Bacterial community and groundwater quality changes in an anaerobic aquifer during groundwater recharge with aerobic recycled water

R E S EA RCH AR T I C L E

Bacterial community and groundwater quality changes in ananaerobic aquifer during groundwater recharge with aerobicrecycled water

Maneesha P. Ginige1, Anna H. Kaksonen1, Christina Morris1, Mark Shackelton1 &Bradley M. Patterson1,2

1CSIRO Land and Water, Wembley, WA, Australia; and 2School of Chemistry and Biochemistry, University of Western Australia, Crawley, WA,

Australia

Correspondence: Bradley M. Patterson,

CSIRO Land and Water, Private Bag No. 5,

Wembley, WA 6913, Australia. Tel.:

+61 8 9333 6276; fax: +61 8 9333 6499;

e-mail: [email protected]

Received 4 January 2013; revised 12 April

2013; accepted 24 April 2013.

Final version published online 20 May 2013.

DOI: 10.1111/1574-6941.12137

Editor: Tillmann Lueders

Keywords

aquifer; bacterial diversity; groundwater

replenishment; managed aquifer recharge;

qPCR; 16S rRNA gene.

Abstract

Managed aquifer recharge offers the opportunity to manage groundwaterresources by storing water in aquifers when in surplus and thus increase theamount of groundwater available for abstraction during high demand. The WaterCorporation of Western Australia (WA) is undertaking a Groundwater Replen-ishment Trial to evaluate the effects of recharging aerobic recycled water (second-ary treated wastewater subjected to ultrafiltration, reverse osmosis, andultraviolet disinfection) into the anaerobic Leederville aquifer in Perth, WA.Using culture-independent methods, this study showed the presence of Actino-bacteria, Alphaproteobacteria, Bacilli, Betaproteobacteria, Cytophaga, Flavobacteria,Gammaproteobacteria, and Sphingobacteria, and a decrease in microbial diversitywith an increase in depth of aquifer. Assessment of physico-chemical and micro-biological properties of groundwater before and after recharge revealed thatrecharging the aquifer with aerobic recycled water resulted in elevated redoxpotentials in the aquifer and increased bacterial numbers, but reduced microbialdiversity. The increase in bacterial numbers and reduced microbial diversity ingroundwater could be a reflection of an increased denitrifier and sulfur-oxidizingpopulations in the aquifer, as a result of the increased availability of nitrate, oxy-gen, and residual organic matter. This is consistent with the geochemical datathat showed pyrite oxidation and denitrification within the aquifer after recycledwater recharge commenced.

Introduction

Water from dams, borefields, and desalination plantscollectively meets all of Perth’s (Western Australia) drinkingwater requirements. Due to climate change and populationincrease, the pressures on groundwater and desalinationplants will inevitably increase to maintain supply. Managedaquifer recharge (MAR) offers an opportunity to managethe groundwater resource by storing water in a suitableaquifer when in surplus (Pyne, 1995). Among a range ofwater sources, excess surface water (Bouwer, 2002), recycledwastewater (Greskowiak et al., 2005; Sheng, 2005), desali-nated seawater (Mukhopadhyay et al., 1998), and excesspotable water (Stuyfzand, 1998) have been recommendedsuitable for groundwater replenishment. The replenishment

is restrictive to aquifers that are physically or chemicallyconfined, and the approach has been recognized to furtherimprove overall recharge water quality (Pyne, 1995; Appeloet al., 1999). These beneficial aspects of groundwater replen-ishment have helped improve its social acceptance.

The Leederville formation is a major confined anaero-bic sedimentary aquifer in Perth. The aquifer, whichprovides 20% of Perth’s water supply, is proposed forgroundwater replenishment operations using highly trea-ted recycled water. Descourvieres et al. (2010) investi-gated consequences of potable water storage and recoveryfrom the Leederville aquifer. According to the study,dominant oxidation reactions, buffering, and weatheringprocesses are factors likely to determine water qualitywhen recovering injected water from the aquifer. One of

FEMS Microbiol Ecol 85 (2013) 553–567 ª 2013 Federation of European Microbiological SocietiesPublished by John Wiley & Sons Ltd. All rights reserved

MIC

ROBI

OLO

GY

ECO

LOG

Y

https://www.researchgate.net/publication/226890684_Artificial_Recharge_of_Groundwater_Hydrogeology_and_Engineering?el=1_x_8&enrichId=rgreq-412ba95c-a08d-4811-a903-04b695f16171&enrichSource=Y292ZXJQYWdlOzIzNjQ1NzA2ODtBUzo5OTAwNDg5NzU2MjY0MEAxNDAwNjE2MDE1Njg2

https://www.researchgate.net/publication/7882765_An_Aquifer_Storage_and_Recovery_System_with_Reclaimed_Wastewater_to_Preserve_Native_Groundwater_Resources_in_El_Paso_Texas?el=1_x_8&enrichId=rgreq-412ba95c-a08d-4811-a903-04b695f16171&enrichSource=Y292ZXJQYWdlOzIzNjQ1NzA2ODtBUzo5OTAwNDg5NzU2MjY0MEAxNDAwNjE2MDE1Njg2

https://www.researchgate.net/publication/248568581_Laboratory_investigations_of_compatibility_of_the_Dammam_Formation_Aquifer_with_desalinated_freshwater_at_a_pilot_recharge_site_in_Kuwait?el=1_x_8&enrichId=rgreq-412ba95c-a08d-4811-a903-04b695f16171&enrichSource=Y292ZXJQYWdlOzIzNjQ1NzA2ODtBUzo5OTAwNDg5NzU2MjY0MEAxNDAwNjE2MDE1Njg2

the anticipated geochemical processes to take place in theLeederville aquifer is pyrite oxidation, resulting in SO2!

4

release and potential acidification. Although major ionsand trace metals, especially Zn, Ni, and Co, wereobserved to leach from sediments due to acidification inthe respirometer incubations, high concentrations werenot predicted in recovered water during field-scale opera-tions. The sedimentary organic matter (SOM) on theother hand far exceeded pyrite abundance in aquifer sedi-ment. However, the authors found pyrite to be the majorO2 reductant.

The ability of subsurface environments to support adiverse microbial community similar to that of surfaceenvironments has been well demonstrated in literature(Fredrickson et al., 1989; Musslewhite et al., 2003; Takeu-chi et al., 2009; Li et al., 2012). Although studies evaluatingthe potential of subsurface microorganisms to bioremedi-ate contaminated aquifers have outnumbered studies thatfocused on microbial ecology of subsurface environments,these studies together demonstrate that pristine subsurfaceenvironments are rich in metabolically diverse microbialcommunities. The number of microbial ecology studies ofreducing aquifer sediments in particular is low becauseremediation studies largely focused on aerobic biodegrada-tion processes (Martino et al., 1998). Hence, groundwaterrecharge operation and design for reduced aquifers such asthe Leederville aquifer is a challenge with reliance to pastliterature alone.

Subsurface microbial communities and their potentialimpact on aquifer geochemistry and groundwater qualityare required to assist in assessing the feasibility ofgroundwater replenishment of the Leederville aquiferusing recycled water. The detailed geochemical character-ization (Descourvieres et al., 2010) of the aquifer hasfacilitated a conceptual understanding of the key geo-chemical processes that will take place with MAR opera-tions and is of value to reliably predict eminentmicrobiological processes. The importance of having agood knowledge of concentrations and species of organiccarbon, different types and concentrations of electron ac-ceptors or other nutrients, hydraulic conductivity, texture,porosity, surface area, and mineralogy of the aquifer hasbeen highlighted as important to develop a holisticunderstanding of subsurface microbial ecology (Pedersen,1993; Bachofen et al., 1998).

Subsurface microbial ecology investigations thus farhave confirmed the basic principle ‘microbes are every-where, the environment selects’ and concurrentlydescribed microbial diversity and distribution of manysubsurface environments (Chandler et al., 1997a, b; Ben-nett et al., 2001; Musslewhite et al., 2003; Takeuchi et al.,2009; Li et al., 2012). Microbial diversity and distributionhave largely been described in the past using

culture-based methods, and with the realization that cul-ture-based approaches only facilitate enumerating 0.0001–0.1% of bacteria from terrestrial subsurface environments(Brockman et al., 1993), much emphasis has gone towardthe use of culture-independent techniques to describemicrobial ecology in subsurface environments (Chandleret al., 1997a, b; Takeuchi et al., 2009). However, the lowbacterial abundance in these environments (ranging from< 104 to 108 cells g!1 (Fredrickson et al., 1989; Chandleret al., 1997a; Martino et al., 1998)) continues to pose achallenge to obtain an accurate reflection of the phyloge-netic identity and diversity. Obtaining a representativesample for microbiological analysis from an aquifer isalso challenging because of sample contamination by dril-ling muds and overlying sediments (Fredrickson et al.,1989). Also, groundwater samples alone may not repre-sent subsurface microbial communities because 95% ofthe bacterial population is found attached to particlesurfaces (Harvey et al., 1984). Aseptically sampling thecenter of an intact core has been suggested effective togain access to a least contaminated subsurface sample formicrobiological analysis (West et al., 1985).

