Community Assembly and Ecology of Activated Sludge under

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Dark/Feast Light/Famine PageEnvironmental 1 of 43 Science & Technology

Ac P N2

PAO CO2

NH4

NOx- Nitrifiers O2

P

PAO CO2

N2

NH4

NOx- Nitrifiers O2

ACS Paragon Plus Environment

Algae

Algae

Environmental Science & Technology

1

Community Assembly and Ecology of Activated Sludge Under Photosynthetic

2

Feast/Famine Conditions

Page 2 of 43

3 4

Ben O. Oyserman1*, Joseph M. Martirano1, Spenser Wipperfurth1, Brian R. Owen1, Daniel R.

5

Noguera1, Katherine D. McMahon1,2

6 7

1

8

Madison, WI, 53706, USA; 2Department of Bacteriology, University of Wisconsin at Madison,

9

Madison, WI, 53706, USA;

Department of Civil and Environmental Engineering, University of Wisconsin at Madison,

10 11

Keyword: Algae, Photosynthesis, Polyphosphate, Phosphorus Cycling, Wastewater Treatment,

12

Low Dissolved Oxygen; Symbiosis, Cyanobacteria,

13 14

Running Title – Light-driven polyphosphate cycling

15 16

* corresponding author

17 18

Address: 3207D Engineering Hall, 1415 Engineering Drive, Madison, WI 53706

19

Tel: (734)-272-1249

20

Fax: (608) 262-5199

21 22

Email: [email protected]

23

1 ACS Paragon Plus Environment

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Abstract

25

Here we demonstrate that photosynthetic oxygen production under light/dark, feast/famine

26

cycles with no mechanical aeration and negligible oxygen diffusion is able to maintain

27

phosphorus cycling activity associated with the enrichment of polyphosphate accumulating

28

organisms (PAOs). We investigate the ecology of this novel system by conducting a time series

29

analysis of prokaryotic and eukaryotic biodiversity using the V3-V4 and V4 region of the 16S

30

and 18S rRNA gene sequences, respectively. In the Eukaryotic community, the initial dominant

31

alga observed was Desmodesmus. During operation, the algal community became a more diverse

32

consortium of Desmodesmus, Parachlorella, Characiopodium and Bacillariophytina. In the

33

Prokaryotic community, there was an initial enrichment of the PAO Candidatus Accumulibacter

34

phosphatis (Accumulibacter) Acc-SG2, and the dominant ammonia-oxidizing organism was

35

Nitrosomonas oligotropha, however these populations decreased in relative abundance,

36

becoming dominated by Accumulibacter Acc-SG3 and Nitrosomonas ureae. Furthermore,

37

functional guilds that were not abundant initially became enriched including the putative

38

Cyanobacterial PAOs Obscuribacterales and Leptolyngbya, and the H2-oxidizing denitrifying

39

autotroph Sulfuritalea. After a month of operation, the most abundant prokaryote belonged to an

40

uncharacterized clade of Chlorobi classified as Chlorobiales;SJA-28 Clade III, the first reported

41

enrichment of this lineage. This experiment represents the first investigation into the ecological

42

interactions and community assembly during photosynthetic feast/famine conditions. Our

43

findings suggest that photosynthesis may provide sufficient oxygen to drive polyphosphate

44

cycling.

45



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46

Keywords: photosynthesis, phosphorus cycling, community assembly, low dissolved oxygen,

47

Chlorobiales


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48

Introduction

49

The success of microbial consortia based biotechnologies is dependent on an understanding of

50

the ecological processes that select for a particular ecosystem function. Once the selective

51

pressures that promote the deterministic assembly of a community with a targeted function have

52

been identified, operational parameters may be optimized to promote these functions, thereby

53

providing stable and economical biotechnologies such as wastewater treatment. Consequently,

54

novel operational parameters hypothesized to promote particular functions may also be assessed

55

and the resulting communities analyzed to identify organisms and interactions that may be

56

selected for or against. With this conceptual framework in mind, a novel operational

57

configuration was analyzed to determine how the oxygen provided by photosynthetic

58

communities might influence the microbial community in a bioreactor subjected to feast and

59

famine cycles analogous to those used in combination with mechanical aeration to encourage

60

polyphosphate (polyP) cycling.

