C0141
Jeremy J. Barr
1,2
, Marcus L. Hastie
3
, Toshikazu Fukushima
1,4
, Manuel R. Plan
3
, Gene Tyson
1
,
Jeffery J. Gorman
4
3
, Phillip L. Bond
1,2*
1
The University of Queensland (UQ), Advanced Water Management Centre (AWMC), QLD 4072, Australia.
2
Environmental Biotechnology Cooperative Research Centre, Sydney, Australia
3
Protein Discovery Centre, Queensland Institute of Medical Research (QIMR), Herston, Qld, Australia
Department of Environmental Engineering, National Cheng Kung University, Tainan 70101, Taiwan R.O.C.
Enhanced biological phosphorus removal (EBPR) is a widely applied process for the removal of carbon and phosphorus from wastewater, which is achieved through the use of polyphosphate accumulating organisms (PAO). Conventional wastewater treatment plants utilise floccular sludge, which form small, suspended biofilm aggregates for the treatment of wastewater. A novel alternative to conventional floccular sludge is the use of aerobic granular sludge, which form large, dense, spherical biofilm aggregates, and offer numerous operational and economic advantages over conventional floccular systems. However, we still do not understand the fundamental microbial mechanisms associated with granular sludge formation and stability. This study monitored a granulating period in a laboratory scale reactor and present the first metaproteomic comparison of abundant proteins present in aerobic floccular and aerobic granular reactors to determine microbial functions important for granulation. We identified 403 unique proteins between floccular and granular sludge. Results indicate that proteins involved in signal transduction, cell motility, EPS formation and cell membrane biogenesis may play crucial roles in granulation.
Aerobic granular activated sludge is a special type of biofilm, that comprises large (0.2 – 2 mm), spherical, densely packed self-immobilised aggregates for the treatment of wastewater 58 . Currently, conventional wastewater treatment plants (WWTP) utilise floccular activated sludge, which form small, suspended biofilm aggregates (30200µm), for the biological treatment of wastewater 59 . However, these activated sludge flocs typically require long settling times, around 30-60 minutes, to separate biomass from treated wastewater. The comparative use of aerobic granular sludge for wastewater treatment offers several economic and operational advantages over conventional floccular sludges such as; excellent sludge settling capabilities, ability to withstand high organic loading, reduced mixing and aeration and high biomass retention 60 . Consequently, granular sludge has been applied to the treatment of high strength wastewaters for the removal of organics, nitrogen and phosphorus among others 61 .
Enhanced biological phosphorus removal (EBPR) is a widely applied wastewater treatment process for the removal of phosphorus (P) and carbon. EBPR is achieved through the cycling of activated sludge biomass and influent wastewater through alternate anaerobic and aerobic conditions 62 . This removal process employs the use of polyphosphate-accumulating organisms (PAO), which are responsible for P removal from the bulk liquid through intracellular accumulation of polyphosphate stores 43 . Net P removal from the system is achieved through the removal of waste activated sludge, typically enriched with PAO, containing high polyphosphate content 63 . When operated successfully, the EBPR process is an economical and environmentally sustainable option for P removal and wastewater treatment.
However, as stated previously, the majority of full- scale EBPR plants are operating as floccular sludges and there is recent interest for the implementation of granular sludge to further reduce operational expense.

Culture-independent studies of lab-scale EBPR identify the Rhodocyclus -related Betaproteobacterium ,
Candiatus “Accumulibacter phosphatis” (henceforth referred to as Accumulibacter) as a major PAO responsible for EBPR 64 . Furthermore, their presence and hypothesised role have been confirmed in a number of full- scale EBPR plants. Studies using the polyphosphate kinase gene ( ppk1 ) as a genetic marker reveal a rich diversity of Accumulibacter organised into two main clades (I and II), each containing several subclades with potentially different metabolic capabilities 65 . Near-complete genome sequence have been obtained from two lab-scale EBPR sludges enriched with Accumulibacter subclade IIA 66 . Currently there is very limited genome sequence of Accumulibacter clade I.
Increased availability of metagenomic sequence and advances in cultivation- independent ‘- omics’ techniques substantially increases the capability to investigate microbial function directly in the mixed culture environment 67 . Although this work greatly advances our understanding of these natural microbial communities, their complexity and diversity can severely limit analyses. By applying these genomicsenabled methods to so called ‘model’ communities containing reduced complexities and known diversity, such as those in acid mine drainage (AMD) or EBPR, it is possible to determine specific microbial interactions utilising a systems level biology approach 67-68 . Compared with more complex systems, the relatively low microbial diversity and ability to resample during periods of functional stability make these model systems especially amenable targets for detailed ‘meta-omic’ studies.
