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Sludge management in lagoons: The role of denitrification as a

function of carbon biodegradation

Heitor Henriques Guedes Mutchamua, Alice Chiapetti Bolsan,

Charline Bonatto, Angélica Chini, Bruno Venturin, Camila Ester
Hollas, Gabriela Bonassa, Fabiane Goldschmidt Antes, Helen
Treichel, Marco Di Luccio, Airton Kunz

PII: S2589-014X(21)00180-8
DOI: https://doi.org/10.1016/j.biteb.2021.100802
Reference: BITEB 100802

To appear in: Bioresource Technology Reports

Received date: 20 May 2021

Revised date: 12 August 2021
Accepted date: 13 August 2021

Please cite this article as: H.H.G. Mutchamua, A.C. Bolsan, C. Bonatto, et al., Sludge
management in lagoons: The role of denitrification as a function of carbon biodegradation,
Bioresource Technology Reports (2018), https://doi.org/10.1016/j.biteb.2021.100802

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© 2018 © 2021 Published by Elsevier Ltd.

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Sludge management in lagoons: the role of denitrification as a function of carbon

biodegradation

Heitor Henriques Guedes Mutchamuaa, b, Alice Chiapetti Bolsanc, Charline Bonattob, d, Angélica

Chinie, Bruno Venturine, Camila Ester Hollase, Gabriela Bonassae, Fabiane Goldschmidt Antesf,

Helen Treicheld, Marco Di Lucciob, Airton Kunze, f*

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a
Agricultural School, Higher Polytechnic Institute of Gaza, Chokwe, Gaza, Moçambique;
b
Department of Chemical and Food Engineering, Federal University of Santa Catarina,

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Florianópolis, SC, Brazil; -p
c
Universidade Tecnológica Federal do Paraná, Dois Vizinhos - PR – Brazil;
d
Laboratory of Microbiology and Bioprocess, Federal University of Fronteira Sul, Erechim, RS,
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Brazil;
e
Department of Agricultural Engineering, PGEAGRI/CCET-UNIOESTE, Cascavel, PR, Brazil;
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f
Embrapa Suínos e Aves, Concórdia, SC, Brazil.
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*Corresponding author: airton.kunz@embrapa.br; Address: Rodovia BR-153, Km 110, Distrito

de Tamanduá Caixa Postal: 321, 89715-899 - Concórdia – SC. Tel.: +55 (49) 3441-0400.
ur
Jo

Abstract: Sludge from the bottom of an anaerobic lagoon storage system contains nutrient residues,
as well as inorganic and organic matter. These wastes have polluting impacts on swine farms, and
beyond the management alternatives, few studies apply economic and sustainable biological
methods to reduce their effects. In this study, denitrification was proposed as an alternative sludge
treatment for settling tanks (ST), biodigesters, and stabilized lagoons. The sludge sources had
different deposition times and bioavailable carbon concentrations. Accordingly, the effects of
agitation and carbon and nitrate concentrations were evaluated. The best removal feasibility was
presented with the ST sludge, corresponding to 25% (v v-1) of the background volume, achieving
higher total organic carbon (1342 mg L-1 d-1) and nitrate (279 mg L-1 d-1) removal. Furthermore,
sludge management by denitrification was affected by carbon bioavailability and proven to be cost-
effective, with 125 days required for processing.

Keywords: carbon removal; organic matter; nitrate; swine wastewater; sludge treatment.

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1 Introduction

Biological wastewater treatment using anaerobic lagoons is a common method of storing

and treating agro-industrial, municipal, and livestock wastes, such as swine manure. This type of
treatment relies on a symbiosis between anaerobic, facultative, and aerobic bacteria to transform
organic matter into gaseous compounds (CO2, N2, NH3, H2S, CH4, and volatile organic
compounds), cellular biomasses, and residual sludge (Owusu-Twum and Sharara, 2020). However,
anaerobic digestion (AD) using bioreactors has the advantages of biogas and fertilizer production,
low cost, ease of operation, high efficiency, reduced odors, and a low demand for land; therefore

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this approach has intensified for manure management in swine farms (Tápparo et al., 2021). Along
with the positive environmental effects of energy production and sustainable waste management, a

