three general categories: healthy,
80
beginning to decompose, and thoroughly decomposed with macroalgal morphology difficult to
distinguish. The samples were then apportioned into multiple, separate 200 µm-mesh white litter
bags (12.7 cm × 17.8 cm) and incubated in flow-through seawater tanks.
Throughout the course of the decomposition experiment, a different litter bag was
subsampled every few days to assess nitrogenase activity associated with aging macroalgal
detritus. These tanks were located either inside a laboratory or outdoors under a shaded canopy.
Seawater from an adjacent site to WMSC was continuously flowing through the tank for the
duration of the entire experiment to maintain ambient temperature, nutrients, and other parameters.
The photosynthetically active radiation (PAR) was measured daily for a month with a handheld
quantum PAR meter (Biospherical Instruments Inc., San Diego, CA, United States). The surface
ocean (< 0.5 m) PAR reading was assumed to be fully saturated and all other measurements are
expressed as a percentage relative to that. Both indoor and outdoor tanks received ambient sunlight
and were under the same dark-light cycles, but the indoor tanks averaged 4.9 % while the outdoor
tanks averaged 73 % of daily surface ocean PAR.
Acetylene reduction (AR) assays
Acetylene reduction (AR) assays as detailed by Capone (1993) were carried out using
similar modifications to the ones reported by Raut et al. (2018). Acetylene gas (C2H2) was
produced by reacting water with calcium carbide and 1 or 2 mL of C2H2 was injected in 14- or 27-
mL serum vials, respectively. The final volume of C2H2 in either vials (~7 % of the total volume
or ~20 – 22 % of the gas phase) was adequate to saturate the nitrogenase enzyme (Flett et al.,
1976). The aqueous to gas phase ratio for 14- and 27-mL serum vials were 64:36 % and 67:33 %,
respectively. AR assays were done in triplicates or quadruplicates and incubated in either the
indoor or outdoor seawater tanks for 24 – 48 hours. The 2019 C. fragile and Ulva incubations took
81
place in an incubator programmed at 18ºC with 12-hour dark/light cycles. The PAR inside the
incubator averaged ~4 % of daily surface ocean PAR.
A Shimadzu Mini 2 gas chromatograph equipped with a flame ionization detector was used
to analyze 100 µL subsamples taken from the headspace of all replicates in conjunction with
PeakSimple chromatography data system calibrated with 100 ppm ethylene (C2H4) standards to
quantify C2H4 peak heights. Upon C2H2 injection, an initial timepoint, T0, was conducted to
establish any background C2H4 peak heights. Following T0, additional timepoints were conducted
every ~3 – 7 hours, depending on how quickly C2H4 production increased between timepoints, for
the duration of the incubation. For all experiments, negative controls without any C2H2 added to
the serum vials were routinely incubated in parallel and yielded no C2H4 production.
AR assays conducted for all macroalgae surveyed in this study included incubations in the
dark with serum vials wrapped in aluminum foil to block all irradiance and light incubations where
serum vials were left unperturbed. For the 2017 16-days decomposition experiments (Cystoseira,
Dictyota, Dictyopteris, Z. farlowii) and 2019 32-days decomposition experiments (C. fragile,
Ulva), parallel AR assays were conducted in separate dark and light serum vials with 20 mM
sodium molybdate added once at the start of the incubation to assess the presence of sulfate
reducers (Oremland and Capone, 1988).
C and N content of macroalgal tissue
Macroalgal tissue samples were collected at the end of the incubation period of AR assays
throughout the 2017 18-days decomposition experiment (A. taxiformis, C. fragile, Cystoseira,
Dictyota, Plocamium), 2017 16-days decomposition experiment (Cystoseira, Dictyota,
Dictyopteris, Z. farlowii), and short-term experiments with drifting and holdfast-intact Laurencia.
For the 5 different seaweeds from the 2017 18-days decomposition experiment, macroalgal detritus
82
were also collected prior to the beginning of AR assays. All tissue samples were dried at 60ºC in
a conventional drying oven for 48 hours at WMSC before being stored in aluminum pockets for
downstream homogenization using a mortar and pestle. Once homogenized to a fine powder,
between ~1 – 1.3 mg of each sample was weighed out using a microbalance and encapsulated in
tin capsules and pelletized for C and N analysis on a Micromass IsoPrime continuous flow isotope
ratio mass spectrometer with CHN analyzer/sample front ends.
Data analysis and statistical methods
All data analysis and statistical tests were carried out using the R (version 3.6.3)
programming language. A similar workflow to the one described by Raut et al. (2018) was used
to generate BNF rates. C2H4 peak heights were converted to nmol of C2H4 (Capone, 1993;
Breitbarth et al., 2004) before fitting a linear regression model, which excluded lags at the
beginning and plateaus at the end of incubations (Raut et al., 2018), to calculate rates of C2H4
production. Lastly, theoretical rates of BNF were estimated using a 3:1 ratio of C2H2 reduction:N2
reduction (Capone, 1993) and multiplied by 2 to express BNF rates as nmol N × g
-1
(dw) × h
-1
.
The Shapiro-Wilk’s test, R function – “shapiro.test”, was used to determine if the BNF
rates dataset from the different experiments were normally distributed. The Fligner-Killeen’s test,
a non-parametric method that accounts for non-normal data, was employed using the R function
“fligner.test” to determine if assumptions of homoscedasticity were met for this dataset. The
Welch’s two sample t-test, R function – “t.test”, was used to determine if average BNF rates were
significantly different between dark and light incubations. Alternatively, when the data was not
normally distributed, the two sample Wilcoxon test (also referred to as the Mann-Whitney test, R
function – “wilcox.test”) was used to compare BNF rates between dark and light incubations.
83
In experiments with a 2 × 2 factorial design where each factor (i.e. light, treatment) had
two levels (i.e. dark/light, control/sodium molybdate), a two-way analysis of variance (ANOVA)
using the R function “aov” was carried out. If the two-way ANOVA resulted in a significant p-
value < 0.05, then multiple pairwise-comparisons between the means of BNF rates of different
groups were carried out using Tukey’s Honest Significant Difference (HSD) method, R function
– “TukeyHSD”. For datasets which violated the assumptions of normality and/or equal variances,
a non-parametric alternative to the two-way ANOVA was performed using the R function “t2way”
from the latest update (Sept., 2020) to the functions stored in “Rallfun-v37.txt” described by
Wilcox (2017), freely downloaded from https://dornsife.usc.edu/labs/rwilcox/software/.
Analogous to Tukey’s HSD, an alternative, non-parametric post hoc test that does multiple
pairwise-comparisons for independent groups using medians (Wilcox, 2017), R function –
“medpb” from the package “Rallfun-v37.txt”, was used to compare BNF rates between different
groups from the 2 × 2 factorial design experiments if the p-value from “t2way” test was less than
0.05. The results from Welch’s two sample t-test (WT), Wilcoxon/Mann-Whitney’s test (W/MW),
Tukey’s HSD method (THSD), or “medpb” method (MPB) are included in the supplementary
tables highlighting BNF rates (Supplementary Tables 1, 5 – 10). Additionally, the p-values from
these tests are depicted in the various figures displaying BNF rates and defined as follows: P <
0.001 (***), P < 0.01 (**), P < 0.05 (*), or not significant (NS).
A generalized additive model was fit to the % N content and C:N ratios of macroalgal
tissue from this study using the R function “gam” from the “mgcv” package with the restricted
maximum likelihood method (k = 10; family = gaussian; link function = identity) (Wood, 2017).
Lastly, principal component analysis (PCA) for different variables (% C, % N, C:N ratio, BNF
rates) measured with macroalgal samples from the 18-days decomposition experiment (A.
84
taxiformis, C. fragile, Cystoseira, Dictyota, and Plocamium), 16-days decomposition experiment
(Cystoseira, Dictyopteris, Dictyota, and Z. farlowii), and short-term experiments with drifting and
substrate bound Laurencia was performed using the R function “prcomp”. The default method for
the function was used with one modification (scale. = TRUE) which standardized the variables
using Z-score normalization before the PCA was performed. The results from the PCA, including
the factor loadings for the different variables, eigenvalues, standard deviation, proportion of
variance, and cumulative proportion, are summarized in Supplementary Table 11.
Acknowledgements
We would like to thank the USC Wrigley Marine Science Center and their staff, especially Lauren
Czarnecki Oudin and Kellie Spafford, for accommodations and support for this research. We thank
all participants from previous Research Experiences for Undergraduates programs (NSF Award
OCE-1559941) for their help: Taylor Dillon, Calyn Crawford, and Sarah Ortiz. We want to thank
members of the Capone lab for their help: Weimin Deng, Suryia Tanjasiri, Miguel Rincon, Troy
Gunderson, and Michael Morando. Yubin Raut was supported by summer Wrigley/Victoria J.
Bertics Fellowships (2017 – 2019) and USC Research Enhancement Fellowship (2020 – 2021).
Conflict of Interest
The authors declare no conflict of interest.