61

Interest in energy efficient and carbon neutral wastewater treatment processes has been

62

stimulated by an increasing awareness that wastewater is a resource from which water, nutrients

63

and energy may be recovered1. Current biological nutrient removal (BNR) technology is often

64

energy intensive in part due to operational requirements such as mechanical aeration. A common

65

method to decrease these energy requirements is by operating treatment systems with minimal

66

aeration2–5, facilitating conditions favorable for simultaneous nitrification, denitrification and

67

phosphorus removal in the absence of differentially aerated zones6. In addition to practices that

68

minimize oxygen requirements, economical alternatives to mechanical aeration such as

69

photosynthetic oxygenation have long been recognized7. Photosynthetic processes are commonly

70

implemented in low tech systems such as high rate algal ponds (HRAP)8, however they are rarely



Page 6 of 43

71

integrated into the predominantly heterotrophic activated sludge-type BNR systems9,10.

72

Integrating photosynthesis and activated sludge processes is an alluring aspiration because

73

oxygen provided from photosynthesis may be sufficient to fulfill treatment requirements11 and

74

photoautotrophic growth has the potential to contribute to carbon neutral wastewater treatment

75

processes12.

76

In photosynthetic wastewater treatment systems, algae and bacteria may form a reciprocal

77

association in which carbon dioxide (CO2) and oxygen (O2) are exchanged. Theoretical

78

calculations for this symbiotic interaction in a closed system have suggested that the CO2 and O2

79

production are sufficient to drive each process respectively, but that nitrogen (N) and phosphorus

80

(P) remain in solution13. This drawback is also commonly identified in practice, as

81

photosynthetic systems are often unable to meet nutrient removal requirements14. One approach

82

to address this stoichiometric imbalance may be to integrate established feast/famine cycles of

83

BNR technologies with photosynthetic processes. However, traditional BNR treatment plants

84

rely on microbial communities operated primarily in the dark. Therefore, there is currently very

85

limited understanding of the communities and interactions that may be selected for in engineered

86

systems that combine light and dark environments. Only after these interactions have been

87

identified, may they be managed through operational parameterization (e.g., to select for or

88

against specific microorganisms).

89

In this experiment, we investigated whether photosynthetic oxygenation could be

90

sufficient to allow the establishment of polyP cycling and PAO enrichment. Specifically, a

91

reactor was inoculated with two activated sludge communities, a photosynthetic nitrifying

92

culture and an Enhanced Biological Phosphorus Removal (EBPR) culture, and operated over

93

approximately two months under light/dark and feast/famine conditions. Furthermore, we


Page 7 of 43


94

tracked the microbial community structure derived from this coupling. While technical and

95

economical challenges associated with optimizing light delivery in photosynthetic systems

96

remain a hurdle, our results reveal that it is possible to couple polyP cycling with photosynthetic

97

oxygen production, that this configuration may maintain an enrichment of PAOs, and that

98

continuous illumination does not negatively impact polyP cycling. We discuss intriguing

99

ecological interactions that may be exploited under photosynthetic feast/famine conditions

100

including the enrichment of a novel uncharacterized lineage within the Chlorobi. Lastly, the

101

community analysis suggests that a key feature of photosynthetic aeration is the avoidance of gas

102

stripping, and consequently, the development of syntrophic communities relying on gaseous

103

metabolites such as H2.

104

Materials and Methods

105

Seed sludge

106

The bioreactor was inoculated with sludge from two lab scale wastewater treatment systems: an

107

EBPR system achieving P removal and a photosynthetic bioreactor achieving nitrification.

108

Operational parameters for the EBPR system are as previously reported15. The operational

109

parameters for the photosynthetic nitrification system were identical to those described below,

110

except with no added acetate16. Mixed liquor from each reactor (500 mL) was collected, allowed

111

to settle, decanted and then rinsed with tap water. Decanting and rinsing was done to remove

112

contaminating nutrients such as carbon, and potential terminal electron acceptors such as nitrite

113

and nitrate. The rinsed biomass was then mixed with ~ 2000 mL of anoxic mineral medium

114

prepared by extended purging of the medium with N2 gas.



