# MICROBIAL ROLE IN THE CARBON CYCLE IN TROPICAL INLAND AQUATIC ECOSYSTEMS

EDITED BY: André Megali Amado and Fábio Roland PUBLISHED IN: Frontiers in Microbiology

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ISSN 1664-8714 ISBN 978-2-88945-127-2 DOI 10.3389/978-2-88945-127-2

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# **MICROBIAL ROLE IN THE CARBON CYCLE IN TROPICAL INLAND AQUATIC ECOSYSTEMS**

Topic Editors:

**André Megali Amado,** Universidade Federal do Rio Grande do Norte, Brazil **Fábio Roland,** Universidade Federal de Juiz de Fora, Brazil

View of Lake Batata margin (Municipality of Oriximiná, Para State, Brazil - Brazilian Amazon) that shows the aquatic macrophytes and the rain forest and their reflection on the water. We propose that the reflected image represent the great importance of these carbon sources to the microbes in the water and to the aquatic ecosystems metabolism. Photo by Fábio Roland

Aquatic microorganisms are tidily related to the carbon cycle in aquatic systems, especially in respect to its accumulation and emission to atmosphere. In one hand, the autotrophs are responsible for the carbon input to the ecosystems and trophic chain. On the other hand, the heterotrophs traditionally play a role in the carbon mineralization and, since microbial loop theory, may play a role to carbon flow through the organisms. However, it is not yet clear how the heterotrophs contribute to carbon retention and emission especially from tropical aquatic ecosystems.

Most of the studies evaluating the role of microbes to carbon cycle in inland waters were performed in high latitudes and only a few studies in the tropical area. In the prospective of global changes where the warm tropical lakes and rivers become even warmer, it is important to understand how microorganisms behave and interact with carbon cycle in the Earth region with highest temperature and light availability. This research topic documented microbial responses to natural latitudinal gradients, spatial within and between ecosystems gradients, temporal approaches and temperature and nutrient manipulations in the water and in the sediment.

**Citation:** Amado, A. M., Roland, F., eds. (2017). Microbial Role in the Carbon Cycle in Tropical Inland Aquatic Ecosystems. Lausanne: Frontiers Media. doi: 10.3389/978-2-88945-127-2

# Table of Contents

# *77 Dynamics of planktonic prokaryotes and dissolved carbon in a subtropical coastal lake*

Maria Luiza S. Fontes, Denise Tonetta, Larissa Dalpaz, Regina V. Antônio and Maurício M. Petrucio

*86 Environmental dynamics as a structuring factor for microbial carbon utilization in a subtropical coastal lagoon*

Cecilia Alonso, Claudia Piccini, Fernando Unrein, Florencia Bertoglio, Daniel Conde and Jakob Pernthaler

# Editorial: Microbial Role in the Carbon Cycle in Tropical Inland Aquatic Ecosystems

André M. Amado1, 2 \* and Fábio Roland2

1 Limnology Laboratory, Department of Oceanography and Limnology, Universidade Federal do Rio Grande do Norte, Rio Grande do Norte, Brazil, 2 Aquatic Ecology Laboratory, Department of Biology, Universidade Federal de Juiz de Fora, Minas Gerais, Brazil

Keywords: metabolism, lakes, lagoons, rivers, latitudinal gradients, microorganisms, plankton, microbial ecology

**Editorial on the Research Topic**

### **Microbial Role in the Carbon Cycle in Tropical Inland Aquatic Ecosystems**

Microorganisms have been recognized as central to nutrient mineralization and recycling in aquatic ecosystems since Lindeman's groundbreaking work on the trophic-dynamic aspect of ecology (Lindeman, 1942). Since the seventies, the development of new analytical technologies led to important conceptual perspectives, such as the microbial loop and the microbial food web (as summarized elsewhere, e.g., Cotner and Biddanda, 2002; Weisse, 2004), which have been important to understanding connections between microbially-mediated allochthonous and autochthonous organic matter decomposition and carbon dioxide (CO2) concentrations and fluxes to the atmosphere (e.g., Cole et al., 2007; Berggren et al., 2012; Fonte et al., 2013). Currently, one of the main foci of microbial ecologists is to open the "microbial playbill" so that we can better understand who is doing "what" and "when" in ecosystem "plays" (Logue et al., 2015).

It is well-documented how seasonal variation of temperature, light incidence, and precipitation affects microbial metabolism (e.g., Simon and Rosenstock, 1992; Berggren et al., 2010) in high latitude ecosystems. Considering that temperature and light incidence are less variable and remain high year-round in the tropics (Lewis, 1996), one could expect differences in metabolic processes among the latitudinal regions (Farjalla et al., 2009) and consequent effects on microbial respiration and carbon emissions (essentially CO2 and methane) to the atmosphere. On the one hand, more intense metabolic processes are expected in lower latitudes. On the other hand, regional (e.g., flood pulse) or local (e.g., landscape characteristics) environmental conditions could be more relevant regulators of microbial metabolism than global factors (e.g., temperature, etc.). For instance, small rather than large planktonic organisms predominate at the base of microbial food webs of tropical aquatic ecosystems (i.e., pico- vs. nano-plankton) in comparison to temperate lakes, which typically means a higher flow of carbon through the microbes in the tropics than in temperate regions (Roland et al., 2010; Sarmento, 2012). Yet, certainly several new microbial ecology fundamentals will arise from asking questions that remain poorly understood, such as: (1) Does the current knowledge derived mostly from temperate ecosystems hold for tropical ecosystems? and (2) Can tropical ecosystems be good models to predict the changes in microbial metabolism and carbon cycling in temperate aquatic systems in light of climate warming scenarios?

The aim of this research topic—Microbial role in the carbon cycle in tropical inland aquatic ecosystems—was to provide a selection of studies that look at the wide variety of aspects of tropical microbial ecology including barely covered outlines (such as viruses–bacteria interactions or hydrodynamic events driven microbial communities) and their role to the carbon cycle. This research topic has documented 13 contributions that advance our knowledge on the microbial

#### Edited by:

Hongyue Dang, Xiamen University, China

#### Reviewed by:

Ryan J. Newton, University of Wisconsin–Milwaukee, USA

\*Correspondence:

André M. Amado<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Aquatic Microbiology, a section of the journal Frontiers in Microbiology

Received: 05 September 2016 Accepted: 04 January 2017 Published: 19 January 2017

#### Citation:

Amado AM and Roland F (2017) Editorial: Microbial Role in the Carbon Cycle in Tropical Inland Aquatic Ecosystems. Front. Microbiol. 8:20. doi: 10.3389/fmicb.2017.00020 responses to natural latitudinal gradients, spatial and temporal patterns within and between ecosystems, and temperature and nutrient effects on microbial processes in the water and in the sediment. In order to better compile the information, the contributions were grouped in the following paragraphs as: (a) how physical and chemical variables (e.g., temperature and nutrients) affect carbon metabolism via microbes; (b) how latitudinal variation affects metabolism in the microbial food web components, and (c) how changes in environmental forces (such as flood pulse) affect microbial interactions.

Two meta-analysis studies focused on bacterial metabolism and top-down control and how they vary across latitudinal gradients. One study demonstrated that bacterial biomass production (BP) and respiration (BR) are higher while bacterial growth efficiency (BGE) is lower in the tropics (Amado et al.). Additionally, the other study demonstrated that bacterial and heterotrophic flagellate (HNF) abundance were lower in tropical than in temperate ecosystems (Segovia et al.). Furthermore, the authors still showed that the coupling between predator and prey did not differ between tropical and temperate zones, suggesting that other factors, such as the higher temperature or even HNF top-down control could be responsible for higher bacterial loss rates in the tropics.

One study that manipulated temperature and nutrients in tropical humic coastal lagoons (Scofield et al.) recorded increased BR and decreased BP and BGE with increasing temperature, confirming the results reported by Amado et al., and showing that temperature can be a valuable carbon metabolism predictor on a regional scale. Similarly, nutrient manipulations resulted in different metabolic responses and CO2 saturation patterns among lagoons, indicating that temperature effects are modified by local ecosystem conditions (Peixoto et al.). Additionally, another study performed in an eutrophic reservoir (Almeida et al.), observed net heterotrophy, net CO2 emission, and high ebullition and diffusive methane (CH4) emissions. Despite high primary production rates, the shallow nature of the ecosystem, the high temperatures, and accumulated organic matter contributed to high decomposition rates. These three studies supported the idea that local factors are strong drivers of aquatic carbon metabolism at low latitudes.

The Amazon River basin is inserted in a low-latitude zone and it is recognized as a hotspot for freshwater CO2 emissions globally (Raymond et al., 2013; Abril et al., 2014). The flood pulse is the major ecological force affecting carbon-related processes and organisms in this region (Amado et al., 2006; Melack and Coe, 2012). Indeed, the flood pulse was the main driver for bacterial metabolism and carbon mobilization in rivers due to changes in the organic matter origin and quality (Vidal et al.). Moreover, one study in floodplain lakes of a large Amazon tributary contained bacterial and viral abundances that increased with increasing distance from the Amazon River (Almeida et al.). The longitudinal gradient was attributed to a backwater effect caused by the Amazon River that increased water turbidity and decreased organic carbon quality (availability) and concentration in the floodplain lakes downstream, which might also reduce the carbon flow through to the microbial food web.

Three studies evidenced that there is a great deal of heterogeneity among coastal lakes in the subtropical region. One study using seasonal and diel approaches in a coastal lagoon in southern Brazil (Peri Lagoon, Santa Catarina state), showed that precipitation and temperature, as well as bacterial abundance were the main regulators of CO2 concentration in the water (Schmitz Fontes et al.). On the other hand, the bacterial community and activity in another coastal subtropical lagoon (Laguna de Rocha, Uruguay), was carbon-limited and was mainly driven by organic matter inputs from the watershed or dilutions by the seawater (Alonso et al.). This resulted in shifts in the microbial community and the microbial food web through HNF and virus increase. In addition, the dominance of wetlands and aquatic macrophytes reduced microbial carbon metabolism and CO2 concentration in the water when compared to the pelagic areas (i.e., higher depths and no macrophytes presence) creating heterogeneous conditions to carbon processing within the lake in a subtropical coastal lagoon in southern Brazil (Lake Mangueira, Rio Grande do Sul state; They et al.). Subtropical lakes are somewhat of a transition type of ecosystem, which consistently share regulation of microbial and carbon processes by local/regional environmental factors with tropical ecosystems, and by climate-driven seasonal environmental factors with temperate ecosystems.

Sediment metabolism has been extensively demonstrated to be relevant to the carbon balance in deep temperate lakes (e.g., Jonsson et al., 2001), but was poorly studied in tropical inland waters. Cardoso et al. observed high mineralization rates in the sediment of a mesotrophic tropical hydroelectric reservoir, which has driven the CO2 variation in the water column and high internal spatial variation in the respiration. Also, Canterle et al. observed both spatial and temporal variation in the microbial CO2 and CH4 production and emission in the sediment of a subtropical shallow wetland ecosystem. Additionally, Liengaard et al. detected high emission of nitrous oxide (N2O) in the largest wetland in the world (Pantanal, a pristine tropical wetland) providing up to 1.7% of global N2O emissions, as result of high organic matter availability processed via anaerobic metabolism.  The intensification of biological processes may increase greenhouse-gas emissions 2–4 times more in the tropics than in temperate sediments (Marotta et al., 2014).

The articles presented in this research topic investigate several aspects of microbial interaction with carbon cycling in tropical inland waters and produced some evidence that it can change across the latitudinal gradient as follows: (a) higher temperature in the tropics enhances bacterial carbon processing and reduces BGE; (b) the energy flow through the microbial food web in the tropics may be either reduced due to low BGE, or enhanced due to a possible high top-down control of HNF; and (c) as in temperate ecosystems it seems consistent that nutrient availability is also a key component of the aquatic metabolism and carbon processing in tropical ecosystems, but local or regional regulation may frequently be equally or even more relevant than globally controlled environmental factors. Nonetheless, several gaps still remain. For example, is absolutely pertinent a better understanding regarding the controls of trophic cascade relationships (e.g., top-down control of bacteria by HNF) in the microbial food web and how landscape conditions (e.g., land use) affect carbon processing in aquatic ecosystems. Certainly, many opportunities exist for research focusing on the relationship of carbon and the microbial components of freshwater. As we commented before, we expect that these papers will stimulate new discussions and investigations to cover the great diversity of ecological processes that still need to be addressed and help formulate more robust general patterns for tropical freshwaters.

# AUTHOR CONTRIBUTIONS

All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.

# REFERENCES

# FUNDING

This research topic received support from National Council of Technological and Scientific Development (CNPq—Brazil) specifically to AA under the grant # 475537/2012-2 and to FR under the grant # 309180/2013-9.

