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Ecological Engineering 60 (2013) 370– 381
Contents lists available at ScienceDirect
Ecological Engineering
j ourna l ho me pa g e: www.elsev ier .com/ locate /eco leng
acterial community structure in a biofilter used as a pretreatmentor seawater desalination
anghyun Jeonga, Hyokwan Baeb, Gayathri Naidua, Dawoon Jeongb, Seockheon Leeb,aravanamuthu Vigneswarana,∗
Faculty of Engineering and IT, University of Technology, Sydney (UTS), PO Box 123, Broadway, NSW 2007, AustraliaCenter for Water Resource Cycle, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, Republic of Korea
r t i c l e i n f o
rticle history:eceived 7 May 2013eceived in revised form 17 July 2013ccepted 11 September 2013vailable online 11 October 2013
a b s t r a c t
In this study, two biofilters with different media, anthracite and granular activated carbon (GAC), wereused to pre-treat seawater for desalination. Both biofilters had the same operating conditions that lastedfor 75 days. The bacterial community structures in the filter media were studied during the biofilters’operation using terminal restriction fragment length polymorphism (T-RFLP) combined with principalcomponent analysis (PCA), clustering of samples and sequencing based on the 16S rRNA gene. Bacterial
eywords:acterial communityiofilterCAretreatment
community structure analyzed from T-RFLP patterns showed a dynamic shift at the top parts of the biofil-ters, while relatively stable bacterial community structures were observed in the middle and bottom partsof biofilters. The GAC biofilter consisted of diverse heterotrophs while the anthracite biofilter was mainlycomposed of sulfur-oxidizing and reducing bacteria, and alkalitrophic heterotrophs. This is associatedwith sulfur being a present impurity in the anthracite medium.
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. Introduction
Seawater is an alternative source for supporting increasinglobal water demands. In this context, seawater reverse osmosisSWRO) desalination technology has shown potential. However,WRO plants face the challenge of microbial and organic adhesiono the membrane surface which causes biofouling and organic foul-ng. In turn, this leads to deterioration in membrane performancend increases overall operational costs. Thus, pretreatment isssential to remove or reduce undesirable materials (or biofoulingotential) such as organic matter and biofoulants in raw seawa-er to acceptable levels (Sutzkover-Gutman and Hasson, 2010). Inrder to effectively control biofouling, organic matter (especially,iodegradable organics) be removed with microbial inactivationFlemming et al., 1997). However, the control of organic and bio-ogical fouling is difficult, requiring advanced treatments.
Deep bed filtration has been used in large-scale desalinationlants due to its relative simplicity, low energy consumption, andelatively low operational costs. The removal of suspended and col-
oidal particles by deep bed filtration is based on their depositionn the surface of filter grains while water flows through a bed ofhese grains (filter media). This can also reduce biofoulants through∗ Corresponding author. Tel.: +61 295142641; fax: +61 295142633.E-mail address: [email protected] (S. Vigneswaran).
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925-8574/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.ecoleng.2013.09.005
© 2013 Elsevier B.V. All rights reserved.
dsorption and biodegradation from feedwater (Hu et al., 2005).revious studies on biofilter as a pretreatment have indicated thatan reduce membrane flux decline and lower the deposit of organ-cs on the membrane surface (Mosqueda-Jimenez and Huck, 2009).
Slow sand filter, with filtration rates ranging between 0.1 and.2 m3/h, has been used as a standard biofiltration treatment forecades in the water industry (Bar-Zeev et al., 2012). Here, theiofilm developed on the packed medium in the filter, helps toecompose the biodegradable organic material. Further degrada-ion can occur due to the presence of a diverse microbial biofilmommunity developed in the filter media (Larsen and Harremoes,994; Baig et al., 2011).
Together with sand, the most common filter medium in waterreatment is anthracite. Anthracite, in essence, is a high quality coal.oal is also one of the sources for making GAC. In comparison toand, anthracite enables higher penetration of suspended matternto the filter bed due to its larger and more effective size. Thisesults in a more efficient filtration and longer intervals betweenleaning (Mitrouli et al., 2008). Removal of organic matter also takeslace in anthracite filter media through a surface phenomenon andiological activity in a long-term operation (Boon et al., 2011; Chinut al., 2009).
Recently, Naidu et al. (2013) showed the possibility of reducingiofouling potential with granular activated carbon (GAC) biofilter.his biofilter also has the capacity to maintain a stable microbialctivity in the filter bed. GAC is processed, often from bituminous
ngineering 60 (2013) 370– 381 371
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t(sismmD1TlPga16TawmUSA) and raw T-RFLP data were exported from PeakScannerTM soft-ware (version 1.0, Applied Biosystems, USA). The forward terminalrestriction fragment (T-RF) patterns produced by 27F-FAM for all
Table 1The codes for biofilm samples taken for DNA extraction.
Packing media Position Day 14 Day 49 Day 70
GAC (G) Top G1.1 G2.1 G3.1Middle G1.2 G2.2 G3.2
S. Jeong et al. / Ecological E
oal, to absorb and remove molecular size particles directly fromhe solution. Anthracite does not function very well in terms of
olecular absorption of chemicals because of its small pores, whichre much smaller than most of the molecules that can be absorbedy GAC media. Backwashing alone will not remove matter that haseen absorbed into the pores of GAC media (Voutchkov, 2010).
Even though carbonaceous materials are efficient materials inhat they can support biofilm with favorable surface properties foracterial adhesion, currently no information is available on the tax-nomic assignment of the marine bacterial community structuren the packing media. Culture-independent analysis is a powerfulool for understanding better the bacterial community structuren biofilters (Ji et al., 2013). Only a few studies have been donen molecular bacterial identification with reference to sand filtra-ion, yet it is the most commonly used pre-treatment system foreawater. The abundance of �-proteobacteria bacteria containingutative biofilm forming bacteria such as Alteromonas, Cowellia andlaciecola sp. were identified in the sand filter effluent by cloningnd sequencing based on the 16S rRNA gene (Bae et al., 2011). Dena-uring gradient gel electrophoresis (DGGE) was conducted for theapid sand filtration with significant biofilm maturation, but theaxonomic information was not driven by sequencing the DGGEands (Bar-Zeev et al., 2012). To our knowledge, this is the firsttudy on the functional bacterial community structure in biofiltersnfluencing the removal of organic matter in seawater. Detailednowledge on the interactions between treatment conditions andiodegradation in filters will significantly contribute to a more effi-ient application of biofilter for seawater pretreatment.
