Article

Disintegrated Waste-Activated Sludge (NO2/FNA Method) as a

Source of Carbon for Denitrification in the Mainstream of a WWTP
Dorota Szypulska 1 , Kamil Janiak 1,2, * , Bartosz Zi˛eba 1 , Anna Wizimirska 1 , Marek Mołczan 1 ,
Sławomir Szerzyna 2 and Renata Tomczak-Wandzel 3

1 Faculty of Environmental Engineering, Wroclaw University of Science and Technology,

Wybrzeże Wyspiańskiego 27, 50-370 Wroclaw, Poland; [email protected] (D.S.);
[email protected] (A.W.); [email protected] (M.M.)
2 Wroclaw Municipal Water and Sewage Company, Na Grobli 14/16, 50-421 Wroclaw, Poland;
[email protected]
3 Aquateam COWI, Karvesvingen 2, 0579 Oslo, Norway; [email protected]
* Correspondence: [email protected]

Abstract: The deficiency of readily biodegradable organic carbon can be a significant limitation to
effective nitrogen removal during wastewater denitrification. Waste-activated sludge (WAS) is a source
of carbon produced directly at wastewater treatment plants (WWTPs). Raw WAS has a large molecular
weight and complex chemical structure molecules that are not easily available for microorganisms.
In this study, easily biodegradable organic fractions were released using pH control and/or nitrites
and nitric acid (NO2 /FNA). The obtained results indicated that WAS can be a sufficient carbon source
for denitrification in WWTPs that are at risk of minor effluent violations. The implementation of WAS
disintegration with the use of pH control and NO2 /FNA allowed for the denitrification of an additional
0.5 and 0.8 mgN-NO3 /L. WAS disintegration, besides being a source of carbon generation, reduces the
volume of sludge and leads to the implementation of a closed-loop system.

Keywords: SCOD biodegradability fractions; waste-activated sludge; nitrogen uptake rate; WWTP
effluent simulations
Citation: Szypulska, D.; Janiak, K.;
Zi˛eba, B.; Wizimirska, A.; Mołczan,
M.; Szerzyna, S.; Tomczak-Wandzel, R.
Disintegrated Waste-Activated Sludge 1. Introduction
(NO2 /FNA Method) as a Source of
Nitrogen is one of the main biogenic compounds and is responsible for eutrophication
Carbon for Denitrification in the
Mainstream of a WWTP. Resources
when it enters water bodies in excessive quantities. Therefore, its efficient removal from
2024, 13, 80. https://doi.org/
wastewater is required [1]. Activated sludge processes (with enhanced nutrient removal)
10.3390/resources13060080
are widely used in wastewater treatment plants (WWTPs) due to their relatively simple
operation, low cost, and high efficiency [2]. The biological removal of nitrogen is conven-
Academic Editor: Barbara Ruffino
tionally carried out via the autotrophic conversion of ammonia to nitrite and nitrate under
Received: 19 April 2024 aerobic conditions (nitrification), which is then followed by heterotrophic reduction to dini-
Revised: 29 May 2024 trogen gas under anoxic conditions (denitrification) [3]. Denitrifiers require biodegradable
Accepted: 5 June 2024 organic compounds as electron donors. Therefore, the potential of denitrification depends
Published: 10 June 2024 on the availability of biodegradable organic carbon in raw wastewater. Efficient nitrogen
removal can be achieved if the C/N ratio is at a minimum level of 6. However, wastewaters
treated by municipal WWTPs are often characterized by lower C/N ratios. In such cases,
the effective removal of nitrogen requires the provision of an external carbon source [4,5].
Copyright: © 2024 by the authors. Methanol, ethanol, acetate, and glucose are the external carbon sources that are of-
Licensee MDPI, Basel, Switzerland.
ten used to enhance nitrogen removal efficiency. Their application leads to a signifi-
This article is an open access article
cant increase in the operating costs of wastewater treatment (USD 0.33–1.03/kgC) [4–6].
distributed under the terms and
In recent years, research on alternative carbon sources as alternatives to expensive commer-
conditions of the Creative Commons
cial solutions has gained significant interest. Industrial wastewater rich in biodegradable
Attribution (CC BY) license (https://
organic compounds (i.e., that coming from ice cream production) and food waste-recycling
creativecommons.org/licenses/by/
4.0/).
wastewater, are among the various proposed solutions [4–6]. However, such approaches

Resources 2024, 13, 80. https://doi.org/10.3390/resources13060080 https://www.mdpi.com/journal/resources

Resources 2024, 13, 80 2 of 15

require the availability of a suitable stream in the vicinity of the WWTP. Potentially, the
most beneficial solution appears to be the harvesting of organic compounds from the
waste-activated sludge (WAS) produced at the WWTP itself. WAS contains high levels
of organic substances such as carbohydrates and proteins, which are too complex to be
consumed by denitrifiers. However, the carbon compounds contained in WAS can be
hydrolyzed to easily biodegradable organic compounds (Ss) such as volatile fatty acids
(VFAs) with the use of WAS disintegration [7]. Many disintegration methods are available,
among which mechanical, chemical, and biological methods can be distinguished [8–11].
Most of these technologies require high energy inputs and large quantities of chemicals.
Nitrite/free nitrous acid (NO2 /FNA) is a biocidal agent that can be produced on-site at a
WWTP through nitritation of anaerobic digestion liquor [12]. The mechanism of NO2 /FNA
disintegration is still uncertain. Wu et al. (2018) indicated a strong effect of FNA on cell
membrane disruption and a limited role in EPS disintegration [1]. Wang et al. (2018) con-
cluded that FNA induces the permeabilization of bacteria and may lead to cell membrane
lysis [2]. Despite the lack of a complete understanding of the mechanism, many studies
have proven the effectiveness of NO2 /FNA as a WAS disintegration agent in terms of
soluble chemical oxygen demand (SCOD) release, and consequently for increased methane
production [12–14]. Ma et al. showed that FNA pretreatment of WAS (concentrations
up to 2 mgN-HNO2 /L) enhanced the exploitation of internal carbon sources during the
simultaneous fermentation denitrification process. Additionally, the denitrification effi-
ciency, as well as the reduction in the volume of sludge in a simultaneous fermentation
and denitrification reactor, were improved as a result of applying NO2 /FNA [15]. Those
studies showed the general potential of NO2 /FNA to improve denitrification, but crucial
data on the application of the process for classical activated sludge operated with vastly
different process parameters still require verification. Nitrogen uptake rates (NURs), which
indicate whether released SCOD is easily or slowly biodegradable, can be seen to be very
important. While slowly biodegradable compounds might be useful in long hydraulic
retention systems such as anaerobic digesters, in activated sludge reactors, they will also
penetrate into the aerobic reactor, in turn increasing oxygen consumption. Moreover, the
overall easily and slowly biodegradable COD yield from WAS, which originates from
reactors with high sludge retention time (SRT), should also be assessed.
This study is involved in the developing technology for low-carbon and clean wastew-
ater treatment, which aims to implement FNA for shortcut nitrification/denitrification in
wastewater treatment plants (WWTPs). The following issues are covered:
1. The WAS from a full biological nutrient removal plant (operated at a high SRT) is
exposed to NO2 /FNA concentrations selected according to previous studies (123, 255,
and 264 mgN-NO2 /L; 2.8 and 5.8 mgN-HNO2 /L, pH 5) [16,17].
2. Released SCOD is tested as a carbon source for denitrification in the classical activated
sludge process. The potential of the obtained carbon sources is evaluated based on NUR
and then compared to those obtained for acetate and mechanically treated wastewater.
Moreover, the SCOD yield per g of the volatile solids (VS) of the WAS is assessed.
3. The obtained experimental results are used in mass balance calculations and process
simulations to show the potential of such an application at the full scale.

2. Materials and Methods

2.1. Wastewater and Sludge Sources
The waste-activated sludge for the disintegration tests, as well as the activated sludge
and mechanically treated wastewater for the denitrification tests, were collected from
a full-scale wastewater treatment plant (WWTP) in southwest Poland (1,000,000 PE).
This WWTP conducts full N and P removal with a sludge retention time (SRT) of 25 days.
WAS was harvested from a secondary sludge thickener, activated sludge from the anoxic
chamber, and mechanically treated wastewater from the primary settler. The characteristics
of the substrates used in this study, including those of the total solids (TS), volatile solids
Resources 2024, 13, 80 3 of 15

(VS), total chemical oxygen demand (TCOD), soluble chemical oxygen demand (SCOD),
and soluble total nitrogen (TN), are presented in Table 1.
Table 1. The average parameters (±standard deviations) of substrates collected at a full-scale WWTP.