# ACKNOWLEDGMENTS

We are grateful to the editorial staff at Frontiers in Microbiology for the invitation and support to this research topic. We thank R. Almeida, N. Barros, and J. Cotner and the reviewer for valuable comments on the manuscript.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Amado and Roland. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Tropical freshwater ecosystems have lower bacterial growth efficiency than temperate ones

#### *André M. Amado1 \*, Frederico Meirelles-Pereira 2, Luciana O. Vidal 3, Hugo Sarmento1, Albert L. Suhett 4, Vinicius F. Farjalla2, James B. Cotner 5 and Fabio Roland3*

*1 Limnology Laboratory, Departamento de Oceanografia e Limnologia, Pós-graduação em Ecologia, Universidade Federal do Rio Grande do Norte, Natal, Brazil*

*2 Departamento de Ecologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil*

*3 Departamento de Ecologia, Universidade Federal de Juiz de Fora, Juiz de Fora, Brazil*

*4 Unidade de Biotecnologia e Ciências Biológicas, Centro Universitário Estadual da Zona Oeste, Rio de Janeiro, RJ, Brazil*

*5 Ecology Evolution and Behavior Department, University of Minnesota, St. Paul, MN, USA*

#### *Edited by:*

*Stefan Bertilsson, Uppsala University, Sweden*

#### *Reviewed by:*

*Guang Gao, Chinese Academy of Sciences, China François Guillemette, Uppsala University, Sweden*

#### *\*Correspondence:*

*André M. Amado, Programa de Pós-Graduação em Ecologia, Laboratório de Limnologia, Departamento de Oceanografia e Limnologia, Universidade Federal do Rio Grande do Norte, Via Costeira Senador Dinarte Medeiros Mariz S/N, Praia de Mãe Luiza, 59014-002, Natal-RN, Brazil e-mail<EMAIL_ADDRESS>
Current models and observations indicate that bacterial respiration should increase and growth efficiency (BGE) should decrease with increasing temperatures. However, these models and observations are mostly derived from data collected in temperate regions, and the tropics are under-represented. The aim of this work was to compare bacterial metabolism, namely bacterial production (BP) and respiration (BR), bacterial growth efficiency (BGE) and bacterial carbon demand (BCD) between tropical and temperate ecosystems via a literature review and using unpublished data. We hypothesized that (1) tropical ecosystems have higher metabolism than temperate ones and, (2) that BGE is lower in tropical relative to temperate ecosystems. We collected a total of 498 coupled BP and BR observations (*N*total = 498; *N*temperate = 301; *N*tropical = 197), calculated BGE (BP/(BP+BR)) and BCD (BP+BR) for each case and examined patterns using a model II regression analysis and compared each parameter between the two regions using nonparametric Mann–Whitney U test. We observed a significant positive linear regression between BR and BP for the whole dataset, and also for tropical and temperate data separately. We found that BP, BR and BCD were higher in the tropics, but BGE was lower compared to temperate regions. Also, BR rates per BP unit were at least two fold higher in the tropics than in temperate ecosystems. We argue that higher temperature, nutrient limitation, and light exposure all contribute to lower BGE in the tropics, mediated through effects on thermodynamics, substrate stoichiometry, nutrient availability and interactions with photochemically produced compounds. More efforts are needed in this study area in the tropics, but our work indicates that bottom-up (nutrient availability and resource stoichiometry) and top-down (grazer pressure) processes, coupled with thermodynamic constraints, might contribute to the lower BGE in the tropics relative to temperate regions.

#### **Keywords: microbial metabolism, BGE, carbon cycle, tropical, temperate**

# **INTRODUCTION**

Freshwater ecosystems are critical bioreactors in the global carbon cycle as they process a large fraction of the organic matter exported from terrestrial ecosystems (Cole et al., 2007; Tranvik et al., 2009). Heterotrophic bacteria play an important role in processing this organic matter and in releasing CO2 and inorganic nutrients (Cole et al., 1994; Odum et al., 2004), and re-integrating dissolved organic matter (DOM) to the food web through the microbial loop (Azam et al., 1983). Thus, bacterial production (BP), and bacterial respiration (BR) are key processes in the carbon cycle of all aquatic systems. Two parameters derived from BP and BR are useful tools to understand the role of bacteria in ecosystem functioning: bacterial carbon demand (BCD), which represents the total amount of carbon processed by bacteria (BCD = BP + BR) and bacterial growth efficiency (BGE), which is the proportion of carbon taken up by bacteria that is converted into biomass (BGE = BP/[BP + BR]) (for review see Del Giorgio and Cole, 1998). Higher BGE leads to higher energy and organic matter availability to higher trophic levels, i.e., the microbial loop. On the other hand, lower in BGE may result in higher carbon mineralization rates through CO2 production.

Several environmental factors may regulate bacterial metabolism and affect BGE and BCD. Water temperature is an important metabolic regulator and frequently correlates positively with BP, BR, and BCD, and negatively with BGE (Rivkin and Legendre, 2001; Biddanda and Cotner, 2002; López-Urrutia and Morán, 2007). Additionally, nutrient availability (such as N and P), DOM quality and stoichiometry, and bacterial predation pressure may also be important regulators of microbial activity (Farjalla et al., 2002, 2006; Hall and Cotner, 2007; Berggren et al., 2010; Vidal et al., 2011; Sarmento, 2012; Sarmento and Gasol, 2012). Considering that the tropical region (between latitudes 23◦26 S and 23◦26 N) presents higher sunlight incidence and mean temperature (Lewis, 1987, 1996) and that additional trophic levels within the microbial food web can persist throughout the year (Sarmento, 2012), one might expect that many aquatic processes, such as BGE and BCD could present differences between tropical and temperate regions.

The metabolic theory of ecology predicts that organisms living in warmer conditions exhibit higher metabolic rates than organisms living at lower temperatures (Brown et al., 2004). On the other hand, the ATP paradox suggests that higher metabolic rates yield lower biomass with higher energy dissipation (lower growth efficiency; Pfeiffer et al., 2001; Pfeiffer and Bonhoeffer, 2002; MacLean, 2008). Thus, it could be deduced that at the higher temperatures in the tropics BGE should be reduced relative to the temperate zones. This relationship of decreased BGE with increasing temperatures has been demonstrated already mostly within temperate ecosystems (Hall and Cotner, 2007; López-Urrutia and Morán, 2007; Berggren et al., 2010). Another recent study comparing tropical humic ecosystems to temperate models observed that BR was higher and more variable in the humic tropical systems, and also that BP tends to stabilize, relative to BR (Farjalla et al., 2009). These findings contradict other studies in temperate ecosystems that observed higher BP variability, rather than BR (Del Giorgio and Cole, 1998; Roland and Cole, 1999). Thus, following the humic-tropical pattern, BR would increase proportionally more in relation to BP, and the average BGE in the tropical ecosystems would be lower than in temperate systems, implying a lower relevance of the microbial loop in terms of biomass, but higher relevance in terms of carbon mineralization in tropical systems. However, a recent review on the role of the microbial food webs in tropical lakes was not able to demonstrate that BP was higher in the tropics, compared to temperate systems in small dataset (Sarmento, 2012).

Therefore, assuming higher and more constant average temperatures in tropical aquatic ecosystems compared to temperate aquatic ecosystems, some important questions arise regarding bacterial function in these ecosystem: (1) Do bacteria grow less efficiently in the tropics than in temperate ecosystems? (2) Is the humic-rich tropical ecosystem pattern relevant in a larger context of tropical ecosystems types? (3) If bacteria do grow less efficiently in the tropics than in temperate regions, is temperature the most important factor driving these differences?

In this paper we hypothesize that (1) tropical inland aquatic ecosystems have higher metabolic rates (e.g., BCD) than temperate ecosystems and, (2) that BGE is lower due to high respiration rates tropics. This paper aims to address and discuss these hypotheses by re-analyzing freshwater BP, BR and BGE data from literature (mainly from the temperate region), and by adding published and unpublished data from the tropical region. This work is an effort to expand current paradigms of microbial biogeochemistry to the tropical inland aquatic ecosystems. We point out the importance of increasing data production on BP and BR in the tropics to achieve a more accurate understanding about carbon cycling and the role of bacteria in this vast and understudied part of the world.

# **METHODS**

We pooled models and bacterial production (BP) and bacterial respiration (BR) data from the literature (**Table 1**) and unpublished data from Lake Superior and northern Minnesota lakes (Sampled by J. Cotner and A. Amado), Swedish lakes (Sampled by L. Vidal and W. Granéli) and Amazonian lakes and rivers (Sampled by L. Vidal, G. Abril, F. Artigas and F. Roland). Lake Superior samples were taken at six sampling stations over five cruises in the western arm of the lake, from May–October 2006. Northern Minnesota lakes were sampled in July 2006 (summer) and January 2007 (winter) in a central point of each lake. Nineteen Swedish lakes were sampled in July 2007 (summer) in the deepest part of each lake over a DOC gradient from 3.7 to 26.8 mg L−1. Twenty Amazonian lakes and rivers were sampled in June and November (2009) at stations throughout the Amazon River basin (for details see **Table 1**). For the literature data we surveyed the papers used in the review by Del Giorgio and Cole (1998) and all papers available from 1998–2012 searching for "BGE" and "aquatic ecosystems" key-words in the ISI Web of Science (**Table 1**). We only used data from inland aquatic ecosystems where both BP and the correspondent BR rates were available. Literature data were extracted from tables and graphs and converted to microgram of carbon per liter per hour (µg C L−1 h−1) and log-transformed to perform statistical analysis. When the data were only available in graphs, they were extracted from the graphs using the Digitizeit and GraphClick softwares.

Regarding the original data presented in this work, BP rates were measured using the method of Smith and Azam, (1992; [3H]-leucine incorporation method). The incubation times varied between 0.5 and 5.0 h according to the local conditions (e.g., temperature, nutrients concentrations). BR rates were measured by dissolved oxygen consumption over a 24-h period. Oxygen concentrations were measured by different methods based on changes in dissolved oxygen concentrations over time. In samples from Lake Superior and from Minnesotan lakes we measured the changes in dissolved oxygen in the same sample over time (as initial and final) using a gold tip micro-probe connected to OXY-meter (Briand et al., 2004) controlled by the MicOx Software (Unisense©, Aarhus, Denmark). Samples from Amazonian ecosystems and Swedish lakes were measured in discrete samples at various time-points with sets of replicated flasks/vials using the Winkler technique and titrations with a potentiometric endpoint using a Mettler DL21 titrator (Granéli and Granéli, 1991). BR rates were transformed to carbon using a respiratory coefficient (RQ) of 1 (shown as being the most commonly used by Del Giorgio and Cole, 1998).

From the literature, we only considered BP data estimated by radioisotope incorporation (using tritium-labeled (3H) leucine or thymidine incorporation). As all BP estimates were measured over similar time scales, we considered that both methods estimated equivalent rates (Kirchman, 1992) and, thus, we did not apply any conversion factor between the results from the two methods. We did not consider BP data estimated from bacterial biomass accumulation in batch cultures. Despite the fact that it has been previously suggested that both radioisotope incorporation and biomass accumulation in batch cultures methods yield

**Table 1 | Database used in the analysis with the literature extracted and novel data.**

*BR, Bacterial respiration; BP, bacterial production; BGE, bacterial growth efficiency; BCD, bacterial carbon demand ranges display the minimum and maximum values from each study.*

*Ntotal* = *498; Ntropical* = *197; Ntemperate* = *301.*

similar results (Del Giorgio and Cole, 1998), the latter method is employed at longer timescales and require grazers-free condition and the radioisotope incorporation methods represent conditions closer to those of the natural system.

We used literature BR data measured via three dissolved oxygen consumption methods [the two mentioned above plus by oxygen estimated by membrane inlet mass spectrometer (MIMS)] and through dissolved organic carbon (DIC) accumulation. The BR rates estimated as oxygen consumption were transformed to a carbon basis by respiratory coefficient (RQ) of 1, as in the description of the novel data above. BR rates are frequently estimated from filtered samples (over a very wide range of pore sizes, frequently from 0.45 to 3.0µm), but also in unfiltered samples. In oligotrophic ecosystems BR accounts for up to 90% of plankton respiration (Biddanda et al., 2001) and, thus, it is acceptable that BR could even be measured in unfiltered water. For the purpose of this study, we used the corrections and assumptions of the authors in estimating BR from whole or filtered water measurements. As our aim was to compare tropical to temperate rates with the same criteria, we assumed that any discrepancies related to the filtration pore-size would be randomly distributed between the data from the two regions and would not affect the overall patterns.

The dataset presented here was constructed with data from inland aquatic ecosystems in both temperate and tropical regions (*N*total = 498; *N*tropical = 197; *N*temperate = 301). Our dataset includes information mostly from lakes, rivers and reservoirs, excluding estuarine and marine data. To our knowledge, this is the largest dataset of BP and BR for freshwaters.

From BP and BR data, we calculated bacterial growth efficiency (BGE = BP/[BP + BR]) and bacterial carbon demand (BCD = BP + BR). Statistical analyses were performed in the R environment (www*.*r-project*.*org) using the "vegan" (Oksanen et al., 2008), "lmodel2" and "smart" packages. We performed model II linear regression using the major axis method (package "lmodel2" by Pierre Legendre) on log-transformed data between BP vs. BR, and BP vs. BGE from the whole data set and with tropical and temperate subsets. Slopes and intercepts for temperate and tropical subsets were compared using the "ma" function (package "smart" by David Warton) that tests hypotheses about slope or elevation ("elev.test") based on confidence intervals comparison. We performed non-parametric Mann–Whitney Rank Sum test to compare median values of bacterial metabolism parameters between tropical and temperate subsets. Figures were made on SigmaPlot v.12 software (Systat Softare Inc.).