In this study, we obtained drill core samples throughdiamond core drilling from the Leederville formation.Using a culture-independent approach, we showed thepresence of chemolithoautotrophic and heterotrophicbacteria, their relation to sediment properties, and theirdistribution, in a profile of intact core samples. Throughcontinuous assessment of physico-chemical and microbio-logical properties of groundwater, we also reveal possibleimplications of groundwater replenishment on the Leed-erville aquifer.

Site description

The Groundwater Replenishment Trial site (Fig. 1) waslocated in Perth, Western Australia (Water Corporation,2009). The site consisted of one recharge bore and 22monitoring bores. The monitoring bores were clustered at20, 60, 120, 180 and 240 m from the recharge bore. Therecharge bore was screened between 120 and 220 mbelow ground level (mbgl), within the permeable zone ofthe confined Leederville aquifer. The sediment in thiszone consisted of discontinuous interbedded sands, silts,and clays (Playford et al., 1976). The mineralogy of thesediment was predominantly quartz (72%), K-feldspar(24%), and minor quantities of pyrite (2%) and Na-feld-spar (2%) (based on X-ray diffraction), with 0.32% ofSOM (Patterson et al., 2010). Based on incubation experi-ments by Descourvieres et al. (2010), the sediment washighly reductive with a measured reductive capacitybetween 29 and 143 lmol O2 g!1, and pyrite oxidationwas the dominant oxygen-consuming reaction.

ª 2013 Federation of European Microbiological Societies FEMS Microbiol Ecol 85 (2013) 553–567Published by John Wiley & Sons Ltd. All rights reserved

554 M.P. Ginige et al.

Recharged water used during the Groundwater Replen-ishment Trial was secondary treated wastewater from amunicipal wastewater treatment plant (Beenyup Wastewa-ter Treatment Plant, Western Australia), which had under-gone further treatment process using ultrafiltration, reverseosmosis (RO), and ultraviolet disinfection prior to rechargeinto the aquifer. This recycled water was recharged into theaquifer for a period of 26 months (commenced November2010) with an average recharge rate of 1.2 GL year!1.

Materials and methods

Sampling of sediments for microbiologicalanalysis

Sediment samples for microbiological and geochemicalanalysis were recovered (during March 2007) as detailed

by Descourvieres et al. (2010) using a deep borehole con-struction through the Leederville Formation. In brief, drillcores were collected through diamond core drilling usinga triple tube coring method to minimize contaminationand core loss. To prevent exposure to oxygen, the barrelswere split in an anaerobic chamber installed next to thedrilling rig. The samples were clustered according to threestandard lithological classes referred to as sands (sands/sandstones), silts (silts/siltstones), and clays (mudstones/shales). Thirty-two of 105 samples collected were selectedfor microbiological analysis and were from 77 to257 mbgl. All core samples were stored at 4 °C in sealedtins until analyzed.

The sealed tins were opened in an anaerobic chamber,and 1 g each of homogenized core material was weighedaseptically into separate 50-mL sterile falcon tubes (Cat.# 2345-050; IWAKI, Japan). Anaerobic sterile RO waterwas introduced into each of the falcon tubes to a volumeof 20 mL, and the tubes were placed in an ultrasonicwater bath at 20 °C (Bransonic 220) for 3 min to detachmicroorganisms from the sediment. Approximately 15–20 mL of supernatant was then decanted into a new setof sterile 50-mL falcon tubes. The sediment samples weresonicated once more for 3 min in another 20 mL of freshanaerobic sterile RO water. The two lots of supernatantswere pooled together, and a centrifugal force of 16 060 g(Biofuge! fresco) was applied for 3 min to concentratebiomass into a volume of 1.5 mL. The FastDNA! spinkit for soil (Cat No. 6560-200; MP Biomedicals LLC,France) was used to individually extract DNA from the32 concentrated aliquots of biomass following manufac-turer’s protocol.

16S rRNA gene amplification, cloning, andsequencing

Half of the DNA from each of the 32 sediment samples waspooled together to build a single clone library. The near-complete bacterial 16S rRNA genes were amplified usingprimers 27f (5′-GAG TTT GAT CCT GGC TCA G-3′) andUniv1492r (5′-ACG G5T ACC TTG TTA CGA CTT-3′),and the primer sequences are modifications of Lane (1991).16S rRNA gene amplification and cloning were carried outas detailed in Ginige et al. (2004). In brief, HotStarTaqDNA polymerase (Qiagen, Valencia, CA) was used forpolymerase chain reaction (PCR) amplification. Thethermal cycle conditions applied included an initialDNA polymerase activation step at 95 °C for 15 minfollowed by 28 cycles of denaturation at 95 °C for1 min, annealing at 48 °C for 1 min, and an extensionat 72 °C for 2 min. On completion of 28 cycles, a finalextension at 72 °C for 10 min was applied. Ten replicatePCRs were carried out on the pooled DNA, and the

Sedimentsamples

Superficial

Mirrabooka

Pinjar

Wanneroo

Mariginiup

SPS

Member

Osb

orne

Fm

.Le

eder

vile

form

aƟon

Sout

h pe

rth

shal

e

FormaƟon0

40

80

120

160

200

240

280

320

Depth(mbgl)

Fig. 1. Stratigraphy and sampling depths of borehole. Depth is

reported as meters below ground level. Ground level is at 46 m

above sea level.


Groundwater recharge with aerobic recycled water 555

PCR products were pooled and purified using the QIA-quick PCR purification kit (Qiagen) to construct theclone library.

Purified PCR amplicons were immediately ligated usingthe pGEM!-T Vector System I (Cat. No. A3600; Pro-mega, Australia) following manufacturer’s instructions.The ligated products were transformed using ultracompe-tent Escherichia coli XL2-Blue MRF’ cells (Stratagene,Sydney, Australia) and screened for positive inserts(clones that contain 16S rRNA gene inserts) usingLuria–Bertani (LB) agar plates containing ampicillin5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside andisopropyl-b-D-thiogalactopyranoside (LB Ampicillin/X-gal/IPTG) (Burrell et al., 1998). Inserts from individual cloneswere amplified using vector primers (T7 and SP6) and werefully sequenced by Macrogen Inc., Korea, using the primersdetailed in Burrell et al. (1998). Compilation of full-lengthsequences and their analysis by BLAST (Altschul et al.,1990) were conducted as previously detailed (Burrell et al.,1998; Crocetti et al., 2000). A total of 200 fully sequencedclonal inserts were checked for chimeras using BELLEROPHOnversion 3 (http://greengenes.lbl.gov/cgi-bin/nph-bel3_inter-face.cgi). Nonchimeric sequences were then aligned, andoperational taxonomic units (OTUs) were defined at 97%sequence similarity using the ribosomal database project(Cole et al., 2009). To assess bacterial coverage (Cx), theformula Cx = [1 ! (ni/N)] (Singleton et al., 2001) wasused, where Cx is the coverage, ni is the number of uniquesequences represented by only one clone, and N is the totalnumber of the clones examined in each library.

Physical, chemical, and microbiologicalassessment of groundwater

Background physical and water chemistry data of thegroundwater before and after recharge were obtained bypersonal communication with Water Corporation ofWestern Australia. The data were extracted from therecords of the Beenyup Groundwater ReplenishmentTrial (GWRT) project. For microbial assessment, sevenmonitoring locations were investigated. Six groundwatermonitoring depths were located around a 20 m radius ofthe recharge bore. The six bores were Bore 1 – 94 m,Bore 2 – 129 m, Bore 3 – 147 m, Bore 4 – 165 m, Bore5 – 187 m, and Bore 6 – 202 m. The seventh monitoringbore (Bore 7 – 146 m) was located 60 m away from therecharge bore. All seven monitoring bores were routinelysampled to collect physical and water chemistry data.Bore 1 – 94 m was located within the permeable Pinjaraquifer, above a ~ 35-m siltstone confining layer. As aresult, this location was not impacted by the rechargedrecycled water and was used to provide natural microbialvariation control data. The other bore locations were

below the confining layer, within the permeable zone ofthe Leederville aquifer, and were impacted by recycledwater. The bores were sampled twice (before and after15 months of recharge) to assess any microbiologicalchanges resulting from recharge. The second microbiol-ogy sampling time of 15 months after recharge com-menced was selected as the groundwater chemistry hadstabilized by this time upon breakthrough of recycledwater. An additional two other monitoring bores werealso sampled during the second microbiology samplingevent. These additional sampling bores were located at240 m from the recharge bore (depths 151 and162 mbgl) and were not impacted by recycled water atthe time of sampling. These two samples were used asmicrobial controls for the permeable zone of Leedervilleaquifer.

