Mechanisms of Unstable Nitrite Inhibition of Aerobic Phosphate Uptake

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Journal of Water and Environment Technology, Vol. 6, No.1, 2008


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Mechanisms of Unstable Nitrite Inhibition of Aerobic
Phosphate Uptake


Toshiaki Saito*, Kohei Takahashi**, Senta Tsuboi*, Kouta Yumoto*, Yukihito Yoshida*

* Department of Civil Engineering, College of Science and Technology, Nihon University, 1-8
Kanda-surugadai, Chiyoda-ku, Tokyo, 101-8308, Japan
** present affiliation Organo Corporation, 2-8, Shinsuna 1-chome, Koto-ku, Tokyo, 136-8631,
Japan


ABSTRACT
Recently, nitrite has been recognized as one of the considerable inhibitors of biological
phosphorus removal. In fact, there are several reports on inhibitory effect of nitrite. While
unfortunately, the reported critical levels of nitrite widely spread. So the real effect of nitrite has
not yet been well understood. In this study, several batch tests were conducted to obtain stable
and quantitative relation between the size of nitrite exposure and the size of inhibition. The
obtained results are as follows; 1) Nitrite inhibits aerobic phosphate uptake of PAOs, but the
inhibition is not direct inhibition by nitrite but indirect inhibition caused by reduced respiration,
2) PAOs with higher anoxic activity can reduce the inhibitory effect of nitrite, possibly because
of aerobic nitrite denitrification, 3) Nitrite inhibition of aerobic phosphate uptake is successfully
expressed by the model including aerobic nitrite denitrification rates. These results strongly
suggest that unstable nitrite inhibition of aerobic phosphate uptake is caused by widely
distributed anoxic activities of PAOs.

Keywords: polyphosphate-accumulating organisms (PAOs), nitrite, inhibition, aerobic
denitrification, biological phosphorus removal, anoxic phosphate uptake, activated sludge


INTRODUCTION
Eutrophication of closed water bodies has been one of the most serious water pollution
problems all over the world. To solve this problem, nitrogen and phosphorus have to be
removed from wastewater. Especially, phosphorus is often a limiting element for
eutrophication so that stable performance of phosphorus removal is eagerly required.
However, it is well known that biological phosphorus removal (BPR) process often
becomes unstable. Therefore, it is an urgent subject to clarify the mechanism of BPR
instability. Although several researchers have revealed the influencing factors (Hascoet
and Florents, 1985; Kuba et al., 1994; Cech and Hartman, 1993; Liu et al., 1996), it is
supposed to be other unknown factors.

This study focuses on nitrite that is known as a strong inhibitor for bacteria (Rowe et al.,
1979; Almeida et al., 1994; Weon et al., 2002). Since nitrite is often present in full-scale
wastewater treatment plants as an intermediate both of nitrification and denitrification,
nitrite could be one of the factors deteriorating biological phosphorus removal. Actually,
it has been reported that nitrite severely inhibits aerobic and anoxic phosphate uptake of
Poly-Phosphate Accumulating Organisms (PAOs). However, the reported critical
concentration of nitrite has been widely varying (Saito et al., 2004; Kuba et al.,
1996;Lee et al., 2001; Meinhold et al., 1999; Ahn et al., 2001). In order to attain stable
BPR performance, the mechanism of unstable response of PAOs to nitrite exposure
Address correspondence to Toshiaki Saito, Department of Civil Eengineering, College of Science and
Technology, Nihon University, Email: saito@civil.cst.nihon-u.ac.jp
Received March 11, 2008, Accepted April 7, 2008.
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must be clarified.

One of the key observations is that an inhibitory effect of nitrite on anoxic phosphate
uptake is less sensitive than aerobic phosphate uptake (Saito et al., 2004). This is not
curious, because some of PAOs can utilize nitrite as an electron acceptor under anoxic
condition (Meinhold et al., 1999; Ahn et al., 2001; Hu et al., 2003; Shoji et al., 2003).
Even though nitrite has an inhibitory effect, some of denitrifying PAOs must be able to
utilize and detoxify nitrite under anoxic condition. But, how about aerobic phosphate
uptake? It is well known that some of ordinary denitrifiers can reduce nitrite under
aerobic condition (Alefounder et. al., 1980), in other words, ‘aerobic nitrite
denitrification’. If some of PAOs can do that, the inhibitory effect of nitrite will be
reduced. Hence, the purpose of this study is to examine the effect of anoxic activity of
PAOs on reduction of inhibitory effect of nitrite.