**Table 2 | Slope, intercept and confidence interval (c.i.) of Type II Linear Regressions between log transformed BP vs. BR, and BP vs. BGE for tropical, temperate and whole dataset.**

# **RESULTS**

Model II linear regressions on log-transformed BP and BR were significant, either considering the whole dataset or data from tropical and temperate regions separately (**Table 2**; **Figure 1**). Similarly, there was a significant positive relationship between log-transformed BP and BGE (**Table 2**; **Figure 2**).

Statistical tests on confidence intervals ("ma" function from "smart" package) showed no significant differences between slopes for temperate and tropical sub-sets, for both regressions (logBR vs. logBP and logBGE vs. logBP). However, the confidence interval for the intercepts was lower in the tropical subset (**Table 2**). These results were confirmed testing the elevation confidence intervals ("ma" function from "smart" package), which showed significant differences between the intercepts from temperate and tropical subsets (logBR vs. logBP: *p <* 0*.*001, Test statistic: *t* = 14*.*73 with 195 degrees of freedom, logBGE vs.

logBP: *p <* 0*.*001, Test statistic: *t* = −17*.*29 with 195 degrees of freedom), indicating that, at similar BP levels, BGE was lower and BR was, at least, two-fold higher in tropical systems.

 It is worth noting that all parameters had a wider range of variation in temperate than the tropical subset (**Figure 3**).

# **DISCUSSION**

This meta-analysis indicated that bacterial communities in tropical inland aquatic ecosystems had higher metabolic rates (BP, BR, and BCD) and lower BGE than temperate ecosystems. These

results confirm the hypothesis presented here and the theoretical predictions from the literature (Lewis, 1987; Farjalla et al., 2009; Sarmento, 2012). We observed that BR rates were at least two fold higher in tropical ecosystems for a given BP, when compared to temperate ones. On one hand, heterotrophic bacteria process organic matter at faster rates in tropical ecosystems, regenerating inorganic nutrients more rapidly, but they do so while converting a smaller proportion of the organic matter into biomass. Even though we observed higher BP in the tropics, BR increased relatively more, resulting in lower BGE.

Higher BR rates in the tropics (**Figures 1**, **3**) indicate that, proportionally, more energy is required to maintain a similar bacterial biomass, when compared to temperate ecosystems. In other words, similar bacterial biomass could recycle a greater quantity of carbon in tropical ecosystems compared to temperate ones. Additionally, previous studies reported a comparatively lower bacterial abundance in tropical systems along a productivity gradient relative to temperate systems (Roland et al., 2010; Sarmento, 2012), reinforcing the idea of lower BGE in the tropics. Thus, regarding the allegedly important role of the microbial food web in energy and nutrient regeneration, (for review see Del Giorgio and Cole, 1998), we argue that remineralization is greatly emphasized in tropical inland aquatic ecosystems.

For the first time, we present here evidence of consistent differences in bacterial metabolic rates between tropical and temperate freshwater ecosystems, with a large and representative dataset. These observations were hypothesized in past tropical limnology literature (Lewis, 1987; Farjalla et al., 2009; Sarmento, 2012), but never demonstrated at large spatial scales. Lower BGE in the tropics is likely due in part to two factors that differ greatly between the tropics and the temperate regions: (1) higher average solar irradiance in the tropics and (2) fundamental differences in trophic structure. It is well established that the higher sunlight irradiance in aquatic ecosystems directly affects water temperature, substrate stoichiometry (e.g., higher C:N, C:P under higher light incidence; see Sterner et al., 1998) and nutrient availability (such as N and P). In turn, these factors are important regulators of bacterial activity in aquatic systems (Del Giorgio and Cole, 1998) and can have effects on metabolic efficiency (i.e., BGE) under different environmental conditions (Hall et al., 2009).

Temperature is considered a critical environmental factor affecting microbial metabolism and seems to play a major role in different aspects, from physiology to community structure and algal excretion rates. Recent studies have shown that temperature positively correlates with BP and BR, but negatively with BGE, especially in systems where nutrients (N and P) are not limiting (Rivkin and Legendre, 2001; López-Urrutia and Morán, 2007; Berggren et al., 2010). Usually, under nutrient limitation, BGE is already low (Berggren et al., 2010). Furthermore, it has been shown that the C:P ratio of heterotrophic bacterial biomass increases with increasing temperature even with no change in growth rates (Cotner et al., 2006). Moreover, bacteria with high biomass C:P ratio, as occur under strong P-limitation conditions present relatively lower BGE than when in low biomass C:P ratio (Phillips, 2012). Therefore, it seems reasonable to conclude that the lower BGE in the tropics could result from higher average temperatures coupled with stronger nutrient, i.e., P limitation.

Previous studies predicted that nutrient recycling rates might be twice as high in tropical than in temperate ecosystems (Lewis, 1987). Our results corroborate this prediction showing that BR rates per BP unit was at least two fold higher in the tropics, consistent with higher temperatures and/or more nutrient-limited growth in the tropics. Although nutrient-limited growth has also been demonstrated in many temperate ecosystems as well, it is possible that the consistently higher BGE observed there could be due in part to less extreme nutrient limitation and perhaps stronger limitations by organic carbon availability (e.g., lower C:P ratios in primary producers). So, although nutrient regeneration rates may be high in warmer conditions, it is also likely that increasing growth demands and competition with autotrophs makes it more likely that bacteria in the tropics are limited by inorganic nutrients (Downing et al., 1999; Flecker et al., 2002).

Physical constraints of a thermodynamic nature should also be considered in order to explain the patterns observed. Considering that ATP production supports most biosynthetic assemblies (De Duve, 1991), a trade-off between rate and yield of ATP production might have important consequences to metabolism (Pfeiffer et al., 2001; Pfeiffer and Bonhoeffer, 2002; Schuster et al., 2008). The ATP paradox suggests that nature does not select for increased molar yield (detailed discussion in Schuster et al., 2008), because the highest thermodynamic efficiencies do not correlate to the highest growth rates (Westenhoff et al., 1983; Pfeiffer et al., 2001; Pfeiffer and Bonhoeffer, 2002) and this was confirmed in natural bacterial communities in aquatic ecosystems (Del Giorgio and Cole, 1998; Carlson et al., 2007). Thus, low metabolic rates lead to more efficient growth, while high metabolic rates (faster growth) results in low efficiency of biomass production (MacLean, 2008). For ecosystems, one of the implications is that one would expect cooler, more structured ecosystems where environmental changes are more predictable to have higher trophic efficiencies (Pfeiffer et al., 2001; Pfeiffer and Bonhoeffer, 2002); and lower efficiencies are expected in warmer, high metabolism ecosystems. Our observations are consistent with these predictions.

The tropics may also contrast with the temperate zone in terms of how sunlight interacts with the terrestrially-derived DOM. Assuming similar DOM export from terrestrial ecosystems and similar DOM concentrations in aquatic ecosystems at both tropical and temperate regions, greater photo-exposure in the tropics should increase production of reactive oxygen species (ROS) (Zepp and Cline, 1977; Cory et al., 2009). These compounds, such as singlet oxygen and hydrogen peroxide typically reduce microbial metabolism (Cory et al., 2010) by affecting cell structures activating repair mechanisms increasing the energy expenses (Madigan et al., 2010), and reducing BGE. ROS may also consume biologically available substrates, such as some amino acids, reducing bacterial growth and potentially BGE (Amado et al., 2007; Cory et al., 2010). Thus, the effects of higher DOM photochemical degradation and ROS production on microbial metabolism might be more pronounced in the tropics, also contributing to lower BGE (**Figures 1**, **3**) due to higher sunlight irradiance throughout the year.

In studying global phenomena, such as using meta-analysis, one needs to be cautious to avoid misinterpretation of data as a consequence of methodological artifacts. Regarding BR data, one could argue that using RQ equal to 1 to convert BR from oxygen consumption to carbon basis is not adequate to a large dataset with distinct ecosystems from different regions, because it has recently been shown that RQ can vary between 0.8 and 1.4 depending on the origin and oxidation state of the DOM (Berggren et al., 2011). However, without further information about each ecosystem and/or differences in RQs between the temperate and tropical regions, applying a global RQ of 1 seems to be the most logical and conservative solution. Nonetheless, the highest RQ values have been observed when bacteria use DOM compounds that are highly oxidized, such as photochemically degraded DOM (Berggren et al., 2011). Taking into account that tropical ecosystems are exposed to higher sunlight incidence (Lewis, 1987, 1996) and that DOM is more photochemically oxidized compared to temperate ecosystems (Farjalla et al., 2009), we should expect the highest RQ in the tropical bacterial communities. Higher RQ values would increase BR rates in tropical ecosystems even more, and lower BGE.

Finally, biotic interactions might also contribute to explain the differences observed in bacterial metabolism along the latitudinal gradient. The fact that tropical ecosystems have slightly higher BP and lower bacterial abundance (Roland et al., 2010; Sarmento, 2012) is a strong indication that bacterial loss factors (such as grazing by protists or virus-induced lysogeny) might be more relevant at low latitudes. Despite the few data available for tropical lakes, recent studies provided strong indications that grazing on bacteria by microzooplankton (mainly heterotrophic nanoflagellates) in tropical lakes should be relatively high (Tarbe et al., 2011; Sarmento, 2012). This expected high grazing pressure on bacteria would explain the lower bacterial abundances in the tropics along the productivity gradient (Roland et al., 2010; Sarmento, 2012). Alternatively, strong grazing control might likely maintain bacterial communities in a rapid growth condition (e.g., as in the exponential growth phase), resulting in high-energy metabolic expenditure (such as in the ATP paradox, see discussion above). Concerning viral infection, there is almost no information available on how viral infection varies with latitude or temperature, but it has been suggested that higher bacterial metabolic rates are usually associated with low viral induced lysogeny (Maurice et al., 2010).

# **CONCLUDING REMARKS**

Including data from tropical freshwaters in a conventional (mainly temperate) bacterial metabolism database did not change radically the relationships among BP, BR and BGE previously reported. However, a comparative analysis of tropical against temperate data indicated that BP, BR, and BCD were higher in tropical than in temperate ecosystems. Moreover, comparing both regions, the difference was more pronounced in BR rates than in BP, and consequently BGE in tropical ecosystems was lower. Furthermore, BR rates per BP unit were at least two fold higher in the tropics than in temperate ecosystems indicating higher nutrients turnover rates. Higher annual temperatures, increased nutrient limitation and different food web configurations all help to explain the higher energy dissipation in tropical regions. Lastly, higher sunlight exposure in the tropics contributes to higher photochemical DOM degradation and oxidation states and higher photochemical production of ROS. The current model of bacterial metabolism based on the relationship of BR and BP (Del Giorgio and Cole, 1998) is not adequate to predict the relationships between these two metabolic parameters efficiently at low latitudes. The addition of a large set of tropical data did not improve the general model, but highlighted some relevant factors that regulate microbial metabolism. Finally, we point out the need for intensive data collection on this topic, particularly in tropical regions in multiple biomes and ecosystem types.