In this study, we evaluated the potential of anthracite (A) andAC (G) media filter as a biofilter for pretreating seawater prior
o its application to RO. We assessed the change and the devel-pment of the microbial community in filter media using terminalestriction fragment length polymorphism (T-RFLP) combined withrincipal component analysis (PCA), clustering of samples andequencing based on the 16S rRNA gene.
. Materials and methods
.1. Biofiltration
.1.1. SeawaterThe biofiltration experiments were conducted at Sydney Insti-
ute of Marine Science (SIMS), Chowder Bay, Sydney, Australia. Theeawater was collected from 1 m below the sea surface level anded into biofilters continuously after the removal of large particleshrough 140 �m filter. During the operations which lasted for 75ays, the average pH, dissolved oxygen (DO), turbidity and DOCalues of seawater used in the experiments were 7.8 (±0.2), 4.9±0.3) mg/L, 0.5 (±0.3) NTU and 2.31 (±0.35) mg/L, respectively.
.1.2. Experimental set-upThe biofiltration experimental set-up is shown in Fig. 1. Two
ransparent acrylic filter columns were operated in parallel (at down-flow mode) for 75 days. The columns were packed upo a depth of 65 cm from the bottom with anthracite and gran-lar activated carbon (GAC), respectively. The nominal size (d50)f anthracite (1.00 mm) was larger than that of GAC (0.41 mm)hereas surface area of GAC (1000 ± 50 m2/g) was much larger
han that of anthracite (150 ± 50 m2/g). These specifications ofedia were provided from the supplier (James Cumming & Sons,
ustralia). Both filters were operated at a relatively low filtrationelocity of 5.0 m/h to maintain a stable bacterial activity. The emptyed contact time (EBCT) of the biofilter was 7.8 min. The biofiltersere backwashed occasionally at the same rate of the filtrationFig. 1. Experimental set-up of two biofilters (GAC and anthracite).
elocity (5.0 m/h) for 2 min. Sampling ports were placed at 60 cm,0 cm and 10 cm from the bottom of the medium for collecting theedia specimen.
.2. Bacterial community structure
.2.1. PCR, T-RFLP and statistical analysisIn this study, representative biofilm samples were collected at
hree stages for DNA extraction as shown in Table 1; 14th dayinitial stage), 49th day (intermediate stage) and 70th day (finaltage). These stages were classified according to the bacterial activ-ty (Jeong et al., 2013a; Naidu et al., 2013). Once the mediumamples are collected, the biofilm (biomass) was taken from theedia by voltexing and sonication. For the application of a poly-erase chain reaction (PCR), DNA was extracted using genomicNA extraction kit (Promega, USA). For the amplification of the6S rRNA gene for T-RFLP, two primers of 27F (5′-AGA GTT TGACC TGG CTC AG-3′) and 518R (5′-(A/T)TT ACC GCG GCT GCT GG-3′)abeled by FAM and HEX fluorophores, respectively, were used. TheCR mixture consisted of 12.5 �L of 2× Multiplex PCR Pre-Mix (Sol-ent, Korea), 1 �L of each primer (10 �M), 1 �L of DNA template,nd 9.5 �M of deionized water. The PCR cycles were as follows:
cycle of 15 min at 95 ◦C, 30 cycles of 20 s at 95 ◦C, 40 s at 53 ◦C,0 s at 72 ◦C and then 1 cycle of 3 min at 72 ◦C using MyCyclerTM
hermal Cycler (Bio-Rad, USA). The PCR product was purified using gel extraction and purification kit (Qiagen, USA) and digestedith endonuclease TaqI (10 U) (Takara, Japan) at 65 ◦C for 3 h. Frag-ents were run on an ABI 371X sequencer (Perkin-Elmer Corp.,
Bottom G1.3 G2.3 G3.3
Anthracite (A) Top A1.1 A2.1 A3.1Middle A1.2 A2.2 A3.2Bottom A1.3 A2.3 A3.3
3 ngineering 60 (2013) 370– 381
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72 S. Jeong et al. / Ecological E
amples were simple with only one T-RF of 56 bp. Hence, reverse-RFs of 518R-HEX were utilized for analyzing the dynamics ofacterial community structure. Signal intensities below fluores-ence unit 20 were excluded from analysis. Peaks around 20 bpere regarded as the remaining primers after the gel extraction.
eak intensities were normalized by the sum of intensities for thetatistical analysis and less than 5% were not considered to beajor species for further analysis. The similarity between T-RF pat-
erns for various samples was examined through PCA and clusternalyses were done using SPSS software (version 13.0, SPSS Inc.,SA).
.2.2. Sequencing of 16S rRNA gene and phylogenetic treeAn amplification of the 16S rRNA gene was conducted with
rimers of 27F and 1492R (5′-TAC GG(C/T) TAC CTT GTT ACGCT T-3′) and this amplification was identical to that of T-FLP. Sequences of the 16S rRNA gene analyzed (by Solgent Inc.)ere aligned with 3% cut-off and operational taxonomic units
OTUs) were selected using MOTHUR suite of programs (ver-ion 1.7.2). The closest relatives of OTUs were identified usinghe EzTaxon server (http://www.eztaxon.org) and the taxonomicnformation of OTUs was determined based on the relative withhe highest similarity. Confirming the taxonomic assignment ofTUs was conducted by constructing phylogenetic trees using
he neighbor-joining method (NJ) of OTUs and MEGA 3.1 soft-are. The OTUs that were affiliated with different lineages based
n the taxonomic information from EzTaxon were manuallyxcluded from the analysis to minimize the error of taxonomicssignment. The 16S rRNA gene sequences have been depositedn the NCBI database under accession numbers (KC238320 toC238401).
