energies
Article
Methane Production from Confectionery Wastewater Treated in
the Anaerobic Labyrinth-Flow Bioreactor
Marcin D˛ebowski 1, * , Marta Kisielewska 1 , Joanna Kazimierowicz 2
Citation: D˛ebowski, M.; Kisielewska,
M.; Kazimierowicz, J.; Zieliński, M.
Methane Production from
Confectionery Wastewater Treated in
the Anaerobic Labyrinth-Flow
Bioreactor. Energies 2023, 16, 571.
https://doi.org/10.3390/en16010571
Academic Editor: Seung Gu Shin
Received: 16 November 2022
Revised: 30 December 2022
Accepted: 30 December 2022
Published: 3 January 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Energies 2023, 16, 571. https://doi.org/10.3390/en16010571
and Marcin Zieliński 1
1 Department of Environmental Engineering, Faculty of Geoengineering, University of Warmia and Mazury in
Olsztyn, 10-719 Olsztyn, Poland
2 Department of Water Supply and Sewage Systems, Faculty of Civil Engineering and Environmental Sciences,
Bialystok University of Technology, 15-351 Bialystok, Poland
* Correspondence: marcin.debowski@uwm.edu.pl; Tel.: +48-89-523-4124
Abstract: Production and consumption of confectionery products have increased worldwide, thus, ef-
fective management of wastewater produced is now an important issue. The confectionery high-load
sewage was explored for biogas production in an innovative-design anaerobic reactor with labyrinth
flow. The experimental studies were focused on determining the best technological parameters of
anaerobic digestion for the effective removal of pollutants and obtaining high CH4 production effi-
ciency. It was found that organic loading rate (OLR) of 5.0–6.0 g COD/L·d contributed to the highest
CH4 generation of 94.7 ± 6.1 to 97.1 ± 5.1 L CH4 /d, which corresponded to a high COD removal
of 75.4 ± 1.5 to 75.0 ± 0.6%. Under such conditions the FOS/TAC ratio was below 0.4, indicating
reactor stability, and pH was on the level of 7.15 ± 0.04 at OLR 5.0 g COD/L·d and 7.04 ± 0.07 at
OLR 6.0 g COD/L·d.
Keywords: confectionery industry; wastewater treatment; anaerobic digestion; biogas; hybrid
anaerobic reactor
1. Introduction
The confectionery industry is a group of companies that produces various types of
sweets and products made from cocoa. It is one of the most widespread factories around the
world and manufactured 37 billion tons of products, which corresponded to 4% of the whole
food industry in 2017 [1]. There is constant and dynamic development in the confectionery
industry worldwide [2]. In global comparison, most revenue is generated in the United
States which amounted to USD 186.50 billion in 2022, and the global confectionery market
is expected to grow annually by 3.03% [3].
The increase in confectionery production and the variety produced directly affect
the quantity and quality of wastewater. The activity of confectionery plants requires a
considerable amount of freshwater used both in the production process and for equipment
washing, which generates large amounts of wastewater [4]. Wastewater is characterized
by daily and seasonal variations in composition and quantity which adversely affect the
disposal process [2]. Confectionery plants discharge about 300–500 m3 per month of
wastewater that predominantly consists of organic compounds and suspensions, such as
dissolved sugars, coarse impurities, emulsified fat particles, organic colloidal and solutes,
surfactants, and dyes as well as other chemical additives [5,6]. The values of chemical
oxygen demand (COD) vary from 2500 to 20,025 mg O2 /L, while biological oxygen de-
mand (BOD5 ) concentrations are up to 500–8000 mg O2 /L, which allows the classification
of this sewage as biodegradable [2,5,7]. Confectionery wastewater is comprised of low
nutrient concentrations (nitrogen and phosphorus compounds) at 30–120 mg N-NH4 /L
and 3.2–157 mg TP/L [2]. The confectionery sewage has a mostly acidic pH of five due to
the presence of readily soluble fermentable organic compounds [8]. Moreover, washing and
disinfecting substances in wastewater can cause changes in the pH values within the limits
https://www.mdpi.com/journal/energies
Energies 2023, 16, 571 2 of 18

of 3.2–9.5 [2]. A high load of organic compounds can create a problem with the treatment,
thus improper or insufficient treatment can enhance the processes of eutrophication and
degradation of natural water reservoirs when wastewater is discharged into them [9].
According to the literature, various methods are used to treat wastewater from the con-
fectionery industry, and due to the variety of production, there is no versatile method for its
treatment [2,10]. Effluents from small confectionery plants are usually discharged directly
into the municipal sewerage system where they are diluted by household wastewater and
then treated in a municipal sewage treatment plant [2]. However, this can cause some tech-
nological problems in treatment plants and is quite expensive [11]. Thus, it is recommended
to treat confectionery sewage at the place of its formation to meet standards for industrial
effluent discharge into the municipal sewerage system or natural water reservoirs [8]. Both
aerobic and anaerobic biological treatment systems are used to treat confectionery wastew-
ater [1,12]. Aerobic systems are generally based on the activated sludge process, however
high concentrations of readily biodegradable organic matter in confectionery wastewater
can cause rapid dissolved oxygen depletion in aeration tanks [5]. Another drawback of
this method is the need to manage wastewater sewage sludge, which is produced in large
amounts in aerobic treatment systems [13,14]. Thus, high-rate anaerobic technologies
are more feasible for the treatment of confectionery wastewater [1,10,15,16]. Nowadays,
high-rate anaerobic reactors are used for the treatment of different types of food-processing
industrial wastewater [17–21]. The successful application of anaerobic technology to the
treatment of industrial wastewater is mainly related to the resistance of anaerobic microflora
to a high load of organic compounds [16,22]. Moreover, high-rate anaerobic technologies are
characterized by low energy consumption, low excess sludge production, aerosol, and odor
spread reduction, and quick start-up even after a long outage in operation [23]. The most
important advantage of using anaerobic digestion to treat high-load industrial effluents is
biogas production, which is used for heating and electricity generation [24,25].
Different types of anaerobic reactors are used to treat confectionery wastewater and
upflow anaerobic sludge blanket (UASB) reactors are the most widely used [2,26,27]. A high
flow wastewater velocity and mixing resulting from biogas production ensure the granular
biomass formation at a concentration of approx. 80 g/L, which allows the treatment of high-
load confectionery wastewater with concentrations ranging from 0.5 to 16 g COD/L [28].
The major limitation of the use of the UASB reactor in the treatment of confectionery
wastewater is the need for pre-treating wastewater with a high content of suspended solids
and fats, which inhibit the development of granular sludge [29]. Long-term dynamic
performance of a full-scale anaerobic expanded granular sludge bed (EGSB) reactor treating
confectionery effluent was simulated by using Anaerobic Digestion Model No. 1 (ADM1) by
Dereli [1]. Although the author found good performance of EGSB reactor, it is unsuitable to
treat confectionery wastewater with high concentrations of fats and suspended solids [30].
Other types of most commonly used reactors for confectionery wastewater treatment are
down-flow anaerobic filters (DAFs) and up-flow anaerobic filters (UAFs) [31,32]. However,
the operational problems of AFs result from system clogging and the stability of filter
material that can be washed out from the reaction chamber, thus the reactors are operated
at relatively low OLR up to 10 kg COD/m3 ·d [33].
Problems with the operation of typical anaerobic reactors have prompted the search for
improvements to make them more effective and economical; therefore, hybrid reactors are
designed to combine the positive features of individual anaerobic reactors to provide more
effective treatment [34]. In most cases, hybrid reactors offer the separation of acidogenesis
and methanogenesis phases into two stages to enhance biogas production, because each
metabolic pathway requires specific conditions for a smooth and efficient operation [35].
In this study, an anaerobic labyrinth-flow bioreactor (AL-FB) was constructed as the com-
bination of UASB reactor with methanogenesis phase separation and a settling tank. The
reactor design enhanced the longer retention time of anaerobic biomass, provided a larger
exchange surface between the liquid and gas phases, and minimized problems related to
sludge flotation and preventing sludge loss.
 operation [35]. In this study, an anaerobic labyrinth-flow bioreactor (AL-FB) was con-
structed as the combination of UASB reactor with methanogenesis phase separation and
a settling tank. The reactor design enhanced the longer retention time of anaerobic bio-
mass, provided a larger exchange surface between the liquid and gas phases, and mini-
Energies 2023, 16, 571 3 of 18
mized problems related to sludge flotation and preventing sludge loss.
The aim of this work was to study the process of anaerobic digestion of wastewater
from a The
confectionery plant.was
aim of this work The
to experimental
study the process studies were focused
of anaerobic digestionon
of determining
wastewater the
technological parameters
from a confectionery of The
plant. anaerobic digestion
experimental for were
studies the effective
focused onremoval of pollutants
determining the
andtechnological
obtaining high methane production efficiency. The research was carried
parameters of anaerobic digestion for the effective removal of pollutantsout
andin two
stages, on a high
obtaining laboratory
methanescale, and then
production in the AL-FB
efficiency. operated
The research on a semi-technical
was carried scale.
out in two stages,
on a laboratory scale, and then in the AL-FB operated on a semi-technical scale.
2. Materials and Methods
2. Materials and Methods
2.1.2.1.
Design of ofthe
Design theStudy
Study
The study
The studywas
wasdivided
divided into twostages
into two stages(Figure
(Figure
1).1).

