Responses of Active Ammonia Oxidizers and NitrificationActivity in Eutrophic Lake Sediments to Nitrogen andTemperature

Ling Wu,a,b,d Cheng Han,a,d Guangwei Zhu,c Wenhui Zhonga,d

aJiangsu Provincial Key Laboratory of Materials Cycling and Pollution Control, School of Geography Sciences, Nanjing Normal University, Nanjing, ChinabChangzhou Vocational Institute of Engineering, Changzhou, ChinacState Key Laboratory of Lake Science and Environment, Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing, ChinadJiangsu Center for Collaborative Innovation in Geographical Information Resource Development and Application, Nanjing, China

ABSTRACT Ammonium concentrations and temperature drive the activities ofammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), buttheir effects on these microbes in eutrophic freshwater sediments are unclear. In thisstudy, surface sediments collected from areas of Taihu Lake (China) with differentdegrees of eutrophication were incubated under three levels of nitrogen input andtemperature, and the autotrophic growth of ammonia oxidizers was assessed using13C-labeled DNA-based stable-isotope probing (SIP), while communities were charac-terized using MiSeq sequencing and phylogenetic analysis of 16S rRNA genes. Nitrifi-cation rates in sediment microcosms were positively correlated with nitrogen inputs,but there was no marked association with temperature. Incubation of SIP micro-cosms indicated that AOA and AOB amoA genes were labeled by 13C at 20°C and30°C in the slightly eutrophic sediment, and AOB amoA genes were labeled to amuch greater extent than AOA amoA genes in the moderately eutrophic sedimentafter 56 days. Phylogenetic analysis of 13C-labeled 16S rRNA genes revealed that theactive AOA were mainly affiliated with the Nitrosopumilus cluster, with the Ni-trososphaera cluster dominating in the slightly eutrophic sediment at 30°C with lowammonium input (1 mM). Active AOB communities were more sensitive to nitrogeninput and temperature than were AOA communities, and they were exclusivelydominated by the Nitrosomonas cluster, which tended to be associated withNitrosomonadaceae-like lineages. Nitrosomonas sp. strain Is79A3 tended to dominatethe moderately eutrophic sediment at 10°C with greater ammonium input (2.86 mM).The relative abundance responses of the major active communities to nitrogen inputand temperature gradients varied, indicating niche differentiation and differences inthe physiological metabolism of ammonia oxidizers that are yet to be described.

IMPORTANCE Both archaea and bacteria contribute to ammonia oxidation, whichplays a central role in the global cycling of nitrogen and is important for reducing eutro-phication in freshwater environments. The abundance and activities of ammonia-oxidizing archaea and bacteria in eutrophic limnic sediments vary with different ammo-nium concentrations or with seasonal shifts, and how the two factors affect nitrificationactivity, microbial roles, and active groups in different eutrophic sediments is unclear.The significance of our research is in identifying the archaeal and bacterial responses toanthropogenic activity and climate change, which will greatly enhance our understand-ing of the physiological metabolic differences of ammonia oxidizers.

KEYWORDS active ammonia oxidizers, nitrogen inputs, stable-isotope probing,temperature

Citation Wu L, Han C, Zhu G, Zhong W. 2019.Responses of active ammonia oxidizers andnitrification activity in eutrophic lake sedimentsto nitrogen and temperature. Appl EnvironMicrobiol 85:e00258-19. https://doi.org/10.1128/AEM.00258-19.

Editor Haruyuki Atomi, Kyoto University

Copyright © 2019 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Wenhui Zhong,zhongwenhui@njnu.edu.cn.

Received 31 January 2019Accepted 19 June 2019

Accepted manuscript posted online 28June 2019Published

ENVIRONMENTAL MICROBIOLOGY

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Inland lakes are sensitive to human activities that increase nutrient loads and causeaquatic eutrophication. Trophic gradients within lakes reflect differences in nutrient

input, such as ammonium levels within a habitat, with reduction in eutrophicationbeing achieved through removal of ammonia via nitrification and anaerobic ammoniaoxidation or denitrification and via the volatilization of ammonia (1).