TN
Substrate TSgTS/kg VSgVS/kg TCODmgO2 /L SCODmgO2 /L
mgN/L
WAS 60.2 ± 1.0 44.2 ± 1.7 75 650 ± 1 774 276 ± 24 34 ± 11
Activated sludge 5.9 ± 0.5 4.1 ± 0.4 not determined 38 ± 7 not determined
Mechanically treated wastewater 0.3 ± 0.1 0.2 ± 0.1 700 ± 147 229 ± 31 62 ± 10

2.2. NO2 /FNA Disintegrated WAS

The FNA disintegration tests were conducted according to the methodology presented
in our previous study [17]. Two concentrations and two retention times were tested (R6-R8;
Table S2). In addition, two reference samples (NO2 /FNA free) were run parallelly (R4-R5;
Table S2). The pH level and nitrite concentration were controlled three times a day and
maintained manually with 24% H2 SO4 and NaNO2 stock solutions (20 gN/L), respectively.
The temperature was recorded during all the tests. The average FNA concentrations
presented in Table 2 were calculated according to Equation (1) [18] with regard to the
average values of temperature (T), nitrite concentration (SNO2 −N ), and pH. Immediately
after disintegration, the samples were filtrated through a glass fiber filter with a pore
diameter of 1.2 µm and then used as a substrate in the denitrification tests.

SNO2 −N
SHNO2 −N =   (1)
1 + exp−2300/(273+T) · 10pH

Table 2. Characteristics of additional carbon sources for denitrification.

Disintegration Parameters before Denitrification

Number of
Carbon Source NO2 , FNA, Exposure
pH Repetitions
mg N-NO2 /L mg N-HNO2 /L Time, h
R1 Acetate Not applicated 3
Mechanically
R2 Not applicated 3
treated wastewater
R3 Endogenous Not applicated 3
R4 WAS 0.0 ± 0.0 0.0 ± 0.0 7.0 ± 0.0 48 3
R5 WAS 0.0 ± 0.0 0.0 ± 0.0 5.1 ± 0.0 48 3
R6 WAS 123.0 ± 29.7 2.8 ± 0.1 5.1 ± 0.0 48 2
R7 WAS 254.6 ± 25.2 5.8 ± 0.9 5.1 ± 0.1 24 2
R8 WAS 263.5 ± 17.0 5.8 ± 0.0 5.1 ± 0.2 48 2

2.3. Denitrification
The denitrification tests were conducted on supernatants after WAS disintegration
(with and without NO2 /FNA) (R4-R8; Table 2), as well as on various substrates that were
taken as references (R1-R3; Table S2). The activated sludge was collected directly from
an anoxic chamber without biomass starvation. This allowed for the simulation of the
process conditions, where an external carbon source was added to the real biological reactor.
Due to the high SRT (25d), the content of the slowly biodegradable fraction (XS) in the
tested sludge was very low. The denitrification tests were conducted in glass reactors with
a working volume of 5 L. The reactors were equipped with a mechanical stirrer, pH probe
(Endress + Hauser Ceragel CPS76D-7BB21), and oxygen probe (Endress + Hauser COS61D).
Before each test, the activated sludge was flushed with nitrogen gas in order to maintain
an oxygen concentration below 0.2 mgO2 /L. During all the tests, the temperature was
controlled at the level recorded in the full-scale chamber (14–18 ◦ C). The denitrification
tests were started when the residual nitrite from disintegration (if present) was depleted,
Resources 2024, 13, 80 4 of 15

as it was assumed that the carbon required for nitrite removal should not be included.
The concentration of nitrate at the beginning of each test was maintained by adding KNO3 to
reach a level of 35–40 mgN-NO3 /L. The volume of the carbon source was calculated for each
substrate individually to add 200 mgO2 /L. The experiment was conducted at a constant pH of
7.0 ± 0.5 (manually adjusted with HCl 25%). Samples were taken at a time interval of 30 min,
with the first sample being taken 5 min after the initial parameters were adjusted. Each
sample measured 8.0 mL and was syringed through a glass fiber filter with a pore diameter of
1.2 µm. NURs were calculated on the basis of the decrease in the N-NO3 concentration over
time, which was then divided by the grams of the VS. The results were converted according
to Equation (2) for a standardized temperature of 20 ◦ C. NUR20 and NURT are the nitrogen
uptake rates at the temperature of 20 ◦ C and that at which the test was conducted, respectively.
The temperature coefficient was assumed to be equal to θ = 1.072 [19].

NUR20 = NURT · θ(20−T) (2)

2.4. Analytical Methods

Nitrite, nitrate, ammonium, total nitrogen, and volatile fatty acids (VFAs) were measured
using HACH LCK kuvete tests. The chemical oxygen demand (COD), TS, and VS were
established according to Standard Methods (2540) [20]. During the disintegration test, the
temperature and pH were measured with Hach–Lange LDO and PHC probes, respectively.
The addition of nitrite to the sludge resulted in increased COD results. Therefore,
the COD values were corrected using the theoretical oxygen demand for nitrite oxidation.
The Standard Methods mention that nitrite exerts a COD of 1.1 mgO2 /mgN-NO2 [21].
However, the stoichiometric equation shows that this is precisely 1.14 mg O2 /mg NO2 -N,
and this is the value that was applied.

2.5. Simulation of WWTP Operation

Simulations of WWTP operation were based on the SUMO2 model, which includes
phosphorus removal and a two-stage nitrification process [22,23]. All input data are
presented in the Supplementary Materials (Table S1). The input parameters were the same
for all the scenarios and were taken from a full-scale WWTP, which was the source of the
substrates for this study. The influent parameters were the average values recorded during
the experimental period. The additional carbon source (WAS disintegration supernatant)
was added upstream of anoxic reactor 1, as shown in the WWTP scheme (Figure S2 in the
Supplementary Materials). Simulations were run for each WAS disintegration supernatant
(R4-R8). A reference simulation (without an additional carbon source) was also performed.
Default kinetic parameters were used. The most important kinetic parameters and their
values are shown in Table S2.

3. Results and Discussion

Figure 1 presents the scheme of experimental steps, with the main results indicated for each.

3.1. Disintegration Efficiency—SCOD

The average concentrations of the soluble organic compounds (SCOD and VFA) ob-
tained after the disintegration of WAS (R4-R8) are presented in Figure 2. The exposition of
WAS to pH 7 (R4) and 5 (R5) without the addition of nitrite resulted in comparable increases
in the SCOD concentration of 160 and 157 mgSCOD/gVS, respectively, which indicated that
pH had no influence. The lowest tested NO2 /FNA concentration (R6, 123 mgN-NO2 /L,
pH 5) after 48 h of exposure resulted in a 14% higher SCOD release than when there was
only the pH control (R5), which confirms previous studies stating that the implementation
of FNA increases the effectiveness of sludge disintegration [17]. The highest SCOD release
(200 mgO2 /gVS, R8) occurred when the NO2 /FNA concentration (264 mgN-NO2 /L, pH 5)
was in the range reported in the previous study as being most beneficial [17]. The exposure
of WAS to a concentration of 255 mgN/L for 24 h resulted in a 25% lower SCOD release
 Resources 2024, 13, 80 5 of 15

when compared to 48 h of exposure to a comparable NO2 /FNA concentration (264 mgN/L).

This,
Resources 2024, 13, x FOR PEER REVIEW again, confirms the results of previous studies which indicated that a longer exposure
5 of 16
time was beneficial. Moreover, the addition of NO2 /FNA could not overcome a shorter
retention time, as the value recorded for R7 (255 mgN-NO2 /L, pH 5, 24 h) was lower than
that observed for the reactors with only pH control (no nitrite addition, but a retention
Figure 1 presents the scheme of experimental steps, with the main results indicated
time of 48 h). Overall, the obtained results are in accordance with previous studies, which
for each.
indicates that the obtained disintegrated streams are reliable [16,17].

