VARIABILITY IN BIOMASS DECAY RATES AND NUTRIENT LOSS IN BLOOM-FORMING MACROALGAL SPECIES

Decaying macroalgae release nitrogen and other nutrients into the surrounding marine environment, providing nutrients for future generations of primary producers, as well as fueling a complex web of decomposer microorganisms. There are relatively few studies that examine macroalgal decomposition rates in areas impacted by macroalgal blooms, although fast-growing macroalgal bloom species typically decay more quickly than slow-growing perennial species. We studied whole tissue, organic content, and nutrient decay rates for five macroalgal species in Narragansett Bay, RI in the summer of 2010 using an intertidal litterbag design; four of these species are frequently present in macroalgal blooms in this system. Our results, which we present as logistic decay rates (-k d-), illustrate that the red alga Gracilaria vermiculophylla decomposes most rapidly, followed by green algae (Ulva rigida, Ulva compressa) and the red alga Agardhiella subulata; decay rates for these four species were significantly higher than that for the perennial, non-bloom forming brown alga Fucus vesiculosus. We did not observe refractory pools for any species, though we did not follow F. vesiculosus through the end of its decay process. Decay rates were dependent upon water temperature, with faster decomposition rates occurring during peak temperatures. We speculate that the slow decomposition of F. vesiculosus may be attributable to its relatively


Chapter 1
Variability in biomass decay rates and nutrient loss in bloom-forming macroalgal species

Abstract
Decaying macroalgae release nitrogen and other nutrients into the surrounding marine environment, providing nutrients for future generations of primary producers, as well as fueling a complex web of decomposer microorganisms.
There are relatively few studies that examine macroalgal decomposition rates in areas impacted by macroalgal blooms, although fast-growing macroalgal bloom species typically decay more quickly than slow-growing perennial species. We studied whole tissue, organic content, and nutrient decay rates for five macroalgal species in Narragansett Bay, RI in the summer of 2010 using an intertidal litterbag design; four of these species are frequently present in macroalgal blooms in this system. Our results, which we present as logistic decay rates (-k d-1 ), illustrate that the red alga Gracilaria vermiculophylla decomposes most rapidly, followed by green algae (Ulva rigida, Ulva compressa) and the red alga Agardhiella subulata; decay rates for these four species were significantly higher than that for the perennial, non-bloom forming brown alga Fucus vesiculosus. We did not observe refractory pools for any species, though we did not follow F. vesiculosus through the end of its decay process. Decay rates were dependent upon water temperature, with faster decomposition rates occurring during peak temperatures. We speculate that the slow decomposition of F. vesiculosus may be attributable to its relatively high cell wall phenolics, which have been shown in previous studies to retard decay by microorganisms. We observed nitrogen spikes during initial decay of F. vesiculosus due to chemical or biological immobilization of allochthonous nitrogen in the algal tissue. Nitrogen and organic material were lost from red species at a faster rate than green or brown species, likely due to faster leaching and/or greater decomposer action. Initial δ 15 N values varied greatly among species, collection sites, and collection dates and generally did not exhibit predictable changes over the period of decay. Our results are of particular importance in eutrophied systems, where shifting productivity regimes may lead to changes in total nutrient cycling rates, and where changes in stable nitrogen