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Supplementary Information
Supplementary Figure 1. (A) Daily sea surface temperature (SST, ºC) obtained from a National
Oceanic and Atmospheric Administration (NOAA) buoy in the San Pedro Channel (Station 46222,
33.618 ºN, 118.317 ºW ) from 01/01/2017 – 12/31/2018, (B) estimated nitrate (µM) concentrations
predicted from the SST (A) using a generalized additive model fit to 38 years of in situ seawater
temperature and nitrate concentration measurements (Snyder et al., 2020) , and (C) estimate of
vertical nitrate flux (mmol × m
-1
× s
-1
) using the Biologically Effective Upwelling Transport Index
(BEUTI) for the U.S. west coast (33 ºN) for the same time period. The NOAA buoy data (1) and
BEUTI data (2) are both publicly available datasets downloaded from:
1) https://www.ndbc.noaa.gov/station_page.php?station=46222
2) https://oceanview.pfeg.noaa.gov/products/upwelling/dnld
91
Supplementary Figure 2. Biological nitrogen fixation (BNF) rates derived from acetylene
reduction assays with a green macroalga, C. fragile (n = 3), throughout the 18-days decomposition
experiment in 2017. C. fragile subsampled on days 0, 5, 12, 15, and 18 were incubated in dark and
light serum vials without any further amendments. BNF rates that were below the detection limit
are labeled as BD. The dark and light BNF rates (mean + SE) are represented by the dark and light
green bar graphs. The mean ± SE for all BNF rates from the C. fragile experiment are presented
in Supplementary Table 9. Significance stars between bar graphs highlight the difference in BNF
rates between dark and light incubations. P < 0.001 (***), P < 0.01 (**), P < 0.05 (*)
92
Supplementary Figure 3. Biological nitrogen fixation (BNF) rates measured using acetylene
reduction assays with initially drifitng brown macroalgae that were allowed to decompose in litter
bags for 16 days: Dictyopteris (A, n = 3) and Z. farlowii (B, n = 3). Control treatments constituing
of Dictyopteris subsampled on days 0, 5, 12, 15, and 18 (A) and Z. farlowii subsampled on days
0, 5, 12, 15, and 18 (B) were incubated in dark and light serum vials without any further
amendments. The control BNF rates (mean + SE) are represented by the dark or light brown bar
graphs. Excess biomass of both macroalgae on day 16 allowed for 20 mM sodium molybdate
amendments parallel to control treatments resulting in reduced BNF rates (mean ± SE) as
represented by dotted lines overlaying the corresponding dark and light incubations. The mean ±
SE for all BNF rates from the Dictyopteris and Z. farlowii experiments are presented in
Supplementary Tables 10 and 10.1. Significance stars between bar graphs highlight the difference
in BNF rates between dark and light incubations for control treatments. Significance stars inside
the bar graphs highlight the difference in BNF rates in the corresponding dark or light incubations
between control and sodium molybdate treatments. P < 0.001 (***), P < 0.01 (**), P < 0.05 (*)
93
Supplementary Figure 4. Carbon (C) to nitrogen (N) ratios of post-incubation macroalgal detritus
from dark and light acetylene reduction assays throuhgout the 16-days decomposition experiment
with drifitng brown macroalgae (Dictyopteris – B and Z. farlowii – A). The data is presented in a
Tukey’s box and whisker plot. The complete dataset consisting of % C, % N, and C:N ratios are
listed in Supplementary Table 3.
94
Supplementary Figure 5. Biological nitrogen fixation (BNF) measured using acetylene reduction
assays with red macroalgae throughout the 18-days decomposition period: A. taxiformis (A, n = 3)
and Plocamium (B, n = 3). A. taxiformis subsampled on days 0, 5, 12, 15, and 18 (A) and
Plocamium subsampled on days 0, 5, 12, 15, and 18 (B) were incubated in dark and light serum
vials without any further amendments. BNF rates that were below the detection limit are labeled
as BD. The dark and light BNF rates (mean + SE) are represented by the dark and light red bar
graphs. The mean ± SE for all BNF rates from the A. taxiformis and Plocamium experiments are
presented in Supplementary Table 5. The light BNF rate associated with A. taxiformis on day 5 (†)
only shows the mean for biological replicates A and C; replicate B consisted of the holdfast and
fronds, exhibiting much higher nitrogenase activity (21.9 nmol N × g
-1
(dw) × h
-1
) and was excluded
from the mean.
95
Supplementary Figure 6. Carbon (C) to nitrogen (N) ratios of post-incubation macroalgal detritus
from dark and light acetylene reduction assays throuhgout the 16-days decomposition experiment
with brown macroalgae (Cystoseira – A and Dictyota – B). The data is presented in a Tukey’s box
and whisker plot. The complete dataset consisting of % C, % N, and C:N ratios are listed in
Supplementary Table 3.
96
Supplementary Figure 7. Generalized additive model (GAM) fit to % nitrogen (N) and carbon (C)
to N ratios (represented by the black line) acquired from the 2017 18-days decomposition
experiment with red (A. taxiformis, Plocamium), green (C. fragile), and brown (Cystoseira,
Dictyota) macroalgae, 2017 16-days decomposition experiments with brown macroalgae
(Cystoseira, Dictyota, Dictyopteris, Z. farlowii), and 2017 short-term experiments with drifting
and substrate bound red macroalga (Laurencia). The GAM accounts for 84.6 % of the observed
deviance with an adjusted R
2
of 0.842 and p-value < 2 e
-16
. The points (brown – Phaeophyceae,
green – C. fragile, and red – Rhodophyta) represent measured C:N ratios against % N values.
97
Supplementary Table 1. Mean biological nitrogen fixation (BNF) rates, expressed as nmol N × g
-
1
(dw) × h
-1
± SE, for dark/light (D/L) incubations from short-term single experiments with different
macroalgae. C. fragile was collected in the adult stage as well as juveniles (less than 7 cm).
Macroalgae were collected as drifitng (D) or holdfast-intact (HI) samples, consisitng of healthy
(H) and degrading (DG) macroalgal tissue. BNF rates that were below the detection limit are
labeled as BD. The two statistical methods, Welch’s Two Sample t-Test (WT) or Wilcoxon/Mann-
Whitney (W/MW) test, used to compare D/L incubations are accompanied by their specific p-
value and significance level in the D/L column: P < 0.001 (***), P < 0.01 (**), P < 0.05 (*), or not
significant (NS).
Date Species Clade Light BNF Rate D/L
7/25/2017 Laurencia
Rhodophyta
(Red)
Dark
89 ± 35.7 D – DG
7/25/2017
Laurencia Light
108 ± 44.7
WT: 0.75 (NS)
7/27/2017
Laurencia Dark
17.6 ± 7.2
HI – H
7/27/2017
Laurencia Light
54.9 ± 2.1
WT: 0.03 (*)
6/15/2018
C. fragile Chlorophyta
(Green)
Dark
2.95 ± 1.03
HI – H (Adult)
6/15/2018
C. fragile Light
0.06 ± 0.01
W/MW: 0.1 (NS)
7/1/2018 C. fragile
Dark
2.01 ± 1.2
HI – H (Adult)
7/1/2018
C. fragile Light
BD
W/MW: 0.06 (NS)
7/1/2018
C. fragile Dark
14.4 ± 6.76
HI – H (Juvenile)
7/1/2018
C. fragile Light
BD
W/MW: 0.06 (NS)
6/24/2018 C. sinuosa
Phaeophyceae
(Brown)
Dark
20.1 ± 5.13
HI – DG
6/24/2018
C. sinuosa Light
BD
WT: 0.06 (NS)
6/24/2018
C. sinuosa Dark
BD
HI – H
6/24/2018
C. sinuosa Light
14.6 ± 6.1
W/MW: 0.06 (NS)
7/3/2018 Cystoseira
Dark
6.45 ± 3.13
D – DG
7/3/2018 Cystoseira
Light
2.45 ± 0.61
W/MW: 0.4 (NS)
7/15/2018
C. sinuosa Dark
52.3 ± 8.59
D – DG
7/15/2018
C. sinuosa Light
5.75 ± 2.81
WT: 0.02 (*)
7/15/2018 S. horneri
Dark
28.7 ± 0.96
D – DG
7/15/2018 S. horneri
Light
58.4 ± 15.4
W/MW: 0.1 (NS)
98
Supplementary Table 2. Theoretical calculations estimating potential biological nitrogen fixation
(BNF) contributions (%) to nitrogen (N) immobilization in post-incubation detritus (TF). Due to a
lack in consistent sampling coverage with macroalgal tissue prior to the start of acetylene reduction
incubations (T0), only the dataset from the 2017 18-days decomposition experiments with red (A.
taxiformis, Plocamium), green (C. fragile), and brown (Cystoseira, Dictyota) macroalgae were
used for these calculations. Individual values of % carbon (C), % N, BNF rates, and length of
incubation (hours) were used for each biological replicate when carrying out the calculation and
only replicates that resulted in Δ g N ≥ 0.001 (1 mg) were used to calculate the BNF contribution
to N immobilization (%). If more than one replicate was viable, the data for the 3 calculations
represent an average of all the replicates.