Figure 1. The
Figure study
1. The studyorganization.
organization.

In In stage
stage 1,1,batch
batchanaerobic
anaerobic biodegradability
biodegradability teststests were
wereperformed
performed in semi-continuous
in semi-continuous
respirometricreactors.
respirometric reactors.The
The experiment
experiment waswasdivided
divided into four
into series
four identified
series depending
identified depending
on an organic loading rate (OLR) per unit respirometer volume,
on an organic loading rate (OLR) per unit respirometer volume, as follows: series as follows: series 1: 2.01:
g 2.0 g
COD/L·d; series 2: 5.0 g COD/L·d; series 3: 7.0 g COD/L·d; series 4: 8.0 g COD/L·d. Stage
COD/L·d; series 2: 5.0 g COD/L·d; series 3: 7.0 g COD/L·d; series 4: 8.0 g COD/L·d. Stage 1
1 focused on selecting the most effective OLRs ensuring the highest efficiency of organic
focused
compoundon selecting
removal andthe efficient
most effective OLRs
biomethane (CHensuring the highest efficiency of organic
4 ) production.
compound removal and efficient biomethane (CH 4 ) production.
The selected value of the most effective OLR was the basis for determination of
The selected
technological value of the
parameters most
of the effective
AL-FB OLR
exploited in was
stagethe basispart
2. This forofdetermination
the research was of tech-
nological
focused on the anaerobic digestion efficiency assessment of confectionery wastewater inwas
parameters of the AL-FB exploited in stage 2. This part of the research a fo-
continuous
cused on the reactor
anaerobic on a digestion
semi-technical scale. The
efficiency changes inofconcentrations
assessment confectionery of wastewater
organic and in a
biogenic compounds were monitored during the study, and the indicators
continuous reactor on a semi-technical scale. The changes in concentrations of organic and characterizing
the methane
biogenic fermentation
compounds process, such
were monitored as pH,
during thevolatile
study,fatty
and acids (VFAs), the
the indicators ratio of
characterizing
free organic acids (FOS) to total alkaline capacity (TAC), as well as biogas production
the methane fermentation process, such as pH, volatile fatty acids (VFAs), the ratio of free
and composition.
organic acids (FOS) to total alkaline capacity (TAC), as well as biogas production and
composition.
2.2. Materials
Confectionery wastewater was collected from the confectionery plant producing
2.2.gingerbreads,
Materials jelly biscuits, shortbread biscuits, and biscuits. The average daily wastewater
flows were approximately
Confectionery 20 m3was
wastewater /day.collected
The physicochemical characteristicsplant
from the confectionery of confectionery
producing gin-
wastewater used in the study are presented in Table 1.
gerbreads, jelly biscuits, shortbread biscuits, and biscuits. The average daily wastewater
flows were approximately 20 m3/day. The physicochemical characteristics of confection-
ery wastewater used in the study are presented in Table 1.
Energies 2023, 16, 571 4 of 18

Table 1. Characteristics of confectionery wastewater used in the study (±std. dev., n = 96).

Parameter Units Value

COD mg O2 /L 11,200 ± 1700
BOD5 mg O2 /L 5100 ± 600
TOC mg/L 3750 ± 190
TN mg N/L 198 ± 27
N-NH4 mg N-NH4 /L 147 ± 14
TP mg P/L 19 ± 9
P-PO4 mg P-PO4 /L 14 ± 7
lipids mg/L 370 ± 120
suspended solids mg TS/L 890 ± 420
pH - 6.71 ± 0.16

The anaerobic granular sludge for respirometer inoculation was collected from a full-
size fruit and vegetable processing wastewater treatment plant. The operation parameters of
anaerobic digesters were: OLR of approx. 10 kg COD/m3 ·d, hydraulic retention time (HRT)
of 24 h, and temperature of 37 ◦ C. At the beginning of the batch anaerobic biodegradability
tests, the respirometers were inoculated with 500 mL of anaerobic granular sludge with
characteristics presented in Table 2. After inoculation, anaerobic granular sludge was
worked out over a period of 45 days to adapt the biomass to wastewater composition.

Table 2. Characteristics of anaerobic granular sludge used as inoculum in the study (±std. dev., n = 16).

Parameter Units Value

hydration % 97.3 ± 0.3
capillary suction time sec 680 ± 27
total solids g TS/L 44.6 ± 1.8
mineral solids g MS/L 15.8 ± 0.8
volatile solids g VS/L 28.7 ± 1.4
filtrate COD mg O2 /L 710 ± 80
filtrate P-PO4 mg P-PO4 /L 41.3 ± 7.2
filtrate TN mg TN/L 112 ± 13.7
filtrate N-NH4 mg N-NH4 /L 97 ± 9.2
pH - 7.42 ± 0.17

2.3. Experimental Station Construction and Exploitation in Stage 1

The Automatic Methane Potential Test System (AMPTS II, BPC Instruments AB,
Sweden) was used to evaluate the biogas potential of confectionery wastewater in stage 1.
The schematic of the experimental station is shown in Figure 2.
The AMPTS II has an automatic temperature-controlled water bath (sample incuba-
tor) for 15 parallel 500 mL respirometric reactors. The reactors were incubated at 35 ◦ C
(mesophilic conditions), while the process of anaerobic digestion was performed for 25 days.
The bioreactors were equipped with a methane recording system attached to the acquisition
system. The produced biogas was transferred by a capillary to a CO2 trap vessel containing
alkaline scrubbing solution (3M NaOH) for CO2 absorption. Then, the cleaned biogas was
sent to a volumetric measuring system to analyze CH4 concentrations. Biomethane concen-
trations were recorded automatically and converted to normal conditions (1013 hPa, 273 K,
dry gases). Since negative control samples were run in parallel to a primary experiment
and with the same procedures. The samples consisted of 200 g of the inoculum without the
substrate (confectionery wastewater).
 Energies 2023, 16, x FOR PEER REVIEW 5 of 20
Energies 2023, 16, 571 5 of 18

Flow cell array and DAQ unit

Sample incubation unit
Reactor volume: 1000 mL
Active volume: 500 mL
Dimensions: 59 × 34 × 28 cm
Temperature control: 35 ± 0.2 °C
Mixing of reactors: 50 rpm
Ex-situ CO2 absorption unit
Measurement resolution: 9 mL
CO2 trap bottles: 15
Detection capacity: up to 900 L
Volume of bottles: 100 mL
Measuring range: 1 to 6000 mL/h
Dimensions of unit: 44 × 30 × 6 cm
Dimensions: 51 x 44 x 18 cm
Absorption liquid: 3M NaOH
Housing: plastic
Absorption efficiency: >98%
Measuring precision: CV ≤ 1%

Figure2.2.Schematic
Figure Schematicdiagram
diagramofofthe
theexperimental
experimentalstation
stationininstage
stage1.1.

