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Seasonal dynamics of ammonia-oxidizing microorganisms in freshwater aquaculture ponds

Seasonal dynamics of ammonia-oxidizing microorganisms in freshwater aquaculture ponds Ann Microbiol (2015) 65:651–657 DOI 10.1007/s13213-014-0903-2 ORIGINAL ARTICLE Seasonal dynamics of ammonia-oxidizing microorganisms in freshwater aquaculture ponds Shimin Lu & Mingjun Liao & Congxin Xie & Xugang He & Dapeng Li & Lulu He & Jin Chen Received: 19 January 2014 /Accepted: 21 April 2014 /Published online: 25 May 2014 Springer-Verlag Berlin Heidelberg and the University of Milan 2014 Abstract An annual investigation into the abundance of Introduction ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in fresh water aquaculture ponds was per- Aquaculture production has been increasing at an average formed by quantitative PCR of the amoA gene. The results growth rate of 8.3 % per year, which makes aquaculture one showed that AOB were the main ammonia-oxidizing micro- of the fastest growing segments of the food economy in organisms in water, and significantly higher copy numbers of modern times (FAO 2012). According to the Fishery Bureau the AOB amoA gene were observed in the summer (Aug of the Chinese Ministry of Agriculture (MOA), the total 2012), while no significant differences were detected among aquaculture production of China in 2012 amounted to the other three seasons. AOA showed low abundances 59.1 million metric tons, and the production of freshwater throughout the year. The predominance of AOB in aquacul- aquaculture was 26.4 million metric tons (Fishery National ture water was suggested to be related to photoinhibition. Both Bureau of Statistics of China 2013, http://www.stats.gov.cn/ the AOB and AOA amoA genes in aquaculture pond sedi- english/statisticaldata/Quarterlydata/). Undoubtedly, China’s ments showed typical seasonal patterns. The maximum den- freshwater aquaculture plays an important role in the global sity of AOB was observed in the autumn (Nov 2012) and supply of aquatic products. winter (Jan 2013), while the maximum density of AOA was Currently, there are 2,566,900 ha freshwater aquaculture observed in winter. The minimum densities of both AOA and ponds, and pond culture is the major freshwater culture method AOB occurred in the summer. The concentration of the AOA in China (National Bureau of Statistics of China 2013, http:// amoA gene was higher than that of the AOB amoA gene in www.stats.gov.cn/english/statisticaldata/Quarterlydata/). sediments by almost one order of magnitude, which indicates However, along with increasing development, concerns are that AOA are the dominant ammonia-oxidizing microorgan- evoked about the possible effects of ever-increasing aquaculture isms in the aquaculture pond sediments. Dissolved oxygen is waste, both on the productivity of aquaculture systems and on suggested to be the key factor determining the predominance the ambient aquatic ecosystem. It was estimated that 9. of AOA in pond sediments. 5 kg phosphorous (P) and 78 kg of nitrogen (N) are released into the aquaculture environment per ton of fish produced. Approximately 72 % of N and 70 % of Keywords Ammonia-oxidizing archaea Ammonia- P in the feed are released into the aquaculture environ- . . . oxidizingbacteria amoAgene Aquaculturewater Sediment ment (Ackefors and Enell 1994). Ammonia is one of the most toxic substances produced by intensive fish farming (Handy and Poxton 1993). Ammonia is toxic Shimin Lu and Mingjun Liao contributed equally to this work. to all vertebrates and can cause convulsions, coma and : : : : : : S. Lu M. Liao C. Xie (*) X. He D. Li L. He J. Chen death, probably because elevated ammonium (NH ) College of Fisheries, Huazhong Agricultural University, ions displace potassium (K ) and depolarizes neurons, Wuhan 430070, China e-mail: xiecongxin@mail.hzau.edu.cn causing activation of the N-methyl-d-aspartate (NMDA) type glutamate receptor, which leads to an influx of excessive M. Liao 2+ calcium (Ca ) ions and subsequent cell death in the central College of Resource and Environmental Engineering, Hubei nervous system (Randall and Tsui 2002). University of Technology, Wuhan 430068, China 652 Ann Microbiol (2015) 65:651–657 Nitrification is a key process in the cycling of nitrogen in pore water. The pore water was then filtered through a + − aquatic ecosystems (Merbt et al. 2012). The oxidation of 0.22-μm membrane. Field measurements of NH -N, NO - 4 3 − − ammonia (NH ) to nitrite (NO )—the first and rate-limiting Nand NO -N concentrations were performed by the Nessler 3 2 2 step of nitrification—has traditionally been considered to be reagent photometric method, photometry with phenol-2- carried out solely by ammonia-oxidizing bacteria (AOB) sulfonic acid method, and the N (1-naphty1)-ethylenediamine (Koops and Pommerening-Röser 2001), which fall into two dihydrochloride spectrophotometric method, respectively, ac- phylogenetic lineages within the β-and γ-Proteobacteria cording to the protocols described in Chinese Standard (Kowalchuk and Stephen 2001). However, this notion has Methods (Editorial Board of Monitoring and Analytical changed owing to the recent discovery of ammonia- Method of Water and Wastewater 2002). oxidizing archaea (AOA). Based on genomic level compari- sons, AOA were classified into the newly proposed phylum DNA extraction from fresh water and sediments Thaumarchaeota (Pester et al. 2011). AOA play important roles in the nitrogen cycle and have been found in various DNA from water- and sediment-dwelling microorganisms habitats, including hot springs (Hatzenpichler et al. 2008), was extracted as described by Lu et al. (2012)withsome oceans (Wuchter et al. 2006; Horak et al. 2013), freshwater modifications. Water (50 mL) from each pond was filtered (Auguet et al. 2011), and soil (Leininger et al. 2006). directly through a 0.22-μm membrane using negative pres- Although AOB and AOA are affiliated with different phylo- sure. After filtration, the membranes were cut into pieces and genetic domains, they both contain homologous ammonia placed in a 2-mL sterile centrifuge tube and stored at −20 °C monooxygenase (AMO), which is responsible for catalyzing until DNA extraction. The pieces of filtered membranes or the first step in ammonia oxidation. The amoA gene, encoding 0.3 g sediment were lysed using 0.7 μL lysis buffer (Zhou the alpha subunit of AMO, has been used widely as a func- et al. 1996), 100 μL 10 % SDS, and two glass beads. The tional gene marker for tracking ammonia oxidizers in envi- lysate was homogenized for 2 min on a vortexer, and then ronmental samples. Nonetheless, there is a great lack of stud- incubated at 70 °C for 1 h. After incubation, the tube was ies on AOA and AOB in aquaculture ponds. In this paper, we centrifuged at 13,000 g for 10 min and 0.7 mL supernatant report on the seasonal dynamics of the abundance of was transferred into a new tube. Then, 0.6 mL crenarchaeotal and betaproteobacterial amoA genes, and in- phenol:chloroform:isoamyl alcohol (25:24:1) was added and vestigated changes in temperature and concentration of am- shaken gently for 1 min, and the tube was centrifuged at + − − monium (NH ), nitrite (NO ) and nitrate (NO )in aqua- 13,000 g for 5 min. After centrifugation, the supernatant was 4 2 3 culture water and sediment, aiming to achieve a better under- transferred to a new tube, followed by the addition of a 0.5 standing of the