Metaproteomics, employing liquid chromatography (LC) coupled with tandem mass-spectrometry (MS), is used for the detection, identification and relative abundance of proteins from these model environmental systems 68 . This is particularly successful when coupled to metagenomic sequence obtained from the same system. Wilmes, et al.
used high -resolution community proteomics to identify key metabolic pathways in an Accumulibacter-enriched EBPR system. The study highlighted the importance of denitrification, fatty acid cycling and the glyoxylate bypass within EBPR. Here we present a shotgun LC-MS/MS metaproteomic analysis of a floccular and a granular laboratory-scale activated sludge. Both were enriched with Accumulibacter and performing EBPR at a high level. Metagenomic sequences, obtained from previously laboratory-grown EBPR sludges enriched with Accumulibacter, were employed for protein identification. This allowed for the first metaproteomic analysis of protein abundance between floccular and granular Accumulibacter-enriched EBPR sludges.
Reactor operation
Two laboratory-scale sequencing batch reactors (SBR), called Floc and Gran, were seeded with floccular activated sludge from a domestic WWTP and operated for EBPR for 160 days. The Floc reactor had a working volume of 4 l, a 6 hr cycle time (10 min decant, 6 min feed, 120 min anaerobic,
180 min aerobic, 4 min waste and 40 min settle), 24 hr hydraulic retention time (HRT) and a 10-day solids retention time (SRT). The Gran reactor had a working volume of 8 l, a 6 hr cycle time (as above except 218 min aerobic and 2 min settle), a 24 hr HRT and 18-day SRT. Both reactors were fed synthetic wastewater containing acetate as the main carbon source and orthophosphate (PO
4
3) for the enrichment of PAO. All other operating conditions are as previously described 69 .
Accumulibacter population characterisation
Fluorescence in-situ hybridisation (FISH) was performed as previously described hybridised with PAOMix 71 and EUBMix
70 . Samples were
70 probes for Accumulibacter abundance and with probes Acc-I
-444 and Acc -II-444 72 for Accumulibacter clade I and clade II abundance respectively. Fluorescent
DNA probes were visualised as previously described 69 . Quantification of Accumulibacter abundance was carried out using DAIME version 1.2 73 .
Protein extraction and purification
Sludge samples were collected 15 min from the end of the aerobic phase via centrifugation at 3500 rpm for 5 min. To ensure comparable extractions, all samples were concentrated to 10 g volatile suspended solids (VSS)/l prior to extraction. Samples were manually homogenised using a 15 ml glass homogeniser in an icebox and total cellular extract was performed using B-PER (Thermo-Scientific, IL,

USA), and an extracellular extract was obtained using a cation exchange resin (CER) method. For the total cellular extraction, sludge was resuspended at 10 gVSS/l in 50 mM Tris-HCl, pH 7 and 1 ml of sample was transferred to a micro-centrifuge tube followed by centrifugation at 15,000 g for 15 min at 4
ºC. Supernatant was discarded and the sample pellet was resuspended in 1 ml of B -PER extraction buffer containing 77 mg of dithiothreitol (DTT) and 1 tablet of Complete Mini (protease inhibitor) per 10 mL of B-PER reagent. Samples were placed at 80 ºC for 1 hr to freeze then thawed on ice and this was repeated for 3 cycles. Samples were centrifuged at 15,000 g for 15 min at 4 ºC to pellet the cell debris and the supernatant was removed for purification. For the extracellular extractions, samples were resuspended at 10 gVSS/l in 30 ml of 2 mM Na
3
PO
4
, 4 mM NaH
2
PO
4
, 9 mM NaCl, 1 mM KCl, pH 7.0 at 4 ºC. The resuspended sample was added to 21 g of Dowex -Marathon CER (Sigma-Aldrich, St.
Louis, MO, USA), 231 mg DTT and 3 tablets of Complete Mini within an extraction vessel which was stirred at 700 rpm for 5 hr at 4 ºC. Following extraction, 2 ml of sample was transferred to a microcentrifuge tube then centrifuged at 15,000 g for 15 min at 4 ºC to remove cell debris and the supernatant was retained for purification. From both the B-PER and CER extracts proteins were purified by precipitation in 13% w/v trichloroacetic acid (TCA) at 4 ºC overnight followed by centrifugation at 15,000 g for 15 min at 4 ºC. The protein pellet was then washed twice in 80% (v/v) ice-cold acetone, air-dried and then stored at 80 ºC.