# **ACKNOWLEDGMENTS**

We acknowledge Fabíola Dantas and Caroline G. B. Moura for help with data collecting and to José Luiz de Attayde and

# **REFERENCES**

streams. *Microb. Ecol.* 57, 170–178. doi: 10.1007/s00248-008-9423-6

the organizing committee of the XIII Brazilian Meeting of Limnology for the organization of the Symposium on bacterial metabolism and carbon cycling in the tropics that motivated this paper. Dr. Wilhelm Granéli provided access to Brownification project data (FORMAS 217-2006-547), and Dr. Gwenael Abril provided CARBAMA Project data (ANR, French National Agency for research). Funding was provided by CNPq Universal Programs proc. 472820/2008-7 and 477655/2010-6, the CAPES/FAPERJ PAPD Program (for Frederico Meirelles-Pereira; Proc.: E-26/102.567/2010), the Brazilian *"Ciência sem Fronteiras"* Program from CAPES/CNPq (Hugo Sarmento; BJT 013/2012), the USA National Science Foundation (James B. Cotner and André M. Amado, OCE-0527196 and DEB-9977047), CNPq (Vinicius F. Farjalla and Fabio Roland fellowship), and CAPES (Luciana O. Vidal post-doctoral fellowship). Two reviewers provided invaluable criticisms that improved the manuscript quality.

doi: 10.1890/0012-9658(2002)083 [1831:IBHFAL]2.0.CO;2

Bacterioplankton metabolism in hydroelectric reservoirs. *Oecol. Australis* 15, 605–617. doi: 10.4257/oeco.2011.1503.12

carbon cycling and climate. *Limnol. Oceanogr.* 54, 2298–2314. doi: 10.4319/lo.2009.54.6\_part\_2.2298

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 14 February 2013; accepted: 03 June 2013; published online: 21 June 2013.*

*Citation: Amado AM, Meirelles-Pereira F, Vidal LD, Sarmento H, Suhett AL, Farjalla VF, Cotner JB and Roland F (2013) Tropical freshwater ecosystems have lower bacterial growth efficiency than temperate ones. Front. Microbiol. 4:167. doi: 10.3389/fmicb.2013.00167*

*This article was submitted to Frontiers in Aquatic Microbiology, a specialty of Frontiers in Microbiology.*

*Copyright © 2013 Amado, Meirelles-Pereira, Vidal, Sarmento, Suhett, Farjalla, Cotner and Roland. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

# Coupling Between Heterotrophic Nanoflagellates and Bacteria in Fresh Waters: Does Latitude Make a Difference?

*Bianca T. Segovia1\*, Carolina D. Domingues2, Bianca R. Meira1, Fernando M. Lansac-Toha1, Paulina Fermani3, Fernando Unrein3, Lúcia M. Lobão4, Fabio Roland4, Luiz F. M. Velho1 and Hugo Sarmento5*

*1 Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura, Universidade Estadual de Maringá, Maringá, Brazil, 2 Departamento de Botânica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 3 Instituto Tecnológico de Chascomús, Universidad Nacional de San Martín, Chascomús, Argentina, 4 Departamento de Ecologia, Universidade Federal de Juiz de Fora, Juiz de Fora, Brazil, 5 Departamento de Hidrobiologia, Universidade Federal de São Carlos, São Carlos, Brazil*

#### *Edited by:*

*James Cotner, University of Minnesota Twin Cities, USA*

#### *Reviewed by:*

*Guang Gao, Nanjing Institute of Geography and Limnology, China Nathalie Niquil, Centre National de la Recherche Scientifique, France*

> *\*Correspondence: Bianca T. Segovia<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Aquatic Microbiology, a section of the journal Frontiers in Microbiology*

*Received: 05 August 2015 Accepted: 22 January 2016 Published: 11 February 2016*

#### *Citation:*

*Segovia BT, Domingues CD, Meira BR, Lansac-Toha FM, Fermani P, Unrein F, Lobão LM, Roland F, Velho LFM and Sarmento H (2016) Coupling Between Heterotrophic Nanoflagellates and Bacteria in Fresh Waters: Does Latitude Make a Difference? Front. Microbiol. 7:114. doi: 10.3389/fmicb.2016.00114*

Recent studies reported comparatively lower heterotrophic bacteria (HB) abundances in tropical regions, indicating that factors involved in bacterial losses could be more relevant in the tropics. Heterotrophic nanoflagellates (HNF) are considered the main predators of HB in aquatic ecosystems, and one should expect higher abundances in the tropics because of differences in the food web configuration (absence of large daphnids). However, there are no comprehensive studies comparing HB and HNF abundances in a latitudinal gradient. We hypothesized that HB abundance would be lower in the tropics because HNF abundance would be higher, resulting in a tighter HNF–HB coupling. To test this hypothesis, we compiled a large dataset of HB and HNF abundances from tropical and temperate freshwater environments. We found that both HB and HNF abundances were lower in the tropical region, and that HNF-HB coupling does not differ between temperate and tropical regions. The lower HNF abundance and lack of coupling may be explained by a strong top-down control on HNF and/or their herbivory preference. Besides, no relationship was found between bacterial specific growth rate and either chlorophyll-*a* and HB abundance, indicating that bacterial losses may have an important role in tropical freshwaters. Thus, we found that HNF is likely not the main controllers of HB abundance, and that grazing by ciliates and cladocerans, together with the physiological effects of higher temperatures, may explain the high bacterial loss rates in the tropics.

Keywords: bacterioplankton, cladocera, protist, predation, latitude

# INTRODUCTION

Inland aquatic ecosystems play a relevant role in the global carbon cycle (Cole et al., 2007; Tranvik et al., 2009; Raymond et al., 2013). Low latitude freshwaters, particularly wetlands, represent a high percentage of global CO2 evasion to the atmosphere compared to colder counterparts located in temperate regions (Marotta et al., 2009; Aufdenkampe et al., 2011; Barros et al., 2011; Abril et al., 2014; Borges et al., 2015). The disproportional importance of tropical fresh waters in CO2 net diffusion would be due to the high input of organic terrestrial carbon and further microbial heterotrophic respiration (Cole et al., 1994; del Giorgio et al., 1999), together with the higher temperatures (Kosten et al., 2010). In fact, bacterial biomass and production has been related to CO2 lake concentrations (Tadonléké et al., 2012; Fontes et al., 2013), evidencing the importance of bacterioplankton in CO2 emission dynamics. Thus, it is essential to identify the patterns and drivers of bacterial abundance, production and respiration across latitudinal gradients.

In this way, recent studies pointed out that, despite the slightly higher bacterial production in lower latitudes (Amado et al., 2013), the bacterial abundance found in those regions is lower, compared to temperate environments (Roland et al., 2010; Sarmento, 2012). This indicates that factors involved in bacterial loss would be more important in the tropics, since bacterial biomass does not seem to increase with increasing bacterial production in similar rates in both regions (Billen et al., 1990). The low bacteria:chlorophyll*-a* ratios found in warm waters suggest that grazing might be an important mechanism limiting bacterial abundance (Sarmento et al., 2008; Roland et al., 2010; Özen et al., 2013). These differences in HB abundance at different latitudes have been attributed, at least in part, to a higher top–down control of rotifers, ciliates, and nanoflagellates in warmer regions (Roland et al., 2010; Sarmento et al., 2010; Sarmento, 2012; Vázquez-Domínguez et al., 2012; Amado et al., 2013).

Because heterotrophic nanoflagellates (HNF) are considered the main responsible for channeling bacterial production to higher trophic levels (Fenchel, 1982; Sanders et al., 1989, 1992; Berninger et al., 1991), one should expect a higher top–down control on bacteria by the HNF in the tropics. Factors known to exert an influence on the predator–prey relationship between HNF and bacteria, such as temperature, bacterial, and HNF abundance (Peters, 1994; Vaqué et al., 1994; Gasol et al., 2002), vary widely with latitude. As temperature alters metabolic rates, it also influences all the other factors above cited, as predicted by the metabolic theory of ecology (MTE; Brown et al., 2004), which might also provide some insights on differences of microbial metabolic rates and trophic interactions between tropical and temperate regions.

The cornerstone of MTE is that metabolic rates, including grazing rates (Sarmento et al., 2010) and population growth rates (Savage et al., 2004), increase exponentially with temperature (Brown et al., 2004). For instance, bacterial abundance and production is thought to increase with increasing temperatures (White et al., 1991). However, the effects of temperature are not always straightforward, and increased temperatures may actually lead to a decrease in the abundance of the organisms, because the increased metabolic cost per individual means that a given supply of energy will support a smaller number of individuals (Brown et al., 2004; Savage et al., 2004; Sarmento et al., 2010). Yet, this assumption does not consider the effects of trophic interactions. For example, Jiang and Morin (2004) found that competition between the populations of two protists changed the outcome of temperature effects on their abundances, when compared with the isolated temperature effect on those populations. Also, Vasseur and McCann (2005) model states that temperature alone would not affect resource density in the absence of predators, implying that the effects of trophic interactions should also be taken into account.

Temperature has also been positively correlated with feeding rates, thereupon protist grazing rates on bacteria are expected to be higher with raised temperatures, since more food is required to fulfill their energy demand (Peters, 1994; Vaqué et al., 1994; Sarmento et al., 2010). Considering that tropical regions experience elevated temperatures throughout the year, bacteria might suffer a higher predation pressure, so that a larger proportion of bacterial production is taken by grazers (Sarmento et al., 2010), outbalancing bacterial growth stimulation by temperature. Indeed, in the few studies available for tropical region, HNF grazing on bacteria was found to be relatively high (Pirlot et al., 2007; Tarbe et al., 2011).

It is believed that HNF abundance in warm environments should be higher than in colder ones, owing to consistent differences in the food web structure along the latitudinal gradient (Sarmento, 2012; Özen et al., 2013). This is because in temperate environments there is a typical prevalence of largebodied cladocerans, which are able to suppress the abundance of HNF (Gasol et al., 1995; Jürgens and Stolpe, 1995; Kalinowska et al., 2015). Actually, the predation pressure of *Daphnia* on HNF was found to result in a lack of coupling between HNF-bacteria in temperate systems, highlighting zooplankton as crucial regulators of bacterial abundance (Gasol and Vaqué, 1993; Jürgens et al., 1994). Meanwhile in the tropics, both temperature (Havens et al., 2015) and the high predation pressure exerted by the juvenile fishes, which are almost permanently present due to fish reproduction throughout the year (Fernando, 1994; Lazzaro, 1997; Iglesias et al., 2011), favor the development of smallbodied zooplankton. Those, in turn, would not be as efficient in reducing microbial abundances as their relatives of the temperate regions, thus the assumed greater HNF abundance would account for a tighter coupling between bacteria and HNF in tropical environments (Sarmento, 2012). Accordingly, elevated temperatures increasing microbial metabolism, along with the higher abundance of HNF and lower abundance of bacteria, all concur to the idea that HNF-bacteria coupling should differ across latitudinal gradients, being stronger in the tropics.

The aim of this study was to compare HNF and HB abundances in different latitudes (temperate vs tropical), as well as the HNF–HB coupling. Taking into account that HNF grazing pressure is thought to be the main explanation for lower bacterial abundance in tropical regions, and that the lack of HNF–bacterial coupling seems to be a widespread phenomenon in the temperate ones, we hypothesized that, in the tropics, (i) HB abundance would be lower, because (ii) HNF abundance would be higher, and consequently (iii) HNF–HB coupling would be stronger. We also investigated the importance of other predators and resources (i.e., chlorophyll-*a*) in explaining bacterial abundance in tropical environments. In order to test these hypotheses, we compiled a large dataset of HB and HNF abundances from tropical and temperate freshwater environments and compared their abundances and the HNF-HB coupling, besides exploring other possible causes involved in bacterial losses in the tropics.

# MATERIALS AND METHODS

# Data Compilation

The dataset consists of 1047 observations of heterotrophic bacteria (HB) and HNF abundances from the literature in both tropical (*N*trop = 381) and temperate (*N*temp = 666) freshwater inland aquatic ecosystems. The data was gathered from some of the vast literature found for temperate environments as well as studies performed so far in the tropics, and encompasses a broad range of environment types, including shallow lakes, deep lakes, and reservoirs of various trophic status (**Table 1**). We also used abundance data of ciliates, rotifers, cladocerans, and copepods from tropical environments.

# Data Analysis

# HB and HNF Abundance and Relationship

To test whether HB and HNF abundances differ among tropical and temperate freshwater environments, we used nonparametric Mann–Whitney Rank Sum test. In addition, we also performed non-parametric Mann–Whitney Rank Sum test to compare median values of HB:HNF ratios between tropical and temperate environments. To examine the relationship between HNF and HB on tropical and temperate datasets, we performed model II linear regression using the major axis (MA) method (Legendre, 2014), and verified the normal distribution of the log-transformed data. We compared the slopes and intercepts for both regions using the "ma" function of the "smatr" package (Warton et al., 2012), that tests hypotheses about slope or elevation ("elev.test") based on confidence intervals comparison.

# Bacterial Specific Growth Rate (SGR) Relationship with Chlorophyll-*a* and Bacterial Abundance

We used a dataset comprehending several tropical environments sampled in different seasons (Lobão et al., in preparation) to verify if bacterial SGR was more related to resources or predators. We estimated bacterial SGR using the equation proposed by Kirchman (2002): SGR = P/B, where *P* = bacterial production (μgC L−1 h−1) and B = bacterial biomass (μgC L−1). We performed linear regressions to test the relationship between SGR and HB abundance, which might provide some hints about the factors controlling their abundance. The rationale is that, considering the density-dependent logistic growth of bacteria, SGR is low when bacterial abundance is reaching the carrying capacity, meaning that they are limited by resource availability. Hence, a negative relationship between SGR and abundance indicates bottom-up control. Conversely, SGR is high when bacterial abundance is far from reaching the carrying capacity. Thus, the lack of relationship between SGR and abundance indicates top-down control, so that predators could be consuming bacteria at rates equal to or higher than their production (Wright and Coffin, 1984; Gasol et al., 2002).

# Impact of Other Communities on HB and HNF Abundance

We examined the effects of potential predators on HB and HNF in the tropical region. We considered the abundances of HB and HNF as response variables separately, and performed multiple regressions for each one. For HB, we used the abundance of the predators HNF, ciliates, rotifers and cladocerans as explanatory variables, excluding copepods, which have a very low capture efficiency of picoplankton (Wilson, 1973; Finlay and Roff, 2004; Sommer and Sommer, 2006). For HNF, we used the abundance of the predators known to exploit them as food, such as ciliates, rotifers, cladocerans, and copepods.

Data was log-transformed and all analyses were performed in R Development Core Team (2013) using the libraries "vegan" (Oksanen et al., 2015), "lmodel2" (Legendre, 2014), and "smatr" (Warton et al., 2012). Figures were made on SigmaPlot v.12 software (Systat Softare Inc.).