Ammonia oxidization, the first step of nitrification, is mainly carried out byammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), which areubiquitous in freshwater ecosystems (2, 3). Research has demonstrated that NO2

�

accumulation, which indicates archaeal and bacterial nitrification activity, is affectedmainly by ammonium levels in culture media or river sediments (4). Differences in cellsize, activity, and physiology of archaea and bacteria drive differences in their nitrifi-cation activities and, although AOA tend to be more abundant than AOB in soils andmarine ecosystems (2, 5–8), AOB growth correlates with nitrification activity, whilearchaea do not respond to increased levels of nitrogen fertilizer (9, 10). Moreover, theconcentration of ammonium is a key driver of population dynamics and communitycomposition for ammonia oxidizers in sediments and soils (11–14); the activity of AOBis always enhanced in soils with high concentrations of ammonium (9, 10), AOB growth(measured as dynamic activity) increases in sediments or biofilms with high concen-trations of ammonium (15, 16), and Nitrosomonas AOB are found in ammonium-richcoastal sediments (17). In contrast, AOA occur where ammonium concentrations arelow (�10 �M), such as in marine ecosystems and in oligotrophic or slightly eutrophiclakes (2, 5), while Nitrosopumilus maritimus AOA show high rates of ammonia oxidiza-tion in low-ammonia environments (14).

Nitrification is a key process in ammonium accumulation, and the lowest ammo-nium concentrations, highest nitrate concentrations, and maximum nitrification activ-ities occur at intermediate temperatures (15°C to 25°C) in hypereutrophic lake andarctic sediments (18, 19). Other studies have also demonstrated that the optimaltemperature for nitrification activity is between 30°C and 40°C in soils (20). Moreover,studies showed that temperature was the most important factor for archaeal andbacterial community abundance and structures in sediments or soils, and AOB abun-dance was always stimulated by increasing temperature (21–23). Research on RushanBay revealed that AOB dominated in ammonia oxidization in surface sediment duringthe summer, whereas AOA played a dominant role during the winter (24), and tem-perature ranges for optimal growth differed within AOA communities (21). However,Tourna et al. found that the relative abundance and transcriptional activity of AOB werenot related to temperature, and only the AOA community structure in soils wasinfluenced by temperature (25). Given that both ammonium concentrations and tem-perature are important factors for the activities of ammonia oxidizers, there is a lack ofclarity regarding the responses of microbial activities to these factors in isolation andin combination.

Taihu Lake (30°55’40� to 31°32’58�N, 119°52’32� to 120°36’10�E) is a large, eutrophic,freshwater lake in China (Fig. 1), and its trophic level decreases continuously fromMeiliang Bay in the north to the central lake, because Meiliang Bay is closer toresidential quarters and under greater nitrogen inputs (26), which lead to the contrast-ing eutrophic statuses of Meiliang Bay and the central lake and different ammoniaoxidation capacities in sediments from the two areas. Furthermore, fluctuations of thefreshwater temperature of Taihu Lake occur throughout the year, with a maximum of31.5°C and a minimum of 5.8°C. Thus, we applied the [13C]DNA-based stable-isotopeprobing (SIP) method to obtain ammonia oxidizer enrichments grown with differentammonium supply levels, representative of the average N input of Taihu Lake and thepotential N oxidation capacity of Meiliang Bay and central lake sediments, and tem-peratures covering the natural freshwater variation, to determine the responses tonitrogen and temperature of their autotrophic growth and community and nitrificationactivity, which would provide evidence for the effects of climate change and anthro-pogenic disturbance on the AOA and AOB in eutrophic aquatic ecosystems.

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RESULTSPhysicochemical properties of sediments and water. Physicochemical properties

varied between the two contrasting surface sediments by the independent-sample ttest (Table 1). Total organic carbon (TOC) content, total nitrogen (TN) content, inorganicnitrogen content, and potential ammonia oxidization (PAO) were greater and pH,oxidation-reduction potential (Eh), and dissolved oxygen (DO) levels were lower in themoderately eutrophic sediments of Meiliang Bay than in the slightly eutrophic sedi-ments from the central lake (P � 0.05).

Levels of eutrophication at the two study sites were estimated using the compre-hensive trophic level index (TLI), which was calculated using indices of chlorophyll a(Chl a) content, total phosphorous (TP) content, TN content, chemiluminescence de-tection of permanganate (CODMn), and transparency (Secchi depth [SD]) (27). The TLIvalues of Taihu Meiliang Bay and the central lake were 65 and 52, respectively, so theareas were classified as moderately and slightly eutrophic, respectively.