. Results and discussion
Based on the trend concerning organics removal and bacterialrowth observed during the filter operation, the experimen-al period was divided into three stages: (i) initial stage (0–30ays of operation); (ii) intermediate stage (31–60 days of oper-tion); and (iii) final (mature) stage (61–75 days of operation).n this study, discussion focused on these three stages and filtered position (top, middle and bottom) (Fig. 1). The particulateemoval in the biofilters showed slightly more removal usinghe GAC biofilter (0.21 ± 0.04 NTU). However, headloss devel-pment (hydraulic resistance to flow) in the GAC biofilter waswice than that in the anthracite biofilter. This may be due tohe higher packing density (packing density of media was esti-
ated by measuring the media weight packed in unit volume ofolumn) with GAC. Anthracite medium had a packing density of400 kg/m3 while GAC medium was almost twice that of anthraciteedium.The changes in bacterial growth and activity when the two
iofilters were at different positions are given in Fig. S1. Organicemoval in terms of dissolved organic carbon (DOC) and assimil-ble organic carbon (AOC) are documented in Fig. S2. The detailedrocedures of DOC and AOC can be found in Naidu et al. (2013) and
eong et al. (2013b), respectively. The organic reduction trend islosely linked to the microbial active biomass. At the initial stagefrom start to 30th day), GAC (approximately 22%) showed a higherapacity for adsorption than anthracite (approximately 8%). Thiss due to the larger surface area of GAC, resulting in higher DOC
emoval. At the intermediate stage (from 31st day to 60th day),he ATP results indicated the significant presence of active biomassf around 238.5 �g ATP/per g media at the top layer of the GACiofilter. At the final stage (from 61st day to 75th day), the DOC Table
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S. Jeong et al. / Ecological Engineering 60 (2013) 370– 381 373
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ig. 2. Classification of T-RFLP patterns for GAC filter by (a) principal component anithin Group G-I.
as reduced by 39% in GAC and 20% in anthracite filter. Comparedo the GAC filter, the anthracite filter demonstrated less abilityo remove DOC. This follows the trend of microbial growth andommunity structure in the two filters. Compared to the GAClter, the anthracite filter showed a slightly lower microbialrend (in terms of ATP and cell count). The AOC also followedame trend. The high AOC concentration at the initial stageould be attributed to the high molecular weight compounds
etting degraded to LMW compounds during the biodegrada-ion process. Upon the GAC filter reaching a mature stage,OC value was reduced to 4.85 (±1.16) �g-C glucose equiva-ents/L.
tTsR
(PCA) and (b) cluster analysis. The dashed line indicates the closely related samples
.1. Dynamics of microbial community structure in GAC biofilterased on T-RFLP
T-RFs of GAC biofilter were identified as shown in Table 2. Allamples shared R51 but compositions of the other T-RFs wereetermined in terms of the similarity of samples obtained fromAC biofilter. Based on the unique compositions of T-RFs, sam-les of GAC biofilter were sub-grouped into Groups G-I and G-II
hrough statistical analysis (PCA) and clustering of samples (Fig. 2).he closely related samples of G1.2, G1.3, G3.2 and G3.3 were clas-ified into Group G-I with diverse T-RFs of R51, R170, R233, R282,366, R408, R423, R426, R430, R454 and R463. Abundance of R423
374 S. Jeong et al. / Ecological Engineering 60 (2013) 370– 381
Table 3Relative abundances of terminal restriction fragments obtained for anthracite biofilter.
Sample R52 R71 R148 R170 R252 R257 R366 R407 R430 R435 R454a R457a R460a R462a R464a
A1.1 21.0 13.6 30.2 10.1 12.4 12.6A1.2 12.7 10.6 5.9 5.9 14.6 6.8 25.8 11.0 6.6A1.3 12.9 6.2 6.1 24.6 39.7 10.6A2.1 9.6 79.3 11.0A2.2 18.3 45.2 36.4A2.3 16.7 14.7 9.5 51.9 7.3A3.1 34.2 36.7 29.2A3.2 12.8 9.4 7.4 18.3 10.1 13.8 8.6 16.0 3.6A3.3 8.0 12.2 10.1 62.3 7.5
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a T-RFs from R454 to R464 were hard to be grouped because of highly diverse p64.5 and 464.6 showed a distance within the tolerance of bias, 4 bp.
as the distinctive feature of the G-I sub-group. The G1.2, G3.2 and3.3 samples shared the minor peaks of R408 and R426. R170 and430 were common in the G1.2 and G3.2 samples. The middle andottom part of the GAC biofilter shared a consistent bacterial com-osition. It may be responsible for the bacterial activity without the
ntrusion of inherent marine bacteria of raw seawater. Thus, Gx.2middle position) and Gx.3 (bottom position) in Group G-I werelosely related as shown in Fig. 2(b) based on the clustering anal-sis. On day 49, however, the bacterial compositions of the middleart (G2.2 in Group G-I) showed less similarity to the G-I sub-group.he T-RFs of R423 and R426, which were the major peaks of theub-group in Group I, were not observed while those of R457, R462nd R465, which are unique in Group G-I, were observed. On theame day, the bottom part shared common T-RFs of R408, R423nd R426, but it contained T-RFs of R444, R458, R460 and R484,hich are unique in Group G-I. In terms of R408, R423 and R426,
t emerged that the bacterial community structure in the middleart experienced a dynamic shift in the intermediate phase andeturned to the original community.
