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Environmental Microbiology Reports (2014) 6(2), 131–135
doi:10.1111/1758-2229.12143
The negative effects of temperature increase on bacterial respiration are independent of changes in community composition Aliny P. F. Pires,* Rafael D. Guariento,† Thais Laque,‡ Francisco A. Esteves‡ and Vinicius F. Farjalla Instituto de Biologia, Departamento de Ecologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. Summary Temporal changes in environmental conditions and in bacterial community composition (BCC) regulate bacterial processes and ecosystem services. An increase in temperature accelerates bacterial processes in polar or temperate regions, but this relationship has not been documented for the tropics. Here, we tested the interactive effects of changing the BCC and increasing the water temperature on tropical bacterial respiration (BR). The BCC was manipulated through successional changes of the bacterial community in a filtered water sample from a tropical coastal lagoon. Four succession incubation periods (120, 240, 288 and 336 h) and four different water temperatures (23, 28, 33 and 38oC) were tested in a full-factorial design microcosm experiment. Both the BCC and the temperature had significant individual, but not interactive, effects on BR. Temperature increasing consistently decreased BR, while there was no clear pattern of successional effects on BR observed. No BCC tested was able to diminish the negative effects of temperature increases on BR. Our results suggest that the effects of an increasing temperature can negatively affect BR, even in tropical ecosystems with different BCC.
Introduction Aquatic bacteria are involved in many key biogeochemical processes. These organisms play a major role in the Received 2 April, 2013; accepted 17 December, 2013. *For correspondence. E-mail [email protected]; Tel. (+55) 21 2562 6319; Fax (+55) 21 2270 4950. Present addresses: †Universidade Federal do Rio Grande do Norte, Centro de Biociências, Departamento de Botânica, Ecologia e Zoologia, Natal, RN, Brazil; ‡ Núcleo em Ecologia e Desenvolvimento Sócio-Ambiental de Macaé/ UFRJ, Macaé, RJ, Brazil.
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd
trophic structure of several aquatic ecosystems and on ecosystem function (Ducklow, 2008). Studies using bacterial communities demonstrated that temperature had a positive effect on bacterial processes, but most of these studies were performed in temperate or polar ecosystems (Robador et al., 2009; Sarmento et al., 2010). In tropical ecosystems, the bacteria functioned at or near their optimal temperature for growth and resource assimilation (Pomeroy and Wiebe, 2001). Thus, changes in temperature could cause a disruption of this optimum, leading to a decrease in bacterial performance in tropical environments (e.g. Farjalla et al., 2005). Bacterial processes are also highly influenced by temporal changes to the bacterial community composition (BCC) and its functional traits (Reinthaler et al., 2005; Iversen and Seuthe, 2011). In natural environments, disturbances may reset the community over the course of ecological succession, which can affect the community structure and the magnitude of the bacterial processes (Prach and Walker, 2010). In microbial ecology, the ecological succession concept has been neglected by studies that intended to verify the ability of bacterial communities to modulate the processes they perform (but see Fierer et al., 2010). However, experimental approaches have recently been proposed to integrate ecological succession issues into microbial ecology (Prach and Walker, 2010). We performed a microcosm experiment that simulated a temperature increase scenario to verify how temperature can affect tropical bacterial respiration (BR) and to determine whether different BCCs from different succession stages can regulate the effect of temperature increase on BR. We used the bacterial community from Cabiúnas lagoon, an oligoaline humic, tropical coastal lagoon that is located on the coastal plains in the northeast Rio de Janeiro State, Brazil. The annual mean temperature of the water in the Cabiúnas lagoon is 23°C, but temperatures as high as 33°C were recorded in the summer season. In addition, a wide range of daily water temperature (27–39°C) was observed in a closely located coastal lagoon (Farjalla et al., 2005). Differences in the BCC were established through different incubation times (i.e. succession stages). It is well known that the differential adjustment of bacterial