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significant fraction of non-biodegradable substances (inorganic constituents, 15%–30% for swine
manure) accumulate as sludge at the bottom of the storage system, which affects lagoon treatment
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performance by reducing lagoon hydraulic retention time and increasing odorous and greenhouse
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gas (GHG) emissions (Owusu-Twum and Sharara, 2020; Varma et al., 2021).
A large volume of sludge is produced in swine manure plants, and its deposition in
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anaerobic lagoons is problematic for farmers and treatment systems. Furthermore, the high content
of humic-type substances, non-biodegradable organic compounds, nitrogen, phosphorus, pathogens,
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heavy metals, and organic matter increases the entire environmental wastewater treatment plant
(WWTP) operation if it is incorrectly discharged into water bodies and soil. Consequently, sludge
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management has become a research focus area in recent years. Alternative treatments include
composting, chemical stabilization, chemical conditioning, drying, pyrolysis, incineration,
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thickening, and the use of wetlands and wet air oxidation (Grobelak et al., 2019). Most of these
physical, chemical, and thermal processes are unattractive because of their high costs, operational
difficulties (the requirement for equipment, infrastructure, or chemical substances), high energy
consumption (for the thermal routes), and environmental pollution levels (GHG emissions)
(Grobelak et al., 2019; Kunz et al., 2009). Thus, alternatives for sludge management escalate the
significant environmental, regulatory, and financial challenges for the sanitation sector of swine
WWTPs.
The digestates from the AD process are organic and inorganic matter contained in the sludge
deposited at the bottom of anaerobic lagoons, and these occur based on the manure pretreatment
strategies (solid-liquid separation by screening and settling) and the anaerobic digestion process

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selected (type of reactor, hydraulic retention time, and processing condition) (Bonassa et al., 2021).
Thus, according to the biodegradability and recalcitrance of the organic carbon (OC) contained in
the sludge and digestate, denitrification can be an alternative biological route to manage the silting-
up of lagoons with high sludge content. Considering a swine farm with AD and anaerobic lagoons,
a biological process with microorganisms reduces onerous costs, complex operations, and
secondary effluent. Wastewater rich in nitrate can be obtained in activated sludge treatment plants,
but few studies present this alternative as a route for sludge treatment. Biological denitrification
reduces nitrate (NO3−) to nitrogen gas (N2) using heterotrophic bacteria. This route requires an OC
source as an electron donor to complete NO3− reduction, and according to the availability of sludge

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OC, sludge stabilization occurs through mineralization and humification (Bortoli et al., 2019;
Hollas et al., 2019).

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In this study, we focused on the applicability of denitrification as a biological method for
managing sludge from swine manure anaerobic lagoons, which were obtained from a settling tank
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(solid-liquid separation), biodigester, and a stabilized pond. Different total organic carbon (TOC)
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concentrations and OC bioavailabilities were evaluated in batch experiments as substrates for
heterotrophic bacteria in the denitrification process. To assess the possibility of this alternative for
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sludge management, the samples were chosen based on sludge age (deposition at the bottom of the
storage system) and OC bioavailability.
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2 Materials and methods

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2.1 Sludge samples

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Sludges from wastewater and swine manure treatment systems (stabilized lagoon (SL),
covered lagoon biodigester (CLB), and settling tank (ST)) with different TOC concentrations were
used to evaluate the effect of the carbon content and bioavailability on the denitrification rate. SL
and CLB were collected from a pig farm in Videira, SC, Brazil, while the ST sludge was obtained
from the primary decanter of a swine wastewater treatment plant (Embrapa Swine and Poultry,
Concórdia, SC, Brazil) (Kunz et al., 2009). The samples were homogenized, stored, and maintained
at 4 °C until use. All the experiments were conducted at room temperature (25 °C). The main
physicochemical characteristics of the sludge samples are presented in Table 1. Figure 1 displays a
flowchart of the experiments performed in this work that are divided into three stages: a) the effect
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of TOC and nitrate on denitrification, b) denitrification rate as a function of TOC, and c)

denitrification rate as a function of TOC and agitation.