Resources 2024, 13, x FOR PEER REVIEW 6 of 16

Figure 1. Scheme
Figure 1. Scheme of
of experimental
experimental steps.
steps.

3.1. Disintegration Efficiency—SCOD

The average concentrations of the soluble organic compounds (SCOD and VFA) ob-
tained after the disintegration of WAS (R4-R8) are presented in Figure 2. The exposition
of WAS to pH 7 (R4) and 5 (R5) without the addition of nitrite resulted in comparable
increases in the SCOD concentration of 160 and 157 mgSCOD/gVS, respectively, which
indicated that pH had no influence. The lowest tested NO2/FNA concentration (R6, 123
mgN-NO2/L, pH 5) after 48 h of exposure resulted in a 14% higher SCOD release than
when there was only the pH control (R5), which confirms previous studies stating that the
implementation of FNA increases the effectiveness of sludge disintegration [17]. The high-
est SCOD release (200 mgO2/gVS, R8) occurred when the NO2/FNA concentration (264
mgN-NO2/L, pH 5) was in the range reported in the previous study as being most benefi-
cial [17]. The exposure of WAS to a concentration of 255 mgN/L for 24 h resulted in a 25%
lower SCOD release when compared to 48 h of exposure to a comparable NO2/FNA con-
centration (264 mgN/L). This, again, confirms the results of previous studies which indi-
cated that a longer exposure time was beneficial. Moreover, the addition of NO2/FNA
could not overcome a shorter retention time, as the value recorded for R7 (255 mgN-
NO2/L, pH 5, 24 h) was lower than that observed for the reactors with only pH control (no
nitrite addition, but a retention time of 48 h). Overall, the obtained results are in accord-
Figure
Figure 2. WAS
2.
ance with WAS disintegration
previous results
studies,
disintegration which(as (as
results SCOD and that
indicates
SCOD VFA VFA
and concentrations),
the obtained presented as mgO
disintegrated
concentrations), per
streams
presented 2as gVS
mgOare2 per
of
gVSthe feedstock
of the[16,17].
reliable (A) and per L of supernatant (B), obtained for various disintegration parameters
feedstock (a) and per L of supernatant (b), obtained for various disintegration parameters
(pH, HNO
(pH, HNO2 dose,
dose, exposure
exposuretime).
time).

3.2. Disintegration Efficiency—Volatile Fatty Acids

VFAs are easily biodegradable organic compounds, and therefore, their significantly
higher concentration observed for R4 (pH 7, 48 h) indicates them to be a potentially better
carbon source for denitrification than other tested disintegration methods. Much lower
values were obtained for all the samples with a pH of 5.0, whether nitrite or FNA were
Resources 2024, 13, 80 6 of 15

3.2. Disintegration Efficiency—Volatile Fatty Acids

VFAs are easily biodegradable organic compounds, and therefore, their significantly
higher concentration observed for R4 (pH 7, 48 h) indicates them to be a potentially better
carbon source for denitrification than other tested disintegration methods. Much lower
values were obtained for all the samples with a pH of 5.0, whether nitrite or FNA were
present or not. This may be associated with the addition of small amounts of NaOH
to maintain pH 7.0, with numerous studies indicating that alkaline reagents are more
effective than acidic reagents in dissolving WAS [24,25]. In a previous study [17], the same
influence of pH on VFA release was observed, but only for samples without the addition of
NO2 /FNA. When NO2 /FNA was applied, VFA concentrations rose significantly, especially
with very high NO2 /FNA concentrations (far above those studied in this paper). Wang et al.
also reported the greatest VFA release from WAS when the pH was under 6.4 (without
NO2 /FNA). The addition of nitrite up to 300 mgN-NO2 /L, as well as WAS acidification
(pH 5.5), resulted in decreased VFA concentrations [12]. Contrary results were presented
by Lu et al., where WAS acidification (pH 5.5) increased VFA release by over 45% when
compared to pH 7 (48 h exposure). The addition of nitrite with a pH of 7 increased the
VFA concentration by over 100%. However, nitrite with a pH of 5.5 decreased the VFA
concentration by nearly 70% when compared to the samples without nitrite [26].
In this study, for all the samples kept under a pH of 5, comparable values of VFA/VS
indices (Figure 2A) were obtained. This indicates that the addition of nitrites did not increase
the availability of VFAs, at least with the tested NO2 /FNA concentrations and exposure times.
In our previous study [17], contrary results were observed, which shows the positive impact
of an increased NO2 /FNA concentration on the release of VFAs. However, the greatest
concentrations of VFAs were observed for significantly higher exposure times and NO2 /FNA
concentrations than those tested in this study. After 48 h of exposure, the concentrations of
VFAs were similar for all the tested FNA concentrations up to 6.2 mgN-HNO2 /L. However,
these values were approximately twice as high as those observed for the reference reactor
(pH 5) [17]. These discrepancies between the results obtained in this and previous studies
might be associated with differences in the composition of the WAS.
It is worth mentioning that NO2 /FNA disintegration of WAS resulted in a VFA/SCOD
ratio in the range of 12–14%. WAS exposure to pH 7 and 5 without the addition of nitrite
significantly increased the VFA/SCOD ratios to 31 and 17%, respectively. This indicates that
WAS disintegration without the addition of nitrite results in low SCOD release; however,
the content of easily biodegradable VFA in SCOD remains relatively high. NO2 /FNA
disintegration of WAS may increase the level of SCOD solubilization, but the content of
VFA in it may be lower. This is confirmed by the results of our previous studies, where
nitrite concentrations of up to 2000 mgN-NO2 /L resulted in VFA/SCOD ratios in the
range of 8–15%. The same WAS exposed to pH in the range of 5.5 to 6.7 resulted in
17–32% VFA/SCOD ratios. Concentrations over 2000 mgN-NO2 /L resulted in increased
VFA/SCOD ratios of up to 24%. Moreover, pH levels lower than 5.5 led to VFA/SCOD
ratios under 10% [16,17].

3.3. Disintegration Efficiency—Nitrogen

NO2 /FNA disintegration of WAS resulted in significantly increased concentrations of
soluble nitrogen forms, as presented in Figure 3. The observed trends varied from those
obtained for SCOD and VFA (Figure 2). Generally, the released TN concentrations were
lower for the reactors with the addition of NO2/ FNA (48 h exposure) when compared
to R4 and R5 (no NO2 /FNA). The exception is R7, where WAS exposed to 5.8 mgN-
HNO2 /L for 24 h released a TN concentration that was greater than R8 by over 100%
(264 mgN-NO2 /L, pH 5, 48 h). Disintegration without the addition of nitrite resulted in
a comparable TN release at pH 7 (R4) and 5 (R5) of approximately 600 mgN/L. How-
ever, pH 7 (R4) was close to being optimal for the ammonification process and brought
about the greatest ammonium release, and consequently an N-NH4 /TN ratio of about 0.5.
WAS exposure to pH 5 (R5) resulted in a lower ammonium and a higher organic nitrogen
 R4 and R5 (no NO2/FNA). The exception is R7, where WAS exposed to 5.8 mgN-HNO2/L
for 24 h released a TN concentration that was greater than R8 by over 100% (264 mgN-
NO2/L, pH 5, 48 h). Disintegration without the addition of nitrite resulted in a comparable
TN release at pH 7 (R4) and 5 (R5) of approximately 600 mgN/L. However, pH 7 (R4) was
Resources 2024, 13, 80 close to being optimal for the ammonification process and brought about the greatest7 am- of 15
monium release, and consequently an N-NH4/TN ratio of about 0.5. WAS exposure to pH
5 (R5) resulted in a lower ammonium and a higher organic nitrogen release by 35% and
39%,
releaserespectively.
by 35% andThe addition
39%, of nitrite
respectively. Thecaused further
addition inhibition
of nitrite caused of further
the microorganisms
inhibition of
conducting the ammonification process, with the greatest effect being
the microorganisms conducting the ammonification process, with the greatest observed forbeing
effect 255
mgN-NO 2/L (R7, pH 5, 24 h). An extended exposure time from 24 h to 48 h (R8) allowed
observed for 255 mgN-NO2 /L (R7, pH 5, 24 h). An extended exposure time from 24 h to
for
48 hthe acclimatization
(R8) of acclimatization
allowed for the microorganismsoftomicroorganisms
the presence of toNO 2/FNA and increased the
the presence of NO2 /FNA
N-NH 4/TN ratio from 0.08 (R7) to 0.16 (R8).
and increased the N-NH /TN ratio from 0.08 (R7) to 0.16 (R8).
4