The
Theexperimental
AMPTS II hasseries were divided
an automatic according to the different
temperature-controlled water bathamounts of confec-
(sample incuba-
tionery
tor) forwastewater
15 paralleldosed
500 mLinto the respirometers,
respirometric which
reactors. Theinfluenced
reactors werethe hydraulic
incubatedretention
at 35 °C
time (HRT) asconditions),
(mesophilic well as determined
while thethe valueof
process ofanaerobic
OLRs (Table 3). Anaerobic
digestion conditions
was performed forin-
25
side
days. The bioreactors were equipped with a methane recording system attached togas
the respirometers were ensured by flushing the reaction chambers with nitrogen the
(15 min, 150 L/h).
acquisition system.Thereafter, the reactors
The produced biogaswere
was closed with abyrubber
transferred stopper
a capillary to and
a COconnected
2 trap ves-
to the mechanical agitators. Agitators of all respirometers were
sel containing alkaline scrubbing solution (3M NaOH) for CO 2 absorption. set to be 30 s on/600
Then, the s
off at 50 rpm
cleaned during
biogas the experimental
was sent to a volumetric studies to provide
measuring systemcontact between
to analyze inoculum and
CH4 concentrations.
wastewater without destroying the cells of microflora. The experiments
Biomethane concentrations were recorded automatically and converted to normal in stage 1condi-
were
performed in triplicate.
tions (1013 hPa, 273 K, dry gases). Since negative control samples were run in parallel to
a primary experiment and with the same procedures. The samples consisted of 200 g of
Table 3. Study organization and exploitation parameters in stage 1.
the inoculum without the substrate (confectionery wastewater).
The experimental series were divided according to the different amounts of confec-
OLR Working Volume of the COD Sewage Amount COD Load
Series HRT (h)
(g COD/L·d) tioneryReactor
wastewater
(mL) dosed into
(g O2the
/L) respirometers,
(mL/d) which influenced
(g/d) the hydraulic reten-
tion time (HRT) as well as determined the value of OLRs (Table 3). Anaerobic conditions
1 2.0 90 1.0 ± 0.15 133
2 5.0
inside the respirometers were ensured by flushing 220
the reaction chambers with54nitrogen
2.5 ± 0.38
gas (15 min,500 11.2 ± 1.7
150 L/h). Thereafter, the reactors were
3 7.0 310 closed with 3.5a±rubber
0.53 stopper39 and con-
4 8.0 nected to the mechanical agitators. Agitators of all
360 respirometers were
4.0 ± 0.61 set to be 30
33s on/600
s off at 50 rpm during the experimental studies to provide contact between inoculum and
wastewater without destroying the cells of microflora. The experiments in stage 1 were
2.4. Experimental Station Construction and Exploitation in Stage 2
performed in triplicate.
Anaerobic digestion of confectionery wastewater in stage 2 was performed in the
AL-FB with a vertical wastewater flow on a semi-technical scale. The total working volume
of the reactor was 110 L. The reactor was constructed in the form of three coaxially placed
tanks, where the inner tank (20 L) was a hydrolyzer and the middle container (80 L) served
as an acidogenesis and methanogenesis tank. In the external tank (10 L), sedimentation of
the anaerobic granular sludge and final clarification of purified wastewater took place. The
 Energies 2023, 16, 571 6 of 18

Energies 2023, 16, x FOR PEER REVIEW 7 of 20

reactor design enhanced the longer retention time of anaerobic biomass, a larger exchange
surface between the liquid and gas phases, and minimized problems related to sludge
flotation. The AL-FB was covered with a gas-tight cover which was flooded in the sewage
(as a water seal) equipped with a biogas inlet and a gas flow meter. The overall and detailed
construction of AL-FB is shown in Figure 3.

Biogas

Gas phase

25 110 Φ 200 110 25

50
Effluent

Fermentation tank
80 L

650 Hydrolizer 20 L

Clarifier 10 L

Recirculation pump

Sewage
dosing
Φ 470 pump

Figure 3. The AL-FB with vertical wastewater flows exploited on a semi-technical scale.

Wastewater flowed from the bottom of the reactor to the central part of the hydrolyzer.
The complete mixing course in the tank was maintained by a recirculation pump working
at a rate of 30 L/min, and the suction pipeline was 5 cm below the liquid level. In the upper
Figure 3. The AL-FB with vertical wastewater flows exploited on a semi-technical scale.
Energies 2023, 16, 571 7 of 18

part of the hydrolyzer, there was a wastewater outflow to the fermentation tank with a
piston flow character from the top to the bottom of the fermentation tank. In the external
tank, an upstream flow was forced. Clarified and treated wastewater was discharged
outside the reactor through the overflow.
The initial OLR was at the level of 5.0 g COD/L·d, which was established on the
results obtained in the first stage of the experiment in series 1, while the average daily
wastewater flow was 50 L/d. Confectionery wastewater from the retention tank was dosed
to the anaerobic reactor by a pump working with a capacity of 5 L/h. The retention tank
was equipped with a recirculation pump for mixing the wastewater. The pumps were
switched on 10 times a day in a time regime of 60 min wastewater dosing pump and
84 min recirculation pump. In subsequent experimental series, the applied OLRs were 6.0 g
COD/L·d (series 2) and 7.0 g COD/L·d (series 3). The study organization in stage 2 is
summarized in Table 4.

Table 4. Study organization and exploitation parameters in stage 2.

Working
OLR COD Sewage Amount Dosing Pump COD Load
Series Volume of the HRT (h)
(g COD/L·d) (g O2 /L) (L/d) Capacity (L/h) (g/d)
Reactor (L)
1 5.0 50 5.0 560 ± 85 53
2 6.0 110 11.2 ± 1.7 60 6.0 670 ± 102 44
3 7.0 70 7.0 780 ± 119 38

The AL-FB was heated by a water jacket consisting of a hot water storage tank with a
capacity of 40 L and an electric heater (2000 W). The circulation pump working at a flow
of 4 m3 /h was associated with the water jacket and was automatically switched on/off
to maintain the temperature inside the chamber of the reactor at the level of 35 ± 1 ◦ C. A
temperature sensor to control the work of the circulation pump was placed at the bottom
of the reactor. The AL-FB was also insulated with a 50 mm layer of mineral wool.

