Synergistic exposure of return-sludge to anaerobic starvation, sulfide

May 22, 2018 - A key step towards energy-positive sewage treatment is the development of mainstream partial nitritation/anammox, a nitrogen removal ...
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PN/A

Nitrite accumulation

IFAS Page 1 ofEnvironmental 32 Science & Technology ratio 0.5

Flocs

0.4

Treatment

0.3 0.2 FA shock

starvation

0.1

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0

5 10 15 Time (d)

Environmental Science & Technology

exposure

1

Synergistic

2

starvation, sulfide and free ammonia to suppress nitrite

3

oxidizing bacteria

4 5

Dries, Seuntjens , Michiel, Van Tendeloo , Ioanna, Chatzigiannidou , Jose Maria, Carvajal

6 7 8

1

CMET – Center for Microbial Ecology and Technology, Ghent University, Coupure Links 653, 9000 Gent

2

Research group of Sustainable Energy, Air and Water Technology, University of Antwerp,

1

1

of

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return-sludge

1,2

1

Arroyo , Sander Vandendriessche , Siegfried Elias, Vlaeminck

to

1

1,2+

& Nico, Boon

1*+

Groenenborgerlaan 171, 2020 Antwerpen, Belgium

9 10 11

+

12

[email protected]

anaerobic

Equal contribution as senior author *Corresponding author. Phone: +32 9 264 59 76; fax: +32 9 264 62 48; E-mail:

13

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Abstract

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A key step towards energy-positive sewage treatment is the development of mainstream partial

17

nitritation/anammox, a nitrogen removal technology where aerobic ammonium-oxidizing bacteria

18

(AerAOB) are desired, while nitrite-oxidizing bacteria (NOB) are not. To suppress NOB, a novel

19

return-sludge treatment was investigated. Single and combined effects of sulfide (0-600 mg S L-

20

1

21

tested for immediate effects and long-term recovery. AerAOB and NOB were inhibited

22

immediately and proportionally by sulfide, with AerAOB better coping with the inhibition, while

23

the short FA shock and anaerobic starvation had minor effects. Combinatory effects inhibited

24

AerAOB and NOB more strongly. A combined treatment of sulfide (150 mg S L-1), 2d anaerobic

25

starvation and FA shock (30 mg FA-N L-1) inhibited AerAOB 14% more strongly compared to

26

sulfide addition alone, while the AerAOB/NOB activity ratio remained constant. Despite no

27

positive change was observed in the immediate-stress response, AerAOB recovered much

28

faster than NOB, with a nitrite accumulation ratio (effluent nitrite on nitrite + nitrate) peak of 50%

29

after 12 days. Studying long-term recovery is therefore crucial for design of an optimal NOB-

30

suppression treatment, while applying combined stressors regularly may lead towards practical

31

implementation.

), anaerobic starvation (0-8d) and a free ammonia (FA) shock (30 mg FA-N L-1 for 1h) were

32

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1. Introduction

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Enabling energy-positive sewage treatment can lower sewage treatment operational costs,

35

while potentially decreasing nitrogen emissions in a cost-effective way. Mainstream partial

36

nitritation/anammox (PN/A) is an essential technology for this new sewage treatment plant

37

(STP), since nitrogen is removed autotrophically. This allows maximum carbon redirection in a

38

prior stage towards sidestream anaerobic sludge digestion, where energy is recovered in the

39

form of biogas (1).

40 41

Operating mainstream PN/A (in the water line of a STP) is challenging, since current operation

42

strategies still allow growth of nitrite oxidizing bacteria (NOB), lowering the nitrogen removal

43

efficiency. Most successful attempts so far were in hybrid reactors, where ON/OFF control

44

strategies (promotion/suppression of desired/undesired micro-organisms) were combined with

45

IN/OUT control (retention/removal of desired/undesired micro-organisms) (2). This IN/OUT

46

control consisted of long sludge retention times (SRT) for the slow growing anoxic ammonium

47

oxidizing bacteria (AnAOB) in the biofilm, and an appropriate lower flocculent SRT to ensure

48

aerobic ammonium oxidizing bacteria (AerAOB) presence and NOB wash-out (3,4). In these

49

systems, the highest N-removal efficiencies were observed when high or almost complete

50

flocculent NOB suppression was achieved (3,4). However, disturbances in reactor operation,

51

e.g. changes in temperature, flocculent SRT, etc. often induce flocculent NOB proliferation (4-6).