132 A. P. F. Pires et al. populations over time is driven by changes in resource quantity and quality (Fierer et al., 2010). Therefore, we expected that the bacterial communities in each succession stage would differ in their composition because of different dominance patterns. Bacterial cultures were divided into four 600 ml glass flasks. Each flask received 6 ml of bacterial inoculum [a Cabiúnas water sample filtered through 0.8 μm pore size-cellulose filters (Advantec, Dublin, CA, USA)] and 594 ml of bacterial medium [a Cabiúnas water sample filtered through 0.2 μm pore sizeVacuCap™ sterile filters (Pall Gelman, Port Washington, NY, USA)]. The control did not receive an addition of inoculum. The bacteria grew for 120, 240, 288 and 336 h in each culture flask, simulating the different stages of succession. Each culture was then divided into four groups in 20 ml glass vials (n = 15 for each group), and each group was incubated in different biochemical oxygen demand (BOD) chambers that were set at 23, 28, 33 or 38°C in the dark. BCC successional changes were verified using denaturing gradient gel electrophoresis (DGGE). The DGGE technique detects the most dominant community members, but it results in the best relationship between the processing time per sample and the amount of information (Muyzer and Smalla, 1998). The details of the DGGE procedures adopted here can be found elsewhere (e.g. Laque et al., 2010). The time-integrated BR was calculated by subtracting the initial from the final dissolved oxygen concentration for each vial, and this value was discounted from the average oxygen consumption calculated from the respective control. The oxygen concentration was evaluated by an oxygen micro-sensor (OX-N, Unisense, Aarhus, Denmark) connected to a picoamperemeter (PA 2000, Unisense), which was modeled after Briand and colleagues (2004). This approach is highly accurate, stable and has a low response time. It has been
widely used to study bacterial metabolism (e.g. Amado et al., 2007; Iversen and Ploug, 2013). Differences between the bacterial communities in the four succession stages were evaluated by the Jaccard similarity index, which was calculated by the presence or absence of different bands in the DGGE gel. An analysis of covariance (ANCOVA) was used to test the independent and interactive effects of temperature increase (continuous variable) and BCC (categorical variable related to the successional stage) on BR. The BCC was used as a categorical variable because there is bias in the information obtained by the fingerprint analysis of bacterial richness or the relative abundance (e.g. Schmalenberger and Tebbe, 2003; Loisel et al., 2006). The magnitude of the effect sizes of increasing temperature and of the successional stage of BCC on BR was calculated by Cohen’s f 2 (Cohen, 1988). All analyses were performed in the R software. Results and discussion The DGGE technique revealed BCC differences between the different succession stages (Fig. 1, Supporting Information Fig. S1), which confirmed the efficacy of the incubation method. We identified 16 operational taxonomic units (OTUs) (different bands positions) for all of the succession stages: 11, 13, 9 and 8 bands at 120, 240, 288 and 336 h treatments (succession stages) respectively. This OTU number may be considered low if compared with soil samples (e.g. Schmalenberger and Tebbe, 2003), but they are in the range found in the water column of nearby tropical coastal lagoons (Laque et al., 2010). The highest BCC similarities were found between the 288 and 336 h treatments (Jaccard similarity index = 0.69), and the lowest was found between the 120 and 240 h treatments (Jaccard similarity index = 0.15). There was no
Fig. 1. The effects of water temperature and BCC from different succession stages on BR, measured as oxygen consumption after 10 days of incubation. Each point represents an individual microcosm. The regression values for each succession stage are described in Table 2. The right-hand panel shows the overall mean (± 95% confidence interval) of BR for each succession stage. The consistent negative effect of increasing temperatures on BR is independent of BCC (greater panel). Different BCC would be able to control for the negative effects of increasing temperature if we had found different slope values for each BCC. The flat lines would suggest that there is no effect of increasing temperature on BR in the respective BCC.