Figure 3.
Figure WASdisintegration
3. WAS disintegration results
results shown
shown asas aa release
release of
of nitrogen
nitrogen (TN,
(TN, N-organic,
N-organic, N-NH
N-NH44)) and
and the
the
ratio of
ratio of N-NH
N-NH4 4/TN
/TN obtained
obtainedforfordifferent disintegration
different disintegrationparameters (pH,(pH,
parameters HNOHNO
2 dose, exposure
2 dose, time).
exposure
time).
The SCOD/TN ratio is an important factor when considering external carbon sources
for denitrification
The SCOD/TNinratiothe mainstream
is an importantof afactor
WWTP. Forconsidering
when the supernatants after
external WASsources
carbon disinte-
gration, the SCOD/TN ratios were 9 (R7), 11 (R4, R5), and 18 (R6, R8). These
for denitrification in the mainstream of a WWTP. For the supernatants after WAS disinte- values were
lower than
gration, thethose reported
SCOD/TN as being
ratios werefavorable
9 (R7), 11for carbon
(R4, R5), sources to enhance
and 18 (R6, nitrogen
R8). These removal
values were
(25–30than
lower SCOD/TN) [2], but as
those reported substantially higherfor
being favorable than the COD
carbon demand
sources for denitrification,
to enhance nitrogen re-
which is
moval 2.86 gCOD/gN.
(25–30 SCOD/TN)The [2],addition of nitrogen
but substantially to activated
higher sludge
than the CODleads
demandto thefor
necessity of
denitrifi-
its nitrification (enhanced oxygen demand) and further denitrification (enhanced
cation, which is 2.86 gCOD/gN. The addition of nitrogen to activated sludge leads to the COD de-
mand). Assuming 2.86 gSCOD per denitrification of each gN, approximately 16–17% (R6, R8),
26–27% (R4, R5), and 31% (R7) of SCOD will be used for denitrification of the TN released
during disintegration, with the remaining part still being available for denitrification of ni-
trogen from inflowing wastewater. However, it should be noted that if WAS is anaerobically
digested instead of being used as an external carbon source, the same amount of nitrogen will
be released and subjected to nitrification and denitrification in the WWTP mainstream.

3.4. Denitrification—Process Rates

3.4.1. Reference Substrates
A typical carbon source for the denitrification process at a WWTP is mechanically
treated wastewater, and as an external carbon source, acetate is commonly used. When these
substrates are exhausted, endogenous denitrification may still be occurring, but at a much
lower rate. Therefore, when evaluating the potential of WAS disintegration, supernatants
such as carbon sources, acetate, mechanically treated wastewater, and endogenous substrate
were used as the references. The average NUR obtained for the mechanically treated
wastewater amounted to 3.7 ± 0.5 gN/kgVS · h (Figure 4). This value was within the
wide range of nitrogen utilization rates (3.0–7.3 gN/kgVS · h) reported in the literature for
mechanically treated wastewater [27–29].
 much lower rate. Therefore, when evaluating the potential of WAS disintegration, super-
natants such as carbon sources, acetate, mechanically treated wastewater, and endoge-
nous substrate were used as the references. The average NUR obtained for the mechani-
cally treated wastewater amounted to 3.7 ± 0.5 gN/kgVS·h (Figure 4). This value was
Resources 2024, 13, 80
within the wide range of nitrogen utilization rates (3.0–7.3 gN/kgVS·h) reported in the8 of 15
literature for mechanically treated wastewater [27–29].

Figure 4. The
Figure 4. average denitrification
The average ratesrates
denitrification (standard deviations
(standard as error
deviations bars)bars)
as error observed for all
observed forthe
all the
tested substrates
tested (pH (pH
substrates 7.0 ±7.0
0.5;±T0.5;
14–18 °C—rates
T 14–18 on the
◦ C—rates onchart converted
the chart to 20to
converted °C;20initial N and
◦ C; initial NSCOD
and SCOD
concentrations—35–40
concentrations—35–40mgN-NO
mgN-NO 3/L and 200 mgO2/L, respectively).
/L and 200 mgO /L, respectively).
3 2

In this study,
In this acetate
study, resulted
acetate in a in
resulted NURa NURvalue in the
value in lower range
the lower of the
range of values found
the values found
in the
inliterature (3.2 ± (3.2
the literature 0.2 gN/kgVS·h).
± 0.2 gN/kgVS Additionally, this value was
· h). Additionally, thisslightly
value lower than that
was slightly lower
obtained
than forthatthe mechanically
obtained for thetreated wastewater
mechanically (R2).wastewater
treated The probable reason
(R2). Thefor the differ-
probable reason
encesforinthe
NUR, which, in
differences inthis
NUR, study, were
which, in observed
this study, between the R1 and
were observed R2 reactors,
between the R1wasand R2
the reactors,
adaptation of the activated sludge microorganisms to mechanically
was the adaptation of the activated sludge microorganisms to mechanically treated treated
wastewater
wastewater (as a(ascarbon source).
a carbon The The
source). phenomenon
phenomenon of biomass adaptation
of biomass and and
adaptation its positive
its positive
influence on NUR
influence on NUR havehave
beenbeen
reported elsewhere
reported [30].[30].
elsewhere
The The
lowest NURs
lowest werewere
NURs provided by endogenous
provided by endogenous carbon sources
carbon and and
sources amounted
amountedto anto an
average
average of 0.7 0.1±gN/kgVS·h.
of ±0.7 0.1 gN/kgVS · h.does
This This not
doesdeviate
not deviate
from from
otherother literature
literature values, values,
whichwhich
covercover the range
the range of 0.6–2.2
of 0.6–2.2 gN/kgVS
gN/kgVS·h · h [28,31–33].
[28,31–33].
According to the range of nitrogen uptake rates obtained for both easily biodegradable
reference substrates, NUR above 2.9 gN/kgVS · h (the lowest recorded NUR for acetate)
was assumed to be characteristic of easily biodegradable organic compounds for further
analyses. Values of approximately 0.8 gN/kgVS · h (the highest recorded NUR for the
endogenous substrate) and below were considered in further analyses to be typical for en-
dogenous carbon sources. In contrast, intermediate NURs between 0.8 and 2.9 gN/kgVS · h
were determined as being representative of slowly biodegradable organic matter.

3.4.2. Disintegration Products

The obtained nitrogen uptake rates for the disintegration products are shown in
Figure 4. Nitrate removal curves for both supernatants obtained after WAS disintegration
without NO2 /FNA (R4 and R5) were characteristic of complex carbon sources, where both
easily and slowly biodegradable fractions could be detected (R4 as an example—Figure S1).
This indicates the presence of easily biodegradable organic compounds released during
WAS disintegration.
The reaction rates based on Ss substrates (R4 and R5) were comparable to the values
observed for the reference carbon sources (R1 and R2) and amounted to 3.2–4.5 gN/kgVS · h.
However, in both cases, the availability of easily biodegradable organic matter was rather low,
and this source was depleted within two sampling times: 0–60 min. The consumption of easily
biodegradable carbon sources was followed by a phase of the experiment with 2–3 times lower
NUR, which indicated the presence of slowly biodegradable substrates (XS). XS nitrogen
uptake rates ranged from 1.3 to 1.7 gN/kgVS · h. After the depletion of XS, an endogenous
phase was observed with significantly lower process rates (0.9–1.0 gN/kgVS · h).
Resources 2024, 13, 80 9 of 15