Nitrification activity in sediment microcosms. Nitrification activity in the sedi-ment microcosms was assessed from changes in inorganic nitrogen contents beforeand after 56 days of incubation (Fig. 2). With the exception of the slightly eutrophicsediment with normal nitrogen (NN) and high nitrogen (HN) input treatments at 10°C,ammonium contents decreased following incubation for 56 days (P � 0.05). Nitrateconcentrations increased with all treatments (P � 0.05), except in the slightly eutrophicsediment with NN treatment at 10°C (P � 0.05), and rates of increases in nitrateconcentrations were lower in the slightly eutrophic sediment (NN and HN treatments)at 10°C than with the other treatments (see Table S1 in the supplemental material).

FIG 1 Locations of sampling sites at Taihu Lake. Moderate-E and Slight-E refer to moderately eutrophic sediments from Meiliang Bay and slightly eutrophicsediments from the central lake, respectively. Map created using ArcGis version 10.2.

TABLE 1 Physicochemical properties of sediments in Taihu Lakea

Sampling siteTOC content(g kg�1)

TN content(g kg�1)

NH4� N content

(mg kg�1)NO3

� N content(mg kg�1) pH Eh (mV)

DO level(mmol liter�1)

PAO (�g NO2�

N g�1 day�1)

Moderately eutrophic 11.4 � 0.35 A 1.82 � 0.18 A 24.2 � 0.24 A 4.20 � 0.59 A 7.69 � 0.18 B 400 � 37 B 91.0 � 5.12 B 5.40 � 0.31 ASlightly eutrophic 7.04 � 0.09 B 1.31 � 0.25 B 13.9 � 1.13 B 3.37 � 0.32 B 7.95 � 0.10 A 450 � 30 A 108 � 8.90 A 2.64 � 0.40 BaData are means � standard errors (n � 3). Different letters within a column indicate site differences (t test, P � 0.05).

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Two-way analysis of variance (ANOVA) showed that the net nitrification rates in the twosediments were affected by N input (P � 0.01).

Abundance of AOA and AOB. Copy numbers of archaeal and bacterial amoA genesin sediments following incubation for 0 and 56 days, as determined by quantitative PCR(qPCR) (Fig. 3), showed significant growth of AOA at 20°C and 30°C in the two eutrophicsediments treated with N during SIP incubation (P � 0.05), except for the slightlyeutrophic sediment with NN treatment at 30°C. Bacterial growth was observed withmost treatments (P � 0.05), except for AOB in the slightly eutrophic sediment at 10°Cand 20°C. Rates of increases in the archaeal amoA gene levels were affected bytemperature (P � 0.01), and rates of increases in the bacterial amoA gene levels wereaffected by N input (P � 0.05) and temperature (P � 0.001).

13C-labeled active AOA and AOB in sediments. Isopycnic density gradient ultra-centrifugation was performed in cesium chloride (CsCl) gradients with DNA extracts toseparate [12C]DNA and [13C]DNA from DNA-SIP microcosms incubated with [12C]bicar-bonate and [13C]bicarbonate. The abundance of AOA and AOB amoA genes in allmicrocosms of treatments with buoyant densities between 1.69 and 1.74 g ml�1 with12C-labeled substrate peaked in fractions with buoyant densities of �1.72 g ml�1, andshifts to “heavy” fractions with buoyant densities of �1.72 g ml�1 indicated theassimilation of inorganic carbon by ammonia oxidizers (Fig. 4).

In the moderately eutrophic sediment, the greatest amounts of amoA gene se-quences in 13C-labeled heavy fractions tended to be AOB with both N treatments alongthe temperature gradient, indicating the assimilation of inorganic carbon by bacterialammonia oxidizers, while there were much lower levels of detection of 13C-labeled AOAamoA genes than AOB genes, except with the HN treatment and at 30°C (Fig. 4). In theslightly eutrophic sediment, 13C-labeled AOA and AOB were detected in the NN and HNtreatments at 20°C and 30°C after the 56-day incubation.

Phylogenetic analysis of ammonia oxidizers in heavy fractions. The tag se-quencing data for the 16S rRNA genes were used for determination of the community

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� N (c and d) in the sediment microcosmsof moderately eutrophic (Moderate-E) (a and c) and slightly eutrophic (Slight-E) (b and d) sedimentscultured under NN and HN inputs, at 10°C, 20°C, and 30°C, for 56 days. Data are means � standard errorsof six replicate microcosms (triplicate 12C and 13C microcosms at each temperature). Different lettersindicate temperature differences (Tukey test, P � 0.05) (n � 3).