2.5. Analytical Methods

The parameters such as COD, total phosphorus (TP), total nitrogen (TN), ammonia
nitrogen (N-NH4 ), TP, and orthophosphates (P-PO4 ) were analyzed once a day using
cuvette tests of the DR 2800 spectrophotometer with mineralizer (HACH Lange, Düsseldorf,
Germany), total solids (TS), and volatile solids (VS) concentration by gravimetric method
(part of EPA Standard Method 2540), TOC using a TOC 1200 analyzer (Thermo Scientific,
Waltham, MA, USA). Determination of biochemical oxygen demand (BOD5 ) was carried
out according to PN-EN 1899-1. The pH in the reactor compartments was determined with
a VWR 1000 L pH meter (Germany). The ether extract was analyzed by the gravimetric
method by determining the total content of organic substances extractable with petroleum
ether (PN-86/C-0457/01). The FOS/TAC ratio was determined by a titration method
(Tritlab AT 1000, Hach, Düsseldorf, Germany). The concentration of lipids was determined
by Soxhlet extraction (B-323 Büchi, Flawil, Switzerland).
A digital gas flow meter (XFM17S, Aalborg Instruments & Controls, Inc., Orangeburg,
NY, USA) measured the instant flow rate and total biogas flow. The biogas composition
was analyzed every 24 h using a gas chromatograph (GC 7890A, Agilent). The device was
equipped with a thermal conductivity detector (TCD), two Hayesep Q columns (80/100
mesh), two molecular sieves columns (60/80 mesh), and a Porapak Q column (80/100).
The temperatures of the injection and detector ports were 150 ◦ C and 250 ◦ C, respec-
tively. Helium and argon were used as carrier gases at 15 mL/min flow. Additionally, the
biogas composition (CO2 and CH4 concentration) was analyzed with the GMF 430 Gas
Data analyzer.
Energies 2023, 16, 571 8 of 18

2.6. Statistical Methods

The Statistica 13.1 PL software package (StatSoft, Inc., Tulsa, OK, USA) was used for
the analysis. The homogeneity of variance in groups was determined using Levene’s test.
Tukey’s HSD test was applied to determine the significance of differences between the
series (p = 0.05).

3. Results and Discussion

Confectionery wastewater used in the study originating from the plant that produced
gingerbreads, jelly biscuits, shortbread biscuits, and biscuits is shown in Table 1. As can be
seen, it contained a high concentration of organic compounds (11,200 ± 1700 mg O2 /L as
COD and 5100 ± 600 mg O2 /L as BOD5 ), a TN value of 198 ± 27 mg/L, lipids concentration
on the level of 370 ± 120 mg/L, and a low content of phosphorus (14 ± 7 mg/L). A balanced
carbon-to-nitrogen (C/N) ratio is necessary for the optimization of methane production
during anaerobic digestion [36]. It was found that the C/N ratio between 20 and 35 is
recommended for maximizing methane generation [37,38]. In the study, the C/N ratio was
over 56, indicating the excess carbon content in the wastewater. According to the literature,
the excess carbon concentration may result in the accumulation of CO2 in the biogas [36,39].
In turn, the TOC/TN ratio was at the level of 18.9. According to the literature, the optimal
ratio of TOC/TN for anaerobic digestion is from 20:1 to 30:1 [40,41], however other authors
found that 15 was also suitable [42]. Confectionery wastewater used in the study had a low
content of dissolved matter and was characterized by a high concentration of suspended
solids (Table 1). The average pH was close to the neutral value of 6.71 ± 0.16, which was
favorable for anaerobic digestion.
The high organic content in confectionery sewage promoted the application of anaer-
obic digestion as a method of treatment. The experiments were performed in 500 mL
respirometric reactors and in semi-technical scaled AL-FB with a working volume of 110 L.
In stage 1, the respirometric reactors were operated at four different OLRs of 2.0, 5.0,
7.0, and 8.0 g COD/L·d. The application of increasing OLRs to respirometric reactors
directly influenced the reduction of its performance. COD reduction was found to be
higher at OLRs 2.0 g COD/L·d and 5.0 g COD/L·d, and respectively were 56.8 ± 4.6% and
54.6 ± 2.3% compared to OLRs of 7.0 g COD/L·d and 8.0 g COD/L·d (Figure 4, Table 5). A
similar relationship was noted for BOD5 and TOC removal (Figure 4, Table 5). However, the
highest organic load removal of 1.37 ± 0.04 g COD/d was noted at OLR of 5.0 g COD/L·d,
which was over two times higher than at the lowest OLR (Table 5). The FOS/TAC ratio
was found to be less than 0.4 for OLRs (0.33 in series 1 and 0.37 in series 2) indicating the
presence of a proper buffering capacity in the reactor chambers, [43,44]. The highest CH4
production of 0.34 ± 0.014 L CH4 /d (0.25 ± 0.010 L CH4 /g COD removed) was achieved
when OLR was 5.0 g COD/L·d (Table 5, Figure 4). There was a significant decrease in CH4
generation (0.25 ± 0.019 L CH4 /d) when the OLR increased to 7.0 g COD/L·d (Table 5). The
pH value in series 1–3 was from 6.73 ± 0.09 in series 3 to 6.94 ± 0.17 in series 1, which was
within the optimum range for methanogens (pH 6.6–7.0) [45,46]. The increase in OLR in
series 4 led to a decrease in pH to the average level of pH 6.31 ± 0.11, growth in FOS/TAC
ratio to 0.51, and a reduction in biogas production to the level of 0.18 ± 0.009 L CH4 /d
(0.16 ± 0.008 L CH4 /g COD removed) (Table 5, Figure 4).
Based on the results from stage 1, biogas productivity, COD removal efficiency, and
organic load removal were the highest at OLR of 5.0 g COD/L·d. Thus, in the second stage
of the experiment, the AL-FB reactor was initially fed at 5.0 g COD/L·d, and then gradually
increased to 6.0 and 7.0 g COD/L·d.
 ± 0.019 L CH4/d) when the OLR increased to 7.0 g COD/L·d (Table 5). The pH value in
series 1–3 was from 6.73 ± 0.09 in series 3 to 6.94 ± 0.17 in series 1, which was within the
optimum range for methanogens (pH 6.6–7.0) [45,46]. The increase in OLR in series 4 led
to a decrease in pH to the average level of pH 6.31 ± 0.11, growth in FOS/TAC ratio to 0.51,
Energies 2023, 16, 571 and a reduction in biogas production to the level of 0.18 ± 0.009 L CH4/d (0.169 ±of0.008
18 L
CH4/g COD removed) (Table 5, Figure 4).

100 1
y = ‒0.026x + 0.275
Organic compounds removal (%)

y = ‒10.652x + 70.313 y = ‒8.1961x + 79.02 y = ‒0.0772x + 0.5366

Methane (L CH4/g removed)

90
R² = 0.9235 R² = 0.8451 R² = 0.7348 R² = 0.8727
80 0.8
y = ‒10.08x + 73.067 y = ‒0.0894x + 0.7953
70 R² = 0.8318 R² = 0.8139
60 0.6
50
40 0.4
30
20 0.2
10
0 0
1 2 3 4 1 2 3 4
Series Series
COD BOD5 TOC COD BOD5 TOC

(a) (b)
Figure 4. The efficiency of anaerobic confectionery wastewater treatment in stage 1: (a) organic
Figure 4. The efficiency of anaerobic confectionery wastewater treatment in stage 1: (a) organic
compound removal; (b) CH4 production per g of organic substances removed (b).
compound removal; (b) CH4 production per g of organic substances removed (b).