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports, 6, 131–135
Bacterial community and temperature changes Table 1. The pairwise Jaccard similarity index for bacterial communities of different successional stages (120, 240, 288 and 336 h) was calculated by the presence of bands observed in the DGGE image. Succession stages (h)
120
240
288
336
120 240 288 336
1.00 0.15 0.46 0.53
1.00 0.42 0.60
1.00 0.69
1.00
clear pattern of increased or decreased similarity throughout the successional stages (Table 1). We observed a significant and independent effect of increasing temperature on BR (ANCOVA, P < 0.05; Ftemperature = 58.47). The increasing temperature had a negative effect on BR (P < 0.05; Table 2, Fig. 1 – greater panel). BR at the highest temperature (38°C – 48.9% of oxygen consumption) was approximately 25% lower than the values observed at 23, 28 or 33°C (68.6%, 66.6% and 62.1% of oxygen consumption respectively). Therefore, the temperature increase had a different effect on BR in tropical ecosystems than it did in temperate or polar systems (Robador et al., 2009; Sarmento et al., 2010). We suggest that this result is a consequence of the specific range of temperature changes in tropical ecosystems. High temperatures may affect bacterial cells and their physiology primarily through denaturation and inactivation of enzymes and through changes on the membrane fluidity (Wertz et al., 2007; Hall et al., 2008). Additionally, well-established evolutionary trade-offs in the regulation of metabolic rates suggest that BR should be lower for organisms that are adapted or acclimated to higher temperature climates (Bradford et al., 2008). Consequently, the bacterial species that live at higher temperature conditions (e.g. 38°C) would be expected to have lower BR than bacterial species that live at lower temperature conditions (e.g. 23°C). However, it is unknown whether the BCC of an ecosystem is capable of regulating the effects of increasing temperature on bacterial processes. Some important aspects of a BCC, such as the diversity and the species richness Table 2. The best fit values of the linear regressions were determined to verify the effect of temperature on bacterial respiration at different BCC (succession stages). Succession stages (h) R2 120 240 288 336
P
Slope
Intercept
0.3029 < 0.0001 −1.603 ± 0.3249 118.6 ± 10.05a 0.1379 0.0035 −0.8697 ± 0.2855a 80.03 ± 8.854b 0.2296 0.0002 −1.395 ± 0.3444a 111.5 ± 10.60a 0.1459 0.0031 −0.9114 ± 0.2946a 82.93 ± 9.140b a
The different letters show significant differences between succession stages [Tukey HSD (honest significant difference) post-hoc test, P < 0.05].
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and abundance, regulate the ecosystem functions. The main mechanisms that are related to these effects include the presence of species that are tolerant to disturbances, the presence of some functional groups and the elevated functional redundancy in the bacterial communities (Carney et al., 2004; Wertz et al., 2006; Bowen et al., 2011). Through these mechanisms, the changes in the BCC could mediate the effects of increased temperature on BR. For example, we might expect that a BCC in the first stages of a bacterial community succession could suppress the negative effects of increasing temperatures by maintaining higher diversity levels, and as a consequence, the temperature would have a smaller impact on BR in these stages. However, we did not find an interactive effect of the BCC and the increasing temperatures on BR (ANCOVA P ≥ 0.05; Finteraction = 1.35). In addition, the negative effect of increasing temperatures on BR were consistent for all successional stages, and we did not observe any significant differences on the regression slopes of the different succession stages (P < 0.05; Table 2, Fig. 1 – greater panel). We observed a significant and independent effect of the BCC successional stage on BR (ANCOVA, P < 0.05; Fsuccession stages = 4.13), but the magnitude of the effect size of BCC was approximately six times lower than the effect size of increased temperature on BR (Cohen’s f 2temperature = 0.96, Cohen’s f 2succession stages = 0.16). Moreover, the magnitude of BR showed no clear pattern in relation to the successional stages. The highest rates were associated with 120 and 288 h of incubation (P < 0.05; Table 2, Fig. 1 – smaller panel). Others studies have also demonstrated that changes in the BCC can result in different changes of ecosystem functions, such as carbon mineralization, nitrification and denitrification in soils (e.g. Carney et al., 2004; Wertz et al., 2006). However, as we observed here, the relation between the changes on BCC and bacterial processes is unpredictable in disturbed systems, and it is one of the most important challenges for microbial ecologists (Östman et al., 2010). Many important aspects should be taken into account in studies that aim to verify the effect of temperature on bacterial processes and the ability of the bacterial community to modulate this effect. In natural ecosystems, the bacterial communities should become more temperature tolerant with changes on BCC because of selective processes (Bradford et al., 2008). Additionally, the effects of temperature on the bacterial communities can add to the effects on other organisms through trophic interactions. As a consequence, complex effects could emerge in more realistic scenarios (Jiang and Morin, 2004). Although studies failed to provide a mechanistic explanation for the patterns observed, more sophisticated molecular tools should be incorporated in studies using bacterial communities to better understand the relationship between BCC
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports, 6, 131–135
134 A. P. F. Pires et al. and ecological processes. Nevertheless, our study provides empirical evidence that increasing temperatures may suppress the BCC regulatory effects in tropical aquatic ecosystems. Acknowledgments A.P.F.P., R.D.G. and T.L. are grateful to CNPq and CAPES for their postgraduate scholarships. This work was part of a project titled “Impact of Global Climate Changes on Tropical Continental Aquatic Ecosystems” coordinated by Dr. Bias M. Faria from the Petrobras Research and Development Center (CENPES). This research was supported by grants from CENPES/PETROBRAS (ANPETRO 9803 to VFF) and CNPq (Project 475961/2007-2 to V.F.F.). V.F.F. and F.A.E are partially supported by CNPq productivity grants.