On the other hand, clustering analysis resulted in distinguish-ble Group G-II in the top part of the GAC biofilter, which iselatively distant from the other samples. The most distinctive fea-ure of the Group G-II was the occurrence of R366 in all samplesnd the absence of R408, R423 and R426 which were abundantn Group G-I. Even though the Group G-II samples are grouped,istances between the three samples were relatively longer thanhose of Group G-I (Fig. 2(b)). This could be due to the occurrencend disappearance of minor peaks which indicates the intrusion ofnherent marine bacteria in raw seawater. Interestingly, Group G-IIhared R170 as did all samples of the middle part. It was estimatedhat bacterial species of R170 was consistently dominant in the topart. They penetrated to the middle part, but were not transportedo the bottom part during the experimental period of 75 days. Inummary, the positions (top to bottom) within the GAC biofilter sig-ificantly influenced the creation of diverse bacteria rather than thelter’s operational period. In the GAC biofilter, R408, R423 and R426re the major classification of the samples based on the position ofhe filter bed.
.2. Dynamics of microbial community structure in anthraciteiofilter based on T-RFLP
T-RFs of anthracite biofilter were identified as shown in Table 3.he bacterial community structure of anthracite biofilter of 15 T-Fs was less diverse than that of GAC biofilter regarding 22 T-RFs.ll samples shared R52. The compositions of the other T-RFs were
etermined in terms of the similarity of samples obtained fromnthracite biofilter. The T-RF patterns of anthracite biofilter werelassified into two major groups of Groups A-I and A-II and samplesf A1.1 and A2.1 were not closely related to the two groups (Fig. 3).trdb
These peaks of 454.0, 454.2, 457.2, 460.5, 462.2, 462.3, 462.5, 462.6, 462.9, 463.0,
n Group A-I, the closely related samples of the bottom part (A1.3,2.3 and A3.3 in the sub-group of A-I) contained R407, R454 and462, which is probably responsible for the bacterial activity. Notehat R408, which could be regarded as R407 with a tolerance of biasf 4 bp, was also the major peak in the middle and bottom part ofAC biofilter and it represents the common biofilm bacteria in bothiofilters in this study. On Days 49 and 70, anthracite biofilter con-ained R430 in the bottom part (A2.3 and A3.3). The minor peaksf R170 and R366 were found in the bottom part on Day 14 (A1.3).n Group A-I, the middle part of A1.2 and A3.2 on Days 14 and 70,espectively, shared the major peaks of R407, R454 and R462. Theseeaks were also observed in the bottom part (A1.3, A2.3 and A3.3).owever, the occurrence of R170, R366 and R464 which were gen-rated from the A1.2 and A3.2 samples was relatively distant fromhe sub-group in A-1. T-RFs of R457 and R460 of A3.2 and R257f A1.2 in the middle part were distinct. The bacterial communitytructures of the middle and bottom parts were relatively similar,xcept for the A2.2 sample which did have the major peaks of R407,454 and R462. In Group A-II, for A2.2 and A3.1, only two T-RFs of170 and R252 represented bacterial species. In the same manners GAC biofilter, the dynamic shifts of bacterial species of the middleart were observed in the intermediate phase. The relative insta-ility of the middle part compared to the bottom part was observedccording to R407, R454 an R462. Temporal observance with A1.1,2.1 and A3.1 showed the dynamic variances and small similari-
ies between samples (Fig. 3(b)). Only R170 was shared by A1.1 and3.1, but the other signals of R71, R148, R252, R407, R435, R462nd R464 were independently present in the top part of anthraciteiofilter depending on the operational periods. In the anthraciteiofilter, similar bacterial community structures in the middle andottom parts appeared consistently compared to the ones in theAC biofilter.
.3. Taxonomic assignment of biofilm bacteria
The sequence of the 16S rRNA gene was retrieved from the sam-les of G1.1 and G3.1 at the top, G1.2 and G2.2 in the middle and2.3 and G3.3 in the bottom parts of the GAC biofilter; and A1.1 and2.1 at the top, A3.2 in the middle and A1.3 in the bottom parts ofnthracite biofilter. The taxonomic information of sequences wasetermined in terms of the highly relative (similarity) and wasummarized in Table 4. The taxonomic assignment was confirmedhrough the construction of the phylogenetic trees (Fig. 4). Bothiofilters were occupied with diverse bacterial community such asctinobacteria, Firmicutes, Planctomycetes, Verrucomicrobia, ˛, ˇ,
and �-proteobacteria, Caldithrix and Bacteroidetes. In terms of
he phylum level, GAC and anthracite biofilters shared all bacte-ia, while the ecological niches in the level of species were entirelyifferent. Sampling positions also have significant effects on theacterial community structure in species level in both the GAC and
S. Jeong et al. / Ecological Engineering 60 (2013) 370– 381 375
F nent as
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ig. 3. Classification of T-RFLP patterns for anthracite filters by (a) principal compoamples within Group A-I.
nthracite biofilters. The relative abundance could not be evalu-ted in terms of the number of 16S rRNA gene sequences since theequencing for all samples was not conducted.
.3.1. GAC biofilterAt the top part of the GAC biofilter, diverse heterotrophic bacte-
ia such as facultative anaerobic Granulicatella adiacens, anaerobiclanctomyces brasiliensis, aerobic Phaeobacter daeponensis and aer-bic Formosa spongicola were found. Also found was one species of
naerobic photolithoautotrophic Thiohalocapsa marina. It is knownhat G. adiacens (formally, Abiotrophia adiacens) grows in the pairr short chains of cocci (Collins and Lawson, 2000). P. daeponensisas isolated in the Yellow Sea off South Korea (Yoon et al., 2007)(pwp
nalysis (PCA) and (b) cluster analysis. The dashed line indicates the closely related
nd Phaeobacter sp. Indicated a tendency to aggregate in a certainedium (Martens et al., 2006). T. marina grows phototrophically
nd several organic substances such as acetate, pyruvate, lactate,umarate, succinate, glucose and Casamino acids can be photo-ssimilated when sulfide and bicarbonate are present (Kumar et al.,009). The combination of aerobic and anaerobic bacteria (whichas responsible for the AOC removal) in the filter were dominant at
he top part of biofilter with the highest ATP level. The capability ofacteria in aggregation (P. daeponensis) and producing aggregates
G. adiacens) would help themselves to adhere to GAC media. Thehoto-assimilation by T. marina can also contribute to AOC removalith the help light penetrating through the acrylic wall. At the topart of the GAC biofilter, they are responsible for the highest cell
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76 S. Jeong et al. / Ecological E
umbers throughout the entire operation. They played significantoles in removing of DOC and AOC during the intermediate andnal phases (Fig. S2). They may originate from raw seawater andecome acclimated on the top part of the filter. They are retained athe top position of GAC filter by the sieving effect of GAC medium.
