This document summarizes a study of the regeneration zone in a wastewater treatment plant in Liberec, Czech Republic. The regeneration zone has three main functions: 1) regenerating the storage capacity of activated sludge cells, 2) increasing the aerobic sludge age, and 3) bioaugmenting nitrification in situ by supplying substrate for nitrifying bacteria. Experimental results from the plant show that the regeneration zone increased nitrification rates and initially fulfilled its role in bioaugmenting nitrification. However, accidents disrupting the supply of reject water stopped the bioaugmentation and regeneration functions.

1. Study of regeneration zone in WWTP Liberec
Vojtěch Pospíšil*, Petr Kelbich*, Lucie Chovancová*, František Martínek*, Martin Pečenka*, Iveta
Růžičková*, Jiří Wanner*
*University of Chemistry and Technology, Prague
Abstract
This paper deals with the study of basic functions of activated sludge regeneration zone, particularly in
its application for bioaugmentation of nitrification in situ.
Keywords
Activated sludge, nitrification, regeneration, bioaugmentation in situ
NITRIFICATION – REGENERATION AND BIOAUGMENTATION IN SITU
The main role of modern activated sludge process for wastewater treatment is to remove organic
pollution, nitrogen and phosphorus to very low residual concentrations to prevent pollution of receiving
waters. The most difficult pollution to deal with is nitrogen and its components present in wastewater.
The main process of nitrogen removal is nitrification when ammonia nitrogen is oxidized to nitrate
nitrogen which is subsequently reduced by other bacteria to nitrogen gas. This process is performed
by chemolithotrophic nitrification bacteria which are slow-growing and very sensitive to external
factors. Therefore it is very difficult to maintain enough nitrification bacteria in activated sludge. The
bioaugmentation of nitrification in situ is one of possible ways how to enhance the nitrification
population in activated sludge. This method is often combined with activated sludge regeneration for
bulking control. Both processes bioaugmentation and regeneration are performed in the side-stream of
return activated sludge in a separate reaction zone.
Function of the regeneration tank. Regeneration zone has three basic functions and these are
(i) regeneration of the storage capacity of cells, (ii) increasing the aerobic sludge age and (iii)
nitrification bioaugmentation in situ. Another feature is the accumulation of sludge / biomass. This is
used for example in WWTP Opava, where there is a potential toxic inflow from local pharmaceutical
industry, where the accumulated sludge from the regeneration can be used to inoculate the aeraton
tank. There is a regeneration tank same as the aeration tank. Generally denitrification function is not in
regeneration zone, but sometimes it does. There is a potential for denitrification of produced nitrate.
For this purpose sometimes branch part of the influent is pumped into anoxic part of regeneration
zone, which is for example in WWTP Liberec.
Regeneration of storage capacity of the cells is happening by passing regeneration tanks and by
consuming storage compounds. This leads to the reduction of the endogenous respiration rate. Higher
endogenous respiration rate at the beginning and lower endogenous respiration rate at the end of
regeneration tank confirms its function to regenerate. In the RDN (Regeneration, Denitrification,
Nitrification) arrangement of WWTP a regeneration tank also has an influence on the formation of floc
forming bacteria and thereby serve to improve the sedimentation properties of sludge.
Reducing the respiration rate is just a sign that regeneration was successful; the main role of
regeneration is thus returning the cells to the main reactor "hungry". Reduction of the concentration of
easily biodegradable substrate proceeds as follows. In the first stage easily biodegradable organic
substrate is removed in the activation tank. When the concentration of easily biodegradable substrate
is nearly constant and the sludge is aerated in the regeneration tank than the additional consumption
of storage compounds happens. The advantage of regeneration tank is that the sludge is concentrated
already.
Another function of the regeneration zone is to increase the aerobic sludge age, which is defined as
the weight of biomass in aerated reactors to the total amount of biomass in the system. It should be
noted that the very high aerobic sludge age is not sufficient to support the growth of nitrifying bacteria,
if there is not enough substrate in the system. This increase is used to create better conditions for
slow-growing nitrifying bacteria.
Bioaugmentation in situ. Bioaugmentation of nitrification in situ is based on the supply of
substrate for nitrifying bacteria. The principle of this method is to pump into the regeneration tanks
substrate for nitrifying bacteria in the form of reject water, which serves as a source of ammonia
nitrogen. It is important not to confuse this method with "simple" bioaugmentation nitrification, when
nitrifying bacteria are dosed into the activation tank directly. The advantage of in situ bioaugmentation
nitrification is that nitrifying bacteria are cultivated in system, so they are used to the environment.