For each of the monitoring bores investigated, thebore was initially pumped for a period of 15 to 60 minto stabilize groundwater chemistry; then, approximately20 L of groundwater was collected in a sterile carboycontainer (Nalgene) and transferred to the laboratoryfor storage at 4 °C prior to analyzing for the abundanceof microorganisms. Within 6 h of collection, the sam-ples were concentrated by a hollow-fiber ultrafiltrationsystem (HFUFS), using Hemoflow HF80S dialysis filters(Fresenius Medical Care, Lexington, MA) as previouslydescribed by Hill et al. (2005). The samples wereconcentrated to approximately 100 mL and further con-centrated by filtering through 0.22-lm filters (Polycar-bonate membrane, Cat No. GTBP02500; Millipore, UK)to recover biomass. Subsequently DNA was extractedusing the Fast DNA! spin kit for soil (Cat No. 6560-200; MP Biomedicals LLC) following manufacturer’sinstructions.

Flow cytometry for total bacterial cell counts

Staining of cells for flow cytometry was carried out using5 lL mL!1 SYBR! Gold (1 : 100 dilution in DMSO; Cat.No. S11494, Invitrogen, Australia Pty. Ltd.), and thestained samples were incubated in the dark for 15 minprior to measurement. A Cell Lab QuantaTM SC (Beck-man Coulter!) flow cytometer fitted with a 488-nm solidstate laser was used for counting. SYBR! Gold has excita-tion/emission maxima at 495/537 nm, respectively. FL1channel (525 nm) was used to collect the green fluores-cence and also was used as the trigger. The data collectedwere processed using Cell Lab Quanta Analysis software(Beckman Coulter!). The water samples were measuredin triplicate, and epifluorescence microscopy was used toconfirm the bacterial nature of stained particles. Thequantification error of flow cytometric measurements was0.2–2% of average reported.



Primer design and quantitative real-time PCR(qPCR)

Sequences that showed highest abundance in the clonelibrary were targeted through primer design. The newlydesigned primers were then used to quantitatively esti-mate the abundance of the respective microorganisms inbulk water and sediment samples using qPCR. To facili-tate primer design, all full-length sequences and theirclose relatives were imported into ARB (Ludwig et al.,2004). Primer designing for the selected sequences wascarried out using Beacon Designer 7.9 software. The ARBdatabase was used to verify whether the designed primersonly target the selected microorganism(s) discriminatingthe others. The designed primers to quantitatively esti-mate 16S rRNA gene copy numbers of microorganismsare listed in Table 1.

The thermocycler conditions used during qPCR for allprimer pairs included an initial denaturation step at95 °C for 15 min followed by 50 cycles of denaturationat 95 °C for 60 s, annealing at 60 °C for 60 s, and anelongation at 72 °C for 45 s. An iQ5 real-time PCRdetection system (Bio-Rad) was used for all qPCR, and

IQ SYBR green supermix (Bio-Rad) was used in all reac-tions following manufacturers’ instructions. Plasmidscarrying the respective cloned genes used as standards forcalibration of the assay are also given in Table 1. A nega-tive control (1- to 5-base mismatches, Table 1) and anegative control with no template DNA were alsoincluded in each qPCR run. qPCR of dissimilatory sulfitereductase (dsrB) genes of sulfate-reducing prokaryotes(SRP) was carried out using primers DSR1F (5′-ACSCAC TGG AAR CAC G-3′) and RH3-dsr-R (5′-GTGGAR CCR TGC ATG TT-3′), and the primer sequencesare modifications of Ben-Dov et al. (2007). A PCR-ampli-fied product of dsrB gene was used as standard for cali-bration of the assay, and the thermocycler conditionsused were similar to those of Ben-Dov et al. (2007). AllqPCRs were performed in triplicate, and at the end ofeach assay, a single band of expected size was observedusing agarose gel electrophoresis. Additionally, the speci-ficity of each qPCR was confirmed by comparing meltingcurve analysis of the sample and its respective referenceclone–derived PCR product. Data analysis was carried outusing IQTM software (version 5.2). The quantificationerror of qPCR was 10–15% of average reported.

Table 1. Oligonucleotide primers used in this study

Forward primer Reverse primer

Product

length

Positive

control

Negative

control

Target bacterial

order

SHEW590f

5′(TGTTAAGCGAGATGTGAA)3′

SHEW647r

5′(CCTCTACAAGACTCTAGTTC)3′

77 KC166793 KC166742 Alteromonadales

ACHRO939f

5′(CGGTGGATGATGTGGATT)3′

ACHRO1018r

5′(TTCTCTTGCGAGCACTTC)3′

99 KC166742 KC166857 Burkholderiales

PSE638f

5′(ATAACTGCTTGGCTAGAG)3′

PSE702r

5′(TGGTGTTCCTTCCTATATC)3′

83 KC166823 KC166751 Pseudomonadales

PSE413f

5′(AAGGTCTTCGGATTGTAA)3′

PSE484r1

5′(TGCTTATTCTGTCGGTAA)3′


PSE413f

5′(AAGGTCTTCGGATTGTAA)3′

PSE484r2

5′(GTGCTTATTCTGTTGGTAA)3′


BEE1069f

5′(TCGTGTCGTGAGATGTTG)3′

BEE1127r

5′(ATTAGAGTGCCCTTTCGTAG)3′


BEE940f

5′(GGTGGATGATGTGGTTTA)3′

BEE1029r

5′(CTGTGTTACGGTTCTCTT)3′


Thau935f

5′(CAAGCGGTGGATGATGTG)3′

Thau996r

5′(TCAGCAAGGTTCCAGACA)3′

79 KC166840 KC166850 Rhodocyclales

Agro402f

5′(CGTGAGTGATGAAGGTCTTA)3′

Agro484r

5′(GGCTTCTTCTCCGACTAC)3′

75 KC166729 KC166835 Rhizobiales

Bee642f

5′(CTGGCTATCTTGAGTATGG)3′

Bee702r

5′(TGGTGTTCTTCCGAATATC)3′

79 KC166768 KC166741 Caulobacterales

Bee636f

5′(TTGATACTGACTGTCTTGAG)3′

Bee697r

5′(GTTCTTCCGAATATCTACGA)3′

81 KC166741 KC166782 Caulobacterales

Flavo674f

5′(AATATGTAGTGTAGCGGTGAA)3′

Flavo745r

5′(GTCCATCAGCGTCAATCA)3′

90 KC166702 KC166782 Flavobacteriales

Blasto144f

5′(TGGGATAACTCCAAGAAAT)3′

Blasto193r

5′(AGCCGATAAATCTTTCCA)3′

81 KC166745 KC166835 Actinomycetales

27f

5′(GAGTTTGATCCTGGCTCAG)3′

EUB338r

5′(GCTGCCTCCCGTAGGAGT)3′

312 Q629738 N/A All bacteria



Nucleotide sequence accession numbers

The 16S rRNA gene nucleotide sequence data of thisstudy were deposited in GenBank database under acces-sion numbers KC166698–KC166861.

Results and discussion

Clone library analysis of the bacterialcommunities in the sediments of Leedervilleaquifer

To investigate the overall bacterial community in Leeder-ville aquifer, we subjected 32 sediment samples (fromdepths of 77–257 mbgl) to a clone library analysis usingthe 16S rRNA gene. While bacterial 16S rRNA geneamplification was successful by a direct PCR step withDNA extractions from most sediment samples, someshowed amplification only upon nested PCR. This indi-cated that core material obtained from certain depthscontained quantitatively a much smaller bacterial com-munity to that of others. The uneven distribution of elec-tron donors and acceptors in different sediment types ofLeederville (Descourvieres et al., 2010) may have contrib-uted toward this observation.

Of 200 full-length sequences, 36 sequences were found tobe chimeric and were removed from the data set. Based on97% sequence similarity, 37 OTUs were identified. Table 2details the phylogenetic analysis of the 16S rRNA genesequences of OTUs and demonstrates the large diversity ofmicroorganisms present in the Leederville aquifer sedi-ment. Phylogenetically, the organisms were spread acrosseight classes, namely Alphaproteobacteria (84 clones), Gam-maproteobacteria (36 clones), Betaproteobacteria (22clones), Flavobacteria (12 clones), Actinobacteria (fourclones), Sphingobacteria (three clones), Bacilli (two clones),and Cytophaga (one clone). Seventy of the 84 Alphaproteo-bacteria clones formed a single OTU and were closelyrelated to an uncultured bacterium identified from beneaththe West Antarctic ice sheets (Lanoil et al., 2009). Someless dominant OTUs also affiliating with Alphaproteobacte-ria showed close resemblance to sequences derived fromenvironments such as Australian gold mining environ-ments (Santini et al., 2002), drinking water (Williamset al., 2004), rice paddy fields (Stubner et al., 1998), andsoil (Yoon et al., 2007). Specifically, the Alphaproteobacte-ria sequences that showed close resemblance to autotrophicthiosulfate-oxidizing bacteria highlight the potential of theindigenous sediment microbial community to oxidizereduced sulfur compounds in the Leederville aquifer. Addi-tionally, the potential of Alphaproteobacteria to producemethane utilizing H2 was realized with the discovery ofsequences that showed close resemblance to sequences

identified from a methane-producing deep aquifer (Kimuraet al., 2010).