Table 5.The
Removal of organic
efficiency compounds
of organic as COD,
compound BODdetermines
removal 5, TOC, and the
production
yield ofof CH4 in stage
methane pro- 1.
duced and generally, a higher COD removal indicates aCH better conversion of organic
4 Production
COD
compounds into biogas components [47]. In stage 2, the highest organic compound re-
OLR
Series moval of aboutInfluent75% as CODLoad was obtained at OLRs ranging from 5.0
4/gto 6.0 g COD/L·d,
(g COD/L·d) Influent Effluent Removal Load Removal
(L CH 4/g COD (L CH COD
(L CH4/d)
(g O2/L) (g O2/L)and at OLR
(%) of 7.0 g COD/L · d it dropped to 61.6 ± 4.4%
introduced) (Table 6,removed) The obtained
Figure 5).
(g COD/d) (g COD/d)
removals were lower than the performances achieved by other researchers. For exam-
2.0 4.84 ± 0.39 56.8 and
ple, Beal ± Raman1.0 ± 0.15[16] 0.57 ± 0.05 a sequential
exploited 0.13 ± 0.011 0.23 ± 0.019
two-stage anaerobic treatment 0.13 ± 0.010
system
1
4.6
of confectionery wastewater consisting of an upflow anaerobic sludge blanket (UASB)
5.0 5.08 ± 0.21
reactor54.6
and± a downflow
2.5 ± 0.38 anaerobic
1.37 ± 0.04 0.14found
filter. They ± 0.007
that the UASB 0.25reactor
± 0.010achieved 0.34 ± 0.014
COD
2
11.2 ± 2.3
removal of 98%, while the downflow filter achieved about 50%. The two-stage anaerobic
7.0 1.7 7.20 ± 0.54
system 35.7 ± 3.5 ±COD
enhanced 0.53 removal
1.25 ± 0.09to an overall0.07 treatment
± 0.005 0.20 of
efficiency ± 0.015
98% as COD 0.25at±an
0.019
3
overall2.7OLR of 12.5 g/L·d. Balcıoğlu et al. [31] reported 99% of COD removal efficiency at
8.0 OLRs27.6
8.11 ± 0.42 ranging
± 4.0 from 1.1 to 7.9
± 0.61 0.03 ·d in0.04
1.10g ±COD/L an Anaerobic
± 0.002 Membrane0.16 ±Bioreactor
0.008 (AnMBR).
0.18 ± 0.009
4
The AnMBR1.4 is an integrated system of the anaerobic reactor and the low-pressure ultra-
filtrationBODor microfiltration
5 membrane filtration, in which CH membranes
4 production retain suspended
OLR solids, thus in the AnMBR system, Load a complete separation of the solid retention time from
Series Influent Effluent Removal Influent load (L CH4/g BOD5 (L CH4/g BOD5
(g BOD5/L·d) the hydraulic retention time is achieved, enhancing an overall COD removal [48].
removal (L CHIn4/d)
(g O2/L) (g O2/L) (%) (g BOD5/d) introduced) removed)
turn, 85.46% COD removal(gwas BOD 5/d)
achieved during anaerobic treatment with the application
1 0.9 1.65 ± 0.22 67.6 ± 0.46
of ultrasound ± 0.05 0.31
pretreatment ± 0.04
[39]. 0.29 ± 0.038
The pretreatment promotes the 0.42disintegration
± 0.057 0.13 ± 0.010
of com-
plex structure
9.0 molecules, which were easier degraded by anaerobic microflora [49]. The
5.1 ± 0.6
2 2.3 treatment
1.58 ± 0.13 69.0 ±of wastewater
1.14 ± 0.13 containing
0.79 ± 0.06sugars0.30 by the UASB reactor0.43
± 0.025 was±also
0.036 investigated
0.34 ±by0.014
Tanksali 5.7et al. [50]. The COD removal efficiency was obtained from 80% to 96% within the
values of OLR between 2 g COD/L·d to 4.67 g COD/L·d.
Energies 2023, 16, 571 10 of 18

Table 5. Removal of organic compounds as COD, BOD5 , TOC, and production of CH4 in stage 1.

COD CH4 Production

OLR Load
Series (g COD/L·d) Influent Effluent Influent Load (L CH4 /g COD (L CH4 /g COD
Removal (%) Removal (L CH4 /d)
(g O2 /L) (g O2 /L) (g COD/d) Introduced) Removed)
(g COD/d)
1 2.0 4.84 ± 0.39 56.8 ± 4.6 1.0 ± 0.15 0.57 ± 0.05 0.13 ± 0.011 0.23 ± 0.019 0.13 ± 0.010
2 5.0 5.08 ± 0.21 54.6 ± 2.3 2.5 ± 0.38 1.37 ± 0.04 0.14 ± 0.007 0.25 ± 0.010 0.34 ± 0.014
11.2 ± 1.7
3 7.0 7.20 ± 0.54 35.7 ± 2.7 3.5 ± 0.53 1.25 ± 0.09 0.07 ± 0.005 0.20 ± 0.015 0.25 ± 0.019
4 8.0 8.11 ± 0.42 27.6 ± 1.4 4.0 ± 0.61 1.10 ± 0.03 0.04 ± 0.002 0.16 ± 0.008 0.18 ± 0.009
BOD5 CH4 production
OLR Load
Series (g BOD5 /L·d) Influent Effluent Influent Load (L CH4 /g BOD5 (L CH4 /g BOD5
Removal (%) Removal (L CH4 /d)
(g O2 /L) (g O2 /L) (g BOD5 /d) Introduced) Removed)
(g BOD5 /d)
1 0.9 1.65 ± 0.22 67.6 ± 9.0 0.46 ± 0.05 0.31 ± 0.04 0.29 ± 0.038 0.42 ± 0.057 0.13 ± 0.010
2 2.3 1.58 ± 0.13 69.0 ± 5.7 1.14 ± 0.13 0.79 ± 0.06 0.30 ± 0.025 0.43 ± 0.036 0.34 ± 0.014
5.1 ± 0.6
3 3.2 2.49 ± 0.36 51.2 ± 7.4 1.59 ± 0.19 0.82 ± 0.12 0.16 ± 0.023 0.31 ± 0.044 0.25 ± 0.019
4 3.6 2.74 ± 0.38 46.3 ± 6.4 1.82 ± 0.21 0.84 ± 0.11 0.10 ± 0.013 0.21 ± 0.029 0.18 ± 0.009
TOC CH4 Production
OLR Load
Series (g TOC/L·d) Influent Effluent Influent Load (L CH4 /g TOC (L CH4 /g TOC
Removal (%) Removal (L CH4 /d)
(g/L) (g/L) (g TOC/d) Introduced) Removed)
(g TOC/d)
1 0.7 1.52 ± 0.14 59.5 ± 5.5 0.33 ± 0.017 0.20 ± 0.02 039 ± 0.04 0.66 ± 0.06 0.13 ± 0.010
2 1.7 1.47 ± 0.21 60.8 ± 8.7 0.84 ± 0.042 0.51 ± 0.07 0.41 ± 0.06 0.67 ± 0.10 0.34 ± 0.014
3.75 ± 0.2
3 2.3 2.34 ± 0.18 37.6 ± 2.9 1.17 ± 0.059 0.44 ± 0.03 0.21 ± 0.02 0.57 ± 0.04 0.25 ± 0.019
4 2.7 2.49 ± 0.27 33.6 ± 3.6 1.34 ± 0.068 0.45 ± 0.05 0.13 ± 0.01 0.39 ± 0.04 0.18 ± 0.009
Energies 2023, 16, 571 11 of 18

Table 6. Removal of organic compounds such as COD, BOD5 , TOC, and production of biogas and CH4 in stage 2.