# RESULTS

At first, we considered all data we gathered from the literature in our analyses. However, some of the studies performed in highly eutrophic environments have found extreme values of HB and HNF abundance, never reported before on the literature (i.e., Fermani et al., 2013, 2015). As we did not found any equivalent conditions in the tropical dataset, and since we noticed that the data from those studies were outliers, we decided to disregard those values from all our analyses. In this way, we maintained a similar distribution of points among trophic states in the temperate (oligotrophic: 16%, mesotrophic: 50%, eutrophic: 34%) and tropical (oligotrophic: 19%, mesotrophic: 45%, eutrophic: 36%) regions. Nevertheless, we show them in the regression figure (**Figure 3**) for comparison purposes.

# HB and HNF Abundance

Comparing HB and HNF abundance in tropical and temperate freshwater environments, we found higher values in the temperate region for both HB (logHB: *p <* 0.001, Mann–Whitney *U* Statistic = 218964.5; **Figure 1A**) and HNF (logHNF: *p <* 0.001, Mann–Whitney *U* Statistic = 189579; **Figure 1B**) communities.

# HNF–HB Relationship

HB:HNF ratios were not significantly different between tropical and temperate environments (HB:HNF: *p* = 0.3049, Mann– Whitney *U* Statistic = 131703; **Figure 2**).

We found a significant positive relationship between HNF and HB for tropical and temperate regions. Comparing the regression models from both regions, we found no significant differences between the slopes, besides no differences in the confidence intervals for the intercepts (**Table 2**; **Figure 3**).

# Factors Controlling HB and HNF Abundances

Linear regressions between SGR and HB abundance (**Figure 4**) were non-significant in most tropical systems

TABLE 1 | Database from each literature data in tropical and temperate environments used in all analysis. **∗**To perform the analysis of the relationship between bacterial specific growth rates, chlorophyll-*a*, and bacterial abundance, we used a different dataset (see below).

∗*Data from those references were considered outliers and were not used in our analysis.*

(six out of eight systems), pointing toward a regulation of bacterial numbers by predation for most systems. Taking all systems together, this relationship was not significant either.

We performed multiple regressions to evaluate the effects of potential predators on HB abundance. The regression model explained 28% of the variation in HB abundance of the tropical data and included the abundances of HNF, ciliates, rotifers and cladocerans (**Table 3**). The standardized regression coefficients of both HNF and rotifers were positive, thus an increase in HNF and rotifer abundance was associated with an increase in HB abundance, suggesting a bottom-up effect. As for the ciliates and cladocerans, we found a negative relationship, suggesting a top down effect, since an increase in ciliate and cladoceran abundance was associated with a reduction in HB abundance.

The best multiple regression model for HNF abundance included only ciliates and cladocerans and explained 32% of the HNF abundance variation. The standard regression coefficient of ciliates was positive, indicating a simultaneously increase in both variables. As for the cladocerans, we found a negative

relationship, indicating a top–down effect of this group on HNF (**Table 3**).

# DISCUSSION

We compiled for the first time a consistent HNF and HB abundance database for tropical freshwaters, and compared the abundances of those communities with the ones from the temperate environments, as well as explored probable causes of lower bacterial abundance in the tropics. We found that both HNF and HB abundances were lower in the tropics and that there is no difference in the HNF-HB coupling between those

FIGURE 2 | Comparison of HB:HNF ratios among tropical and temperate freshwater environments. The central full line indicates the median value, the dotted line indicates the arithmetic mean value, the boxes indicate the lower and upper quartiles, the vertical lines indicate the 10th and 90th percentiles, and the dots represent the 5th and 95th percentiles. HB:HNF ratios were not significantly different between tropical and temperate environments (see text).

regions. Besides, HB abundances were apparently more regulated by predation, especially from ciliates and cladocerans.

(see Materials and Methods).

Temperate 2.48 (2.22:2.81) –13.13 (–15.28:–11.38) 666 0.3 *<*0.0001

TABLE 3 | Regression analyses for HB and HNF abundance of the tropical region.

β*, standard regression coefficient; SE, standard error; HB, heterotrophic bacteria; HNF, heterotrophic nanoflagellates; Cili, ciliates; Rot, rotifers; Clad, cladocerans; Cop, copepods. Bold values are the negative* β *values.*

# Weak Evidence of Resource Limitation

Evidence found in the literature suggests that bacterial growth dependence on phytoplankton derived dissolved organic carbon (DOC) supply might not always be that relevant in low latitudes. Although there is evidence that phytoplankton derived DOC would be important for the bacterioplankton of large African tropical lakes (Stenuite et al., 2009; Morana et al., 2014), low HB:phytoplankton biomass ratios have been found (Sarmento et al., 2008). In a comparative analysis using different types of Brazilian freshwater ecosystems, Roland et al. (2010) found a much weaker HB:chlorophyll-*a* correlation in tropical when compared to the non-tropical environments. In this way, the bacteria–phytoplankton uncoupling seems to be a recurrent situation in south-American lowland lakes (e.g., Carvalho et al., 2003; Gocke et al., 2004; Rejas et al., 2005; Petrucio et al., 2006; Teixeira et al., 2011; Almeida et al., 2015), which are generally smaller and shallower, comparing to the East-African Great Lakes. White et al. (1991) reported a rather weak correlation between SGR and chlorophyll-*a* in freshwaters, and suggested that variations in the importance of grazing pressure may have contributed to this finding.

However, as allochthonous DOC may also constitute an important resource for HB (Tranvik, 1992), a regulation of HB by those carbon sources could also explain the weak dependency of bacteria on phytoplankton. Unfortunately, we do not have data concerning those variables, which would allow us to elucidate this point. Nonetheless, our results of non-significant relationships between bacterial SGR and HB abundance in most of the tropical systems analyzed (**Figure 4**), reinforce the idea that predation might be more relevant than resource limitation, whatever that resource could be. If HB abundance and SGR were not related, grazing was likely consuming HB at such a rate that it was limited by a small range of possible growth rates (Wright and Coffin, 1984; Gasol et al., 2002). Thus, we could infer that resource

limitation was not likely to restrain HB abundance in most tropical freshwater environments, and that a top-down control might prevail in these systems.

# HNF Abundance is Also Lower in Tropical Environments

The assumption of a higher abundance of HNF in tropical, relative to temperate environments, was not corroborated in our study. Although large-bodied cladocerans are relatively low abundant in the tropics, the typical small bodied cladocerans, seem to exert a strong predation pressure on HNF, as evidenced by the negative standard coefficient multiple regression model (**Table 3**).

The impact of small-bodied cladocerans on HNF is somewhat unexpected, since in the tropics there is usually a smaller proportion of Daphniidae, which is replaced by Bosminids, Sidids, and Moinids (Dumont, 1994; Elmoor-Loureiro, 2000). However, the influence of cladocerans on the abundance of HNF was already verified in the bottom layer a tropical floodplain lake where those predators were more abundant, specially represented by *Bosmina hagmanni* and *Ceriodaphnia cornuta* (Segovia et al., 2014). In fact, the small-bodied cladocerans *Bosmina*, *Ceriodaphnia*, and *Diaphanosoma* were found to achieve higher weight-specific clearance rates on HNF than that of *Daphnia* species (Jürgens et al., 1996). Specifically, *Bosmina* have a particular foraging mode, different from filter-feeding, which allows certain selectivity and consequently more efficient removal of small flagellates compared to *Daphnia* (DeMott and Kerfoot, 1982), even at low food concentrations (DeMott, 1982). Thus, even though Daphnids are recognized as the main responsible for hampering the development of HNF in temperate ecosystems (Pace and Vaqué, 1994; Gasol et al., 1995; Jürgens and Stolpe, 1995), their low abundance in the tropics would not result in a weaker predation pressure of cladocerans on HNF, since other small-bodied cladocerans such as the Bosminids may replace *Daphnia*, in the sense that they would also be able to suppress HNF effectively. As for the ciliates, we found a positive relationship with HNF, indicating that both variables are increasing. It is possible that this could be the result of the control of both HNF and ciliates by variables related to their shared resources and predators (Auer et al., 2004; Segovia et al., 2014; Domingues et al., submitted).

HNF–HB coupling in the tropics does not seem to differ from that of the temperate regions. A top–down control by cladocerans on HNF may be keeping them from reaching the high abundances they presumably would have in the tropics, blurring their effects on bacteria (Gasol and Vaqué, 1993; Gasol, 1994; Wieltschnig et al., 2001; Segovia et al., 2014; Kalinowska et al., 2015). Another possible cause for the lack of HNF–HB coupling is the use of an alternative food resource by the HNF, such as the picophytoplankton (PPP). Herbivory preference by nanoflagellates, rather than bacterivory, was verified in the large tropical Lake Tanganyika (Tarbe et al., 2011). The preference of HNF for PPP was also found in shallow floodplain lakes in the tropical region (Meira et al., in preparation). In addition, the biomass of HNF was negatively related to PPP in tropical reservoirs of different trophic states, pointing out the importance of this interaction on these environments as well (Domingues et al., submitted). To sum up, the lower HNF abundance found, together with the similar HNF-HB coupling, suggests that HNF is probably not related to the lower HB abundance in the tropics.

# Grazing by Ciliates and Cladocerans May Explain the Lower HB Abundance in the Tropics

The variables associated with HB abundance in the tropics were HNF, ciliates, rotifers, and cladocerans. HNF and rotifers were positively related with HB abundance, which means that they are likely feeding on bacteria but are not able to suppress their abundance. On the contrary, both ciliates and cladocerans showed a negative relationship, suggesting a top–down control on HB abundance. As stated before, resource limitation or predation by HNF are unlikely to be the reason why bacterial abundance is lower in the tropics. Thus, the negative effect of both ciliates and cladocerans could be part of the explanation for such a pattern.

Although there is a vast literature relating the prevalence of HNF as the major bacterivores (Fenchel, 1982; Sanders et al., 1989, 1992; Berninger et al., 1991), the relatively higher importance of ciliates as predators of bacteria was also documented. The dominance of ciliates as grazers of bacteria has been reported in occasions where HNF abundance is rather low (Kisand and Zingel, 2000; Tadonléké et al., 2005; Zingel et al., 2007). Also, ciliate community structure in the tropics may differ from that of the temperate regions. It is known that bacterivory is predominant among the small oligotrich ciliates (Stabell, 1996; Šimek et al., 2000), thus perhaps features such as ciliate community composition might be playing a role on the impact of ciliates in tropical environments, where there could be a larger proportion of those bacterivorous taxa. However, more studies are necessary to draw such a conclusion. Another overlooked aspect would be the influence of temperature on the ciliate feeding rates. It has been shown that ciliate feeding rates increase considerably with the raise of temperature (Sherr et al., 1988; Rychert, 2011). Therefore, it is plausible to infer that the higher temperatures of the tropics may be a relevant factor.

Similarly to the impact on HNF, weight-specific filtering rates on bacteria were found to be higher for *Ceriodaphnia* and *Bosmina* than for the large *Daphnia magna* (Porter et al., 1983). Vaqué and Pace (1992) found that lakes dominated by large populations of *Bosmina longirostris* showed even slightly higher maximum values of grazing on bacteria (35 × 106 bacteria L−1 h−1) than *Daphnia pulex* (30 × 106 bacteria L−1 h−1), and concluded that, when in large numbers, populations of small cladocerans compensate for the lack of large Daphnids. Thus, if tropical environments are dominated by those small-bodied cladocerans, then their impact on bacterioplankton could be higher than in temperate environments. In addition, a positive relationship has been found between cladoceran filtering rate and temperature (Burns, 1969). For example, Mourelatos and Lacroix (1990) found that at a temperature of 20◦C, a *Daphnia* of 0.5 mm size filtered as much as one twice its size but at a 10◦C temperature, suggesting that at higher temperatures those small-bodied cladocerans should have an even greater impact. Moreover, a recent study found that pelagic cladocerans significantly explained the variation in bacterial community composition in tropical South American shallow lakes (Souffreau et al., 2015), demonstrating that the predation pressure of those microcrustaceans might also be responsible for changes in bacterial community structure.

Thus, ciliates and small cladocerans seem to play a central role in the pelagic food webs of tropical freshwater environments, and the fundamental differences in the food web structure of freshwater environments in temperate and tropical environments, together with the higher temperatures of the tropical ones, likely dictate the fate of bacterial production (**Figure 5**).

It is worth noting that virus lysis is also recognized as a major source of bacterial losses (Fuhrman and Noble, 1995), however, few studies concerning this topic were performed in the tropics. Low virus-to-bacterium ratios and frequency of visible infected cells were found in Amazonian floodplain lakes (Barros et al., 2010; Almeida et al., 2015) and African lakes (Bettarel et al., 2006). Barros et al. (2010)suggested that these low values could be related to the registered low bacterial abundances, which restrain the rates of encounter between the virus and the bacterial host cell, resulting in a low level of viral predation. As a corollary for

# REFERENCES

this explanation, the comparable lower abundances of bacteria in the tropics should result in lower loss rates by viral attack than in the temperate systems. Nonetheless, relatively high values of virus-to-bacterium ratios were found in tropical reservoirs (Peduzzi and Schiemer, 2004) and in a tropical lake (Araújo and Godinho, 2009). Thus, bacterial mortality caused by virus should be taken into account when studying mechanisms controlling bacterial abundance in tropical freshwaters in the future to elucidate this issue.