MATERIALS AND METHODS
Cultivation of PAOs with acetate as a sole carbon source
Three sequencing batch reactors (AO SBR, AO/N SBR and AA SBR) were used to
cultivate the enriched PAOs that have different anoxic phosphate uptake activities. All
reactors were fed with acetate as a sole carbon source. Composition of synthetic
wastewater is listed in Table 1. The cycle operations were shown in Figure 1. AO SBR
and AO/N SBR were operated with 4L of cylindrical reactor under the alternating
anaerobic(180min)-aerobic(130min) conditions. AO/N SBR received a small amount of
nitrite that theoretically resulted in 1 mgN/l in the reactor at initial 1 minute of aerobic
phase, while AO SBR did not. Allylthiourea (ATU) was periodically added to both of
the reactor to suppress nitrification. AA SBR was operated with 1L of cylindrical reactor
under the alternating anaerobic-anoxic conditions. 1 L of cylindrical reactor for AA SBR
was operated with the alternating anaerobic(120min)-anoxic(190min) conditions.
During initial 120 minutes of anoxic phase, nitrate was introduced into the reactor to
theoretically result in 46 mgN/l in the reactor. Other experimental conditions were the
same for all the reactors. HRT, SRT, water temperature and pH were controlled at
12hours, 10 days, 20 ±5 °C and 7.0 ±.1, respectively. Seeded sludge were from a
bench-scale SBR (Yoshida et al., 2005) operated with anaerobic-aerobic-anoxic-aerobic
cycle to remove nitrogen and phosphorus from sewage.




Figure 1 - Cycle Operations of SBRs for Cultivation of the Enriched PAOs


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Table 1 - Composition of Synthetic Wastewater




Methods of nitrite inhibition batch tests
21 sludge from AO SBR (7 sludge), AO/N SBR (11 sludge) and AA SBR (3 sludge)
were tested with the procedure as follows. 200 to 300 ml of sludge was taken from the
reactor at the end of anaerobic phase and was divided into several portions. The one was
provided with air. The others were provided with air and different concentration of
nitrite (0.27 to 4.9mgN
.
gVSS
-1
). They were controlled at around 7.0 ±.1 of pH and 20
°C of water temperature. Some sludge were tested with several concentration of nitrite
so that total number of batch tests were 36. In some of batch tests, oxygen uptake rate
(OUR) was also measured to evaluate nitrite effect on respiration.

Measurement methods of aerobic and anoxic phosphate uptake activities
Evaluation of aerobic and anoxic phosphate uptake activities of PAOs was conducted by
the methods (Wachtmeister et. al., 19997) with some modifications. 100 to 200 ml of
sludge was taken from the reactor at the end of anaerobic phase and was divided into
two portions. The one was provided with air and the other was added with nitrate
anaerobically. Both of pH were controlled at around 7.0 ±.1. The aerobic and anoxic
phosphate uptake activities were calculated with the phosphate-decreasing rate during
initial 20 to 30 minutes and 50 to 60 minutes, respectively.

Characteristics of the enriched PAOs in terms of anoxic activity
Since AO sludge was cultivated with the alternating anaerobic-aerobic cycle operation
without exposure to oxidized nitrogen, they have no anoxic phosphate uptake activity (0
mgP/gVSS.h). AO/N sludge has a little anoxic activity (0 to 6.4 mgP/gVSS.h), because
a small amount of nitrite was added under aerobic condition. This kind of operation is
known to increase anoxic activity (Yoshida et. al., 2005; Saito et. al., 2005). AA sludge
has the largest anoxic activity (12.6 to 25.9 mgP/gVSS.h), because they were cultivated
by nitrate as an electron acceptor instead of oxygen. Thus, the enriched PAOs with
different anoxic activities were obtained. Variation of aerobic phosphorus uptake
activity was caused by rather unstable operation.


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Analytical procedures
Phosphate, MLSS and MLVSS were analyzed in accordance with Japanese Standard
Methods for Examinations of Wastewater. The concentration of nitrite and nitrate were
determined by Shimadzu HPLC system equipped with a Shimadzu CDD-10A detector
and a Shim-pack IC-A3 column. 0.45 m of membrane filters were used to separate
dissolved and particulate matters.