Page 8 of 43

115

Reactor operation

116

A 2.5 L reactor was operated as a sequencing batch reactor with a 12-hour cycle with the

117

following sequence: A 30 minute settling, 8 minute decanting, 8 minute fill (8 minutes for water,

118

2 minutes for media), a 2-hour and 14 minute dark period and a 9-hour light period. The reactor

119

was constructed out of a 20" tall x 4" diameter glass and was illuminated with a General Electric

120

18-Inch Basic Fluorescent Light Fixture (product descriptor UCF18/P/BSC) with an illumination

121

intensity of 1200-1300 lux. The stirring speed was set at level 5 using a CimarecTM stirrer

122

(Thermo Scientific, USA). A hydraulic retention time (HRT) of 0.625 days and a solids retention

123

time (SRT) of 13.3 days were achieved by decanting after settling to a volume of 500 mL

124

(removing 2 L of effluent) twice per day, and wasting approximately 188 mL of the mixed

125

volume once a day. After an initial 10 minutes of the dark period, 25 mL of media A and B,

126

respectively, and 1950 mL of H2O were pumped into the reactor. Media A was a mineral

127

medium composed of 13.1 g/L sodium acetate trihydrate, 0.88 g/L of KH2PO4, 9.1 g/L of

128

NaHCO3 and 0.68 g/L of KHSO4. Media B was composed of 1.96 g/L NH4Cl, 4.44 g/L

129

CaCl2·2H2O, 3.03 g/L MgSO4 and 20mL of a trace element solution composed of 5.51 g/L citric

130

acid, 4.03 g/L hippuric acid, 0.73 g/L Na3NTA.H2O, 0.3 g/L Na3EDTA·4H2O, 3.03 g/L

131

FeCl3·6H2O, 0.5 g/L H3BO3, 0.3 g/L ZnSO4·7H2O, 0.24 g/L MnCl2·4H2O, 0.12 g/L

132

CuSO4·5H2O, 0.06 g/L KI, 0.06 g/L Na2MoO4·2H2O, 0.06 g/L CoCl2·6H2O, 0.06 g/L

133

NiCl2·6H2O, 0.06 g/L Na2WO4·2H2O. Throughout the cycle, pH was monitored using an EW-

134

56700-10 Eutech Instruments pH 190 1/8-DIN pH/ORP controller (Cole-Parmer, Vernon Hills,

135

IL). The pH was automatically adjusted using 5% HCl and 50 g/L of Na2CO3 to a set point of 7.5

136

with a hysteresis value of 0.1. After operating in this fashion for 46 days (~3.5 SRTs), the dark

137

period was eliminated during an additional 21 days of operation (~1.5 SRT), to investigate


Page 9 of 43


138

whether P cycling could be sustained under continuous illumination. The analytical chemistry,

139

DNA extraction, and sequencing methodology followed standard practices and may be found in

140

the Supplemental Methods.

141

Nucleotide accession numbers

142

The full 16S and 18S data sets were deposited to the National Center for Biotechnology

143

Information (NCBI) Sequence Read Archive (SRA) under accession number SRP073389.

144

Results

145

Reactor performance

146

Based on reactor performance and operational parameters, three distinct phases were observed

147

(Figure 1A). Phase 1 was characterized by a rapid stabilization of P removal, polyP cycling, and

148

a slow degradation of N removal. Phase 2 was characterized by continued stability in P removal

149

and increasing N removal. Finally, Phase 3 was defined by the shift in operational mode from

150

dark/light cycles to continuous illumination.

151

Near complete P removal was achieved by day four of reactor operation and was

152

maintained (94±7% removal) for the entirety of reactor operation (Figure 1A, Table 1). The

153

average P removal showed improvement during each phase, however only the difference

154

between Phase 1 and 3 was statistically significant (Supplemental Table 1). Even after initial

155

inoculation (cycle 1), when low P removal efficiency was observed, P release and uptake

156

occurred. P release and uptake then continued for the duration of reactor operation, even when

157

operated under continuous illumination (Figure 1B-D, Table 1). Both TP and dry cell weight

158

increased slightly throughout operation but stabilized between Phase 2 and 3 (Table 1). The ratio

159

of TP/dry cell weight did not vary significantly throughout reactor operation (Table 1).