52

When these events occur, swiftly suppressing the floccular NOB activity by an ON/OFF control

53

strategy would enable to quickly restore treatment efficiency. Furthermore, fast start-up of

54

mainstream PN/A reactors can be facilitated. A number of stressors or inhibitors like free

55

ammonia (FA), free nitrous acid (FNA), sulfide, ClO4-, formic acid or volatile fatty acids have

56

been shown to favor AerAOB activity over NOB activity when in high enough concentrations (7).

57

Unfortunately, high concentrations of relatively selective toxicants cannot be achieved under the

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mainstream conditions. Alternatively, NOB-selective inhibition can be applied in the return-

59

sludge line (8).

60 61

Two readily available inhibitors in the context of a STP are FA (< 50 mg FA-N L-1) and FNA (
150 mg S L-1),

297

decreasing the relative rAerAOB/rNOB, which is comparable to the combined effect of sulfide

298

and FA. A similar pattern was seen when starvation, sulfide and FA were combined. With either

299

no effect at 150 mg S L-1 or a 33% decrease in AerAOB activity and 48% decrease in relative

300

rAerAOB/rNOB at 300 mg S L-1, compared to the treatment with sulfide and starvation. The

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combined effect of starvation, sulfide and FA caused thus a very high initial inhibition, including

302

lag phases of AerAOB, while NOB initially coped better with the perceived stress.

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3.3.Long-term recovery of one single parameter

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The long-term recovery of one single parameter was evaluated over 14 days in parallel to the

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immediate effects assessed in the batch tests. Table 1 summarizes the key nitrite accumulation

306

ratio (NAR = effluent nitrite on nitrite + nitrate) profile parameters (Supplemental S.6-S.8

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depicts the obtained profiles). The control treatment, when no sulfide, starvation, or FA shock

308

was applied showed an anticlinal profile (∩), with a maximum NAR-ratio from 0.19 to 0.32 after

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3 days. This indicates an unexpected imbalance in activity recovery of AerAOB and NOB due

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the reactor conditions, of which NOB swiftly recovered. The effect of an additional FA shock on

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the sludge did not enhance nitrite accumulation, with an almost identical integrated NAR of 2.98

312

compared to 3.01 for the control treatment. This agrees with the batch tests, were an FA shock

313

did not enhance the relative rAerAOB/rNOB.

314 315

Exposure to 150 mg S L-1 for 1h (no starvation) resulted in a different profile, with a maximum

316

NAR of 0.2 at the starting point, which declined over the following 16 days (Figure 4). The

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recovery of AerAOB after sulfide inhibition was thus not faster than NOB, and the initial faster

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AerAOB recovery observed in the batch tests was not amplified on the long-term.

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Table 1 Long-term effects of different stress factors on the nitrite accumulation ratio and AerAOB/NOB activity ratio at its maximum peak. tmax = day when the maximum NAR was measured. Starvation occurred at 15°C, FA shock was 30 mg FA-N L-1 over 1h. Recovery profiles with specific sludge activities are depicted in Figure S.6-8. Trends highlighted in bold represent the different long-term profiles plotted in Figure 4. t0 = day 1 of the experiment. tend = last day of the experiment. FA: Free ammonia. NAR: nitrite accumulation ratio.