TABLE 1

FIGURE 1

2.2 Effect of TOC and nitrate on denitrification

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The influence of NO3--N and TOC concentrations on the efficiency of the denitrification
process was evaluated using a 2-factor central composite design (CCD) with two levels (2²) (Table

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2). First, SL (772 ± 109 mg TOC L-1) and ST (5107 ± 794 mg TOC L-1) sludges were mixed in a
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1:1 ratio to maintain the orthogonality of the experimental design (assumption of the experimental
planning tool) and obtain an intermediate concentration of TOC (2864 ± 1172 mg L-1).
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The sludges were transferred into 50 mL reaction vessels and combined with a nitrate
solution (100, 200, or 300 mg NO3--N L-1). In all tests, the sludge and nitrate solution ratios were
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1:3 (25% sludge and 75% nitrate solution, v v-1).

The denitrification process was monitored by analyzing the concentrations of NO3--N and
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TOC in the supernatants of the mixture (sludge with nitrate solution) as a function of time.
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2.3 Denitrification rate as a function of TOC

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The evaluation of the denitrification rate behavior in the different types of sludge according
to their TOC concentration was conducted in a 4 L cylindrical reaction system to increase the scale
and simulate the conditions of a stabilized lagoon. Denitrification was induced by adding each
sludge sample (CLB, ST, and SL) to a nitrate solution (300 mg NO3--N L-1) in a ratio of 1:3 (25%
sludge and 75% nitrate solution, v v-1).
The determination of nitrate and TOC in samples collected from the supernatants of the
reaction medium (sludge with nitrate solution) was performed at various periods to calculate nitrate
and organic carbon consumption.

2.4 Denitrification rate as a function of TOC and agitation

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The effect of agitation on the denitrification process was evaluated in various sludge
samples using a CCD 22 experimental design (Table 3). Each sludge sample was mixed with nitrate
solution (300 mg L-1) and either stirred 10 times with a spatula once or twice a day, or not agitated,
according to the experimental design. The tests were also performed in a 4 L reaction system, using
the same nitrate and sludge proportions.
The supernatants were daily collected after agitation and sludge sedimentation, and nitrate
consumption rates were determined analytically by the linear regression of concentration versus
time, with the highest nitrate consumption rate as the slope of the curve.

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2.5 Sludge stabilization time

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Based on the TOC and nitrate removal rates obtained in the experiments, the stabilization of
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the sludge from the SL was determined by using an effluent resulting from the treatment of swine
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wastewater by a modified Ludzack-Ettinger (MLE) system. The nitrate concentration of this
effluent is normally between 100 and 200 mg L-1, and this can be used as a nitrogen source for
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simultaneous denitrification and sludge stabilization (Bortoli et al., 2019; Hollas et al., 2019).
The biological stabilization of lagoons is fundamentally based on organic matter
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consumption through mineralization processes (Grobelak et al., 2019). Therefore the initial TOC
mass (CTOC) in the stabilized lagoon was determined using the volume (V) and TOC concentration
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in the sludge ([TOC]) (Equation 1). The mass of nitrate ( - ) necessary to remove the TOC

mass from the sludge was also determined based on the TOC and nitrate removal rates ( and
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) obtained in this study (Equation 2). The daily nitrate load of the MLE effluent ( d -
) was

quantified using the flow rate (Q) (Equation 3). The sludge stabilization time ( ) was calculated as
the ratio between the nitrate loading rate necessary for sludge stabilization and the daily nitrate load
available in the effluent (Equation 4).

( g )= ( g ) ( ) (1)

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( g d ) ( g )
( g )= (2)
( g d )

d ( g d )= ( d ) ( g ) (3)

( g )
(d)= (4)
d ( g d )