All calculations are based on 1 g (dry weight, dw) of macroalgal detritus
Δ g N (g N) = ((% N of TF/100) × 1 g (dw)) - ((% N of T0/100) × 1 g (dw))
BNF production (g N) = (BNF rates (nmol N * g(dw)
-1
* hour
-1
)) × 1 g (dw) × length of incubation (hours) ×
(14.001 g of N/1,000,000,000 nmol of N)
BNF Contribution to N Immobilization (%) = BNF production (g N) × (Δ g N)
-1
× 100
Species Day Light # of
Replicates
Δ g N
(mg N)
BNF production
(mg N)
BNF Contribution to
N Immobilization
(%)
A. taxiformis 5 Dark 1 4.58 < 0.01 < 0.1
A. taxiformis 5 Light 1 3.45 0.022 0.64
Cystoseira 12 Dark 3 3.55 0.094 2.72
Cystoseira 12 Light 3 3.58 0.091 3.49
Cystoseira 15 Dark 3 6.62 0.093 1.44
Cystoseira 15 Light 3 6.99 0.118 1.66
Cystoseira 18 Light 1 13.68 0.052 0.38
Dictyota 5 Dark 1 1.51 0.084 5.54
Dictyota 5 Light 2 1.85 0.084 4.06
Dictyota 15 Dark 3 9.03 0.117 1.32
Dictyota 15 Light 3 4.14 0.093 3.13
Dictyota 18 Dark 3 18.51 0.039 0.22
Dictyota 18 Light 2 14.83 0.025 0.17
Plocamium 5 Dark 2 4.48 < 0.01 < 0.1
Plocamium 5 Light 3 2.97 < 0.01 < 0.1
Plocamium 12 Dark 1 4.42 < 0.01 < 0.1
Plocamium 12 Light 2 1.14 < 0.01 < 0.1
Plocamium 15 Dark 1 5.74 < 0.01 < 0.1
Plocamium 15 Light 1 2.47 < 0.01 < 0.1
Plocamium 18 Dark 3 28.51 < 0.01 < 0.1
Plocamium 18 Light 3 31.45 < 0.01 < 0.1
99
Supplementary Table 3. Mean % carbon (C), % nitrogen (N), and C:N ratios ± SE for dark/light
(D/L) incubations from the 2017 18-days decomposition experiment with red (A. taxiformis,
Plocamium), green (C. fragile), and brown (Cystoseira, Dictyota) macroalgae, 2017 16-days
decomposition experiments with brown macroalgae (Cystoseira, Dictyota, Dictyopteris, Z.
farlowii), and 2017 short term experiment with drifting and substrate bound red macroalga
(Laurencia).
Experiment Species Day Light % C % N C:N ratio
Decomposition Cystoseira
0 Dark
27.8 ± 0.4 1.26 ± 0.06 22.3 ± 1.3
Decomposition Cystoseira
0 Light
28.6 ± 0.63 1.26 ± 0.01 22.6 ± 0.58
Decomposition Cystoseira
5 Dark
29 ± 0.8 1.42 ± 0.08 20.5 ± 0.63
Decomposition Cystoseira
5 Light
30 ± 0.98 1.56 ± 0.1 19.4 ± 1.1
Decomposition Cystoseira
12 Dark
27 ± 0.59 1.74 ± 0.06 15.5 ± 0.4
Decomposition Cystoseira
12 Light
26.6 ± 0.63 1.74 ± 0.07 15.4 ± 1
Decomposition Cystoseira
15 Dark
26.5 ± 0.56 2.06 ± 0.03 12.9 ± 0.28
Decomposition Cystoseira
15 Light
26.5 ± 1 2.09 ± 0.05 12.7 ± 0.5
Decomposition Cystoseira
18 Dark
29 ± 0.35 1.77 ± 0.06 16.5 ± 0.64
Decomposition Cystoseira
18 Light
30.5 ± 1.4 2.5 ± 0.53 13 ± 1.9
Decomposition Dictyota
0 Dark
30.3 ± 1.3 2.03 ± 0.06 14.9 ± 0.31
Decomposition
Dictyota 0 Light
27.5 ± 2 1.68 ± 0.09 16.3 ± 0.41
Decomposition
Dictyota 5 Dark
30.3 ± 1.5 2.18 ± 0.06 13.9 ± 0.37
Decomposition
Dictyota 5 Light
34.1 ± 1.6 2.28 ± 0.06 15 ± 0.32
Decomposition
Dictyota 12 Dark
30.3 ± 1.5 2.55 ± 0.13 11.9 ± 0.64
Decomposition
Dictyota 12 Light
29.2 ± 3 2.52 ± 0.25 11.6 ± 0.54
Decomposition
Dictyota 15 Dark
34.1 ± 1.8 3 ± 0.15 11.4 ± 0.03
Decomposition
Dictyota 15 Light
30.2 ± 0.6 2.51 ± 0.13 12.1 ± 0.84
Decomposition
Dictyota 18 Dark
33.5 ± 0.8 4.45 ± 0.2 7.57 ± 0.45
Decomposition
Dictyota 18 Light
33.8 ± 2.8 4.08 ± 0.004 8.29 ± 0.69
Decomposition A. taxiformis
0 Dark
24.6 ± 1.5 3.94 ± 0.36 6.28 ± 0.2
Decomposition
A. taxiformis 0 Light
20.5 ± 1.5 3.08 ± 0.3 6.68 ± 0.15
Decomposition
A. taxiformis 5 Dark
24.9 ± 1.6 4.42 ± 0.4 5.65 ± 0.15
Decomposition
A. taxiformis 5 Light
24.4 ± 0.84 4.45 ± 0.32 5.52 ± 0.22
Decomposition
A. taxiformis 12 Dark
21.3 ± 0.81 4.01 ± 0.23 5.31 ± 0.09
Decomposition
A. taxiformis 12 Light
20.6 ± 1.7 3.99 ± 0.42 5.18 ± 0.12
Decomposition
A. taxiformis 15 Dark
22.8 ± 1.7 4.41 ± 0.39 5.18 ± 0.08
Decomposition
A. taxiformis 15 Light
20.1 ± 1.6 3.52 ± 0.65 5.98 ± 0.78
Decomposition
A. taxiformis 18 Dark
25.3 ± 0.76 4.69 ± 0.12 5.39 ± 0.1
Decomposition
A. taxiformis 18 Light
NA
NA NA
100
Supplementary Table 3. Continued.
Experiment Species Day Light % C % N C:N ratio
Decomposition Plocamium
0 Dark
25.8 ± 1.7 3.21 ± 0.3 8.09 ± 0.27
Decomposition
Plocamium 0 Light
26.5 ± 1.4 3.52 ± 0.22 7.54 ± 0.19
Decomposition
Plocamium 5 Dark
25.5 ± 1.2 3.74 ± 0.22 6.84 ± 0.11
Decomposition
Plocamium 5 Light
24.7 ± 1.1 3.75 ± 0.097 6.59 ± 0.18
Decomposition
Plocamium 12 Dark
28.4 ± 0.78 3.94 ± 0.28 7.25 ± 0.33
Decomposition
Plocamium 12 Light
28.1 ± 0.84 4.02 ± 0.1 7.01 ± 0.28
Decomposition
Plocamium 15 Dark
26.8 ± 1.1 3.87 ± 0.27 6.95 ± 0.18
Decomposition
Plocamium 15 Light
28 ± 0.72 3.75 ± 0.16 7.46 ± 0.19
Decomposition
Plocamium 18 Dark
33.1 ± 0.87 6.91 ± 0.18 4.79 ± 0.2
Decomposition
Plocamium 18 Light
31.8 ± 1.1 7.21 ± 0.16 4.42 ± 0.19
Decomposition C. fragile
0 Dark
20.5 ± 0.52 1.76 ± 0.06 11.6 ± 0.07
Decomposition
C. fragile 0 Light
21 ± 0.02 1.74 ± 0.03 12 ± 0.2
Decomposition
C. fragile 5 Dark
19.7 ± 0.71 1.64 ± 0.06 12 ± 0.29
Decomposition
C. fragile 5 Light
20.6 ± 0.53 1.66 ± 0.04 12.4 ± 0.09
Decomposition
C. fragile 12 Dark
20.9 ± 0.98 1.9 ± 0.07 11 ± 0.14
Decomposition
C. fragile 12 Light
21.6 ± 0.66 1.9 ± 0.1 11.4 ± 0.51
Decomposition
C. fragile 15 Dark
20.1 ± 1.3 1.86 ± 0.1 10.8 ± 0.09
Decomposition
C. fragile 15 Light
17.9 ± 0.98 1.74 ± 0.1 10.3 ± 0.12
Decomposition
C. fragile 18 Dark
27.6 ± 1.5 2.9 ± 0.13 9.52 ± 0.32
Decomposition
C. fragile 18 Light
27.6 ± 0.25 2.96 ± 0.04 9.33 ± 0.16
Decomposition Cystoseira
0 Dark
32.8 ± 0.69 0.913 ± 0.03 36 ± 1.3
Decomposition
Cystoseira 0 Light
30.5 ± 0.67 0.847 ± 0.03 36 ± 1.9
Decomposition
Cystoseira 3 Dark
31.9 ± 1.6 1.68 ± 0.07 19 ± 0.22
Decomposition
Cystoseira 3 Light
31 ± 1.9 1.63 ± 0.03 19 ± 0.86
Decomposition
Cystoseira 6 Dark
30.2 ± 0.77 1.74 ± 0.18 17.7 ± 1.3
Decomposition
Cystoseira 6 Light
28.5 ± 2.7 1.53 ± 0.1 18.6 ± 0.49
Decomposition
Cystoseira 10 Dark
31.8 ± 0.89 2.85 ± 0.04 11.1 ± 0.22
Decomposition
Cystoseira 10 Light
30.8 ± 0.78 2.52 ± 0.33 12.9 ± 1.8
Decomposition
Cystoseira 14 Dark
30.8 ± 0.74 2.13 ± 0.09 14.5 ± 0.52
Decomposition
Cystoseira 14 Light
31.4 ± 0.55 2.35 ± 0.06 13.4 ± 0.44
Decomposition
Cystoseira 16 Dark
32.1 ± 0.57 3.01 ± 0.26 10.9 ± 1.1
Decomposition
Cystoseira 16 Light
30.3 ± 0.19 2.99 ± 0.15 10.2 ± 0.44
101
Supplementary Table 3. Continued.