# CONCLUSION

Comparing tropical against temperate data reinforced the previous findings that bacterial abundance is lower in the tropics. Moreover, bacterial specific growth rate was not related to either chlorophyll-*a* and HB abundance, pointing to an important role of bacterial losses in the tropics. Besides, we found that HNF abundance is also lower in the tropics and that HNF-HB coupling is not different across latitudes. A top–down control on HNF and their herbivory preference may help explain the lack of HNF–HB coupling, and suggests that HNF is likely not the main cause for bacterial loss. It is possible that grazing by ciliates and cladocerans play a large role in controlling bacterial abundance in the warmer regions. However, this issue should be more investigated in future studies concerning tropical freshwater environments.

# AUTHOR CONTRIBUTIONS

HS and BTS conceived and designed the study. CDD, BRM, FMLT, PF, FU and LFMV sampled and analyzed the abundance data of microbial communities from tropical/temperate freshwaters. LML and FR sampled and analyzed the bacterial specific growth rate data from tropical freshwaters. BTS wrote the manuscript with input from all coauthors.

# ACKNOWLEDGMENTS

BTS is grateful for the funding and fellowships provided by the Coordination for the Improvement of Higher Education Personnel (CAPES) and the Brazilian National Council of Research and Development (CNPq). LFMV and FR have been continuously supported by CNPq grants. HS's work was supported by CNPq (Project: 474759/2013-0), FAPESP (Project: 2014/14139-3) and Furnas.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Segovia, Domingues, Meira, Lansac-Toha, Fermani, Unrein, Lobão, Roland, Velho and Sarmento. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Potential changes in bacterial metabolism associated with increased water temperature and nutrient inputs in tropical humic lagoons

*Vinicius Scofield1,2, Saulo M. S. Jacques1,3, Jean R. D. Guimarães4 and Vinicius F. Farjalla1,5\**

*1 Department of Ecology, Biology Institute, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 2 Post-Graduate Program in Ecology, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 3 Post-Graduate Program in Ecology and Evolution, Federal University of Juiz de Fora, Rio de Janeiro, Brazil, 4 Institute of Biophysics Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 5 Laboratorio Internacional en Cambio Global, Rio de Janeiro, Brazil*

#### *Edited by:*

*André Megali Amado, Universidade Federal do Rio Grande do Norte, Brazil*

#### *Reviewed by:*

*Ng Haig They, Federal University of Rio Grande do Norte, Brazil François Guillemette, Uppsala University, Sweden*

#### *\*Correspondence:*

*Vinicius F. Farjalla, Department of Ecology, Biology Institute, CCS Building, Universidade Federal do Rio de Janeiro, P.O. Box 68020, Rio de Janeiro, RJ, Brazil<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Aquatic Microbiology, a section of the journal Frontiers in Microbiology*

> *Received: 10 December 2014 Accepted: 29 March 2015 Published: 15 April 2015*

#### *Citation:*

*Scofield V, Jacques SMS, Guimarães JRD and Farjalla VF (2015) Potential changes in bacterial metabolism associated with increased water temperature and nutrient inputs in tropical humic lagoons. Front. Microbiol. 6:310. doi: 10.3389/fmicb.2015.00310* Temperature and nutrient concentrations regulate aquatic bacterial metabolism. However, few studies have focused on the effect of the interaction between these factors on bacterial processes, and none have been performed in tropical aquatic ecosystems. We analyzed the main and interactive effects of changes in water temperature and N and P concentrations on bacterioplankton production (BP), bacterioplankton respiration (BR) and bacterial growth efficiency (BGE) in tropical coastal lagoons. We used a factorial design with three levels of water temperature (25, 30, and 35◦C) and four levels of N and/or P additions (Control, N, P, and NP additions) in five tropical humic lagoons. When data for all lagoons were pooled together, a weak interaction was observed between the increase in water temperature and the addition of nutrients. Water temperature alone had the greatest impact on bacterial metabolism by increasing BR, decreasing BP, and decreasing BGE. An increase of 1◦C lead to an increase of ∼4% in BR, a decrease of ∼0.9% in BP, and a decrease of ∼4% in BGE. When data were analyzed separately, lagoons responded differently to nutrient additions depending on Dissolved Organic Carbon (DOC) concentration. Lagoons with lowest DOC concentrations showed the strongest responses to nutrient additions: BP increased in response to N, P, and their interaction, BR increased in response to N and the interaction between N and P, and BGE was negatively affected, mainly by the interaction between N and P additions. Lagoons with the highest DOC concentrations showed almost no significant relationship with nutrient additions. Taken together, these results show that different environmental drivers impact bacterial processes at different scales. Changes of bacterial metabolism related to the increase of water temperature are consistent between lagoons, therefore their consequences can be predicted at a regional scale, while the effect of nutrient inputs is specific to different lagoons but seems to be related to the DOC concentration.

Keywords: environmental changes, bacterial metabolism, bacterial growth efficiency, tropical aquatic ecosystems, coastal lagoons, water temperature

# Introduction

Changes in climate and biogeochemical cycles are among the most important effects of human alterations to ecosystems (Rockström et al., 2009). For instance, nitrogen deposition, and phosphorus loads related to sewage disposal are affecting natural aquatic ecosystems worldwide (Schindler and Vallentyne, 2008). Increases in air temperature and changes in rain patterns are also expected in most regions (IPCC, 2013). For the highly populated Brazilian Southeast in particular, an increase in the average daily temperature and dramatic changes in the intensity and periodicity of rainfall events are expected for the next century (Marengo et al., 2010; PBMC, 2013). These changes will impact the amount of energy and matter flowing into aquatic ecosystems by increasing water temperature and augmenting the input of nutrients from drainage areas, with consequences for the structure and functioning of aquatic ecosystems (Roland et al., 2012). Furthermore, in tropical aquatic ecosystems, synergy between climate-driven impacts and changes in the input of inorganic nutrients is expected, mainly in highly populated areas (Roland et al., 2012). Thus, studies integrating different environmental changes into the same experimental design are necessary to better understand the effects of synergistic interactions on the functioning of aquatic ecosystems.

Bacterioplankton play an essential function in the transfer of energy and materials in aquatic ecosystems. Bacterioplankton respiration (BR) mineralizes large amounts of organic carbon substrates, thereby contributing to CO2 saturation in aquatic ecosystems, whereas bacterioplankton production (BP, i.e., secondary production) is an important source of energy for higher trophic levels through the microbial loop food chain (Fenchel, 2008). The ratio of carbon incorporated into bacterial biomass to total carbon assimilated by bacteria (bacterial growth efficiency, BGE) has been used to evaluate whether and where bacteria act as a carbon source or sink in aquatic ecosystems (for a review, see del Giorgio and Cole, 1998). Several environmental factors regulate BGE in aquatic ecosystems, and any changes in these environmental factors would alter bacterioplankton metabolism, which, in turn, would alter bacterioplankton function in aquatic ecosystems. For instance, low concentrations of N and P limit BP in aquatic ecosystems (e.g., Granéli et al., 2004; Smith and Prairie, 2004; Haubrich et al., 2009) but BR appears to be less affected in oligotrophic aquatic ecosystems. Low BGE is expected in aquatic ecosystems with high C:N and C:P ratios, and the input of inorganic nutrients in oligotrophic waters should increase BGE. Rising water temperature increases the permeability of the cell membrane to carbon substrates, stimulating both BP and BR (Van de Vossenberg et al., 1999; Mansilla et al., 2004). However, higher temperatures could also decrease some enzymatic reactions, increasing the metabolic cost of maintenance and cellular repair and consequently decreasing BGE (Hall and Cotner, 2007). The effect of temperature on BGE can also be mediated by nutrient concentrations, but this is not a general response pattern. For instance, some studies reported a negative relationship between BGE and temperature in nutrient limiting conditions (e.g., Kritzberg et al., 2010), others reported a negative relationship between BGE and temperature when nutrients were abundant (e.g., Hall and Cotner, 2007; Berggren et al., 2010), while others found little or no effect of temperature on BGE, and suggested that BGE was primarily regulated by the availability of nutrients (e.g., López-Urrutia and Morán, 2007; Lee et al., 2009). Most studies on the effects of temperature on bacterial metabolism were performed in temperate ecosystems, but those effects could be more dramatic in tropical systems, where organisms are already in their optimum temperature conditions (Deutsch et al., 2008). In addition, most of these studies failed to disentangle the effects of temperature and nutrients on bacterial metabolism; this article aims to fill this gap in the literature evaluating the main and interactive effects of water temperature and nutrient additions on bacterial metabolism in tropical aquatic ecosystems.

Tropical coastal lagoons of the Brazilian Southeast have several characteristics that make them suitable ecosystems for the evaluation of how changes in environmental condition affect bacterial metabolism. First, these lagoons provide a natural gradient in environmental conditions (for a review, see Esteves et al., 2008). Inorganic N and P concentrations are low, and oligotrophic and dystrophic conditions prevail, though some effects of cultural eutrophication on nutrient availability have also been observed in lagoons located near major cities (Esteves et al., 2008). Water temperature varies synchronously in these lagoons (Caliman et al., 2010), and daily changes of more than 10◦C have been observed in the shallowest lagoons (depth <1 m; Farjalla et al., 2005). Second, climate- and human-driven impacts on these lagoons are predicted to increase during this century (Esteves et al., 2008; Roland et al., 2012; PBMC, 2013). For instance, local population triplicate in the last few years and should double in the next decade driven by the development of the offshore oil industry, and there is good a 1:1 relationship between population growth and cultural eutrophication of coastal aquatic ecosystems in the region (Esteves et al., 2008; Borges et al., 2009). Climate prediction indicates an increase of 0.5–1.0◦C through 2040 and of 2–3◦C between 2041 and 2070 (PBMC, 2013). Third, bacterioplankton metabolism has been extensively studied in these lagoons (e.g., Farjalla et al., 2002, 2005, 2009a). Previous studies have found that BP is usually limited by the availability of inorganic P, though N limitation and N and P co-limitation were also observed (Farjalla et al., 2002). The quality of bulk Dissolved Organic Carbon (DOC) favors BR over BP, and BGE is typically low in these lagoons (Farjalla et al., 2002, 2009a). Under high temperature conditions (above 40◦C), the water temperature regulates bacterial metabolism (Farjalla et al., 2005).

In this study, we evaluated both the main and interactive effects of water temperature increases and inorganic nutrient additions on the bacterial metabolism of tropical coastal lagoons in the Brazilian Southeast. Based on previous results, we expected (i) an increase in BP and BGE after inorganic nutrient additions, (ii) a decrease in both BP and BR with higher temperatures, (iii) synergistic effects between temperature increase and inorganic nutrient additions on bacterial metabolism. We established a full-factorial design in which water temperature and inorganic nutrients were manipulated and BP, BR, and BGE were evaluated following microcosm incubation. The increase in water temperature had a consistent effect on bacterial metabolism across all lagoons, increasing BR and decreasing both BP and BGE. The effects of nutrient additions varied among lagoons and seemed to be related to the concentration of DOC in the systems. We concluded that (i) different environmental stressors impact bacterial processes at different scales, and (ii) the temperature increase predicted for the next century would alter bacterioplankton function in these lagoons, diverting more carbon to bacterial catabolism and CO2 production and/or less carbon to bacterial biomass and higher trophic level through the microbial loop.

# Materials and Methods

# Study Area

The study was conducted in the Cabiúnas, Carapebus, Comprida, Amarra-Boi, and Atoleiro shallow coastal lagoons, all located in Restinga de Jurubatiba National Park in the coastal region of the Brazilian Southeast (−22◦ 17 30, −41◦ 41 30). These lagoons constitute a natural gradient in DOC concentration varying from less than 12 to more than 110 mg L−1 (**Table 1**). More than 90% of the total DOC is composed of allochthonous humic substances mostly derived from an impermeable soil layer rich in organic matter (Farjalla et al., 2009a). Chlorophyll-a concentrations are low, as is the availability of nitrogen and phosphorus (**Table 1**). Despite being located on the coast, salinity is low as is the pH (**Table 1**), which reflects the major contribution of humic-rich freshwater to these systems. Other limnological features can be found in Caliman et al. (2010), and a detailed map of the region and the study lagoons can be found in Laque et al. (2010).

*DOC, Dissolved Organic Carbon; DIN, Dissolved Inorganic Nitrogen; DIP, Dissolved Inorganic Phosphorus. See below information about the analytical methods. pH was obtained using a pH meter (Analion PM 608) and salinity was measured using a portable Thermosalinometer multi-functional probe (YSI-30). For DOC, DIN, and DIP analyses, water samples were filtered through 0.7-*μ*m glass fiber filters (*- = *25 mm, GF/F, Whatman). DOC concentration was determined using a Pt-catalyzed high-temperature combustion method with a TOC-5000 Shimadzu Carbon Analyzer. DIN concentrations were calculated as the sum of N-nitrate and N-ammonium concentrations, which were, in turn, analyzed using a Flow Injection Analysis System (FIA – Asia Ismatec; Zagatto et al., 1980) and spectrophotometry after applying blue indophenol (Koroleff, 1978), respectively. DIP concentrations were determined using the molybdenum blue reaction (Golterman et al., 1978). Chlorophyll-a concentrations were measured through ethanol extraction and spectrophotometry (Nusch and Palme, 1975).*

# Sampling and BP, BR, and BGE Evaluations

Water samples were collected on three alternate days from the central point of each lagoon in January/2009. Each sampling day was treated as a block in the ensuing analyses (see Statistical Analysis below). For sampling, we used 5-l polyethylene bottles previously washed with 10% HCl and rinsed with deionized water. The samples were taken to the laboratory and filtered through 0.7-μm glass fiber filters (- = 47 mm, Macherey-Nagel GF-3) to remove particles and larger organisms. All sampling and filtering procedures were performed in approximately 6 h. Filtered water samples were divided among 20-mL glass vials previously washed with 10% HCl and rinsed with Milli-Q water.

