Influence of Polyferric Sulfate Coagulant on the amoA mRNA Expression of Ammonia Oxidizer in Activated Sludge

Physicochemical coagulation process is one of the most popular techniques to remove phosphorus in wastewater treatment processes where the activated sludge was used. However, the influence of coagulants on biological nitrification is not elucidated in detail. Then, the influence of polyferric sulfate as coagulant on nitrogen conversion by ammonia oxidizer was investigated by using water quality test and transcript analysis. In the batch operation of the nitrification-denitrification process, Fe/P molar ratios of 0, 1 and 5 were tested. Phosphorus concentration was immediately decreased at the beginning of the experiment in the case of Fe/P = 1 and 5, however, 40% of ammonia remained in the case of Fe/P = 5. In this case, the amoA mRNA expression was suppressed because of the excessive addition of polyferric sulfate. Furthermore, T-RFLP profiles of amoA mRNA showed that polyferric sulfate affects various species of ammonia oxidizer, and some species suffer a great loss

Address correspondence to Yoshitaka Ebie, Research Center for Material Cycles and Waste Management,

Influence of Polyferric Sulfate Coagulant on the amoA

mRNA Expression of Ammonia Oxidizer in Activated Sludge

Yoshitaka EBIE*, Hiroshi YAMAZAKI**, Kaiqin XU*

*Research Center for Material Cycles and Waste Management, National Institute for

Environmental Studies, Ibaraki, 305-8506, Japan

**Planning division, Public Health Research Center of Ibaraki Pharmaceutical Association,

3-5-35 Midori, Mito, Ibaraki, 310-0034, Japan

ABSTRACT

Physicochemical coagulation process is one of the most popular techniques to remove phosphorus in wastewater treatment processes where the activated sludge was used However, the influence of coagulants on biological nitrification is not elucidated in detail Then, the influence of polyferric sulfate as coagulant on nitrogen conversion by ammonia oxidizer was investigated by using water quality test and transcript analysis In the batch operation of the nitrification-denitrification process, Fe/P molar ratios of 0, 1 and 5 were tested Phosphorus concentration was immediately decreased at the beginning of the experiment in the case of Fe/P

= 1 and 5, however, 40% of ammonia remained in the case of Fe/P = 5 In this case, the amoA

mRNA expression was suppressed because of the excessive addition of polyferric sulfate

Furthermore, T-RFLP profiles of amoA mRNA showed that polyferric sulfate affects various

species of ammonia oxidizer, and some species suffer a great loss

Keywords: activated sludge, ammonia oxidizer, amoA mRNA, coagulant

INTRODUCTION

Physicochemical coagulation process is one of the most popular techniques to remove phosphorus in wastewater treatment processes where the activated sludge is used Phosphorus is coagulated as aluminum phosphate or iron phosphate by adding PAC (polyaluminum chloride) or polyferric sulfate On the other hand, nitrogen is removed

by biological nitrification-denitrification process in general Activated sludge process with coagulant addition is one of the promising ways to remove nitrogen and phosphorus simultaneously In this process, coagulant is added to the nitrification-denitrification process, and nitrogen and phosphorus can be removed simultaneously in the same tank However, the influence of coagulants on biological nitrification is not elucidated in detail

Previous studies showed the possibility of measuring in situ functional activity of ammonia oxidizer by RT-PCR-DGGE technique that focuses on specific and transient

markers of in situ metabolism, amoA mRNA (Ebie et al., 2004; El Sheikh and Klotz,

2008) In general, mRNA is short-lived and has a unique nucleic acid sequence that can

be identified with a high degree of specificity However, recent evidence suggests that starvation does not correlate with the expression of ammonia monooxygenase and

ammonia oxidation activity (Bollmann et al., 2005; Wei et al., 2006) This might be due

to the induction of stabilization of the amoA mRNA in ammonia-oxidizing bacteria (AOB) by starvation (Bollmann et al., 2005) Although amoA mRNA is not an indicator

of ammonia oxidizing activity in different ammonium concentrations, it still has the

possibility of measuring specific response to some environmental factors In this study,

we introduce an approach to the investigation of inhibition effects of polyferric sulfate

on ammonia oxidizers based on expressed amoA mRNA

MATERIALS AND METHODS

Batch operation of the activated sludge process with coagulant addition

Actual domestic wastewater and activated sludge were collected from a wastewater treatment plant The fill-and-draw of the domestic wastewater was carried out for habituation of the activated sludge Batch operation of the activated sludge process was conducted with 1 L conical reactor at 20°C The MLSS of the activated sludge was adjusted to 1,500 mg/L and the aeration rate was set at 1.0 L/min A commercial coagulant, polyferric sulfate, was used as a coagulation agent The coagulant was added just before starting the operation and the Fe/P molar ratio of the test was set at 1 (Run 1) and 5 (Run 2) Activated sludge without any coagulant added was used as a control During 600 min of operation, activated sludge was collected The parameters NH4+-N, NO2--N, NO3--N, and dissolved total phosphorus (DTP) were measured according to the standard methods of Japan Sewage Works Association (1995) Dissolved organic carbon (DOC) was analyzed as non-purgable organic carbon (NPOC) by TOC-5000 (Shimadzu, Japan)

