inﬂuence of efﬂuent irrigation on community composition ...soil (hamra) which was obtained from...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/01/$04.0010 DOI: 10.1128/AEM.67.8.3426–3433.2001

Aug. 2001, p. 3426–3433 Vol. 67, No. 8

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Influence of Effluent Irrigation on Community Composition andFunction of Ammonia-Oxidizing Bacteria in Soil

TAMAR OVED,1,2 AVI SHAVIV,2 TAL GOLDRATH,2 RAPHI T. MANDELBAUM,1

AND DROR MINZ1*

Institute of Soil, Water and Environmental Sciences, Agricultural Research Organization, The VolcaniResearch Center, Bet-Dagan 50-250,1 and Water-Soil-Environment, Agricultural

Engineering, Technion Institute, Haifa 32-000,2 Israel

Received 8 January 2001/Accepted 30 May 2001

The effect of effluent irrigation on community composition and function of ammonia-oxidizing bacteria(AOB) in soil was evaluated, using techniques of molecular biology and analytical soil chemistry. Analyses wereconducted on soil sampled from lysimeters and from a grapefruit orchard which had been irrigated withwastewater effluent or fertilizer-amended water (FAW). Specifically, comparisons of AOB community compo-sition were conducted using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified fragments ofthe gene encoding the a-subunit of the ammonia monooxygenase gene (amoA) recovered from soil samples andsubsequent sequencing of relevant bands. A significant and consistent shift in the population composition ofAOB was detected in soil irrigated with effluent. This shift was absent in soils irrigated with FAW, despite thefact that the ammonium concentration in the FAW was similar. At the end of the irrigation period, Nitrosospira-like populations were dominant in soils irrigated with FAW, while Nitrosomonas-like populations were domi-nant in effluent-irrigated soils. Furthermore, DGGE analysis of the amoA gene proved to be a powerful tool inevaluating the soil AOB community population and population shifts therein.

Agricultural irrigation with wastewater effluent is a commonpractice in arid and semiarid regions, and it is used as a readilyavailable and inexpensive option to fresh water. However, ir-rigation with effluent has possible public health and environ-mental side effects, as effluents may contain pathogens andhigh levels of sodium, dissolved organic carbon, detergents,and toxic metals (22). Furthermore, the addition of such a“mixed bag” of compounds may cause significant shifts instructure and function of a microbial community, which in turnmay influence the viability of the soil for agriculture. Oneimportant group of organisms that may be affected is thechemolithotrophic ammonia-oxidizing bacteria (AOB). TheAOB, which are responsible for the first, rate-limiting step innitrification in which ammonia (NH3) is transformed to nitrate(NO3

2) via nitrite (NO22), play a critical role in natural ni-

trogen cycling (9, 21).The AOB have previously been demonstrated to be affected

by a variety of chemical conditions including, but not limited tolevels of ammonium, organic matter, O2, toxic metals, salt, andpH (6, 11, 17, 21, 24, 29–31). Traditional biological methods tostudy the AOB are extremely time-consuming and generallyunrepresentative, as the AOB exhibit slow growth rates, lowbiomass yields, and a limited number of distinguishing pheno-typic characteristics (26).

To address these concerns, molecular methods may be im-plemented. Application of molecular techniques targeting the16S ribosomal DNA (rDNA) of the AOB has shown that thisgroup is comprised of two monophyletic lineages within the