Easily biodegradable substrates were not observed during the denitrification process
when supernatants were used as the carbon sources for nitrate removal after WAS exposi-
tion to FNA (Figure 4). This is associated with lower VFA-to-SCOD ratios (Figure 2), as well
as the need to denitrify nitrites remaining after the disintegration process (measurements
of NUR were started after depletion of residual nitrite, as explained in Section 2.3).
The residual nitrite concentrations observed in the reactors prior to the denitrification
tests were approximately 1.4 (R6), 5.1 (R7), and 4.8 mgN-NO2 /L (R8), and their denitrifica-
tion required about 8 (R6), 32 (R7), and 30 mgCOD/L (R8). Please note that nitrite, as well
as the SCOD and VFA concentrations found in the denitrification reactors, were much lower
than those sustained in the disintegration tests due to the dilution of supernatants. Accord-
ing to stoichiometry, the VFA concentrations observed in the denitrification reactors at the
beginning of the denitrification tests were close to those required for nitrite denitrification
in each test and amounted to approximately 23, 28, and 26 mg COD/L for R6, R7, and
R8, respectively. Therefore, it was assumed that the easily biodegradable organic matter
released during the NO2 /FNA disintegration of WAS was sufficient for nitrite removal,
but its availability was too low to be observed during the denitrification tests.
The greatest process rate (1.92 gN/kgVS · h) for slowly biodegradable substrates was
recorded for the highest tested NO2 /FNA concentration and exposure time (R8, 264 mgN-
NO2 /L, pH 5, 48 h). For the other tested NO2 /FNA concentrations (R6, R7), denitrification
of the slowly biodegradable carbon source resulted in comparable average process rates of
approximately 1.4 gN/kgVS · h. The origin of the differences is unknown. The endogenous
process rates recorded when slowly biodegradable substrates were depleted were similar
for all the tests (R6, R7, R8) and amounted to approximately 0.7 gN/kgVS · h, the value
recorded for the reference substrate.
Guo et al. [34] tested carbon sources based on liquids obtained from WAS disintegra-
tion using thermal and acid hydrolysis. The resulting NURs were comparable with those
obtained in this study for easily biodegradable substrates and amounted to 2.8 (thermal
hydrolysis liquid) and 3.2 gN/kgVS · h (acidogenic liquid). Zhang et al. (2020) reported
significantly higher NUR (5.4 gN/kgVS · h in 20 ◦ C, θ = 1.072) for SS for a carbon source
obtained as a result of WAS disintegration with a composite ferrate solution. However,
Zhang et al. [2] did not provide any specific data on WAS parameters, and it is therefore
difficult to assess whether the differences were due to different disintegration methods or
peculiar WAS characteristics (e.g., a low SRT).

3.5. Denitrification—The Biodegradability of Disintegration Products

The content of the easily and slowly biodegradable compounds in the tested carbon sub-
strates was determined based on the recorded consumption of the nitrates and the calculated
NURs. The average values, with standard deviations, are presented in Figure 5. Note that for
R6-R8, the content of the easily biodegradable fraction was assessed based on the residual
nitrites, as that fraction was not detected directly in the NUR tests (see Section 3.4.2).
As a result, 60–97% (or 99–162 mgO2 /gVS) of the total SCOD released during dis-
integration was biodegradable under anoxic conditions. This is consistent with other
disintegration methods. For example, Mancuso et al. demonstrated that, during the disinte-
gration of WAS (hydrodynamic cavitation), biodegradable SCOD (measured under aerobic
conditions) increased with a rise in SCOD and ranged from approximately 50 to 75% of the
SCOD concentration [35].
 The content of the easily and slowly biodegradable compounds in the tested carbon
substrates was determined based on the recorded consumption of the nitrates and the
calculated NURs. The average values, with standard deviations, are presented in Figure
5. Note that for R6-R8, the content of the easily biodegradable fraction was assessed based
Resources 2024, 13, 80 on the residual nitrites, as that fraction was not detected directly in the NUR10tests
of 15 (see
Section 3.4.2).

Figure5.5.The
Figure Theaverage values
average of of
values thethe
SCOD (±standard
SCOD deviations)
(±standard fractions
deviations) (A) and
fractions (a) concentrations
and concentrations
(b)obtained
(B) obtainedfrom
fromthe
the disintegrated
disintegrated WAS.
WAS.

The
As conditions in R4 resulted
a result, 60–97% (or 99–162in the
mgOlowest SCOD biodegradability (both in mgO2 /gVS
2/gVS) of the total SCOD released during disinte-
and percentage). In all instances except
gration was biodegradable under anoxic conditions. R4 (pH 7, no NO2/FNA), it can bewith
This is consistent assumed
otherthatdisinte-
more than 70% of the released SCOD was biodegradable. A lower pH and the presence of
gration methods. For example, Mancuso et al. demonstrated that, during the disintegra-
NO2 /FNA in the remaining reactors led to an overall increase in the biodegradable fraction;
tion of WAS (hydrodynamic cavitation), biodegradable SCOD (measured under aerobic
however, for reactors R5 and R6, it was only the slowly biodegradable fraction that increased.
conditions) increased with a rise in SCOD and ranged from approximately 50 to 75% of
Moreover, this additional yield was fully consumed during the denitrification of the residual
the SCOD
nitrites concentration
(Figure 6). Considering [35].the potential use of WAS disintegration to produce a carbon
sourceThe conditions inNO
for denitrification, R4/FNA
2
resulted
does in
nottheseemlowest SCOD
favorable. biodegradability
It leads (both in
to a lack of an easily
biodegradable organic carbon source and consequently enables a process rate two to threebe as-
mgO 2/gVS and percentage). In all instances except R4 (pH 7, no NO2/FNA), it can
sumed
times thatthan
lower more
thatthan 70% of the released
of mechanically SCOD was
treated wastewater or biodegradable.
acetate. Compared A to
lower
WASpH and the
which
ispresence of NONO
not pretreated, 2/FNA2 /FNAin the remaining
increases the reactors
availability led
of to an overall
biodegradable increase
organic in
carbonthe biode-
for
gradable fraction;
denitrification however,
[15], but for reactors
the characteristics of R5
theseand R6, it wasdo
compounds only
not the slowly
allow such abiodegradable
substrate
to be usedthat
fraction as an alternative
increased. to acetate.
Moreover, thisGuo et al. (2017),
additional yieldwhowas used fullythermal
consumed and during
acidic the
disintegration,
denitrificationreported
of the greater
residual availability of SS in hydrolysis
nitrites (Figure liquid (after
6). Considering the disintegration
the potential use of WAS
of WAS). This intoturn
disintegration enabled
produce larger amounts
a carbon source for of N-NO x (approximately
denitrification, NO2/FNA 17 mgN-NO
does notxseem/L) fa-
to
vorable. It leads to a lack of an easily biodegradable organic carbon source andno
undergo denitrification when compared to the results of this study [32]. There is conse-
significant effect ofaexposition
quently enables process rate time on to
two thethree
biodegradability
times lowerofthan released
that SCOD (Figure 5B R7
of mechanically treated
vs. R8); a longer exposition time results in a higher SCOD release, but more importantly, the
wastewater or acetate. Compared to WAS which is not pretreated, NO2/FNA increases the
additional SCOD is nonbiodegradable. Furthermore, there is no effect of a higher NO2 /FNA
availability of biodegradable organic carbon for denitrification [15], but the characteristics
concentration on available SCOD for denitrification in the mainline (Figure 6). Exposure to a
of these compounds do not allow such a substrate to be used as an alternative to acetate.
higher NO2 /FNA concentration results in a higher biodegradable SCOD release (Figure 5B
Guo
R6 vs.etR8);
al. (2017),
however,who thisused
gainthermal and acidic during
is fully consumed disintegration, reported greater
the denitrification availability
of the residual
of SS in hydrolysis liquid (after the disintegration of WAS). This
nitrites. Please note that if the NO2 /FNA solution is created in situ from digester liquor and in turn enabled larger
no side-stream deammonification is present, the removal of residual nitrites also contributeswhen
amounts of N-NO x (approximately 17 mgN-NO x /L) to undergo denitrification
to a lower nitrogen load to the mainline.
When comparing the easily biodegradable fraction contents (Figure 5B) with the
VFA measurements (Figure 2A), it can be seen that VFA constituted 50–70% of the total
easily biodegradable fraction for R5-R8. The only exception is R4, where the VFA content
was clearly overestimated (compare Figures 2A and 5). Generally, it can be assumed
that, on average, 35 mgO2 /gVS of the easily biodegradable fraction is released during
disintegration, with at least half of that being VFA.
 ble SCOD for denitrification in the mainline (Figure 6). Exposure to a higher NO2/FNA
concentration results in a higher biodegradable SCOD release (Figure 5b R6 vs. R8); how-
ever, this gain is fully consumed during the denitrification of the residual nitrites. Please
note that if the NO2/FNA solution is created in situ from digester liquor and no side-
Resources 2024, 13, 80 11 of
stream deammonification is present, the removal of residual nitrites also contributes to15a
lower nitrogen load to the mainline.