Proteomics
Proteins extracted from the Floc and Gran sludges were proteolytically digested with trypsin and analysed via two-dimensional nano- LC followed by MS/MS (LC-MS/MS) using a LTQ/Orbitrap mass spectrometer. Peptide mass spectra were matched in silico to predicted peptides and, thus, proteins in the sludge using a combined dataset of three EBPR metagenomic databases 66 . For positive protein identification a minimum of two peptides were required per protein, with a minimum protein identification probability of 99% and a minimum peptide identification probability of 95%.

SBR performance and community structure
The Floc and Gran SBRs were inoculated with floccular EBPR sludge from a domestic WWTP and operated until stable EBPR performance and particle size distribution was achieved. Floc SBR demonstrated stable EBPR performance with a mean (±SE) effluent P-PO
4
3-
4
3 concentration of 2.55
(±0.96) mg L - 1 (n=13), a mean P-PO release during the anaerobic phase of 84.34 (±4.96) mg L -1
(n=13). Flocs were the predominant form of biofilm as this maintained a median particleµm size of 176
(n=16). Quantitative FISH employing 16S rRNA oligonucleotide probes of the Floc SBR revealed a mean
Figure 1. Median particle size distribution of Floc and Gran SBR over 160-day period of operation. Blue ellipses indicate protein extraction time points, BP indicates a B-PER extraction procedure was performed, CER indicates a cation exchange resin extraction procedure was performed.
!
Accumulibacter abundance of 84.1%, which was comprised approximately of 45% clade I and 46% clade II. The Gran SBR demonstrated stable EBPR performance with a mean effluent P -PO
4
3concentration of 5.83 (±1.66) mg L -1 (n=13), a P- PO
4
3 release during the anaerobic phase of 55.86
(±4.87) mg L -1 (n=13). The Gran SBR maintained a median particleµm (n=12)sizeof 743 indicating this was a granular system. Quantitative FISH of the Gran SBR revealed Accumulibacter abundance of
75.7%, which was comprised approximately of 63% clade I and 30% clade II. Protein extracts were collected from both the Floc and Gran SBR at numerous time points during the 160 days of operation
( Figure 1 ).
Protein identifications
A total of 403 unique proteins were identified from both the Floc and Gran samples using the combined
EBPR metagenomic database. Overall, 52% of the identified proteins were encoded by genes located on genomic contigs or scaffolds assigned t o ‘ A. phosphatis’ , sequences from the near -complete clade
IIA Accumulibacter genome ( Figure 2 ). In addition, 42% of identified proteins were found to derive from

contigs or scaffolds assigned to ‘not A. phosphatis ’, which are sequences from the partial clade I
Accumulibacter genome which were not included in the assemblage of the clade IIA Accumulibacter genome 27,40 . Finally, the remaining identified proteins were inferred to derive from Betaproteobacter ,
Gammaproteobacteria , Thiothrix and other bacterial species, including Actinobacteria , Sphingobacteria and Aplhaproteobacteria . In total, 11% of identified proteins were associated with information storage and processing, 15% with cellular processes and 58% with metabolism associated proteins.
Furthermore, 16% of all identified proteins were of unknown function, of which 65% exhibited uncharacterised conserved domains.
Figure 2. Taxonomic affiliation of proportion of identified proteins detected from the floccular and granular EBPR sludges, which belong to taxonomic groups based on concatenated genomic contig and scaffold binning.
There were notable differences in protein identifications and abundances between floccular and granular sludges. In total, 9% of identified proteins were unique to floccular sludge compared to 5% for granular sludge. In addition to unique proteins, 48% of all identified proteins exhibited differential abundance between floccular and granular sludge (p-values < 0.05). Specifically 27% of proteins were statistically more abundant in the floccular sludge compared to 21% for granular sludge. A summary or some abundant proteins is provided in Table 1.

Both sludge types were performing a high level of EBPR with similar reactor operational parameters and were both highly enriched for Accumulibacter . The major difference between the sample types being the biofilm morphology. The floccular sludge formed small, loose biofilm aggregates approximatelyµinsize 176compared to granular sludge, which formed large, dense, spherical biofilm aggregatesµminsize.approximatelyConsequently743 , this study detected abundant functional proteins to determine those of importance to the granulation process. It must also be noted that the function of particular genes discussed is inferred, based on sequence homology and annotation provided in the metagenomic databases. Verification of protein function was not performed in this study, but that remains a valuable endeavor.
Using metaproteomics we identified a total of 403 unique proteins from both floccular and granular sludge. In a previous proteomic analysis performed on a similar EBPR system a higher level of protein identifications was achieved 68 . A possible explanation for the lower level of identifications here is that the metagenomic database used was dominated by Accumulibacter clade IIA sequences
66 . However, Accumulibacter clade I was abundant in the Floc and Gran reactors. Nonetheless, we were still able to detect abundant proteins in the floccular and granular sludges. Some of these proteins are inferred to have function important for the biofilm state.