class Proteobacteria. These lineages include the genus Nitroso-coccus in the g subgroup and the two genera Nitrosospira(containing the former genera Nitrosovibrio and Nitrosolobus)and Nitrosomonas in the b subgroup (7). One major drawbackof using the 16S rRNA gene as a molecular marker for culti-vation-independent study of AOB in environmental samples isthat PCR primers targeting the 16S rDNA gene may cross-react with members of other phylogenetic and physiologicalgroups (23, 28, 32). Furthermore, 16S rDNA sequence simi-larity among different AOB is so high that only limited phylo-genetic information can be obtained using this gene as a mo-lecular marker, and none of the published PCR primers andprobes for detection of these organisms in the environmentshow both 100% sensitivity and 100% specificity (1, 27). Alter-natively, the functional gene encoding the a-subunit ammoniamonooxygenase (amoA), found only in AOB, has been shownto be useful as a specific molecular marker in environmentalstudies of AOB (28, 30). The successful use of a highly specificset of PCR primers to amplify a fragment of amoA from avariety of pure cultures of b-subgroup AOB and environmen-tal samples was previously demonstrated (8, 14, 27, 28, 30). Inaddition, Purkhold et al. (27) have demonstrated that the phy-logeny of the amoA gene corresponds with the phylogeny ofthe 16S rDNA of these bacteria. Denaturing gradient gel elec-trophoresis (DGGE) is a powerful tool for analyzing microbialcommunities and has been used to separate mixed PCR prod-ucts (13, 18, 20, 35, 36), including those of AOB 16S rDNAfragments (5, 12, 14–16, 31). However, DGGE analysis of 16SrDNA fragments amplified with AOB-specific primers did notadequately resolve environmental populations of AOB (5, 12,14–16, 31).

In this study we examined the impact of soil irrigation witheffluent on the community composition of autotrophic AOB.Total DNA was extracted from lysimeter and orchard soils

* Corresponding author. Mailing address: Institute of Soil, Waterand Environmental Sciences, Agricultural Research Organization(ARO), The Volcani Research Center, P.O. Box 06, Bet-Dagan 50-250, Israel. Phone: 972-3-9683316. Fax: 972-3-9604017. E-mail: [email protected].

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irrigated with effluent or fertilizer-amended water (FAW) andfrom the effluents used for irrigation. DGGE analyses of PCR-amplified amoA gene fragments from the respective soils wereconducted in tandem with chemical analyses.

MATERIALS AND METHODS

Soil, irrigation, and experimental setup. The studies were conducted in twotypes of experimental systems, lysimeters and orchard soil. The lysimeters con-sisted of asbestos containers that were 120 cm high with a surface area of 0.5 m2,yielding a total soil volume of 600 liters. The lysimeters contained a sandy loamsoil (Hamra) which was obtained from the coastal plain in Israel. The chemicaland physical properties of the soils are summarized in Table 1. The lysimetersreceived one of two water treatments: drip irrigation with secondary effluent oftreated urban sewage from the Shomrat reservoir located near Ackre in Israel, ordrip irrigation with FAW amended with nitrogen (in the form of ammoniumsulfate), phosphorus (monopotassium phosphate), and potassium (potassiumchloride and monopotassium phosphate) at concentrations similar to those in theeffluent. The ammonium concentration in the FAW was designed to be similar tothe sum of ammonium and organic nitrogen concentrations in the effluent. Theproperties of the irrigation waters are summarized in Table 2. Each treatmentwas conducted in triplicate in separate lysimeters, each growing maize. Twolysimeters with no plants, one irrigated with FAW and the other with effluent,were also maintained. Irrigation of the lysimeters began in July 1998, and soil wassampled for AOB analysis during the second season of maize growth, whichbegan in June 1999. The lysimeters were sampled on days 25, 55, and 82 aftermaize seeding during the second growth season (referred to as the first, second,and third samplings, respectively) from different horizontal holes located at twosampling depths, 15 and 65 cm below the upper rim (three holes at each depth).At each sampling date, approximately 100 g of soil was removed per samplingdepth. Molecular analysis was performed on the samples obtained on the lastsampling date.

