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Systematic and Applied Microbiology 34 (2011) 127–138

Contents lists available at ScienceDirect

Systematic and Applied Microbiology

journa l homepage: www.e lsev ier .de /syapm

icrobial ecology of autothermal thermophilic aerobic digesterATAD) systems for treating waste activated sludge

avid Hayes, Leonard Izzard1, Robert Seviour ∗

iotechnology Research Centre, La Trobe University, Bendigo, VIC 3552, Australia

r t i c l e i n f o

rticle history:eceived 3 June 2010

eywords:aste activated sludge

TADhermus thermophilus

a b s t r a c t

Despite their widespread use, our understanding of the microbial ecology of the autothermal thermophilicaerobic digesters (ATAD) used to dispose of sludge from wastewater treatment plants is poor. Applyingboth culture-dependent and molecular methods to two ATAD systems in Victoria, Australia treatingdifferent wastewaters revealed that their communities were highly specialized. Denaturing gradientgel electrophoresis (DGGE) profiling suggested differences in their population compositions and bothchanged over time. However, both showed low level biodiversity, and contained several novel bacterial

ISH/MARGGE

populations. 16S rRNA clone library data and FISH analyses showed that Thermus thermophilus dominatedboth communities and that of a third ATAD plant in NSW (more than 90% of the total bacterial biovolumein repeated samples taken from each of the three ATAD plants). Culture-dependent methods also showedGeobacillus spp. were present in both Victorian communities. Nevertheless, the ecophysiology of thesepopulations and their putative roles in sludge digestion remain unclear. FISH/microautoradiographicstudies did not provide conclusive data elucidating which substrate/s T. thermophilus might utilize in the

C

ATAD reactors.

ntroduction

Most activated sludge plants generate large volumes of sec-ndary sludge, which is generally viewed as a waste product of therocess, and it is expensive to treat [24,68]. Secondary sludge, oraste activated sludge, usually contains heavy metals, pathogenic

iruses and bacteria, all at levels which may limit how it is even-ually handled [27,82]. Different disposal methods have been usedn attempts to maximize the economic value of this material andach has its advantages, but the most popular method in countrieshere land space is available is its application to land as a fertilizer

5,43]. However, land disposal requires some pre-treatment to sta-ilize the sludge to minimize odour production and reduce the levelf pathogens before its application [46,62]. In addition to anaerobicigestion, other popular full-scale stabilization methods in currentse include chemical treatment, thermal drying and composting5,82], each of which varies in its costs, land requirements, and

ffectiveness in reducing the pathogen and toxic chemical levels10,29].

One potentially attractive method is autothermal thermophilicerobic digestion (ATAD). The systems using this method were

∗ Corresponding author. Tel.: +61 354447459; fax: +61 354447476.E-mail address: r.seviour@latrobe.edu.au (R. Seviour).

1 Present address: The Australian Rickettsial Reference Laboratory,eelong Hospital, Geelong, VIC 3220, Australia.

723-2020/$ – see front matter. Crown Copyright © 2011 Published by Elsevier GmbH. Aoi:10.1016/j.syapm.2010.11.017

rown Copyright © 2011 Published by Elsevier GmbH. All rights reserved.

designed originally from work in Europe and North America[37,83] and were commercially patented and implemented first inGermany in 1980 [26]. ATAD relies on the solubilisation of suffi-cient oxygen in a reactor so that the resulting high levels of aerobicbacterial metabolic activity generate a high operating tempera-ture (>60 ◦C), leading to rapid inactivation of viral and mesophilicbacterial pathogens, such as Salmonella and other Enterobacteri-aceae, in the sludge [29,66,86]. Thus, the benefits claimed for ATADsystems include production of a safe, high quality end product suit-able for land application [42,86], and a substantial reduction involatile solids levels [67]. Furthermore, much shorter sludge reten-tion times compared to conventional composting protocols meanthat the system has very low space requirements [77].

However, this system is not without its critics. Operating prob-lems include difficulties in dewatering the sludge from ATADreactors, incurring corresponding increases in costs [1,50]. Fre-quent episodes of foaming in the reactors can also occur [41,42],and difficulties in supplying enough oxygen to maintain aero-bic conditions in bioreactors run at temperatures usually above60 ◦C mean that these ATAD processes become anaerobic, leadingto microbial formation of odorous compounds, such as mercap-tans and dimethyl sulphides [11,33]. While these systems have

attracted the interest of engineers [42], surprisingly little is knownabout the microbial composition of the ATAD communities [68].Culture-dependent studies with laboratory [32], pilot [73], and fullscale ATADs [56], treating domestic waste sludge have all detected

ll rights reserved.

128 D. Hayes et al. / Systematic and Applied

Table 1Operational features for Victorian ATAD plants studied.

Parameter Castlemaine Bendigo

Population equivalent(p.e)

40,000 120,000

Source feed sludge On-site EBPRsecondarysludge

On-site EBPRsecondarysludge

Number of treatmentstages; number ofreactors;volume/reactor (m3)

II; 3; 65 m3 II; 3; 120 m3

Hydraulic retentiontime (HRT)

6–10 days 6–10 days

Operating temperature(◦C)

50–70 50–70

Storage facility forATAD treated sludge

Transported toBendigo

120 m3

concrete Tank

Gfslsuts(bmc

M

D

wfbTaiwitcfibccpfdiartti

S

t

Facility (30 kmnorth)

Biosolids disposalmethod

Landapplication

Landapplication

eobacillus spp. and Bacillus spp., while Thermus ruber was isolatedrom one system [32]. In the only published culture-independenttudy of a full scale batch-fed ATAD system, the 16S rRNA cloneibrary generated contained sequences closely related to Bacilluspp. and Paenibacillus spp. [58]. This study therefore undertook tose culture-dependent and culture-independent methods to iden-ify which bacterial populations were present in two full scale ATADystems and, by a combination of fluorescence in situ hybridizationFISH) and microautoradiography (MAR), attempted to understandetter their in situ physiology in the hope that this informationight eventually enable these processes to be better operated and

ontrolled.