Biogas
COD CH4 Production
Production
OLR
Series (g COD/L·d) Load
Influent Effluent Influent Load (L CH4 /g COD
Removal (%) Removal (L/d) (L CH4 /d)
(g O2 /L) (g O2 /L) (g COD/d) Removed)
(g COD/d)
1 5.0 2.76 ± 0.17 75.4 ± 1.5 560 ± 85 422 ± 8.4 135.0 ± 12.6 0.22 ± 0.01 94.7 ± 6.1
2 6.0 11.2 ± 1.7 2.80 ± 0.06 75.0 ± 0.6 670 ± 102 503 ± 4.0 145.8 ± 10.0 0.19 ± 0.01 97.1 ± 5.1
3 7.0 4.30 ± 0.48 61.6 ± 4.4 780 ± 118 480 ± 32.1 129.6 ± 9.6 0.16 ± 0.01 76.4 ± 4.8

BOD5 Biogas CH4 Production

Production
OLR
Series (g BOD5 /L·d) Load
Influent Effluent Removal Influent Load (L CH4 /g BOD5
Removal (L/d) (L CH4 /d)
(g O2 /L) (g O2 /L) (%) (g BOD5 /d) Removed)
(g BOD5 /d)
1 2.3 0.82 ± 0.03 83.9 ± 2.7 250 ± 29 210 ± 6.7 135.0 ± 12.6 0.45 ± 0.04 94.7 ± 6.1
2 2.7 5.1 ± 0.6 0.91 ± 0.04 82.2 ± 3.6 300 ± 35 247 ± 10.8 145.8 ± 10.0 0.39 ± 0.02 97.1 ± 5.1
3 3.2 1.49 ± 0.13 70.7 ± 6.2 350 ± 41 248 ± 21.7 129.6 ± 9.6 0.31 ± 0.02 76.4 ± 4.8

TOC Biogas CH4 Production

Production
OLR
Series (g TOC/L·d) Load
Influent Effluent Influent Load (L CH4 /g TOC
Removal (%) Removal (L/d) (L CH4 /d)
(g O2 /L) (g O2 /L) (g TOC/d) Removed)
(g TOC/d)
1 1.7 0.85 ± 0.04 77.4 ± 3.4 190 ± 9.6 148 ± 6.5 135.0 ± 12.6 0.64 ± 0.04 94.7 ± 6.1
2 2.0 3.75 ± 0.2 0.93 ± 0.06 75.2 ± 5.2 220 ± 11.1 165 ± 11.4 145.8 ± 10.0 0.59 ± 0.03 97.1 ± 5.1
3 2.3 1.32 ± 0.12 64.9 ± 8.7 250 ± 12.7 162 ± 20.1 129.6 ± 9.6 0.47 ± 0.02 76.4 ± 4.8
Energies 2023, 16, x FOR PEER REVIEW 12 of 20

Energies 2023, 16, 571 12 of 18

100 85
y = ‒6.95x + 84.533
90 R² = 0.7716 80

COD removal (%)

80
COD removal (%)

70 75
60 70
50
40 65
30 60
20
55
10
0 50
1 2 3 0 2 4 6 8 10 12 14 16 18 20 22 24
Series Time (weeks)
series 1 series 2 series 3
(a) (b)
Figure 5. 5.The
Figure Theefficiency
efficiency of anaerobicconfectionery
of anaerobic confectionery wastewater
wastewater treatment
treatment in2:stage
in stage 2: (a) average
(a) average COD
COD removal with the standard deviation; (b) COD removal during experimental
removal with the standard deviation; (b) COD removal during experimental time. time.

Table 6.InRemoval
the study, of any
organic compounds
pretreatment such
was as to
used COD, BOD5COD
enhance , TOC,removal.
and production of biogas and
As the sewage
CHwas
4 in rich
stagein2.suspended solids, a relatively low removal of organic compounds could be
attributed to their poor decomposition. According to Ramanathan et al. [51], particulate
COD Biogas Production CH4 Production
organic material limited the hydrolysis stage of anaerobic digestion. Moreover, lipids,
OLR which are usually Influent
abundant in Load
confectionery wastewater, may also be the cause. Some
Series
(g COD/L·d) Influent Effluent Removal
Load Removal (L/d)
(g O2/L) authors
(g O2/L) noted
(%) that lipid hydrolysis is the rate-limiting step(L
ofCH
the4/g COD removed)
overall (L CH4/d)
anaerobic digestion
(g COD/d) (g COD/d)
process and the rate of lipid hydrolysis is affected by their chemical and physical properties
5.0 2.76 ± (i.e.,
0.17 degree
75.4 ± of un-saturation,
560 ± 85 422fluidity,
± 8.4 available
135.0 ± 12.6
surface area,0.22 ± 0.01size) [52,53].
particle 94.7For
± 6.1
1
1.5
example, saturated lipids and long-chain fatty acids (LCFA) in mesophilic temperatures are
6.0 11.2 ± 2.80 ± in
0.06
the 75.0
solid±phase,670therefore
± 102 503
their± availability
4.0 145.8
to ±microorganisms
10.0 0.19 ± 0.01 [54,55]. The
is limited 97.1size
± 5.1
2
1.7 of LCFA0.6 also affects its bioavailability [56]. In addition, it was found that LCFAs suppress
7.0 4.30 ± microbial
0.48 61.6 activity
± 780 by adsorbing
± 118 onto the129.6
480 ± 32.1 cells±of9.6biomass limiting
0.16 ± 0.01access to substrate
76.4 ± 4.8
3 and nutrients [56].
4.4
When BODOLR
5 was maintained at 5.0–6.0 g COD/L·d, a FOS/TAC
Biogas production ratio was below
CH4 production
OLR 0.4, indicating reactor stability (Figure 6). The average pH in the chamber of the reactor
Load
Series Influent Effluent Removal Influent load
(g BOD5/L·d) was on the level of 7.15 ± 0.04 at OLR 5.0 (L/d)
removal g COD/L·d, and
(L CH BOD±
4/g7.04 0.07 at OLR(L6.0
5 removed) CHg 4/d)
(g O2/L) (g O2/L) (%) (g BOD5/d)
COD/L·d (Figure 6). Under(gsuch BODconditions,
5/d) biogas production achieved 145.8 ± 10.0 L/d
1 2.3 0.03 ±83.9
0.82 ± (0.29 0.02± L/g250COD ± 29removed)
210 ± in6.7series135.0 12.6 ± 12.6 L/d
2 and± 135.0 ± 0.04± 0.03 L/g94.7
0.45 (0.32 COD ± 6.1
removed) 2.7series 1 (Table 6, Figure 7). The highest daily methane production of 97.1 ± 5.1 L
2 2.7 0.91 ± CH
0.044 was
82.2obtained
± 300when
± 35 OLR 247was maintained
± 10.8 145.8 at OLR 6.0 g COD/L
± 10.0 0.39 ±·d0.02(Table 6). However,
97.1 ± 5.1
5.1 ± 0.6 the highest CH concentration in biogas of 70.1 ± 0.7% was at the lowest OLR (Figure 7).
3.6 4
3 3.2 1.49 ± An
0.13increase
70.7 ± in OLR350 ± to
417.0 g248 ± 21.7 ·d resulted
COD/L 129.6 ± in
9.6a significant0.31reduction
± 0.02 in biogas 76.4and
± 4.8
CH4 production,
6.2 the pH value dropped to 6.37 ± 0.17, while the FOS/TAC ratio increased
to 0.48, which
TOC
indicates the accumulation of volatile fatty acids in a CH
Biogas production
reactor chamber and
4 production

OLR overloading the reactor (TableLoad 6, Figures 6 and 7) [43,57,58].