Orchard sampling. In addition to soil sampling from lysimeters, soil was alsosampled (at a depth of 15 cm) from a sandy-loam grapefruit orchard located inRamat-Hakovesh in northern Israel. Three samples of FAW-irrigated orchardsoil and three samples of effluent-irrigated soil were taken for molecular analysison January 2000, at the end of a 9-month irrigation period of four irrigations per

week. The physical and chemical characteristics of the orchard soil were similarto those of the lysimeters (data not shown). The grapefruit orchard was firstplanted in 1980, and since 1995 the plots have been irrigated either with effluentor FAW with nitrogen, phosphorus, and potassium content similar to that of theeffluent. The ammonium concentration in the FAW was designed to be similar tothe sum of ammonium and organic nitrogen concentrations in effluent. Theproperties of the irrigation waters are summarized in Table 2.

Nitrogen compounds in waters and lysimeters soils. Ammonium and nitrateconcentrations were determined in the effluent, in the FAW, and in 1 N KClextracts of soil with a Lachat System IV flow injection autoanalyzer (Lachat,Milwaukee, Wis.). The sum of organic nitrogen and ammonium concentrationsin effluents used to irrigate the lysimeters was measured using a rapid wetdigestion method for N and P, as described previously (33). Total nitrogenconcentrations in effluents used to irrigate the orchard were measured using theKjeldahl method (4).

BOD. Biological oxygen demand (BOD) was measured using a HACH BODapparatus (model 2173B; HACH Company, Loveland, Colo.) in accordance withthe manufacturer’s instructions.

pH and EC in soil. pH and electroconductivity (EC) in soil were measured inwater extracts of a 1:2 soil-to-water ratio. pH was measured with an El-Hama pHmeter (El-Hama Instruments, Rosh Pina, Israel). EC was measured with an El-Hama conductometer TH 27.

DNA extraction and purification from soil and effluent. DNA was extractedeither from 0.4 g of soil or from a pellet of a 500-ml effluent sample centrifugedat 4,000 3 g for 5 min. DNA was extracted using a FastPrep machine (Bio 101,Holbrook, N.Y.) with the FastSoil DNA purification kit (Bio 101, Vista, Calif.)in accordance with the manufacturer’s instructions.

PCR and DGGE analysis of amoA fragments. Fragments (491 bp) of the amoAgene were enzymatically amplified using a 1650 Air Thermo-Cycler (Idaho Tech-nology, Idaho Falls, Idaho) with the primers amoA-1F and amoA-2R (28). A GCclamp (59-CCGCCGCGCGGCGGGCGGGGCGGGGGCACGGGG-39) wasattached to the forward primer in order to increase DGGE gel separation (18).Reactions were performed in accordance with the manufacturer’s recommenda-tions in a total volume of 50 ml by using 40 mM MgCl2 reaction buffer (IdahoTechnology), 1.5 U of Taq DNA polymerase (RedTaq; Sigma, St. Louis, Mo.),1 ml of template DNA, 50 pmol of each primer, 20 nmol of each deoxynucleosidetriphosphate, and 12.5 mg of bovine serum albumin. Thermal cycling was carriedout with an initial denaturation step of 94°C for 30 s followed by 35 cycles ofdenaturation at 94°C for 15 s, annealing at 55°C for 20 s, and elongation at 72°Cfor 40 s; cycling was completed by a final elongation step of 72°C for 2 min. Thepresence and size of the amplification products were determined by agarose(2%) gel electrophoresis of the reaction product and ethidium bromide staining.

DGGE analysis was performed with a D-Gene system (Bio-Rad, Hercules,Calif.) using the following ingredients and conditions: 13 TAE (40 mM Tris, 20mM acetic acid, 1 mM EDTA [pH 8.3]); 1-mm thick gels; a denaturant gradientof 20 to 50% urea-formamide at 60°C (19); 250 V for 3 h. Ethidium bromide-stained DGGE gels were photographed on a UV transillumination table (302nm) with a Kodak digital camera, and inverse images were prepared with Pho-toshop version 4.0 (Adobe). Alternatively, gels were photographed using a Po-laroid camera, and pictures were scanned and prepared with Photoshop.