nitrogen cycle in the aquaculture environment. volume of PEG 6000 solution (50 % in m/v) and incubated for 60 min at ambient temperature. The tube was then centrifuged at 13,000 g for 15 min, the supernatant was discarded, and the Materials and methods pellet was re-dissolved in 0.6 mL distilled water before being extracted with 0.4 mL chloroform by several gentle end-over- Sample collection and hydration index determination end inversions. After extraction, the tube was centrifuged at 12,000 g for 5 min and 0.45 mL of the supernatant was Experimental samples were collected from an aquaculture transferred to a new tube and mixed with 0.9 mL ethanol farm [29°55′ N, 112°(17–18)′ E], Gong’an, Hubei, China. and 45 μL NaCl solution (5 M) and precipitated for 15 min Ten aquaculture ponds were selected for investigation. The on ice. After precipitation, the tube was centrifuged at average depth of the sampling ponds (350 m×20 m) was 13,000 g for 15 min, and the pellet was washed twice with about 1.5 m, in which grass carp, silver carp, bighead carp 70 % ethanol and air-dried. The final pellet was dissolved in and soft-shelled turtle were raised for commercial use since 50 μLTE buffer. DNA concentrations were quantified using a the 1980s. The ponds have never been dredged and are typical ND-2000 UV-vis spectrophotometer (http://www.nanodrop. of a eutrophic environment. The samples were collected on 14 com). April, 22 August, 2 November 2012, and 15 January 2013. The water samples were collected with a sterile glass bottle qPCR analysis of bacterial and archaeal amoA about 30 cm below the water surface. The top 0–5cm of black color sediment samples were collected using a Peterson Grab Quantitative PCR (qPCR) was used to estimate the abundance Sampler in the center of the aquaculture ponds, and the of bacterial and archaeal amoA genes. qPCR was performed homogenized sediment was transferred immediately to a ster- using a Qiagen Q thermocycler (Qiagen, Hilden, Germany) ile plastic bag on the fishing boat. The water and sediment and primers amoA-1F/amoA-2R (specific for the AOB amoA samples were stored at 4 °C and treated within 48 h. The gene) (Rotthauwe et al. 1997) and CrenamoA23f/ CrenamoA616r (specific for the AOA amoA gene) (Tourna sediment was centrifuged at 7,000 g for 10 min to extract bulk Ann Microbiol (2015) 65:651–657 653 et al. 2008). The qPCR was conducted in a total volume of significantly higher than in other seasons (1.55±8.41×10 in 3 −1 20 μLcontaining 10 μL SYBR Premix Ex Taq II for AOB or spring, 1.04±2.28×10 copy mL in autumn and 3.83± 2 −1 SYBR Premix Ex Taq for AOA (Takara, Dalian, China), 1 μL 2.24×10 copy mL in winter) (Fig. 1e; P<0.01, one-way −1 DNA template, 0.2 μM of each primer and 0.2 mg mL ANOVA). No significant differences were detected between bovine serum albumin (BSA). The qPCR thermocycling steps the other three seasons. AOB amoA gene copy numbers were as follows: an initial denaturation of 95 °C for 30 s, showed significant positive correlation with temperature (r= followed by 35 cycles of 95 °C for 5 s, 54 °C for 35 s or 53 °C 0.597, P<0.01; nonparametric correlations). These results for 60 s, and 72 °C for 60 s. Plates were read at 81 °C for AOB indicate that temperature plays a more important role in the or 80 °C for AOA, respectively. The negative control without seasonal dynamics of AOB than aquaculture activity, includ- DNA template was subjected to the same procedures to ex- ing the application of probiotics and fish medicine and aera- clude or detect any possible contamination. After qPCR, the tion, etc. A similar phenomenon was observed in a construct- specificity of amplification was verified by melting curve ed wetland, where the amoA gene copy numbers of AOB 4 6 −1 analysis and agarose gel electrophoresis. All measurements ranged from 5.3±0.6×10 to 8.1±0.5×10 copy mL in were performed in triplicate. water samples in summer, while AOB was not detected at Standard curves for qPCR were developed as described the outlet of the first wetland treatment unit in the winter previously with some modifications (He et al. 2007). The season (Sims et al. 2012a). bacterial or archaeal amoA gene was PCR-amplified from In the aquaculture water, the concentration of NH corre- the extracted DNA using primers amoA-1F/amoA-2R or lated roughly with the copy numbers of the AOB amoA gene CrenamoA23f/CrenamoA616r, and the PCR amplification throughout the year (r=0.251, P<0.01), which indicates that products were purified and cloned into the pMD18-T Easy other routes of NH removal in aquaculture water, such as Vector (Takara, Dalian, China). Plasmid extracted from the absorption by phytoplankton, also play a very important role correct clones was used as a standard for quantitative analysis. in addition to the oxidation by AOB. No significant correla- The plasmid DNA concentration was determined using a ND- tion was observed between the copy numbers of the AOB − − 2000 UV-vis Spectrophotometer and the copy numbers of the amoA gene and the concentrations of NO and NO in 2 3 target genes were calculated directly from the concentration of water, which was probably due to the fact that NO and the extracted plasmid DNA. Ten-fold serial dilutions of plas- NO levels are not only influenced by the amount and activ- 2 7 mid DNA ranging from 2.0×10 to 2.0×10 copies of the ity of nitrifying bacteria, but are also determined by denitrifi- 1 6 AOA amoA gene or 2.0×10 to 2.0×10 copies of the AOB cation intensity. amoA gene were subjected to qPCR in triplicate to generate an The AOA amoA gene was detected by PCR throughout the external standard curve. year in the aquaculture water based on our research over Two controls were performed to estimate the possible 2 years. However, because the copy number of the AOA inhibition of qPCR performance by the co-extracted polyphe- amoA gene was below the minimum level of detection (20 −1 nolic compounds or humic acids in sediment or water extracts: copy mL water) in the freshwater column, the exact copy (1) different amounts of amoA standards were mixed with number of the AOA amoA gene could not be determined. The equal amounts of sediment or water DNA extract, and (2) availability of NH does not appear to be the limiting eco- two different volumes of DNA extract from each sediment or logical factor for this phenomenon. It was shown that enriched water sample used in this study were applied for the quantifi- AOA cultures could grow well when the NH concentration −1 cation of amoA copy number. In most cases, inhibition was ranged from 0.22 to 70 mg mL (French et al. 2012). In this not detected or was negligible. study, the NH concentrations of aquaculture water ranged −1 −1 from 1.02±0.86 mg L to 2.80±0.89 mg L during the year, which falls within the aforementioned range of NH Results and discussion concentrations. The fact that AOB were the predominant ammonia- Seasonal changes and abundance of amoA gene oxidizer in the surface water could be explained by in ammonia-oxidizing microorganisms in aquaculture water photoinhibition because the sampling point was at a depth of only 30 cm. A similar study, in which water samples were Figure 1a–e shows the seasonal changes in temperature, collected from 50 cm below the surface, found that AOB were + − − NH ,NO and NO concentrations and AOB amoA gene the only ammonia-oxidizer in surface water in freshwater of 4 2 3 copy number in aquaculture water in April, August, Lake