Series Influent Effluent Removal Influent load
(g TOC/L·d) removal (L/d) (L CH4/g TOC removed) (L CH4/d)
(g O2/L) (g O2/L) (%) (g TOC /d)
(g TOC /d)
1 1.7 0.85 ± 0.04 77.4 ± 190 ± 9.6 148 ± 6.5 135.0 ± 12.6 0.64 ± 0.04 94.7 ± 6.1
3.4
2 2.0 3.75 ± 0.93 ± 0.06 75.2 ± 220 ± 11.1 165 ± 11.4 145.8 ± 10.0 0.59 ± 0.03 97.1 ± 5.1
0.2 5.2
3 2.3 1.32 ± 0.12 64.9 ± 250 ± 12.7 162 ± 20.1 129.6 ± 9.6 0.47 ± 0.02 76.4 ± 4.8
8.7

In the study, any pretreatment was used to enhance COD removal. As the sewage
was rich in suspended solids, a relatively low removal of organic compounds could be
attributed to their poor decomposition. According to Ramanathan et al. [51], particulate
organic material limited the hydrolysis stage of anaerobic digestion. Moreover, lipids,
 Energies 2023, 16, x FOR PEER REVIEW 14 of 20
Energies 2023, 16, 571 13 of 18

8.0 7.4
y = ‒0.39x + 7.6333
7.5 7.2
R² = 0.8534
7.0 7.0
6.5

pH
6.8
pH

6.0
6.6
5.5
6.4
5.0
4.5 6.2

4.0 6.0
1 2 3 0 2 4 6 8 10 12 14 16 18 20 22 24
Series Time (weeks)
series 1 series 2 series 3

(a) (b)
0.6

0.5
FOS/TAC

0.4

0.3

0.2
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (weeks)
series 1 series 2 series 3
(c)
Figure6.6.The
Figure Theefficiency
efficiencyof
ofanaerobic
anaerobicconfectionery
confectionerywastewater
wastewatertreatment
treatmentininstage
stage2:2:(a)
(a)average
averagepHpH
with the standard deviation; (b) pH in a chamber during experimental time; (c) FOS/TAC ratio in aa
with the standard deviation; (b) pH in a chamber during experimental time; (c) FOS/TAC ratio in
chamber during experimental time.
chamber during experimental time.

The treatment of confectionery0.34 wastewater was also studied by Balcıoğlu et al. [31].
0.40
They operated an anaerobic membrane bioreactor (AnMBR) at various OLRs of 1.1, 2.2, 4.4,
Biogas (L/g COD removed)

0.35 y = ‒0.025x + 0.3433 0.33

6.6, and 7.9 g COD/L ·d. The maximum methane production of 0.26 L/g COD removed was
Biogas (L/g COD removed)

R² = 0.9868
0.32
0.30 achieved at OLR 7.9 g COD/L·d, which was lower than the values obtained in the study
0.25 (Table 6). In turn, the average biogas0.31 production in the EGSB reactor treating confectionery
wastewater achieved 1730 m3 /d and0.30 88% of COD removal at OLR 2.9 ± 0.8 g COD/L·d [12].
0.20
Rusín et al. [59] obtained the biogas production from confectionery wastes (waste wafer
0.29
0.15
material) of 0.703 L/g volatile solids removed with an average methane content in biogas
0.28
0.10 of 55.1% at the average load of 8.230.27 g volatile solids/L·d.
0.05 During anaerobic digestion, the complex compounds of nutrients in the sewage were
0.26
0.00
decomposed into simple particles 0.25 that could be easily absorbed by microorganisms. It
1 was observed
2 that in series
3 1 and 2, the concentration
0 2 4 6 8 of10total 12 nitrogen
14 16 18 in
20digestate
22 24 was on
Series
the level of 170–172 mg/L, while in series 3 it was higher (188.7
Time (weeks) ± 5.2 mg/L) (Figure 8).
There were no differences in phosphorusseries contents
1 in digestate
series 2 in all series
series3 (Figure 8).
During anaerobic digestion, the removal of nitrogen and phosphorus from sewage is
(a) (b)
low and mainly related to the biomass cells growing [60]. Thus, generally, the biological
degradation of the organic matter leads to an increase in the concentration of soluble
nutrient species in digestate, in the form of ammonia nitrogen and orthophosphate [61].
 series 1 series 2 series 3
(c)
Figure 6. The efficiency of anaerobic confectionery wastewater treatment in stage 2: (a) average pH
with the standard deviation; (b) pH in a chamber during experimental time; (c) FOS/TAC ratio in a
Energies 2023, 16, 571 14 of 18
chamber during experimental time.

0.40 0.34

Biogas (L/g COD removed)

0.35 y = ‒0.025x + 0.3433 0.33
Biogas (L/g COD removed)

R² = 0.9868
0.32
0.30
0.31
0.25
0.30
0.20
0.29
0.15
0.28
0.10 0.27
0.05 0.26
0.00 0.25
1 2 3 0 2 4 6 8 10 12 14 16 18 20 22 24
Energies 2023, 16, x FOR PEER REVIEW Series Time (weeks) 15 of 20

series 1 series 2 series 3

(a) (b)
74 74
72 72
70 y = ‒5.6063x + 76.438 70
68 R² = 0.9561
68
Methane (%)

66
Methane (%)

66
64
64
62
62
60
60
58
56 58
54 56
52 54
50 52
1 2 3 0 2 4 6 8 10 12 14 16 18 20 22 24
Series Time (weeks)
series 1 series 2 series 3
(c) (d)
Figure 7.
Figure 7. The
Theefficiency
efficiencyofofanaerobic
anaerobicconfectionery
confectionerywastewater
wastewatertreatment
treatmentin in
stage 2: (a)
stage average
2: (a) bi-
average
ogas production per g of COD removed with the standard deviation; (b) biogas production
biogas production per g of COD removed with the standard deviation; (b) biogas production per per g of
COD removed during experimental time; (c) CH4 concentration in biogas with the standard devia-
g of COD removed during experimental time; (c) CH4 concentration in biogas with the standard
tion; (d) CH4 concentration in biogas during experimental time.
deviation; (d) CH4 concentration in biogas during experimental time.
The results
The treatment of confectionery
of this work could bewastewater was also the
taken for optimizing studied by Balcıoğlu
operational et al.of[31].
conditions the
They operated an anaerobic membrane bioreactor (AnMBR) at various
full-scale plant and also as a starting basis for scaling up the process to the industrial OLRs of 1.1,scale.
2.2,
4.4, 6.6,
Due andhigh
to the 7.9 g organic
COD/L·d. The maximum
content methaneeffluents,
of confectionery production theofnew
0.26 L/g COD removed
construction of the
anaerobic reactor is suitable to treat such kinds of sewage. Anaerobic digestion takesin
was achieved at OLR 7.9 g COD/L·d, which was lower than the values obtained the
place
study
in four(Table 6). Inphases
metabolic turn, the average biogas
(hydrolysis, production
acidogenesis, in the EGSBand
acetogenesis, reactor treating con-
methanogenesis),
fectionery wastewater achieved 1730 m 3/d and 88% of COD removal at OLR 2.9 ± 0.8 g
and each of them requires specific conditions for a smooth and efficient run [62,63]. The
COD/L·d
most [12]. Rusín
popular reactorset al.
used [59]inobtained
full-scalethe tobiogas production from
treat confectionery confectionery
sewage wastes
are single-stage
(waste wafer material) of 0.703 L/g volatile solids removed with
tank biogas reactors, such as UASB and EGSB reactors, in which all phases of anaerobic an average methane con-
tent in biogas of 55.1% at the average load of 8.23 g volatile solids/L·d.
digestion are conducted in parallel in one digestion chamber. The disadvantage of using
During tank
single-stage anaerobic digestion,
reactors the complex
is the possibility compounds of
of destabilizing thenutrients in the sewage
process when treating were
high
decomposed
loads into substrate
of organic simple particles
due tothat could be easily
acidification absorbed
[1]. This can leadby microorganisms.
to process upsets It was
and
observed thatdowntimes
considerable in series 1 and[58].2,Inthetheconcentration
AL-FB, individual of total nitrogen
phases in digestate
of anaerobic was ontake
digestion the
level of 170–172 mg/L, while in series 3 it was higher (188.7 ± 5.2
place in separate chambers which promotes the treatment of highly loaded confectionery mg/L) (Figure 8). There
were no differences
wastewater. in phosphorus
In the study, contents in
biogas production was digestate in all series
high regardless (Figure
of OLRs, 8). though
even During
anaerobic
the digestion,was
COD removal thelower
removal than of noted
nitrogen by and
otherphosphorus
researchersfrom usingsewage is low tank
single-stage and
mainly related
anaerobic to theAccording
reactors. biomass cells to thegrowing [60].the
literature, Thus, generally,
separation the biological
of metabolic degrada-
pathways of
tiondigestion
the of the organic matter
enhanced to leads
produce to an increase
biogas, in thethe
because concentration
products of of thesoluble nutrient
hydrolysis could spe-
be
cies in digestate,
added dynamically in the form
to the of ammoniastage
methanogenic nitrogen and orthophosphate [61].
[1,64,65].