Excision and reamplification of DGGE bands. DGGE bands were carefullyexcised on an UV transilluminator with a scalpel and subsequently transferred toa 1.5-ml tube containing 500 ml of TE and 500 mg of glass beads (0.75- to 1.0-mmdiameter). The acrylamide was disrupted by bead beating (FastPrep, Bio101) atmaximum speed for 1 min. The samples were incubated for 30 min at 37°C, and

TABLE 1. Properties of the soil used in the lysimeter experiment

Component or property Value

Clay (%a) .......................................................................................... 10Silt (%a) ............................................................................................ 6.9Sand (%a) ......................................................................................... 83.1Organic matter (%a) ....................................................................... 0.3Calcium carbonate (%a) ................................................................. 2.1CEC (meq/100 g of soil)b ............................................................... 3EC (dS m21)c ................................................................................... 0.12pHc ..................................................................................................... 6.6

a Percent composition (wt/wt).b CEC, cation exchange capacity.c Measured in aqueous extract with 1:2 soil:water ratio.

TABLE 2. Properties of irrigation water used in the experimentsa

Source EC(dS m21) pH N-NH4

(mg liter21)N-NO3 (mg

liter21)Total nitrogen(mg liter21)b

Phosphate(mg liter21)

Total P(mg liter21)

Potassium(mg liter21)

BOD(mg liter21)

LysimeterEffluent 1.4 6 0.1 7.6 6 0.4 18.8 6 8.6 2.8 6 5.0 28.8 6 12.0 6.2 6 2.2 11.1 6 4.4 29.8 6 0.5 44.6 6 22.4FAW 1.1 6 0.2 7.6 6 0.4 31.0 5.5 6 3.3 36.5 6 3.3 6.0 6.0 30.0 0

OrchardEffluent 1.7 6 0.1 7.6 6 0.2 30.8 6 5.2 0.28 6 0.7 32.2 6 5.0 6.2 6 2.8 NDc 32.1 NDc

FAW 1.1 6 0.1 7.4 6 0.2 9.8 6 3.6 23.8 6 5.2 33.6 6 8.7 4.65 6 1.6 4.65 20.0 0

a Average or mean (6 standard deviation) values for 1999.b Calculated as the sum of the nitrate concentration (determined with a Lachat System IV flow injection autoanalyzer) and ammonium and organic nitrogen

concentrations (measured using the rapid wet digestion method for N and P [33]).c ND, not determined.

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1 ml of the supernatant was subsequently used for reamplification of the DNAfragment. A second DGGE analysis was performed to confirm that only oneband was reamplified and that it had the same position in the gel as the excisedband.

Cloning of PCR products. The pGEM-T Easy Vector System (Promega, Mad-ison, Wis.) was used for cloning of excised and reamplified DGGE bands.Ligation and transformation reactions were performed according to the manu-facturer’s instructions. Clones were screened by suspending colony material inthe PCR mixture and amplifying as described before. Plasmids from selectedcolonies were purified with the Wizard Plus Miniprep DNA Purification System(Promega) according to the manufacturer’s instructions.

Sequencing. Clones were sequenced using the Applied Biosystems PRISMDye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNApolymerase. The sequencing products were analyzed with an Applied Biosystems377 DNA sequencer.

Phylogenetic analyses. Nucleotide and deduced amoA amino acid sequenceswere analyzed using the program package ARB (http://www.biol.chemie.tu_munchen.de/Pub/ARB). Phylogenetic analyses were performed by applyingprotein parsimony (PHYLIP 3.55), neighbor-joining (using the PAM correc-tion), and maximum likelihood programs implemented in the ARB softwarepackage. The topologies of the resulting trees were compared. Branching orderwas supported by all methods. Primer sequences were not included in the phy-logenetic analyses.

Nucleotide sequence accession numbers. The sequences discussed in this studyare available from EMBL under accession numbers AF353246 to AF353264.