Taihu, which is hypertrophic and has a mean depth of November 2012, and January 2013, respectively. The AOB 1.89 m (Ye et al. 2009). Another study found a greater abun- content in water showed a typical seasonal pattern: the highest dance of amoA genes at lower light intensities in the ocean copy number of the AOB amoA gene, 8.56±11.3×10 copy (Church et al. 2010). Merbt et al. (2012)showed that both −1 mL , was observed in summer (August 2012), and was AOA and AOB could be completely inhibited by continuous 654 Ann Microbiol (2015) 65:651–657 Fig. 1 a Water temperatures in April, August, November 2012 and 2013, respectively. Identical letters above the bars indicate means with no January 2013. b–e Results are means ± standard deviations of the statistically significant differences. Different letters indicate statistically + − − NH ,NO ,NO and AOB amoA gene concentrations in the overlying significant differences at the 95 % confidence level 4 2 3 water of ten aquaculture ponds in April, August, November 2012 and Jan −2 −1 illumination at high intensity (500 μmol photons m s )in growth was much more photosensitive than bacterial growth, −2 −1 the laboratory. However, at lower light intensities, archaeal with greater inhibition occurring at 60 μmol photons m s Ann Microbiol (2015) 65:651–657 655 −2 −1 than at 15 μmol photons m s , whereas bacteria were unlike bacterial strains, showed no evidence of recovery dur- unaffected by these intensities. Archaeal ammonia oxidizers ing dark phases (Merbt et al. 2012). In addition, French et al. −2 −1 also were more sensitive to cycles of 8-h light/16-h darkness at (2012) showed that white light (30 μmol photons m s ) −2 −1 two light intensities (60 and 15 μmol photons m s )and, strongly inhibited the growth of AOA, but had no effect on the + − Fig. 2 a–e Results are means ± standard deviations of the NH ,NO , respectively. Identical letters above the bars indicate means with no 4 2 NO ,AOB andAOA amoA gene concentrations in the sediment of ten statistically significant differences. Different letters indicate statistically aquaculture ponds in April, August, November 2012 and January 2013, significant differences at the 95 % confidence level 656 Ann Microbiol (2015) 65:651–657 growth of AOB. Furthermore, it was shown that variations in detected in winter, and the lowest AOA content (4.21± −2 −1 5 light intensity ranged from 50 to 2,000 μmol photons m s 2.00×10 ) in summer. Thus, it can be stated that temperature between 07:00 and 19:00 hours on sunny days during the is an important factor that regulates AOA populations in summer at Lake Taihu (30°56′–31°34′ N, 119°54′–120°36′ aquaculture pond sediments. The AOA amoA gene was more E) (Wang et al. 2011), which is located at the same latitude as abundant in winter and spring than in summer and autumn. the aquaculture farm investigated in our study. Therefore, Similar results were was also observed in the sand of an AOA in the surface water of aquaculture ponds was probably eelgrass zone (Ando et al. 2009). The fact that the max- inhibited by light. imum abundance of AOA was observed in winter is in accordance with the findings in estuarine sediments and Seasonal changes and abundance of the amoA gene subtropical coastal mangrove sediments (Caffrey et al. of ammonia-oxidizing microorganisms in aquaculture 2007;Wanget al. 2013). In addition, it has been found pond sediments that AOA is the major driver of nitrification in four cold- water sponges (Radax et al. 2012). + − Figure 2a–c shows the seasonal changes of NH ,NO and As shown in Fig. 2d, e, the concentration of the AOA 4 2 NO concentrations in sediment pore water in April, August amoA gene was greater than that of the AOB amoA gene in and November 2012, and January 2013, respectively. The sediments by almost one order of magnitude. This result concentrations of the AOB amoA gene in the aquaculture contradicts some previous observations, which suggested that pond sediments ranged from 4.05±3.83×10 to 3.11±1.65× AOB outnumbered AOA in the sediments of Eastern Taihu 5 −1 10 copy g (Fig. 2d). In sediments, the maximum densities Bay, which was likely due to the presence of rich organic of the AOB amoA gene were observed in autumn (November substances resulting from intensive pen aquaculture (Wu et al. 2012) and winter (January 2013). There was no significant 2010). Moreover, AOA tend to grow in oligotrophic environ- difference in the density of the AOB amoA gene between ments with low NH concentrations (Stahl and de la Torre autumn and winter (P=0.432, one-way ANOVA). The lowest 2012). Significant negative correlations were observed be- density was observed in summer, which was in contrast with tween the copy numbers of the AOA amoA gene and NH the high densities observed in aquaculture water during this concentrations in sediments in Lake Taihu, China (Wu et al. season. Similar seasonal changes have been observed in estu- 2010). AOA were more abundant than AOB in freshwater arine and eelgrass zone studies (Bernhard et al. 2007; Ando ecosystems and nutrient-depleted oligotrophic wetlands, et al. 2009). The low copy number of the AOB amoA gene in where NH was quite limited (Sims et al. 2012b;Hugoni summer could be attributed to the decrease in the availability et al. 2013). However, under conditions with rich organic + + of NH in the eelgrass zone studies (Ando et al. 2009). Based substances and high NH concentrations in the aquaculture 4 4 on AOB growth experiments, increasing NH concentrations pond sediments, AOA, as opposed to AOB, were the predom- could increase the growth rates and shorten the lag phases of inant ammonia-oxidizing organisms. This also can be attrib- AOB. However, high concentrations of NH pore water were uted to the fact that AOA are more resistant to low levels of observed during the summer in both the estuarine study dissolved oxygen (Coolen et al. 2007;Molina et al. 2010; (Bernhard et al. 2007) and this study (Fig. 2a). Obviously, Bouskill et al. 2012). NH concentration is not a key factor for the growth of AOB in pond sediment. It is possible that AOB experience a greater oxygen limitation in summer when sediment oxygen demand Conclusion peaks. Oxygen dynamics in aquaculture ponds differ substan- tially from those in natural aquatic systems, since pond envi- The annual investigation of freshwater aquaculture ponds ronments are smaller in area and shallower in depth, have showed that AOB were the main ammonia-oxidizing micro- limited water circulation, and are subject to large depositions organisms in water, while the AOA were predominant in of feeding debris. The hypolimnion dissolved oxygen concen- sediments. Both AOB and AOA densities showed typical −1 tration was rarely greater than 2 mg L in summer, although seasonal changes in both water and sediments, and the super-saturation of oxygen usually occurs during the day- photoinhibition and dissolved oxygen levels were suggested light period (Chang and Ouyang 1988). Therefore it can be to be the main regulating factors. speculated that the pond sediment is an extremely oxygen- deficient environment. There were also distinct seasonal changes in the abundance Acknowledgments This study was supported financially by the Special Fund for National Technology System for Conventional Freshwater of the AOA amoA gene in pond sediment. AOA amoA gene Industries (No. nycytx-49-09), Agro-scientific Research in the Public copy numbers showed significant negative