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composition of ammonia oxidizers at 0 days and the active ammonia oxidizers in theheavy fractions after the 56-day incubation. After subsampling, we obtained an averageof 19,484 high-quality reads per sample, for about 392 bp in the V4-V5 regions of the16S rRNA genes. AOA and AOB were found to represent minor proportions of themicrobe communities, with �3.0% of sequences in the 16S rRNA gene pools found inthe two surface sediments, regardless of N treatment.

There were a total of 17 archaeon-related gene sequences in the slightly eutrophicsediment cultured with HN and NN treatments at 20°C and 30°C and the moderatelyeutrophic sediment with HN treatment at 30°C following incubation for 56 days and inthe two eutrophic sediments at 0 days. The number of PCR-amplified products in othergroups was insufficient for 16S rRNA gene tag sequencing, and the DNA fractions wereundetectable. The gene sequences clustered into 9 operational taxonomic units (OTUs)(97% nucleotide identity), mainly containing Nitrosopumilus and Nitrososphaera; theformer was associated with Nitrosopumilus maritimus SCM1 and “Candidatus Nitroso-tenuis cloacae” SAT1, and the latter was affiliated with “Candidatus Nitrosocosmicusexaquare” G61 and Nitrososphaera sp. strain JG1. Nitrosopumilus strains mainly affiliatedwith the Nitrosopumilus maritimus SCM1 lineage dominated in most treatments, exceptfor the NN-treated slightly eutrophic sediment at 30°C (Fig. 5; also see Fig. S1a). Therelative abundance of Nitrosopumilus strains in the slightly eutrophic sediment in-creased with N input and with a decrease in temperature, in contrast to the abundanceof Nitrososphaera strains.

We identified 58 sequences of AOB-related genes, which formed 21 OTUs (97%nucleotide identity) dominated by Nitrosomonas strains, including Nitrosomonadaceae-like, Nitrosomonas sp. strain Is79A3, Nitrosomonas europaea, and Nitrosomonas commu-nis lineages (Fig. 6; also see Fig. S1b). Dominance of the Nitrosomonadaceae-like lineageoccurred in the slightly eutrophic sediment before and after incubation, whereas AOBdominance in the moderately eutrophic sediment changed from Nitrosomonas sp.strain Is79A3 at 0 days to the Nitrosomonadaceae-like lineage after incubation for 56days in most groups (Fig. 6). Variations in the relative abundance of the active

FIG 3 Changes in archaeal (a and c) and bacterial (b and d) amoA gene copy numbers in the microcosmsof moderately eutrophic (Moderate-E) (a and b) and slightly eutrophic (Slight-E) (c and d) sedimentscultured under NN and HN inputs, at 10°C, 20°C, and 30°C, for 56 days. Data are means � standard errorsof six replicate microcosms (triplicate 12C and 13C microcosms at each temperature). Different lettersindicate temperature differences (Tukey test, P � 0.05) (n � 3).

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Nitrosomonadaceae-like lineage under the different nitrogen inputs included an in-crease in the slightly eutrophic sediment with the improvement of N input but adecrease in the moderately eutrophic sediment; in contrast, the relative abundance ofthe Nitrosomonadaceae-like lineage increased with temperature in both eutrophicsediments. Furthermore, most sequences of this lineage were closely related to thefamily Nitrosomonadaceae, whose relative proportions were higher in Meiliang Baysamples (�90% of the Nitrosomonadaceae-like lineage) than in central lake samples(�73%) (Fig. S1b). Nitrosomonas sp. strain Is79A3 dominated in the moderately eutro-phic sediment at 0 days and in the HN treatment at 10°C after 56 days; changes in itsrelative abundance with the N treatments differed between the two eutrophic sedi-ments, while the relative abundance always decreased with increasing temperature inthe sediments. Nitrosomonas communis (�7.0% of total AOB OTUs) was detected onlyin the moderately eutrophic sediment. Under short-term (8-week) input of N, therelative abundances of the Nitrosomonadaceae-like lineage and the Nitrosomonas sp.strain Is79A3 lineage were affected by temperature (P � 0.001 and P � 0.01, respec-tively), while that of the Nitrosomonas communis lineage was affected by N input(P � 0.001).