Experiment Species Day Light % C % N C:N ratio
Decomposition Dictyota
0 Dark
30.3 ± 1.3 2.03 ± 0.06 14.9 ± 0.31
Decomposition
Dictyota 0 Light
27.5 ± 2 1.68 ± 0.09 16.3 ± 0.41
Decomposition
Dictyota 3 Dark
30.3 ± 1.5 2.18 ± 0.06 13.9 ± 0.37
Decomposition
Dictyota 3 Light
34.1 ± 1.6 2.28 ± 0.06 15 ± 0.32
Decomposition
Dictyota 6 Dark
30.3 ± 1.5 2.55 ± 0.13 11.9 ± 0.64
Decomposition
Dictyota 6 Light
29.2 ± 3 2.52 ± 0.25 11.6 ± 0.54
Decomposition
Dictyota 10 Dark
34.1 ± 1.8 3 ± 0.15 11.4 ± 0.03
Decomposition
Dictyota 10 Light
30.2 ± 0.6 2.51 ± 0.13 12.1 ± 0.84
Decomposition
Dictyota 14 Dark
33.5 ± 0.8 4.45 ± 0.2 7.57 ± 0.45
Decomposition
Dictyota 14 Light
33.8 ± 2.8 2.72 ± 1.4 8.29 ± 0.69
Decomposition
Dictyota 16 Dark
30.3 ± 1.3 2.03 ± 0.06 14.9 ± 0.31
Decomposition
Dictyota 16 Light
27.5 ± 2 1.68 ± 0.09 16.3 ± 0.41
Decomposition
Dictyopteris 0 Dark
30 ± 0.51 1.64 ± 0.13 18.5 ± 1.4
Decomposition
Dictyopteris 0 Light
29.7 ± 0.98 1.85 ± 0.07 16.1 ± 0.26
Decomposition
Dictyopteris 4 Dark
31.4 ± 1.9 2.09 ± 0.07 15 ± 0.67
Decomposition
Dictyopteris 4 Light
27.9 ± 0.74 1.88 ± 0.13 14.9 ± 0.66
Decomposition
Dictyopteris 8 Dark
33.2 ± 0.91 1.91 ± 0.14 17.6 ± 0.95
Decomposition
Dictyopteris 8 Light
32.9 ± 1.1 1.88 ± 0.1 17.7 ± 1.4
Decomposition
Dictyopteris 10 Dark
31.4 ± 2.6 2.22 ± 0.23 14.2 ± 0.75
Decomposition
Dictyopteris 10 Light
31.8 ± 4 1.74 ± 0.08 18.2 ± 01.5
Decomposition
Dictyopteris 14 Dark
33.2 ± 0.41 2.4 ± 0.16 13.9 ± 0.74
Decomposition
Dictyopteris 14 Light
30.5 ± 0.72 2.06 ± 0.17 15 ± 1.2
Decomposition
Dictyopteris 16 Dark
29.1 ± 0.35 1.9 ± 0.15 15.5 ± 0.96
Decomposition
Dictyopteris 16 Light
30.7 ± 0.32 2 ± 0.07 15.4 ± 0.56
Decomposition
Z. farlowii 0 Dark
26.9 ± 0.81 0.891 ± 0.09 30.9 ± 3.6
Decomposition
Z. farlowii 0 Light
27 ± 3.4 0.815 ± 0.09 33.1 ± 3.2
Decomposition
Z. farlowii 4 Dark
34.7 ± 0.29 1.73 ± 0.15 20.4 ± 1.8
Decomposition
Z. farlowii 4 Light
35.5 ± 0.49 1.48 ± 0.04 24.1 ± 0.37
Decomposition
Z. farlowii 8 Dark
32.7 ± 1.3 1.83 ± 0.05 17.8 ± 0.33
Decomposition
Z. farlowii 8 Light
35.8 ± 0.62 2.05 ± 0.02 17.5 ± 0.24
Decomposition
Z. farlowii 10 Dark
32.2 ± 1.5 1.71 ± 0.16 19.1 ± 1.2
Decomposition
Z. farlowii 10 Light
32.3 ± 0.45 1.61 ± 0.11 20.3 ± 1.3
Decomposition Z. farlowii 14 Dark
29.9 ± 2.8 1.33 ± 0.14 22.5 ± 0.42
Decomposition Z. farlowii 14 Light
33.4 ± 1.1 1.17 ± 0.03 28.7 ± 1.6
Decomposition Z. farlowii 16 Dark
32.5 ± 1.9 1.96 ± 0.09 16.6 ± 0.93
Decomposition Z. farlowii 16 Light
34.8 ± 1.3 2.04 ± 0.13 17.1 ± 0.57
Short term Laurencia
7/25/2017 Dark
23.2 ± 0.67 1.19 ± 0.09 19.9 ± 2
Short term
Laurencia 7/25/2017 Light
23.4 ± 0.92 1.78 ± 0.22 13.4 ± 2.2
Short term Laurencia
7/27/2017 Dark
20 ± 1.4 1.26 ± 0.1 16 ± 1.4
Short term
Laurencia 7/27/2017 Light
20.7 ± 1.5 1.32 ± 0.1 15.7 ± 0.43
102
Supplementary Table 4. List of macroalgal samples collected including the species, date, location,
latitude, longitude, notes about the physiological state of the macroalgal tissue, healthy (H) or
degrading (DG), upon collection and whether it was holdfast-intact (HI) or drifitng (D), and
estimated NO3
-
(µM) concentrations derived from sea surface temperatures (Supplementary Figure
1).
Species Date Location Latitude Longitude Notes Estimated
NO3
-
(µM)
A. taxiformis 6/15/2017
Isthmus Reef 33.449 °N 118.489 °W
HI – H
0.089
Plocamium
6/15/2017 Isthmus Reef 33.449 °N 118.489 °W HI – H 0.089
Cystoseira
6/15/2017 Isthmus Reef 33.449 °N 118.489 °W HI – H 0.089
Dictyota
6/15/2017 Lion Head 33.453 °N 118.501 °W HI – H 0.089
C. fragile
6/15/2017 Lion Head 33.453 °N 118.501 °W HI – H 0.089
Cystoseira 7/11/2017
Isthmus Reef 33.449 °N 118.489 °W
HI – H
0.076
Dictyota 7/11/2017
Isthmus Reef 33.449 °N 118.489 °W
HI – H
0.076
Z. farlowii 7/17/2017
Lion Head 33.453 °N 118.501 °W D – DG 0.073
Dictyopteris 7/17/2017
Lion Head 33.453 °N 118.501 °W D – DG 0.073
Laurencia 7/25/2017
Isthmus Reef 33.449 °N 118.489 °W
D – DG
0.074
Laurencia
7/27/2017
Isthmus Reef 33.449 °N 118.489 °W HI – H 0.067
C. fragile
6/15/2018
Chalk Cove 33.445 °N 118.489 °W HI – H 0.064
C. sinuosa 6/24/2018
Isthmus Reef 33.449 °N 118.489 °W HI – DG 0.064
C. sinuosa
6/24/2018
Isthmus Reef 33.449 °N 118.489 °W HI – H 0.064
C. fragile 7/1/2018
Lion Head 33.453 °N 118.501 °W HI – H 0.065
C. fragile
7/1/2018
Lion Head 33.453 °N 118.501 °W HI – H 0.065
Cystoseira 7/3/2018
Isthmus Reef 33.449 °N 118.489 °W D – DG 0.064
C. sinuosa
7/15/2018
Isthmus Reef 33.449 °N 118.489 °W D – DG 0.077
S. horneri 7/15/2018
Isthmus Reef 33.449 °N 118.489 °W D – DG 0.077
C. fragile 7/12/2019
Lion Head 33.453 °N 118.501 °W HI – H 0.066
Ulva 7/12/2019
Port of LA 33.736 °N 118.269 °W HI – H 0.066
103
Supplementary Table 5. Mean biological nitrogen fixation (BNF) rates, expressed as nmol N × g
-
1
(dw) × h
-1
± SE, for dark/light (D/L) incubations from the 2017 18-days decomposition
experiment with two red (A. taxiformis, Plocamium) and two brown (Cystoseira and Dictyota)
macroalgae. BNF rates that were below the detection limit are labeled as BD. The light BNF rate
associated with A. taxiformis on day 5 (†) only shows the mean for biological replicates A and C;
replicate B consisted of the holdfast and fronds, exhibiting much higher nitrogenase activity (21.9
nmol N × g
-1
(dw) × h
-1
) and was excluded from the mean. The two statistical methods, Welch’s
Two Sample t-Test (WT) or Wilcoxon/Mann-Whitney (W/MW) test, used to compare D/L
incubations are accompanied by their specific p-value and significance level in the D/L column: P
< 0.001 (***), P < 0.01 (**), P < 0.05 (*), or not significant (NS). In the event that all BNF rates
were BD, neither statistical methods were utilized and it’s denoted as not applicable (NA).