Introduction
The availability of nitrogen in coastal systems is of critical importance for the ecological dynamics of primary producers and higher trophic levels ). Because nitrogen is frequently the limiting nutrient in estuarine and marine systems, elevated levels of nitrogen are of particular importance in such areas . Nitrogen is absorbed by primary producers as dissolved inorganic nitrogen (DIN) and retained in their tissues until it is transferred to other trophic levels via leaching or decomposition , or via herbivory. The length of time that a producer retains nitrogen in its tissues, determined by its lifespan and its relative consumption by herbivores, as well as total producer biomass, is important to the total nitrogen cycling rate of an estuary ).
Because different groups of primary producers (including phytoplankton, annual and perennial macroalgae, and seagrasses) vary in their nitrogen retention times , the primary production regime in a system can strongly impact system-wide nutrient cycling rates.
Macroalgal blooms are typically found in low wave-energy systems such as estuaries, and may persist for several days or weeks, depending on physical conditions and nutrient availability . Bloom dynamics in response to nitrogen inputs have been well studied, as they are frequently associated with eutrophic conditions . Aside from washing up as wrack and entering terrestrial food webs , bloom biomass typically is retained in marine food webs either through herbivore consumption  or microbial decay processes Cebrian 1996, Hardison et al. 2010).
Particularly in systems where herbivore consumption of blooms is limited, e.g. , understanding decay rates and processes is crucial.
Decomposing macroalgae can cause localized hypoxia and decreased benthic macrofaunal diversity  and are frequently considered a public nuisance ). Because decaying macroalgae increase decomposer populations Stenton-Dozey 1981, Inglis 1989) and release nitrogen and other nutrients into the surrounding marine environment, they fuel a complex web of decomposer microorganisms and provide nutrients for future generations of primary producers ). Overall decay is characterized by microbial decomposition following an initial leaching of soluble compounds Wetzel 1978, Valiela et al. 1985). Microbial decay may be selective ; in other words, some types of molecules may be metabolized from algal tissues more quickly during the microbial phase depending upon decomposer communities and preferences, therefore changing the chemical makeup of the remaining biomass. Generally, tissue nitrogen concentration increases over the period of decay (see  for a review) due to relatively slower loss of nitrogen-rich compounds or mobilization of surrounding nitrogen by microbes.
The rate at which macroalgae decompose and release their nutrients is a key component of their total nutrient recycling time. Although there are few studies that compare decay rates among bloom-forming macroalgal species (but see , in general, fast-growing drift macroalgae decay more quickly than perennial, k-selected macroalgal species such as Fucus . Slow decay of perennial species such as fucoids may be due to their high phenolic content ). In addition, macroalgae with high nitrogen content should decompose quickly because decomposer fauna have increased demand for nutrients and bacterial growth efficiency increases when substrate nutrient content is high .
Therefore, eutrophied systems may have an acceleration effect on the speed of nutrient turnover, because they are frequently characterized by an ephemeral, algal-dominated productivity regime, and second, they may facilitate faster bacterial decomposition if the nitrogen concentration of algal tissue is elevated compared to algae in non-eutrophied systems .
Nitrogen exists in two stable isotopic forms: the lighter 14 N isotope is much more abundant than the heavier 15 N isotope. Secondary treated wastewater typically has a higher 15 N: 14 N (δ 15 N) isotope ratio than oceanic water because bacteria used in the wastewater-treatment process preferentially take up the lighter isotope ). While macroalgae absorb and incorporate both nitrogen isotopes into their tissues , they preferentially take up the lighter isotope (Fry 2006). By analyzing the δ 15 N in algal tissues, it may be possible in some cases to determine the source of the nitrogen used by macroalgae ) and, as a result, macroalgae are commonly used in studies to track nitrogen flow in estuarine food webs ).
However, comparatively little is known about the relative release of 15 N vs. 14 N in macroalgal decay processes. Because the microbial decomposer community takes up molecules and isotopes preferentially (mineralization; , the δ 15 N signature of a decaying alga may change over time depending on the rates of nitrogen mineralization and incorporation ).
We studied the dynamics of macroalgal decay and nutrient release in Narragansett Bay, RI, a highly eutrophied estuarine system .
The largest anthropogenic nutrient inputs are from sewage treatment plants located in the Providence River estuary, at the northern end of the Bay, while smaller treatment plants and other point-source anthropogenic nutrient inputs are distributed throughout the Bay . As the human population increased in this region over the past 150 years, nitrogen inputs from sewage, manufacturing, and atmospheric deposition dramatically elevated nitrogen release from 35-50 million moles/year in the 1860's to 605 million moles/year in the 1980's . Increased nutrient load has contributed to hypoxic events, particularly in the summer months and in geographically restricted areas such as Greenwich Bay . However, planned sewage treatment improvements project decreases in released N up to 30-50% from point sources during the summertime months by 2014 .
In this study, we quantified biomass decay rates for four major bloomforming species and one perennial species, expecting the perennial species (Fucus) to experience the slowest rate of decay, in Narragansett Bay's current (2011) early nutrient reduction regime. We quantified and compared rates of nitrogen and organic content loss in order to determine the chemical composition of the molecules consumed most readily by decomposers, expecting the most labile tissues to be consumed first, shown by an organic content decrease (as light sugars are released or metabolized) and a change in total N. The δ 15 N signature of the tissue was expected to increase over time as decomposer microorganisms preferentially mineralized the lighter N isotope. We interpret our data in the context of increasing anthropogenic stresses on coastal systems such as water warming from climate change and continued eutrophication.