References Amado, A.M., Cotner, J.B., Suhett, A.L., Esteves, F.A., Bozelli, R.L., and Farjalla, V.F. (2007) Contrasting interactions mediate dissolved organic matter decomposition in tropical aquatic ecosystems. Aquat Microb Ecol 49: 25– 34. Bowen, J.L., Ward, B.B., Morrison, H.G., Hobbie, J.E., Valiela, I., Deegan, L.A., et al. (2011) Microbial community composition in sediments resists perturbation by nutrient enrichment. ISME J 5: 1540–1548. Bradford, M.A., Davies, C.A., Frey, S.D., Maddox, T.R., Melillo, J.M., Mohan, J.E., et al. (2008) Thermal adaptation of soil microbial respiration to elevated temperature. Ecol Lett 11: 1316–1327. Briand, E., Pringault, O., Jacquet, S., and Torreton, J.P. (2004) The use of oxygen microprobes to measure bacterial respiration for determining bacterioplankton growth efficiency. Limnol Oceanogr Methods 2: 406–416. Carney, K.M., Matson, P.A., and Bohannan, B.J.M. (2004) Diversity and composition of tropical soil nitrifiers across a plant diversity gradient and among land-use types. Ecol Lett 7: 684–694. Cohen, J. (1988) Statistical Power Analysis for the Behavioral Sciences, 2nd edn. Hillsdale, NJ, USA: Lawrence Erlbaum Associates. Ducklow, H. (2008) Microbial services: challenges for microbial ecologists in a changing world. Aquat Microb Ecol 53: 13–19. Farjalla, V.F., Laque, T., Suhett, A.L., Amado, A.M., and Esteves, F.A. (2005) Diel variation of bacterial abundance and productivity in tropical coastal lagoons: the importance of bottom-up factors in a short-time scale. Acta Limnologica Brasiliensis 17: 373–383. Feld, C.K., da Silva, P.M., Sousa, J.P., de Bello, F., Bugter, R., Grandin, U., et al. (2009) Indicators of biodiversity and ecosystem services: a synthesis across ecosystems and spatial scales. Oikos 118: 1862–1871. Fierer, N., Nemergut, D., Knight, R., and Craine, J.M. (2010) Changes through time: integrating microorganisms into the study of succession. Res Microbiol 161: 635– 642.