160

After the initial seeding event (Cycle 1), total soluble N removal was relatively high but

161

decreased until reaching a minimal efficiency at the end of Phase 1 (Figure 1A). During Phase 2, 8 ACS Paragon Plus Environment


Page 10 of 43

162

without any modification in operational conditions, N removal rebounded, reaching peak

163

efficiency during Phase 3 where, on average, approximately 90% of the soluble N was removed

164

(Figure 1A, Table 1). During Phase 1, NH4+-N levels increased during the feast period (Figure

165

1B, Table 1) relative to at the beginning of the cycle. The change in concentration of NH4+-N at

166

the anaerobic feast (∆ NH4+-N AF) was significantly different than in Phase 2 and 3, where this

167

behavior was not observed (Table 1, Figure 1B-D). NO2--N and NO3--N levels were low at the

168

end of the feast period during Phases 1 and 2, and completely absent during Phase 3, a difference

169

that was statistically significant (Table 1 and Supplemental Table 1). The NO2--N levels at the

170

end of the cycle increased significantly from Phase 1 to 3, with intermediate values at Phase 2

171

(Table 1 and Supplemental Table 1). NO3--N was completely absent in Phase 3 (Table 1), a

172

statistically significant difference from Phase 1 (Supplemental Table 1). The summary of

173

chemical data collected at the beginning, end of anaerobic feast, and end of the cycle may be

174

found in Supplemental Spreadsheet 2. A summary of chemical data from the days in which a full

175

chemical profile was conducted (Figures 1B-D) may be found in Supplemental Spreadsheets 3-7.

176

During all three phases, dissolved oxygen (DO) concentrations were near saturation (~8

177

mg/L) immediately after fill. This oxygen was depleted rapidly and remained below 0.05 mg/L

178

until the end of the cycle when oxygen would occasionally begin to accumulate until the settling

179

period began (Figures 2A and B). Specifically, DO concentrations were below 0.05 mg/L

180

approximately 70% of the time, between 0.05-0.6 mg/L approximately 17% of the time, and

181

above 0.6 mg/L only 13% of the time (Supplemental Figure 1C). DO accumulation at the end of

182

the cycle differed between phases as well, especially during Phase 3, when continuous

183

illumination resulted in frequent accumulation of DO at the end of the cycle (Supplemental

184

Figure 1D). Nitrite and nitrate concentrations were positively correlated with end-of-cycle DO


Page 11 of 43


185

concentrations. In contrast, P concentrations at the end of the anaerobic feast period were

186

negatively correlated with end-of-cycle DO concentrations. A complete DO profile and analysis

187

of end of cycle concentrations may be found in Supplemental Spreadsheet 8.

188

Oxygen diffusion and theoretical oxygen demand

189

In this experiment the primary purpose was to investigate whether photosynthetic oxygenation

190

was sufficient to drive polyP cycling, if a PAO enrichment could be maintained, and what

191

flanking community developed under such conditions. Here we demonstrate that oxygen

192

diffusion into the reactor and the introduction of oxygen through the influent was a negligible

193

source of oxygen. A negative control using tap water showed diffusion into the reactor occurring

194

at a rate of approximately 0.04 mg/L/hr. This suggests that reactor was nearly airtight and that

195

diffusion accounted for approximately 0.5 mg/L over a 12-hour cycle. An additional abiotic

196

source of oxygen is through the addition of the influent, which had oxygen at saturation and

197

contributed approximately 8.5 mg/L at the beginning of each cycle. Together, these abiotic

198

sources of oxygen account for approximately 9 mg/L. In contrast, the theoretical oxygen demand

199

(ThOD) of the influent, primarily in the form of carbonaceous and nitrogen species was 246

200

mg/L O2, in the form of ThODAcetic acid (103 mg/L O2), ThODHippuric acid (79 mg/L O2), ThODCitric

201

acid

202

that does not include the oxygen demand from the degradation of autochthonous carbon

(41 mg/L O2) and ThODAmmonia (23 mg/L O2), respectively. Thus, a conservative estimate

) of oxygen required to achieve nutrient removal in the

203

demonstrates that less than 5% (

204

bioreactor had abiotic origin.