The second most abundant group of bacteria in theclone library belonged to class Gammaproteobacteria. TheOTU that contained the most number of Gammaproteobac-teria sequences was closely related to sequence of Pseudo-monas xinjiangensis (Liu et al., 2009), which is a knownthermotolerant microorganism. The second largest OTUcontaining Gammaproteobacteria sequences shared similar-ities to Pseudomonas stutzeri (Yu et al., 2011), which isknown to fix atmospheric nitrogen. In an aquifer such asthe Leederville where nitrogen and phosphorous are limit-ing, the role of these nitrogen-fixing microorganismsbecomes important to sustain microbial abundance anddiversity in the aquifer. Additionally, some Gammaproteo-bacteria sequences showed similarities to alkali-tolerantbenzene-degrading bacteria sequences derived from con-taminated aquifers (Fahy et al., 2008), and the potential ofthe Leederville microbial community to denitrify wasfurther realized with the discovery of Gammaproteobacteriasequences closely relating to known Pseudomonas sp. con-taining denitrifying genes (Heylen et al., 2006).

The third largest bacterial class of the clone library wasBetaproteobacteria with sequences spread across six OTUs.The most dominant OTU of Betaproteobacteria containedsequences that showed close resemblance to sequences ofgenus Achromobacter of order Burkholderiales derivedfrom soils contaminated with polycyclic aromatic hydro-carbons (La Rosa et al., 2006). Burkholderiales are wellknown for their ability to utilize a diverse range of carbonsources including polycyclic aromatic hydrocarbons, andthe ability of these microorganisms to denitrify has alsobeen well demonstrated (Saito et al., 2008; Guo et al.,2011; Yoshida et al., 2012). Sequence similarities werealso found to bacterial sequences derived from a Lakeplaya and drinking water (Williams et al., 2004; Navarroet al., 2009). The close resemblance of one Betaproteobac-teria OTU affiliated to Thauera (Scholten et al., 1999), abacterial genus known to contain denitrifying microor-ganisms, highlights the potential of the sediment micro-bial community to denitrify in the event nitrate becomesabundant in the aquifer. The OTU that contained the sec-ond largest number of Betaproteobacteria sequences hadclose similarities to sequences derived from a study byMaimaiti et al. (2007). In this study, Maimaiti et al.(2007) report the discovery of hydrogen-oxidizing bacte-ria from soil. With abiotic interactions of ferromagne-sium and groundwater capable of facilitating hydrogenproduction (Stevens, 1997), the discovery of sequencessimilar to hydrogen-oxidizing bacteria in the Leedervillesediment hints the potential for primary production tooccur in the aquifer via chemolithoautotrophic microbialcommunities.



Table

2.16SrRNA

gen

esequen

cean

alysisofLeed

erville

sedim

entbacterial

communities

Accession

number

Noof

OTU

s

Phylogen

etic

group

Nearest

neighbor

Iden

tity

(%)

Sourceoftheclosest

relative

Nearest

isolate

Iden

tity

(%)

KC166745

1Actinobacteria

Blastococcussp.CNJ868PL04(DQ448697)

98

Diversity

ofgram-positive

bacteria

culturedfrom

marinesedim

ents

Blastococcussaxobsiden

sDD2(FO117623)

98

KC166849

1Actinobacteria

Cellulomonas

sp.ANA-W

S2(EU303275)

98

Cellulosicbiohydrogen

production

Actinotaleasp.CF5-4

(HQ730135)

99

KC166727

2Actinobacteria

Geo

dermatophilussp.–isolate

G1S(X92364)

99

Isolatedfrom

dry

soil,

rocks,

and

monumen

tsurfaces

Blastococcusag

gregatus(AJ430193)

99

KC166741

2Alpha

Phen

ylobacteriumfalsum

typestrain

AC-49T

(AJ717391)

99

Nonsalinealkalineen

vironmen

t:

heterotrophic

aerobic

populations

Phen

ylobacterium

falsum

(NR_0

42277)

99

KC166783

1Alpha

Devosiainsulaestrain

DS-56(EF012357)

98

Isolatedfrom

soil

DevosiainsulaeDS-56(NR_0

44036)

99

KC166814

1Alpha

Boseastrain

5Z-2111(AJ224610)

99

Autotrophic

thiosulfate-oxidizing

bacteria

Boseamassiliensis(AF288307)

99

KC166838

70

Alpha

Unculturedbacterium

(FJ477326)

99

Bacteriaben

eath

theWestAntarctic

icesheet

Brevundim

onas-likesp.LM

G11050

(NR_0

41991)

99

KC166705

1Alpha

Caedibactermacronucleo

rum

partial

(AM236091)

90

Obligateen

dosymbiont

EndosymbiontofAcantham

oeb

asp.AC305

(AY549548)

90

KC166768

2Alpha

Drinkingwater

bacterium

MA11(AY328832)

99

Drinkingwater

bacteria

Brevundim

onas

basaltis(JX094172)

99

KC166719

1Alpha

Unculturedbacterium

-clone:

SMD-B09

(AB478001)

99

Microbialmethan

eproductionin

deepaq

uifer

Parvibaculum

lavamen

tivoransDS-1

(CP0

00774)

99

KC166725

3Alpha

Rhizobium

selenitired

ucensstrain

B1(EF440185)

98

Red

uctionofseleniteto

elem

ental

redselenium

byRhizobium

sp.

Rhizobium

sp.P4

9(HQ652582)

99

KC166729

2Alpha

Arsen

ite-oxidizingbacterium

BEN

-5(AY027505)

98

Isolatedfrom

Australiangold

mining

environmen

ts

Agrobacterium

albertimag

ni(GU947873)

99

KC166752

1Bacilli

Unculturedlow

G+C

Gram-positive

bacterium

(DQ206425)

95

Sulfideoxidationcoupledto

arsenatereduction

Low

GCGram-positive

bacterium

strain

AHT2

8(HM046584)

96

KC166762

1Bacilli

Paen

ibacillussp.WPC

B173(EU939688)

93

From

afreshwater

wetland

Paen

ibacillussp.YIM

016(JQ314346)

95

KC166787

3Beta

Bacterium

isolate

SL3.41(DQ517100)

98

Bacterial

inan

Ephem

eral

Hypereu

trophic

Mojave

Desert

PlayaLake

Betaproteobacterium

1E1

(HM587246)

96

KC166818

10

Beta

Alcaligen

esxylosoxidan

sstrain

F.(AJ491845)

99

From

soils,contaminated

with

polycyclic

aromatic

hydrocarbons

Alcaligen

essp.(AJ002804)

99

KC166840

1Beta

Thau

eramechernichen

sisstrain

TL1

(NR_0

26473)

99

Anaerobic

den

itrifier/leachate

treatm

entplant

Thau

erasp.27(AY838760)

99

KC166723

6Beta

Variovoraxparad

oxusisolate

Jm63(DQ256487)

99

Hydrogen

-oxidizingbacteria

Variovoraxparad

oxus(AB680784)

96

KC166857

1Beta

Drinkingwater

bacterium

MB16(AY328846)

98

Drinkingwater

bacteria

Limnobacterthiooxidan

s(NR_0

25421)

99

KC166839

1Cytophag

aAlgoriphag

usornithinivoransstrain

JC2052(NR_0

25745)

92

Isolatedfrom

tidal

flat

sedim

ent

Algoriphag

ussp.IM

SNU

14015(AY264841)

93

KC166739

1Flavobacteria

Flavobacteriaceaebacterium

NHD080

(DQ993338)

96

Bacterium

isolatedfrom

marine

environmen

ts

Flavobacteriaceaebacterium

NHD080

(DQ993338)

96

KC166848

10

Flavobacteria

Flavobacterium

filum

strain

EMB34

(DQ372981)

95

Isolatedfrom

awastewater

treatm

entplantin

Korea

Antarcticbacterium

R-9033(AJ441001)

95



Table

2.Continued

Accession

number

Noof

OTU

s

Phylogen

etic

group

Nearest

neighbor

Iden

tity

(%)

Sourceoftheclosest

relative

Nearest

isolate

Iden

tity

(%)

KC166702

1Flavobacteria

Flavobacterium

cucumisstrain

R2A45-3

(EF126993)

97

Isolatedfrom

soil

Bacterium

SM3-6

(AY773135)

98

KC166807

1Gam

ma

Pseu

domonas

sp.strain

R-24261

(AM231055)

99

Den

itrifier

Pseu

domonas

nitroreducensssp.