Day Species Light BNF Rate D/L Species Light BNF Rate D/L
0 A. taxiformis Dark
BD
NA
Plocamium
Dark
BD
NA
0 A. taxiformis Light
BD
Plocamium Light
BD
5 A. taxiformis Dark
0.91 ± 0.3
W/MW Plocamium Dark
0.61 ± 0.2
WT
5 A. taxiformis Light
0.86 †
0.9 (NS) Plocamium Light
0.78 ± 0.32
0.67 (NS)
12 A. taxiformis Dark
0.33 ± 0.09
WT Plocamium Dark
1.22 ± 0.15
WT
12 A. taxiformis Light
0.23 ± 0.07
0.46 (NS) Plocamium Light
1.37 ± 0.23
0.60 (NS)
15 A. taxiformis Dark
0.49 ± 0.11
WT Plocamium Dark
1.72 ± 0.20
WT
15 A. taxiformis Light
0.49 ± 0.13
0.99 (NS) Plocamium Light
1.52 ± 0.14
0.46 (NS)
18 A. taxiformis Dark
BD
NA Plocamium Dark
0.34 ± 0.02
W/MW
18 A. taxiformis Light
BD
Plocamium Light
0.46 ± 0.13
0.9 (NS)
0 Cystoseira Dark
BD
NA
Dictyota
Dark
BD
NA
0 Cystoseira Light
BD
Dictyota Light
BD
5 Cystoseira Dark
55.8 ± 21.4
WT Dictyota Dark
142 ± 12.6
WT
5 Cystoseira Light
63.7 ± 16.7
0.78 (NS) Dictyota Light
151 ± 50.8
0.87 (NS)
12 Cystoseira Dark
186 ± 11.4
WT Dictyota Dark
145 ± 22.4
WT
12 Cystoseira Light
181 ± 85.1
0.96 (NS) Dictyota Light
143 ± 25
0.96 (NS)
15 Cystoseira Dark
159 ± 25.9
WT Dictyota Dark
200 ± 34.7
WT
15 Cystoseira Light
200 ± 53.5
0.54 (NS) Dictyota Light
158 ± 20.4
0.37 (NS)
18 Cystoseira Dark
80.4 ± 13.2
WT Dictyota Dark
68.8 ± 5.86
WT
18 Cystoseira Light
75.9 ± 13.4
0.82 (NS) Dictyota Light
47.3 ± 4.78
0.05 (*)
104
Supplementary Table 6. Mean biological nitrogen fixation (BNF) rates, expressed as nmol N × g
-
1
(dw) × h
-1
± SE, for control and sodium molybdate (SM) additions and its inhibition (no inhibition
– NI) on nitrogenase activity under dark/light (D/L) treatment during the 2017 16-days
decomposition experiment of brown macroalgae (Cystoseira and Dictyota). % Inhibition was
calculated as (Control BNF Rate – SM BNF Rate) * (Control BNF Rate)
-1
* 100. Tukey’s HSD
(THSD) or medpb (MPB) were used for multiple comparisons between the different groups when
applicable. The statistical method used is specified in the Control D/L column alongisde the p-
value and significance level for the comparison between D/L incubations under control treatment.
The specific p-value and significance level for comparisons between Control/Dark:SM/Dark and
Control/Light:SM/Light are in the Treatment column. Significance levels: P < 0.001 (***), P <
0.01 (**), P < 0.05 (*), or not significant (NS). Not applicable is denoted as NA.
Day Species Light Control BNF
Rate
Control
D/L
SM BNF
Rate
Treatment % I
0
Cystoseira Dark
2.65 ± 0.57
MPB
2.06 ± 0.57
0.92 (NS)
22
0
Cystoseira Light
BD
0.000 (***)
3.21 ± 2.05
0.26 (NS)
NA
3
Cystoseira Dark
310 ± 16.3
THSD
93 ± 7.19
0.003 (**)
70
3
Cystoseira Light
169 ± 50
0.03 (*)
95.1 ± 20.1
0.32 (NS)
44
6
Cystoseira Dark
140 ± 18.6
NA
206 ± 22.1
NA
NI
6
Cystoseira Light
144 ± 42
191 ± 26.4
NI
10
Cystoseira Dark
188 ± 2.89
THSD
73.2 ± 14.7
0.000 (***)
61
10
Cystoseira Light
218 ± 12.5
0.31 (NS)
71.3 ± 10.9
0.000 (***)
67
14
Cystoseira Dark
330 ± 80.4
THSD
259 ± 49.8
0.82 (NS)
22
14
Cystoseira Light
404 ± 44.5
0.81 (NS)
157 ± 51.8
0.07 (NS)
61
16
Cystoseira Dark
155 ± 48.8
NA
99.6 ± 23.1
NA
36
16
Cystoseira Light
172 ± 15
120 ± 25.4
30
0 Dictyota
Dark
BD
MPB
292 ± 2.92
0.000 (***)
NA
0
Dictyota Light
BD
0.74 (NS)
99.6 ± 51.5
0.000 (***)
NA
3
Dictyota Dark
33.4 ± 19
NA
128 ± 35.6
NA
NI
3
Dictyota Light
76.5 ± 21.7
75.2 ± 31.6
1.6
6
Dictyota Dark
339 ± 7.47
THSD
118 ± 22.4
0.002 (**)
65
6
Dictyota Light
396 ± 42.5
0.50 (NS)
118 ± 26.8
0.000 (***)
70
10
Dictyota Dark
170 ± 18.6
THSD
87.9 ± 7.01
0.20 (NS)
48
10
Dictyota Light
125 ± 43.4
0.64 (NS)
70.5 ± 22.9
0.50 (NS)
44
14
Dictyota Dark
192 ± 36.9
THSD
42.7 ± 20.6
0.005 (**)
78
14
Dictyota Light
210 ± 6.37
0.93 (NS)
128 ± 6.57
0.10 (NS)
39
16
Dictyota Dark
77.3 ± 22.3
THSD
25.8 ± 4.28
0.07 (NS)
67
16
Dictyota Light
82.9 ± 7.85
0.99 (NS)
8.89 ± 4.64
0.01 (**)
89
105
Supplementary Table 7. Mean biological nitrogen fixation (BNF) rates, expressed as nmol N × g
-
1
(dw) × h
-1
± SE, for dark/light (D/L) incubations from the 2019 32-days decomposition
experiment of green macroalga C. fragile. BNF rates for sodium molybdate (SM) additions and its
inhibition on nitrogenase activity under D/L treatment are also included. % Inhibition (% I) was
calculated as (Control BNF Rate – SM BNF Rate) * (Control BNF Rate)
-1
* 100. Tukey’s HSD
(THSD) or medpb (MPB) were used for multiple comparisons between the different groups when
applicable. The statistical method used is specified in the Control D/L column alongisde the p-
value and significance level for the comparison between D/L incubations under control treatment.
The specific p-value and significance level for comparisons between Control/Dark:SM/Dark and
Control/Light:SM/Light are in the Treatment column. Significance levels: P < 0.001 (***), P <
0.01 (**), P < 0.05 (*), or not significant (NS).
Day Species Light Control
BNF Rate
Control
D/L
SM BNF
Rate
Treatment % I
0
C. fragile
Dark 5.77 ± 1.07
MPB
1.3 ± 0.6
0.000 (***)
77
0
C. fragile
Light BD
0.000 (***)
0.65 ± 0.3
0.24 (NS)
NA
2
C. fragile
Dark 38.9 ± 4.75
NA
30.1 ± 7.17
NA
23
2
C. fragile
Light 18 ± 3.96
41.9 ± 16.8
NI
4
C. fragile
Dark 62.6 ± 13.6
MPB
44.6 ± 12.5
0.40 (NS)
29
4
C. fragile
Light 41.5 ± 22.9
0.39 (NS)
2.9 ± 0.95
0.000 (***)
93
7
C. fragile
Dark 44.4 ± 3.71
MPB
20.8 ± 9.09
0.03 (*)
53
7
C. fragile
Light 0.87 ± 0.19
0.000 (***)
1.9 ± 0.49
0.09 (NS)
NI
10
C. fragile
Dark 49.6 ± 11.2
MPB
15.4 ± 5.57
0.02 (*)
69
10
C. fragile
Light 5.03 ± 2.93
0.000 (***)
0.87 ± 0.33
0.60 (NS)
83
13
C. fragile
Dark 173 ± 46.8
NA
362 ± 162
NA
NI
13
C. fragile
Light 123 ± 34.2
56.2 ± 38.9
54
17
C. fragile
Dark 226 ± 49.2
MPB
236 ± 15.7
0.79 (NS) NI
17
C. fragile
Light 33.4 ± 11.2
0.000 (***)
55 ± 15.3
0.24 (NS) NI
22
C. fragile
Dark 89.6 ± 25.7
NA
115 ± 25.8
NA NI
22
C. fragile
Light 126 ± 65.4
72.6 ± 49.2
42
26
C. fragile
Dark 103 ± 47.3
NA
124 ± 43.1
NA NI
26
C. fragile
Light 26.3 ± 12
210 ± 79.2
NI
32
C. fragile
Dark 31 ± 14.7
NA
6.98 ± 2.25
NA
77
32
C. fragile
Light 7.8 ± 1.6
45.8 ± 11.1
NI
106
Supplementary Table 8. Mean biological nitrogen fixation (BNF) rates, expressed as nmol N × g
-
1
(dw) × h
-1
± SE, for sodium molybdate (SM) additions and its inhibition on nitrogenase activity
under dark/light (D/L) treatment during the 2019 32-days decomposition experiment of green
macroalga Ulva. % Inhibition (% I) was calculated as (Control BNF Rate – SM BNF Rate) *
(Control BNF Rate)
-1
* 100. Tukey’s HSD (THSD) or medpb (MPB) were used for multiple
comparisons between the different groups when applicable. The statistical method used is specified
in the Control D/L column alongisde the p-value and significance level for the comparison between
D/L incubations under control treatment. The specific p-value and significance level for
comparisons between Control/Dark:SM/Dark and Control/Light:SM/Light are in the Treatment
column. Significance levels: P < 0.001 (***), P < 0.01 (**), P < 0.05 (*), or not significant (NS).