correlation with Interest Project (No. 201203083), and National Key Technology R&D temperature (r=− 0.637, P<0.01; nonparametric correla- Program (2012BAD25B01). We also thank Prof. Liu Zuo-xiong for tions). The highest AOA content (1.71±0.76×10 )was reading the manuscript. Ann Microbiol (2015) 65:651–657 657 Koops H-P, Pommerening-Röser A (2001) Distribution and ecophysiol- References ogy of the nitrifying bacteria emphasizing cultured species. FEMS Microbiol Ecol 37:1–9 Ackefors H, Enell M (1994) The release of nutrients and organic matter Kowalchuk GA, Stephen JR (2001) Ammonia-oxidizing bacteria: a from aquaculture systems in Nordic countries. J Appl Ichthyol 10: model for molecular microbial ecology. Annu Rev Microbiol 55: 225–241 485–529 Ando Y, Nakagawa T, Takahashi R, Yoshihara K, Tokuyama T (2009) Leininger S, Urich T, Schloter M, Schwark L, Qi J, Nicol GW, Prosser JI, Seasonal changes in abundance of ammonia-oxidizing archaea and Schuster SC, Schleper C (2006) Archaea predominate among ammonia-oxidizing bacteria and their nitrification in sand of an ammonia-oxidizing prokaryotes in soils. Nature 442:806–809 eelgrass zone. Microbes Environ 24:21–27 Lu SM, Liao MJ, Zhang M, Qi PZ, Xie CC, He XG (2012) A rapid DNA Auguet J-C, Nomokonova N, Camarero L, Casamayor EO (2011) extraction method for quantitative real-time PCR amplification from Seasonal changes of freshwater ammonia-oxidizing archaeal assem- fresh water sediment. J Food Agric Environ 10:1252–1255 blages and nitrogen species in oligotrophic alpine lakes. Appl Merbt SN, Stahl DA, Casamayor EO, Marti E, Nicol GW, Prosser JI Environ Microbiol 77:1937–1945 (2012) Differential photoinhibition of bacterial and archaeal ammo- Bernhard AE, Tucker J, Giblin AE, Stahl DA (2007) Functionally distinct nia oxidation. FEMS Microbiol Lett 327:41–46 communities of ammonia-oxidizing bacteria along an estuarine Molina V, Belmar L, Ulloa O (2010) High diversity of ammonia- salinity gradient. Environ Microbiol 9:1439–1447 oxidizing archaea in permanent and seasonal oxygen-deficient wa- Bouskill NJ, Eveillard D, Chien D, Jayakumar A, Ward BB (2012) ters of the eastern South Pacific. Environ Microbiol 12:2450–2465 Environmental factors determining ammonia-oxidizing organism Pester M, Schleper C, Wagner M (2011) The Thaumarchaeota: an emerg- distribution and diversity in marine environments. Environ ing view of their phylogeny and ecophysiology. Curr Opin Microbiol 14:714–729 Microbiol 14:300–306 Caffrey JM, Bano N, Kalanetra K, Hollibaugh JT (2007) Ammonia Radax R, Hoffmann F, Rapp HT, Leininger S, Schleper C (2012) oxidation and ammonia-oxidizing bacteria and archaea from estuar- Ammonia-oxidizing archaea as main drivers of nitrification in ies with differing histories of hypoxia. ISME J 1:660–662 cold-water sponges. Environ Microbiol 14:909–923 Chang WYB, Ouyang H (1988) Dynamics of dissolved oxygen and Randall DJ, Tsui TKN (2002) Ammonia toxicity in fish. Mar Pollut Bull vertical circulation in fish ponds. Aquaculture 74:263–276 45:17–23 Church MJ, Wai B, Karl DM, DeLong EF (2010) Abundances of Rotthauwe JH, Witzel KP, Liesack W (1997) The ammonia crenarchaeal amoA genes and transcripts in the Pacific Ocean. monooxygenase structural gene amoA as a functional marker: mo- Environ Microbiol 12:679–688 lecular fine-scale analysis of natural ammonia-oxidizing popula- Coolen MJL, Abbas B, Bleijswijk JV, Hopmans EC, Kuypers MMM, tions. Appl Environ Microbiol 63:4704–4712 Wakeham SG, Damsté JSS (2007) Putative ammonia-oxidizing Sims A, Gajaraj S, Hu ZQ (2012a) Seasonal population changes of Crenarchaeota in suboxic waters of the Black Sea: a basin-wide ammonia-oxidizing organisms and their relationship to water quality ecological study using 16S ribosomal and functional genes and in a constructed wetland. Ecol Eng 40:100–107 membrane lipids. Environ Microbiol 9:1001–1016 Sims A, Horton J, Gajaraj S, McIntosh S, Miles RJ, Mueller R, Reed R, Editorial Board of Monitoring and Analytical Method of Water and Hu ZQ (2012b) Temporal and spatial distributions of ammonia- Wastewater, State Environmental Protection Administration of China oxidizing archaea and bacteria and their ratio as an indicator of (2002) Monitoring and analytical method of water and wastewater, 4th oligotrophic conditions in natural wetlands. Water Res 46:4121– edn (in Chinese). China Environmental Science Press, Beijing 4129 FAO (2012) The state of world fisheries and aquaculture 2012. Food and Stahl DA, de la Torre JR (2012) Physiology and diversity of ammonia- Agricultural Organization of the United Nations, Rome, pp 3–4 oxidizing archaea. Annu Rev Microbiol 66:83–101 French E, Kozlowski JA, Mukherjee M, Bullerjahn G, Bollmann A Tourna M, Freitag TE, Nicol GW, Prosser JI (2008) Growth, activity and (2012) Ecophysiological characterization of ammonia-oxidizing ar- temperature responses of ammonia-oxidizing archaea and bacteria chaea and bacteria from freshwater. Appl Environ Microbiol 78: in soil microcosms. Environ Microbiol 10:1357–1364 5773–5780 Wang ZC, Zuo M, Wang YC, Liu YD, Li DH (2011) Dynamics of Handy RD, Poxton MG (1993) Nitrogen pollution in mariculture: toxicity chlorophyll fluorescence and eco-morphological properties of and excretion of nitrogenous compounds by marine fish. Rev Fish Microcystis bloom in Meiliang Bay of Lake Taihu, China. Biol Fish 3:205–241 Fresenius Environ Bull 20:2295–2305 Hatzenpichler R, Lebedeva EV, Spieck E, Stoecker K, Richter A, Daims Wang Y-F, Feng Y-Y, Ma XJ, Gu J-D (2013) Seasonal dynamics of H, Wagner M (2008) A moderately thermophilic ammonia- ammonia/ammonium-oxidizing prokaryotes in oxic and anoxic wet- oxidizing crenarchaeote from a hot spring. Proc Natl Acad Sci land sediments of subtropical coastal mangrove. Appl Microbiol USA 105:2134–2139 Biotechnol 97:7919–7934 He J-Z, Shen J-P, Zhang L-M, Zhu Y-G, Zheng Y-M, Xu M-G, Di HJ Wu YC, Xiang Y, Wang JJ, Zhong JC, He JZ, Wu QL (2010) (2007) Quantitative analyses of the abundance and composition of Heterogeneity of archaeal and bacterial ammonia-oxidizing commu- ammonia-oxidizing bacteria and ammonia-oxidizing archaea of a nities in Lake Taihu, China. Environ Microbiol Rep 2:569–576 Chinese upland red soil under long-term fertilization practices. Wuchter C, Abbas B, Coolen MJL, Herfort L, van Bleijswijk J, Timmers Environ Microbiol 9:2364–2374 P, Strous M, Teira E, Herndl GJ, Middelburg JJ, Schouten S, Damste Horak REA, Qin W, Schauer AJ, Armbrust EV, Ingalls AE, Moffett JW, JSS (2006) Archaeal nitrification in the ocean. Proc Natl Acad Sci Devol AH (2013) Ammonia oxidation kinetics and temperature USA 103:12317–12322 sensitivity of a natural marine community dominated by Archaea. Ye WJ, Liu XL, Lin SQ, Tan J, Pan JL, Li DT, Yang H (2009) The ISME J 7:2023–2033 vertical distribution of bacterial and archaeal communities in the Hugoni M, Etien S, Bourges A, Lepere C, Domaizon I, Mallet C, Bronner water and sediment of Lake Taihu. FEMS Microbiol Ecol 70: G, Debroas D, Mary I (2013) Dynamics of ammonia-oxidizing 263–276 Archaea and Bacteria in contrasted freshwater ecosystems. Res Zhou J, Bruns MA, Tiedje JM (1996) DNA recovery from soils of diverse Microbiol 164:360–370 composition. Appl Environ Microbiol 62:316–322 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Seasonal dynamics of ammonia-oxidizing microorganisms in freshwater aquaculture ponds

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Springer Journals