aterials and methods

esign and performance criteria of the ATAD systems

Two ATAD systems were used for most of this study. Theyere operating to dispose of the waste activated sludge (WAS)

rom two enhanced biological phosphate removal (EBPR) plants,oth of which were configured as modified University of Capeown (MUCT) systems. The ATAD at Castlemaine, Victoria treatedmixture of domestic wastes and effluent from a meat process-

ng plant (approximately 80% domestic and 20% meat processingastewater), while that at Bendigo, Victoria handled predom-

nantly domestic sewage. Both ATAD facilities were configuredo operate as two stage continuous, and when required, semi-ontinuous systems. In both plants, the waste activated sludge wasrst thickened using conventional gravity settling (CGS) followedy dissolved air flotation (DAF), assisted by addition of Ciba® Zetag®

ationic polyelectrolyte [51], to give an influent total solids (TS)ontent of 6.3 (±0.5)%. The key operational features of these twolants are compared in Table 1. Regular monthly analyses for theaecal indicator organisms E. coli and Enterococci always failed toetect their presence, suggesting that the process successfully elim-

nated enteric pathogenic bacteria. The microbial community fromthird two stage ATAD plant in Gerroa, NSW incorporating spi-

al aeration technology, where mixers can also act to draw air intohe reactor, as opposed to the Victorian ATAD plants that main-ained aerobic conditions by mechanical mixing of pure oxygenntroduced through static aerators, was also examined.

ample collection

Sludge samples were collected from ATAD stages I and II (reac-ors 2 and 3) from the plants at Bendigo, Castlemaine, and Gerroa,

Microbiology 34 (2011) 127–138

NSW. All were immediately placed on ice. Additional samples forlater analysis using FISH, were collected at the same locations, andfixed immediately with ethanol or paraformaldehyde [84]. The pH,temperatures, and redox potentials of each reactor system weremeasured continuously in situ. Sludge samples for DNA extractionwere stored at 4 ◦C for no more than 24 h before being analysed,while those fixed for FISH were kept at −20 ◦C. Samples for denatur-ing gradient gel electrophoresis (DGGE) analysis and ecophysiologystudies were taken from ATAD reactor 2 (stage I) at Bendigo andCastlemaine. Samples collected from reactor 2 were chosen sinceregular microscopic examination of samples from reactor 1 alwaysrevealed the presence of many viable aerobic mesophiles and fil-amentous bacteria carried over in the secondary sludge from thefinal aerobic basin of the treatment plants.

Isolation and identification of pure cultures from ATAD sludge

Isolates were cultured from ATAD samples by streaking onto acomplex medium previously used to isolate Thermus sp. [81], whichwas chosen because clone library and FISH analyses (see later) sug-gested that Thermus spp. were dominant community members.Plates were incubated at 65 ◦C for 24 h. Single, often spread-ing, colonies, differing in their appearance, were then re-streakedrepeatedly onto the same medium until eventually Gram stainingsuggested they were pure. Their genomic DNA was extracted witha MoBio Laboratories UltraClean® Soil DNA Extraction Kit and 16SrRNA genes were PCR amplified with the universal primers 27F and1525R [40], subjected to agarose gel electrophoresis and the bandsof the appropriate size (about 1500 bp) were cut from the gel andpurified with Qiagen QIAquick® Gel Extraction kits. The genes werecloned using the pGEM®-T Easy Vector system (Promega, USA).Inserts were then sequenced with the M13F and M13R plasmid spe-cific primers [49], and 926F, 907R, 519R and 530F primers [40], andthe Applied Biosystems PRISM BigDye 3.1 Terminator ChemistryKit. Amplicons were sent to the Australian Genome Research Facil-ity (AGRF), University of Queensland for sequencing, and the resultswere analysed with Biomatters Ltd, Geneious Pro 4.0.4 softwareand the BLAST [2] facility available from www.ncbi.nih.gov/blast.Phylogenetic trees were constructed with the maximum likeli-hood [21], maximum parsimony [38] and neighbour joining [63]algorithms. Using the Jukes and Cantor [36] distance model, anevolutionary distance tree was created from the neighbour joiningmethod. The SEQBOOT and CONSENSE options from PHYLIP [22]were used to determine bootstrap values (as percentages of 1000replications).

DNA extraction and amplification from ATAD biomass

ATAD sludge samples were pre-washed following the proto-col of Purohit et al. [59], in order to remove humic substances.Genomic DNA was then extracted from each sample with a MoBioLaboratories UltraClean® Soil DNA Extraction Kit following themanufacturer’s protocol and stored at −20 ◦C. This extractionmethod provided large amounts of high quality non-sheared DNAfrom all ATAD samples examined. Amplification of 16S rRNA geneswas performed with Applied Biosystems Amplitaq Gold® DNApolymerase, PCR primers 27F and 1525R [40] and the followingprotocol: 94 ◦C (7 min), followed by 35 cycles of 94 ◦C (1 min), 50 ◦C(50 s), 72 ◦C (90 s) and a final extension step of 72 ◦C for 10 min. PCRamplicons were cloned with the Promega pGEM®-T Easy VectorSystem.

16S rRNA gene clone library construction

Plasmids were extracted with the Promega Wizard® SVMiniprep DNA purification kit and inserts (one hundred for each

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lone library) were sent for sequencing. Sequences were anal-sed and phylogenetic trees constructed as described previously.perational taxonomic units (OTUs) were generated with distanceatrices obtained using the Jukes and Cantor [36] model in PHYLIP

22] as input for DOTUR [64]. Sequences assigned to individualTUs were those sharing 99% sequence similarity using the furthesteighbour algorithm. Assembled sequences were checked individ-ally for chimeras against closest matches from GenBank by visual

nspection of ClustalW [78] alignments of all sequences from cloneibraries and the pure cultures. Pintail [6] was used after collectivenalyses of sequences with Bellerophon version 3 [35], and the Mal-ard program [7]. When similarities of sequenced inserts from eachlone library to all other sequence entries in GenBank was less than0%, PCR primers targeting these were designed with the primeresign feature of Geneious. If a positive match occurred repeatedlyetween the sequence in the clone library and an amplicon from theifferent biomass samples, the probability of this cloned sequenceeing chimeric was considered to be reduced.