326 Sulfide dose

-1

mg S L 0

10

150

150

Sewage matrix

Nitrite accumulation ratio (NAR) Activity Starvation FA- Profile Max. tmax Gain Integrated AOB/NOB AOB time shock (tmax- t0) NAR activity activity (at tmax) gain (tend/t0) days day NAR*t 0

2

0

2

2

No

0.32

3

0.13

3.01

1.7

9.8

Yes

0.32

3

0.09

2.98

1.7

8.8

No

0.20

0

0

1.39

1.7

7.7

Yes

0.18

0

0

1.46

1.7

7.4

No

0.17

0

0

1.17

1.4

10.1

Yes

0.19

0

0

1.55

1.5

11.4

No

0.20

5

0.14

1.66

1.5

7.2

Yes

0.47

12

0.34

5.59

2.3

10.0

No

0.24

6

0.17

1.78

1.3

8.2

Yes

0.37

8

0.37

3.28

1.7

9.8

327 328

3.4.Long-term recovery of the combined parameters

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The combination of starvation (2d) and sulfide exposure enhanced the NAR in the long term. A

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two-day starvation while exposed to 150 mg S L-1 resulted in an anticlinal profile, in which the

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NAR peaked after 5 days (Figure 4, empty circles). AerAOB recovered faster than NOB, with a

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maximum NAR of 0.2 after 5 days, leading to a higher integrated NAR of 1.66 vs. 1.17 when no

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starvation took place (Table 1). The results from the initial recovery batch tests and long-term

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experiments are in this case contradicting. Initial recovery from the batch test decreased the

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relative rAerAOB/rNOB, while long-term recovery showed that longer exposure to sulfide

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inhibition is more toxic to NOB than AerAOB. This effect was further enhanced when an

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additional FA shock (Figure 4, filled circles) was applied after 2d of exposure. AerAOB

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recovered much faster than NOB. The NAR peak was 0.47 after 12 days of starvation, and

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compared to the treatment without FA shock, about 3.3 times as much nitrite was accumulated.

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In this case again, the initial recovery from the batch test could not predict the outcome of the

341

long-term recovery.

342 343 344 345 346 347

Figure 4 Long-term recovery of AerAOB and NOB in Ossemeersen STP after exposure to 150 mg S L-1 in combination with other stressors. The NAR profile was measured in a membrane bio-reactor under previously mentioned operational conditions Starvation was executed at 15°C under stirring conditions. -1 FA shock was 30 ppm FA-N L for 1h. Unless starvation occurred, long-term effects were measured after ~1h of sulfide exposure.

348 349

3.5.Community analysis

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Community analysis (Figure S.9) revealed a nitrifying community in Ossemeersen STP

351

containing the genus Nitrosomonas (9 OTUs), Nitrospira (1 OTU) and Ca. Nitrotoga (3 OTUs).

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Ca. Nitrotoga increased in relative abundance over time, when sewage temperatures decreased

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from 14.8 to 11.3 °C, yet Nitrospira had the highest relative abundance on all sampling dates. In

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Nieuwveer STP, no Ca. Nitrotoga was detected (T= 16.3 °C), and only Nitrospira (1 OTU) was

355

present.

356 357

For three long-term recovery experiments (10 mg S L-1 + 2d starvation, 150 mg S L-1 + 2d

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starvation with/without FA shock), the nitrifying community was monitored (See Figure S.10).

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The reactor conditions in all cases enriched the NOB Ca. Nitrotoga instead of NOB Nitrospira,

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suggesting a selective Ca. Nitrotoga preference for the combination of a shorter SRT of 10d, the

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absence of oxygen and nitrite limitations (> 2 mg O2 L-, >1.67 mg NO2--N L-1) and lower

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temperatures of 15°C. The combined stress parameters 150 mg S L-1 + 2d starvation

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with/without FA shock, which shared the same inoculum, were compared. In contrast to 150 mg

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S L-1 and 2d starvation, the extra FA shock inhibited Ca. Nitrotoga as well, resulting in a slower

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enrichment of Ca. Nitrotoga in this reactor, with Nitrospira being the most dominant NOB after

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14 days.