Hall, E.K., Neuhauser, C., and Cotner, J.B. (2008) Toward a mechanistic understanding of how natural bacterial communities respond to changes in temperature in aquatic ecosystems. ISME J 2: 471–481. Iversen, K.R., and Seuthe, L. (2011) Seasonal microbial processes in a high-latitude fjord (Kongsfjorden, Svalbard): I. Heterotrophic bacteria, picoplankton and nanoflagellates. Polar Biol 34: 731–749. Iversen, M.H., and Ploug, H. (2013) Temperature effects on carbon-specific respiration rate and sinking velocity of diatom aggregates – potential implications for deep ocean export processes. Biogeosciences 10: 4073– 4085. Jiang, L., and Morin, P.J. (2004) Temperature-dependent interactions explain unexpected responses to environmental warming in communities of competitors. J Anim Ecol 73: 569–576. Laque, T., Farjalla, V.F., Rosado, A.S., and Esteves, F.A. (2010) Spatiotemporal variation of bacterial community composition and possible controlling factors in tropical shallow lagoons. Microb Ecol 59: 819–829. Loisel, P., Harmand, J., Zemb, O., Latrille, E., Lobry, C., Delgenès, J.-P., and Godon, J.-J. (2006) Denaturing gradient electrophoresis (DGE) and single-strand conformation polymorphism (SSCP) molecular fingerprintings revisited by simulation and used as a tool to measure microbial diversity. Environ Microbiol 8: 720–731. Muyzer, G., and Smalla, K. (1998) Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie Van Leeuwenhoek 73: 127–141. Östman, O., Drakare, S., Kritzberg, E.S., Langenhander, S., Logue, J.B., and Lindström, E.S. (2010) Regional invariance among microbial communities. Ecol Lett 13: 118– 127. Pomeroy, L.R., and Wiebe, W.J. (2001) Temperature and substrates as interactive limiting factors for marine heterotrophic bacteria. Aquatic Microbial Ecology 23: 187– 204. Prach, K., and Walker, L.R. (2010) Four opportunities for studies of ecological succession. Trends Ecol Evol 26: 119–123. Reinthaler, T., Winter, C., and Herndl, G.J. (2005) Relationship between bacterioplankton richness, respiration, and production in the southern North Sea. Appl Environ Microbiol 71: 2260–2266. Robador, A., Bruchert, V., and Jorgensen, B.B. (2009) The impact of temperature change on the activity and community composition of sulfate-reducing bacteria in arctic versus temperate marine sediments. Environ Microbiol 11: 1692–1703. Sarmento, H., Montoya, J.M., Vazquez-Dominguez, E., Vaque, D., and Gasol, J.M. (2010) Warming effects on marine microbial food web processes: how far can we go when it comes to predictions? Philos Trans Royal Soc Lond B Biol Sci 365: 2137–2149. Schmalenberger, A., and Tebbe, C.C. (2003) Bacterial diversity in maize rhizospheres: conclusions on the use of genetic profiles based on PCR-amplified partial small subunit rRNA genes in ecological studies. Mol Ecol 12: 251–262.
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports, 6, 131–135
Bacterial community and temperature changes Wertz, S., Degrange, V., Prosser, J.I., Poly, F., Commeaux, C., Freitag, T., et al. (2006) Maintenance of soil functioning following erosion of microbial diversity. Environ Microbiol 8: 2162–2169. Wertz, S., Degrange, V., Prosser, J.I., Poly, F., Commeaux, C., Guillaumaud, N., et al. (2007) Decline of soil microbial diversity does not influence the resistance and resilience of key soil microbial functional groups following a model disturbance. Environ Microbiol 9: 2211–2219. Wetzel, R.G. (2001) Limnology: Lake and River Ecosystems, 3rd edn. San Diego, CA, USA: Academic Press.
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Supporting information Additional Supporting Information may be found in the online version of this article: Fig. S1. Denaturing gradient gel electrophoresis (DGGE) shows the different BCCs used in the experiment. The bands were visualized using the STORM (Amersham) image capture system. Cabiúnas Lagoon (CL), 120, 240, 288 and 336 h of incubation time.
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports, 6, 131–135
Environmental Microbiology Reports (2014) 6(2), 131–135
doi:10.1111/1758-2229.12143
The negative effects of temperature increase on bacterial respiration are independent of changes in community composition Aliny P. F. Pires,* Rafael D. Guariento,† Thais Laque,‡ Francisco A. Esteves‡ and Vinicius F. Farjalla Instituto de Biologia, Departamento de Ecologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. Summary Temporal changes in environmental conditions and in bacterial community composition (BCC) regulate bacterial processes and ecosystem services. An increase in temperature accelerates bacterial processes in polar or temperate regions, but this relationship has not been documented for the tropics. Here, we tested the interactive effects of changing the BCC and increasing the water temperature on tropical bacterial respiration (BR). The BCC was manipulated through successional changes of the bacterial community in a filtered water sample from a tropical coastal lagoon. Four succession incubation periods (120, 240, 288 and 336 h) and four different water temperatures (23, 28, 33 and 38oC) were tested in a full-factorial design microcosm experiment. Both the BCC and the temperature had significant individual, but not interactive, effects on BR. Temperature increasing consistently decreased BR, while there was no clear pattern of successional effects on BR observed. No BCC tested was able to diminish the negative effects of temperature increases on BR. Our results suggest that the effects of an increasing temperature can negatively affect BR, even in tropical ecosystems with different BCC.