Accession numbers for sequences submitted to EMBLhttp://www.ebi.ac.uk/embl/) were: clone otuB1 (FN687452);lone c13 (FN687453); clone c18 (FN687454); clone otuB2FN687450); clone otuC2 (FN687451); clone c26 (FN687455); clone51 (FN687456); clone b32 (FN687457); clone c22 (FN687458).

iodiversity analysis

The homogenous coverage (C) was calculated, where C%) = [1 − n1/N] × 100 [69], and where n1 is the total number ofTUs with only one sequence and N is the total number of 16S

RNA cloned genes analysed. The minimum number of species (S)as estimated by dividing the number of OTUs into (C) [39].

enerating 16S rRNA DGGE profiles

The hypervariable V3 region of bacterial and archaeal 16S rRNAenes was PCR amplified from template DNA using Amplitaq®

old DNA polymerase. This region was preferred to the V6 or V9egions. DGGE based DNA fragment separation depends on differ-nces in the G + C mol% content, and analysis of 16S rRNA clonedequences from the ATAD communities (see later) revealed larger+ C mol% variations in the V3 region than the V6 or V9 regions.

urthermore, other studies have shown that this V3 region pro-ides improved resolution [23,85]. Primers 341F, with a 40 bp GClamp [52] attached to the 5′ end, and 534R were used for the Bac-eria, while PARCH340F, with a 40 bp GC clamp [52] attached to the′ end, and PARCH519R were used for Archaea [57]. The PCR proto-ol was: 94 ◦C (4 min), followed by 33 cycles of 94 ◦C (1 min), 53 ◦C30 s), 72 ◦C (50 s) and a final extension step of 72 ◦C for 8 min. DGGErofiling was performed with a Bio-Rad DCode Universal Mutationetection System. Denaturing gradient gels were formed using aio-Rad 475 gradient delivery system. An electric motor mountedn an adjustable platform connected to the rotation wheel with aubber drive belt was incorporated in attempts to improve gra-ient reproducibility and quality. Approximately 320 ng of eachCR product was loaded into each well of a 12% (w/v) acrylamideel (acrylamide/bis acrylamide solution, 37.5:1) containing a lin-ar chemical gradient of 40% to 90% denaturant (with 100% = 7 Mrea and 40% (v/v) formamide). Electrophoresis was for 18 h at0 V (1080 V.h) in 1 × TAE electrophoresis buffer at 57 ◦C. Gels weretained subsequently with Invitrogen SYBR Gold DNA stain andhotographed on an Alpha Innotech UV transilluminator at 302 nm.

tatistical analysis of DGGE profiles

Statistical analysis of DGGE band patterns was conducted withhe similarity coefficient (SD) of Dice [20]. GelComparII software

Microbiology 34 (2011) 127–138 129

(Applied Maths, Sint-Martens-Latem, Belgium) was used for bandallocation, and a dendrogram was constructed with NCSS statisticalsoftware and unweighted pair-group arithmetic average clustering(UPGMA) was conducted [71]. Levels of correlation between theoriginal similarity (SD) matrix and the chosen clustering methodwere assessed with the cophenetic correlation index of Sokal andRohlf [72]. Similarity estimations between the Castlemaine andBendigo DGGE profiles were calculated using (SD), and means of(SD) values ± one standard deviation were determined.

Identification of bacteria by fluorescence in situ hybridisation(FISH)

ATAD samples and cultured isolates were probed for bacte-ria with the 16S or 23S rRNA targeted FISH probes (Table 2). Thedesign of probe A180 used sequence data from the clone librariesand ARB software [45]. This probe was validated by testing it onseveral ATAD samples at formamide concentrations increasing in5% increments from 0% to 60%. Unlabelled oligonucleotide helperprobes [25] were also designed to target sequence positions adja-cent to the A180 target site spanning helices 8–11 in order toimprove in situ probe accessibility [25]. The probe Tth for iden-tifying Thermus thermophilus [12] was validated using a culturedisolate obtained from the Bendigo ATAD. Semi-quantitative analy-ses of FISH images were conducted by counting a minimum of 500cells responding to the EUBmix probes and using the calculationoverlay feature on Adobe® Photoshop® to estimate the percent-age contribution of cells responding to the Tth probe to the totalcell biovolume estimated by using EUBmix probes [65]. All probeswere labelled with either Fluos or Cy3 fluorochromes purchasedfrom ProOligo, Australia. The method for FISH was modified fromAmann [4]. In brief, 2 �L sludge samples were smeared onto eachcircular well of microscope slides treated with VectabondTM andleft to air dry. Smears were dehydrated with ethanol and 10 �Lof appropriate hybridization buffer were then placed on each welltogether with 1 �L of a 50 ng �L−1 stock solution of the appropriatefluorescent probe. Samples were incubated for 2 h in a hybridi-sation chamber at 46 ◦C, before being viewed microscopically.The FISH procedure used for ATAD sludge during microautora-diography (MAR), see later, varied where microscope slides with0.1% gelatine (w/v) coated cover slips were used, as described byLee et al. [44].