DISCUSSIONEffects of nitrogen input and temperature on nitrification activity. Measure-

ment of nitrification activity is of great importance for linking microbial communitymetabolism to the phylogeny of physiologically distinct microorganisms in complexfreshwater environments (28). Our results showed that N input and temperature alteredthe nitrification activities in two contrasting eutrophic sediments from Taihu Lake;however, interaction effects of these factors on nitrification activities were unclear. Wefound that nitrification rates increased with N input (Fig. 2; also see Table S1 in the

FIG 4 Relative abundance of archaeal and bacterial amoA genes recovered from each gradient fraction of DNA-SIP sediment microcosms from Meiliang Bay(Moderate-E) and the central lake (Slight-E) that were incubated under NN and HN inputs, at 10°C, 20°C, and 30°C, with [12C]NaHCO3 and [13C]NaHCO3 for 56days. The y axes indicate the relative proportions of amoA genes of the total quantity detected from a gradient set equal to 1.0.

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supplemental material), in contrast to previous studies that reported that nitrificationactivity decreases with high concentrations of ammonium, especially in acidic soils (28,29), because nitrite accumulates in acidic or neutral soil (30). Supporting our study ofslightly alkaline sediments (Fig. 1), high ammonium input is known to increase oxida-tion reaction substrates in soils with higher pH (31), which probably enhances theoxidation rate. We found that sediment nitrification rates did not change markedlywithin the temperature range of 10°C to 30°C. Other studies reported that the nitrifi-cation activities were related to temperature (20, 32), and Wu et al. showed a negativecorrelation between nitrate accumulation and culture temperature (4°C to 37°C) in

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FIG 5 Relative abundance of active AOA groups, based on archaeal 16S rRNA genes identified fromsediments collected from the central lake (light-E) and Meiliang Bay (middle-E), under NN and HN inputs,at 20°C and 30°C, following incubation for 0 and 56 days.

FIG 6 Relative abundance of active AOB groups, based on bacterial 16S rRNA genes identified from sediments collected from thecentral lake (Slight-E) and Meiliang Bay (Moderate-E), under NN (a) and HN (b) inputs, at 10°C, 20°C, and 30°C, following incubationfor 0 and 56 days. ND, no data.

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sediment from one of our study areas (33), while Stark and Thamdrup and Fleischerdemonstrated positive correlations between them within the range of 10°C to 30°C (19,32). The responses of nitrification activity to temperature in both sediments wereprobably attributable to the fact that temperature changes not only the activity ofnitrifiers but also possibly that of denitrifiers and anammox (anaerobic ammonia-oxidizing) bacteria that consume produced nitrate (19, 33), because their relatedsequences have been detected in Taihu Lake sediments (34) (Fig. S2 and S3).

Effects of nitrogen and temperature on ammonia oxidizers. Changes in patternsof archaeal and bacterial 16S rRNA genes were reflected in their phylogeny, which wasalways found to be consistent with amoA genes (35), highlighting the effects of N inputand temperature on the active ammonia-oxidizing communities. Autotrophic growth ofAOA and AOB under the contrasting experimental conditions was supported bybicarbonate assimilation, as shown by 13C labeling of the archaeal and bacterial amoAgene pools (Fig. 4).

Archaeal assimilation of 13C was observed mainly in the slightly eutrophic sedimentunder HN and NN inputs at 20°C and 30°C and in the moderately eutrophic sedimentunder HN input at 30°C. [13C]Bicarbonate assimilation by AOA tended to be detectedin the slightly eutrophic sediment, probably due to its lower ammonium concentration,as observed previously in North Sea water (36) and in a Scottish agricultural soil (7), andculture temperature, as detected previously in sediment from the same study area thatwas cultured at 37°C using the same 13C method (33). We found that the majority ofarchaeal 16S rRNA genes in the heavy fraction of CsCl gradients fell into the Nitros-opumilus cluster, which comprises the Nitrosopumilus maritimus SCM1 and “CandidatusNitrosotenuis cloacae” SAT1 lineages, and were mainly affiliated with the formerlineage. The two lineages were isolated from a marine environment and activatedsludge in wastewater treatment plants, respectively, and both have been demonstratedto be mesophilic (37, 38), which resulted in their main distribution under the temper-atures of 20°C and 30°C. The Nitrososphaera cluster, which involved mesophilic “Can-didatus Nitrosocosmicus exaquare” strain G61 from a municipal wastewater treatmentsystem (39) and moderately thermophilic Nitrososphaera sp. strain JG1 from agriculturalsoil (40), tended to dominate the slightly eutrophic sediment in the NN treatment at30°C, consistent with findings reported by Wu et al. (33), which demonstrated that thesediment from Taihu Lake cultured at �37°C was exclusively dominated by this AOAcluster under NN input.