Study Site
Narragansett Bay, Rhode Island, is a modest-sized, temperate coastal estuary (370 km 2 ) with several smaller embayments. The most frequently bloomaffected   ), while blooms of macroalgae are more common during the summer months (French et al. 1992, Granger et al.).
Our field experiments were conducted from June to October 2010.

Species and Design
We used five algal species, all of which are abundant in Narragansett Bay.
Four were common bloom-forming species growing subtidally, the green algae Papenfuss (a recent invader in this system); the fifth species was the perennial, brown alga Fucus vesiculosus Linnaeus. We collected fresh algae in Greenwich Bay with the exception of F. vesiculosus, which we collected from rocky shores in the lower Narragansett Bay, where it is more reliably found.
We cleaned fresh thalli in order to remove epiphytes and small animals.
Then, the thalli were frozen in a -80ºC freezer for at least two days to induce tissue senescence . Thawed individuals were subsampled for determinations of wet:dry mass, initial organic content and nitrogen concentration and nitrogen isotopic ratio (see techniques below). We placed each individual preweighed algal thallus in a mesh litter bag measuring 20 x 20 cm, with a mesh size of 0.25 mm, and secured the bag with a cable tie for deployment in the field. This mesh size was designed to exclude meso-and micro-herbivore effects, and thus is a study of decay (not decomposition), as defined by .

Statistical Analyses
We analyzed all data using JMP v 8.0 (SAS Institute, Inc., Cary, NC USA; www.sas.com). To determine the rate of change in mass over time for each experimental replicate (stake), we performed a log transformation on the calculated % mass remaining data and fit a linear equation to the transformed data; all masses used were dry mass. Any increase in mass greater than 120% (n = ~20 of 327 total samples) was excluded from the analysis; these samples frequently had sediment indistinguishable from decaying tissue on them, which would bias our results. We determined the k (rate of change in mass over time) from the slope of the regression for each replicate stake, for each species during each trial. We calculated 'absolute' values for organic content and total nitrogen; these absolute values represent the amount of organic content or total nitrogen remaining in the tissue, expressed as a percentage of the initial amount .
We determined k from these absolute values using the same regression process used for mass loss. We compared k values and initial values using one-way ANOVAs and post-hoc Tukey-Kramer HSD tests. Relative change in isotopic signature was calculated using the following equation: [[After δ 15 N -Before δ 15 N] / Before δ 15 N] x 100 = relative change δ 15 N Total and isotopic nitrogen data are not available for G. vermiculophylla due to mass spectrometer equipment failure.

Biomass Decay
All bloom-forming macroalgal species decayed three to five times faster than Fucus, the non-bloom forming species (Figure 1,  (Table 1).
We found significant variation in U. rigida decay rates throughout the summer months (F 6, 21 = 9.7547, p < 0.0001). The slowest decay rate occurred in the June 14 trial (mean k = 0.21) and the fastest occurred during the July 23 trial (mean k = 0.67). Rates of U. rigida biomass decay were positively correlated with mean water temperature (r = 0.636, p = 0.0003). Mean trial period water temperature ranged from 22.64°C to 24.48°C, with the highest mean temperature occurring in late July ( Figure 2).

Organic Content
Absolute organic content, expressed as a percentage of the original organic content remaining in the tissue, decreased over the period of decay in all species, though the loss rates (k) differed among species (F 4, 51 = 26.62, p < 0.0001, Table   1). Post-hoc analysis (Tukey HSD) revealed that red species (A. subulata and G.  Table 2). Post-hoc analysis does not reveal significant differences among species. Similarly, we found significant variation in initial total nitrogen for U. rigida among starting dates; tissues collected at the end of the summer (Trial 8-2) had at least one and a half times as much nitrogen (3.3%) than earlier in the summer (F 3, 42 = 24.89, p < 0.0001) and is significantly higher according to post-hoc analysis.

Isotopic Nitrogen
Changes in isotopic nitrogen ratios over the period of decay generally followed a trend of increasing variability; no consistent trends were observed except for F. vesiculosus, which was positively enriched in 15 N over the decay period (r = 0.63, p = 0.01; Figure 3). A. subulata had a slight negative correlation (r = 0.47, p = 0.04), though late in the decay period samples sizes for this species were extremely low (n = 2). Initial δ 15 N values varied significantly among species (F 3, 95 = 6.41, p = 0.0005; Table 2).