In situ physiology of populations in ATAD communities bycombination of FISH with microautoradiography (FISH/MAR)

The method was modified from that described by Lee et al.[44]. As ATAD sludge samples were thick, they were dilutedwith filtered ATAD supernatant to a suspended solids (SS) con-centration of 2 mg mL−1. Labelled compounds used for the MARexperiments included [1-14C]acetic acid, l-[2,3-3H]aspartic acid,[6-3H]glucose, [9,10(n)-3H]oleic acid, [1(3)-3H]glycerol, and l-[G-3H]glutamic acid. Final concentrations of 2 mM for the combinedlabelled and unlabelled stocks of substrates were used, with sub-strate specific activities all standardised at 20 �Ci each. Sampleswere pre-incubated for 30 min to reduce any residual substratelevels [44], before unlabelled and labelled substrates were added.Initially aerobic and anaerobic MAR incubations were performedat 65 ◦C, which was the mean operating temperature of the sec-ond thermophilic reactor for both Bendigo and Castlemaine ATADplants. Substrate uptake rates were determined at several tem-

peratures by measuring �-emissions with a Wallac, Finland 1450Microbeta PLUS Liquid Scintillation counter. Sterile oxygen wasbubbled into each incubation mix to maintain aerobic conditions.MAR reactions were terminated and samples were prepared foranalysis as described by Lee et al. [44]. Smears were prepared on

130 D. Hayes et al. / Systematic and Applied Microbiology 34 (2011) 127–138

Table 2List of 16S rRNA and 23S rRNA targeted FISH probes used in this study.

Probe Name Target Organism Sequence 5′–3′ Ref. FA (%)

EUB338 I Most Bacteria GCTGCCTCCCGTAGGAGT [3] 0–60EUB338 II Planctomycetales GCAGCCACCCGTAGGTGT [19] 0–60EUB338 III Verrcomicrobiales GCTGCCACCCGTAGGTGT [18] 0–60LGC354a Firmicutes TGGAAGATTCCCTACTGC [48] 35LGC354b Firmicutes CGGAAGATTCCCTACTGC [48] 35LGC354c Firmicutes CCGAAGATTCCCTACTGC [48] 35HGC69a Actinobacteria TATAGTTACCACCGCCGT Competitor:

TATAGTTACGGCCGCCGT[61] 25

ALF968 Alphaproteobacteria GGTAAGGTTCTGCGCGTT [47] 20BET42a Betaproteobacteria GCCTTCCCACTTCGTTT Competitor:

GCCTTCCCACATCGTTT[47] 35

GAM42a Gammaproteobacteria GCCTTCCCACATCGTTT Competitor:GCCTTCCCACTTCGTTT

[47] 35

Delta495a Deltaproteobacteria AGTTAGCCGGTGCTTCCT [47] 35Tbcil832 Bacillus spp. and Geobacillus spp. GGGTGTGACCCCTCTAAC [30] 40THUS438 Thermus spp. GGGTTTCGTCCCGGGTTC [30] 60Tth T. thermophilus TCCCCGTTGCCGGGTGGC [12] 25Trub T. ruber TCCCCGTTACCGGGTCGC [12] 25Taq T. aquaticus TCCCTGTTGCCAGGTCGC [12] 20

CCTGTCACCCGGGCGGGA

0a

R

Cp

fiormitfpbitofl

bypmsbBuflJsstpCDsm

Tfili T. filiformis TCA180 OTUs B1 and C1 TA

TGAA

.1% gelatine coated coverslips and left to air dry for later FISHnalysis, as detailed above.

esults

omposition of ATAD communities in Bendigo and Castlemainelants by DGGE profiling

DGGE profiling of 16S rRNA V3 region fragments was used toollow changes in the composition of the bacterial communitiesn reactor 2 (ATAD stage I) at Bendigo and Castlemaine plantsver a 12-month period. No amplicons were produced when 16SRNA primers targeting the Archaea were used. Examples of DGGEonthly profiles from both ATAD bacterial communities are shown

n Fig. 1, and include a dendrogram representing the UPGMA clus-ering results for the Castlemaine similarity (SD) data. While manyainter bands in these profiles varied in their occurrence during thiseriod, more emphasis is placed here on the strongly fluorescingands, since these were those whose identity was sought. However,

t is recognised that PCR biases mean these are not necessarily fromhe dominant populations in the communities [23,34]. The majorityf bands marked with the prefix ‘a’ are those adjudged as stronglyuorescing.

While all DGGE profiles were similar, clear differences existedetween and within the two communities over the course of theear. For example, band a1 appeared consistently in all Bendigorofiles, yet was absent from all but one (July) of those from Castle-aine, and band a2 was detected only in Bendigo profiles from

amples taken in May, August, and April. Band a3 was common tooth communities but more strongly fluoresced in samples fromendigo, while Band a4 was visible in Castlemaine profiles for Jan-ary and February (February data not shown) and showed lessuorescence intensity in profiles of several other samples (e.g.,

uly and August). Band a6 was seen in profiles generated fromamples taken in July and September (data for September nothown) only, while bands a7 and a8 were present exclusively inhe Bendigo sample profiles, with one exception being in the July

rofile of the Castlemaine samples. Band a9 was present only inastlemaine profiles for January, February, August, November andecember (November and February data not shown). While the

trongly fluorescing band a10 was present from both ATAD com-unities throughout, band a11, with a similar mobility to band

CACCAGGTCGC [12] 20AAAAGGTGAC Helper 3′–5′:

TAATACCCG Helper 5′–3′:GGCGGAAGC

This study –

a10, appeared with less frequency and fluorescence intensity inboth. When all available plant operational data were examined forthis sampling period, no obvious explanation for why there weremarked changes in community composition was forthcoming. Forexample, no corresponding shifts in continuously monitored oper-ating temperature, redox or loading rates (inflows, COD) coincidedwith the appearance or otherwise of any of these major bands.