In situ hybridization

In situ hybridization was conducted to monitor the number of ammonia oxidizers

Activated sludge samples were fixed according to the previous study (Ebie et al., 2002)

For quantitative analysis, the same volume of 400 mg/L of tripolysodiumphosphate was added to the fixed sample and was dispersed by a sonic generator for 5 min Ammonia

oxidizer specific probe, NEU (Wagner et al., 1995) was labeled with Cy3 Hybridization

was performed according to the standard protocol described by Amann (1995) Cell number of ammonia oxidizers was directly counted using confocal laser scanning microscope (Leica, Germany)

RNA extraction and reverse transcription

Samples for RNA extraction were immediately stored in equal volumes of RNAlater (Ambion) which is an RNA stabilization reagent Total RNA extraction and reverse

transcription reaction were performed according to the previous study (Ebie et al., 2004)

except for the cell disruption procedure Homogenization was performed with Mini-Beadbeater (BioSpec Products) at 5,000 rpm for 2 min in the presence of 1 g of Zirco/Silica beads (diameter, 0.1 mm; BioSpec Products, Bartlesville, Oklahoma, USA)

Real time Polymerase Chain Reaction (PCR)

The collected cDNA was used as a template of amoA-targeted real time PCR with

LightCycler-FastStart DNA Master SYBR Green I (Roche Diagnostics, Germany)

Amplification of a 491-bp fragment of the amoA gene was carried out using the

amoA-1F and amoA-2R primer set specific for amoA gene of AOB belonging to the ß

subclass of Proteobacteria The thermal profilesincluded an initial denaturing step at 95°C for10 min followed by 45 cycles of denaturation at 95°C for 20s, annealing at 60°C for 8 s, and elongation at 72°C for 20 s

0 200 400 600

Time (min)

-1 )

0 2 4 6 0 20 40 60

-1 )

Control Run 1 Run 2

Fig 1 - Time course of DOC and DTP in batch experiment

Terminal Restriction Fragment Length Polymorphism (T-RFLP)

The collected cDNA was used as a template of amoA-targeted T-RFLP with TaqI as the restriction enzyme The primers amoA-1F-6FAM and amoA-2R were used for this PCR

The reaction mixture was prepared according to the manufacturer’s instruction of TaKaRa Ex Taq (TaKaRa, Shiga, Japan) The thermal cycling comprised an initial denaturation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 20 s, annealing at 58°C for 10 s, elongation at 72°C for 20 s, and a final extension at 72°C for

1 min After agarose gel electrophoresis, amplicon was exiced and purified using Wizard SV Gel and PCR Clean-Up System (Promega, USA) The restriction reaction

was performed at 65°C for 4 hrs according to the manufacturer’s instruction of TaqI

(TaKaRa, Shiga, Japan) The fluorescently labeled T-RFs were analyzed by electrophoresis on 3100-Avant Genetic Analyzer (Applied Biosystems)

RESULTS AND DISCUSSION

Effects of the addition of polyferric sulfate on phosphorus removal and nitrification

Phosphorus concentration was immediately decreased at the beginning of the experiment in Runs 1 and 2 (Fig 1) Final phosphorus concentrations in Runs 1 and 2 were below 1 mg/L, whereas that in control was over 2 mg/L Although the lowest phosphorus concentration was observed in Run 2, there was no significant difference in the phosphorus removal efficiency in Runs 1 and 2 Effect of polyferric sulfate on DOC removal was also examined Almost all DOC was removed in 200 min, and there was

no difference in DOC removal rates in all runs This means that polyferric sulfate has no effect on DOC removal by heterotrophic bacteria in these conditions

The time courses of NH4+-N and NO2+3--N in batch experiment are shown in Fig 2 Gradual decrease in NH4+-N and increase in NO2+3--N were observed just after the beginning of the experiment in control and both runs However, decrease in nitrification efficiency was observed as an effect of the addition of polyferric sulfate from 100 min after starting the experiment Although complete nitrification occurred in control and Run 1 at 600 min, nitrification rate was slow in Run 1 and 40% of ammonia remained in Run 2 Therefore, nitrification rate is affected by the addition of polyferric sulfate, and polyferric sulfate dosage was obviously an important parameter In this experiment,

0 200 400 600 0

10 20 30

O2+

- -N (m

-1 )