Day Species Light Control
BNF Rate
Control
D/L
SM BNF Rate Treatment % I
0
Ulva
Dark 0.54 ± 0.30
NA
BD
NA NA
0
Ulva
Light BD
BD
NA
2
Ulva
Dark 18.7 ± 3.7
MPB
5.18 ± 0.31
0.000 (***)
72
2
Ulva
Light 2.26 ± 0.85
0.000 (***)
2.68 ± 0.88
0.89 (NS)
NI
4
Ulva
Dark 44.5 ± 15.2
MPB
2.23 ± 1.79
0.000 (***)
95
4
Ulva
Light 3.86 ± 1.51
0.000 (***)
2.86 ± 1.18
0.77 (NS)
26
7
Ulva
Dark 26.5 ± 4.69
NA
24.1 ± 12.5
NA
8.8
7
Ulva
Light 14.7 ± 14.6
0.49 ± 0.1
97
10
Ulva
Dark 17.1 ± 5.16
MPB
3.46 ± 1.42
0.000 (***)
80
10
Ulva
Light 3.89 ± 2.07
0.000 (***)
BD
0.26 (NS)
NA
13
Ulva
Dark 9.68 ± 3.84
MPB
1.38 ± 0.79
0.000 (***)
86
13
Ulva
Light BD
0.000 (***)
BD
0.26 (NS)
NA
17
Ulva
Dark 15.2 ± 7.79
MPB
1.93 ± 0.76
0.000 (***)
87
17
Ulva
Light BD
0.000 (***)
3.75 ± 1.38
0.000 (***)
NA
22
Ulva
Dark 43.5 ± 13.3
MPB
5.91 ± 1.1
0.000 (***)
86
22
Ulva
Light BD
0.000 (***)
0.27 ± 0.12
0.22 (NS) NA
32
Ulva
Dark BD
NA
BD
NA NA
32
Ulva
Light BD
BD
NA
107
Supplementary Table 9. Mean biological nitrogen fixation (BNF) rates, expressed as nmol N × g
-
1
(dw) × h
-1
± SE, for dark/light (D/L) incubations from the 2017 18-days decomposition
experiment with green macroalga C. fragile. Welch’s Two Sample t-Test (WT) or
Wilcoxon/Mann-Whitney (W/MW) test were used to compare D/L incubations and are
accompanied by their specific p-value and significance level in the D/L column. Significance
levels: P < 0.001 (***), P < 0.01 (**), P < 0.05 (*), or not significant (NS).
Day Species Light Control BNF
Rate
D/L
0
C. fragile
Dark 9.46 ± 5.27
W/WM
0
C. fragile
Light BD
0.06 (NS)
5
C. fragile
Dark 51 ± 12.2
WT
5
C. fragile
Light 0.77 ± 0.11
0.05 (*)
12
C. fragile
Dark 17.8 ± 11.7
WT
12
C. fragile
Light 30.3 ± 25.5
0.69 (NS)
15
C. fragile
Dark 5.26 ± 2.97
WT
15
C. fragile
Light 3.96 ± 1.35
0.72 (NS)
18
C. fragile
Dark BD
NA
18
C. fragile
Light BD
108
Supplementary Table 10. Mean biological nitrogen fixation (BNF) rates, expressed as nmol N ×
g
-1
(dw) × h
-1
± SE, for dark/light (D/L) incubations from the 2017 16-days decomposition
experiment with two drifting brown macroalgae: Dictyopteris and Z. farlowii. Welch’s Two
Sample t-Test (WT) or Wilcoxon/Mann-Whitney (W/MW) test were used to compare D/L
incubations and are accompanied by their specific p-value and significance level in the D/L
column. Significance levels: P < 0.001 (***), P < 0.01 (**), P < 0.05 (*), or not significant (NS).
Day Species Light BNF Rate D/L
0
Dictyopteris Dark
152 ± 78.2
WT: 0.99 (NS)
0
Dictyopteris Light
154 ± 21.2
4
Dictyopteris Dark
204 ± 29.2
WT: 0.22 (NS)
4
Dictyopteris Light
150 ± 22.2
8
Dictyopteris Dark
63.4 ± 30.1
WT: 0.74 (NS)
8
Dictyopteris Light
79.1 ± 32.8
10
Dictyopteris Dark
20.2 ± 10.2
WT: 0.36 (NS)
10
Dictyopteris Light
45.2 ± 20.9
14
Dictyopteris Dark
93.8 ± 31.5
WT: 0.57 (NS)
14
Dictyopteris Light
68.9 ± 24.1
0 Z. farlowii
Dark
88 ± 27.4
WT: 0.66 (NS)
0
Z. farlowii Light
104 ± 17.9
4
Z. farlowii Dark
273 ± 28.4
WT: 0.65 (NS)
4
Z. farlowii Light
289 ± 14.6
8
Z. farlowii Dark
279 ± 30
WT: 0.04 (*)
8
Z. farlowii Light
114 ± 43.8
10
Z. farlowii Dark
26.5 ± 10
WT: 0.55 (NS)
10
Z. farlowii Light
35.1 ± 8.48
14
Z. farlowii Dark
48.6 ± 17.4
WT: 0.12 (NS)
14
Z. farlowii Light
2.02 ± 0.982
Supplementary Table 10.1. Excess biomass of both macroalgae on day 16 allowed for sodium
molybdate (SM) amendments parallel to control treatments. % Inhibition (%I) was calculated as
(Control BNF Rate – SM BNF Rate) * (Control BNF Rate)
-1
* 100. Tukey’s HSD (THSD) or
medpb (MPB) were used for multiple comparisons between the different groups. The statistical
method used is in the Control D/L column alongisde the p-value and significance level for the
comparison between Control/Dark:Control/Light. The specific p-value and significance level for
comparisons between Control/Dark:SM/Dark and Control/Light:SM/Light are in the Treatment
column. Significance levels: P < 0.001 (***), P < 0.01 (**), P < 0.05 (*), or not significant (NS).
Day Species Light Control
BNF Rate
Control
D/L
SM BNF
Rate
Treatment % I
16
Dictyopteris Dark
234 ± 84.5
MPB
40.9 ± 4.91
0.000 (***)
82
16
Dictyopteris Light
305 ± 86.1
0.62 (NS)
84.1 ± 30
0.000 (***)
72
16 Z. farlowii Dark 693 ± 63.3 MPB 225 ± 69.4 0.000 (***) 68
16 Z. farlowii Light 520 ± 132 0.40 (NS) 233 ± 9.06 0.000 (***) 55
109
Supplementary Table 11. Principal component analysis (PCA) results including the factor loadings
for the different variables (% carbon (C), % nitrogen (N), C:N ratio, and biological nitrogen
fixation (BNF) rate), eigenvalues, standard deviation, proportion of variance, and cumulative
proportion. Principal component 1 and 2 account for ~79% of the variance in the dataset. The first
component is negatively correlated with % N and positively correlated with the C:N ratio. The
second component is most correlated with % C and BNF rate, both in a negative direction. The
bold values represent the highly significant (> |0.5|) factor loadings of the variables for the different
PCA axes.