200 200
190 y = 9.3x + 158.27
R² = 0.8179 195
180
170 190
/L)

160 185
Energies 2023, 16, x FOR PEER REVIEW 16 of 20
Energies 2023, 16, 571 15 of 18

200 200
190 158.27x + 9.3y =
0.8179 =² R 195
180
170 190

TN (mg/L)
160 185
TN (mg/L)

150
140 180
130 175
120
110 170
100
165
90
80 160
1 2 3 0 2 4 6 8 10 12 14 16 18 20 22 24
Series Time (weeks)

series 1 series 2 series 3

(a) (b)

16 16
y = x + 9.02 15
14 R² = 0.9945 14
12 13
TP (mg/L)

12
10
TP (mg/L)

11
8 10
9
6
8
4 7
6
2
5
0 4
1 2 3 0 2 4 6 8 10 12 14 16 18 20 22 24
Series Time (weeks)
series 1 series 2 series 3
(c) (d)
Figure 8. The efficiency of anaerobic confectionery wastewater treatment in stage 2: (a) average total
Figure 8. The efficiency of anaerobic confectionery wastewater treatment in stage 2: (a) average total
nitrogen (TN) concentration with the standard deviation; (b) total nitrogen (TN) concentration dur-
nitrogen (TN) concentration with the standard deviation; (b) total nitrogen (TN) concentration during
ing experimental time; (c) total phosphorus (TP) concentration with the standard deviation; (d) total
experimental
phosphorus time;
(TP) (c) total phosphorus
concentration (TP) concentration
during experimental time. with the standard deviation; (d) total
phosphorus (TP) concentration during experimental time.
The results of this work could be taken for optimizing the operational conditions of
4. Conclusions
the full-scale plant and also as a starting basis for scaling up the process to the industrial
A high content of organic compounds in confectionery wastewater promotes the
scale. Due to the high organic content of confectionery effluents, the new construction of
application of anaerobic digestion as a method of treatment. Moreover, biogas production
the anaerobic reactor is suitable to treat such kinds of sewage. Anaerobic digestion takes
is a benefit that can be used as a source of renewable energy. The experimental studies were
place in four metabolic phases (hydrolysis, acidogenesis, acetogenesis, and methanogen-
focused on determining the technological parameters of anaerobic digestion for effective
esis), and each of them requires specific conditions for a smooth and efficient run [62,63].
organic compound removal with simultaneous high methane production efficiency.
The most popular reactors used in full-scale to treat confectionery sewage are single-stage
The research was carried out in two stages. In the first stage, respirometric reactors
tank biogas reactors, such as UASB and EGSB reactors, in which all phases of anaerobic
were used in batch anaerobic biodegradability tests to select the most effective OLR. There
digestion are conducted in parallel in one digestion chamber. The disadvantage of using
were identified four series depending on OLR ranging from 2.0 g COD/L·d to 8.0 g
single-stage tank reactors is the possibility of destabilizing the process when treating high
COD/L·d. On the basis of the results from stage 1, biogas productivity, COD removal
loads of organic substrate due to acidification [1]. This can lead to process upsets and
efficiency, and organic load removal were the highest at OLR of 5.0 g COD/L·d.
considerable downtimes [58]. In the AL-FB, individual phases of anaerobic digestion take
In the second stage, the anaerobic labyrinth-flow bioreactor with innovative con-
place in separate
struction chambersvolume
and a working which promotes the treatment
of 110 L was operated ofonhighly loaded confectionery
a semi-technical scale. The
wastewater.
reactor was In the study,
initially fed atbiogas production
5.0 g COD/L ·d, andwasthen
highgradually
regardless of OLRs,toeven
increased though
6.0 and 7.0 g
the COD removal was lower than noted by other researchers using
COD/L·d. When OLR was maintained at 5.0–6.0 g COD/L·d, biogas production rangedsingle-stage tank an-
aerobic reactors. According to the literature, the separation of metabolic pathways
from 135.0 ± 12.6 L/d (0.32 ± 0.03 L/g COD removed) to 145.8 ± 10.0 L/d (0.29 ± 0.02 L/g of the
digestion
COD removed). Under such conditions, the FOS/TAC ratio was below 0.4, and the pHbe
enhanced to produce biogas, because the products of the hydrolysis could in
added dynamically
the chamber of the to the methanogenic
reactor stage [1,64,65].
was 7.04 ± 0.07–7.15 ± 0.04, indicating reactor stability. The
Energies 2023, 16, 571 16 of 18

highest daily methane production of 97.1 ± 5.1 L CH4 was obtained at OLR 6.0 g COD/L·d,
while the highest CH4 concentration in biogas of 70.1 ± 0.7% was noted at OLR 5.0 g
COD/L·d. An increase in OLR to 7.0 g COD/L·d resulted in a significant reduction in
biogas and methane production. Anaerobic digestion of confectionery wastewater did not
contribute to the significant removal of nitrogen and phosphorus.

Author Contributions: Conceptualization, M.D. and M.Z.; Data curation, M.D., M.K., J.K. and
M.Z.; Formal analysis, M.D. and M.Z.; Funding acquisition, M.D. and M.Z.; Investigation, M.K.;
Methodology, M.Z.; Project administration, M.D.; Resources, M.D., M.K., J.K. and M.Z.; Supervision,
M.D.; Writing—original draft, M.K. and J.K.; Writing—review & editing, M.D. All authors have read
and agreed to the published version of the manuscript.
Funding: This project was financially supported by Minister of Education and Science in the range
of the program entitled “Regional Initiative of Excellence” for the years 2019–2023, project no.
010/RID/2018/19, amount of funding: PLN 12,000,000, and project no. 29.610.023-110 of the Univer-
sity of Warmia and Mazury in Olsztyn funded by the Minister of Education and Science.
Data Availability Statement: Not applicable.
Acknowledgments: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study, in the collection, analyses and or interpretation of data, in the writing of the manuscript
and in the decision to publish the results.

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