205

Raw read processing

206

A total of 8,149,404 16S and 11,581,146 18S raw reads were generated. After quality filtering,

207

adapter trimming and merging paired end reads, a total of 3,988,393 16S and 5,134,217 18S 10 ACS Paragon Plus Environment


Page 12 of 43

208

contigs remained that were further processed using the Mothur MiSeq standard operating

209

procedure (SOP) pipeline17. Following the SOP guidelines, preclustering and chimera removal

210

were conducted on the contigs resulting in 2,850,182 16S and 4,616,271 18S total reads

211

remaining in each data set. These reads were represented by 104,266 unique 16S and 54,226

212

unique 18S sequences, respectively. The number of reads per sample ranged from 110,901 to

213

161,901 in the 16S dataset and 192,198 to 266,630 in the 18S dataset, and therefore, each sample

214

was subsampled to a depth of 110,901 and 192,198 respectively. These values are summarized in

215

Supplemental Spreadsheet 9.

216

16S and 18S rRNA gene based microbial community analysis

217

Principal components analysis demonstrated tight clustering of triplicate samples for both 16S

218

and 18S datasets (Figure 2A and B), and this was corroborated by hierarchical clustering analysis

219

(Supplemental Figure 2). Thus all downstream analysis on relative abundance for each date was

220

conducted using the average OTU read abundance obtained from the triplicates.

221

The clustering analysis of the top 20 OTUs revealed that two distinct dominant Bacterial

222

communities were found in the time series. Initially, the community was characterized by an

223

abundance

224

Flavobacterium, Chryseobacterium, Inhella, Lysobacter and Ignavibacterium (Figure 2C). As

225

time progressed, a new community developed in the reactor characterized by Ferruginibacter,

226

Dechloromonas,

227

Sediminibacterium,

228

Accumulibacter (OTU000005). Notable differences between the early and late communities

229

included selection for different Accumulibacter OTUs, and the enrichment of the Cyanobacterial

of

Accumulibacter

(OTU000001),

Saprospiraceae,

Chitinophagaceae,

Leptolyngbya,

Obscuribacterales,

Sulfuritalea,

Lewinella,

Chlorobiales;SJA-28 Accumulibacter

Thauera,

(OTU000002),

(OTU000003)

and


Page 13 of 43


230

lineages Leptolyngbya18 and Obscuribacterales19, H2-oxidizing facultative autotrophic

231

Sulfuritalea, and uncharacterized Chlorobiales;SJA-28 (Figure 2C).

232

In contrast to the Bacterial community (based on 16S rRNA), much of the Eukaryotic

233

community (based on 18S rRNA) was unclassified and no specific lineages displayed a distinct

234

pattern of either increasing or decreasing across the time series (Figure 2D). However, a clear

235

pattern in the photosynthetic eukaryotic community was identified, which was initially

236

dominated by Desmodesmus and transitioned to a more diverse assemblage, which included

237

Desmodesmus, Parachlorella, Bacillariophytina, and Characiopodium. Numerous potential

238

predatory eukaryotic organisms were also identified including Gymnophrys, Rotifera and

239

Alveolata. However, their presence was sporadic and no pattern was detected (Figure 2D).

240

We examined the relative abundance of OTUs classified as taxa likely to be involved in

241

nitrification. The nitrifying community relative abundance decreased over the course of the

242

experiment (Figure 3). Initially, the ammonia oxidizing community was dominated by

243

OTU000120 and OTU000327, however after day 28 (cycle 57) OTU000322 increased in relative

244

abundance (Figure 3A). In contrast, the nitrite oxidizing community was composed of only a

245

single identified OTU (OTU000052) within the Nitrospira, which saw a large relative decrease

246

from initial concentrations (Figure 3B). No Nitrosospira, Nitrosococcus, ammonia oxidizing

247

archaea, Nitrobacter, Nitrococcus, and Nitrotoga, were detected.

