water

Article
Anaerobic Digestion of Dye Wastewater and Agricultural Waste
with Bio-Energy and Biochar Recovery: A Techno-Economic and
Sustainable Approach
Albert Tumanyisibwe 1 , Mahmoud Nasr 1,2 , Manabu Fujii 3, * and Mona G. Ibrahim 1,4

1 Environmental Engineering Department, Egypt-Japan University of Science and Technology (E-JUST),

Alexandria 21934, Egypt; [email protected] (A.T.); [email protected] (M.N.);
[email protected] (M.G.I.)
2 Sanitary Engineering Department, Faculty of Engineering, Alexandria University, Alexandria 21544, Egypt
3 Department of Civil and Environmental Engineering, Tokyo Institute of Technology, Meguro-Ku,
Tokyo 152-8552, Japan
4 Environmental Health Department, High Institute of Public Health, Alexandria University,
Alexandria 21544, Egypt
* Correspondence: [email protected]

Abstract: While several researchers have investigated the anaerobic digestion (AD) of textile wastewa-
ter for dye degradation, their studies suffer from lower biogas productivity due to substrate inhibition
and the occurrence of secondary pollution from digestate disposal. Hence, this study focuses on
using the extract of wheat straw (WS) as a co-substrate to facilitate the dye AD process, followed
by recycling the digestate sludge for biochar production. In the first study, the batch digesters were
operated at different dye wastewater (DW)/WS ratios (0–50% v/v), substrate-to-inoculum ratio
of 0.28–0.50 g/g, pH 7.0 ± 0.2, and 37 ◦ C. The digester operated at a DW/WS fraction of 65/35%
(v/v) showed the best chemical oxygen demand (COD) removal efficiency of 68.52 ± 3.40% with
bio-CH4 of 270.52 ± 19.14 mL/g CODremoved . About 52.96 ± 3.61% of the initial COD mass was
converted to CH4 , avoiding inhibition caused by volatile fatty acid (VFA) accumulation. In the second
experiment, the dry digestate was thermally treated at 550 ◦ C for 2 h under an oxygen-deprived
Citation: Tumanyisibwe, A.; Nasr, M.; condition, yielding 0.613 ± 0.031 g biochar/g. This biochar exhibited multiple functional groups,
Fujii, M.; Ibrahim, M.G. Anaerobic mineral contents, and high stability (O/C = 0.193). The combined digestion/pyrolysis scenario
Digestion of Dye Wastewater and treating 35 m3 /d (106.75 kg COD/d) could maintain profits from pollution reduction, biogas, biochar,
Agricultural Waste with Bio-Energy and carbon trading, obtaining a 6.5-year payback period.
and Biochar Recovery: A Techno-
Economic and Sustainable Approach. Keywords: biogas recovery; co-substrate; methylene blue; profits; pyrolysis; sludge pyrolysis
Water 2024, 16, 2025. https://doi.org/
10.3390/w16142025

Academic Editor: Andreas Angelakis

1. Introduction
Received: 6 June 2024
Textile industries generate large volumes of wastewater containing organic and in-
Revised: 10 July 2024
Accepted: 15 July 2024
organic pollutants, mainly dyes, surfactants, detergents, solvents, and recalcitrant com-
Published: 17 July 2024
pounds [1]. The informal disposal of dye wastewater (DW) without proper treatment
imposes adverse effects on ecology, agriculture, and public health [2]. For example, a dye
layer can float on the water surface and hamper natural processes of sunlight penetration
into water bodies [3]. This condition negatively influences the photosynthetic activity of
Copyright: © 2024 by the authors. most aquatic plants, further disrupting the availability of dissolved oxygen and nutrients
Licensee MDPI, Basel, Switzerland. in aquacultures [4]. Furthermore, exposure to toxic pollutants in DW is associated with
This article is an open access article various health concerns, including vomiting, stomach pain, and damage to the eyes, liver,
distributed under the terms and skin, and kidneys [5]. Moreover, contaminating the water bodies with textile dyes tends
conditions of the Creative Commons to deteriorate the water resource systems available for landscape irrigation; hence, tex-
Attribution (CC BY) license (https:// tile wastewater should be adequately treated before reaching the aquatic environment to
creativecommons.org/licenses/by/ alleviate the associated risks.
4.0/).