Figure 6.
Figure 6. SCOD
SCOD available
availableforfordenitrification
denitrificationininthe
themainline:
mainline:data onon
data average SCOD
average SCOD (±standard
(±standard de-
viations) fractions (a) and concentrations (b) obtained from disintegrated WAS without
deviations) fractions (A) and concentrations (B) obtained from disintegrated WAS without SCOD SCOD re-
quired for the denitrification of residual nitrites.
required for the denitrification of residual nitrites.

When comparing
Additionally, the easily
on average, biodegradable
100 mgO2 /gVS of fraction contents
the slowly (Figure 5b) with
biodegradable the VFA
fraction was
measurements (Figure 2a), it can be seen that VFA constituted 50–70%
released. Although the concentration of the total SCOD obtained after the disintegra- of the total easily
biodegradable
tion process was fraction for R5-R8.
the greatest for R8The only
(264 exception
mgN-NO is pH
2 /L, R4, 5,
where
48 h),the VFA content
amounting was
to over
200 mgOoverestimated
clearly 2 /gVS, nearly half of thatFigures
(compare (45%) was2a not
andavailable for denitrification
5). Generally, in the mainline
it can be assumed that, on
(Figure
average, 6A).
35 Nitrogen
mgO2/gVS uptake
of therates
easilylower than that for
biodegradable the acetate
fraction attained
is released for supernatants
during disintegra-
after
tion, WAS
with atdisintegration were
least half of that also VFA.
being reported by Walczak et al. when using the hydrody-
namicAdditionally,
method [27]. on average, 100 mgO2/gVS of the slowly biodegradable fraction was
released. Although the concentration of the total SCOD obtained after the disintegration
3.6. Assessment
process was theof Full-Scale
greatest forPotential
R8 (264 mgN-NO2/L, pH 5, 48 h), amounting to over 200
mgOThe concept
2/gVS, nearlyofhalf
full-scale
of thatimplementation of the discussed
(45%) was not available technology in
for denitrification is presented
the mainlinein
Figure
(Figure7.6a).
TheNitrogen
supernatant, which
uptake provides
rates an external
lower than that forcarbon sourceattained
the acetate for denitrification, has to
for supernatants
be produced
after through centrifugation
WAS disintegration were also since the separation
reported by Walczak of theetsolid and liquid
al. when usingphases requires
the hydrody-
anamic
Resources 2024, 13, x FOR PEER REVIEW highermethod
energy [27].
input. In the course of the study, a 60% efficiency of supernatant production 12 of 16
was achieved, and while this can be considered as a threshold, it should also be noted that in
process conditions,
3.6. Assessment this valuePotential
of Full-Scale will be dependent on the respective centrifuge properties.
The concept of full-scale implementation of the discussed technology is presented in
Figure 7. The supernatant, which provides an external carbon source for denitrification,
has to be produced through centrifugation since the separation of the solid and liquid
phases requires a higher energy input. In the course of the study, a 60% efficiency of su-
pernatant production was achieved, and while this can be considered as a threshold, it
should also be noted that in process conditions, this value will be dependent on the re-
spective centrifuge properties.

Figure 7. Full-scale implementation concept of an external carbon source from NO2 /FNA disinte-
Figure 7. Full-scale implementation concept of an external carbon source from NO2/FNA disinte-
grated WAS.
grated WAS.

Simulation Results
To assess the potential of using WAS disintegration supernatants as carbon sources
for denitrification in a full-scale WWTP (scheme in Figure S2), simulations for each tested
disintegration product were conducted. The most important parameters of the WWTP ef-
Resources 2024, 13, 80 12 of 15

Simulation Results
To assess the potential of using WAS disintegration supernatants as carbon sources
for denitrification in a full-scale WWTP (scheme in Figure S2), simulations for each tested
disintegration product were conducted. The most important parameters of the WWTP
effluent (treated wastewater) obtained for each scenario are presented in Table 3, while the
input data are shown in Table S1. Total nitrogen, as well as nitrate concentrations, were the
highest in the reference scenario (without the addition of an external carbon source). Each
of the analyzed carbon sources enhanced the efficiency of the nitrogen removal, with the
highest improvement being up to 0.8 gN/m3 (Scenario R8). The addition of supernatants
from R4 and R5 (no nitrite addition) to the WWTP mainline had a comparable effect on the
wastewater treatment process. The achieved improvement in the denitrification process
was approximately 0.5 gN/m3 in those cases. The lack of significant differences between
pH 7 (R4) and 5 (R5) indicates that it is not reasonable to acidify WAS in order to produce
a carbon source for denitrification. Acidification of WAS (pH 5) with the addition of
nitrite resulted in a slightly higher efficiency of the total nitrogen removal (0.7–0.8 gN/m3 ).
It should be noted that both nitrite concentration and exposure time had no significant
influence on the nitrogen removal efficiency (compare R6-R8).
Table 3. Quality of WWTP effluent according to the simulation results.

Scenarios
Parameter Unit Reference R4 R5 R6 R7 R8
Total ammonia (NHx ) gN/m3 0.64 0.64 0.64 0.64 0.64 0.64
Nitrate (NO3 ) g N/m3 7.98 7.39 7.48 7.23 7.19 7.15
Total nitrogen g N/m3 9.81 9.25 9.33 9.08 9.03 9.00
Orthophosphate (PO4 ) g P/m3 0.15 0.15 0.15 0.15 0.15 0.15
Air flow rate m3 /d 1,785,649 1,785,649 1,783,686 1,784,468 1,773,731 1,789,684

The reason for the improvement of the total nitrogen concentration in the effluent
was the greater load of readily biodegradable substrates that flowed into anoxic reactor
1 (Table S3). This is due to the fact that additional readily biodegradable substrates were
depleted in the first anoxic reactor in all the scenarios. The availability of Ss at the input
to anoxic reactor 2 was comparable for all the scenarios and was around 1400 kg/d.
The lower nitrogen concentrations observed in the effluent for scenarios R6-R8, when
compared to R4-R5, were related to the higher concentrations of the slowly biodegradable
SCOD available in the supernatants, which in turn led to higher hydrolysis and the release
of easily biodegradable matter in the anoxic chambers (Table S4). A higher inflow of Xs
into the denitrification process also resulted in a higher load of Xs into the aerobic reactors
(Table S4), and consequently in an increase in the uptake of air (Table 1). However, this was
of a minor scale (ca. 3%).
Considering the economic and environmental benefits of using WAS as a carbon
source instead of external carbon sources (sourced outside the WWTP), the disintegration
of WAS by controlling the pH or NO2 /FNA may be an advantageous solution. According
to the simulation results, Figure 8 presents the potential of disintegrated WAS as a carbon
source for denitrification. By assuming that recovery efficiency in supernatant separation
from WAS is at a level of 60%, about 0.5–0.8 gN/m3 can be additionally denitrified in the
WWTP mainstream. This denitrification potential is relatively low, but can be sufficient for
those WWTPs that are at risk of minor effluent violations. It should also be noted that if
recovery efficiency could be enhanced, these values would increase. Furthermore, the use
of WAS will enable both a reduction in sludge volume for further management and the
implementation of a closed-loop system in practice.
 source for denitrification. By assuming that recovery efficiency in supernatant separation
from WAS is at a level of 60%, about 0.5–0.8 gN/m3 can be additionally denitrified in the
WWTP mainstream. This denitrification potential is relatively low, but can be sufficient
for those WWTPs that are at risk of minor effluent violations. It should also be noted that
if recovery efficiency could be enhanced, these values would increase. Furthermore, the
Resources 2024, 13, 80 13 of 15
use of WAS will enable both a reduction in sludge volume for further management and
the implementation of a closed-loop system in practice.