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Copyright © 2014 by Springer-Verlag Berlin Heidelberg and the University of Milan
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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10.1007/s13213-014-0903-2
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Abstract

Ann Microbiol (2015) 65:651–657 DOI 10.1007/s13213-014-0903-2 ORIGINAL ARTICLE Seasonal dynamics of ammonia-oxidizing microorganisms in freshwater aquaculture ponds Shimin Lu & Mingjun Liao & Congxin Xie & Xugang He & Dapeng Li & Lulu He & Jin Chen Received: 19 January 2014 /Accepted: 21 April 2014 /Published online: 25 May 2014 Springer-Verlag Berlin Heidelberg and the University of Milan 2014 Abstract An annual investigation into the abundance of Introduction ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in fresh water aquaculture ponds was per- Aquaculture production has been increasing at an average formed by quantitative PCR of the amoA gene. The results growth rate of 8.3 % per year, which makes aquaculture one showed that AOB were the main ammonia-oxidizing micro- of the fastest growing segments of the food economy in organisms in water, and significantly higher copy numbers of modern times (FAO 2012). According to the Fishery Bureau the AOB amoA gene were observed in the summer (Aug of the Chinese Ministry of Agriculture (MOA), the total 2012), while no significant differences were detected among aquaculture production of China in 2012 amounted to the other three seasons. AOA showed low abundances 59.1 million metric tons, and the production of freshwater throughout the year. The predominance of AOB in aquacul- aquaculture was 26.4 million metric tons (Fishery National ture water was suggested to be related to photoinhibition. Both Bureau of Statistics of China 2013, http://www.stats.gov.cn/ the AOB and AOA amoA genes in aquaculture pond sedi- english/statisticaldata/Quarterlydata/). Undoubtedly, China’s ments showed typical seasonal patterns. The maximum den- freshwater aquaculture plays an important role in the global sity of AOB was observed in the autumn (Nov 2012) and supply of aquatic products. winter (Jan 2013), while the maximum density of AOA was Currently, there are 2,566,900 ha freshwater aquaculture observed in winter. The minimum densities of both AOA and ponds, and pond culture is the major freshwater culture method AOB occurred in the summer. The concentration of the AOA in China (National Bureau of Statistics of China 2013, http:// amoA gene was higher than that of the AOB amoA gene in www.stats.gov.cn/english/statisticaldata/Quarterlydata/). sediments by almost one order of magnitude, which indicates However, along with increasing development, concerns are that AOA are the dominant ammonia-oxidizing microorgan- evoked about the possible effects of ever-increasing aquaculture isms in the aquaculture pond sediments. Dissolved oxygen is waste, both on the productivity of aquaculture systems and on suggested to be the key factor determining the predominance the ambient aquatic ecosystem. It was estimated that 9. of AOA in pond sediments. 5 kg phosphorous (P) and 78 kg of nitrogen (N) are released into the aquaculture environment per ton of fish produced. Approximately 72 % of N and 70 % of Keywords Ammonia-oxidizing archaea Ammonia- P in the feed are released into the aquaculture environ- . . . oxidizingbacteria amoAgene Aquaculturewater Sediment ment (Ackefors and Enell 1994). Ammonia is one of the most toxic substances produced by intensive fish farming (Handy and Poxton 1993). Ammonia is toxic Shimin Lu and Mingjun Liao contributed equally to this work. to all vertebrates and can cause convulsions, coma and : : : : : : S. Lu M. Liao C. Xie (*) X. He D. Li L. He J. Chen death, probably because elevated ammonium (NH ) College of Fisheries, Huazhong Agricultural University, ions displace potassium (K ) and depolarizes neurons, Wuhan 430070, China e-mail: xiecongxin@mail.hzau.edu.cn causing activation of the N-methyl-d-aspartate (NMDA) type glutamate receptor, which leads to an influx of excessive M. Liao 2+ calcium (Ca ) ions and subsequent cell death in the central College of Resource and Environmental Engineering, Hubei nervous system (Randall and Tsui 2002). University of Technology, Wuhan 430068, China 652 Ann Microbiol (2015) 65:651–657 Nitrification is a key process in the cycling of nitrogen in pore water. The pore water was then filtered through a + − aquatic ecosystems (Merbt et al. 2012). The oxidation of 0.22-μm membrane. Field measurements of NH -N, NO - 4 3 − − ammonia (NH ) to nitrite (NO )—the first and rate-limiting Nand NO -N concentrations were performed by the Nessler 3 2 2 step of nitrification—has traditionally been considered to be reagent photometric method, photometry with phenol-2- carried out solely by ammonia-oxidizing bacteria (AOB) sulfonic acid method, and the N (1-naphty1)-ethylenediamine (Koops and Pommerening-Röser 2001), which fall into two dihydrochloride spectrophotometric method, respectively, ac- phylogenetic lineages within the β-and γ-Proteobacteria cording to the protocols described in Chinese Standard (Kowalchuk and Stephen 2001). However, this notion has Methods (Editorial Board of Monitoring and Analytical changed owing to the recent discovery of ammonia- Method of Water and Wastewater 2002). oxidizing archaea (AOA). Based on genomic level compari- sons, AOA were classified into the newly proposed phylum DNA extraction from fresh water and sediments Thaumarchaeota (Pester et al. 2011). AOA play important roles in the nitrogen cycle and have been found in various DNA from water- and sediment-dwelling microorganisms habitats, including hot springs (Hatzenpichler et al. 2008), was extracted as described by Lu et al. (2012)withsome oceans (Wuchter et al. 2006; Horak et al. 2013), freshwater modifications. Water (50 mL) from each pond was filtered (Auguet et al. 2011), and soil (Leininger et al. 2006). directly through a 0.22-μm membrane using negative pres- Although AOB and AOA are affiliated with different phylo- sure. After filtration, the membranes were cut into pieces and genetic domains, they both contain homologous ammonia placed in a 2-mL sterile centrifuge tube and stored at −20 °C monooxygenase (AMO), which is responsible for catalyzing until DNA extraction. The pieces of filtered membranes or the first step in ammonia oxidation. The amoA gene, encoding 0.3 g sediment were lysed using 0.7 μL lysis buffer (Zhou the alpha subunit of AMO, has been used widely as a func- et al. 1996), 100 μL 10 % SDS, and two glass beads. The tional gene marker for tracking ammonia oxidizers in envi- lysate was homogenized for 2 min on a vortexer, and then ronmental samples. Nonetheless, there is a great lack of stud- incubated at 70 °C for 1 h. After incubation, the tube was ies on AOA and AOB in aquaculture ponds. In this paper, we centrifuged at 13,000 g for 10 min and 0.7 mL supernatant report on the seasonal dynamics of the abundance of was transferred into a new tube. Then, 0.6 mL crenarchaeotal and betaproteobacterial amoA genes, and in- phenol:chloroform:isoamyl alcohol (25:24:1) was added and vestigated changes in temperature and concentration of am- shaken gently for 1 min, and the tube was centrifuged at + − − monium (NH ), nitrite (NO ) and nitrate (NO )in aqua- 13,000 g for 5 min. After centrifugation, the supernatant was 4 2 3 culture water and sediment, aiming to achieve a better under- transferred