Factor Loadings
Variables PC1 PC2 PC3 PC4
% C 0.3509766 -0.6671762 0.5645765 -0.3360726
% N -0.5735945 -0.4748052 0.1783777 0.6432191
C:N ratio 0.6304053 0.2555806 0.3064767 0.6658376
BNF rate 0.3878065 -0.5139208 -0.7453233 0.1731611
Eigenvalues 2.0135011 1.1555674 0.6984755 0.1324560
Standard deviation 1.4190 1.0750 0.8357 0.36395
Proportion of Variance 0.5034 0.2889 0.1746 0.03311
Cumulative Proportion 0.5034 0.7923 0.9669 1.00000
110
Chapter 4: Conclusion and Future Directions
Nitrogen is an essential macronutrient required by all living organisms. Despite making up
~78 % of the Earth’s atmosphere, this large reservoir of nitrogen gas (N2) is virtually inaccessible
to most organisms, resulting in nitrogen limitation across many terrestrial and marine ecosystems
(Vitousek and Howarth, 1991). Biological nitrogen fixation (BNF) is the process in which
specialized microbes, known as nitrogen fixers or diazotrophs, use the nitrogenase enzyme to
catalyze the conversion of N2 gas into a biologically usable form of nitrogen. In this way,
diazotrophs play an essential role in the global food web by directly contributing a new source of
bioavailable nitrogen that other organisms can utilize (Sohm et al., 2011). Many diazotrophs are
also cyanobacteria, photosynthetic organisms that further play a role as primary producers in the
base of the food web. This has guided most of our research efforts over the last decades (Figure
1), with the vast majority of N2 fixation studies investigating the sunlit portion of our global oceans,
particularly the extensive, oligotrophic open ocean regions in the tropics (Zehr and Capone, 2020).
Figure 1. Current perspective on marine N2 fixation. Adapted from Zehr and Capone (2020).
111
Due to appreciable amounts of nitrogen (e.g. nitrate, ammonium) in the neritic zone, N2
fixation has not been a primary focus of investigation. This thesis identified diazotrophic
macroalgal associations (DMAs), using acetylene reduction assays, to persist in the coastal waters
around Santa Catalina Island, particularly during periods of nitrogen deficiency. Not surprisingly,
cyanobacterial DMAs have been observed globally, most often with living macroalgae from all 3
major clades (Capone et al., 1977; Hanson, 1977; Rosenberg and Paerl, 1981; Gerard et al., 1990;
Tilstra et al., 2017), to alleviate nitrogen limitation across different macroalgal ecosystems. At the
moment, direct molecular studies (e.g., nifH amplicon sequencing) characterizing the diazotrophic
communities associated with living macroalgae remain sparse. The macroalgal samples collected
for BNF rate measurements for this thesis will provide a unique opportunity to carry out nifH
amplicon sequencing in the future to link biogeochemical measurements with the diazotrophic
community composition associated with various living macroalgae.
Living macroalgae require other macronutrients, e.g., phosphorous (Pedersen et al., 2010),
and micronutrients, e.g., cobalt, copper, iron, manganese, zinc (Kuwabara, 1982; Viaroli et al.,
2005), which can also limit their growth and productivity. The same nutrients (e.g., phosphorous)
and trace metals (e.g., iron) are recognized to be critically important to diazotrophs and can also
limit their activity and define their biogeographical distributions (Sañudo-Wilhelmy et al., 2001;
Sohm et al., 2008, 2011; Ward et al., 2013). The influence of these macro- and micro- nutrients
on diazotrophic activity associated with living macroalgae was not investigated in this thesis.
Interestingly, it has already been established that low trace metal concentrations (e.g., cobalt and
manganese limitation) in Southern California waters can directly impede on growth rates of
macroalgal gametophytes such as the endemic giant kelp, Macrocystis pyrifera (Kuwabara, 1982;
Manley, 1984). Considering the high metal (e.g., iron) requirement for the nitrogenase
112
metalloenzyme (Howard and Rees, 1996), it is possible that trace metal limitation could influence
lower diazotrophic activity associated with living macroalgae observed in this study. More
specifically, we hypothesize that DMAs within macroalgal habitats in iron limited regions could
exhibit lower biological nitrogen fixation rates compared to DMAs found in ecosystems with
greater iron availability.
However, it is important to consider that co-limitation of several different nutrients could
also influence diazotrophic activity. Indeed, there are vast regions of the Atlantic Ocean where
phosphorus and iron limitation or co-limitation have been well investigated and shown to impact
diazotrophic activity of various cyanobacteria including Trichodesmium and Crocosphaera
(Sañudo-Wilhelmy et al., 2001; Mills et al., 2004; Saito et al., 2011; Walworth et al., 2016).
Pelagic brown macroalgae, Sargassum spp., also inhabit the same waters and are similarly
impacted by nutrient (e.g., phosphorous, nitrogen) limitation (Lapointe, 1995). Moreover,
diazotrophic associations in these pelagic Sargassum communities can be a critical source of fixed
nitrogen with contributions of 40 % to excess of 100 % of required nitrogen by Sargassum spp.
(Hanson, 1977; Phlips et al., 1986). Thus, it is very likely that co-limitation by multiple nutrients
(e.g., phosphorus, iron) are important controlling factors for DMAs with living macroalgae
throughout the global oceans such as pelagic Sargassum communities in the Atlantic and warrants
attention in future investigations.
Interestingly, significantly higher BNF rates, up to two orders of magnitude higher
(Hamersley et al., 2015), have been observed with decomposing macroalgal tissue (Hanson, 1977;
Zuberer and Silver, 1978). This thesis, for the first time, provides a more comprehensive insight
into the prevalence of diazotrophic associations throughout macroalgal decomposition and
proposes macroalgal detrital systems as an overlooked, potentially global, niche for heterotrophic
113
N2 fixers. Recent oceanographic voyages have employed molecular techniques to uncover an
ample and diverse community of heterotrophic, non-cyanobacterial diazotrophs (NCDs)
throughout the global oceans (Bombar et al., 2016; Moisander et al., 2017). However, beyond
identifying the presence of these microbes, we are still lacking a fundamental understanding of the
ecology, physiology, and biogeochemical impact of these heterotrophic N2 fixers, resulting in an
incomplete understanding of the global marine nitrogen cycle.
Abundant nifH phylotypes of heterotrophic diazotrophs from the Gammaproteobacteria
clade during a macroalgal bloom (Zhang et al., 2015) corroborate the importance of NCDs
recognized via BNF rate measurements during macroalgal decomposition (Hamersley et al., 2015;
Raut et al., 2018; Raut and Capone, 2021). Furthermore, the cosmopolitan distribution of
macroalgae along global coastlines adds another ecological niche to recent efforts which are
uncovering the relevance of heterotrophic N2 fixation in coastal waters (Mulholland et al., 2012;
Messer et al., 2016) and sediments (Fulweiler et al., 2013; Newell et al., 2016). Taken together,
these results suggest DMAs might serve as a potential model system to explore heterotrophic N2
fixation.
In fact, utilizing previously published empirical data throughout decomposition of various
macrophytes (Figure 2), we propose a theoretical three-step model of diazotrophic activity
throughout marine particulate organic matter (POM) degradation: 1) initially lower nitrogenase
activity coinciding with rapid microbial colonization, 2) an intermediate period of relatively higher
BNF rates as much of the labile carbon and nitrogen is heavily utilized by the microbial
community, and 3) return to lower diazotrophic activity due to labile carbon limitation from the
refractory nature of the remaining detrital particle. Indeed, a robust mathematical model recently
reported sinking marine particles to provide a suitable niche for heterotrophic, NCDs (Chakraborty
114
et al., 2021). In this model, phase I is characterized by high cellular growth rates and phase II is
distinguished by high community respiration and an organic solute trail comprised of excess
carbon and nitrogen from degradation of the POM detritus (Chakraborty et al., 2021). These two
phases are comparable to our first phase which predicts accelerated leaching of particulate organic
Figure 2. Compilation of empirical observations of nitrogenase activity throughout
decomposition of various macrophytes. A) Mangrove leaf litter decomposition: blue diamonds
– Avicennia marina detritus (Mann and Steinke, 1992), red squares – A. germinans, Rhizophora
mangle (Pelegri and Twilley, 1998), brown triangles – Ceriops tagal, R. mucronata during dry
season (Woitchik et al., 1997), and green circles – Ceriops tagal, R. mucronata during rainy
season (Woitchik et al., 1997). B) Brown macroalgae decomposition: squares – Zonaria
farlowii and Dictyopteris (Raut and Capone, 2021), “X” – Sargassum horneri (Raut et al.,
2018), circles and triangles – Cystoseira and Dictyota (Raut and Capone, 2021), “*” –
Macrocystis pyrifera (Hamersley et al., 2015), and diamonds – S. horneri and S. palmeri (Raut
et al., 2018). C) Red and green macroalgal decomposition: red square (Asparagopsis
taxiformis) and circle (Plocamium) and green “X” (Codium fragile) and triangle (Ulva) (Raut
and Capone, 2021).
115
carbon and particulate organic nitrogen to support rapid microbial colonization. Similarly, both
models suggest minimal nitrogenase activity during the early phase of POM decomposition, most
likely because diazotrophs are outcompeted by faster growing microbes for available resources,
namely labile carbon (Chakraborty et al., 2021).
Interestingly, phase III (Chakraborty et al., 2021) and the second phase of our model both
predict increased diazotrophic activity due to several factors: a) microbial community depletes
labile nitrogen faster than labile carbon, providing an advantage to NCDs which can supply their
own nitrogen source for growth, b) formation of anoxic zones in the detrital particle due to high
community respiration which prevents O2 inactivation of the nitrogenase enzyme, and c) ability to
use other electron acceptors (e.g. NO3
-
, SO4
2-
) to carry out respiration due to insufficient O2
concentrations. The latter suggests a potentially important role for sulfate reducing diazotrophs
during POM decomposition which has been confirmed during macroalgal decomposition (Zhang
et al., 2015; Raut et al., 2018; Raut and Capone, 2021). Notably, sulfate reducing diazotrophs have
also been identified with marine sinking particles and can even dominate the diazotrophic
community, contributing up to 87% of the nifH sequences (Farnelid et al., 2019), further
corroborating this hypothesis.