Thermotolerans(AB681730)

99

KC166809

15

Gam

ma

Pseu

domonas

xinjiangen

sisstrain

S3-3

(EU286805)

98

Moderatelythermotolerant

bacterium

isolatedfrom

desert

sand

Pseu

domonas

sp.K2B-6

(FJ889564)

97

KC166699

4Gam

ma

Pseu

domonas

sp.strain

Rs87(AM905943)

100

Alkali-tolerantben

zene-deg

rading

bacteriafrom

acontaminated

aquifer

Pseu

domonas

stutzeristrain

M16-9-4

(HM030754)

99

KC166858

5Gam

ma

Pseu

domonas

stutzeriDSM

4166

(CP0

02622)

99

Nitrogen

-Fixingan

dRhizosphere-

Associated

Bacterium

Pseu

domonas

stutzeri(GQ402828)

99

KC166714

1Gam

ma

Pseu

domonas

sp.AB42(EF554871)

96

Alkaliphilic(pH

9–1

1)Bacteria

towardsChloroaromatic

Substrates

Pseu

domonas

sp.DAC9(JF708185)

96

KC166773

4Gam

ma

Shew

anella

putrefaciens(X81623)

99

Halotolerant,facultativelyiron-

reducingbacterium

Shew

anella

putrefaciensCN-32(CP0

00681)

99

KC166732

3Sp

hingobacteria

Pontibacterko

rlen

sisstrain

X14-1

(DQ888330)

94

Isolatedfrom

thedesertsand

Pontibacterko

rlen

sis(AB682651)

94

KC166815

2Gam

ma

Pseu

domonas

stutzeri(FR667909)

99

Roots

offield-growncereal

crops

Pseu

domonas

stutzeriDSM

4166

(CP0

02622)

99

KC166730

1Gam

ma

Unculturedbacterium

(AB369174)

99

Riser

drillingmudfluid

Alishew

anella

jeotgaliKCTC

22429

(EU817498)

99

KC166822

1Gam

ma

Pseu

domonas

sp.DSM

Z141-No.

4(AB733584)

97

Subsurfacewater

Pseu

domonas

sp.DSM

Z141-No.615-4

(AB733406)

97

KC166713

1Alpha

Unculturedbacterium

(FJ230894)

99

River

water

Devosiariboflavina(AB680451)

99

KC166747

1Gam

ma

Unculturedbacterium

(EU979078)

99

Soil

Sten

otrophomonas

sp.R-41388(FR682931)

99

KC166781

1Beta

Unculturedbacterium

(DQ248271)

99

Carbontetrachloride–

contaminated

soil

Variovoraxparad

oxusS1

10(NR_0

74654)

99

KC166766

1Gam

ma

Unculturedbacterium

(DQ336973)

99

Subsurfacewater

Pseu

domonas

sp.G-R2A7(EF554919)

96



Bacterial sequences belonging to class Flavobacteriaspanned across three OTUs, and one OTU contained 10sequences. All sequences showed close similarities tosequences derived from soil and water (including waste-water) (Ryu et al., 2007; Weon et al., 2007). Althoughsome Flavobacteria strains are pathogenic to humans andanimals, the largest number of Flavobacterium sequencesidentified showed close similarity to Flavobacterium filum(Ryu et al., 2007), and this species has not been reportedas an opportunistic pathogen toward human or animals.

The sequences of the OTUs that belonged to the classActinobacteria showed close resemblance to sequencesderived from marine sediments, dry rocks, and monu-ment surfaces (Eppard et al., 1996; Gontang et al., 2007).The sequences of class Bacilli highlighted the potential forarsenate reduction (electron acceptor) to take place cou-pled with sulfide oxidation (electron donor). The closesimilarity found to sequences derived from a study byHollibaugh et al. (2006) that investigated sulfide oxida-tion coupled to arsenate reduction in a soda lake indi-cated the possibility for this geochemical process to takeplace in the Leederville aquifer.

According to the clone library, the microbial diversity ofLeederville aquifer is noteworthy, and the identified micro-bial communities demonstrate potential to facilitate a largenumber of biological processes. Although a pooled DNAsample representing the entire depth of aquifer was usedfor the construction of the clone library, only 77% of bac-terial diversity was revealed, indicating possible existence

of other microbial communities that may impact aquiferrecharge. Pyrosequencing the DNA of individual sedimentsamples, on the other hand, could have revealed a near100% diversity of the Leederville aquifer.

The distribution of the identified communitiesin the Leederville sediment

Figure 2 reveals the distribution of the identified micro-bial communities between depths of 77–257 mbgl ofLeederville sediment as analyzed by qPCR. It is apparentfrom the figure that not all identified microorganisms areuniformly distributed along the entire depth studied.Further, the bacterial coverage with the designed primersappeared incomplete where in some instances, 80%(e.g. sediment sample 101 and 257 mbgl) of communityabundance was not revealed. Although lack of primers totarget smaller OTUs may have largely contributed towardthis observation, the 77% coverage of diversity by theclone library may also not have facilitated the capture of100% community abundance at each of the depths ofLeederville aquifer. Considering multiple copies of the16S rRNA genes in some bacteria, the percentage copynumbers of the 16S rRNA gene reported may also reflecta reduced abundance (under estimation), further contrib-uting toward the report of an incomplete bacterial cover-age of sediments at different depths.

Bacteria of order Burkholderiales (belonging to theclass Betaproteobacteria) are spread across the entire

0

10

20

30

40

50

60

70

80

90

100

77 79 93 96 101 103 116 135 152 153 165 174 202 204 225 230 238 239 251 257

% R

elat

ive

abun

danc

e

Depth (mbgl)

Alteromonadales (Gamma) Burkholderiales (Beta) Pseudomonadales (Gamma)

Rhodocyclales (Beta) Rhizobiales (Alpha) Caulobacterales (Alpha)

Flavobacteriales (Flavo) Actinomycetales (Actino)

Fig. 2. The abundance of the 16S rRNA gene

copy number of identified bacterial order

relative to the total bacterial copy numbers

determined using qPCR for depths between of

77–257 mbgl of Leederville sediment

[% relative abundance = (copy numbers of

16S rRNA gene targeted by specific primer/

16S rRNA gene copy numbers of all bacteria

in sample) 9 100]. The texts within brackets

are the phylogenetic classes of the quantified

bacterial orders.



depth of sediment that was subjected to the study(Fig. 2). Considering that this group of bacteria was sec-ond dominant in the clone library, this result can beexpected. Bacteria of order Caulobacterales, belonging tothe class Alphaproteobacteria, on the other hand wereonly observed dominant at selected depths of the aquifer.The order Pseudomonadales representing bacterial classGammaproteobacteria and the order Rhizobiales repre-senting the class Alphaproteobacteria were also foundwide spread and showed dominance as indicated by theclone library.

When the distribution of the sediment microbial com-munity was investigated with respect to the lithology ofLeederville sediment, clear preference of certain microbialcommunities toward certain lithological classes wasrevealed (Fig. 3). The highest bacteria diversity wasobserved on sand and sandstone, while the lowest wasnoted on shale. Burkholderiales (belonging to class Beta-proteobacteria) was dominant in all but siltstone, and Alt-eromonadales of Gammaproteobacteria was only observedin shale. With respect to the finding of Descourviereset al. (2010), the lithology of the aquifer is largely ofsands (~ 90%) and silts in the first 200 mbgl. The lithol-ogy of sediments between 200 and 257 mbgl on the otherhand is largely of clay (mudstones/shales). According toFig. 3, it is apparent that sands and silts facilitate a largerdiversity of microorganisms compared with clays.

Chemical, physical, and microbiologicalchanges before and during groundwaterreplenishment

Groundwater microbial cell numbers

Groundwater microbial cell numbers were measured priorto recharge and after 15 months of recharge to assess themicrobial impact of recharging recycled water into theLeederville aquifer. Within the Pinjar aquifer (Bore1 – 94 m), above the confining layer, only minor increase(2.2 9 104 to 3.1 9 104 cell mL!1) in microbial cellnumbers (Fig. 4) was observed between the two samplingevents. As this location was not impacted by recycledwater, this change was likely due to natural microbial var-iation over time (~ 2 years between sampling events).

Within the permeable zone of the Leederville aquifer,below the confining layer, the average microbial cell num-bers were 2.2 " 1.2 9 103 cell mL!1 prior to recharge.These values were lower than that in the zone abovethe confining layer, but consistent (4.0 " 1.4 9 103

cell mL!1) with microbial cell numbers from groundwatersamples collected during the second sampling (after15 months of recharge) from locations below the confininglayer (bores located 240 m from the recharge bore – depths151 and 163 mbgl) that were not impacted by recycledwater. These data suggest that lower bacterial numbers exist

0

10

20

30

40

50

60

70

80

% R

elat

ive

abun

danc

e

Alteromonadales (Gamma) Burkholderiales (Beta) Pseudomonadales (Gamma)

Rhodocyclales (Beta) Rhizobiales (Alpha) Caulobacterales (Alpha)

Flavobacteriales (Flavo) Actinomycetales (Actino)




determined using qPCR for different

lithological classes of Leederville sediment [%

relative abundance = (copy numbers of 16S

rRNA genes targeted by specific

primer 9 100/16S rRNA gene copy numbers

of all bacteria in sample)]. The texts within

brackets are the phylogenetic classes of the

quantified bacterial orders.



within the permeable zone below the confining layer,compared with above the confining layer. Data from thesecond microbial sampling event (after 15 months ofrecharge) showed a substantial increase in microbial cellnumbers in groundwater samples from locations impactedby recycled water (Fig. 4). Average microbial cell numberswithin the Leederville aquifer increased to6.3 " 4.2 9 104 cell mL!1 (an average increase of 30times). These data suggest that increased microbial cellnumbers were a result of the recharged recycled water.