Introduction Aquatic bacteria are involved in many key biogeochemical processes. These organisms play a major role in the Received 2 April, 2013; accepted 17 December, 2013. *For correspondence. E-mail [email protected]; Tel. (+55) 21 2562 6319; Fax (+55) 21 2270 4950. Present addresses: †Universidade Federal do Rio Grande do Norte, Centro de Biociências, Departamento de Botânica, Ecologia e Zoologia, Natal, RN, Brazil; ‡ Núcleo em Ecologia e Desenvolvimento Sócio-Ambiental de Macaé/ UFRJ, Macaé, RJ, Brazil.
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd
trophic structure of several aquatic ecosystems and on ecosystem function (Ducklow, 2008). Studies using bacterial communities demonstrated that temperature had a positive effect on bacterial processes, but most of these studies were performed in temperate or polar ecosystems (Robador et al., 2009; Sarmento et al., 2010). In tropical ecosystems, the bacteria functioned at or near their optimal temperature for growth and resource assimilation (Pomeroy and Wiebe, 2001). Thus, changes in temperature could cause a disruption of this optimum, leading to a decrease in bacterial performance in tropical environments (e.g. Farjalla et al., 2005). Bacterial processes are also highly influenced by temporal changes to the bacterial community composition (BCC) and its functional traits (Reinthaler et al., 2005; Iversen and Seuthe, 2011). In natural environments, disturbances may reset the community over the course of ecological succession, which can affect the community structure and the magnitude of the bacterial processes (Prach and Walker, 2010). In microbial ecology, the ecological succession concept has been neglected by studies that intended to verify the ability of bacterial communities to modulate the processes they perform (but see Fierer et al., 2010). However, experimental approaches have recently been proposed to integrate ecological succession issues into microbial ecology (Prach and Walker, 2010). We performed a microcosm experiment that simulated a temperature increase scenario to verify how temperature can affect tropical bacterial respiration (BR) and to determine whether different BCCs from different succession stages can regulate the effect of temperature increase on BR. We used the bacterial community from Cabiúnas lagoon, an oligoaline humic, tropical coastal lagoon that is located on the coastal plains in the northeast Rio de Janeiro State, Brazil. The annual mean temperature of the water in the Cabiúnas lagoon is 23°C, but temperatures as high as 33°C were recorded in the summer season. In addition, a wide range of daily water temperature (27–39°C) was observed in a closely located coastal lagoon (Farjalla et al., 2005). Differences in the BCC were established through different incubation times (i.e. succession stages). It is well known that the differential adjustment of bacterial
132 A. P. F. Pires et al. populations over time is driven by changes in resource quantity and quality (Fierer et al., 2010). Therefore, we expected that the bacterial communities in each succession stage would differ in their composition because of different dominance patterns. Bacterial cultures were divided into four 600 ml glass flasks. Each flask received 6 ml of bacterial inoculum [a Cabiúnas water sample filtered through 0.8 μm pore size-cellulose filters (Advantec, Dublin, CA, USA)] and 594 ml of bacterial medium [a Cabiúnas water sample filtered through 0.2 μm pore sizeVacuCap™ sterile filters (Pall Gelman, Port Washington, NY, USA)]. The control did not receive an addition of inoculum. The bacteria grew for 120, 240, 288 and 336 h in each culture flask, simulating the different stages of succession. Each culture was then divided into four groups in 20 ml glass vials (n = 15 for each group), and each group was incubated in different biochemical oxygen demand (BOD) chambers that were set at 23, 28, 33 or 38°C in the dark. BCC successional changes were verified using denaturing gradient gel electrophoresis (DGGE). The DGGE technique detects the most dominant community members, but it results in the best relationship between the processing time per sample and the amount of information (Muyzer and Smalla, 1998). The details of the DGGE procedures adopted here can be found elsewhere (e.g. Laque et al., 2010). The time-integrated BR was calculated by subtracting the initial from the final dissolved oxygen concentration for each vial, and this value was discounted from the average oxygen consumption calculated from the respective control. The oxygen concentration was evaluated by an oxygen micro-sensor (OX-N, Unisense, Aarhus, Denmark) connected to a picoamperemeter (PA 2000, Unisense), which was modeled after Briand and colleagues (2004). This approach is highly accurate, stable and has a low response time. It has been