RESULTS

Ammonium input. Ammonium concentration in the FAWused for irrigation was 31.0 mg liter21 N-NH4

1 (Table 2).Assuming a fast mineralization rate of organic nitrogen toammonium in the effluents, the average ammonium concen-tration in the effluents used for irrigation was 26.0 mg liter21

N-NH41 (Table 2) (2).

Inorganic nitrogen compounds in soil. Average nitrate con-centrations in the soil samples obtained from the effluent- andFAW-irrigated lysimeters with plants were less than 1.5 mg/g ofsoil at both depths, on the first and second sampling dates (Fig.1). However, during the last sampling, nitrate concentrations inthe lysimeters increased at a depth of 15 cm in both the efflu-ent- and the FAW-irrigated soils, to 17 and 16 mg/g of soil,respectively. A similar trend was observed in the lysimeterswith no plants (Fig. 1). In these lysimeters, nitrate concentra-tions on the first and second sampling dates were similarly low(less than 5 mg/g of soil) at both depths. On the last samplingdate, nitrate concentrations at the depth of 15 cm increased to16 and 37 mg/g of soil in the effluent- and FAW-irrigatedlysimeters, respectively. At a depth of 65 cm, nitrate concen-trations remained low during the entire irrigation season, in allirrigation treatments.

The soil ammonium concentrations in the lysimeters withand without plants increased from a range of 6 to 10 mg/g ofsoil during the first sampling to a range of 13 to 21 mg/g of soilduring the second sampling, and then they decreased to arange of 0 to 10 mg/g of soil during the last sampling (Fig. 2).Ammonium concentrations in the lysimeters with plants at alldepths approached near-identical values in the final analysis.

In brief summary, the highest ammonium concentrations atthe depth of 15 cm were observed in all of the samples duringthe second sampling, and the highest nitrate concentrationswere observed during the last sampling.

pH and EC in soil. pH measurements of replicate lysimetersoils taken during the final sampling period indicated that theaverage pH values of the effluent- and FAW-irrigated lysim-

eters differed by no more than 0.1 pH units, being between 7.4and 7.6 (data not shown).

A similar trend was observed in the orchard soil. AveragepH values of the effluent-irrigated orchard soil were 0.1 pHunits higher than the pH values of the FAW-irrigated orchardsoil (data not shown).

EC measurements of replicate lysimeter soils taken duringthe last sampling period revealed that the EC of both effluent-and FAW-irrigated lysimeters with plants were 0.17 dS m21.Similarly, average EC values for orchard soil at the end of theirrigation period were 0.19 and 0.18 dS m21 in the effluent- andFAW-irrigated soils, respectively.

AOB community composition in lysimeter soil. Analysis ofthe AOB community composition by PCR-DGGE was per-formed on all lysimeters, with and without plants. Figure 3presents the DGGE banding pattern of amoA fragments am-plified from total DNA extracted during the third sampling,from all the lysimeters and depths studied, with the exceptionof one sample from a depth of 65 cm which amplified poorly.Examination of the DGGE showed that banding patterns ob-tained from identically treated lysimeters were similar andthat two main general distributions of dominant bands were

FIG. 1. Changes in nitrate concentrations (microgram per gram ofsoil) in soil of lysimeters with (A) and without (B) maize. Analysis wasconducted on FAW-irrigated soil at depths of 15 (■) and 65 (h) cmand on effluent-irrigated soils at depths of 15 (F) and 65 (E) cm.Values in panel A are mean of three independent measurements, andvalues in panel B are from one measurement.

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present. Dominant DGGE bands detected in the FAW-irri-gated soils (w2521, w627, and w2404) migrated to the lowerthird of the gel, while those detected in the effluent-irrigatedsoils migrated both to the lower (w2521 and w2404) and to theupper (w637, w614, w735, w716, w1203 and w537) third of thegel (Fig. 3). In only one of the FAW-irrigated lysimeters was aband detected in the upper third of the gel (w2406).