The total organic carbon in recharge water was< 1 mg L!1. The total organic carbon in bulk groundwa-ter before and after recharge was 1.7 " 2.1 and1.2 " 0.9 mg L!1, respectively. With recharge water notintroducing much total organic carbon into the aquifer,the bulk water total organic carbon concentration afterrecharge continued to remain low. Descourvieres et al.(2010) found SOM not to be the main oxygen reductant,although more abundant than pyrite in the Leedervilleaquifer. This could be due to SOM largely being recalci-trant or stable toward any chemical or physical break-down. The clone library, however, indicated theavailability of diverse metabolic pathways among residentmicroorganisms (e.g. order Burkholderiales) suggestingoxidizing capabilities of even complex aromatic hydrocar-bons. In the event the microbial turnover of SOM waslimited by the availability of electron acceptor and/ormicro- or macronutrients, with groundwater recharge

and an increased availability of basic essentials such asnitrogen, phosphorous, and electron acceptors such asoxygen, nitrate, and sulfate, the resident microbial com-munities are likely to overcome growth barriers andincrease in numbers.

Major biogeochemical reactions

The average physical and chemical water quality data for(1) background groundwater prior to the commencementof recharge, (2) recycled water that was recharged into theaquifer, and (3) groundwater (Bores 2–7) after 15 monthsof recharge are given in Table 3. The chemical screeningfor recycled water specifically included monitoring ofapproximately 300 chemicals (e.g. pesticides, pharmaceuti-cals, etc.), and measured concentrations were well belowhealth guidelines. A limited number of major groundwaterbiogeochemical reactions could be identified in the Leeder-ville aquifer, based on observed groundwater qualitychanges between the recycled water and post-breakthroughgroundwater (Table 3). These biogeochemical reactionswere (1) pyrite oxidation and (2) denitrification. Thesegeochemical reactions are consistent with previous labora-tory incubation experiments (Descourvieres et al., 2010)and large-scale column experiments (Patterson et al., 2010)using sediment collected from the Leederville aquifer.

Pyrite oxidation would explain the decrease indissolved oxygen from an average 8.4 mg L!1 in the

1.0E+02

2.0E+04

4.0E+04

6.0E+04

8.0E+04

1.0E+05

1.2E+05

1.4E+05

1.6E+05

0

10

20

30

40

50

60

70

80

90

Befo

re

Afte

r

Befo

re

Afte

r

Befo

re

Afte

r

Befo

re

Afte

r

Befo

re

Afte

r

Befo

re

Afte

r

Befo

re

Afte

r

Bore -1 20 /94 m

Bore -2 20 /129 m

Bore -3 20 /147 m

Bore -4 20 /165 m

Bore -5 20 /187 m

Bore -6 20 /202 m

Bore -7 60 /146 m

Cells

mL–1

% R

elat

ive

abun

danc

e

Actinobacteridae(Gamma)

Rhodocyclales(Beta)

Flavobacteriales(Flavo)

Burkholderiales(Beta)

Rhizobiales(Alpha)

Alteromonadales(Actino)

Pseudomonadales(Gamma)

Caulobacterales(Alpha)

Cell Count Before After)/(




determined using qPCR and flow-cytometric

cell counts in ground water before and after

recharge [% relative abundance = (copy

numbers of 16S rRNA gene targeted by

specific primer/16S rRNA gene copy numbers

of all bacterial in sample) 9 100].



recycled water to < 0.5 mg L!1 in the post-breakthroughgroundwater (Table 3) along with the increased sulfateconcentrations from 0.2 to 11 mg L!1. These data areconsistent with the microbiological data. There are OTUsin the clone library that have shown sequences (BC0153and BC022, see Table 2) similar to microorganisms thatare cable of oxidizing different sulfur species. One OTUshowed close similarities to Bosea massiliensis(AF288307), which is known to be able to oxidize thio-sulfate (La Scola et al., 2003). Thiosulfate is often a resultof sulfite ions reacting with elemental sulfur, and this canbe observed with an incomplete oxidation of sulfides(pyrite oxidation) (Druschel et al., 2004). Further, theOTU that showed close sequence similarities to low GCGram-positive bacterial strain AHT28 (HM046584) islikely to facilitate sulfide oxidation while reducing arse-nate. These data indicate that the bacterial community inthe Leederville aquifer could microbialy mediate sulfuroxidation.

The decrease in introduced nitrate in the recycled waterfrom 2.5 mg L!1 – N to 0.53 mg L!1 – N in the postbreak-through groundwater (Table 3) is possibly a result of bio-logical denitrification taking place, making use of organiccarbon in the Leederville aquifer. Denitrification can how-ever also be driven by pyrite oxidation (Zhang et al., 2012),and the presence of nitrate in the recycled water mayextend the pyrite oxidation beyond the oxic zone, with anincrease in population of the pyrite oxidizing denitrifyingcommunities. The close resemblance of BetaproteobacteriaOTUs [affiliated to Thauera (Scholten et al., 1999) andAlcaligenes xylosoxidans (La Rosa et al., 2006)] and aGammaproteobacteria OTU [affiliated to Pseudomonasnitroreducens ssp. thermotolerans (AB681730)] to knowndenitrifiers strongly supports the hypothesis that bacterialcommunities in the Leederville aquifer could mediatenitrate reduction in the aquifer. The order Burkholderiales,which showed a significant presence in the clone library,showed dominance particularly in bulk water upon aquiferrecharge. While bacteria belonging to Burkholderiales havebeen reported to be able to denitrify (Saito et al., 2008;Guo et al., 2011; Yoshida et al., 2012), the closeresemblance of one dominant Burkholderiales OTU affili-ated to a well-studied denitrifier [Alcaligenes xylosoxidans(AJ491845)] confirms that nitrate in recharge water (elec-tron acceptor) is influencing the observed increase in Burk-holderiales in the aquifer (Fig. 3). With geochemical datasuggesting pyrite oxidation and denitrification to be thetwo major reactions taking place in the Leederville aquifer,the increase in microbial numbers in groundwater is likelya reflection of an increased denitrifier and/or sulfur-oxidiz-ing microbial populations in the aquifer. Upon recharge,the increase in cell numbers of Burkholderiales also indi-cates a rapid assimilation of SOM by these microorganisms,T

able

3.Averagephysical

andchem

ical

characteristicsofsourcewatersusedforrecharge

Parameter

Conductivity

(msm

!1)

pH

Red

ox

(mV)

Totalorgan

ic

carbon

(mgL!

1)

Dissolved

oxygen

(mgL!

1)

Fe(m

gL!

1)

Mn(m

gL!

1)

NOx-N*

(mgL!

1)

TotalP

(mgL!

1)

SO2!

4

(mgL!

1)

Groundwater

quality–

priorto

recharge

125"

41

6.7

"0.1

!65"

91.7

"2.1

<0.5

6.6

"2.5

0.052"

0.011

0.03"

0.001

0.21"

0.06

35"

15

Rechargewater

quality

6.7

"1.3

6.9

"0.3

n.d.

<1

8.4

<0.01

<0.001

2.5

"0.7

0.02"

0.01

0.2

"0.06

Groundwater

qualityafter

rechargebreakthrough

9.4

"2.8

6.9

"0.3

74"

90

1.2

"0.9

<0.5

0.67"

0.91

0.031"

0.048

0.53"

0.56

0.16"

0.16

11"

2

*NOx-N¼

NO

! 3-N

þNO

! 2-N

:



indicating biogeochemical processes to be as dominant asgeochemical processes in the Leederville aquifer.

Increases in bacterial numbers may not have occurredrapidly. Long-term monitoring of groundwater (data notshown) showed an initial breakthrough of nitrate at 60 mfrom the recharge bore. After approximately 12 monthsof recharge, nitrate concentrations were observed todecrease over a period of ~ 4 months to concentrationsbelow detection. These data suggested that denitrificationwas not rapid initially, but the rate of denitrificationincreased over time until it complemented with the rateof nitrate injection into the aquifer.