367 368

Plotting the AerAOB and NOB sludge-specific activity in function of the relative abundance of

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summed AerAOB or NOB OTUs in Figure S.11, could point out whether the observed rates are

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solely due to growth, or a combined effect of recovery (e.g. cell repair), wash-out of dead

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biomass, community shifts (and shifts in kinetics) or other unknown effects. For AerAOB, with

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the treatment of 10 and 150 mg S L-1 + 2d anaerobic starvation without FA shock, the ratio of

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increase in AerAOB sludge-specific activity over increase in relative abundance in week 1 was

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1.4-3.42. This is higher than the ratio of 0.25-0.51 in week 2. For NOB, this difference was not

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apparent, indicating that AerAOB and NOB community reacted differently towards the applied

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stress conditions.

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4. Discussion

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Sulfide inhibition

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Sulfide inhibition played an essential role in the observed AerAOB and NOB inhibition,

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being the factor that had the most inhibitory effect, and the one that increased

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susceptibility for FA. Although most literature reported the impact of sulfide on AerAOB,

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only a few studies differentiated for AerAOB and NOB (11-13, 27). In agreement with

384

those studies, NOB were more sensitive for direct sulfide inhibition than AerAOB. These

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immediate effects were not amplified on the long term. This is in accordance with

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Erguder et al. (2008), who reported that after 2 days contact time with 45 mg S L-1, the

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NAR ratio (no anticlinal-profile) declined from max. 0.75 to 0 in 10 days (12).

388 389

A wide range of sulfide inhibitory concentrations are reported for AerAOB, ranging for

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50% inhibition from 5.64 – 200 mg S L-1 (11-15, 27). This large difference in sensitivity

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can be due to the complex nature of sulfide chemistry. Depending on the conditions

392

during sulfide addition, i.e. pH, or the oxidative conditions, the sulfur speciation (sulfide,

393

sulphite, sulfate, Q), toxicity will change. Furthermore, sulfide is a very reactive

394

compound, and directly reacts away unless excess of sulfide is provided, which was

395

also observed in our tests (Figure S.12). Depending on the biomass concentration or

396

matrix composition, e.g. metal precipitates from iron dosing for phosphate removal (28),

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sulfide inhibition levels will differ from test to test. To account for these effects, the

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bioreactive sulfide dose that reacted per g of biomass was calculated (Supplemental

399

Information S.13) and plotted in Figure S.14. AerAOB and NOB sulfide inhibition

400

linearly correlated with amount mg bioreactive S per g VSS, with AerAOB having a

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lower slope than NOB. Inhibitory concentrations for 50% inhibition were calculated as

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20.6 mg S g VSS-1 and 15.8 mg S g VSS-1 for AerAOB and NOB respectively. These

403

results indicated that necessary sulfide doses might differ from sludge to sludge due to

404

its matrix. Expressing sulfide inhibition in bioreactive mg S g VSS-1 appeared to be more

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correct than in mg S L-1, which is advised for comparison with further studies or when

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designing a return-sludge treatment.

407 408

Combinatory or synergetic effects

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Combined or synergetic effects appeared to be necessary to obtain an effective long-

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lasting NOB inhibition, as the effect of the stressor alone was not sufficient. The best

411

results were obtained with a triple synergy of 150 mg S L-1 (= 13.5 mg S g VSS-1), 2

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days of anaerobic starvation, and a FA-shock of 30 mg FA-N L-1 for 1h. Literature also

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indirectly hinted that the combination of FA and sulfide is more toxic to nitrifiers.

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Bejarano Ortiz et al. (2012) reported a very low 50% inhibitory concentration of 1.2 mg

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S L-1 for AerAOB (29).

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unintentionally present because the pH was not corrected after Na2S addition (pH 8.8,

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30°C, max. 100 mg NH+4-N L-1). Similarly, Erguder et al. (2008) obtained a NAR value

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of 0.95 for more than 30 days after repeated exposure with increasing sulfide doses (2d

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anaerobic contact time of 1.3 up to 80 mg sulfide-S L-1) when pH was not corrected

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(max. pH 9.98), resulting in higher FA-concentrations (max. 47 mg FA-N L-1) (11). In

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contrast, the NAR value dropped within 10 days to 0 when pH was corrected and sulfide

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was the only major inhibitory compound. Confirming our results, AerAOB thus

In their batch activity tests, max. 33 mg FA-N L-1 was

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recovered faster from the combined (synergetic) stress conditions than NOB, enabling

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long-term NOB suppression.