2. Wastewater treatment plant Liberec - Full-scale plant with bioaugmentation in
situ
Samples for experimental measurements were taken at the wastewater treatment plant in
Liberec. This is a mechanical-biological treatment plant with a capacity of 190 000 PE in the city of
Liberec and Jablonec nad Nisou. WWTP is organized into six parallel lines containing primary settling
tank, anoxic selector, denitrification, nitrification and rectangular secondary settling tank. For each pair
of lines there is one assigned regeneration tank. Samples were taken from five sampling points. The
first sampling point was an anoxic selector. Wastewater after primary settling flowing from the well was
collected here. This sample was used to dilute the kinetic denitrification tests and served as a source
of easily biodegradable substrate. The second sampling point was nitrification tank. Activated sludge
and foam was collected here. They were used both for FISH analysis and for microscopic tests. The
third and fourth sampling point was at the beginning and at the end of the regeneration tank. Activated
sludge was collected for nitrification and denitrification kinetic tests and respirometry tests. The fifth
sampling point was effluent from the WWTP. Collected water was used for fractionation tests of
COD Cr.
RESULTS:
The experiments were obtained in the period of December 2013 – April 2014 at WWTP Liberec. The
results from 16th April at WWTP Liberec are shown in charts 1.1, 1.2, 1.3; they are showing positive
effect of regeneration zone on nitrification rate. This statement is confirmed by table 1.1, where it can
be seen higher nitrification rate at the end of the regeneration zone. Charts 1.4, 1.5, 1.6 are showing
denitrification in regeneration zone from 4th February at WWTP Liberec. From our previous
experiments we have found out that the denitrification rate is the same throughout the regeneration
zone, which can be seen from chart 1.4 and table 1.2.
Chart 1.1 N-NO3- concentration during the kinetic
test of nitrification with sludge from nitrification zone
of regeneration tank WWTP Liberec
Chart 1.2 N-NO2 concentration during the kinetic test
of nitrification with sludge from nitrification zone of
regeneration tank WWTP Liberec
Chart 1.3 N-NH4+ concentration during the kinetic
test of nitrification with sludge from nitrification zone
of regeneration tank WWTP Liberec
Chart 1.4 N-NO3 concentration during the kinetic test
of denitrification with sludge from denitrification zone
of regeneration tank WWTP Liberec

3. Chart 1.5 N-NO2 concentration during the kinetic
test of denitrification with sludge from denitrification
zone of regeneration tank WWTP Liberec
Chart 1.6 COD concentration during the kinetic test of
denitrification with sludge from denitrification zone of
regeneration tank WWTP Liberec
Table 1.1 Specific rates of nitrification in regeneration tank at WWTP Liberec
Table 1.2 Specific rates of denitrification in regeneration tank at WWTP Liberec
The chart 1.7 shows that in March the regeneration tank fulfilled its function to regenerate the storage
capacity of cells. There is a noticeable difference between the higher endogenous respiration rate at
the beginning of the regeneration tank and the lower endogenous respiration rate at the end of the
regeneration tank. However, after an accident occurred in the supply of reject water into the system,
bioaugmentation of nitrification in situ stopped working correctly, because of this there was not
sufficiently high concentration of nitrates in the regeneration tank. Denitrification and oxidation of easily
biodegradable organic compounds could not take place. Due to constant inflow of branched
wastewater after primary settling, a further increase in the concentration of easily biodegradable
substances occurred. Under these conditions, it was quite impossible to regenerate the storage
capacity of cells, thus regeneration tank did not fulfil this function. This fact is proved by the
convergence of endogenous respiration rates. The accident of blowers also contributed to the lack of
oxidation of easily biodegradable organic compounds.
The chart 1.8 shows that from January to April regeneration tank fulfilled its function bioaugmentation
nitrification in situ. This statement is proved by noticeable difference between the nitrification rates.
The nitrification rate is lower at the beginning of the regeneration tank and higher at the end of the
regeneration tank. However, after the aforementioned accident in dosing the reject water into the
system bioaugmentation nitrification in situ completely disappeared. The nitrification rate in the
regeneration tank was identical to the nitrification rate in the aeration tank. After dosing reject water
into the system again, bioaugmentation nitrification in situ was restored, but the difference between
nitrification rates and nitrification rates itself were significantly smaller. The second accident in dosing
the reject water into the system again stopped bioaugmentation nitrification in situ. The general trend
of the development of specific nitrification rates was reported as rising from December 2013 to 25th
February 2014. Then, due to blower’s accident, which was in operation until 1. 4. 2014 and by
following reject water pump’s accident, specific nitrification rate decreased. Reject water was not
pumped from 14th march 2014 to 5th May 2014; this event affected the last two samplings. It can be
assumed that the regeneration tank at this time did not fulfil its function, because it was the overdosed
by organic substrate with the wastewater after primary settling and it was also poorly aerated.