to a new tube, followed by the addition of a 0.5 standing of the nitrogen cycle in the aquaculture environment. volume of PEG 6000 solution (50 % in m/v) and incubated for 60 min at ambient temperature. The tube was then centrifuged at 13,000 g for 15 min, the supernatant was discarded, and the Materials and methods pellet was re-dissolved in 0.6 mL distilled water before being extracted with 0.4 mL chloroform by several gentle end-over- Sample collection and hydration index determination end inversions. After extraction, the tube was centrifuged at 12,000 g for 5 min and 0.45 mL of the supernatant was Experimental samples were collected from an aquaculture transferred to a new tube and mixed with 0.9 mL ethanol farm [29°55′ N, 112°(17–18)′ E], Gong’an, Hubei, China. and 45 μL NaCl solution (5 M) and precipitated for 15 min Ten aquaculture ponds were selected for investigation. The on ice. After precipitation, the tube was centrifuged at average depth of the sampling ponds (350 m×20 m) was 13,000 g for 15 min, and the pellet was washed twice with about 1.5 m, in which grass carp, silver carp, bighead carp 70 % ethanol and air-dried. The final pellet was dissolved in and soft-shelled turtle were raised for commercial use since 50 μLTE buffer. DNA concentrations were quantified using a the 1980s. The ponds have never been dredged and are typical ND-2000 UV-vis spectrophotometer (http://www.nanodrop. of a eutrophic environment. The samples were collected on 14 com). April, 22 August, 2 November 2012, and 15 January 2013. The water samples were collected with a sterile glass bottle qPCR analysis of bacterial and archaeal amoA about 30 cm below the water surface. The top 0–5cm of black color sediment samples were collected using a Peterson Grab Quantitative PCR (qPCR) was used to estimate the abundance Sampler in the center of the aquaculture ponds, and the of bacterial and archaeal amoA genes. qPCR was performed homogenized sediment was transferred immediately to a ster- using a Qiagen Q thermocycler (Qiagen, Hilden, Germany) ile plastic bag on the fishing boat. The water and sediment and primers amoA-1F/amoA-2R (specific for the AOB amoA samples were stored at 4 °C and treated within 48 h. The gene) (Rotthauwe et al. 1997) and CrenamoA23f/ CrenamoA616r (specific for the AOA amoA gene) (Tourna sediment was centrifuged at 7,000 g for 10 min to extract bulk Ann Microbiol (2015) 65:651–657 653 et al. 2008). The qPCR was conducted in a total volume of significantly higher than in other seasons (1.55±8.41×10 in 3 −1 20 μLcontaining 10 μL SYBR Premix Ex Taq II for AOB or spring, 1.04±2.28×10 copy mL in autumn and 3.83± 2 −1 SYBR Premix Ex Taq for AOA (Takara, Dalian, China), 1 μL 2.24×10 copy mL in winter) (Fig. 1e; P<0.01, one-way −1 DNA template, 0.2 μM of each primer and 0.2 mg mL ANOVA). No significant differences were detected between bovine serum albumin (BSA). The qPCR thermocycling steps the other three seasons. AOB amoA gene copy numbers were as follows: an initial denaturation of 95 °C for 30 s, showed significant positive correlation with temperature (r= followed by 35 cycles of 95 °C for 5 s, 54 °C for 35 s or 53 °C 0.597, P<0.01; nonparametric correlations). These results for 60 s, and 72 °C for 60 s. Plates were read at 81 °C for AOB indicate that temperature plays a more important role in the or 80 °C for AOA, respectively. The negative control without seasonal dynamics of AOB than aquaculture activity, includ- DNA template was subjected to the same procedures to ex- ing the application of probiotics and fish medicine and aera- clude or detect any possible contamination. After qPCR, the tion, etc. A similar phenomenon was observed in a construct- specificity of amplification was verified by melting curve ed wetland, where the amoA gene copy numbers of AOB 4 6 −1 analysis and agarose gel electrophoresis. All measurements ranged from 5.3±0.6×10 to 8.1±0.5×10 copy mL in were performed in triplicate. water samples in summer, while AOB was not detected at Standard curves for qPCR were developed as described the outlet of the first wetland treatment unit in the winter previously with some modifications (He et al. 2007). The season (Sims et al. 2012a). bacterial or archaeal amoA gene was PCR-amplified from In the aquaculture water, the concentration of NH corre- the extracted DNA using primers amoA-1F/amoA-2R or lated roughly with the copy numbers of the AOB amoA gene CrenamoA23f/CrenamoA616r, and the PCR amplification throughout the year (r=0.251, P<0.01), which indicates that products were purified and cloned into the pMD18-T Easy other routes of NH removal in aquaculture water, such as Vector (Takara, Dalian, China). Plasmid extracted from the absorption by phytoplankton, also play a very important role correct clones was used as a standard for quantitative analysis. in addition to the oxidation by AOB. No significant correla- The plasmid DNA concentration was determined using a ND- tion was observed between the copy numbers of the AOB − − 2000 UV-vis Spectrophotometer and the copy numbers of the amoA gene and the concentrations of NO and NO in 2 3 target genes were calculated directly from the concentration of water, which was probably due to the fact that NO and the extracted plasmid DNA. Ten-fold serial dilutions of plas- NO levels are not only influenced by the amount and activ- 2 7 mid DNA ranging from 2.0×10 to 2.0×10 copies of the ity of nitrifying bacteria, but are also determined by denitrifi- 1 6 AOA amoA gene or 2.0×10 to 2.0×10 copies of the AOB cation intensity. amoA gene were subjected to qPCR in triplicate to generate an The AOA amoA gene was detected by PCR throughout the external standard curve. year in the aquaculture water based on our research over Two controls were performed to estimate the possible 2 years. However, because the copy number of the AOA inhibition of qPCR performance by the co-extracted polyphe- amoA gene was below the minimum level of detection (20 −1 nolic compounds or humic acids in sediment or water extracts: copy mL water) in the freshwater column, the exact copy (1) different amounts of amoA standards were mixed with number of the AOA amoA gene could not be determined. The equal amounts of sediment or water DNA extract, and (2) availability of NH does not appear to be the limiting eco- two different volumes of DNA extract from each sediment or logical factor for this phenomenon. It was shown that enriched water sample used in this study were applied for the quantifi- AOA cultures could grow well when the NH concentration −1 cation of amoA copy number. In most cases, inhibition was ranged from 0.22 to 70 mg mL (French et al. 2012). In this not detected or was negligible. study, the NH concentrations of aquaculture water ranged −1 −1 from 1.02±0.86 mg L to 2.80±0.89 mg L during the year, which falls within the aforementioned range of NH Results and discussion concentrations. The fact that AOB were the predominant ammonia- Seasonal changes and abundance of amoA gene oxidizer in the surface water could be explained by in ammonia-oxidizing microorganisms in aquaculture water photoinhibition because the sampling point was at a depth of only 30 cm. A similar study, in which water samples were Figure 1a–e shows the seasonal changes in temperature, collected from 50 cm below the surface, found that AOB were + − − NH ,NO and NO concentrations and AOB amoA gene the only ammonia-oxidizer in surface water in freshwater