widely used to study bacterial metabolism (e.g. Amado et al., 2007; Iversen and Ploug, 2013). Differences between the bacterial communities in the four succession stages were evaluated by the Jaccard similarity index, which was calculated by the presence or absence of different bands in the DGGE gel. An analysis of covariance (ANCOVA) was used to test the independent and interactive effects of temperature increase (continuous variable) and BCC (categorical variable related to the successional stage) on BR. The BCC was used as a categorical variable because there is bias in the information obtained by the fingerprint analysis of bacterial richness or the relative abundance (e.g. Schmalenberger and Tebbe, 2003; Loisel et al., 2006). The magnitude of the effect sizes of increasing temperature and of the successional stage of BCC on BR was calculated by Cohen’s f 2 (Cohen, 1988). All analyses were performed in the R software. Results and discussion The DGGE technique revealed BCC differences between the different succession stages (Fig. 1, Supporting Information Fig. S1), which confirmed the efficacy of the incubation method. We identified 16 operational taxonomic units (OTUs) (different bands positions) for all of the succession stages: 11, 13, 9 and 8 bands at 120, 240, 288 and 336 h treatments (succession stages) respectively. This OTU number may be considered low if compared with soil samples (e.g. Schmalenberger and Tebbe, 2003), but they are in the range found in the water column of nearby tropical coastal lagoons (Laque et al., 2010). The highest BCC similarities were found between the 288 and 336 h treatments (Jaccard similarity index = 0.69), and the lowest was found between the 120 and 240 h treatments (Jaccard similarity index = 0.15). There was no
Fig. 1. The effects of water temperature and BCC from different succession stages on BR, measured as oxygen consumption after 10 days of incubation. Each point represents an individual microcosm. The regression values for each succession stage are described in Table 2. The right-hand panel shows the overall mean (± 95% confidence interval) of BR for each succession stage. The consistent negative effect of increasing temperatures on BR is independent of BCC (greater panel). Different BCC would be able to control for the negative effects of increasing temperature if we had found different slope values for each BCC. The flat lines would suggest that there is no effect of increasing temperature on BR in the respective BCC.
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports, 6, 131–135
Bacterial community and temperature changes Table 1. The pairwise Jaccard similarity index for bacterial communities of different successional stages (120, 240, 288 and 336 h) was calculated by the presence of bands observed in the DGGE image. Succession stages (h)
120
240
288
336
120 240 288 336
1.00 0.15 0.46 0.53
1.00 0.42 0.60
1.00 0.69
1.00
clear pattern of increased or decreased similarity throughout the successional stages (Table 1). We observed a significant and independent effect of increasing temperature on BR (ANCOVA, P < 0.05; Ftemperature = 58.47). The increasing temperature had a negative effect on BR (P < 0.05; Table 2, Fig. 1 – greater panel). BR at the highest temperature (38°C – 48.9% of oxygen consumption) was approximately 25% lower than the values observed at 23, 28 or 33°C (68.6%, 66.6% and 62.1% of oxygen consumption respectively). Therefore, the temperature increase had a different effect on BR in tropical ecosystems than it did in temperate or polar systems (Robador et al., 2009; Sarmento et al., 2010). We suggest that this result is a consequence of the specific range of temperature changes in tropical ecosystems. High temperatures may affect bacterial cells and their physiology primarily through denaturation and inactivation of enzymes and through changes on the membrane fluidity (Wertz et al., 2007; Hall et al., 2008). Additionally, well-established evolutionary trade-offs in the regulation of metabolic rates suggest that BR should be lower for organisms that are adapted or acclimated to higher temperature climates (Bradford et al., 2008). Consequently, the bacterial species that live at higher temperature conditions (e.g. 38°C) would be expected to have lower BR than bacterial species that live at lower temperature conditions (e.g. 23°C). However, it is unknown whether the BCC of an ecosystem is capable of regulating the effects of increasing temperature on bacterial processes. Some important aspects of a BCC, such as the diversity and the species richness Table 2. The best fit values of the linear regressions were determined to verify the effect of temperature on bacterial respiration at different BCC (succession stages). Succession stages (h) R2 120 240 288 336
P
Slope
Intercept
0.3029 < 0.0001 −1.603 ± 0.3249 118.6 ± 10.05a 0.1379 0.0035 −0.8697 ± 0.2855a 80.03 ± 8.854b 0.2296 0.0002 −1.395 ± 0.3444a 111.5 ± 10.60a 0.1459 0.0031 −0.9114 ± 0.2946a 82.93 ± 9.140b a
The different letters show significant differences between succession stages [Tukey HSD (honest significant difference) post-hoc test, P < 0.05].