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2.6 Analytical methods

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Sludge sample characterization for biochemical oxygen demand (BOD), alkalinity, pH,
sludge density, total solids (TS), fixed solids (FS), and volatile solids (VS) were determined
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according to standard methods (Rice et al., 2017).
Alkalinity was determined using an automatic titrator (model 848 Titrino Plus, Metrohm,
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Herisau, Switzerland). The TS, VS, and FS were established by the gravimetric method, and the pH
was measured using a portable probe from Hanna (model HI 8424). An elemental analyzer was
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used for TOC and total nitrogen (TN) concentrations, following he anufac urer’
recommendations (TOC-LCPH/CPN, Shimadzu, Kyoto, Japan). Total ammonia nitrogen (TAN:
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NH4+-N + NH3-N, expressed as NH3-N), nitrate (NO3−-N), and nitrite (NO2−-N) were measured
using the colorimetric method in a flow injection analysis system (model 2500, Fialab Instruments,
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Seattle, WA, USA) (Hollas et al., 2019; Rice et al., 2017). All chemical analyses were performed in
triplicate.

2.7 Data processing and statistical analysis

The effects of agitation and TOC and NO3--N concentrations on denitrification were
evaluated using statistical planning in a central composite design (CCD). Statistical analysis of
these responses was performed using the Statistica v. 12 trial (Statsoft, Tulsa, OK, USA). The
results were analyzed using analysis of variance (ANOVA), with confidence levels of 85%
(p < 0. 5) and 95% (p < 0.05) becau e of he ub an ial variabili y inheren in he deni rifica ion
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process. The statistical significance of the regression model was determined using the Fisher test
(Ftest), and the model was considered predictive when the Fcalculated was more significant than the
Ftabulated.

3 Results and Discussion

3.1 Effects of nitrate concentration and organic carbon bioavailability

Higher removal rates of TOC and nitrate were obtained in experiments conducted with

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larger C/N ratios (12 and 4.2) using sludge with the greatest availability of organic matter (15725 ±
3336 mgBOD L-1) (Table 2). Correspondingly, lower C/N ratios and organic matter availabilities

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(7760 ± 1427 mgBOD L-1) decreased the TOC and nitrate removal rates. The same behavior was
observed by Silva et al. (2018), who attributed this result to the increased activity of heterotrophic
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denitrifying bacteria in the media with the greatest carbon bioavailability from the electron donors
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of organic carbon sources for the denitrification process and cell growth. Several studies have
purported the ideal C/N ratio for wastewater denitrification (Tan et al., 2020; Xu et al., 2018);
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however, according to Bonassa et al. (2021), the appropriate C/N ratio for denitrification depends
on the nature of the carbon source. Therefore, it is specific to each type of wastewater as a function
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of carbon bioavailability, which justifies the lower rates for SL and CLB sludge, since they present
more recalcitrant material compared to ST sludge.
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TABLE 2
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Effect analysis verified that the interaction between nitrate concentration and TOC
negatively affected TOC removal (Figure 2), denoting an inverse proportionality between the
concentrations of nitrate and organic carbon. A lower nitrate concentration leads to less carbon
removal, which has direct implications for denitrification. Nevertheless, the TOC concentration
positively affects the consumption of this parameter, suggesting that the higher bioavailability of
TOC in the sludge promoted greater TOC consumption. All variables (nitrate and TOC
concentrations and their interactions) positively affected TOC and NO3--N in relation to nitrate
removal. TOC and NO3--N availability promoted higher rates of nitrate removal by denitrification.
These results suggest that the denitrification potential is primarily governed by the availability of

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biodegradable organic carbon, by which the readily available fraction promoted improved metabolic
activity of the denitrifying bacteria (Tan et al., 2020)

FIGURE 2

Based on the experimental design results, empirical mathematical models were proposed
(Equations 5 and 6) to predict the carbon ratios required for the denitrification process. The
mathematical models were confirmed with 85% confidence (Fcalculated TOC ꞊ 4.2 > Ftabulated TOC ꞊ .82;
Fcalculated nitrate ꞊ 7. > Ftabulated nitrate ꞊ .82) and presented determination coefficients (R2) of 81% and

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88%, respectively, thereby showing an excellent predictive capacity.

( g d ) = 442.0 48. [ ] 6 5. [
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] 8.5 [ ] [ ]
-p (5)

( g d ) = 86.6 45. [ ] 8 . [ ] 8.5 [ ] [ ] (6)

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-
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where and - are the TOC and nitrate removal rates; * + is the nitrate

concentration and [ ] is the TOC concentration.