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The effect of a longer anaerobic sulfide contact time on AerAOB inhibition was

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previously studied by Zhou et al. (2014) (13). An increased anaerobic contact time from

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1h to 4h with 10 mg S L-1 exponentially increased inhibition from 50% to 91%. This

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confirmed that longer exposure to sulfide changed the toxic effect -or chemistry of the

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sulfide inhibition, hypothetically leading to more irreversible inhibition.

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Salem et al. (2006) reported no increased inhibition after a 3-day starvation with 50-60

431

mg S L-1 at 20°C and pH 7.5, compared to a control without sulfide inhibition (30). Yet,

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the above mentioned complex nature of sulfide chemistry could have led to the

433

contradicting results. Overall, further optimization of sludge-dependent sulfide doses,

434

contact times and FA-concentrations could guide us towards an optimally designed

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return-sludge NOB suppression treatment.

In contrast,

436 437

Factors influencing recovery

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The immediate effect on AerAOB and NOB after anaerobic starvation and their

439

response to an FA shock was dissimilar for the two different STP, with starvation time,

440

sulfide, pH or other unknown factors potentially influencing recovery. Nieuwveer sludge

441

was after 2 and 8 days of starvation more susceptible to FA shocks than Ossemeersen

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sludge. This response was similar to that when sulfide inhibited sludge was exposed to

443

a FA shock, indicating that sulfide inhibition was most likely the culprit during Nieuwveer

444

starvation. Volatile fatty acids were most likely not influencing inhibition, since their

445

concentrations were well below reported inhibition values (Figure S.15) (19). One

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surprising result was measured for the long-term recovery experiment, where

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Ossemeersen sludge was anaerobically starved in its effluent matrix for 2 days. The FA

448

shock in this case elevated nitrite accumulation, with an integrated NAR twice as high

449

as without FA shock (Table 1). NOB recovered with the same speed as the control, yet

450

AerAOB were recovering faster. From the few samples taken during anaerobic

451

starvation (See Table S.1), it was unclear which parameters could have influenced this

452

outcome. Salem et al., (2006) pointed out that different starvation conditions influenced

453

the decay rate of AerAOB and NOB, and further research is thus necessary to elucidate

454

what parameters can steer faster recovery of AerAOB over NOB (18).

455 456

Long-term repeated exposure and microbial adaptation

457

The observed long-term recovery did not always reflect the immediate stress response.

458

For design of a return-sludge NOB-suppression treatment, short-term batch tests are

459

thus not sufficient, and long-term recovery must be studied. Furthermore, repeated

460

exposure should be assessed in follow up studies to understand potential effects of

461

microbial adaptation (both AerAOB and NOB). From the community analysis, the

462

increase in AerAOB activity was not proportional to cell concentration, while for NOB

463

this was proportional. Since no large shifts in AerAOB community were detected that

464

could influence the measured kinetics, it is possible that AerAOB where recovering

465

reversibly from the sulfide inhibition, while NOB were not.

466 467

Recurrent exposure to the combined stress factors might select for more resilient

468

AerAOB or NOB. Previous reports have shown niche differentiation of some AerAOB

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469

due to different abilities to recover after ammonium starvation (20). In the case of NOB,

470

Ca. Nitrotoga recovered and grew faster than Nitrospira after a treatment of 150 mg S L-

471

1

472

from 0.04 to 4.63 in the first week. Although reactor operation might have selected for

473

Ca. Nitrotoga over Nitrospira, this strong increase hinted to a better sulfide resistance of

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Ca. Nitrotoga. The extra FA shock appeared to be inhibitory for both Ca. Nitrotoga and

475

Nitrospira, indicating that the right cocktail of stress conditions could potentially inhibit

476

both NOB genera.