Incubation of SIP microcosms showed that AOB were 13C labeled to a much greaterextent than AOA in sediments with higher levels of nutrient enrichment during activenitrification between 10°C and 30°C (Fig. 4). These results imply that bacteria domi-nated the ammonia oxidization process in the more eutrophicated sediment withrelatively high nitrogen loads, supporting previous studies of estuarine ecosystems withhigh nutrient loads (33, 41), soil microcosms (10, 42), and biofilm enrichments ofsimulated creek ecosystems (1) with large amounts of added ammonium. Our studyalso revealed that AOB were not adapted to low temperatures in the slightly eutrophicsediment, because the weakened activity of AOB in this sediment was probably a resultof the relatively lower level of ammonium than in the moderately eutrophic sedimentat Meiliang Bay.

Temperature affected the ammonia-oxidizing activity through its effect on commu-nity composition, as observed by Urakawa et al. (43). We found that AOB in thecontrasting eutrophic sediments were exclusively dominated by the Nitrosomonascluster following SIP culture at different nitrogen inputs and temperatures, asreported in previous studies (11, 33) but in contrast to the findings of Dai et al. (21),possibly due to differences in sampling season. Our study illustrated that thecompositions of communities of active AOB within the Nitrosomonas clusterchanged with N input and temperature; in most sediments, they were mainlyassociated with the Nitrosomonadaceae-like lineage, which represents a monophyleticphylogenetic group within the betaproteobacteria, all of whose cultivated representa-

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tives are lithoautotrophic ammonia oxidizers (44). The sequences affiliated withNitrosomonadaceae-like AOB had low similarity to the known species of AOB and werealso found to dominate in the organic-matter-rich wastewater (45). We found that therelative proportion of this AOB lineage increased with N input in the slightly eutrophicsediment, in contrast to effects in the moderately eutrophic sediment, probably as aresult of different AOB sequences in Nitrosomonadaceae-like lineages among thesediments, which were marked by the sequence with GenBank accession no. AM934971in Meiliang Bay and that with GenBank accession no. EU449746 in center of the lake(Fig. S1b), and their ecophysiological properties remain unclear. Temperature had amuch stronger effect on this lineage than ammonium availability, because the abun-dance of this lineage was positively affected by temperature in the two eutrophicsediments; this finding supported previous work, especially in wastewater bioreactors,in which the Nitrosomonadaceae-like lineage was dominant (18, 46). The Nitrosomonassp. strain Is79A3 lineage was the second most dominant and is globally distributed infreshwater habitats (47, 48), mainly in low-temperature environments. This lineageenrichment occurs in microcosms at low temperatures (4°C and 15°C) in freshwaterenvironments (33), and Nitrosomonas oligotropha-like AOB, mostly related to the Nitro-somonas sp. strain Is79A3 lineage (97% sequence identity), predominate in cold fresh-water habitats in Europe (49, 50); however, their ecophysiological traits remain to bedefined. The active Nitrosomonas communis-like AOB were mainly detected in themoderately eutrophic sediment with the HN treatment, where abundance was greatestat 20°C. This lineage belongs to the mesophilic aerobic betaproteobacteria, its growthis inhibited at higher temperatures (42°C), and it thrives in environments with highammonium concentrations (17, 51).

MATERIALS AND METHODSStudy sites and sample collection. Sampling sites were located in the Meiliang Bay (31°30=N,

120°12=36�E) (moderately eutrophic) and central lake (31°16=N,120°11=24�E) (slightly eutrophic) of TaihuLake in Wuxi, with water depths of 2.7 m and 3.1 m, respectively (Fig. 1). Sediment cores were collectedfrom nonduplicate plots at approximately 50-m intervals at each sampling station, using a handheldsediment sampler, at the end of November 2016. The sampling scheme was carried out as describedpreviously (52). Briefly, the polyvinyl chloride (PVC) tube was inserted vertically into the sediment; afterbeing plugged into the required sediment thickness, the sampler was slowly lifted and a completesediment-interface-water column could be obtained. The bottom of the sample column was sealed witha large rubber plug, and the interfacial water in the sampling tube was removed by siphoning. Thesediment column was pushed out of the PVC tube with the bracket rod, and a 2-cm surface sedimentsample was cut from the top of the sampling tube. The sliced sediments were preserved in cleanzipper-lock bags with nitrogen gas, and the contents of the three bags were pooled into a single sample.Overlying water was collected using a water sampler and was filtered through a 0.2-�m microfiltrationmembrane (Whatman GmbH, Dassel, Germany) prior to chemical analysis within 24 h after collection. TheChl a content and SD of the overlying water were measured in situ using a 6600 V2 multisensor sonde(Yellow Springs Instruments, Yellow Springs, OH, USA). The sliced and unsliced sediments and watersamples were then transported to the laboratory on ice. The unsliced sediment cores were divided intothree parts; one was air dried and used for physicochemical analysis, the second was processed forDNA-SIP analysis, and the third was stored at �80°C for molecular analysis.