Discussion
The overall biomass and total nitrogen decay rates we recorded are near the maximum of those reported in a literature review by , but do not exceed previously observed measurements. Water temperature was significantly positively correlated with U. rigida decay rates. Initial organic content and total nitrogen tissue values did not influence decay rate.
As we expected, fast-growing, bloom-forming ephemeral macroalgae are also fast-decaying. These findings are consistent with other studies comparing similar ephemeral and perennial species . Many morphological and chemical qualities differentiate ephemeral and perennial species, including but not limited to morphological complexity, nutrient content, and cell wall constituents. Ephemeral species tend to have simpler cell wall constituents and low phenolics , both of which could potentially hasten the microbial decay process compared to perennial macroalgae.
Phenolic compounds such as those present in Fucus are known to impede decay of marine algae  and are likely responsible, in part, for the lower decay rate of Fucus compared with ephemeral species low in phenolics.  found that decomposition (defined to include microbial decay and detritivore feeding) was more responsible for mass loss than was tissue leaching in Ulva lactuca; in Fucus the opposite was true, and leaching had a greater effect on mass loss. Antimicrobial compounds in Fucus may explain the lowered role of microbes in Fucus decomposition .
We found no refractory pool in any of the ephemeral species, which is consist with other studies of macroalgae ). While our experiments did not last long enough to test for a Fucus refractory pool,  tested Fucus decay characteristics and did not find a refractory pool.

Water temperature and decay
Higher water temperatures frequently hasten bacterial metabolism in coastal waters , increasing rates of colonization and decomposition . Large temperature differences have been shown by  and by  to strongly influence decomposition, however the relatively small temperature differences (~2°C) experienced in this study do not overcome the effect of species on decay rate. As water temperatures rise-as they have in Narragansett Bay )-and decay rates experience a corresponding increase, there will likely be accelerated nutrient turnover from macroalgal detritus.

Organic content changes during decay
The change in organic content over the period of decay is consistent with the overall rate of biomass decay: species that decayed at a fast rate also lost organic content at a faster rate that slower-decaying species. All species except A. subulata lost organic content at a slower rate than they lost total biomass. By contrast, A. subulata lost organic content much faster relative to biomass, possibly indicating that the organic molecules in A. subulata are more available to bacterial colonizers than in other species, or that bacterial colonizers select organic molecules more aggressively on A. subulata.
Our findings indicate that for many species, the rate of biomass loss is underestimated when total carbon loss alone is used. Thus, data from studies in which C loss is treated as biomass loss (e.g. ) should be interpreted with caution.

Total nitrogen
Out of all five species examined, U. rigida and U. compressa had the highest and lowest initial total nitrogen values, respectively. These species were collected from the same location in each trial and are morphologically identical in the field and had similar natural biomass abundance, however, in this study U.
rigida lost its nitrogen twice as fast as U. compressa, perhaps because it had a higher initial N content and thus had more nitrogen available in its tissues for removal by leaching or microbial colonizers. Tissue enriched with nitrogen has accelerated biomass decay rates ; here it also may affect nitrogen loss rates.  found that Ulva lactuca and Gracilaria tikvahiae decay rapidly and speculate that this may be due to low cell wall phenolics and large nitrogen pools. They found that F. vesiculosus decayed at half the rate of U. lactuca and G. tikvahiae and speculate that its slower decay rate was a result of a smaller total nitrogen pool and high initial phlorotannin levels that reduce activities of microbial cellulases ).
All species experienced a net loss of nitrogen as they decayed; this was expected because as tissue degrades and disperses, so will the nitrogen-containing molecules. The proportion of nitrogen in the remaining tissue, however, increased during the decay process. This increase has been observed previously and consistently in vascular plants in estuarine systems ).  attribute the nitrogenous compound increase not to an accumulation of soluble proteins, but instead to nitrogen-structural complexes (phenol-proteins), resistant amino groups, and nitrogenous humic acids. Rice (1982) observed increased reactive phenols and humic material for the nonliving macroalgal detritus complex over the decay period. The high phenolic levels of Fucus (the most structurally and chemically similar to vascular plants of the species studied) may be partially responsible for the very high early-decay nitrogen levels it experiences.
As tissues decay, they may experience net nutrient mineralization or net immobilization . We observed immobilization only in one species, F. vesiculosus, when absolute nitrogen values exceed 100% of the original tissue nitrogen on Day 1 and 2, indicating that early in the decay process F.
vesiculosus incorporates more nitrogen into its tissues than it loses to the surrounding environment.  attribute this immobilization effect to phenolic compounds that slow nitrogen loss by either binding directly to proteins in the water column  or by slowing decay by inhibiting bacterial enzymes , though they observed immobilization only in marsh grasses. We examined total nitrogen on a much finer time scale to find the immobilization effect.