RESULTS
Typical results of nitrite inhibition batch tests
Among 36 batch tests, the results of AA sludge with 4.9mgN/L of nitrite addition are
shown in Figure 2 as representatives. As shown in the figure ‘(a) control’, in the case
without nitrite addition, initial OUR was the highest and OUR decreased with time. This
OUR curve resembles the curve reported by Smolders et al. (1994). Phosphate
concentration also decreased linearly with time. Hence, the amount of oxygen
respiration and the amount of phosphate uptake was not linear. This might be because
the energy produced by oxygen respiration is used for several activities of PAOs,
namely polyphosphate storage, glycogen restoration, cell growth and maintenance. On
the other hand, in the case with nitrite addition, we observed the characteristic curves of
phosphate concentration and OUR. Initially, low OUR was observed as compared to
those without nitrite addition (‘control’), and later, OUR once increased and again
decreased. These fluctuations are significantly corresponding to the course of phosphate
concentration. Phosphate concentration initially showed the gradual decrease
(corresponding to low OUR). Then, the sudden decrease was monitored (in other words,
sudden increase of phosphate uptake). This behavior synchronized with increase of
OUR. Moreover, this sudden increase of both phosphate uptake and OUR indeed
synchronized with nitrite disappearance. These results suggest that suppression of
phosphate uptake by nitrite is caused by suppression of oxygen respiration to some
extent, because phosphate uptake requires energy produced by respiration. The other
important observation is that both of phosphate uptake and oxygen respiration are not
fully recovered. Even after nitrite completely disappeared, inhibition still remained.
Moreover, nitrite disappeared under aerobic condition without nitrate production.

Time (min)Time (min)
OUR (mgO
.
gVSS
-1
.
h
-1
)
Phosphate (mgP/gVSS)
Phosphate (mgP/gVSS)
OUR (mgO
.
gVSS
-1
.
h
-1
)
(a) cont rol (b) with nitrite addition


Figure 2 - Results of Nitrite Inhibition Batch Test (AA sludge with 4.9mgN/L of nitrite
addition) (a) ‘control’ without nitrite addition, (b) with nitrite addition, Grey
circle: phosphate, Empty square: OUR, Grey triangle: nitrite
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Different response of PAOs with different cultivation career
The effect of cultivation career on the size of inhibition was examined by comparing the
responses of AO sludge, AO/N sludge and AA sludge with the same nitrite
concentration. The results are shown in Figure 3 and Table 2.

In the case of AO sludge (Figure 3(a)) with 2.3 mgN/gVSS of initial nitrite
concentration, the initial phosphate uptake was completely inhibited. The phosphate
uptake activity was almost 0 mg
.
gVSS
-1.
h
-1
. After 60 min, the phosphate uptake activity
recovered to some degree (23 % of that in the absence of nitrite). Interesting observation
is that nitrite concentration decreased without nitrate production (data not shown) and
the recovery of phosphate uptake activity started just after nitrite disappearance.
Similarly, in the case of AO/N with 2.0 mgN/gVSS of initial nitrite concentration,
phosphate uptake was initially inhibited, but recovered soon at the time when nitrite
disappeared. The phosphate uptake rate in the presence of nitrite was 13 mgP
.
gVSS
-1.
h
-1

and the % activity of phosphate uptake was 39%. On the other hands, in the case of AA
with 1.9 mgN/gVSS of initial nitrite concentration, the size of inhibition was the
smallest among them. The phosphate uptake rate in the presence of nitrite was 20
mgP
.
gVSS
-1.
h
-1
and the % activity of phosphate uptake was 60 %. These results indicate
that the size of inhibition is clearly different on different PAOs, even if they are exposed
to the same range of nitrite concentrations. Interestingly, the rest of batch tests also
indicate that the sizes of inhibition are AA sludge < AO/N sludge < AO sludge in order.
And this order is the same as the order of their anoxic activity. Moreover, in all tests,
nitrite disappeared without nitrate production even under aerobic condition and, the
order of nitrite decreasing rates was also consistent with that of anoxic activity of PAOs.
These observations imply a mutual relation among the size of inhibition, the anoxic
activity and the nitrite-decreasing rate.



Figure 3 - Comparison of Cultivation Career (AO, AO/N and AA) and Nitrite Inhibition
at The Same Range of Nitrite Exposure. (a) AO with 2.3mgN/gVSS of nitrite,
(b) AO/N with 2.0mgN/gVSS of nitrite, (c) AA with 1.9mgN/gVSS, Empty
circle: phosphate in control, Grey circle: phosphate with nitrite addition,
Triangle: nitrite

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Table 2 – Summary of Comparison of Cultivation Career


Relationship between anoxic activity of PAOs and aerobic nitrite disappearance
It is so interesting that we observed nitrite disappearance under aerobic condition
without nitrate production in all batch tests, since there are no reports on aerobic nitrite
denitrification by PAOs. However, our results strongly suggest that unstable response of
PAOs to nitrite exposure, must come from their different anoxic phosphate uptake
activities, in other words, PAOs with higher anoxic activity can reduce nitrite under
aerobic condition (aerobic denitrification!) and, hence, can reduce nitrite inhibition.