4. Chart 1.7 The course of the endogenous
respiration rate during the time
Chart 1.8 The course of the nitrification rate
expressed as rX;N-NH4+ during the time
At 4th February 2014 denitrification rates in different profiles of a regeneration zone were compared.
From the resulting values it is clear that the rate of denitrification in various profiles regeneration zone
not differ significantly. Overdose organic substrate and insufficient aeration could lead to increased
specific rate of denitrification in 16th April 2014.
On dosing period without reject water can be documented hypothesis that even high aerobic sludge
age itself is not sufficient to increase the intensity of nitrification, if it is not enough substrate for nitric
bacteria in the regeneration zone.
From the resulting specific nitrification rates on the beginning and end of the regeneration zone we
can conclude that bioaugmentation nitrification in situ was not completely suppressed due to the
presence of ammonium ions in the activated sludge.
Regeneration function of regeneration zone was studied by specific endogenous respiration rates.
Table 1.3 shows that in 16th April 2014 was more than 50% higher specific respiration rate than in 4th
February 2014, which means that regeneration function of regeneration tank was decreasing. Dosage
of wastewater after the primary settling is not enough, because there is no sufficiently high
concentration of nitrates in order to enable the organic compounds oxidation by denitrifying bacteria.
Increasing the concentration of nitrate is achieved by dosing the reject water with a high concentration
of ammonium ions to the beginning of the regeneration zone and by subsequent oxidation to nitrates
by nitrifying bacteria.
In order to verify the specific denitrification rates and specific respiration rates, recalculation to oxygen
units was made. When specific denitrification rate was multiplied oxygen equivalent of 2.86 and 0.8.
Coefficient 0.8 is there due to the fact that 80% of the microorganisms are capable to respire under
anoxic conditions.
Table 1.3 Comparison recalculated specific denitrification rate r X, red; N-NO3 specific exogenous respiratory rate R ex
Date of collection February 4, 2014 February 25, 2014 April 16, 2014 Oxygen unit
rX;red;N-NO3- 6.46 12.89 22.96
mg / (h.g)
rex 6.93 13.85 22.65
Chart 1.9 shows the progress of sedimentation properties of activated sludge expressed by sludge
index, which were in the optimum range between 80 to 100 ml / g. Stable sedimentation properties of
activated sludge wastewater treatment plant Liberec can be related to the presence of a regeneration
zone in the activation system.
Chart 1.10 shows the increase in the concentration of total nitrogen in the influent, which was
accompanied by a trend of increasing CODCr, this prevents the degradation of ratio CODCr / N tot.
Chart 1.11 shows the mentioned ratios CODCr / N tot. In 2014 was smaller inflow to the WWTP Liberec,
but the amount of pollution was the same. From this follows that the ratio CODCr / N tot is relatively
stable. This situation is good for the dosage of external substrate.

5. Chart 1.9 Dependence of the sludge index in the
nitrifying tank at the time
Chart 1.10 Increase in the concentration of total
nitrogen
Chart 1.11 The ratio of CODCr to the concentration
of total nitrogen in the influent for screens at the
time
FISH analysis was performed, which showed that the representation of AOB and NOB bacteria in the
profile regeneration tanks was similar. AOB occurred in small and medium-sized compact clusters and
their amounts after passing the regeneration zone did not increase. The increased amount of NOB is
noticeable, but not sufficiently conclusive. Results are influenced by the fact that samples were taken
at the time when the flow of reject water into the regeneration was not working for one month. Results
are in table 1.4.
Table 1.4 Analysis of AOB and NOB at the beginning and at the end of the regeneration tank..
AOB NOB
[%] [%]
Beginning 6,0 2,7
End 5,6 2,9
AOB occurred in small and medium-sized compact cluster. High amount of free bacteria were detected (level 3).
NOB occurred in small and medium-sized compact cluster, free bacteria were not detected.
Figure 1.11 AOB (Cy3, red/light grey) and total
biomass (DAPI, blue/dark grey), the beginning of the
regeneration tank, 320x.
Figure 1.2 AOB (Cy3, red/light grey) and total biomass
(DAPI, blue/dark grey), the end of the regeneration
tank, 320x.