A full factorial design was established for each lagoon, with four levels of addition of two different nutrients (nutrient treatment) and three temperature manipulations (temperature treatment). The nutrient treatment consisted of N addition (addition of KNO3, 50 μM final concentration), P addition (KH2PO4, 5 μM final concentration), N and P addition (additions of KNO3, 50 μM final concentration, and KH2PO4, 5 μM final concentration) and a control with no nutrients added. Added nutrients represented an increase from ambient conditions (control treatments) of 82 to more than 300% for nitrogen and 270–500% for phosphorus. The temperature treatment included incubations at 25, 30, or 35◦C in BOD incubation chambers. The average water temperature is 25◦C in the studied systems (see Caliman et al., 2010 for review) but values up to 40◦C were observed in sporadic drought events in summer months (Farjalla et al., 2005), which should occur more frequently according to projections for the region (Roland et al., 2012). Six replicates were performed for each treatment in each lagoon, yielding a total of 360 bacterial cultures. All incubations were performed in the dark for 48 h. Previous tests and other studies showed bacterial cultures established from water samples from these lagoons responded rapidly to nutrient additions or temperature changes. For instance, daily changes in bacterial production related to changes in the water temperature were observed in another lagoon located in the same area (Farjalla et al., 2005), while bacterial metabolism responded in 24–48 h after nutrient additions in Cabiúnas, Carapebus, and Comprida lagoons (Farjalla et al., 2002).

Bacterioplankton production was evaluated in each bacterial culture at the beginning and end of the 48-h incubation using the 3H-leucine incorporation method (Kirchman et al., 1985) and trichloroacetic acid (TCA) protein extraction (Smith and Azam, 1992), as modified by Miranda et al. (2007). An intracellular isotopic dilution factor of two was used in the calculations (Simon and Azam, 1989). Incubations were performed in the dark for 45 min with 20 nM of 3H-leucine (specific activity 150 Ci mmol−1). The concentration of 20 nM of 3H-leucine was previously established from saturation curves (unpublished data). We set up negative controls by adding 90 μL of TCA before starting the incubations. After the incubation, 3H-leucine incorporation was stopped by the addition of 90 μL TCA. Bacterial protein was extracted by washing with 5% TCA and 80% ethanol. After protein extraction, we added a liquid scintillation cocktail [EcoLite(+) TM] to each sample, and the samples were radio-assayed by liquid scintillation counting (Beckman LS – 6500) after 2 days in the dark to reduce the chemiluminescence. Bacterial protein production was converted to BP (g C 1−1 h−1) using a C:protein ratio of 0.86, according to Wetzel and Likens (1991). BP ratio unit was further converted to μMCh−1 to comparisons with other nutrient concentrations.

Bacterioplankton respiration was assessed by measuring oxygen consumption in each bacterial culture at the beginning and end of the 48-h incubation using an oxygen micro-sensor (Clark-type sensors, OX-N, Unisense) connected to a picoamperimeter (PA 2000, Unisense), following Briand et al. (2004). This approach is highly accurate, stable and has a low response time, and has been widely used to study bacterial metabolism (steering sensitivity <2%, response time <10 s; Briand et al., 2004). A respiratory quotient of 1.2 was used to convert oxygen measurements to carbon values (Berggren et al., 2012). Based on the results of BP and BR, we calculated BGE according to the formula BGE = BP/(BP+BR) (del Giorgio and Cole, 1998).

# Statistical Analyses

The main and interactive effects of each factor on BP, BR, and BGE were assessed using linear models (LMs), linear mixed effect models (LMMs), or non-linear mixed effect models (NLMMs). First, to assess the global effects of treatments and their interactions we pooled together the data of all lagoons. We used LMM and NLMM with additions of N and P set as categorical explanatory factors, temperature as the continuous explanatory variable, sampling blocks nested within lagoons as the random categorical factor, and BP, BR, and BGE as the dependent variables. Because using NLMM had not improved the results (verified by using the Akaike's information criterion), we kept and showed only the LMM results. The Gaussian family (link = "identity") was used to fit the model. To meet test assumptions, BR and BGE were log-transformed. Because only the temperature treatment showed consistent effects on bacterial metabolism in this first analysis (see results below), we (i) ran LMs relating BP, BR, and BGE with the water temperature, and (ii) ran different LMMs for each lagoon to analyze the effects of N and P addition treatments and their interaction on each system separately, searching for common responses to nutrient additions among lagoons. In this case, N and P were set as categorical explanatory factors, sampling blocks nested within temperature treatments as the random categorical factor, and BP, BR, and BGE as dependent variables. Again BR and BGE data were log-transformed to meet test assumptions and the Gaussian family (link = "identity") was used to fit the model. Finally, as we observed two different groups of lagoons with similar types of bacterial responses to N and P additions (low and high DOC concentration ecosystems; low <1.5 μM C, high >4.0 μM C), we re-ran the latter analysis, pooling together the bacterial data for each group. We used contrast analysis to distinguish significant differences between the levels of N and P additions. Tests were performed using the "nlme" (Pinheiro et al., 2014) and "multcomp" (Hothorn et al., 2008) libraries in the R statistical software (R Development Core Team, 2014), and an α criterion level of 0.05 was used.

# Results

Bacterioplankton respiration varied from 0.01 to 0.97 μM C h−1, BP varied from less than 0.01–0.25 μMCh−1, and BGE varied from values as low as 0.01 to values as high as 0.93 (Supplementary Table S1). The overall means were 0.36 μM C h−1 for BR, 0.10 μMCh−1 for BP and 0.15 for BGE. In general, Cabiúnas and Carapebus lagoons showed higher BP rates (means = 0.16 and 0.17 μMCh−1, respectively) than Comprida, Amarra-Boi, and Atoleiro lagoons (means = 0.08, 0.07, and 0.03 μMCh−1, respectively). The Carapebus lagoon showed higher BR rates (mean = 0.45 μMCh−1) than the others (means = 0.37, 0.34, 0.27, and 0.30 μMCh−1 for Cabiúnas, Comprida, Amarra-Boi, and Atoleiro, respectively). BGE was highest in Cabiúnas lagoon (mean = 0.38), followed by Carapebus lagoon (mean = 0.29), Amarra-Boi and Comprida lagoons (means = 0.24 and 0.21, respectively), and Atoleiro lagoon (mean = 0.09; Supplementary Table S1).

In the first analysis, we pooled the data from all lagoons together to observe general patterns of response to nutrient additions and increase of water temperature. Water temperature alone had the greatest effect on bacterial metabolism (**Table 2**). BR increased with increasing temperatures while BP slightly decreased with increasing temperatures. Consequently BGE decreased with increasing temperatures mainly related to the highest BR in the highest incubation temperatures. Neither nutrient additions (N or P) nor their interaction (N and P) had consistent general effects on bacterial metabolism when data from all lagoons were pooled together (i.e., there was no general pattern of nutrient limitation among lagoons, **Table 2**). The only significant interaction between water temperature and nutrient addition was observed for bacterial respiration, in which N additions interacted with the increasing water temperatures, resulting in higher BR rates. Based on these results, we established simple LMs relating bacterial metabolism to water temperature for these lagoons (**Figure 1**). An increase of about 1◦C in water temperature in the lagoons lead to an increase in BR rates of approximately 12.8 nM C h−1, a slight decrease in BP rates of approximately 0.9 nM C h−1, and a decrease in BGE of approximately 0.0116.

We next analyzed the data for each lagoon separately, searching for common responses to nutrient additions among lagoons. Lagoons with the lowest DOC concentrations (Cabiúnas and Carapebus) showed the strongest responses to nutrient additions: BP increased in response to N, P and the interaction between N and P, BR increased in response to N and the interaction between N and P additions, and BGE was negatively affected – mainly by the interaction between N and P additions (**Figure 2**). On the other hand, lagoons with the highest DOC concentrations (Comprida, Amarra-Boi, and Atoleiro) showed almost no significant relationship with nutrient additions: N and P additions slightly stimulated BR in Atoleiro bacterial cultures, the interaction between N and P had a strong positive effect on BR in Comprida bacterial cultures, and neither nutrient addition nor their interaction affected BP or BGE in any of these lagoons (**Figure 2**). We then pooled together

TABLE 2 | Results of linear mixed model (LMM) analysis for bacterial respiration, bacterial production and bacterial growth efficiency (BGE).

*We calculated the main and interactive effects of manipulated factors (nutrient additions and temperature) on bacterial parameters for the complete dataset (all lagoons are pooled together). The block and lagoon identity were inserted as a random factors in the models. Negative or positive signals indicate a negative or positive significant effect of the variable (or interaction) on the bacterial process. Significant effects are in bold.*

the datasets for different lagoons in relation to DOC concentrations and observed that: (1) In the lagoons with the lowest DOC concentrations, BR was enhanced by the N addition and the interaction between N and P, BP was enhanced by N or P alone, and BGE decreased in response to the interaction between N and P additions, and (2) In the lagoons with the highest DOC concentrations, BR was enhanced by the N addition (**Table 3**).

# Discussion

The main goal of this study was to evaluate how bacterioplankton metabolism and its relative importance as a carbon link or sink is singly and/or synergistically influenced by the environmental changes predicted for tropical lagoons of the Brazilian Southeast. We verified that anabolic and catabolic bacterial processes have different trends in response to temperature and nutrient manipulation, and that the strength and generality of the response varies according to the process evaluated and the factor manipulated. For instance, the increase

in water temperature increased BR, decreased BP and, consequently, decreased BGE. Furthermore, the addition of nutrients did not yield a consistent change in bacterial metabolism among all lagoons, but its impact was stronger for lagoons with lower DOC concentrations. Overall, we observed few interactions between water temperature and nutrient concentration, indicating these factors almost always impacted bacterial processes independently.

0.128, and −0.0009 (BP), and 0.488, slope = −0.0116 (BGE).

An increase in the average global temperature is a major environmental change expected for this century. The Brazilian

Southeast is expected an increase of temperature of 0.5–1.0◦C through 2040 and of 2–3◦C between 2041 and 2070 (PBMC, 2013). In temperate systems, increases in temperature are associated with an overall increase in bacterial metabolism with a positive effect on BGE (Hall et al., 2008). This effect may be direct, via increased cellular activity, or indirect, via input of nutrients from the melting of drainage basins in aquatic systems. On the other hand, temperature is consistently higher in tropical systems where tropical organisms are living close to optimum thermal conditions (Deutsch et al., 2008). Even a small increase in water temperature in tropical ecosystems is expected to drive organisms beyond optimal growing conditions (Deutsch et al., 2008), increasing energetic requirements for cell maintenance as a consequence of changes in the membrane fluidity or in the functioning of the cellular enzymatic machinery (Nedwell, 1999; Pomeroy and Wiebe, 2001). The temperature manipulations in this experiment are in the upper temperature limit found in inland waters, and are inserted in the regular temperature range found in these lagoons (Caliman et al., 2010). Therefore, the observed increase in BR and decrease in BGE related to the increase in water temperature in this experiment were expected based on known changes in bacterial TABLE 3 | Results of LMM analysis for bacterial respiration, bacterial production and BGE for two partial datasets pooled in relation to dissolved organic carbon (DOC) concentration in the lagoons.

*Cabiúnas and Carapebus lagoons were categorized as low DOC concentration lagoons, while Comprida, Amarra-Boi, and Atoleiro were categorized as high DOC concentration lagoons (see Table 1 for DOC concentrations). We calculated the main and interactive effects of nutrient additions on bacterial parameters. Incubation temperature and lagoon identity were inserted as a random factors in the models. Negative or positive signals indicate a negative or positive significant effect of the variable (or interaction) on the bacterial process.* ∗*p* < *0.05,* ∗∗*p* < *0.01, and* ∗∗∗*p* < *0.001.*

physiology at high temperatures and based on the temperature range of our manipulations. In fact, higher BR rates and lower rates of carbon incorporation into bacterial biomass associated with higher temperatures were found in a survey of bacterial metabolism in tropical aquatic ecosystems (Amado et al., 2013).