To confirm that aerobic nitrite disappearance is caused by a biologically mediated
reaction by PAOs, several experiments were conducted (data not shown). First, the
filtrates of culture solution and sludge were aerated with nitrite. However, nitrite never
disappeared. This fact suggests that the aerobic nitrite disappearance is not a chemical
reaction but a biologically mediated reaction. Next, the enriched PAOs was provided
with more than 4 hours of excessive aeration to deplete internally stored organic
substances completely. Then, nitrite was added. However, nitrite again never
disappeared. This result suggests that the observed aerobic nitrite disappearance
requires an internally stored organic substance.

Next, to confirm the participation of PAOs, the relationship between anoxic phosphate
uptake activity and aerobic nitrite decrease that are obtained from all batch tests was
examined. The result is shown in Figure 4. As shown in the figure, AO sludge that has
little anoxic activity has lower nitrite decreasing rate. AO-N sludge that has small
anoxic activity has relatively higher rate and AA sludge that has the highest anoxic
activity has the highest nitrite consumption rate. And interestingly even PAOs with no
anoxic phosphate uptake activity have a little aerobic nitrite denitrification activity.

Finally, the possibility of ordinary denitrification inside thick floc of the enriched PAOs
was examined by adding nitrate instead of nitrite. Since nitrate concentration did not
change, while nitrite disappeared, it was verified that nitrite was not reduced under
anoxic condition inside floc. If anoxic condition was formed, nitrate also must have
been reduced. From these results, it is concluded that the enriched PAOs in this study
can do aerobic nitrite denitrification.


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0
5
10
15
20
25
30
0246810
Anoxic Phoaphate Uptake Rate
(mgP
.
gVSS
-1
.
h
-1
)
Aerobic Nitrite Consumption Rat e
(mgN
.
gVSS
-1
.
h
-1
)


Figure 4 – Relationship between Anoxic Phosphate Uptake Rate and Aerobic Nitrite
Consumption Rate, Empty circle : AO sludge, Grey circle : AO/N sludge,
Dark sludge : AA sludge


Dependency of nitrite inhibition on aerobic nitrite denitrification by PAOs
From above, it is suggested that nitrite inhibition not only depends on nitrite
concentration, but also on aerobic nitrite denitrification. In this paragraph, an
appropriate expression of unstable inhibitory responses is studied. First, all tested sludge
were classified into three groups in terms of aerobic nitrite denitrification rate as shown
in Table 3.

Table 3 - Classification of PAOs

PUA : phosphate uptake activity

To know the effect of aerobic nitrite denitrification activity on relieving inhibitory effect
quantitatively, inhibitory effect of nitrite is expressed as non-competitive inhibition as
follows;


r = r
max
⋅
K
NO 2
K
NO 2
+ (C
NO 2
)
α


r
: aerobic phosphate uptake rate (mgP
.
gVSS
-1.
h
-1
)
, r
max
: aerobic phosphate uptake
activity (mgP
.
gVSS
-1.
h
-1
) without nitrite addition (control),
K
NO2
: inhibition constant
of nitrite on phosphate uptake,
C
NO2
: concentration of nitrite (mgN/gVSS),  :
inhibition coefficient

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The obtained constants are listed in Table 4. As shown in the table, the inhibition
constant of Gourp-3 is the largest among them, since they have the largest anoxic
activity and aerobic nitrite denitrification activity. Next is Goup-2 and the most sensitive
group is Goup-1 that has little anoxic activity. K
NO2
is 0.1 and  is 3.6. The fact that
obtained values in the case of Group-1 and 2 are more than 1.0 may indicate that there
are not one but several inhibition mechanisms. Anyway, since mechanism of nitrite
inhibition has not yet been clarified, the used inhibition formula is not theoretical but
practical one. In order to develop the adequate expression of nitrite inhibition, further
research is required especially on inhibition mechanisms of phosphate uptake.


Table 4 – Determined Inhibition Constants


Figure 5 shows the responses of PAOs to nitrite exposure with the obtained curves of
modified non-competitive model. Although plots are somewhat scattering, the
classification of tested sludge into three groups does make clear the size of inhibition. It
is concluded that the aerobic nitrite denitrification rate is the key parameter to express
the size of inhibition. Plots of Group-2 are rather scattering. Although the reason of the
scattering is not clear, this may be caused by fragile denitrification potential of PAOs
and/or other unknown reasons.