Stable isotopic nitrogen findings
Initial stable isotope values were extremely variable across time and between collection sites. This is consistent with previous studies that have documented differences in stable isotope values in biota in estuaries exposed to varying amounts of nitrogen from different sources (e.g., atmospheric nitrogen versus human wastewater  Previous studies on stable isotopic nitrogen found slight decreases in δ 15 N during decay and attributed the change to bacterial immobilization . It is possible that bacterial immobilization was occurring in our study, but only F. vesiculosus had an initial signal discernibly different from the (higher) allochthonous nitrogen value, though this difference is not significant.  mechanism for nitrogen accumulation suggests that the allochthonous nitrogen originates from microbial exoenzymes whose nitrogen binds to reactive carbohydrates and phenols in the detritus, thus raising the isotopic nitrogen signature of Fucus spp. detritus. The high variability in the isotopic nitrogen signal over time and among species is consistent with  and , who found significant fluctuations in signal change directionality in decomposing plants. The prevalence of these fluctuations in decaying plants and algae underscores the importance of large sample sizes in isotopic sampling.
An awareness of the nitrogen signal in decaying macroalgae is important because the signal is not static, and a single measurement cannot capture the isotopic signature of the entire process. Also, decaying algae should not be treated as if they have a δ 15 N consistent with living tissue because of bacterial processes and immobilization. To test the mechanism of bacterial incorporation of isotopic nitrogen by phenolic binding, a low δ 15 N alga with low phenolic constituents should be observed over the course of decay in high δ 15 N waters.

Comparison with perennial algae and vascular plant decay
Seagrass beds have declined worldwide and have frequently been accompanied by an increase in macroalgal blooms ). This regime shift may have also impacted nutrient cycling dynamics associated with decay. Of the major estuarine primary producers, phytoplankton decay most quickly, followed by macroalgae, then seagrasses and finally marsh grasses, the order of which is primarily determined by the size of the initial tissue nitrogen pool . The cell walls of algae do not accumulate nitrogen like vascular plants do and, in addition, lose their initial cell wall nitrogen very quickly. Green and red algal detritus have faster rates of nutrient mineralization and are more nutrient-rich than detritus from brown algae or sea-and marsh grasses . A second important determinant of algal decay rates is their less complex cell wall polysaccharide structures compared with vascular plants . A byproduct of this fast decay is that macroalgal regimes lead to less producer-derived organic matter accumulation (see  for a review).
As a result of their rapid and complete decay, macroalgae transfer important quantities of nutrients to the surrounding water, which has important implications for the sediment and water column microphytobenthos that utilize those nutrients in the estuarine regime . In oligotrophic conditions such as reefs, the rapid turnover supplies nutrients necessary for production ), but in eutrophied waters, nutrients are made rapidly available to an already nutrient-rich environment characterized by nutritional dynamism and trophic instability caused by macroalgal dominance ).  observe greater herbivory in systems dominated by macroalgae. The balance between decay and herbivory in these systems may influence the transfer of nutrients from macroalgae to other trophic levels via different pathways. Understanding the coupled effects of faster nutrient turnover through rapid decay and greater rates of herbivory is crucial to understanding system-wide shifts in nutrient cycling brought about by macroalgal productivity regimes in eutrophied coastal systems. grateful to N. Millette and N. Mikkelsen for their help in the field. We thank R.
McKinney and the US EPA Atlantic Ecology Division for use of a mass spectrometer. Funding for this project was provided by the Quebec-Labrador Fund, Bay Window (NOAA), and the URI Coastal Fellows Program.   The relative change in signal is expressed as a percent change from the initial δ 15 N value measured in F. vesiculosus tissue before decay. There is a significant positive correlation between the change in isotopic signature and decay time (r = 0.63, p = 0.01).
Organic content decay graphs follow. The absolute organic content (y axis) per day of decay (x axis) is displayed for each macroalgal species.

Fucus vesiculosus
Total nitrogen decay curves follow. The absolute total nitrogen (y axis) per day of decay (x axis) is displayed for each macroalgal species.