+ 2d anaerobic starvation. The Ca. Nitrotoga/Nitrospira abundance ratio increased

477 478

To obtain a stable, well-performing process, the necessary contact frequency must be

479

identified. Piculell et al. (2016) reported for a 1-day exposure to varying FA/FNA

480

concentrations, a stable NAR (75-85%) for 10 to 30 days, depending on the stress

481

perceived during the switch from sidestream to mainstream conditions (10). Wang et al.

482

(2016 and 2017) used very high FA/FNA concentrations (1.82 mg FNA-N L-1 and 230

483

mg FA-N L-1), and reported a NAR of 80-90% with contact frequencies of 1 time in 3-5

484

days (8-9). From our experiments, the minimum contact frequency would be about 1

485

time in 12 days, since NAR peaked at this value, yet might be shorter to achieve full

486

NOB suppression. Within this timeframe, AerAOB would also fully recover (Figure

487

S.6.1), considering biomass washout (-50%) and immediate activity loss due to the

488

return-sludge treatment. The applied FA shock (30 mg FA-N L-1 for 1h) with a frequency

489

of 1 time in 12 days would require around 60-100% of the produced filtrate from

490

anaerobic digestion (numbers STP Nieuwveer, NL: Temperature STP = 10-25°C;

491

sludge-return flow rate: 23,534 m3 d-1; thickened sludge concentration = 10 g VSS L-1;

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filtrate production = 419 m3 d-1; NH4+-N concentration filtrate = 1033 mg N L-1;

493

temperature filtrate = 33°C; pH after mixing with a digestate:return-sludge ratio of 0.8 =

494

8.12). This could render a sidestream PN/A system redundant, eliminating the

495

possibility of AnAOB bioaugmentation from the sludge line on the one hand side, yet

496

lowering the bCOD/N ratio in the water line on the other hand side, which might enable

497

to increase the AnAOB to heterotrophs ratio.

498 499

The proposed return-sludge treatment can periodically expose the floccular sludge of a

500

nitritation, nitritation/denitritation or hybrid PN/A reactor to boost the floccular AerAOB

501

over NOB activity. AnAOB are very sensitive towards sulfide (30), and should not be

502

exposed to this treatment. As they typically prevail in biofilm on carriers or in the larger

503

or more heavy flocs or granules, those should be separated from the smaller flocs prior

504

to return-sludge treatment involving sulfide. The proposed return-sludge treatment is

505

therefore likely limited to systems with proper retention of AnAOB in the mixed liquor,

506

e.g. the IFAS (Integrated fixed-film activated sludge) approach. This also implies that

507

NOB in the biofilm will elude the treatment and additional suppression strategies might

508

be needed. The sulfide concentrations that were effective in this study cannot be

509

produced in situ. Sulfide needs to be added from an external source, i.e. Na2S. Further

510

optimization and implementation studies should include long-term recurrent exposure of

511

the floccular sludge. This optimization of the proposed treatment might lead to a more

512

efficient use of sulfide and starvation time, and therefore practically implementable as

513

an add-on tool for successful NOB-suppression in mainstream shortcut nitrogen

514

removal processes.

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5. Supporting information

517

S.1, S.4 Conditions during anaerobic starvation. S.2 Calculations (M&M).

518

physicochemical analysis (M&M). S.5-8 Short- and long-term recovery nitrite

519

accumulation profiles. S.9-11 Community analysis. S.12-14 Calculations and graphs for

520

bioreactive sulfide concentration. S15 VFA toxicity.

S.3

521 522

6. Acknowledgments

523

D.S was supported by a PhD grant from the Institute for the promotion of Innovation by

524

Science and Technology in Flanders (IWT-Vlaanderen, SB-131769).