Figure 8. The potential of WAS disintegration products as a carbon source for denitrification in the
Figure 8. The potential of WAS disintegration products as a carbon source for denitrification in the
mainstream of a WWTP (Designed by macrovector / Freepik).
mainstream of a WWTP (Designed by macrovector/Freepik).
3.7.Summary
3.7. Summary
Biodegradablematter
Biodegradable matter released
released during
during NONO22/FNA
/FNA disintegration
disintegration of of
WAS WAScancan
be be
used
used
for denitrification or methane production, or even both. In this study, we proposed the
for denitrification or methane production, or even both. In this study, we proposed the use
use of a centrifuge and the direct transfer of 60% of released SCOD to the denitrification
of a centrifuge and the direct transfer of 60% of released SCOD to the denitrification reactor,
reactor, with the other 40% being anaerobically digested. In most studies to date
with the other 40% being anaerobically digested. In most studies to date [12,13,17,36,37],
[12,13,17,36,37], it has been assumed that the entire stream is directed to the fermentation
it has been assumed that the entire stream is directed to the fermentation reactors, which
reactors, which results in a substantial increase in the methane yield—a 16–52% surplus
results in a substantial increase in the methane yield—a 16–52% surplus in comparison to
in comparison to sludge that is not pretreated.
sludge that is not pretreated.
The results obtained in this study, which show the relatively low potential of
The results obtained in this study, which show the relatively low potential of NO2 /FNA-
NO2/FNA-disintegrated WAS as a carbon source for denitrification, as well as the results
disintegrated WAS as(indicated
from other studies a carbon above)
source showing
for denitrification, as well as
the high potential ofthe
thisresults from other
disintegration
studies (indicated above) showing the high potential of this disintegration
method in enhancing the biodegradability of WAS, lead to the conclusion that the condi- method in en-
hancing the biodegradability of WAS, lead to the conclusion that the conditions
tions in anaerobic digesters are more suitable for the degradation of released organic mat- in anaerobic
digesters
ter. Under areanaerobic
more suitable for the
digestion degradation
process of released
conditions, organic
their potential canmatter.
be fullyUnder anaero-
used. This
bic digestion process conditions, their potential can be fully used. This is due
is due to the long retention time (typically over 20 days), the higher processing tempera- to the long
retention time (typically
ture (approximately overand
37 °C), 20 days),
the factthe higher
that processing
the biomass temperature
is adapted (approximately
to the slowly biode-
◦ C), and the fact that the biomass is adapted to the slowly biodegradable carbon sources.
37gradable carbon sources. The denitrification process in full-scale WWTPs is characterized
The
by denitrification process
completely different in full-scale
conditions. TheWWTPs
wastewateris characterized
temperature by completely different
is significantly lower
conditions. The wastewater temperature is significantly lower (mostly up to 20 ◦ C), and
therefore, biological processes are slower. Moreover, the retention time in the denitrification
chamber is short, and due to the regular influent of mechanically treated wastewater or easily
biodegradable external carbon sources, the activated sludge biomass is not able to utilize the
slowly degradable organic carbon.
It can therefore be concluded that WAS disintegration by NO2 /FNA is suitable for
producing additional organic carbon for the denitrification process in cases where a slight
reduction in the nitrogen concentration in the plant effluent is required in order to meet the
requirements for treated wastewater.

4. Conclusions
Disintegration of WAS by 264 mgN-NO2 /L (48 h) resulted in the highest release of
SCOD. The VFA content in this sample was lower (13%) compared to pH-controlled reactors
(without NO2 /FNA), where 17% (pH 5) and 31% (pH 7) VFA/SCOD ratios were recorded.
The nitrogen uptake rates recorded for the disintegration products indicated a rela-
tively low availability of easily biodegradable compounds.
According to the simulation results, the implementation of WAS disintegration allows
for the denitrification of an additional 0.5–0.8 mgN/L. The denitrification could be enhanced
to ca. 1 mgN/L if there were a complete recovery of SCOD during centrifugation.
Resources 2024, 13, 80 14 of 15

Supplementary Materials: The following supporting information can be downloaded at:

https://www.mdpi.com/article/10.3390/resources13060080/s1, Table S1. Technological parameters;
Table S2. Key kinetic parameters used in simulations; Table S3. Easily biodegradable substrates
[kgCOD/d]; Table S4. Slowly biodegradable substrates [kgCOD/d]; Figure S1. Run of the denitrifica-
tion process with the supernatant after WAS disintegration (R4, only pH adjustment) as carbon source;
Figure S2. Scheme of WWTP.
Author Contributions: Conceptualization, K.J. and A.W.; methodology, K.J., M.M. and S.S.; software,
B.Z.; validation, K.J., D.S. and B.Z.; formal analysis, D.S. and K.J.; investigation, A.W. and S.S.;
resources, K.J.; data curation, K.J.; writing—original draft preparation, D.S.; writing—review and
editing, K.J., D.S., B.Z., M.M. and R.T.-W.; visualization, B.Z. and D.S.; supervision, K.J.; project
administration, K.J.; funding acquisition, K.J. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by National Centre for Research and Development and Develop-
ment in the Norway Grants POLNOR19 programme (grant no. NOR/POLNOR/SNIT/0033/2019-00).
Data Availability Statement: The original contributions presented in the study are included in the
article/Supplementary Materials. Further inquiries can be directed to the corresponding author/s.
Acknowledgments: The authors gratefully acknowledge co-funding from the National Centre for
Research and Development and Development.
Conflicts of Interest: Author Kamil Janiak and Sławomir Szerzyna were employed by the MPWiK
company. Renata Tomczak Wandzel was employed by Aquateam COWI. The remaining authors
declare that the research was conducted in the absence of any commercial or financial relationships
that could be construed as a potential conflict of interest. The remaining authors declare that the
research was conducted in the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.

References
1. Li, C.; Liu, S.; Ma, T.; Zheng, M.; Ni, J. Simultaneous nitri fi cation, denitri fi cation and phosphorus removal in a sequencing
batch reactor (SBR) under low temperature. Chemosphere 2019, 229, 132–141. [CrossRef] [PubMed]
2. Zhang, Y.; Lu, G.; Zhang, H.; Li, F.; Li, L. Enhancement of nitrogen and phosphorus removal, sludge reduction and microbial
community structure in an anaerobic/anoxic/oxic process coupled with composite ferrate solution disintegration. Environ. Res.
2020, 190, 110006. [CrossRef] [PubMed]
3. Winkler, M.K.H.; Straka, L. New directions in biological nitrogen removal and recovery from wastewater. Curr. Opin. Biotechnol.
2019, 57, 50–55. [CrossRef] [PubMed]
4. Sun, S.-P.; Carles, P.I.N.; Merkey, B.; Zhou, Q.; Xia, S.-Q.; Yang, D.-H.; Sun, J.-H.; Smets, B.F. Effective Biological Nitrogen Removal
Treatment Processes for Domestic Wastewaters with Low C/N Ratios: A Review. Environ. Eng. Sci. 2010, 27, 111–126. [CrossRef]
5. Kim, E.; Gu, S.; Hanifa, A.; Vincent, J. Use of food waste-recycling wastewater as an alternative carbon source for denitri fi cation
process: A full-scale study. Bioresour. Technol. 2017, 245, 1016–1021. [CrossRef] [PubMed]
6. Qi, S.; Yuan, S.; Wang, W.; Xiao, L.; Zhan, X.; Hu, Z. Effect of solid-liquid separation on food waste fermentation products as
external carbon source for denitri fi cation. J. Clean. Prod. 2021, 284, 124687. [CrossRef]
7. Shao, M.; Guo, L.; She, Z.; Gao, M.; Zhao, Y.; Sun, M.; Guo, Y. Enhancing denitrification efficiency for nitrogen removal using
waste sludge alkaline fermentation liquid as external carbon source. Environ. Sci. Pollut. Res. 2019, 26, 4633–4644. [CrossRef]
[PubMed]
8. Nzila, A. Mini review: Update on bioaugmentation in anaerobic processes for biogas production. Anaerobe 2017, 46, 3–12.
[CrossRef] [PubMed]
9. Bonilla, S.; Choolaei, Z.; Meyer, T.; Edwards, E.A.; Yakunin, A.F.; Allen, D.G. Evaluating the effect of enzymatic pretreatment on
the anaerobic digestibility of pulp and paper biosludge. Biotechnol. Rep. 2018, 17, 77–85. [CrossRef]
10. Sun, D.; Qiao, M.; Xu, Y.; Ma, C.; Zhang, X. Pretreatment of waste activated sludge by peracetic acid oxidation for enhanced
anaerobic digestion. Environ. Prog. Sustain. Energy. 2018, 37, 2058–2062. [CrossRef]
11. Lippert, T.; Bandelin, J.; Vogl, D.; Tesieh, Z.A.; Wild, T.; Drewes, J.E.; Koch, K. Full-Scale Assessment of Ultrasonic Sewage Sludge
Pretreatment Using a Novel Double-Tube Reactor. ACS ES T Eng. 2020, 1, 298–309. [CrossRef]
12. Wang, Q.; Ye, L.; Jiang, G.; Jensen, P.D.; Batstone, D.J.; Yuan, Z. Free nitrous acid (FNA)-based pretreatment enhances methane
production from waste activated sludge, Environ. Sci. Technol. 2013, 47, 11897–11904. [CrossRef] [PubMed]
13. Zahedi, S.; Icaran, P.; Yuan, Z.; Pijuan, M. Assessment of free nitrous acid pre-treatment on a mixture of primary sludge and waste
activated sludge: Effect of exposure time and concentration. Bioresour. Technol. 2016, 216, 870–875. [CrossRef] [PubMed]
Resources 2024, 13, 80 15 of 15