The functional bacterial niche which contributes to the develop-ent of biofilm and subsequent degradation of organic compounds
s described as follows: During the intermediate stage, the ATPesults indicated the significant presence of active biomass atround 238.5 �g ATP/per g media at the top layer of the GACiofilter (Fig. S1). The presence of microbes in the media may biode-rade the large molecular weight compounds into lower moleculareight (LMW) compounds. This is associated with the increase in
vailable labile organics (or AOC) that contribute to further micro-ial growth (Fig. S1). Biofilm (which consists of biopolymers suchs polysaccharide and protein) formed by microbial activity maylso have contributed to the slight increase in biopolymer concen-ration (Jeong et al., 2013a). This led to an increase in DOC level athe intermediate stage.
The middle part of the GAC biofilter contained heterotrophsf anaerobic Natranaerobius thermophilus, anaerobic P. brasilien-is, aerobic Maritalea mobilis, aerobic Maritimibacter alkaliphilus,erobic Ruegeria halocynthiae, anaerobic Desulfomonile limimaris,erobic Alcanivorax jadensis, aerobic Haliea mediterranea, aero-ic Marinobacter adhaerens, aerobic Owenweeksia hongkongensis
nd aerobic Ulvibacter antarcticus. N. thermophilus is one speciesf extremophiles of halophilic alkalithermophiles (Mesbah et al.,007). P. brasiliensis was the only common bacterial species foundt the top part and middle part of the biofilter. A large differencesGSh
ig. 4. Phylogenetic trees based on the 16S rRNA gene sequences showing the phylogenomycetes and Verrucomicrobia, (b) �-proteobacteria, (c) Caldithrix and �-proteobacterinto OTUs using a definition of 97% similarity. The numbers at the nodes are percentages000 re-sampled data sets. One unit of scale bar represents 2% nucleotide substitution. Tharentheses.
ering 60 (2013) 370– 381
as indicated in the bacterial community structure between theop and middle parts of the biofilter. M. mobilis (formally, Zhangellaobilis) has the ability to reduce nitrate under anaerobic conditions
Xu et al., 2009). A combination of diverse heterotrophs may con-ribute to the increase in AOC removal and biofilm development.. limimaris is a dehalogenating bacterium with electron acceptorsf fumarate, sulfate, sulfite, thiosulfate and nitrate isolated fromarine sediments (Sun et al., 2001). A. jadensis (formally, Fundibac-
er jadensis) is a moderately halophilic hydrocarbon-degradingacterium isolated from intertidal sediments (Bruns and Berthe-orti, 1999).
The formation of biofilm is related to the filamentous celltructure and the production of biopolymer. H. mediterranea is
bacterium showing the causing the production of extracellularlaments and material, but it was not aggregated (Lucena et al.,010). M. adhaerens was isolated from aggregates taken from sur-ace waters and exhibited high potential to produce transparentxopolymeric particles (TEPs) (Kaeppel et al., 2012). O. hongkon-ensis was isolated from a tank storing sand-filtered sea water andhowed rod- to filamentous-shaped morphology (Lau et al., 2005).t is noted that the M. adhaerens producing TEPs could support othererobic heterotrophs to adhere on the GAC granules. Filamentous. honkongensis may also have contributed to the formation of atable biofilm structure in the GAC granule.
Distinctive heterotrophic bacteria which are responsible for the
ignificant removal of organic components in the bottom part of theAC biofilter (Fig. S1) were found to be: aerobic M. mobilis, aerobichinella daejeonensis, facultatively anaerobic Thalassospira xian-ensis, anaerobic Caldithrix palaeochoryensis, facultative anaerobicetic affiliation of microorganisms grouped in (a) Actinobacteria, Firmicutes, Planc-a, (d) �- and �-proteobacteria, and (e) Bacteroidetes. The sequences were grouped
indicating the levels of bootstrap support based on a neighbor-joining analysis ofe numbers of clones that are included in corresponding OTUs are indicated within
S. Jeong et al. / Ecological Engineering 60 (2013) 370– 381 377
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hewanella colwelliana, and aerobic Reichenbachiella agariperforans.. mobilis was found commonly in both the middle and bottom
arts. Heterotrophic metabolic activities of bacteria residing in theottom part of the filter were identified. S. daejeonensis is a het-rotrophic nitrate-reducing bacterium which was isolated from theludge of a leachate treatment plant (Lee et al., 2011). Heterotrophic. xianhensis has the capability of polycyclic aromatic hydrocarbonsPAHs) and facultative anaerobic growth with nitrate and nitrite aslectron acceptors (Zhao et al., 2010).