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After mathematical model validation with ANOVA, surface curves were constructed that
showed a direct relationship between carbon and nitrate removal and the concentrations of these
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compounds in the denitrification system (Figure 2). Higher carbon removal was observed when the
TOC concentration remained high (5107 ± 794 mg L-1) and the nitrate concentration was low (100
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mg L-1). However, efficient removal was also obtained with a nitrate concentration of 300 mg L-1.
Nitrate removal depended on its concentration (at the first effluent) and that of the TOC , and larger

values of these parameters (5107 ± 794 mgTOC L-1 and 300 mg -

- L-1, respectively) promoted
higher nitrate removal rates (Figure 2).
Sludge from biological wastewater treatment has a low concentration of readily degradable
substrates; therefore, the organic matter typically constitutes the particulate fraction, which is
compatible with CLB and ST sludge characteristics. Depending on the bioavailability of these
fractions, these sludges were able to promote the consumption of nitrate and carbon in the medium,
which reflects directly on the denitrification rates and supports the possibility of using this strategy
as a way to manage these wastes (Owusu-Twum and Sharara, 2020; Xu et al., 2018).
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3.2 Sludge denitrification rates

As already discussed, sludge characteristics greatly affect the denitrification process,

particularly the denitrification rate. A high denitrification rate was obtained on the ST sludge (111.8

mg -
- L-1 d-1), whereas lower rates were acquired with SL (26.5 mg -
- L-1 d-1) and CLB sludge

(21.7 mg -
- L-1 d-1).
The chemical characteristics of the carbon source affect the mechanism of substrate
consumption, leading to varying denitrification rates, which is supported in the literature. Zou et al.

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(2020) obtained a denitrification rate of 1.2 kg -
2-
m-3 d-1 using a COD of 400–600 mgCOD L-1

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when evaluating nitrogen removal rates from raw wastewater from an anaerobically digested sludge
lagoon. Forbis-Stokes et al. (2020) obtained a denitrification rate of 0.03 kg m-3 d-1 with an
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effluent having a COD of 4000–6000 mgCOD L-1, while Zhang et al. (2019) achieved a
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denitrification rate of 0.0034 kg -
2-
m-3 d-1, with a COD of 1700 mgCOD L-1, and the removal rate
improved as carbon was added to the system; with a 9.6% increase in COD, the system nitrogen
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removal rate increased to 0.36 kg m-3 d-1, corroborating the information previously presented.
The ST sludge used in this work was sampled from the primary decanter of a swine
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wastewater treatment plant in which relatively fresh swine manure was chemically treated.
Therefore, this sludge endured less biological degradation than those obtained from anaerobic
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digestion (CLB) and the anaerobic lagoon (SL), and the organic carbon was proportionally more
readily metabolized by heterotrophic bacteria.
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3.3 Effect of agitation on the denitrification rates of different sludges

One dynamic of a lagoon is that the sludge accumulates on the bottom; therefore,
experiments were performed to check if agitation would have an effect on the denitrification rates
by increasing the sludge contact with the nitrate solution, thereby allowing further TOC and nitrate
accessibility to the denitrifying microorganisms. However, it was revealed that the denitrification
rate was affected by the TOC concentration, as opposed to the stirring action (Table 3).

TABLE 3

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Through effect analysis, with 95% confidence, it was observed that the TOC concentration
was the only significant parameter affecting the denitrification rate. Based on the results of the
experimental design, an empirical model was proposed (Equation 7), confirmed by analysis of
variance (ANOVA), since p < 0.05 (Fcalculated ꞊ 2. > Ftabulated ꞊ 9.27), with a determination
coefficient (R2) of 97%, presented an excellent predictive capacity.

( g d ) = 28.6 4. 9. [ ] 0.7 [ ] (7)

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where - is the nitrate removal rate; [ ] is the TOC concentration, and A is the
-

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number of daily agitations.
The response surface generated after model validation (Figure 3) revealed that agitation did
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not favor nitrate consumption, regardless of the carbon concentration, and the best denitrification
rate was obtained without agitation using the ST sludge. Beccari et al. (1983) also concluded that
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reactor denitrification rates did not differ according to the presence or absence of system agitation if
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the carbon source had bioavailability, suggesting that organic matter consumption was greater in
sludge with readily biodegradable organic matter, corroborating the findings of the present study.
This is a significant result in relation to cost and feasibility in practice, and thereby can be
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considered an attractive strategy (Ouedraogo et al., 2016).