248

Phylogenetic analysis of Accumulibacter, Nitrosomonas and Chlorobi OTUs

249

A phylogenetic analysis of each operational taxonomic unit (OTU) classified within

250

Accumulibacter, Nitrosomonas and Chlorobi was conducted to further resolve their taxonomy

251

(See Supplemental Methods). The phylogenetic analysis revealed that the dominant

252

Accumulibacter OTUs each had different taxonomies including Clade IIC Acc-SG2

253

(OTU000001), Clade IIC Acc-SG3 (OTU000003), and Clade IIB (OTU000005)20,21 12 ACS Paragon Plus Environment


Page 14 of 43

254

(Supplemental Figure 3). A majority of the Nitrosomonas-related OTUs clustered with

255

Nitrosomonas oligotropha (OTU000327, OTU000120, OTU000714 and OTU000512), however

256

the dominant Nitrosomonas OTU at the end of the investigation was Nitrosomonas ureae

257

(OTU000322) (Supplemental Figure 4).

258

The

phylogenetic

analysis

of

sequences

affiliated

with

Chlorobiales;SJA-28

259

(OTU000002) revealed a

lineage composed of at least 3 distinct monophyletic groups

260

henceforth referred to as Chlorobiales;SJA-28 Clade I, Clade II, and Clade III (Figure 4).

261

Together, these three clades form an uncharacterized monophyletic lineage sister to the

262

Chlorobea. Clade I was composed of sequences identified in anaerobic digesters, hydrothermal

263

soils and termite hindguts. When reported, the relative abundance of Clade I type sequences were

264

always below 5%. Clade II type sequences contained clone SJA-2822, for which the current

265

taxonomical classification is based. The habitats for which Clade II type sequences were detected

266

included microbial fuel cells, anaerobic digesters and gas-polluted soil. Relative abundance

267

estimates for Clade II type sequences were always below 3.1%. Sequences within Clade III

268

originate predominately from wastewater treatment systems (11/13), of which EBPR-type

269

systems23–25 with acetate, propionate or municipal wastewater as a primary carbon source were

270

the most abundant. Other habitats included anaerobic digesters with carbon sources of municipal

271

wastewater26 and benzene27, Anammox reactors (unpublished), a flow through column reactor28,

272

vermiculture system29, deep groundwater (unpublished) and rhizosphere30. In contrast to other

273

studies, Chlorobiales;SJA-28 Clade III type sequences were identified with high relative

274

abundance (>25%) in this investigation, and in an nitrate reducing, anaerobic benzene degrading

275

culture amended with H227.

276

Discussion


Page 15 of 43


277

Assessing reactor function under light/dark cycles

278

One of the basic failures of algal-based treatment systems is their variable and inconsistent

279

ability to completely remove nutrient contaminants such as P14. Conversely, while BNR systems

280

generally achieve stable nutrient removal, they currently lack a substantial mechanism for CO2

281

sequestration. A potential solution would be to integrate photosynthetic and polyP accumulating

282

technologies, thus improving nutrient removal and CO2 sequestration by exploiting the

283

feast/famine physiology of PAOs and the photosynthetic capabilities of diverse bacterial and

284

eukaryotic lineages. In this investigation, the primary question was whether photosynthetic

285

oxygen production was sufficient to drive polyP cycling and enrich for PAOs. We demonstrated

286

that polyP accumulation, polyP cycling, and enrichment of PAOs under photosynthetic

287

conditions and with no mechanical aeration were achieved (Figure 1, Figure 2). Given the low

288

cost of light-emitting diode (LED) technology and its increasingly sophisticated application in

289

wastewater biotechnology31, these results suggest that polyP cycling may be successfully

290

coupled with photosynthetic oxygenation to improve P removal in algal systems while

291

simultaneously improving carbon sequestration in activated sludge-type systems.