Figure 5 - Relationship between Nitrite Exposure and Inhibition of Phosphate Uptake,
Empty circle: Group-1, grey triangle: Group-2, dark triangle: Group-3.




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DISCUSSION
Aerobic nitrite denitrification by PAOs
Recent developments of molecular biology techniques, e.g., fluorescent in situ
hybridization (FISH) and polymerase chain reaction (PCR), revealed that
Rhodocyclus-related PAOs are predominant in a lab-scale anaerobic-aerobic reactor fed
with acetate (Hesselmann et al., 1999; Crocetti et.al., 2000). Rhodocyclus-related PAOs
are also identified as denitrifying PAOs cultivated with acetate (Zeng et. al., 2003).
Since, in this study, PAOs were enriched by acetate as sole carbon source,
Rhodocyclus-related PAOs were probably predominant. Hence, the enriched PAOs in
this study could perform aerobic nitrite denitrification. Because, it is well known that
some of gram-negative heterotrophic denitrifiers can perform aerobic denitrification of
nitrite, since they have nitrite reductase on the periplasmic side of cell membrane. So at
least some of the PAOs enriched in this study most likely have nitrite reductase on the
periplasmic side of cell membrane and can reduce nitrite under aerobic condition. So far,
there are no reports on aerobic nitrite denitrification by PAOs. However, aerobic nitrite
disappearance observed in this study is probably an aerobic nitrite denitrification by
PAOs.

Mechanism of buffering nitrite inhibition by PAOs
Nitrite is a well-known inhibitor of aerobic metabolism of bacteria (Rowe et. al., 1979).
Main mechanism of inhibitory effect of nitrite is cytochrom oxidation by nitric oxide
produced by aerobic nitrite reduction, and is not by a direct attack by nitrite (Kucera et.
al., 1986; Carr et. al., 1990). Hence, nitric oxide concentration inside cell is important to
control inhibitory effect of nitrite. One of the ways is to supply full amount of COD. If
readily biodegradable COD is abundantly available, aerobically produced nitric oxide
will be easily removed by aerobic reduction and inhibitory effect can be significantly
reduced (Casey et. al., 1999). In this study, PAOs with higher anoxic activity performed
higher aerobic nitrite denitrification and is less sensitive to nitrite. That is because they
have enough ability to remove nitric oxide by denitrification. Mechanism of buffering
phosphate uptake inhibition is most likely aerobic denitrification ability of PAOs.

Another interesting point is that aerobic phosphate uptake of AO sludge was strongly
inhibited, though it has no anoxic phosphate uptake activity and, hence no nitric oxide
production potential. In the case of Paracoccus denitrificans without nitrite reductase,
aerobic respiration was not inhibited even in the presence of nitrite (Kucera et. al.,
1986). While, aerobic activity of AO sludge was strongly inhibited. One of the reasons
is that nitric oxide was produced with nitrite reductase. AO sludge with no anoxic
phosphate uptake activity has aerobic denitrification activity of nitrite (see Figure 4).
Anoxic activity of PAOs is measured by nitrate denitrification. Possible explanation is
that they had nitrite reductase, but did not have nitrate reductase. Regardless of
inhibition mechanism (attack by nitric oxide or direct attack by nitrite), phosphate
uptake inhibition is expectedly buffered by aerobic denitrification that can reduce nitrite
or nitric oxide concentration inside cell. Observations of phosphate uptake and OUR
recovery from inhibition soon after nitrite disappearance (Figure 2) strongly supports
the hypothesis mentioned above.



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CONCLUSIONS
In order to clarify the mechanism of unstable response of PAOs to nitrite exposure,
nitrite inhibition batch tests were conducted with the enriched PAOs that have different
anoxic activity.
•
Nitrite inhibits aerobic phosphate uptake of PAOs, but the inhibition is not direct
inhibition by nitrite but indirect inhibition caused by reduced respiration.
•
PAOs with higher anoxic activity can reduce the inhibitory effect of nitrite, possibly
because of aerobic nitrite denitrification.
•
Nitrite inhibition of aerobic phosphate uptake is successfully expressed by the
model including aerobic nitrite denitrification rates.
From these results, we concluded that unstable response of PAOs to nitrite is caused by
widely distributed anoxic activities of PAOs.


ACKNOWLEDGMENTS
This study was funded by Grant-in-Aid for Scientific Research by Japan Society for the
Promotion of Science (No.17560489).


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