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FIGURE 3
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3.4 Practical implications and final considerations

As previously observed, the sludge that accumulates in silted ponds is a source of organic
matter for the denitrification process that is commonly used in agro-industrial and livestock
wastewater treatment plants for organic matter removal. A system that combines anaerobic
digestion for energy production and an MLE process for nitrogen removal has been developed
(patent number PI 1100464-9 A2 (Kunz et al., 2015)), and is in use in a full-scale treatment plant
located in Videira, SC, Brazil. The volume of daily effluent generated on this plant is 200 m³, with a
residual nitrate concentration of 100–200 mg L-1. This effluent could be utilized for anoxic sludge

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stabilization, such as in the application of the present study with silted lagoons, rendering it an
efficient and low-cost alternative.
Considering this opportunity and based on the TOC and nitrate removal rates (34 and 22 mg
L-1 d-1, respectively) when applying this method to sludge of silted lagoons (Table 1), the time
necessary to complete TOC removal, that is, to stabilize the sludge from the silted pond, was
predetermined, considering that the silted lagoon has a volume of 5000 m3. With that, the mass of
TOC to be removed would be 3860 g with a daily nitrate load of the MLE effluent of 20

g - d- resulting in a required mass of nitrate of 2498 g - .

- -

According to the results obtained through the calculations, it is possible to remove a

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considerable amount of organic matter (TOC) and nitrate simultaneously from the effluent in 125

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days. However, according to Ouedraogo et al. (2016), the efficiency of pond treatment depends on
sludge accumulation and its constituents (which also include solids, flocs, extracellular polymeric
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compounds, and recalcitrant proteins and lipids).
According to Owusu-Twum and Sharara (2020), the simple process of removing and
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disposing of the material deposited at the bottom of the lagoon has a related cost of US$ 1.32–13.21
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per m3, with the variability dependent on the technological level employed for the treatment of this
fraction. The proposed procedure is therefore an economical and technically favorable alternative
for the management of this waste, as the costs involved relate only to the transference of NO3--N-
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rich wastewater to the sludge reservoir.

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4 Conclusion
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The approaches summarized in the present work are valuable strategies for process
maintenance and alternatives for the management of accumulated sludges. Additionally, sludges
with shorter deposition ages were proven to present higher TOC and N-NO3- removal rates,
suggesting that the best strategy would be to apply the denitrification process for sludge treatment
as early as possible to avoid the reduction of carbon bioavailability due to organic matter
degradation.

CRediT authorship contribution statement

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Heitor Henriques Guedes Mutchamua: Conceptualization; Formal analysis; Investigation,

Writing - original draft. Alice Chiapetti Bolsan: Conceptualization; Formal analysis; Investigation;
Writing - review & editing. Charline Bonatto: Conceptualization; Formal analysis; Investigation;
Writing - review & editing. Angélica Chini: Conceptualization; Formal analysis; Investigation;
Writing - review & editing. Bruno Venturin: Conceptualization; Formal analysis; Investigation;
Writing - review & editing. Camila Ester Hollas: Conceptualization; Formal analysis;
Investigation; Writing - review & editing. Gabriela Bonassa: Conceptualization; Formal analysis;
Investigation; Writing - review & editing. Fabiane Goldschmidt Antes: Conceptualization;
Investigation; Writing - review & editing. Helen Treichel: Conceptualization; Investigation;

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Writing - review & editing Marco Di Luccio: Conceptualization; Investigation; Writing - review &
editing Airton Kunz: Conceptualization; Funding acquisition; Investigation; Project administration,

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Writing - review & editing.
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Conflicts of Interest
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The authors declare no conflict of interest.

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Funding
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This research was funded by SISTRATES FUNTEC-BNDES (grant number 15.2.0837.1).