Ten bands were excised from the DGGE gel and sequenced.All excised fragments were confirmed as amoA by sequenceanalysis, with the exception of one fragment which was de-tected in the upper third of the gel in only one of the FAW-irrigated lysimeters (w2406). Sequence analysis also revealedthat the bands in the lower third of the DGGE gel (w2521,w627, and w2404) were phylogenetically most closely related toNitrosospira species, while the bands in the upper third of thegel (w637, w614, w735, w716, w1203, and w537) were phyloge-netically most closely related to Nitrosomonas species (Fig. 3and 4).

AOB community composition in orchard soils. Figure 5 pre-sents the DGGE banding pattern of amoA fragments amplifiedfrom orchard soil samples. Since banding patterns obtained

from similar irrigation treatments were similar, only one rep-resentative sample of each treatment is represented.

In FAW-irrigated orchard soil samples, a single DGGEband was detected in the lower third of the DGGE gel (w923),while in the effluent-irrigated soil bands were detected both inthe upper (w1710) and the lower (w923, w1009, and w2215)third (Fig. 5). Sequence analyses of these bands confirmedtheir phylogenetic affiliation with the sequences obtained fromthe lysimeter experiment (Fig. 4). A single Nitrosospira-likesequence was detected in the FAW-irrigated orchard (w923),while both Nitrosomonas-like (w1710) and Nitrosospira-like(w1009, w923, and w2215) sequences were found in the efflu-ent-irrigated orchard.

Six of the seven Nitrosomonas-like sequences obtained fromthe effluent-irrigated soils of the lysimeter and the orchardexperiment were grouped into one cluster with Nitrosomonasnitrosa. The only Nitrosomonas-like sequence which did notcluster within this group was w735, obtained from the 65-cmdepth of an effluent-irrigated lysimeter, which grouped withNitrosomonas europaea (Fig. 4). All seven sequences related toNitrosomonas obtained from the effluent-irrigated soils be-longed to the N. europaea-Nitrosococcus mobilis cluster (27)(Fig. 4). Three of the six Nitrosospira-like sequences obtainedfrom the lysimeters and orchard soils grouped into one clustertogether with Nitrosospira (formerly Nitrosolobus) multiformis.The other three bands (w627, w2521, and w923) clustered withthe soil isolate Nitrosospira sp. AHB1.

AOB community composition in the effluent. DGGE analy-sis of PCR products obtained from two samples of effluentsused to irrigate the lysimeters (taken from different dates)indicated that only one strong band in the middle third of thegel was detected for each effluent sample (w915 and w1101)(Fig. 5). Both sequences migrated differently from all of thebands in soil samples. The two bands, although migratingsimilarly on the DGGE gel, were determined to be differentorganisms, based on amoA sequence analysis. This analysisrevealed that both were closely related to Nitrosomonas oligo-tropha of the Nitrosomonas marina cluster and were distinctlydifferent from the Nitrosomonas-like sequences obtained fromthe effluent-irrigated soils (27) (Fig. 4).

DISCUSSION

New developments in the application of molecular methodsto the study of AOB have increased our ability to characterizemicrobial diversity and population shifts in environmental sys-tems. These developments include the design of a new set ofPCR primers that specifically amplify a fragment of amoA, afunctional molecular marker for the b-subgroup of AOB (27,28, 30), and also the use of DGGE to separate the PCR-amplified fragments on the basis of sequence composition. Inthis study, we present the first application of amoA DGGE tothe study of AOB in complex environmental systems. Thepredominant AOB populations identified in this study, Ni-trosospira-like species and Nitrosomonas-like species, werereadily separated by DGGE analyses. Specifically, all six ana-lyzed amoA bands which migrated to the lower third of theDGGE gel were determined to be phylogenetically related toNitrosospira species, while all amoA bands migrating to theupper third of the gel were phylogenetically associated with

FIG. 2. Changes in ammonium concentrations (microgram pergram of soil) in soil of lysimeters with (A) and without (B) maize.Analysis was conducted on FAW-irrigated soil at depths of 15 (■) and65 (h) cm and on effluent-irrigated soils at depths of 15 (F) and 65 (E)cm. Values in panel A are means of three independent measurements,and values in panel B are from one measurement.