Isolation and identification of cultured bacteria from ATADcommunities

Nine strains from Bendigo and eight from Castlemaine were iso-lated into pure cultures. 16S rRNA sequence analyses revealed thatthose from both ATAD systems were very similar, with 16 of the17 isolates being identified as members of the genus Geobacillus,which was constructed to accommodate members of the RNA group5 thermophilic Bacillus spp. [53]. All isolates were identified asexisting species, with most being closely related to G. thermodenitri-ficans originally isolated from compost [54]. Three isolates emergedas being most closely related to G. caldoxylosilyticus strain BGSCW98A2 and G. toebii, isolated from soil and composting hay, respec-tively [76]. The other isolate obtained only from Bendigo was T.thermophilus strain L1 with an identical 16S rRNA sequence (acces-sion number AY788091) to that of the T. thermophilus detectedin both clone libraries (Fig. 3), and whose 16S rRNA sequencediffered from the T. thermophilus HB8 type strain (99% similar-ity). A phylogenetic tree based on 1449 bp of the 16S rRNA genesof these isolates and validly described species of Geobacillus isshown in Fig. 2.

16S rRNA gene clone library analyses of Bendigo and CastlemaineATAD communities

A total of 200 clones (100 from each ATAD plant) were generatedfrom DNA extracted from samples collected in August 2004 fromthe second reactor at the Bendigo and Castlemaine plants. Phyloge-netic trees constructed for the cloned 16S rRNA gene sequences and

closest affiliates obtained from the GenBank database are shownin Fig. 3. Both clone libraries were dominated by 16S rRNA genesequences from T. thermophilus (OTU C1 – 43 sequences and OUTB2 – 57 sequences), all of which were identical (>99%) to thesequence obtained from the T. thermophilus isolate L1 (AY788091)

D. Hayes et al. / Systematic and Applied Microbiology 34 (2011) 127–138 131

F pling f1 tor 2)

cctSnC(

ig. 1. (a) UPGMA cluster dendrogram presented as distances (1 − SD) for each sam6S rRNA gene fragments extracted from biomass samples from ATAD stage I (reac

ultured from the Bendigo community (see above). They were lesslosely related (98%) to sequences from the T. thermophilus HB8

ype strain (M26923), isolated from a Japanese thermal spa [55].equences only distantly related (87%) to Thermaerobacter subterra-eous species also featured dominantly in both clone libraries (OTU3 – 17 sequences and OTU B1 – 24 sequences). Those from OTU C219 sequences) were most closely related to uncultured bacterial

rom the Castlemaine ATAD stage I (reactor 2), (b) DGGE profiles of the V3 region ofat Bendigo and Castlemaine taken at monthly intervals.

sequences with a high (>97%) similarity to those from members ofthe Gemmatimonadetes. Sequences occurring less frequently in the

Castlemaine and Bendigo clone libraries (Fig. 3) were also detected.These included that from clone cas18, which was closely related(>97%) to Thermobacillus xylanticus, originally isolated from a com-posting manure heap [80]. The clone cas26 sequence was closelyrelated (>98%) to an uncultured bacterial clone SMC37 (AM183105)

132 D. Hayes et al. / Systematic and Applied Microbiology 34 (2011) 127–138

Geobacillus thermoleovorans isolate T70 (AJ489328) Geobacillus lituanicus strain BGSC W9A89 (AY608945)

Geobacillus kaustophilus strain BGSC 90A1 (AY608934) Geobacillus zalihae strain T1 (AY166603)

Geobacillus bogazici (AY323205) Geobacillus stearothermophilus strain BGSC 9A21 (AY297092)

Geobacillus thermocatenulatus isolate hs6 1 (AY550104) Geobacillus thermocatenulatus strain BGSC 93A1 (AY608935)

Geobacillus uzenensis strain U (T) (AF276304) Geobacillus jurassicus strain DS1 (AY312404)

Geobacillus subterraneus strain BGSC 91A1 (AY608956) Geobacillus uzenensis strain BGSC 92A2 (AY608959)

Geobacillus anatolicus strain SB (AF411064) Geobacillus thermodenitrificans strain S64 (AB116115)

Geobacillus a (10 isolates) Geobacillus toebii strain T1671 (AB116119)

Geobacillus toebii strain BGSC 99A1 (AY608982)

Geobacillus b (3 isolates) Geobacillus caldoxylosilyticus isolate Ti (AJ564617) Geobacillus caldoxylosilyticus isolate B70 (AJ489326)

Geobacillus caldoxylosilyticus strain BGSC W98A2 (AY608950)

Geobacillus c (3 isolates)

99

99

97

70

89

88

80

84

92

99

100

95

0.002

F ip be( % sequD .2% d

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ig. 2. Neighbour joining phylogenetic tree of 16S rRNA genes showing relationshidentified by accession numbers). Isolates are grouped into OTUs on the basis of 99OTUR program. Bootstrap values >70% are given at the nodes. Scale bar indicates 0

rom composting swine manure [28], while the sequence fromlone cas22 was most similar (98%) to Sporichthya polymorpha, aember of the Actinobacteria [60]. The clone ben32 sequence was

losely related to an uncultured bacterial clone 2B06 (DQ346478)rom composting swine slurry [14].

Sequences from both clone libraries revealed 33 putativehimeras. With representatives of OTUs C3, C2, and B1 sharing veryow similarity to other 16S rRNA gene sequences, current statistical

eans for chimera checking (e.g. [6]) can produce unreliable out-omes at large evolutionary distances [6]. Consequently, a primerTR526F’ (5′TAGGCGGCCTGTGAAGTC3′) targeting nucleotide posi-ion 526 of sequences representing OTUs C3 and B1 was designednd, in combination with the 16S rRNA primer 1492R, was used todentify sequences identical to those from OTUs C3 and B1 by PCR

ith template DNA extracted from all Bendigo and Castlemaineamples and another from the Gerroa NSW plant. Each PCR pro-uced amplicons of the predicted size (∼800 bp) whose sequencesere identical to the cloned representative sequences from OTUs3 and B1. This outcome suggested that the probability of theequences of members of these OTUs being chimeric was low.