of 4 2 3 copy number in aquaculture water in April, August, Lake Taihu, which is hypertrophic and has a mean depth of November 2012, and January 2013, respectively. The AOB 1.89 m (Ye et al. 2009). Another study found a greater abun- content in water showed a typical seasonal pattern: the highest dance of amoA genes at lower light intensities in the ocean copy number of the AOB amoA gene, 8.56±11.3×10 copy (Church et al. 2010). Merbt et al. (2012)showed that both −1 mL , was observed in summer (August 2012), and was AOA and AOB could be completely inhibited by continuous 654 Ann Microbiol (2015) 65:651–657 Fig. 1 a Water temperatures in April, August, November 2012 and 2013, respectively. Identical letters above the bars indicate means with no January 2013. b–e Results are means ± standard deviations of the statistically significant differences. Different letters indicate statistically + − − NH ,NO ,NO and AOB amoA gene concentrations in the overlying significant differences at the 95 % confidence level 4 2 3 water of ten aquaculture ponds in April, August, November 2012 and Jan −2 −1 illumination at high intensity (500 μmol photons m s )in growth was much more photosensitive than bacterial growth, −2 −1 the laboratory. However, at lower light intensities, archaeal with greater inhibition occurring at 60 μmol photons m s Ann Microbiol (2015) 65:651–657 655 −2 −1 than at 15 μmol photons m s , whereas bacteria were unlike bacterial strains, showed no evidence of recovery dur- unaffected by these intensities. Archaeal ammonia oxidizers ing dark phases (Merbt et al. 2012). In addition, French et al. −2 −1 also were more sensitive to cycles of 8-h light/16-h darkness at (2012) showed that white light (30 μmol photons m s ) −2 −1 two light intensities (60 and 15 μmol photons m s )and, strongly inhibited the growth of AOA, but had no effect on the + − Fig. 2 a–e Results are means ± standard deviations of the NH ,NO , respectively. Identical letters above the bars indicate means with no 4 2 NO ,AOB andAOA amoA gene concentrations in the sediment of ten statistically significant differences. Different letters indicate statistically aquaculture ponds in April, August, November 2012 and January 2013, significant differences at the 95 % confidence level 656 Ann Microbiol (2015) 65:651–657 growth of AOB. Furthermore, it was shown that variations in detected in winter, and the lowest AOA content (4.21± −2 −1 5 light intensity ranged from 50 to 2,000 μmol photons m s 2.00×10 ) in summer. Thus, it can be stated that temperature between 07:00 and 19:00 hours on sunny days during the is an important factor that regulates AOA populations in summer at Lake Taihu (30°56′–31°34′ N, 119°54′–120°36′ aquaculture pond sediments. The AOA amoA gene was more E) (Wang et al. 2011), which is located at the same latitude as abundant in winter and spring than in summer and autumn. the aquaculture farm investigated in our study. Therefore, Similar results were was also observed in the sand of an AOA in the surface water of aquaculture ponds was probably eelgrass zone (Ando et al. 2009). The fact that the max- inhibited by light. imum abundance of AOA was observed in winter is in accordance with the findings in estuarine sediments and Seasonal changes and abundance of the amoA gene subtropical coastal mangrove sediments (Caffrey et al. of ammonia-oxidizing microorganisms in aquaculture 2007;Wanget al. 2013). In addition, it has been found pond sediments that AOA is the major driver of nitrification in four cold- water sponges (Radax et al. 2012). + − Figure 2a–c shows the seasonal changes of NH ,NO and As shown in Fig. 2d, e, the concentration of the AOA 4 2 NO concentrations in sediment pore water in April, August amoA gene was greater than that of the AOB amoA gene in and November 2012, and January 2013, respectively. The sediments by almost one order of magnitude. This result concentrations of the AOB amoA gene in the aquaculture contradicts some previous observations, which suggested that pond sediments ranged from 4.05±3.83×10 to 3.11±1.65× AOB outnumbered AOA in the sediments of Eastern Taihu 5 −1 10 copy g (Fig. 2d). In sediments, the maximum densities Bay, which was likely due to the presence of rich organic of the AOB amoA gene were observed in autumn (November substances resulting from intensive pen aquaculture (Wu et al. 2012) and winter (January 2013). There was no significant 2010). Moreover, AOA tend to grow in oligotrophic environ- difference in the density of the AOB amoA gene between ments with low NH concentrations (Stahl and de la Torre autumn and winter (P=0.432, one-way ANOVA). The lowest 2012). Significant negative correlations were observed be- density was observed in summer, which was in contrast with tween the copy numbers of the AOA amoA gene and NH the high densities observed in aquaculture water during this concentrations in sediments in Lake Taihu, China (Wu et al. season. Similar seasonal changes have been observed in estu- 2010). AOA were more abundant than AOB in freshwater arine and eelgrass zone studies (Bernhard et al. 2007; Ando ecosystems and nutrient-depleted oligotrophic wetlands, et al. 2009). The low copy number of the AOB amoA gene in where NH was quite limited (Sims et al. 2012b;Hugoni summer could be attributed to the decrease in the availability et al. 2013). However, under conditions with rich organic + + of NH in the eelgrass zone studies (Ando et al. 2009). Based substances and high NH concentrations in the aquaculture 4 4 on AOB growth experiments, increasing NH concentrations pond sediments, AOA, as opposed to AOB, were the predom- could increase the growth rates and shorten the lag phases of inant ammonia-oxidizing organisms. This also can be attrib- AOB. However, high concentrations of NH pore water were uted to the fact that AOA are more resistant to low levels of observed during the summer in both the estuarine study dissolved oxygen (Coolen et al. 2007;Molina et al. 2010; (Bernhard et al. 2007) and this study (Fig. 2a). Obviously, Bouskill et al. 2012). NH concentration is not a key factor for the growth of AOB in pond sediment. It is possible that AOB experience a greater oxygen limitation in summer when sediment oxygen demand Conclusion peaks. Oxygen dynamics in aquaculture ponds differ substan- tially from those in natural aquatic systems, since pond envi- The annual investigation of freshwater aquaculture ponds ronments are smaller in area and shallower in depth, have showed that AOB were the main ammonia-oxidizing micro- limited water circulation, and are subject to large depositions organisms in water, while the AOA were predominant in of feeding debris. The hypolimnion dissolved oxygen concen- sediments. Both AOB and AOA densities showed typical −1 tration was rarely greater than 2 mg L in summer, although seasonal changes in both water and sediments, and the super-saturation of oxygen usually occurs during the day- photoinhibition and dissolved oxygen levels were suggested light period (Chang and Ouyang 1988). Therefore it can be to be the main regulating factors. speculated that the pond sediment is an extremely oxygen- deficient environment. There were also distinct seasonal changes in the abundance Acknowledgments This study was supported financially by the Special Fund for National Technology System for Conventional Freshwater of the AOA amoA gene in pond sediment. AOA amoA gene Industries (No. nycytx-49-09), Agro-scientific