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Nitrosomonas species. Furthermore, bands consisting of highlysimilar sequences (i.e., bands w614 and w716, differing by 2 bpand identical in amino acid sequence) were clearly separatedon the DGGE gel. The only band which migrated to the upperthird of the gel and was not phylogenetically associated withNitrosomonas species was determined not to be an amoA se-quence (w2406). It is important to note that the amoA primershave two degenerate nucleotide positions (28); thus, generally,one PCR-amplified amoA sequence might be represented intwo separate DGGE bands. However, no such case was ob-served in this research.

The findings of this study suggest that irrigation with waste-water effluent altered the predominant AOB population in thesoil, as compared to soil irrigated with FAW. Towards the endof the irrigation period, Nitrosospira-like sequences were dom-inant in FAW-irrigated soil (Fig. 3, 4, and 5) and Nitrosomonaspopulations were absent, while in the effluent-irrigated soilboth Nitrosospira-like and Nitrosomonas-like sequences werepresent (Fig. 3, 4, and 5). This community change manifesteditself in a consistent manner in an established orchard and inlysimeters with and without plants.

Three of the six Nitrosospira-like sequences obtained in thisstudy were phylogenetically related to N. multiformis (Fig. 4),which is known to be among the most common soil AOB (25).The other three bands clustered with a Nitrosospira speciesisolated from fertilized heath soil (Nitrosospira sp. AHB1) (3).Six of the seven Nitrosomonas-like sequences obtained fromthe effluent-irrigated soils in the lysimeter and orchard exper-iments clustered with N. nitrosa (Fig. 4), an AOB commonlyfound in eutrophic environments (10). The only Nitrosomonas-like sequence which did not cluster within this group was ob-tained from a depth of 65 cm of an effluent-irrigated lysimeter(w735) and grouped with N. europaea (Fig. 4). This finding mayreflect the different conditions at the 65-cm depth.

In addition to population shifts, we also noted that effluent-irrigated systems supported more complex communities ofAOB. In the FAW-irrigated systems, only a few Nitrosospira-like sequences were detected from the last sampling event (Fig.3, 4, and 5), while in the effluent-irrigated systems additionalNitrosospira-like and Nitrosomonas-like sequences were de-tected.

The Nitrosomonas-like sequences identified in the effluent-irrigated soil in this study were clearly different than the Ni-trosomonas-like sequences identified in the effluent used forirrigation. The sequences obtained from the effluents fromdifferent dates were closely related to each other and to N.oligotropha, which is commonly detected in industrial sewagedisposal systems (10), but they were divergent from the efflu-ent-irrigated soil clones (Fig. 4). Two possible mechanisms forthis scenario may be considered. First, the Nitrosomonas-likespecies present in the effluent-irrigated soil originated from thesoil and were selected for by the environmental conditions,which changed after irrigation with the effluent. The secondpossibility is that these species originated from the effluentitself but were either below the PCR-DGGE detection limit(approximately 1% of the target community [19]) or originatedfrom the effluent used for irrigation during the first year ofirrigation (this period was not analyzed). Currently, there is noinformation to resolve this issue. Regardless of the origin ofthe Nitrosomonas species, it is clear that the dominant Nitro-somonas species in the wastewater were unable to dominate inthe effluent-irrigated soil.

As was demonstrated, the addition of effluent to the soilsystems consistently favored the development of alternate Ni-trosomonas populations in these soil communities, in additionto the already-present Nitrosospira populations. Several factorsare considered in examining this shift in the microbial popu-lation from that found in the soil irrigated with FAW and that

FIG. 3. DGGE analysis of amoA fragments obtained from soils of lysimeters from the last sampling period. Samples were analyzed from depthsof 15 and 65 cm. Five samples were from FAW-irrigated soil from lysimeters with plants (FeS1P); six samples were from effluent-irrigated soilfrom lysimeters with plants (EfS1P); two samples were from FAW-irrigated soil from lysimeters without plants (FeS); and two samples were fromeffluent-irrigated soils from lysimeters without plants (EfS).