The method was repeated for representative sequences fromTU C2, using a primer ‘GEM664F’ (5′GGTAGAGGCCGGTGGAAT′) designed to target their nucleotide position 664. Again, DNAxtracted from all samples, including those from Gerroa NSW, wassed as template for PCR. Only DNA from the Gerroa plant providedn amplicon whose sequence was identical to the cloned represen-ative sequence, but this result again suggested that the 16S rRNAequences from OTU C2 were not chimeric.

dentification of main DGGE fragments

Repeated attempts to excise and sequence these individualands were unsuccessful because of contamination problems, ando other approaches were used to identify them. The V3 regions

f the 16S rRNA genes of inserts in both clone libraries were PCRmplified and run on DGGE gels (Fig. 1). DGGE bands a11 and a10ad the same relative mobilities as bands clC2 and clB2, thus rep-esenting sequences from the distantly related Gemmatimonadetesnd T. thermophilus L1, respectively. Band c18, identified as Ther-

tween isolates cultured from ATAD samples to those from the GenBank databaseence similarity using the distance matrix from the PHYLIP package as input for the

ivergence.

mobacillus xylanticus, had the same migratory properties as banda6b, which appeared infrequently and with low fluorescence duringthe sampling period. Band a5, visible in Castlemaine profiles only,had the same mobility as band c26, identified in the clone libraryas being most similar to an uncultured bacterium clone found in asample of composting swine manure. The band c1C3 found in allprofiles, with the same mobility as the strongly fluorescing banda3, was closely related to that of Thermaerobacter subterraneous,a member of the Firmicutes. Bands c18, c26, and c13 (identifiedas being from Nitrospira spp.) co-migrated with bands a6b, a6,and a4, respectively. As both plants nitrify, their presence was notunexpected, but whether they were active at the ATAD operatingtemperatures was not resolved. Band b51 that was identified inthe clone library as an unidentified member of the Chloroflexi waspresent in all Bendigo DGGE profiles as band a1. The band u1 wasunidentified in the clone library and did not appear in any DGGEprofiles.

Levels of biodiversity in ATAD digesters

The statistical indicators C and S for the clone libraries sug-gested 93% homogenous coverage (C) of the species present. Basedon this value, the proposed minimum number (S) was 8 and 4 forthe Castlemaine and Bendigo plants, respectively, suggesting verylow population diversity within both communities. Analyses of theDGGE profiles detailed over 12 months (Fig. 1) from Castlemaineand Bendigo, showed the mean similarity (SD) between bacterialcommunities in each ATAD plant was 0.6 ± 0.1. Although thesevalues only indicated biodiversity similarity levels, a cautious inter-pretation might suggest a positive correlation between differencesin the predicted number of species (S) for each ATAD community(50%) from the clone library data and the consistency of similaritiesbetween DGGE profiles from the Castlemaine and Bendigo plants(60 ± 10%).

Analysis of ATAD communities with FISH

When repeated samples taken from communities of theBendigo, Castlemaine, and Gerroa ATAD systems were examined

plied

wshFrltpeowatBrnqdie

Fru

D. Hayes et al. / Systematic and Ap

ith FISH probes targeting members of the main bacterial divi-ions, (85 ± 5%) of the cells responding to the EUB338mix probeybridised with probe Tth targeting T. thermophilus. The lack of anyISH signals with probes targeting populations of the Proteobacte-ia in the ATAD communities supported the data from the cloneibrary analyses, which was not surprising in systems operating athese high temperatures [19,74]. The finding supports the monthlylant analysis data that enteric bacterial pathogens were probablyffectively eliminated during the ATAD process. A lack of cells flu-rescing with probe HGC69a also suggested that the Actinobacteriaere not numerically important in these communities. The LGC354

, b, and c probes showed few positive cells despite the presence ofhe Thermaerobacter-related organism (OTUs B1 and C3) in both theendigo and Castlemaine clone libraries. However, the cloned 16SRNA gene sequences for members of both these OTUs contained 2ucleotide mismatches to those of all three LGC354 probes. Conse-

uently, a 16S rRNA targeted FISH probe, A180 (see Table 2), wasesigned using ARB [46] to identify these populations in situ, but

ts application failed to detect any cells, despite several attempts toliminate any possible FISH permeabilisation problems [13]. Bear-

aUncultured bacterium clone SMG

Uncultured compost bacterium c

Clone ben32 (1 sequence)

Uncultured compost bacterium

Uncul UUncultured ThermoanaerobacteOTU B1 (24 sequences)

Uncultured bacterium clone 7

Thermaerobacter litoralis strain KW1

Thermaerobacter nagasakiensis strain

Thermaerobacter subterraneus strain

Thermaerobacter subterraneus strain m

Uncultured bacterium

Uncultured ChlorofleUncultured bacterium

Uncultured bacteriu

Uncultured ChlorofleUncultured ChloroflexUncultured bacterium

Clone ben51 (1 sequ

Thermus filiformis (L09667)

Thermus rehai (AF331969)

Thermus yunnanensis strain RH071

Thermus scotoductus strain ITI-252T

Thermus igniterrae strain GE-2 (Y18

Thermus brockianus strain 15038T (Y

Thermus aquaticus strain YT-1NR (02

Thermus flavus AT-62 (L09660)Thermus thermophilus HB8 – type str

OTU B2 (57 sequences)

Thermus thermophilus strain L1 (AY

Thermus thermophilus strain CS (AJ2

Thermus thermophilus strain X6 (AJ2

Thermus thermophilus strain CT1 (AJ

100

100

96

97

100

96

93

100

87

91

80

94

100

99

99

100

90

99

100

0.05

ig. 3. Neighbour joining phylogenetic tree of 16S rRNA clone library genes for: (a) Castleecovered sequences to those from the GenBank database (identified by accession numbesing the distance matrix from the PHYLIP package as input for the DOTUR program. Boo

Microbiology 34 (2011) 127–138 133

ing in mind the large number of cloned sequences in the libraryagainst which the A180 probe was designed, and the methods usedto resolve whether this cloned sequence was chimeric, its failure toimpart fluorescence to any cells in all three communities was sur-prising but suggested a PCR bias where its 16S rRNA gene sequencewas more readily amplified and cloned than others. Equally, no cellsresponded in any of the three ATAD communities to the Tbcil832probe targeting the thermophilic Bacillus spp. [30], members ofwhich are now placed in the genus Geobacillus [53], despite someof these being readily cultured in this study from both the Victorianplants.