.3.2. Anthracite biofilterAt the top part of the anthracite biofilter, the bacterial commu-
ity structure showed a significantly distinctive niche consisting ofulfur-related bacteria as a major consortium favoring the alkaliticondition. Anaerobic haloalkaliphilic sulfate-reducing Desulfona-ronum thiosulfatophilum utilized elemental sulfur as an electroncceptor and H2, formate, EtOH, lactate and pyruvate as electrononors during the growth period (Sorokin et al., 2011). Alkaliphilicutotrophic sulfur-oxidizing Thioalkalivibrio denitrificans can alsorow in anaerobic conditions through the denitrification of N2Ond NO2 growing on thiosulfate as an electron donor (Sorokin et al.,001). Autotrophic sulfur-oxidizing Thioalkalivibrio thiocyanodeni-rificans grows on thiosulfate and thiocyanate as an electron donornder oxic and anoxic conditions using nitrate or nitrite as an elec-ron acceptor with high optimum pH over 9.6–10.0 (Sorokin et al.,
004). Furthermore, autotrophic sulfur-oxidizing Thiohalomonasitratireducens utilizes sulfide and thosulfate as an electron donorith sulfate as the final oxidate product with nitrate as the electroncceptor. It can grow with oxygen as an electron acceptor under
(rou
nued ).
icro-oxic conditions. The optimum pH ranges ranged between 7.8nd 8.0 (Sorokin et al., 2007). Autotrophic Thioprofundum hispidums mesophilic, facultative anerobic, sulfure-oxidizing bacteriumsolated from a surface rock sample from a hydrothermal fieldMori et al., 2011). Bacteria related to the sulfur oxidation and itseduction were not be expected to contribute to the degradation ofrganic pollutants because the majority of them are autotrophs.
Aerobic heterotrophs sharing the same ideal state of relativelyigh pH were also found. Aerobic hetetotrophic Ilumatobacter flu-inis was isolated from the sediment sample of the mouth of theuriragawa River, Japan (Matsumoto et al., 2009). Aerobic het-rotrophic Parvularcula lutaonensis was isolated from a coastal hotpring having an optimal temperature of 37 ◦C and pH of 7 and 9Arun et al., 2009).
P. brasiliensis and aerobic heterotrophic M. mobilis were theommon bacteria in the anthracite biofilter. Aerobic heterotrophiclcanivorax dieselolei able to degrade n-alkane as a carbon sourceas isolated from an oil-contaminated seawater. Aerobic het-
rotrophic Haliea salexigens is closely related to H. mediterraneaausing the production of extracellular filaments and materialLucena et al., 2010).
In the top and middle part of the anthracite biofilter, sulfur-xidizing T. thiocyanodenitrificans and T. hispidum were found. Theost common bacterium found in the biofilter was P. brasilien-
is. Brocadia fulgida which is an anaerobic ammonium oxidizing
ANAMMOX) bacteria consuming ammonium and nitrite at theatio of 1:1.32 and producing nitrogen gas and a little amountf nitrite as by-products in Planctomycetes phylum. It is able totilize organic acids in contrast to the other ANAMMOX bacteria
378 S. Jeong et al. / Ecological Engineering 60 (2013) 370– 381
Table 4Taxonomic assignment of 16S rRNA gene between 27F and 1492R and in-silico T-RFLP of TaqI for fragments of 27F-518R.
No. Group Phylum Name Similarity GAC Anthracite T-RFLP (TCGA) T-RFLP (TCGA)
1 I Actinobacteria Ilumatobacter fluminis YM22-133(T) 91.2 A32-13, A21-02 56 2292 Firmicutes Granulicatella adiacens ATCC 49175(T) 99.6 GA11-08 56 3553 Natranaerobius thermophilus JW/NM-WN-LF(T) 79.6 GA22-02 56 1744 Planctomycetes Brocadia fulgida 81.7 A32-12 56 1745 Planctomyces brasiliensis DSM 5305(T) 89.4 GA22-16 56 1576 Planctomyces brasiliensis DSM 5305(T) 82.4 GA31-01 352 1727 Planctomyces brasiliensis DSM 5305(T) 88.8 A11-16 352 1728 Planctomyces brasiliensis DSM 5305(T) 89.2 A32-22 96 1729 Verrucomicrobia Roseibacillus ishigakijimensis MN1-741(T) 89.7 A32-02 56 175
10 II Proteobacteria Antarctobacter heliothermus EL-219(T) 92.5 A32-19 56 44111 (�-proteobacteria) Maricaulis maris ATCC 15268(T) 97.6 A13-03 56 41412 Maritalea mobilis E6(T) 89.7 GA12-02 56 42813 Maritalea mobilis E6(T) 89.1 GA23-01 56 42814 Maritalea mobilis E6(T) 91.1 A11-19 56 41415 Maritimibacter alkaliphilus HTCC2654(T) 97.1 GA12-04 56 41416 Parvularcula lutaonensis CC-MMS-1(T) 92.6 A11-17 56 6917 Phaeobacter arcticus 20188(T) 99.2 A13-17 56 41418 Phaeobacter daeponensis TF-218(T) 93.6 GA31-03 56 41419 Phaeobacter inhibens T5(T) 95.9 A32-07 56 41420 Rickettsia peacockii Skalkaho(T) 85.3 A11-10 56 44121 Ruegeria atlantica IAM 14463(T) 98.3 A13-06 56 41422 Ruegeria halocynthiae MA1-6(T) 95.8 GA22-22 56 41223 Shinella daejeonensis MJ02(T) 88.7 GA33-02 56 42824 Sulfitobacter pontiacus DSM 10014(T) 99.9 A13-28 56 41225 Thalassospira xianhensis P-4(T) 99.7 GA33-03 480 480
26 III Caldithrix Caldithrix palaeochoryensis MC(T) 83.7 GA23-04 56 35527 Proteobacteria Desulfomonile limimaris DCB-M(T) 80.1 GA22-05 56 46128 (�-proteobacteria) Desulfomonile tiedjei ATCC 49306(T) 86.4 A13-30 56 35529 Desulfonatronum thiosulfatophilum ASO4-2(T) 82.6 A11-11 56 11330 Desulfovibrio alkalitolerans DSM 16529(T) 82.1 A13-01 56 45231 Sandaracinus amylolyticus NOSO-4(T) 88.2 A13-29 56 394