14. Karimi, R.; Hallaji, S.M.; Siami, S.; Torabian, A.; Aminzadeh, B.; Eshtiaghi, N.; Zahedi, S. Synergy of combined free nitrous
acid and Fenton technology in enhancing anaerobic digestion of actual sewage waste activated sludge. Sci. Rep. 2020, 10, 5027.
[CrossRef] [PubMed]
15. Ma, B.; Peng, Y.; Wei, Y.; Li, B.; Bao, P.; Wang, Y. Free nitrous acid pretreatment of wasted activated sludge to exploit internal
carbon source for enhanced denitrification. Bioresour. Technol. 2015, 179, 20–25. [CrossRef]
16. Szypulska, D.; Miodoński, S.; Muszyński-Huhajło, M.; Zi˛eba, B.; Janiak, K. Determination of the major factor responsible
for soluble organic matter release during nitrite/free nitrous acid pre-treatment of waste activated sludge. Bioresour. Technol.
2021, 329, 124917. [CrossRef]
17. Szypulska, D.; Miodoński, S.; Janiak, K.; Muszyński-Huhajło, M.; Zi˛eba, B.; Cema, G. Waste activated sludge disintegration with
free nitrous acid—Comprehensive analysis of the process parameters. Renew. Energy 2021, 172, 112–120. [CrossRef]
18. Park, S.; Bae, W. Modeling kinetics of ammonium oxidation and nitrite oxidation under simultaneous inhibition by free ammonia
and free nitrous acid. Process Biochem. 2009, 6, 631–640. [CrossRef]
19. Kadlec, R.H.; Reddy, K.R. Temperature Effects in Treatment Wetlands. Water Environ. Res. 2001, 73, 543–557. [CrossRef]
20. APHA. 2540 G. Total, fixed, and volatile solids in solid and semisolid samples. In Standard Methods for the Examination of Water
and Wastewater; American Public Health Association: Washington, DC, USA, 2012.
21. APHA. 5220 Chemical Oxygen Demand (COD). In Standard Methods for the Examination of Water and Wastewater; American Public
Health Association: Washington, DC, USA, 2000.
22. Hauduc, H.; Takács, I.; Smith, S.; Szabo, A.; Murthy, S.; Daigger, G.T.; Spérandio, M. A dynamic physicochemical model for
chemical phosphorus removal. Water Res. 2015, 73, 157–170. [CrossRef]
23. Varga, E.; Hauduc, H.; Barnard, J.; Dunlap, P.; Jimenez, J.; Menniti, A.; Schauer, P.; Vazquez, C.M.L.; Gu, A.Z.; Sperandio, M.; et al.
Recent advances in bio-P modelling—A new approach verified by full-scale observations. Water Sci. Technol. 2018, 78, 2119–2130.
[CrossRef] [PubMed]
24. Atay, Ş.; Akbal, F. Classification and Effects of Sludge Disintegration Technologies Integrated into Sludge Handling Units:
An Overview. Clean-Soil Air Water 2016, 44, 1198–1213. [CrossRef]
25. de Sousa, T.A.T.; Monte, F.P.D.; Silva, J.V.D.N.; Lopes, W.S.; Leite, V.D.; van Lier, J.B.; de Sousa, J.T. Alkaline and acid solubilisation
of waste activated sludge. Water Sci. Technol. 2021, 83, 2980–2996. [CrossRef] [PubMed]
26. Lu, Y.; Xu, Y.; Dong, B.; Dai, X. Effects of free nitrous acid and nitrite on two-phase anaerobic digestion of waste activated sludge:
A preliminary study. Sci. Total Environ. 2019, 654, 1064–1071. [CrossRef] [PubMed]
27. Walczak, J.; Zubrowska-Sudol, M. The rate of denitrification using hydrodynamically disintegrated excess sludge as an organic
carbon source. Water Sci. Technol. 2018, 77, 2165–2173. [CrossRef] [PubMed]
28. Naidoo, V.; Urbain, V.; Buckley, C.A. Characterization of wastewater and activated sludge from European municipal wastewater
treatment plants using the NUR test. Water Sci. Technol. 1998, 38, 303–305. [CrossRef]
29. Rodríguez, L.; Villaseñor, J.; Fernández, F.J.J. Use of agro-food wastewaters for the optimisation of the denitrification process.
Water Sci. Technol. 2007, 55, 63–70. [CrossRef] [PubMed]
30. Kowal, P.; Ciesielski, S.; Otieno, J.; Majtacz, J.B.; Czerwionka, K.; Makinia,
˛ J. Denitrification process enhancement and diversity
of the denitrifying community in the full scale activated sludge system after adaptation to fusel oil. Energies 2021, 14, 5225.
[CrossRef]
31. Sage, M.; Daufin, G.; Ge, G. Denitrification potential and rates of complex carbon source from dairy effluents in activated sludge
system. Water Res. 2006, 40, 2747–2755. [CrossRef]
32. Świniarski, M. The Effect of External Carbon Cources on Enhancing the Denitrification Process in Activated Sludge Systems.
Ph.D. Thesis, Gdańsk University of Technology, Gdańsk, Poland, 2011.
33. Henze, M.; Gujer, W.; Mino, T.; Van Loosdrecht, M.C.M. Activated Sludge Models ASM1, ASM2, ASM2d and ASM3; IWA Publishing:
London, UK, 2000.
34. Guo, Y.; Guo, L.; Sun, M.; Zhao, Y.; Gao, M.; She, Z. Effects of hydraulic retention time (HRT) on denitrification using waste
activated sludge thermal hydrolysis liquid and acidogenic liquid as carbon sources. Bioresour. Technol. 2017, 224, 147–156.
[CrossRef] [PubMed]
35. Mancuso, G.; Langone, M.; Andreottola, G. Ultrasonics Sonochemistry A swirling jet-induced cavitation to increase activated
sludge solubilisation and aerobic sludge biodegradability. Ultrason.-Sonochem. 2017, 35, 489–501. [CrossRef] [PubMed]
36. Guerrero, A.; Duan, H.; Chen, X.; Wu, Z.; Yu, W.; Silva, C.E.; Li, Y.; Shrestha, S.; Wang, Z.; Keller, J.; et al. Enhancing anaerobic
digestion using free nitrous acid: Identifying the optimal pre-treatment condition in continuous operation. Water Res. 2021, 205, 117694.
[CrossRef] [PubMed]
37. Pijuan, M.; Wang, Q.; Ye, L.; Yuan, Z. Improving secondary sludge biodegradability using free nitrous acid treatment. Bioresour. Technol.
2012, 116, 92–98. [CrossRef] [PubMed]

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