A number of proteins involved in the regulation of the signal mess enger molecule 3´,5´-cyclic diguanylic acid (c-di-GMP) were detected within our floccular and granular sludges. This is thought to be a key intracellular molecule regulating the transition between sessile and motile lifestyles for bacteria 74 . High levels cause a signal transduction cascade promoting the switch between a motile, single -cell state to an adhesive, surface-attached, EPS-producing, multicellular biofilm state. Low levels of c-di-GMP promote release of cells from a biofilm towards a motile, single-cell state. The intracellular levels of c-di-GMP are regulated by various proteins; proteins that contain GGDEF domains will cause increased levels, and EAL domain proteins will decrease those levels. Both
GGDEF and EAL domain proteins were detected in the granular sludge, with GGDEF domain proteins being more abundant, promoting cells toward biofilm formation. In the floccular sludge two
EAL domain proteins were detected, even though the majority of biomass within floccular sludge will exist as small biofilm structures. These results suggest there are differences in the regulation of c-di-
GMP between floccular and granular sludge and the control of this could be important for determining the type of biofilm forming. Further research is needed to determine details involved in the stimuli and regulation mechanisms, which may facilitate understanding and possible control of the granular biofilm state.
Flagella associated proteins were abundant within the granular sludge, indicating they may play a role in granule formation or stability. This seems in contrast to biofilm formation as flagella are typically associated with motile, planktonic cells. However, it has been demonstrated that highly regulated motility by flagella is required for the development of sophisticated biofilm architecture 75 , and the movement of individual cells via flagella may have a role here in granule formation and stability. An alternative explanation is that motile based dispersal of cells is more active in the granular biofilm. Regardless, the apparent tight regulation of c-di-GMP, the role of flagella and the switching between motile and sessile phase appears important in granulation, and further research is required to understand these complex mechanisms.
Genes encoding outer membrane proteins, such as porins, transporters and secretory systems were also among the most abundant transcripts and proteins identified, with differences of abundance between the floccular and granular sludges. ABC- type amino acid and branched amino acid transport systems were detected in high abundance. These outer membrane transporters appeared to be more abundant in granular sludge and were among the unique granular proteins. This suggests that

30
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granular sludge appears to be adapted for scavenging extra-cellular amino acids, possibly for metabolism or protein synthesis, which may be further excreted as extracellular polymeric substances (EPS). It is plausible that in the granular state there may be a higher turnover of extracellular proteins associated with EPS synthesis and the abundance of these amino acid transporters may further reflect the importance of the protein component within EPS.
EPS an important in biofilm component, is thought to play a major role in the granulation process.
Three EPS gene cassettes have been identified within the Accumulibacter genome, however these cassettes are found to be hyper variable 66 . We detected two proteins involved in EPS production, glycosyltransferase (GT) and glucose-6-phosphate isomerase (GPI), however neither appeared to be different in abundance between the floccular and granular sludges. In addition to the
Accumulibacter clade differences making protein detection difficult, it is likely that the hypervariability of these modules would cause low detection of these EPS production proteins within our sludges.
A unique ABC-type xylose transport system protein was identified from granular sludge. These can facilitate the uptake or excretion of the polysaccharide xylose, which may play a structural role in
EPS, however, the specific role xylose plays within granular sludge biofilms is unknown. A number of outer membrane peptidoglycan-associated lipoproteins were also identified. These are typically involved in the transport and assemblage of peptidoglycan and lipoproteins into the bacterial cell wall for growth and structural integrity. These will affect the charge and hydrophobicity of the cell membrane which is important for biofilm stability. Lipoproteins were detected in both the floccular and granular sludges, and likely these are important for cell growth and biofilm structure, but the results do not implicate these with a role specific for the granular sludge.
This study reports the first proteomic investigation to investigate functional differences between aerobic granule and flocullar activated sludge. Differences have been detected in abundance of particular proteins, and these may be important for physiological differences between the sludges biofilm state.
Acknowledgements
This work received funding support from the Environmental Biotechnology Cooperative Research
Centre (EBCRC), which was established and funded by the Australian Government together with industry and university partners. Jeremy Barr acknowledges EBCRC for funding of PhD Scholarship.
Philip Bond acknowledges EBCRC, Waste Technologies of Australia, The University of Queensland, and the Queensland Government Smart State Fellowship Program for funding of a senior research fellowship.
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