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Acknowledgements
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Authors thanks financial support from Centro Nacional de Aperfeiçoamento de Pessoal de

Nível Superior (CAPES), Conselho Nacional de Pesquisa (CNPq) and Projeto SISTRATES
FUNTEC-BNDES (grant number 15.2.0837.1). H.H.G.M. also thanks to the Government of
Mozambique, particularly the Ministry of Science and Technology, Higher Education and
Professional Technician, for granting the scholarship.

5 References

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1. Beccari, M., Passino, R., Ramadori, R., 1983. Kinetics of dissimilatory nitrate and nitrite reduction in
suspended growth culture. J. Water Pollut. Control Fed. 55, 58–64. www.jstor.org/stable/25041798
2. Bonassa, G., Bolsan, A.C., Hollas, C.E., Venturin, B., Candido, D., Chini, A., De Prá, M.C., Antes, F.G.,
Campos, J.L., Kunz, A., 2021. Organic carbon bioavailability: Is it a good driver to choose the best biological
nitrogen removal process? Sci. Total Environ. 786, 147390. https://doi.org/10.1016/j.scitotenv.2021.147390
3. Bortoli, M., Kunz, A., Prá, M.C. De, Silva, M.L.B. Da, Cé, A., Soares, H.M., 2019. Simultaneous removal of
nitrogen and organic carbon from swine wastewater using the pre-denitrification/nitrification process.
Ambient. e Agua - An Interdiscip. J. Appl. Sci. 14, 1. https://doi.org/10.4136/ambi-agua.2241
4. Forbis-Stokes, A.A., Miller, G.H., Segretain, A., Rabarison, F., Andriambololona, T., Deshusses, M.A., 2020.
Nutrient removal from human fecal sludge digestate in full-scale biological filters. Chemosphere 257, 127219.
https://doi.org/10.1016/j.chemosphere.2020.127219

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5. Grobelak, A., Grosser, A., Kacprzak, M., Kamizela, T., 2019. Sewage sludge processing and management in
small and medium-sized municipal wastewater treatment plant-new technical solution. J. Environ. Manage.

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234, 90–96. https://doi.org/10.1016/j.jenvman.2018.12.111
6. Hollas, C.E., Chini, A., Antes, F.G., do Prado, N. V., Bortoli, M., Kunz, A., 2019. Modified Ludzack–Ettinger
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system role in efficient nitrogen removal from swine manure under high total suspended solids concentration.
Int. J. Environ. Sci. Technol. 16, 7715–7726. https://doi.org/10.1007/s13762-019-02326-2
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7. Kunz, A., Bortoli, M., Miele, M., Steinmetz, R.L.R., Soares, H.M., 2015. Sistema de Tratamento de Efluentes-
SISTRATES. PI 1100464-9 A2.
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8. Kunz, A., Miele, M., Steinmetz, R.L.R., 2009. Advanced swine manure treatment and utilization in Brazil.
Bioresour. Technol. 100, 5485–5489. https://doi.org/10.1016/j.biortech.2008.10.039
9. Ouedraogo, F.R., Zhang, J., Cornejo, P.K., Zhang, Q., Mihelcic, J.R., Tejada-Martinez, A.E., 2016. Impact of
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sludge layer geometry on the hydraulic performance of a waste stabilization pond. Water Res. 99, 253–262.
https://doi.org/10.1016/j.watres.2016.05.011
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10. Owusu-Twum, M.Y., Sharara, M.A., 2020. Sludge management in anaerobic swine lagoons: A review. J.
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CRediT authorship contribution statement

Heitor Henriques Guedes Mutchamua: Conceptualization; Formal analysis; Investigation,

Writing - original draft. Alice Chiapetti Bolsan: Conceptualization; Formal analysis; Investigation;

Writing - review & editing. Charline Bonatto: Conceptualization; Formal analysis; Investigation;

Writing - review & editing. Angélica Chini: Conceptualization; Formal analysis; Investigation;

Writing - review & editing. Bruno Venturin: Conceptualization; Formal analysis; Investigation;

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Writing - review & editing. Camila Ester Hollas: Conceptualization; Formal analysis;

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Investigation; Writing - review & editing. Gabriela Bonassa: Conceptualization; Formal analysis;

Investigation; Writing - review & editing. Fabiane Goldschmidt Antes: Conceptualization;

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Investigation; Writing - review & editing. Helen Treichel: Conceptualization; Investigation;
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Writing - review & editing Marco Di Luccio: Conceptualization; Investigation; Writing - review &
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editing Airton Kunz: Conceptualization; Funding acquisition; Investigation; Project administration,

Writing - review & editing.