Water 2024, 16, 2025. https://doi.org/10.3390/w16142025 https://www.mdpi.com/journal/water

Water 2024, 16, 2025 2 of 19

While various physical [6], chemical [7], and electrochemical [1] methods have been
employed in DW treatment, these systems entail some drawbacks, especially in low-income
countries. For instance, DW filtration using membrane technologies requires high amounts
of energy to overcome the osmotic pressure accompanied by the high concentrations of
dissolved ions. Moreover, the electrochemical techniques used to capture cationic dyes are
infeasible in developing countries with limited access to energy services, and they might
generate toxic degradation byproducts [8]. The chemical processes require the addition
of reagents that might be expensive and unavailable in some countries, and they could
generate sludge containing unfavorable dye byproducts [9]. The biological treatment of
DW under anaerobic conditions is considered environmentally friendly because of less
chemical consumption and resource (e.g., energy) utilization [10]. Moreover, it could earn
profits from bioenergy recovery (based on renewable feedstock resources) owing to dye
degradation under the action of some methanogenic activities [11].
The anaerobic digestion (AD) process undergoes multiple biological reactions using
microorganisms (e.g., archaea, bacteria) to decompose the biodegradable material of DW
under an oxygen-deprived condition [12]. This process can be employed for DW because it
involves a consortium of microbes that perform a sequence of biochemical and metabolic
pathways [13], including enzymatic hydrolysis, acidogenesis/fermentation, acetogenesis,
and methanogenesis [14]. The main products of this AD process include biogas that can
be used for electricity production, while the released biogenic CO2 is considered “carbon
neutral” [12]. Digestate, including nutrients and undigested biomass, can also be obtained
from the biogas systems, and it can be managed to generate fertilizers [15]. Another study
showed that the thermal treatment of solid digestate could generate biochar used for carbon
storage and the enhancement of soil texture [16]. The marketing of these outputs could
gain revenues that could be used to overcome the initial investment of the AD project.
More research is required to estimate the time consumed to recoup the construction and
equipment costs of the digesters and determine the amount of money that can be earned
per year per investment.
Due to the recalcitrant nature of DW, adding a co-substrate to the fermentation medium
has been used to enhance the overall biodegradation process [17]. This co-substrate (e.g.,
lactate and glucose) serves as an electron donor to facilitate the breakdown of complex
dye compounds, avoiding volatile fatty acid (VFA) accumulation in the reactors. Some
examples of co-substrates/electron donors used in the AD of textile dyes include henna
plant [18], honey processing wastewater [15], pineapple wastewater [19], and ethanol [17].
Supplementing the digester with low-cost co-substrates, e.g., extracts of agricultural wastes,
would stimulate microbial activity to stabilize sludge and convert more organic dyes into
bio-methane (bio-CH4 ) [20].
Different lignocellulosic waste materials, e.g., wheat straw (WS), paddy straw, sugar-
cane bagasse, wood shavings, and rice husk, have been reported to enhance the biodegra-
dation of DW under anaerobic conditions [2]. Moreover, these crop residues are rich in
multiple macro-nutrients, such as potassium (K), phosphorous (P), nitrogen (N), and alu-
minum (Al), along with carbohydrates and polysaccharides, which could serve as food for
microorganisms’ growth [20]. Because the AD process involves a diversity of microorgan-
isms, optimizing the substrate-to-inoculum ratio (SIR) is a crucial factor for facilitating the
hydrolytic acidification of poor biodegradable compounds [21]. The adaptation of SIR not
only promotes process efficiency but also avoids the accumulation of inhibitory compounds
in the AD systems [10].
The resultant digestate from the anaerobic co-digestion of DW could further cause en-
vironmental pollution, such as soil/groundwater contamination via nutrient leaching [22].
Therefore, the thermal treatment of this secondary waste could eliminate the remaining
pollutants (e.g., inorganic elements, pathogens, and aromatic compounds) [3]. In addi-
tion, this thermal decomposition has been recognized as a waste-to-resource technique,
obtaining a final product that has successful environmental applications. For instance,
pyrolysis of digestate from the AD of petrochemical wastewater at 500 ◦ C generates biochar
Water 2024, 16, 2025 3 of 19