As water temperature affected bacterial metabolism in all lagoons in a similar way, we established negative linear relationships between water temperature, BP and BGE, as well as a positive linear relationship between water temperature and BR. On one hand, the predictive slight negative effect of higher water temperatures on BP indicates that less carbon will be assimilated into bacterial biomass under future climate scenarios. Consequently, the microbial loop is expected to be slightly less influential as an alternative energy source for higher trophic levels in tropical aquatic ecosystems. On the other hand, the positive effect of higher temperatures on BR indicates a positive feedback between a climate change outcome (increase in average temperature) and a climate change driver (release of greenhouse gasses from natural systems). The positive and significant relationship between increasing temperature and BR has been observed previously in temperate regions, albeit, the magnitude of this effect on bacterial metabolism is relatively low compared to tropical aquatic ecosystems (Marotta et al., 2014). This marked effect of temperature in tropical inland waters is strongest in the 35◦C treatment, where BR increased by 2.3–10.5-fold compared with BR in temperate aquatic ecosystems (Berggren et al., 2010; Kritzberg et al., 2010). Therefore, increases in water temperature may be more important for driving bacterial metabolism in tropical rather than

temperate aquatic ecosystems, but this needs further investigation.

Bacterioplankton production is usually limited by the low P concentration in the water column of inland aquatic ecosystems (Granéli et al., 2004; Smith and Prairie, 2004; Bertoni et al., 2008), including the Carapebus and Comprida lagoons (Farjalla et al., 2002). Conversely, the nitrogen limitation of BP is far less common in inland aquatic ecosystems, yet has been observed in some Amazonian ecosystems (Rai and Hill, 1984), in the water accumulated in tank-bromeliads (Haubrich et al., 2009) and in the Cabiúnas lagoon (Farjalla et al., 2002), which is also studied here. Therefore, the observed lack of a clear change in BP in response to the addition of a specific nutrient may be associated with different limiting nutrients in different lakes. This explanation is partially corroborated by the results obtained after the dataset was divided based on DOC concentration in the lagoons: N or P stimulated BP in the lagoons with lowest DOC concentration while there was no clear pattern of change in the lagoons with highest DOC concentration. An alternative hypothesis is based on the colimitation of BP by N and P in a given system, which may have occurred at Cabiúnas and Carapebus lagoons. However, this hypothesis is apparently contrary to the well-established Law of the Minimum (Liebig, 1843), which was first established to describe the patterns of growth limitation in individual plants but not natural communities. In natural communities, each individual or species can specifically respond to the availability of different nutrients, leading to a possible co-limitation by different nutrients of the entire community (for a review, see Harpole et al., 2011). In contrast to BP, BR was strongly influenced by N or NP additions in our study, which is an unexpected result because inorganic nutrients are more important to BP than to BR (e.g., Smith and Prairie, 2004). The nitrate added in the N and NP treatments could be anaerobically respired by denitrifying bacteria, though this scenario is unlikely because the microcosms remained under aerobic conditions throughout the experiment (data not shown) and this process would not be detected by our BR method.

The DOC concentration in the lagoons apparently influences the outcome of nutrient additions on bacterial metabolism. Ecosystems with high carbon concentrations usually show a large imbalance between nutrient availability in the system and nutrient demand by bacteria (Hessen, 1992; Jansson et al., 2006). Despite slightly higher nutrient concentration in the DOC-richest lagoons than in the DOC-poorest lagoons (**Table 1**), all lagoons showed relative greater carbon than nitrogen and phosphorus concentration (greater C:N and C:P ratios) in relation to bacterial demands (for bacterial elemental composition see Fagerbakke et al., 1996). Those ecosystems are usually characterized by strong nutrient limitation, low BP rates and low carbon conversion into CO2 by respiration into bacterial cells (Farjalla et al., 2009a). In this experiment, lagoons with lower carbon concentrations had stronger responses to nutrient addition than lagoons with higher carbon concentrations, which was an unexpected result because of the largest imbalance between nutrient concentration and bacterial demands found in DOC-richest lagoons. We suggest that the quality of DOC bulk for bacterial growth is inversely related to DOC concentration in these lagoons, and nutrient additions have a restricted influence in low quality DOC bulk ecosystems (Farjalla et al., 2002). Besides receiving highly humic DOC from the drainage basin, Cabiúnas and Carapebus lagoons contain extensive macrophyte stands, dominated by *Typha domingensis* and *Elecocharis interstincta*. These plants contribute high quality DOC to the water column by exuding excess organic carbon produced by photosynthesis or by leaching during the early stages of decomposition (Stepanauskas et al., 2000; Farjalla et al., 2009b). Periphytic primary production is severely limited by light availability in these coastal lagoons (Sanches et al., 2011) and light penetrates further down the water column of Cabiunas and Carapebus lagoons than in the highly humic lagoons. On the other hand, macrophyte stands are restricted or absent and autochthonous primary production is very low in Comprida, Amarra-Boi, and Atoleiro lagoons. The input of DOC is exclusively external via the drainage basin, and its composition is almost exclusively of highly refractory humic compounds. Therefore, the increase in bacterial metabolism after nutrient addition in the lowest DOC concentration lagoons was most likely related to the ease of consumption and assimilation of more labile autochthonous DOC compounds, while in the highest DOC concentration lagoons the refractory characteristics of the DOC bulk restricted the responses of bacterial metabolism after nutrient additions, at least over the duration of the experiment (48 h).

Some peculiarities of the experimental design may have influenced our results. For example, some delayed physiological and re-growth responses of bacterial communities related to the treatments could have occurred and been observed under longer incubation periods than those used in our experiment. Thus, our results represent only a snapshot of rapid responses of bacterial metabolism to changes in environmental conditions. Follow-up studies focused on longer incubation periods should be performed to determine whether these responses would be maintained for long time periods. Changes in the bacterial community composition during the experiment related to the different treatments may have occurred; however, as such changes were previously observed in longer experiments, we did not expect any significant effect here. The spatiotemporal representation of the study may have been compromised, as we sampled only once from each lagoon. Nonetheless, we have observed in previous studies that the greatest changes in bacterial processes occur between lagoons, not within the same lagoon over time or between sampling stations in the same lagoon (MacCord et al., 2013). The use of artificial microcosms was necessary for a thorough evaluation without the confounding effects of varying the concentrations of nutrients and water temperature on bacterial

# References

Amado, A. M., Meirelles-Pereira, F., Vidal, L. O., Sarmento, H., Suhett, A. L., Farjalla, V. F., et al. (2013). Tropical freshwater ecosystems have lower bacterial growth efficiency than temperate ones. *Front. Microbiol.* 4:167. doi: 10.3389/fmicb.2013. 00167

metabolism; regardless, extrapolation from studies in artificial microcosms to natural systems should be performed with caution. It is noteworthy that the nutrient concentrations used in this study were the same as those observed in the actual lagoons or in similar systems with domestic sewage input (Caliman et al., 2010). Finally, although an increase of 10◦C is not predicted in any future climate scenario, this change in water temperature is usually found throughout a single day in summer months in shallow lagoons (Farjalla et al., 2005). The impact of these episodic events on long term microbial metabolism and carbon cycling in these ecosystems remain to be evaluated.

We conclude that increase in water temperature impacted microbial metabolism in the tropical coastal lagoons by increasing BR, slightly decreasing BP, and decreasing BGE. Nutrient inputs also changed the bacterial metabolism, but the magnitude of alterations varied among lagoons and seemed to be related to the availability and quality of carbon substrates in each system. Overall, both nutrient additions and increases in water temperature decreased BGE. This result points to important changes in the future carbon cycle of aquatic ecosystems related to the role of bacterioplankton. In the future, it is expected that the predicted environmental changes will favor processes related to bacterial catabolism, whereby relatively more carbon is converted into CO2 than into bacterial biomass available to higher trophic levels through the microbial loop.

# Acknowledgments

The authors thank A. M. Amado for constructive comments that improved the final version of this manuscript and R. R. C. Silva for her kind radiometric assistance. A. D. Letaw revised the English language. This work was part of the project "Impact of Global Climate Changes on Tropical Continental Aquatic Ecosystems" coordinated by B. M. Faria and supported by Petrobras Research and Development Center (CENPES). This research was supported by grants from CENPES/PETROBRAS (Project ANPETRO 9803) and CNPq (Processes 475961/2007-2 and 479437/2010-6) to VF. VF and JG are partially supported by productivity grants provided by CNPq. Post-graduate scholarships were provided by CAPES and CENPES/PETROBRAS to VS and SJ., respectively.

# Supplementary Material

The Supplementary Material for this article can be found online at: http://www.frontiersin.org/journal/10.3389/fmicb.2015.00310/ abstract

macrophyte stands of a tropical coastal lake. *Hydrobiologia* 436, 241–248. doi: 10.1023/A:1026537905947

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2015 Scofield, Jacques, Guimarães and Farjalla. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Experimental evidence of nitrogen control on pCO2 in phosphorus-enriched humic and clear coastal lagoon waters

# *Roberta B. Peixoto1, Humberto Marotta2 and Alex Enrich-Prast1\**

1 Laboratory of Biogeochemistry, Department of Ecology, Institute of Biology, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil 2 Sedimentary and Environmental Processes Laboratory (LAPSA/UFF), Department of Geography, Institute of Geosciences, Universidade Federal Fluminense, Niterói, Brazil

#### *Edited by:*

André Megali Amado, Universidade Federal do Rio Grande do Norte, Brazil

*Reviewed by:* James Cotner, University of Minnesota, USA Luciana De Oliveira Vidal, Federal University of Juiz de Fora, Brazil

#### *\*Correspondence:*

Alex Enrich-Prast, Laboratory of Biogeochemistry, Department of Ecology, Institute of Biology, Universidade Federal do Rio de Janeiro, Cidade Universitária s/n, Rio de Janeiro 68020, Brazil. e-mail<EMAIL_ADDRESS>
Natural and human-induced controls on carbon dioxide (CO2) in tropical waters may be very dynamic (over time and among or within ecosystems) considering the potential role of warmer temperatures intensifying metabolic responses and playing a direct role on the balance between photosynthesis and respiration. The high magnitude of biological processes at low latitudes following eutrophication by nitrogen (N) and phosphorus (P) inputs into coastal lagoons waters may be a relevant component of the carbon cycle, showing controls on partial pressure of CO2 (pCO2) that are still poorly understood. Here we assessed the strength of N control on pCO2 in P-enriched humic and clear coastal lagoons waters, using four experimental treatments in microcosms: control (no additional nutrients) and three levels of N additions coupled to P enrichments. In humic coastal lagoons waters, a persistent CO2 supersaturation was reported in controls and all nutrient-enriched treatments, ranging from 24- to 4-fold the atmospheric equilibrium value. However, both humic and clear coastal lagoons waters only showed significant decreases in pCO2 in relation to the controlled microcosms in the two treatments with higher N addition levels. Additionally, clear coastal lagoons water microcosms showed a shift from CO2 sources to CO2 sinks, in relation to the atmosphere. Only in the two more N-enriched treatments did pCO2 substantially decrease, from 650 μatm in controls and less N-enriched treatments to 10 μatm in more N-enriched microcosms. Humic substrates and N inputs can modulate pCO2 even in P-enriched coastal lagoons waters, thereby being important drivers on CO2 outgassing from inland waters.

**Keywords: eutrophication,** *p***CO2, nitrogen, humic coastal lagoons, clear water coastal lagoons**

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# **INTRODUCTION**

Carbon dioxide (CO2) is one of most important greenhouse gas in terms of global warming (IPCC, 2007; Royer et al., 2007; Solomon et al., 2010). The terrestrial biomass represents a relevant global stock of carbon (C), which is removed from the atmosphere by primary production (Gough et al., 2008). However, a significant part of this terrestrial organic matter leaches into aquatic ecosystems, where it may be buried in bottom sediments (Downing et al., 2008) or remineralized to CO2 by aquatic biological decomposition (Aufdenkampe et al., 2011). In the watershed, most natural inland waters are relatively small, but their wide geographic distribution, high abundance, and common location at low altitudes make them a typical fate for the water inflow from broad areas, playing a crucial role on the global C cycle (Cole et al., 2007).

Coastal lagoons are ecosystems often altered by the human land use (Marotta et al., 2010b), which show intense C fluxes (Duarte et al., 2008; Marotta et al., 2010b) The terrestrial inputs from leaching and groundwaters enhance CO2 in lakes by the contribution of inorganic C (Raymond et al., 1997; Marotta et al., 2010b), or organic substrates supporting the aquatic respiration (del Giorgio et al., 1997; Jonsson et al., 2003). Photosynthesis and respiration are the major metabolic pathways determining whether what level organic matter is produced and destroyed (Cole et al., 2000). Indeed, high terrestrial organic inputs may explain the positive general trend reported between dissolved organic carbon (DOC) and the partial pressure of CO2 (*p*CO2) in lake waters (Jonsson et al., 2003). Several studies have showed positive relationships in DOC and *p*CO2 in lakes in high latitudes and even globally (Sobek et al., 2005) supporting the idea that lakes are an important source of CO2 globally (Cole et al., 1994, 2007; Duarte and Prairie, 2005; Tranvik et al.,2009). Mean areal rates of CO2 evasionfrom lakes are higher at low latitudes, probably by the potential positive effect of warmer conditions on the organic decomposition (Marotta et al., 2009; Kosten et al., 2010). In this way, the degradation of organic matter to CO2 by bacteria shows important fluxes in the carbon cycling in natural aquatic ecosystems (Azam, 1998).