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Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal

relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:

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Figure 1. Schematic representation of the experiments conducted for the different tested

sludge. a) effect of TOC and nitrate on denitrification; b) denitrification rate as a function of TOC

and c) denitrification rate as a function of TOC and agitation.

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Figure 2. Response surfaces of the effect of TOC and nitrate concentration to remove (a)

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TOC.
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Figure 3. Response surface of the effect of carbon concentration and agitation on the
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denitrification process.
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Table 1. Chemical and physical properties of sludge used in denitrification assays.

Table 2. CCD 2² design matrix, with the coded and actual values of the independent

variables, and the experimental responses C/N, TOC and nitrate removal rate.

Table 3. CCD 2² design matrix to evaluate the influence of agitation and carbon content in

the denitrification processa.

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Sources of sludge
Parameter
ST CLD SL
BOD (mg L-1) 15725 ± 3336 11642 ± 2634 7760 ± 1427
TOC (mg L-1)a 2504 ± 354 – 5107 ± 794 520 ± 66 – 575 ± 98 260 ± 40 – 772 ± 109
TN (mgN L-1) 1733 ±172 1833 ± 156 959 ± 81
NH3 (mgN L-1) 1290 ± 91 1321 ± 106 783 ± 62
Alkalinity (mg CaCO3 L-1) 5284 ± 501 4190 ± 427 4233 ± 359
TS (g kg-1) 65 ± 3 29 ± 2 123 ± 7
-1
FS (g kg ) 18 ± 1 10 ± 1 66 ± 4

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VS (g kg-1) 47 ± 2 19 ± 1 57 ± 3

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Density (g L-1) 1.02 ± 0.06 1.02 ± 0.10 1.04 ± 0.04
pH 7.3 ± 0.8 7.0 ± 0.9 7.4 ± 0.9
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TOC values showed high variability according to the point and collection date, in the different
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systems studied. This variability is inherent to the microbiological process and the results are
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averages from the collected dates.

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TOC Nitrate TOC removal rate Nitrate removal

-1 -1 -1 -1
C/N
(mgTOC L ) (mgN L ) (mgTOC L d ) rate (mgN L-1 d-1)
772 (-1) 100 (-1) 34 22 1.5
5107 (1) 100 (-1) 1342 112 12
772 (-1) 300 (1) 15 35 0.4
5107 (1) 300 (1) 1168 279 4.2
a
2864 (0) 200 (0) 167 54 3.1
2864 (0)a 200 (0) 185 52 3.6
2864 (0)a 200 (0) 183 54 3.4

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a
Due to the heterogeneity of the samples, it was not possible to precisely adjust the orthogonality of

the factorial tests. Therefore, the tests were realized with 2864 ± 1172 mg TOC L-1 at the central

point (0).
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Homogenization TOC Nitrate consumption rate (mg L-1d-1)

-1 (without agitation) 8.86 (2,504) 111.8
1(two daily agitations) 8.86 (2,504) 108.7
-1 (without agitation) -1 (260) 23.8
1(two daily agitations) -1 (260) 33.8
0 (a daily agitation) 0 (543) 22.0
0 (a daily agitation) 0 (543) 21.4
0 (a daily agitation) 0 (543) 21.7
a
Due to the heterogeneity of the samples, it was not possible to precisely adjust the orthogonality of

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the factorial tests.

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Highlights

Denitrification is a promising alternative for sludge management from lagoons;

Sludge stabilization is not affected by agitation;

Sludge stabilization is affected by the carbon bioavailability and NO3- concentration;

With cost-effectiveness, 125 days are required to reach sludge stabilization.

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Graphics Abstract
Figure 1
Figure 2
Figure 3