As clone library data indicated that members of the genusThermus were well represented, the THUS438 probe targetingall Thermus spp. was applied to all three ATAD communities.The majority (∼85%) of all the EUB338mix positive cells in eachresponded to it. These Thermus cells, appearing as typically thin

rods [70], were then all further identified as belonging to the speciesT. thermophilus, since only the Tth probe of all the available species-targeted Thermus probes (Table 2) imparted fluorescence to them(Fig. 4). No cells responding to the ‘Trub’ probe that targeted Ther-

94 (AM930302)

lone 1B07 (DQ346486)

clone 2B06 (DQ346478)

riaceae clone MRE50b19 (AY684095)

025P1B12 (EF562038)

(AY936496)

JCM 11223 (NR 024776)

C21 (NR 028814)

t-14 (EU652084)

clone MSB-2B11 (EF125425)

xi bacterium clone QEDS3AB06 (CU921045)

clone FGL7S_B100 (FJ437962)

m clone53 (FJ623314)

xi bacterium clone QEDQ1CA04 (CU923706)

i bacterium clone TDNWbc97233 (FJ517062)

clone 1-20 (AY548939)

ence)

3 (AY862388)

(Y18410)

408)

18409)

5900)

ain (AP008226)

788091)

51938)

51939)

251940)

Thermus -D

ienococcus

Chloroflexi

Su

b g

rou

p 2

Su

b g

rou

p 1 F

irmicutes

maine ATAD plant and (b) Bendigo ATAD plant, showing the relationship betweenrs). OTU sequences are grouped into OTUs on the basis of 99% sequence similarity

tstrap values >70% are given at the nodes. Scale bars indicate 5% divergence.

134 D. Hayes et al. / Systematic and Applied Microbiology 34 (2011) 127–138

b Cohnella fontinalis (AB362828)

Paenibacillus sp. R-7652 (AY382189)

Thermobacillus sp. clone 8 (GQ471932)

Thermobacillus xylanilyticus strain XE (NR 025282)

Clone cas18 (1 sequence)

Clone cas26 (1 sequence)

Uncultured bacterium clone SMC37 (AM183105)

Uncultured bacterium clone E16 (AM500770)

Uncultured Clostridium sp. clone EHF (EU071533)

OTU C3 (17 sequences)

Uncultured bacterium clone 7025P1B12 (EF562038)

Thermaerobacter litoralis strain KW1 (AY936496) Thermaerobacter subterraneus strain mt-14 (EU652084)

Uncultured bacterium clone M1-5 (EU015101)

Nitrospira sp. strain GC86 (Y14644)

Uncultured Nitrospira sp. clone9 (AB252940)

Clone cas13 (1 sequence)

Actinomycetales bacterium Gsoil 1632 (AB245397)

Clone cas22 (1 sequence)

Sporichthya polymorpha (X72377)

Sporichthya polymorpha strain IFO 12702 (NR 024727)

Uncultured actinomycete clone (AB006163)

Uncultured bacterium clone 3y-46 (FJ444711)

Uncultured Gemmatimonadetes bacterium clone g54 (EU979063) Uncultured bacterium clone TX1A_90 (FJ152642)

OTU C2 (19 sequences) Thermus yunnanensis strain RH0713 (AY862388)

Thermus scotoductus strain ITI-252T (Y18410)

Thermus rehai (AF331969)

Thermus igniterrae strain GE-2 (Y18408)

Thermus brockianus strain 15038T (Y18409)

Thermus thermophilus strain CS (AJ251938)

OTU C1 (43 sequences)

Thermus thermophilus strain L1 (AY788091) Thermus flavus AT-62 (L09660)

ermo

80 100

100

86

100

100

81 100

100

99

94

100

100

100

100

84

95

100

100

100

92

76

89

100

Thermus - D

ienococcusF

irmicutes

Su

b g

rou

p1 S

ub

gro

up

2 S

ub

gro

up

3 S

ub

gro

up

4

Actinobacteria

(Cont

m‘m

SF

lsas2mitpimgnpr

Thermus th89 0.02

Fig. 3.

us ruber, the ‘Taq’ probe that targeted Thermus aquaticus and theTfili’ probe that targeted Thermus filiformis were seen in these com-

unities.

ubstrate assimilation by populations in ATAD communities withISH/MAR

As T. thermophilus appeared from FISH to be the dominant popu-ation in both ATAD communities, attempts were made to assess itsubstrate uptake patterns in situ with MAR. The incubation temper-ture used in these experiments markedly affected overall levels ofubstrate assimilation by the ATAD sludges. As sludge from reactorwas used in both aerobic and anaerobic MAR experiments, theean operating temperature for this reactor (65 ◦C) was selected

nitially for the incubations. However, when FISH/MAR was appliedo the ATAD sludge with the substrates chosen together with the Tthrobe targeting T. thermophilus, none of the substrates were assim-

lated by this population in situ. Therefore, their assimilation was

easured at several temperatures, which produced results sug-

esting that an incubation temperature of 45 ◦C was optimal (dataot presented here), based on �−-emission levels. However, whenrobe Tth was applied to ATAD samples incubated at 45 ◦C no cellsesponding to this probe showed unequivocal evidence of chosen

philus HB8 – type strain (AP008226)

inued).

substrate assimilation, even after 12 days of emulsion develop-ment. On the other hand, many bacteria responding to FISH probesLGC354 a, b, and c, and showing assimilation of glycerol, glutamicacid, aspartic acid, and acetic acid, were present (SupplementaryFig. S1). FISH/MAR was then applied with probe Tbcil832 to testif these cells were Bacillus spp. or Geobacillus spp. However, nocells responded to this probe. This result did not demonstrate anabsence of all Geobacillus species in these ATAD communities sincesome, including G. caldoxylosilyticus, do not carry the full RNA targetsequence of the Tbcil832 probe.