The third phase in our model predicts a decrease in nitrogenase activity due to labile carbon
limitation. Similarly, the output from the final phase (IV) of the mathematical model attributes the
reduction in nitrogenase activity to an inability to maintain low O2 to perform BNF due to
insufficient labile carbon (Chakraborty et al., 2021). Furthermore, the refractory nature of the
remaining detritus results in a decrease in bacterial populations, reducing community respiration
and allowing O2 influx to excess consumption, which further eliminates the microaerobic niche
necessary to support nitrogenase activity. Although the availability of labile carbon has been
116
thoroughly investigated, the impact of nutrient limitation by other key elements, e.g., phosphorous
and iron, on the diazotrophic community throughout macroalgal degradation was not explored in
this thesis. There are multiple reports of decreasing phosphorous and nitrogen concentrations
throughout decomposition of macroalgae and other coastal macrophytes (Paalme et al., 2002;
Banta et al., 2004). Furthermore, it has been suggested that iron release during macroalgal bloom
decomposition alongside sulfide production can yield insoluble iron sulfide compounds resulting
in overall decrease in soluble iron leading to iron limitation (Viaroli et al., 2005). Combined with
the numerous reports of phosphorous and iron limitation on diazotrophic activity as discussed
previously, we hypothesize that the decreasing availability of these nutrients throughout
macroalgal decomposition could similarly limit diazotrophic activity as observed with labile
carbon limitation.
More direct molecular studies employing nifH amplicon sequencing and ‘omics-based
approaches are necessary to investigate the taxonomic diversity of diazotrophs throughout
different macroalgal life stages and during decomposition. The macroalgal detritus subsampled
throughout long term (16 – 32 days) litter bag decomposition and degrading macroalgal tissue
sampled from coastal sites for this thesis will provide an additional opportunity to expand our
fundamental understanding of DMAs. In the future, sequencing this sample set will allow us to
identify which microbes have the metabolic capability for N2 fixation as indicated by the presence
of nifH genes, encoding for the nitrogenase enzyme. In light of recent efforts to better understand
the role of NCDs in the global oceans (Bombar et al., 2016), identifying the diazotrophic diversity
will be crucial to determine whether macroalgal detrital systems are indeed a model system for
marine heterotrophic N2 fixation, a potentially important and uninvestigated part of the marine
nitrogen cycle.
117
Lastly, a combination of biogeochemical rate measurements and molecular studies suggest
the benthic microbial community found in marine sediments of various macrophyte systems also
host an ample and diverse assemblage of heterotrophic diazotrophs (Capone, 1982; O’Donohue et
al., 1991; Welsh et al., 1996; Romero et al., 2015; Sun et al., 2015; Lehnen et al., 2016). Previous
investigations have reported increased availability of organic matter (e.g. burrowing shrimp,
macrophyte deposition) can stimulate benthic BNF rates (Chisholm and Moulin, 2003; Bertics et
al., 2010). Considering a substantial portion of macroalgal productivity is exported to the benthos
(Krumhansl and Scheibling, 2012; Krause-Jensen and Duarte, 2016), this raises questions about
how macroalgal detritus may impact benthic BNF. It is likely that remineralization of macroalgal
detritus in marine sediments could further support benthic heterotrophic N2 fixation, particularly
in productive coastal macroalgal ecosystems, and also contribute significantly to the global
nitrogen budget. However, much more extensive surveys of benthic heterotrophic N2 fixation are
necessary to approximate its impact to the global nitrogen cycle.
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Abstract (if available)
Abstract
Macroalgae, or seaweed, are cosmopolitan organisms that populate global coastlines ranging from the poles to the tropics. They contribute significantly to photosynthetic primary production and create diverse habitats, providing food and shelter for a wide array of micro- and macro-organisms. Macroalgae require nitrogen (N) and typically obtain N (e.g., ammonium, nitrate) via direct uptake from the surrounding water. Biological nitrogen fixation (BNF) is the process in which specialized prokaryotes, termed diazotrophs, use the nitrogenase enzyme to convert dinitrogen (N₂) gas into a bioavailable form of N. The most striking advantage for macroalgae to host diazotrophs would be direct access to a N source. While microbial macroalgal interactions have been well documented and identified to play an important role in the life cycle of macroalgae, the prevalence of diazotrophic macroalgal associations (DMAs) are not well characterized, especially in coastal ecosystems. ❧ BNF rates were measured, using the acetylene reduction assay (ARA), with an invasive benthic brown seaweed, Sargassum horneri, around Santa Catalina Island, CA. BNF associated with S. horneri was absent throughout most of its life cycle, but higher BNF rates, supporting ~3 – 36% of the required N demand of juvenile S. horneri during the late summer suggests BNF may be important during periods of N deficiency. A larger investigation consisting of macroalgae from all 3 clades corroborates this hypothesis, as DMAs persisted during periods of low nitrate availability in the waters surrounding Santa Catalina Island. While it is unlikely that these lower BNF rates provide a significant contribution towards the host macroalgal N demand, these results further solidify that DMAs with living macroalgae are also prevalent in coastal environments. ❧ Seaweeds continue to serve a myriad of ecological functions after death as they enter the detrital food web, support benthic coastal ecosystems, and contribute to carbon (C) sequestration. Interestingly, diazotrophic activity was more pronounced throughout the decomposition of S. horneri, suggesting another role for N₂ fixers during the microbial breakdown of organic rich macroalgal detritus. Diazotrophic activity was measured using the ARA during several long-term litter bag decomposition experiments with a diverse set of brown, green, and red macroalgae. The results suggest that decomposing brown and green macroalgal systems support higher rates of BNF than red macroalgae. ❧ This may largely be linked to the availability of N throughout decomposition, as inferred from the C:N ratio of the aging macroalgal detritus. The higher C:N content of brown macroalgae may result in faster utilization of the available N by the microbial community degrading the macroalgal tissue. This can potentially lead to N limitation throughout decomposition, promoting diazotrophs, who supply their own N, to facilitate the breakdown of the N limited, organic macroalgal detritus. Accordingly, the 2 green and 1 red macroalgae which had higher C:N content also exhibited greater diazotrophic activity throughout decomposition. Lastly, a series of sodium molybdate amendments resulted in severe inhibition of nitrogenase activity, suggesting the importance of sulfate reducing diazotrophs in macroalgal detrital systems. ❧ Taken together, the results from this dissertation greatly expand upon our previously limited understanding of DMAs with both living and decomposing macroalgae, further identifying macroalgal detrital systems as a novel, globally relevant niche for BNF. These data were also used in combination with a literature review of diazotrophic activity during decomposition of other macrophytes to propose a theoretical model that predicts nitrogenase activity throughout macrophyte degradation in three phases. Finally, the results from this thesis raises questions about the influence of macroalgal detritus on benthic diazotrophic communities found in coastal marine sediments.
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The connection of the phosphorus cycle to diazotrophs and nitrogen fixation
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Comparative behavior and distribution of biologically relevant trace metals - iron, manganese, and copper in four representative oxygen deficient regimes of the world's oceans
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The distribution and speciation of copper across different biogeochemical regimes
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Effects of global change on the physiology and biogeochemistry of the N₂-fixing cyanobacteria Trichodesmium erythraeum and Crocosphaera watsonii
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Marine biogenic halocarbons: potential for heterotrophic bacterial production and seasonality at San Pedro Ocean Time-series
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Photophysiological parameters for CO2 and N2 fixation in trichodesmium spp. in natural populations and culture nutrient limitation experiments
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Oxygen uptake rates in the thermocline of coastal waters: assessing the role of carbon and inorganic nutrient inputs
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The distributions and geochemistry of iodine and copper in the Pacific Ocean
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Unexplored microbial communities in marine sediment porewater
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Asset Metadata
Creator
Raut, Yubin
(author)
Core Title
Biological nitrogen fixation associated with living and decomposing macroalgae
School
College of Letters, Arts and Sciences
Degree
Doctor of Philosophy
Degree Program
Ocean Sciences
Degree Conferral Date
2021-12
Publication Date
10/11/2021
Defense Date
06/22/2021
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
coastal ecosystems,decomposition,heterotrophic,macroalgae,microbes,nitrogen fixation,OAI-PMH Harvest
Format
application/pdf
(imt)
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Capone, Douglas G. (
committee chair
), Heidelberg, John (
committee member
), Hutchins, David A. (
committee member
), Sañudo-Wilhelmy, Sergio (
committee member
)
Creator Email
yubinrau@usc.edu,yubinraut@gmail.com
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-oUC16208009
Unique identifier
UC16208009
Legacy Identifier
etd-RautYubin-10159
Document Type
Dissertation
Format
application/pdf (imt)
Rights
Raut, Yubin
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 author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given.
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
Repository Email
cisadmin@lib.usc.edu
Tags
coastal ecosystems
decomposition
heterotrophic
macroalgae
microbes
nitrogen fixation