Minor biogeochemical reactions

Other possible biogeochemical reactions such as biodegra-dation of disinfection byproducts and sulfate reductionwere also investigated. Reduction in the disinfectionbyproduct N-nitrosodimethylamine in the recycled waterfrom 2.5 to 1.3 ng L!1 in the postbreakthrough groundwa-ter was observed (Patterson et al., 2012). The most domi-nant OTU of Betaproteobacteria sequences showed closeresemblance to sequences of genus Achromobacter of orderBurkholderiales, and these organisms were identified frompolycyclic aromatic hydrocarbons-contaminated soils (LaRosa et al., 2006). There is a strong possibility that theAchromobacter spp. is mediating the biodegradation ofpolycyclic aromatic hydrocarbons in these soils. Achromo-bacter spp. could be mediating the biodegradation ofdisinfection byproducts N-nitrosodimethylamine in theLeederville aquifer and N-nitrosomorpholine and N-nitros-odimethylamine in column experiments carried out utiliz-ing Leederville aquifer sediment and recycled water (Pitoiet al., 2011; Patterson et al., 2012).

From the microbiological sediment data, sequences withsimilarities to known sulfate-reducing microorganismswere not detected in the clone library. This is consistentwith groundwater microbiological data, as qPCR of sulfate-reducing genes in groundwaters at the six monitoring sitesshowed an average relative abundance of only 0.01% [per-centage relative abundance = (copy numbers of dsrBgenes/16S rRNA gene copy numbers of all bacterial in sam-ple) 9 100]. With groundwater recharge, there was achange in groundwater chemistry from a reduced state to amore oxidized state (Table 3). The increase in redox poten-tial in groundwater suggests that sulfate reduction is unli-kely to occur in the Leederville aquifer, as sulfate reductiontypically only takes place under reduced conditions.

Microbial growth-limiting factors

Due to the complex physical, chemical, and biologicalinteractions involved, it is often difficult to determine

microbial growth-limiting factors in aquifers such as theLeederville aquifer. The increase in the availability of elec-tron acceptors (e.g. oxygen, nitrate) is likely to influenceincreased microbial activity in the aquifer. However,nitrogen or phosphorous is often the limiting nutrients inaquifers. An increase in phosphate concentrations (aver-age 0.16 mg L!1 total phosphate) was observed in theaquifer compared with the recycled water (0.02 mg L!1)indicating potential P release from mineral dissolution orcolloid release from the aquifer sediment. If P was thelimiting nutrient, the increase in P may also increasemicrobial activity in the aquifer.

Conclusion

Using a culture-independent approach, this study showedthe presence of a diverse bacterial community and thedistribution of various phylogenetic groups in a profile ofintact core samples obtained from the Leederville aquiferat the Groundwater Replenishment Trial site inPerth, WA. The bacteria were affiliated with classes Actino-bacteria, Alphaproteobacteria, Bacilli, Betaproteobacteria,Cytophaga, Flavobacteria, Gammaproteobacteria, andSphingobacteria. Data also suggested that with an increase indepth, there was a decrease in microbial diversity in theLeederville aquifer. Through assessment of physico-chemicaland microbiological properties of groundwater before andafter recycled water recharge, the study also revealed possibleimplications of recharging aerobic recycled water into theanaerobic aquifer. These included elevated redox potentialand microbial numbers, but reduced microbial diversity.The elevated microbial numbers could be related to theincreased availability of electron acceptors (oxygen andnitrate) and/or possibly phosphate. With geochemical dataproviding evidence of pyrite oxidation and also denitrifica-tion of the nitrate in groundwater, the increase in bacterialnumbers and reduced microbial diversity in groundwatercould be a reflection of an increased denitrifier and sulfur-oxidizing populations in the aquifer.

References

Altschul SF, Gish W, Miller W, Myers EW & Lipman DJ(1990) Basic local alignment search tool. J Mol Biol 215:403–410.

Appelo CAJ, Drijver B, Hekkenberg R & de Jonge M (1999)Modeling in situ iron removal from ground water.Groundwater 37: 811–817.

Bachofen R, Ferloni P & Flynn I (1998) Microorganisms in thesubsurface. Microbiol Res 153: 1–22.

Ben-Dov E, Brenner A & Kushmaro A (2007) Quantificationof sulfate-reducing bacteria in industrial wastewater, byreal-time polymerase chain reaction (PCR) using dsrA andapsA genes. Microb Ecol 54: 439–451.



Bennett PC, Rogers J & Choi WJ (2001) Silicates, silicateweathering, and microbial ecology. Geomicrobiol J 18: 3–19.

Bouwer H (2002) Artificial recharge of groundwater:hydrogeology and engineering. Hydrogeol J 10: 121–142.

Brockman FJ, Ringelberg DB, White DC, Fredrickson JK,Balkwill DL, Kieft TL, Phelps TJ & Ghiorse WC (1993)Estimates of intact but nonviable and viable butnonculturable microorganisms in subsurface sediments fromsix boreholes located in wet and dry climatic regions of theUnited States. 1993 International Symposium on SubsurfaceMicrobiology B-32.

Burrell PC, Keller J & Blackall LL (1998) Microbiology of anitrite-oxidizing bioreactor. Appl Environ Microbiol 64:1878–1883.

Chandler DP, Brockman FJ & Fredrickson JK (1997a) Use of16S rDNA clone libraries to study changes in a microbialcommunity resulting from ex situ perturbation of asubsurface sediment. FEMS Microbiol Rev 20: 217–230.

Chandler DP, Li SM, Spadoni CM, Drake GR, Balkwill DL,Fredrickson JK & Brockman FJ (1997b) A molecularcomparison of culturable aerobic heterotrophic bacteria and16S rDNA clones derived from a deep subsurface sediment.FEMS Microbiol Ecol 23: 131–144.

Cole JR, Wang Q, Cardenas E et al. (2009) The RibosomalDatabase Project: improved alignments and new tools forrRNA analysis. Nucleic Acids Res 37: D141–D145.

Crocetti GR, Hugenholtz P, Bond PL, Schuler A, Keller J,Jenkins D & Blackall LL (2000) Identification ofpolyphosphate-accumulating organisms and design of 16SrRNA-directed probes for their detection and quantitation.Appl Environ Microbiol 66: 1175–1182.

Descourvieres C, Hartog N, Patterson BM, Oldham C &Prommer H (2010) Geochemical controls on sedimentreactivity and buffering processes in a heterogeneousaquifer. Appl Geochem 25: 261–275.

Druschel GK, Baker BJ, Gihring TM & Banfield JF (2004) Acidmine drainage biogeochemistry at Iron Mountain,California. Geochem Trans 5: 13–32.

Eppard M, Krumbein WE, Koch C, Rhiel E, Staley JT &Stackebrandt E (1996) Morphological, physiological, andmolecular characterization of actinomycetes isolated fromdry soil, rocks, and monument surfaces. Arch Microbiol 166:12–22.

Fahy A, Ball AS, Lethbridge G, Timmis KN & McGenity TJ(2008) Isolation of alkali-tolerant benzene-degradingbacteria from a contaminated aquifer. Lett Appl Microbiol47: 60–66.

Fredrickson JK, Garland TR, Hicks RJ, Thomas JM, Li SW &McFadden KM (1989) Lithotrophic and heterotrophicbacteria in deep subsurface sediments and their relation tosediment properties. Geomicrobiol J 7: 53–66.

Ginige MP, Hugenholtz P, Daims H, Wagner M, Keller J &Blackall LL (2004) Use of stable-isotope probing, full-cyclerRNA analysis, and fluorescence in situ hybridization-microautoradiography to study a methanol-fed denitrifyingmicrobial community. Appl Environ Microbiol 70: 588–596.

Gontang EA, Fenical W & Jensen PR (2007) Phylogeneticdiversity of gram-positive bacteria cultured from marinesediments. Appl Environ Microbiol 73: 3272–3282.

Greskowiak J, Prommer H, Vanderzalm J, Pavelic P & DillonP (2005) Modeling of carbon cycling and biogeochemicalchanges during injection and recovery of reclaimed water atBolivar, South Australia. Water Resour Res 41: W10418.

Guo GX, Deng H, Qiao M, Mu YJ & Zhu YG (2011) Effect ofpyrene on denitrification activity and abundance andcomposition of denitrifying community in an agriculturalsoil. Environ Pollut 159: 1886–1895.

Harvey RW, Smith RL & George L (1984) Effect of organiccontamination upon microbial distributions andheterotrophic uptake in a cape-cod, mass, aquifer. ApplEnviron Microbiol 48: 1197–1202.

Heylen K, Gevers D, Vanparys B, Wittebolle L, Geets J, BoonN & De Vos P (2006) The incidence of nirS and nirK andtheir genetic heterogeneity in cultivated denitrifiers. EnvironMicrobiol 8: 2012–2021.

Hill VR, Polaczyk AL, Hahn D, Narayanan J, Cromeans TL,Roberts JM & Amburgey JE (2005) Development of a rapidmethod for simultaneous recovery of diverse microbes indrinking water by ultrafiltration with sodium polyphosphateand surfactants. Appl Environ Microbiol 71: 6878–6884.

Hollibaugh JT, Budinoff C, Hollibaugh RA, Ransom B & BanoN (2006) Sulfide oxidation coupled to arsenate reduction bya diverse microbial community in a Soda Lake. ApplEnviron Microbiol 72: 2043–2049.