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and abundance, regulate the ecosystem functions. The main mechanisms that are related to these effects include the presence of species that are tolerant to disturbances, the presence of some functional groups and the elevated functional redundancy in the bacterial communities (Carney et al., 2004; Wertz et al., 2006; Bowen et al., 2011). Through these mechanisms, the changes in the BCC could mediate the effects of increased temperature on BR. For example, we might expect that a BCC in the first stages of a bacterial community succession could suppress the negative effects of increasing temperatures by maintaining higher diversity levels, and as a consequence, the temperature would have a smaller impact on BR in these stages. However, we did not find an interactive effect of the BCC and the increasing temperatures on BR (ANCOVA P ≥ 0.05; Finteraction = 1.35). In addition, the negative effect of increasing temperatures on BR were consistent for all successional stages, and we did not observe any significant differences on the regression slopes of the different succession stages (P < 0.05; Table 2, Fig. 1 – greater panel). We observed a significant and independent effect of the BCC successional stage on BR (ANCOVA, P < 0.05; Fsuccession stages = 4.13), but the magnitude of the effect size of BCC was approximately six times lower than the effect size of increased temperature on BR (Cohen’s f 2temperature = 0.96, Cohen’s f 2succession stages = 0.16). Moreover, the magnitude of BR showed no clear pattern in relation to the successional stages. The highest rates were associated with 120 and 288 h of incubation (P < 0.05; Table 2, Fig. 1 – smaller panel). Others studies have also demonstrated that changes in the BCC can result in different changes of ecosystem functions, such as carbon mineralization, nitrification and denitrification in soils (e.g. Carney et al., 2004; Wertz et al., 2006). However, as we observed here, the relation between the changes on BCC and bacterial processes is unpredictable in disturbed systems, and it is one of the most important challenges for microbial ecologists (Östman et al., 2010). Many important aspects should be taken into account in studies that aim to verify the effect of temperature on bacterial processes and the ability of the bacterial community to modulate this effect. In natural ecosystems, the bacterial communities should become more temperature tolerant with changes on BCC because of selective processes (Bradford et al., 2008). Additionally, the effects of temperature on the bacterial communities can add to the effects on other organisms through trophic interactions. As a consequence, complex effects could emerge in more realistic scenarios (Jiang and Morin, 2004). Although studies failed to provide a mechanistic explanation for the patterns observed, more sophisticated molecular tools should be incorporated in studies using bacterial communities to better understand the relationship between BCC
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports, 6, 131–135
134 A. P. F. Pires et al. and ecological processes. Nevertheless, our study provides empirical evidence that increasing temperatures may suppress the BCC regulatory effects in tropical aquatic ecosystems. Acknowledgments A.P.F.P., R.D.G. and T.L. are grateful to CNPq and CAPES for their postgraduate scholarships. This work was part of a project titled “Impact of Global Climate Changes on Tropical Continental Aquatic Ecosystems” coordinated by Dr. Bias M. Faria from the Petrobras Research and Development Center (CENPES). This research was supported by grants from CENPES/PETROBRAS (ANPETRO 9803 to VFF) and CNPq (Project 475961/2007-2 to V.F.F.). V.F.F. and F.A.E are partially supported by CNPq productivity grants.
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Supporting information Additional Supporting Information may be found in the online version of this article: Fig. S1. Denaturing gradient gel electrophoresis (DGGE) shows the different BCCs used in the experiment. The bands were visualized using the STORM (Amersham) image capture system. Cabiúnas Lagoon (CL), 120, 240, 288 and 336 h of incubation time.
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports, 6, 131–135