292

P removal rates and polyP cycling remained stable in our experiment. In particular,

293

polymer cycling was demonstrated by high levels of P release (~30 mg/L) after acetate addition

294

and subsequent uptake. Additionally, the TP/TSS ratios (~0.16) demonstrate that the P fraction

295

of the biomass were approximately 10 times greater than in non-polyP accumulating systems

296

(~0.01-0.02). In comparison, the N removal rates decreased initially during phase 1, rebounded

297

during phase 2, finally reaching N removal rates averaging 90% during phase 3. Two

298

observations may explain the N removal dynamics. First, during Phase 1 of operation, there was

299

a slight release of ammonium during the dark period (Figure 1B). As operation progressed, and



Page 16 of 43

300

as the community shifted, this accumulation diminished and then ceased during Phase 2 and 3

301

(Figure 1C-D, Table 1). The second observation that may explain the depressed N-removal early

302

in this experiment was a shift in the nitrifying community (Figure 3). The seed nitrification

303

community originated from a photobioreactor to which no acetate was added. The oxygen

304

demand from acetate addition likely resulted in greater competition for oxygen than the

305

conditions to which the nitrifying community had originally adapted. While nitrifying

306

populations are sensitive to oxygen, they have been previously shown to adapt to low-DO

307

conditions2. Indeed, we observed a shift in the dominant Nitrosomonas OTU during this period

308

(Figure 3) upon which a diverse assemblage of Nitrosomonas oligotropha OTUs was replaced by

309

Nitrosomonas ureae. The increase in Nitrosomonas ureae relative abundance may be linked with

310

the initial increases in ammonium during the dark period in Phase 1, as urea release from

311

photosynthetic organisms may have eventually selected for organisms capable of urea

312

degradation. Given the low relative abundance of N. oligotropha and N. ureae, additional

313

investigations are required to determine the ecological significance of these populations and the

314

importance of alternative mechanisms such as assimilation for N removal in low-DO

315

photosynthetic systems. However, it is important to note that these results are consistent with

316

investigations of low-DO nitrifying systems, in which the low abundance of recognized

317

ammonia oxidizers is not sufficient to explain the extent of nitrification achieved in these

318

reactors2,32.

319

The nitrite oxidizing community did not exhibit such an adaptive response, as

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demonstrated by an increased accumulation of nitrite and loss of nitrate accumulation during

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Phase 3 (Table 1). This is in contrast to what is generally observed in BNR systems, where

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nitrate often accumulates. The deterioration of nitrite oxidation may be explained by an extended


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lag in the adaptive response of the nitrite oxidizing community, or environmental and ecological

324

conditions that selected against nitrite oxidation in favor of denitrification. Investigations into

325

low-DO systems have shown that there is a lag phase between altered oxygen concentrations and

326

community response2,33. Taken together, the efficient and stable N-removal rates achieved during

327

phase 3 of operation, previous reports that nitrification may be successfully coupled with

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photosynthetic oxygenation16, and may be achieved at low-DO33, even with low abundance of

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recognized ammonia oxidizers2, suggest that low-DO photosynthetic systems may be designed

330

achieve N removal.

331

Due to the limited scope and duration of this experiment, additional long-term ecological

332

research is needed into photosynthetic feast/famine systems operated under stable state. In

333

particular while ammonium, nitrite and nitrate measurements taken in this study demonstrate N

334

removal under photosynthetic conditions, the relative contribution of N removal through

335

assimilation versus nitrification/denitrification must be investigated in the future. Furthermore,

336

additional experiments are necessary to determine if photosynthetic feast/famine conditions

337

provide a selective mechanism for the denitrification of nitrite, as this would lower the total

338

oxygen demand required for N removal.

339

Assessing reactor function under continuous illumination

340

One of the central tenets of successful polyP accumulation in BNR technologies is the strict

341

separation of carbon and terminal electron acceptor availability. However, simultaneous

342

anaerobic and aerobic processes, such as nitrification, denitrification and EBPR, have been

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achieved under continuously aerated conditions in what is termed simultaneous biological

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nutrient removal (SBNR)6. In this experiment when the dark cycle was eliminated and the

345

reactor was operated under continuous illumination (Phase 3), P removal, N removal, and polyP



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cycling dynamics remained unperturbed (Figure 1 D, Table 1). This suggests that, as seen in

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some mechanically aerated systems6, photosynthetic communities may achieve SBNR by

348

operating without strict separation of anaerobic/aerobic (dark/light) cycles.

349

In BNR systems that are mechanically aerated, oxygen must diffuse from the gas phase,

350

to the liquid phase and finally to the cell34. Thus, mechanically aerated BNR processes rely upon

351

habitat heterogeneity across the macro and microenvironment in order to achieve SBNR6.