32 IV Proteobacteria Alcanivorax dieselolei B-5(T) 90.6 A11-13 56 43533 (�-proteobacteria) Alcanivorax jadensis T9(T) 97.7 GA22-17 56 43534 Haliea mediterranea 7SM29(T) 95.5 GA22-21 56 46235 Haliea salexigens 3X/A02/235(T) 94.7 A11-01 56 46236 Marichromatium bheemlicum JA124(T) 90.7 A32-23 56 43437 Marinobacter adhaerens HP15(T) 98.4 GA12-07 56 32438 Marinobacter sediminum R65(T) 99.4 A13-13 56 46939 Pseudoteredinibacter isoporae SW-11(T) 92.3 A32-18 56 46240 Shewanella colwelliana ATCC 39565(T) 99.7 GA33-01 56 39341 Thioalkalivibrio denitrificans ALJD(T) 90.9 A11-09 56 46942 Thioalkalivibrio thiocyanodenitrificans ARhD1(T) 92.7 A11-18 56 46943 Thioalkalivibrio thiocyanodenitrificans ARhD1(T) 92.3 A32-05 56 36944 Thiohalocapsa marina JA142(T) 90.3 GA11-03 56 43545 Thiohalomonas nitratireducens HRHd 3sp(T) 89.5 A11-04 56 45946 Thiohalomonas nitratireducens HRHd 3sp(T) 88.8 A13-22 56 43947 Thioprofundum hispidum gps61(T) 90.2 A11-15 56 43548 Thioprofundum hispidum gps61(T) 91.3 A32-16 56 36949 (�-proteobacteria) Neisseria mucosa M5 99.7 GA11-05 151 308
50 V Bacteroidetes Aequorivita antarctica SW49(T) 92.3 A32-03 56 46151 Bizionia echini KMM 6177(T) 96.2 A11-14 56 46952 Candidatus Aquirestis calciphila MS-Falk1-L 85.4 A11-12 56 46953 Fabibacter halotolerans UST030701-097(T) 85.3 A32-17 56 46254 Formosa spongicola A2(T) 94.8 GA11-07 56 46755 Lacinutrix algicola AKS293(T) 95.7 A11-02 56 46556 Owenweeksia hongkongensis UST20020801(T) 89.5 A13-08 56 46457 Owenweeksia hongkongensis UST20020801(T) 89.2 GA12-03 56 467
5(T)
(opmidas
opsi
58 Reichenbachiella agariperforans KMM 35259 Roseivirga spongicola UST030701-084(T)
60 Ulvibacter antarcticus IMCC3101(T)
Kartal et al., 2008). At the middle part of the biofilter, a numberf the heteroprophs favoring mesophilic temperature and alkaliticH for growth were also found: aerobic Roseibacillus ishigakiji-ensis, aerobic Antarctobacter heliothermus, aerobic Phaeobacter
nhibens, anaerobic Marichromatium bheemlicum, aerobic Pseu-
oteredinibacter isoporae, psychrotolerant aerobic Aequorivitantarctica, aerobic Fabibacter halotolerans, and aerobic Roseivirgapongicola.spb
92.6 GA33-04 56 46488.8 A32-11 56 46494.9 GA22-12 56 465
In the bottom part of the anthracite biofilter, common sulfur-xidizing T. nitratireducens (which was also present in the topart) was discovered. Sulfitobacter pontiacus is a heterotrophiculfur-oxidizing bacterium (SOB) affiliated with ˛-proteobacterian this study (Sorokin, 1995). The consortia of autotrophic SOB and
ulfitobacter-type organotrophs which oxidize thiosulfate com-letely to sulfate in natural microbial communities was suggestedy Sorokin (2003).
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S. Jeong et al. / Ecological E
Sulfur-reducing anaerobic autotrophic Desulfomonile tiedjei waslso found (DeWeerd et al., 1990). It is phylogenetically similar to D.imimaris. Another sulfur-reducing bacterium Desulfovibrio alkali-olerans (which is alkali-tolerant at an optimum pH of 9.0–9.4 withn optimum temperature of 43 ◦C utilizing sulfate as an electroncceptor and lactate, pyruvate, formate and hydrogen/acetate as anlectron acceptor) was found in the bottom part (Abildgaard et al.,006). The occurrence of only sulfur-reducing bacteria indicatedhat the bottom part of the biofilter had at a reduction environmentith oxidized sulfur sources. The diverse aerobic heterotrophic
acteria identified were: aerobic Maricaulis maris, psychrophilicerobic Phaeobacter arcticus, aerobic Ruegeria atlantica, aerobicandaracinus amylolyticus, aerobic Marinobacter sediminum, anderobic O. hongkongensis.
. Discussion
The degree of biofilm stability could be the main factor in main-aining a consistent bacterial community structure. Consistency inhe bacterial community structure can be maintained if biofilmxperiences controlled but sufficient shear stress caused by occa-ional backwashing.
The stability of biofilm varied with the locations in GAC andnthracite biofilters. Even though the total cell numbers wereigher at the top part than the middle and bottom parts (Fig. S1), theacterial community structure did not follow the same trend. Theigher cell number does not essentially guarantee the formation of
firmer biofilm structure. Since one of the main functions of a deeped filtration is the mechanical separation and reduction in turbid-
ty, inherent marine bacteria could be predominantly acclimated athe top part during the filtration process and get quickly washed-ut during the backwashing process. In spite of the instability ofacterial community structure, the bacteria in the top part still play
major role in AOC degradation (Fig. S2) which is represented byighest ATP concentrations (Fig. S1).
In both GAC and anthracite biofilters, the middle parts of thelter during intermediate phase (31–60 days of operation) showed
relatively larger shift in the bacterial community structure com-ared to the bottom part of the filter. In contrast, the fact that theacterial community structure at the bottom part of the filter wasonsistent implies that biofilm structure was relatively stable. Tonhance the AOC removal efficiency, the biofilm’s stability shoulde monitored and optimized by controlling frequency and intensityf backwashing.