Physicochemical analysis and determination of potential ammonia oxidization. Sediment TOClevels were determined using the potassium dichromate oxidation-reduction titration method, and TNlevels were determined using the Kjeldahl method (53). Sediment pH was measured with a water/soilratio of 2.5. Ammonium and nitrate were extracted from the sediment using 2.0 M KCl, and theirconcentrations were measured using a Skalar SAN�� continuous-flow analyzer (Skalar Inc., Breda, theNetherlands). DO levels and Eh were determined from unsliced sediment samples using needle-typeOX-100-16146 and RD-N-9251 microelectrodes, respectively (Unisense FetiliTech, Aarhus, Denmark).Sediment PAO was measured using the nitrite oxidation inhibition method, as described by Wu et al. (2),within 36 h after sampling. TN and TP levels and CODMn of water samples measured by spectropho-tometry, as described previously (54), together with Chl a levels and SD, were used to determine levelsof eutrophication at the two sampling areas. Physicochemical properties of the sediments are listed inTable 1.

DNA-SIP microcosms. We constructed DNA-SIP microcosms of the sediments following the methodused by Wang et al. (28). All SIP incubations were performed in triplicate, including the controlincubations with unlabeled substrate. Sediment equivalent to 5.0 g of dry weight of sediment was addedto sterilized 120-ml serum bottles, which were then sealed and preincubated at 28°C in the dark. Theconcentration of CO2 in the headspace of the bottles was measured weekly, using an Agilent 7890B GCsystem (Agilent Technologies, Palo Alto, CA, USA), until CO2 production was �0.5% (5,000 ppm [vol/vol]).

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Following preincubation, 5.0 ml of synthetic lake water (0.58 mM CaCl2, 0.45 mM MgSO4 [pH 8.0]) wasadded to the bottles to simulate field conditions (55). The headspace of each bottle was flushed with 80%N2/20% O2 for 2 min to replenish O2, and then [12C]bicarbonate or [13C]bicarbonate (Isotec, Sigma-Aldrich, St. Louis, MO, USA) was supplemented and refreshed weekly to a final concentration in theoverlying water of 1 mg ml�1.

Effects of ammonium levels and temperature on active ammonia oxidizers were assessed by adding1.43 and 2.86 mM ammonium chloride weekly to the slightly and moderately eutrophic sediments,respectively, as HN input, which represented the potential N requirements based on their PAO; micro-cosms were treated weekly with 1 mM N to reflect the NN input rate at Lake Taihu (56). SIP incubationsof sediment samples, which were at an average temperature of 14.1°C when collected from twoeutrophic areas in November, were performed in the dark at 10°C, 20°C, and 30°C to reflect within-yearvariations in water temperatures at the lake. The pH of the overlying water was monitored and remainedstable during the incubation. Destructive sampling following preincubation and incubation was per-formed in triplicate, following the 8-week incubation, with 20-g sediment samples that were storedimmediately at �80°C prior to molecular analysis; the rest was used to determine inorganic nitrogencontent.

Nucleic acid extraction and SIP DNA fractionation. DNA was extracted from 0.5 g of each sedimentsample using a FastDNA Spin kit for soil (MP Biomedicals, Cleveland, OH, USA). The concentration andpurity of extracted DNA were measured using a Nanodrop ND-2000 UV-visible spectrophotometer(Thermo Fisher, Wilmington, DE, USA).