6. Figure 1.32 NOB (Cy3, red/light grey) and total
biomass (DAPI, blue/dark grey), the beginning of the
regeneration tank, 320x.
Figure 1.4 NOB (Cy3, red/light grey) and total biomass
(DAPI, blue/dark grey), the end of the regeneration
tank, 320x.
Microscopic analysis showed that the regeneration tank does not affect the growth of foaming
filamentous microorganisms, while the number of filamentous organisms causing classic sludge
bulking decreased. This results in improved stable sedimentation properties of the
sludge. Microscopic analysis confirmed the presence of relatively well developed flocs that are
predominantly solid and compact and 80% are about the size of 150 microns. The microscopic
analysis also shows that intermittent dosing of iron and aluminum ions has a positive effect against
excessive growth of Microthrix parvicella, so the observed foam at WWTP Liberec is rather formed by
nocardioform actinomycetes type GALO. Microscopic analysis revealed the presence of relatively well-
developed clusters of poly-P bacteria. Although the technological line does not have real anaerobic
zone, the mechanism of biological phosphorus removal can occur thanks to anoxic selector, in which
there is a significant decrease in redox potential. The reason for low redox potential is rapid
consumption of the nitrates, which happens due to dosage of wastewater and returned sludge, as a
result of this the system switches to anaerobic.
Figure 1.5 Figure 1.6
Liberec BP 210114-GS750-character-BP-GALO Liberec 210114-NAT125 character AS + Rotifer
Figure 1.7 Figure 1.8
Liberec BP 210114-GS750-character-BP-GALO II Liberec 210114-NS1250- cluster poly-P

7. Figure 1.9 Figure 1.10
Liberec 210114-NAT125 character AS Liberec 210114-NAT125 character AS II
Figure 1.11 Figure 1.123
Liberec 210114-GS750- filamentous bacteria Liberec 210114-GS750- filamentous bacteria II
Fractionation of CODCr was performed for wastewater after primary settling and on effluent by
physicochemical characterization method according Mamais et al 1993. The results are summarized in
table 1.4. Although the wastewater contains a high concentration of easily biodegradable substrate
SS, but in overall it is not too concentrated. For this reason it is necessary intermittent dosing of
external substrate..
Table 1.4 Fractionation of CODCr in wastewater after primary settling and in effluent
April 2, 2014
CODCr
Wastewater unfiltered 210
mg/lWastewater coagulated 90 SS 41
Effluent 58 SI 49
Effluent coagulated 49 XS +XI 120
April 16, 2014 CODCr
Wastewater unfiltered 156
mg/l
Wastewater coagulated 55 SS 25
Effluent 37 SI 30
Effluent coagulated 30 XS +XI 101
CONCLUSION:
From these measurements we can formulate the following main conclusions. Additional function of
denitrification in the regeneration tank was confirmed. Furthermore, it was confirmed by respirometric
measurements was confirmed that if there is no sufficient nitrification in regeneration tank, the
branched supply of wastewater after primary settling to the regeneration tank is harmful. The
accumulation storage capacity is deteriorating.
The actual effect of bioaugmentation nitrification in situ was directly confirmed by lower nitrification rate
in the beginning and by higher nitrification rate at the end of the regeneration tank. It was indirectly
confirmed by approximating these nitrification speeds after accident in dosing reject water. It is
important to note that there must not be long-term disruptions in dosing the reject water or other
sources of ammonia nitrogen into regeneration tank.

8. In the initial stages of our laboratory experiments, kinetic tests managed to trace positive effect of the
regeneration zone to the level of nitrification. The gradual increase of nitrification speed over time
occurred and the positive difference between nitrification rate at the beginning and end of the
regeneration zone was observed. This positive development was interrupted by prolonged
discontinuation of dosing reject water into the regeneration tank. An important finding was that even
after discontinuation of dosing the reject water, the rapid collapse of nitrification did not happen,
because nitrification bacteria could also use ammonia nitrogen present in lower concentration in
activated sludge.
The dosing period without reject water can document the validity of the hypothesis that the high
aerobic sludge age is not sufficient to provide stable nitrification unless there is not enough substrate
for nitrification bacteria.
Continuous dosing of wastewater after primary settling to the regeneration tank to promote
denitrification proved to be unsuitable, because the organic substrate was not completely used in
anoxic part of regeneration tank due to lack of nitrates. A low concentration of nitrates was due to
insufficient nitrification. As a result the gradual slow increase of endogenous respiration rate during the
experiments occurred.
Despite the increase in endogenous respiration rates, regeneration zone still showed a positive effect
on the sedimentation properties of activated sludge, which was composed of well-formed flocs and
number of filamentous organisms causing bulking, has been reduced. The observed increased
incidence of certain foaming organisms is unrelated to the function of regeneration zone because their
presence in the activated sludge is determined by characteristics which the regeneration zone cannot
influence.
Acknowledgment:
This paper was supported by the contract for SčVK a.s., as contract no. 217 61 31 09. The financial
support of the company is therefore highly appreciated. Financial support from specific university
research (MSMT No 20/2015)
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