Time (min)

Fig 2 - Time course of NH4+-N and NOx--N in batch experiment

Fe/P molar ratio of 1 was enough for phosphorus removal and tolerable for nitrification

In this study, Fe/P is used as index because of the following three reasons: N/P ratio of influent domestic wastewater is constant in general, excess polyferric sulfate would affect nitrification, and Fe/P is a usual index for phosphorus removal processes However, the effect of polyferric sulfate on nitrification might be due to the total amount of the added coagulant, and similar experiment with different N/P ratio in influent might be important for further investigation

Number of ammonia oxidizers and copy number of amoA mRNA

The time courses of the copy number of amoA mRNA and the number of ammonia

oxidizer are shown in Fig 3 No significant change and difference in the number of ammonia oxidizer was observed in each run During this short batch experiment, polyferric sulfate has no effect on the increase or decrease in the number of ammonia oxidizer On the other hand, significant change and obvious difference in the amount of

amoA mRNA were observed in each run Vertical axis of this graph shows amoA

mRNA copy number in a PCR-reaction tube, and the quantity of the sample in each

tube was the same Amount of amoA mRNA increased in all runs after starting the experiment The highest amount of amoA mRNA was observed in control at 200 min

after starting the experiment and only 30% of that in Run 2 was observed at 100 min These results suggest that addition of polyferric sulfate does not affect the number of ammonia oxidizer, instead, the amount of amoA mRNA is affected in a short time

Because amoA mRNA is one of the key templates of ammonia monooxygenase, copy number of amoA mRNA should reflect ammonia oxidizing activity This low amount of

amoA mRNA in Run 2 should be related to the fact that 40% of ammonia remained at

the end of batch experiment Small decrease in the amount of amoA mRNA in Run 1

was observed due to low dosage of polyferric sulfate This means that the impact of low

concentration of polyferric sulfate on amoA mRNA expression is limited, and dosage of

polyferric sulfate must be optimized not only for phosphorus removal but also for ammonia oxidation

Although the possibility that amoA mRNA would still be detectable after 12 days of starvation was reported (Bollmann et al., 2005), the result of this study definitely indicated that the copy number of amoA mRNA is related to the ammonia oxidation performance Aoi et al (2004) reported that an increase in amoA mRNA level can be

detected within 1 - 2 h in response to an initiation of cell activity whereas a decrease in

amoA mRNA level is detected within 24 h in response to a cessation of activity This

range of time-lag is reasonable to explain the result of this study Certainly, there must

be some other factors affecting the ammonia oxidation performance and/or the amount

of amoA mRNA, then continuing care should be taken into consideration in order to

make a direct correlation between mRNA detection and in situ activity of ammonia oxidation

Community analysis of ammonia oxidizer based on amoA mRNA

The difference of the effect of polyferric sulfate on each species of ammonia oxidizer

was demonstrated by T-RFLP analysis of cDNA of amoA mRNA The profiles of

T-RFLP in Run 2 are shown in Fig 4 Table 1 shows the species of ammonia oxidizer

of the major T-RFs (219, 283, 354 and 491 bp) Although almost all of the heights of the T-RFs were decreased over time, T-RFs of 283 and 491 bp remarkably decreased These profiles suggest that polyferric sulfate affect various species of ammonia oxidizer, and some species suffer a great loss

7 cell

-1 )

0

Time (min)

1 2 3

6 C

-1 )

0.5 1.0 1.5

0

Control Run 1 Run 2

Fig 3 - Time course of the amount of amoA mRNA and number of AOB

Fragment size (bp)

0.5 min

400 min

600 min

1.0 0.5 0

1.0 0.5

1.0 0.5

Fig 4 - T-RFLP profiles of cDNA derived from amoA mRNA in Run 2 after digestion

with TaqI

Table 1 Ammonia oxidizers categorized by amoA fragment size analyzed by

TaqI-based T-RFLP

219bp

283bp

491bp

CONCLUSIONS

Phosphorus removal using polyferric sulfate was effective and equal molar of polyferric sulfate to influent phosphorus was enough to achieve phosphorus concentration below 1 mg/L There was no impact on the number of ammonia oxidizer regardless of Fe/P

molar ratio However, in the case of Fe/P molar ratio of 5, copy number of amoA

mRNA obviously decreased and 40% of ammonia remained at the end of the batch experiment According to the result of T-RFLP analysis, there is great impact of

polyferric sulfate on amoA mRNA expression for some species of ammonia oxidizers

Therefore, in the activated sludge process of nitrification-denitrification with coagulant addition, Fe/P molar ratio should not exceed 1, and equal number of moles of polyferric sulfate to influent phosphorus is sufficient for phosphorus removal and is suited for simultaneous removal of nitrogen and phosphorus

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