Research in the Public copy numbers showed significant negative correlation with Interest Project (No. 201203083), and National Key Technology R&D temperature (r=− 0.637, P<0.01; nonparametric correla- Program (2012BAD25B01). We also thank Prof. Liu Zuo-xiong for tions). The highest AOA content (1.71±0.76×10 )was reading the manuscript. Ann Microbiol (2015) 65:651–657 657 Koops H-P, Pommerening-Röser A (2001) Distribution and ecophysiol- References ogy of the nitrifying bacteria emphasizing cultured species. FEMS Microbiol Ecol 37:1–9 Ackefors H, Enell M (1994) The release of nutrients and organic matter Kowalchuk GA, Stephen JR (2001) Ammonia-oxidizing bacteria: a from aquaculture systems in Nordic countries. J Appl Ichthyol 10: model for molecular microbial ecology. Annu Rev Microbiol 55: 225–241 485–529 Ando Y, Nakagawa T, Takahashi R, Yoshihara K, Tokuyama T (2009) Leininger S, Urich T, Schloter M, Schwark L, Qi J, Nicol GW, Prosser JI, Seasonal changes in abundance of ammonia-oxidizing archaea and Schuster SC, Schleper C (2006) Archaea predominate among ammonia-oxidizing bacteria and their nitrification in sand of an ammonia-oxidizing prokaryotes in soils. Nature 442:806–809 eelgrass zone. Microbes Environ 24:21–27 Lu SM, Liao MJ, Zhang M, Qi PZ, Xie CC, He XG (2012) A rapid DNA Auguet J-C, Nomokonova N, Camarero L, Casamayor EO (2011) extraction method for quantitative real-time PCR amplification from Seasonal changes of freshwater ammonia-oxidizing archaeal assem- fresh water sediment. J Food Agric Environ 10:1252–1255 blages and nitrogen species in oligotrophic alpine lakes. Appl Merbt SN, Stahl DA, Casamayor EO, Marti E, Nicol GW, Prosser JI Environ Microbiol 77:1937–1945 (2012) Differential photoinhibition of bacterial and archaeal ammo- Bernhard AE, Tucker J, Giblin AE, Stahl DA (2007) Functionally distinct nia oxidation. FEMS Microbiol Lett 327:41–46 communities of ammonia-oxidizing bacteria along an estuarine Molina V, Belmar L, Ulloa O (2010) High diversity of ammonia- salinity gradient. Environ Microbiol 9:1439–1447 oxidizing archaea in permanent and seasonal oxygen-deficient wa- Bouskill NJ, Eveillard D, Chien D, Jayakumar A, Ward BB (2012) ters of the eastern South Pacific. Environ Microbiol 12:2450–2465 Environmental factors determining ammonia-oxidizing organism Pester M, Schleper C, Wagner M (2011) The Thaumarchaeota: an emerg- distribution and diversity in marine environments. Environ ing view of their phylogeny and ecophysiology. Curr Opin Microbiol 14:714–729 Microbiol 14:300–306 Caffrey JM, Bano N, Kalanetra K, Hollibaugh JT (2007) Ammonia Radax R, Hoffmann F, Rapp HT, Leininger S, Schleper C (2012) oxidation and ammonia-oxidizing bacteria and archaea from estuar- Ammonia-oxidizing archaea as main drivers of nitrification in ies with differing histories of hypoxia. ISME J 1:660–662 cold-water sponges. Environ Microbiol 14:909–923 Chang WYB, Ouyang H (1988) Dynamics of dissolved oxygen and Randall DJ, Tsui TKN (2002) Ammonia toxicity in fish. Mar Pollut Bull vertical circulation in fish ponds. Aquaculture 74:263–276 45:17–23 Church MJ, Wai B, Karl DM, DeLong EF (2010) Abundances of Rotthauwe JH, Witzel KP, Liesack W (1997) The ammonia crenarchaeal amoA genes and transcripts in the Pacific Ocean. monooxygenase structural gene amoA as a functional marker: mo- Environ Microbiol 12:679–688 lecular fine-scale analysis of natural ammonia-oxidizing popula- Coolen MJL, Abbas B, Bleijswijk JV, Hopmans EC, Kuypers MMM, tions. Appl Environ Microbiol 63:4704–4712 Wakeham SG, Damsté JSS (2007) Putative ammonia-oxidizing Sims A, Gajaraj S, Hu ZQ (2012a) Seasonal population changes of Crenarchaeota in suboxic waters of the Black Sea: a basin-wide ammonia-oxidizing organisms and their relationship to water quality ecological study using 16S ribosomal and functional genes and in a constructed wetland. Ecol Eng 40:100–107 membrane lipids. Environ Microbiol 9:1001–1016 Sims A, Horton J, Gajaraj S, McIntosh S, Miles RJ, Mueller R, Reed R, Editorial Board of Monitoring and Analytical Method of Water and Hu ZQ (2012b) Temporal and spatial distributions of ammonia- Wastewater, State Environmental Protection Administration of China oxidizing archaea and bacteria and their ratio as an indicator of (2002) Monitoring and analytical method of water and wastewater, 4th oligotrophic conditions in natural wetlands. Water Res 46:4121– edn (in Chinese). China Environmental Science Press, Beijing 4129 FAO (2012) The state of world fisheries and aquaculture 2012. Food and Stahl DA, de la Torre JR (2012) Physiology and diversity of ammonia- Agricultural Organization of the United Nations, Rome, pp 3–4 oxidizing archaea. Annu Rev Microbiol 66:83–101 French E, Kozlowski JA, Mukherjee M, Bullerjahn G, Bollmann A Tourna M, Freitag TE, Nicol GW, Prosser JI (2008) Growth, activity and (2012) Ecophysiological characterization of ammonia-oxidizing ar- temperature responses of ammonia-oxidizing archaea and bacteria chaea and bacteria from freshwater. Appl Environ Microbiol 78: in soil microcosms. Environ Microbiol 10:1357–1364 5773–5780 Wang ZC, Zuo M, Wang YC, Liu YD, Li DH (2011) Dynamics of Handy RD, Poxton MG (1993) Nitrogen pollution in mariculture: toxicity chlorophyll fluorescence and eco-morphological properties of and excretion of nitrogenous compounds by marine fish. Rev Fish Microcystis bloom in Meiliang Bay of Lake Taihu, China. Biol Fish 3:205–241 Fresenius Environ Bull 20:2295–2305 Hatzenpichler R, Lebedeva EV, Spieck E, Stoecker K, Richter A, Daims Wang Y-F, Feng Y-Y, Ma XJ, Gu J-D (2013) Seasonal dynamics of H, Wagner M (2008) A moderately thermophilic ammonia- ammonia/ammonium-oxidizing prokaryotes in oxic and anoxic wet- oxidizing crenarchaeote from a hot spring. Proc Natl Acad Sci land sediments of subtropical coastal mangrove. Appl Microbiol USA 105:2134–2139 Biotechnol 97:7919–7934 He J-Z, Shen J-P, Zhang L-M, Zhu Y-G, Zheng Y-M, Xu M-G, Di HJ Wu YC, Xiang Y, Wang JJ, Zhong JC, He JZ, Wu QL (2010) (2007) Quantitative analyses of the abundance and composition of Heterogeneity of archaeal and bacterial ammonia-oxidizing commu- ammonia-oxidizing bacteria and ammonia-oxidizing archaea of a nities in Lake Taihu, China. Environ Microbiol Rep 2:569–576 Chinese upland red soil under long-term fertilization practices. Wuchter C, Abbas B, Coolen MJL, Herfort L, van Bleijswijk J, Timmers Environ Microbiol 9:2364–2374 P, Strous M, Teira E, Herndl GJ, Middelburg JJ, Schouten S, Damste Horak REA, Qin W, Schauer AJ, Armbrust EV, Ingalls AE, Moffett JW, JSS (2006) Archaeal nitrification in the ocean. Proc Natl Acad Sci Devol AH (2013) Ammonia oxidation kinetics and temperature USA 103:12317–12322 sensitivity of a natural marine community dominated by Archaea. Ye WJ, Liu XL, Lin SQ, Tan J, Pan JL, Li DT, Yang H (2009) The ISME J 7:2023–2033 vertical distribution of bacterial and archaeal communities in the Hugoni M, Etien S, Bourges A, Lepere C, Domaizon I, Mallet C, Bronner water and sediment of Lake Taihu. FEMS Microbiol Ecol 70: G, Debroas D, Mary I (2013) Dynamics of ammonia-oxidizing 263–276 Archaea and Bacteria in contrasted freshwater ecosystems. Res Zhou J, Bruns MA, Tiedje JM (1996) DNA recovery from soils of diverse Microbiol 164:360–370 composition. Appl Environ Microbiol 62:316–322

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Annals of Microbiology – Springer Journals

Published: May 25, 2014

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