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7. References

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(14) Sears, K; Alleman, JE; Barnard, JL & Oleszkiewicz, J. A.. Impacts of reduced sulfur components on active and resting ammonia oxidizers. Journal of Industrial Microbiology and Biotechnology 2004. 31(8), 369-378. (15) Zhou, Z; Xing, C; An, Y; Hu, D; Qiao, W. Inhibitory effects of sulfide on nitrifying biomass in the anaerobic–anoxic–aerobic wastewater treatment process. Journal of Chemical Technology and Biotechnology 2014. 89(2), 214-219. (16) Kornaros, M; Dokianakis, SN; Lyberatos, G. Partial nitrification/denitrification can be attributed to the slow response of nitrite oxidizing bacteria to periodic anoxic disturbances. Environmental science & technology 2010. 44 (19), 7245–7253. (17) Gilbert, E. M.; Agrawal, S.; Brunner, F.; Schwartz, T.; Horn, H.; Lackner, S. Response of Different Nitrospira Species To Anoxic Periods Depends on Operational DO. Environmental science & technology 2014. 48 (5), 2934–2941. (18) Salem, S.; Moussa, M. S.; & Van Loosdrecht, M. C. M. Determination of the decay rate of nitrifying bacteria. Biotechnology and bioengineering 2006. 94(2), 252262. (19) Eilersen, A.; Henze, M.; Kløft, L. Effect of volatile fatty acids and trimethylamine on nitrification in activated sludge. Water research 1994. 29 (5), 1259–1266. (20) Bollmann; Bär-Gilissen. Growth at low ammonium concentrations and starvation response as potential factors involved in niche differentiation among ammonia-oxidizing bacteria. Applied and Environmental Microbiology. 2002. 68(10), 4751-4757. (21) Torà, J. A.; Lafuente, J.; Baeza, J. A.; & Carrera, J. Combined effect of inorganic carbon limitation and inhibition by free ammonia and free nitrous acid on ammonia oxidizing bacteria. Bioresource technology 2010. 101(15), 6051-6058. (22) Vilchez‐Vargas, R.; Geffers, R.; Suárez‐Diez, M.; Conte, I.; Waliczek, A.; Kaser, V. S.; ... & Pieper, D. H. Analysis of the microbial gene landscape and transcriptome for aromatic pollutants and alkane degradation using a novel internally calibrated microarray system. Environmental microbiology. 2014. 15(4), 1016-1039. (23) Klindworth, A.; Pruesse, E.; Schweer, T.; Peplies, J.; Quast, C.; Horn, M.; & Glöckner, F. O. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic acids research. 2013. 41(1), e1 (24) Schloss, P. D.; Westcott, S. L.; Ryabin, T.;Hall, J. R.; Hartmann, M.; Hollister, E. B.; ... & Sahl, J. W. Introducing mothur: open-source, platform-independent, communitysupported software for describing and comparing microbial communities. Applied and environmental microbiology. 2009. 75(23), 7537-7541. (25) Edgar, R. C.; Haas, B. J.; Clemente, J. C.; Quince, C.; & Knight, R. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics. 2011. 27(16), 2194-2200. (26) McMurdie, P. J.; & Holmes, S. Waste not, want not: why rarefying microbiome data is inadmissible. PLoS computational biology. 2014. 10(4), e1003531. (27) Delgado Vela, J.; Love, N. G.; Dick, G. J. The impact of sulfide on nitrification: implications for nitritation processes; Fifth International Conference on Nitrification and Related Processes (ICoN5) 2017. Vienna, Austria (28) Wilfert, P; Mandalidis, A; Dugulan, AI; Goubitz, K. Vivianite as an important iron phosphate precipitate in sewage treatment plants. Water research 2016. 104, 449460.

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(29) Bejarano Ortiz, D. I.; Thalasso, F.; Cuervo López, F. D. M.; & Texier, A. C. Inhibitory effect of sulfide on the nitrifying respiratory process. Journal of Chemical Technology and Biotechnology 2013. 88(7), 1344-1349. (30) Oshiki, M., Satoh, H., & Okabe, S. Ecology and physiology of anaerobic ammonium oxidizing bacteria. Environmental microbiology 2016. 18(9), 2784-2796.

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