For better AOC removal efficiency, the functional bacteria forOC removal could be enriched in the biofilters. However, the maineterotrophic bacteria for the AOC removal in GAC and anthraciteiofilters could not be determined in this study due to the highiversity of aerobic and anaerobic heterotrophs observed. Thexistence of diverse heterotrophic bacteria in GAC and anthracites natural. Any bacterial growth is only possible with the extra-ellular enzymatic hydrolysis of organic content attached on theAC. The adsorption of organic contents from seawater on GACnd anthracite is non-specific. Additionally, the GAC has more sur-ace area than anthracite, and there is not much selective pressureor a specific bacterial species to be attached. Therefore, no singlepecies can out-compete the other bacteria. This implies that theptimization of GAC and anthracite biofilters could not be focusedn due to the limited number of marine bacteria.
The optimization process should therefore deal with a wide
ange of bacteria that incorporate the build-up of heterotrophiciofilm. The development of biofilm is a complex process: initialttachment of cells to a surface, production of extracellular poly-eric substances (EPS), early development of biofilm architecture,citt
ering 60 (2013) 370– 381 379
aturation of biofilm architecture and dispersion of single cellsrom the biofilm (Stoodley et al., 2002).
The precursors of GAC are hard coal, brown coal, wood,oconut shells and some polymers and the quality of GAC isvaluated based on the adsorption properties for soluble organicnd inorganic substances (Heschel and Klose, 1995; Yadav et al.,013). The adsorption of organic substances on carbon mate-ial is a complex reaction governed by non-electrostatic andlectrostatic interactions (Moreno-Castilla, 2004). Because theunctional groups of activated carbon are phenolic, lactone, car-oxylic, carbon–hydrogen bond and carbon–carbon double bondshich are strongly associated with backbone carbons, there is no
vailable carbon source for bacterial growth in the activated car-on. Because GAC is biologically inert, the bacterial communitytructure is not directly influenced by GAC.
In contrast to GAC, the anthracite, which is called hard coalith a high content of fixed carbon as compared to bitumi-ous and lignite coal, is identical to activated carbon in havinghe same mechanism for removing organic compounds. How-ver, the remaining materials or impurities after the naturaloalification can react with bacteria and impact on the bacterialommunity structure. In particular, sulfur is the major inorganicmpurity of coal, which is reactive to bacteria. Anthracite con-ains 0.60–0.77 (%w) weight percentage of sulfur (Classificationf coal, http://www.engineeringtoolbox.com/classification-coal-d64.html). Sulfur has a potentially toxic effect that may lead to
imited growth of heterotrophic bacterial mass that in turn mayemove AOC or competition for oxygen between heterotrophsnd SOB might cause a reduction in the anthracite biofilter’sOC removal efficiency. However, the anthracite granule is stilln attractive option for the biofilter due to less flow-resistancedue to its larger granule size) and consequently less mechan-cal energy requirement. Diverse heterotrophs were found inhe anthracite biofilter, but alkaliphilic bacteria that were dom-nant out-competed the normal heterotrophic biofilm bacteria.he latter was dominant in the GAC biofilter. However, it wasot possible to evaluate the influence of typical heterotrophs andlkaliphiles on AOC removal due to limited sets of data available.ulfur-related denitrifiers in anthracite biofilter would be advan-ageous in limiting the nitrogen compositions (NO2
− and NO3−)
or bacterial growth after filtration. To confirm the presence oflkaliphilc and mesophilic bacteria in the anthracite biofilter, moreetailed investigations are required at constant pH and temper-ture. In this study, a slight decline in pH from 8.0 to 7.1 wasbserved on Day 70. Furthermore the temperature was not con-rolled because it was kept at room temperature. Investigation ofppropriate/optimum conditions and especially concerning sulfur-elated and alkaliphilic heterotrophic bacteria in the anthraciteiofilter could help to maximize anthracite biofilter’s AOC removalfficiency.
The community structure of heterotrophic bacteria in anthraciteiofilter including a heterotrophic sulfur-oxidizing bacterium, S.ontiacus, which is responsible for the removal of organic matters,s extremely different to that of the GAC biofilter in the middle andhe bottom parts. This is despite their use of the same organic com-onents in the influent. The residual sulfur content in the anthracitelearly influenced the marine bacterial community structure. Therecise reason for the different heterotrophic bacterial community
s unclear at present and the niche differentiation for heterotrophsaused by the sulfur elements has not been characterized. The cellumbers and ATP concentrations of the anthracite biofilter are
omparable to those of the GAC biofilter. However, the reductionn the relative abundance of heterotrophs in the anthracite biofil-er results in lower DOC and AOC removal efficiencies compared tohat of the GAC biofilter.
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80 S. Jeong et al. / Ecological E
. Conclusion
This study investigated the sustainability of biofilters (GAC andnthracite as media) applied as pretreatment to a RO-based desali-ation plant. Detailed microbial community structure using T-RFLPt three stages of the operational period lasting 75 days was ana-yzed and discussed in this study. A dynamic shift in the bacterialommunity was observed at the top part of the biofilter during theltration experiment. The change in the bacterial community struc-ure in the filter bed may be associated with biofilm stability. BothAC and anthracite biofilters consisted of diverse heterotrophs.he anthracite biofilter indicated predominantly sulfur-oxidizingnd reducing bacteria and alkalitrophic heterotrophs due to theresence of sulfur as an impurity in anthracite. Relatively lowbundance of heterotrophic bacteria in the anthracite, which wasnfluenced by the sulfur metabolism, contributed to a lower organicemoval from seawater. It is desirable in the future to undertakeetailed characterization of the bacterial community structure onrganic removal under different conditions (such as different fil-ration rates, backwash velocities and media grain sizes).
cknowledgment
The authors acknowledge the financial support of the Nationalentre of Excellence in Desalination Australia which is funded byhe Australian Government through the Water for the Future ini-iative.
ppendix A. Supplementary data
Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.ecoleng.013.09.005.
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