Kimura H, Nashimoto H, Shimizu M, Hattori S, Yamada K,Koba K, Yoshida N & Kato K (2010) Microbial methaneproduction in deep aquifer associated with the accretionaryprism in Southwest Japan. ISME J 4:531–541.

La Rosa G, De Carolis E, Sali M, Papacchini M, Riccardi C,Mansi A, Papa E, Alquati C, Bestetti G & Muscillo M(2006) Genetic diversity of bacterial strains isolated fromsoils, contaminated with polycyclic aromatic hydrocarbons,by 16S rRNA gene sequencing and amplified fragmentlength polymorphism fingerprinting. Microbiol Res 161:150–157.

La Scola B, Mallet MN, Grimont PAD & Raoult D (2003)Bosea eneae sp nov., Bosea massiliensis sp nov and Boseavestrisii sp nov., isolated from hospital water supplies, andemendation of the genus Bosea (Das et al. 1996). Int J SystEvol Microbiol 53: 15–20.

Lane DJ (1991) 16S/23S rRNA sequencing. Nucleic Acid Techniquesin Bacterial Systematics (Goodfellow M & Stackebrandt E, eds),pp. 115–175. John Wiley & Sons, New York.

Lanoil B, Skidmore M, Priscu JC, Han S, Foo W, Vogel SW,Tulaczyk S & Engelhardt H (2009) Bacteria beneath theWest Antarctic Ice Sheet. Environ Microbiol 11: 609–615.

Li D, Sharp JO, Saikaly PE, Dai J, Wang Y, Tang XL, Li J, SunTJ & Fang CX (2012) Dissolved organic carbon influencesmicrobial community composition and diversity inmanaged aquifer recharge systems. Appl Environ Microbiol78: 6819–6828.



Liu M, Luo XS, Zhang L, Dai J, Wang Y, Tang YL, Li J, SunTJ & Fang CX (2009) Pseudomonas xinjiangensis sp nov., amoderately thermotolerant bacterium isolated from desertsand. Int J Syst Evol Microbiol 59: 1286–1289.

Ludwig W, Strunk O, Westram R et al. (2004) ARB: asoftware environment for sequence data. Nucleic Acids Res32: 1363–1371.

Maimaiti J, Zhang Y, Yang J, Cen YP, Layzell DB, Peoples M& Dong ZM (2007) Isolation and characterization ofhydrogen-oxidizing bacteria induced following exposure ofsoil to hydrogen gas and their impact on plant growth.Environ Microbiol 9: 435–444.

Martino DP, Grossman EL, Ulrich GA, Burger KC,Schlichenmeyer JL, Suflita JM & Ammerman JW (1998)Microbial abundance and activity in a low-conductivityaquifer system in east-central Texas.Microb Ecol 35: 224–234.

Mukhopadhyay A, Al-Awadi E, AlSenafy MN & Smith PC(1998) Laboratory investigations of compatibility of theDammam Formation Aquifer with desalinated freshwater ata pilot recharge site in Kuwait. J Arid Environ 40: 27–42.

Musslewhite CL, McInerney MJ, Dong HL, Onstott TC,Green-Blum M, Swift D, Macnaughton S, White DC,Murray C & Chien YJ (2003) The factors controllingmicrobial distribution and activity in the shallow subsurface.Geomicrobiol J 20: 245–261.

Navarro J, Moser D, Flores A, Ross C, Rosen M, Dong HL,Zhang GG & Hedlund B (2009) Bacterial succession withinan ephemeral hypereutrophic Mojave Desert Playa Lake.Microb Ecol 57: 307–320.

Patterson BM, Shackleton M, Furness AJ, Pearce J,Descourvieres C, Linge KL, Busetti F & Spadek T (2010)Fate of nine recycled water trace organic contaminants andmetal(loid)s during managed aquifer recharge into aanaerobic aquifer: column studies. Water Res 44: 1471–1481.

Patterson BM, Pitoi MM, Furness AJ, Bastow TP &McKinley AJ(2012) Fate of N-Nitrosodimethylamine in recycled water afterrecharge into anaerobic aquifer.Water Res 46: 1260–1272.

Pedersen K (1993) The deep subterranean biosphere. Earth SciRev 34: 243–260.

Pitoi MM, Patterson BM, Furness AJ, Bastow TP & McKinleyAJ (2011) Fate of N-nitrosomorpholine in an anaerobicaquifer used for managed aquifer recharge: a column study.Water Res 45: 2550–2560.

Playford PE, Cockbain AE & Low GH (1976) Geology of thePerth Basin, Western Australia. Geological Survey of WA,Bulletin, 311p.

Pyne RDG (1995) Groundwater Recharge and Wells: A Guide toAquifer Storage Recovery. Lewis Publishers, Boca Raton.

Ryu SH, Park M, Jeon Y, Lee JR, Park W & Jeon CO (2007)Flavobacterium filum sp nov., isolated from a wastewatertreatment plant in Korea. Int J Syst Evol Microbiol 57: 2026–2030.

Saito T, Ishii S, Otsuka S, Nishiyama M & Senoo K (2008)Identification of novel Betaproteobacteria in a succinateassimilating population in denitrifying rice paddy soil byusing stable isotope probing. Microbes Environ 23:192–200.

Santini JM, Sly LI, Wen AM, Comrie D, De Wulf-Durand P &Macy JM (2002) New arsenite-oxidizing bacteria isolatedfrom Australian gold mining environments – phylogeneticrelationships. Geomicrobiol J 19: 67–76.

Scholten E, Lukow T, Auling G, Kroppenstedt RM, Rainey FA& Diekmann H (1999) Thauera mechernichensis sp nov., anaerobic denitrifier from a leachate treatment plant. Int J SystBacteriol 49: 1045–1051.

Sheng ZP (2005) An aquifer storage and recovery system withreclaimed wastewater to preserve native groundwaterresources in El Paso, Texas. J Environ Manag 75: 367–377.

Singleton DR, Furlong MA, Rathbun SL & Whitman WB(2001) Quantitative comparisons of 16S rRNA genesequence libraries from environmental samples. ApplEnviron Microbiol 67: 4374–4376.

Stevens T (1997) Lithoautotrophy in the subsurface. FEMSMicrobiol Rev 20: 327–337.

Stubner S,WindT&ConradR (1998) Sulfur oxidation in rice fieldsoil: activity, enumeration, isolation and characterization ofthiosulfate-oxidizing bacteria. Syst ApplMicrobiol 21: 569–578.

Stuyfzand PJ (1998) Quality changes upon injection into anoxicaquifers in the Netherlands: evaluation of 11 experiments. 3rdInternational Symposium. Artificial Recharge of Groundwater.(Peters JH, ed), pp. 283–291. Balkema, Rotterdam.

Takeuchi M, Komai T, Hanada S, Tamaki H, Tanabe S,Miyachi Y, Uchiyama M, Nakazawa T, Kimura K &Kamagata Y (2009) Bacterial and archaeal 16S rRNA genesin late pleistocene to holocene muddy sediments from theKanto plain of Japan. Geomicrobiol J 26: 104–118.

Water Corporation (2009) Site Characterisation Report:Groundwater Replenishment Trial. Water Corporation, Perth.

Weon HY, Song MH, Son JA, Kim BY, Kwon SW, Go SJ &Stackebrandt E (2007) Flavobacterium terrae sp nov andFlavobacterium cucumis sp nov., isolated from greenhousesoil. Int J Syst Evol Microbiol 57: 1594–1598.

West JM, Christofi N & Mckinley IG (1985) An overview ofrecent microbiological research relevant to the geologicaldisposal of nuclear waste. Radioact Waste Manag 6: 79–95.

Williams MM, Domingo JWS, Meckes MC, Kelty CA & RochonHS (2004) Phylogenetic diversity of drinking water bacteria ina distribution system simulator. J Appl Microbiol 96: 954–964.

Yoon JH, Kang SJ, Park S & Oh TK (2007) Devosia insulae spnov., isolated from soil, and emended description of thegenus Devosia. Int J Syst Evol Microbiol 57: 1310–1314.

Yoshida M, Ishii S, Fujii D, Otsuka S & Senoo K (2012)Identification of active denitrifiers in rice paddy soil by DNAand RNA based analyses. Microbes Environ 27: 456–461.

Yu HY, Yuan ML, Lu W et al. (2011) Complete genomesequence of the nitrogen-fixing and rhizosphere-associatedbacterium Pseudomonas stutzeri strain DSM4166. J Bacteriol193: 3422–3423.

Zhang YC, Slomp CP, Broers HP, Bostick B, Passier HF,Bottcher ME, Omoregie EO, Lloyd JR, Polya DA & VanCappellen P (2012) Isotopic and microbiological signaturesof pyrite-driven denitrification in a sandy aquifer. ChemGeol 300: 123–132.



Documents

Bacterial community and groundwater quality changes in an anaerobic aquifer during groundwater recharge with aerobic recycled water