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from the original effluent. Previous work has suggested thatammonium concentration determines which AOB species willdominate an environmental niche (29). However, ammoniumconcentration does not appear to explain solely the communityshift in the systems examined in this study. Assuming a fastmineralization rate of organic nitrogen to ammonium in theeffluents (2), ammonium inputs in the FAW- and effluent-irrigated soils were similar (Table 2). One of the differences inthe constitution of the effluent and FAW was the BOD. Theeffluent water contained high organic levels (BOD, 44.6 mgliter21) compared to the FAW (BOD, 0 mg liter21). Previousstudies have indicated that the growth of Nitrosomonas popu-lations are encouraged in soil plots amended with swine ma-nure (6), and in other organic-rich environments, such as ac-tivated sludge and biofilm reactors, Nitrosomonas species arereadily detected (28, 34). It is possible that in effluent-irrigatedsoil, as in other organic-rich environments, environmental con-ditions favor Nitrosomonas species.

Additional determinants of the AOB population structure inenvironmental systems may be soil pH and salt concentration.Specifically, different Nitrosospira species have been shown todevelop in neutral and acid soils (31, 32). However, no signif-icant differences in pH and salt concentration were detected inthe different irrigation systems (data not shown).

Surprisingly, the salient shifts in microbial community be-tween the effluent- and FAW-irrigated soils seem not to havebeen accompanied by significant shifts in community function.

In the lysimeters with plants, measurements of nitrate andammonium were similar, particularly in the final sampling pe-riod. Significant nitrate development in the lysimeters with orwithout plants was seen only at the depth of 15 cm on the thirdsampling date (82 days), although it may have begun to accreteanytime after the second sampling period (55 days). Likewise,a decrease in the soil ammonium concentration was not seenuntil the third sampling period (Fig. 1 and 2). These resultsmay suggest higher nitrification rates only toward the end ofthe 3-month irrigation period and are consistent with the slowgrowth rates of AOB. The chemical analysis has also demon-strated that nitrate concentrations were significantly lower atthe depth of 65 cm. Several factors may result in this difference,including a smaller AOB population. In general, it should benoted, however, that both ammonium and nitrate concentra-tions may also be affected by other processes, such as denitri-fication and leaching.

Similar trends in population shifts were noted between ly-simeters with or without plants, suggesting that the plants werenot critical factors in the determination of AOB community.The observation is remarkable since plant roots are generallyknown to affect soil microflora (21). This study has demon-strated that DGGE of the amoA fragment is a powerful tool inevaluating AOB populations in natural samples. Such studiesmay provide not only insight into the distribution and diversityof AOB but may also help in assessing their response to chang-ing environmental conditions.

FIG. 4. Phylogenetic tree constructed for partial amoA gene sequences, based upon 151 deduced amino acid sites. The tree was produced byusing a neighbor-joining algorithm. The tree shows the relationship between cultured AOB and environmental sequences obtained fromeffluent-irrigated soil in lysimeters (EfLS) or orchard (EfOS), exclusively; effluent- or FAW-irrigated soil from lysimeters (LS) or orchard (OS);and effluents used for irrigation (EfW). The bar indicates an estimated 10% sequence divergence. The cluster nomenclature is that of Purkholdet al. (27).



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ACKNOWLEDGMENTS

We thank Larissa Kautsky and Ori Haras for technical assistance.We greatly appreciate the help of Stefan Green and Edouard Ju-rkevitch in reviewing the manuscript and discussion.

The study was supported by the Israeli Ministry of Science (grant8868) and by the Chief Scientist of the Israeli Ministry of Agricultureand Rural Development.

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inﬂuence of efﬂuent irrigation on community composition ...soil (hamra) which was obtained from...

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