Isopycnic centrifugation of total DNA along CsCl gradients was performed as described previously(28), with 3.0 �g of extracted sediment DNA being mixed with CsCl solution and GB buffer (0.1 M Tris-HCl[pH 0.8], 0.1 M KCl, 1.0 mM EDTA) to achieve an initial buoyant density of 1.725 g ml�1. The ultracen-trifugation of the mixed solution was performed in 5.1-ml OptiSeal polyallomer tubes using an NTV-100vertical rotor (Beckman Coulter, Palo Alto, CA, USA), at 55,000 g for 44 h at 20°C. A total of 15 layersof equal DNA volumes (312.5 �l) were fractionated from centrifuged gradients, by displacement withsterile water using an NE-1000 single-syringe pump (New Era Pump Systems Inc, Farmingdale, NY, USA);the buoyant density of each fraction was determined according to the refractive index, using an AR200digital handheld refractometer (Reichert Technologies, Buffalo, NY, USA). Fractionated DNA was purifiedusing polyethylene glycol (PEG) 6000 precipitation (9) and was dissolved in 30 �l of DES buffer (pH 8.0;MP Biomedicals, Cleveland, OH).

amoA gene abundance. We used real-time qPCR to quantify the abundance of archaeal andbacterial amoA genes from the fractionated DNA across the entire buoyant density gradient, to estimate13C-labeled ammonia oxidizers in the DNA-SIP microcosms, using a CFX96 real-time PCR system (Bio-RadLaboratories. Inc., Hercules, CA, USA); there were three replicate analyses per sample. We used primerpairs Arch-amoAF/Arch-amoAR (57) and amoA1F/amoA2R (58) for archaeal and bacterial amoA genes,respectively. The reaction was performed in a 20-�l mixture, containing 10 �l of SYBR Premix Ex Taq(TaKaRa, Dalian, China), 0.5 �M each primer, and 1 �l of DNA template, at 95°C for 3 min, followed by 40cycles of 95°C for 10 s, 55°C for 30 s, and 72°C for 30 s; plate reads were at 83°C. Melting curve andagarose gel electrophoresis analyses confirmed the specificity of amplification products. The qPCRstandard was generated using plasmid DNA from representative clones containing the archaeal andbacterial amoA genes (29), and we used a serial dilution of the standard templates across 7 orders ofmagnitude (7.16 102 to 7.16 109 for archaeal amoA genes and 8.60 102 to 8.60 109 for bacterialamoA genes) for each assay. Amplification efficiencies of 98.8 to 100.6% were obtained with R2 values of0.998 to 1.000, and data were analyzed using Bio-Rad CFX Manager (version 1.6).

Tag sequencing and phylogenetic analysis. Archaeal and bacterial 16S rRNA genes in the heavyfraction of CsCl gradients obtained from ultracentrifugation were amplified with the primer pair515F/907R, and the real-time PCR conditions were 94°C for 3.0 min, 30 cycles of 95°C for 30 s, 55°C for30 s, and 72°C for 30 s, and 72°C for 6 min, on an ABI9700 thermocycler (Applied Biosystems, Carlsbad,CA, USA) using TransStart FastPfu DNA polymerase (TransGen, Beijing, China). The products of triplicatePCR amplicons were pooled and gel purified, and tag sequencing was performed on an Illumina MiSeqPE300 system (Illumina Corp., San Diego, CA, USA) by analyzing the V4-V5 regions of 16S rRNA genes. Weidentified and removed chimeras using the Chimera-uchime command in mothur (59). The averageneighbor algorithm was used to cluster sequences into OTUs, and representative sequences from eachOTU, as defined by 97% sequence identity, were obtained to search the NCBI database with BLAST toobtain the closest published sequences. Neighbor-joining phylogenetic trees were constructed with theJukes-Cantor correction in MEGA 4.0 (60).

Statistical analysis. Independent-sample t tests were performed to test for differences in physico-chemical properties between two eutrophic sediments, and two-way ANOVA with the Tukey multiple-comparison method was used to test for effects of nitrogen input and temperature. Data were analyzedusing SPSS 18.0, with a P value of �0.05.

Data availability. Nucleotide sequences have been deposited in the Sequence Read Archive, withaccession no. SRP176634 for the 16S rRNA genes derived from the DNA-SIP experiment.

SUPPLEMENTAL MATERIALSupplemental material for this article may be found at https://doi.org/10.1128/AEM

.00258-19.SUPPLEMENTAL FILE 1, PDF file, 0.8 MB.

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ACKNOWLEDGMENTSWe thank Mengyuan Zhu and the staff at the Taihu Laboratory for Lake Ecosystem

Research for help with sediment sampling, as well as the technical innovation group ofChangzhou Vocational Institute of Engineering.

This work was supported by the National Natural Science Foundation of China(grants 41771286, 41271255, and 41401293).

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