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Conversely, when oxygen is produced at the cellular level through photosynthesis, oxygen

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measured in the bulk fluid must first diffuse through the boundary layer around a cell, or floc, in

354

which it was produced (Supplemental Figure 5). Thus, while both mechanically aerated and

355

photosynthetically oxygenated treatment processes rely upon habitat heterogeneity between

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macro and microenvironments, they are fundamentally different. Mechanically aerated systems

357

have oxygenated macro-environments with diffusion limitations into the microenvironments,

358

whereas photosynthetic systems have anaerobic macro-environments with oxygenated

359

microenvironments. Furthermore, mechanical aeration purges native gas production, potentially

360

limiting the development of communities based on metabolite exchange of gasses such as H2. As

361

a result, photosynthetic treatment systems achieve simultaneous N and P removal in a novel, and

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fundamentally different, SBNR arrangement than traditional mechanical aeration under which

363

distinct microbial communities are expected to develop.

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Community assembly

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The cyclic feast/famine conditions typical of EBPR systems result in a strong selective pressure

366

for PAO enrichment35. In these traditional treatment systems, the carbon pool is primarily

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allochthonous, in the form of organic carbon introduced with the influent. The cyclic

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feast/famine environment found under photosynthetic-polyP accumulating conditions have a


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similarly strong selective pressure for PAO. However, the addition of light and the removal of

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mechanical aeration significantly alter the flow of energy within photosynthetic-polyP

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accumulating treatment systems, resulting in the co-enrichment of diverse functional guilds.

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Eukaryotic photosynthesis and aerobic heterotrophs

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The assemblage of eukaryotic photosynthetic organisms supported a relatively large (~24%)

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assemblage of putatively aerobic polymer hydrolyzing heterotrophs (Figure 2). Especially well

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represented were lineages within the Sphingobacteriales (two Chitinophagaceae OTUs,

376

Lewinella, Ferruginibacter and two Saprospiraceae OTUs), which are commonly found as

377

epiphytic polymer hydrolyzing bacteria in activated sludge systems36,37. Interestingly, both the

378

dominant photosynthetic Eukaryote, and aerobic hydrolyzing bacteria shifted after initial

379

inoculation, suggesting that species-specific interactions between photosynthetic and associated

380

heterotrophs may exist. Identifying strategies and operational parameters to manage oxygen

381

transfer between oxygen-producers and different guilds of oxygen-consumers may be important

382

to further develop photosynthetically oxygenated systems. For example, reducing oxygen

383

demand by algal associated heterotrophic aerobes would increase the availability of oxygen for

384

processes such as nitrification or for PAO metabolism. One strategy to achieve these may be to

385

identify operational parameters that limit algal exudates, or algal turnover (e.g., senescence and

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decay). Incorporating measurements that quantify algal-based heterotrophy in comparison to

387

simple operational changes such as the SRT represent an important first step for future

388

investigations.

389

Polyphosphate accumulation

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Throughout reactor operation the Accumulibacter relative abundance decreased and was

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accompanied by a concomitant shift in the dominant Accumulibacter OTU. Initially, Clade IIC 18 ACS Paragon Plus Environment


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Acc-SG2 dominated but was eventually replaced by Clade IIC Acc-SG3 and Clade IIB (Figure

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2). Despite the dynamic nature of the Accumulibacter community relative abundance, reactor

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performance remained stable with regards to P removal aerobically, release anaerobically, and

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the TP/dry cell weight (Table 1). Furthermore, when the reactor was operated under continuous

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illumination, these functional parameters remained stable despite the simultaneous availability of

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both organic carbon sources (acetate) and light, which are conditions favorable for

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photoheterotrophy. This suggests that under photosynthetic conditions, Accumulibacter out-

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competed other heterotrophic/photoheterotrophic organisms for acetate, likely because of the

400

evolution of numerous mechanisms for the acquisition of acetate and rapid acetate uptake

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kinetics38. Two common organisms capable of photoheterotrophic growth in wastewater

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treatment systems, Rhodopseudomonas39 and Rhodobacter40, were absent or at very low relative

403

abundance (

Community Assembly and Ecology of Activated Sludge under

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