Discussion

Only very limited published data derived from culture-dependent methods [56] and culture-independent methods [58]are available on the microbiology of full scale ATAD systems fortreatment of domestic waste sludge. Therefore, this study, employ-ing both culture-dependent and culture-independent methods, is

the first detailed attempt to elucidate the microbial compositionof communities in two full scale ATAD sludge digestion systems.One treated sludge from a plant handling predominantly domesticwastes, while the other was fed with a proportion of its influentderived from a meat processing plant. The molecular data sug-

D. Hayes et al. / Systematic and Applied Microbiology 34 (2011) 127–138 135

F ictoris

gphwsbutaa

ig. 4. Photomicrographs of images of ATAD sludge from reactor 2, Castlemaine, Vame field of view. Indicator bar = 20 �m.

est that ATAD communities are highly specialized with very lowopulation biodiversity, which is not too surprising given theirigh operating temperatures. The DGGE profiling data support thisith clustering analyses agreeing with the expected number of

pecies (S = 8 and 4 for Castlemaine and Bendigo, respectively)

ased on clone library analyses. Given the consistent levels of pop-lation diversity emerging with FISH, 16S rRNA gene cloning, andhe culture-dependent methods used in this study, the statisticalnalysis of the DGGE profiles was considered a suitably cautiouspproach. DGGE profile band counts are often used as direct mea-

a, (a) EUBmix FISH image and (b) Tth probe for Thermus thermophilus showing the

sures (presence/absence analysis) of total community biodiversity,while band intensity has been used for abundance measurementswithout supporting phylogenetic data [57,85]. These were not con-ducted here because of the recognised problems associated withthe generation of identical bands with variable electrophoretic

mobility or single bands containing different fragments from a sin-gle PCR [17,79].

Both communities and that from a NSW ATAD plant were dom-inated by rod-shaped T. thermophilus. On the other hand, the 16SrRNA gene sequences of cultured isolates belonged predominantly

1 plied

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36 D. Hayes et al. / Systematic and Ap

o previously identified thermophilic Geobacillus spp. These haveeen isolated previously from a full scale ATAD facility [56] androm other laboratory scale systems [32,73] and industrial com-osting facilities [9,75]. However, despite identifying Bacillus sp.,hese were not identified in the only other culture-independenttudy of a full scale ATAD facility [58].

Although only one medium was used here, the incubation tem-erature was selected to encourage organisms that were able torow at 65 ◦C and above, because these were considered as theost likely active ATAD community members [32,73]. No Geobacil-

us spp. were detected in either clone libraries, which instead wereominated by T. thermophilus. Semi-quantitative analysis of theISH data supported the trends shown with the clone library datand supported their dominance in all three ATAD communities.his discrepancy in ATAD community structure with the two dif-erent approaches was not unexpected. It is quite possible that

any Geobacillus spp. may be present in the ATAD samples asndospores rather than vegetative cells. These would probably note detected by FISH probes targeting them (the LGC354 mix andbcil842 probes) as their highly impermeable walls would preventrobes from gaining access to their target sites. The endosporealls would also make DNA extraction difficult with the methodsed here, and may explain the absence of Geobacillus spp. fromhe ATAD clone libraries. However, it would also mean that, if true,

any of the Geobacillus spp. in ATADs would be dormant and notctively involved in the chemical degradation of the sludge underhese conditions.

The detection of Thermus sp. in such large numbers in theseommunities and its isolation, albeit rarely, as shown in earlierTAD studies [32], was also not completely unexpected, since itas been isolated from thermophilic aerobic composting systems8], which is essentially what these ATAD systems are. Beffa et al.8] suggested, on the basis of their temperature requirements,hat Thermus spp. are probably the most important active degrad-ng populations in compost communities, but without producingny in situ physiological data to support that claim. Despite theiew that most Thermus spp. are nutritionally versatile [16,31], theISH/MAR data obtained here suggest that their ATAD ecophysiol-gy is more specialized. Our data failed to show convincingly whatight be supporting their growth and so their role in sludge diges-

ion remains unclear. Thus, of the limited number of substratesested on the ATAD samples, none were apparently assimilatedy T. thermophilus, despite the prolonged exposure and develop-ent of samples. The negative response for uptake of glucose and

cetate in situ by T. thermophilus strain L1 was not unexpected, andlthough da Costa et al. [16] claimed that T. thermophilus can utiliseoth, they were not assimilated by the pure culture of the T. ther-ophilus L1 strain isolated here either (data not presented). Both

hese substrates, and most other readily biodegradable materials,re unlikely to persist into the ATAD systems, as they are almostertainly rapidly taken up or, in the case of glucose, fermenteduch earlier by other bacterial populations in these EBPR acti-

ated sludge processes [15]. Unidentified bacterial cells respondingo the LGC354 a, b, and c FISH probes were MAR positive for thessimilation of all these substrates, except for glucose, but theseells failed to respond to the Tbcil842 probe, eliminating all ther-ophilic Geobacillus spp., except G. caldoxylosilyticus, identified in

he culture-dependant work as the dominant identified MAR activeacteria.

This work has shown that the ATAD systems in this study usedor treatment of domestic and industrial waste activated sludge,

espite their low biodiversity, are complex in their ecophysiol-gy and require further work, especially in clarifying the rolesf dominant bacteria, such as T. thermophilus, in sludge degrada-ion. Furthermore, work is urgently needed to relate changes inTAD communities in response to changes in operational factors

[

[

Microbiology 34 (2011) 127–138

like sludge loading rates, oxygen levels and temperature, and howboth relate to odour production and pathogen reduction rates. Suchstudies are currently underway.

Acknowledgements

We acknowledge financial support by means of ARC LinkageGrant # LP0346946, Coliban Water, Simon Engineering P/L and thekind assistance of Veolia P/L.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.syapm.2010.11.017.

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