with multiple functional groups, improved surface morphology, and a large surface area
suitable for adsorbing dyes and heavy metals from aqueous solutions [12]. Biochar has
also been applied to improve soil fertility and physical properties, further maximizing
the crop yield pattern [23]. Because of its high carbon content, biochar can be a suitable
candidate for mitigating greenhouse gas (GHG) emissions via carbon sequestration. As
such, the thermal treatment of solid digestate for generating biochar can add profits to the
AD process because the market value of biochar would reach up to 1.6 USD/kg [7].
Although previous studies have demonstrated the anaerobic treatment of DW by co-
digestion, there is still a research gap in the economic feasibility of the treatment system, as
well as the recyclability of the sludge digestate. Therefore, this study focused on combining
the co-digestion/pyrolysis approach for the dual benefit of reducing pollution from textile
wastewater and maintaining a sustainable strategy from digestate recycling. Methylene
blue (MB) was selected as a cationic and primary thiazine dye because it is extensively used
in various industries (e.g., as a colorant) and medical applications [9], and direct exposure
to MB results in serious health complexities [4]. The study objectives were threefold: (1) use
the extract of WS as a co-substrate to enhance biogas productivity from the AD of MB-laden
solutions, (2) determine the characteristics, surface morphology, and surface functional
groups of biochar derived from the pyrolysis of solid digestate after DW treatment, and
(3) estimate the techno-economic feasibility from implementing the digestion/pyrolysis
integrated process, regarding biogas recovery, biochar selling, chemical oxygen demand
(COD) shadow price, and carbon credit trading.

2. Materials and Methods

2.1. Substrate and Inoculum Preparation
Synthetic DW was prepared to simulate the composition of real textile wastewater [6].
The components of this DW feed included (in mg/L) methylene blue (50), NH4 Cl (203),
KH2 PO4 (22), K2 HPO4 (28), CaCl2 (100), NaCl (204), and sodium acetate (205). NaHCO3
solution was used to buffer the fermentation medium and maintain the desirable pH
condition, whereas urea was added as a nitrogen source [24]. Trace elements (in mg/L)
of CoCl2 (0.1), MgCl2 (4), NiSO4 (0.48), FeCl3 ·6H2 O (0.24), and KI (10) were used for
nutrient supply [25]. The solution recorded an initial total COD of 1810 ± 131 mg/L. All
the chemicals were of analytical grade.
Wheat straw (WS) was collected from an agricultural field located in Borg El Arab City
in Alexandria, Egypt. Cleaning was performed carefully with distilled water to eliminate
any foreign material adhering to the WS surface. The cleaned samples were reduced into
small sizes and dried at 105 ◦ C for 24 h to obtain dry biomass. The dried matter was
milled and passed through a 0.1 mm sieve, and the resultant fine powder was stored in
plastic bags for further utilization. The biomass was subjected to a pretreatment process
to increase the bio-availability of monomeric sugars required to promote the formation of
CH4 , following the previously established hypothesis [26]. For this purpose, the samples
were dissolved in an alkaline solution containing 1% NaOH and heated at 120–130 ◦ C
for 2 h. After pretreatment, the biomass was filtered and washed with deionized water.
The solid biomass was air-dried at room temperature and kept for further utilization in
subsequent AD experimentations.
Activated sludge as the inoculum was collected from a sewage treatment plant located
in Alexandria, Egypt, treating domestic wastewater. The collected sludge was initially
settled for 8 h, and the supernatant was withdrawn to acquire well-concentrated microor-
ganisms. Further, the concentrated sludge passed through a 200 µm pore screen for sieving.
The inoculum recorded total solids (TS: 21.35 ± 1.00 g/L) and volatile suspended solids
(VSS: 13.88 ± 0.71 g/L), leading to a VSS/TS ratio of 0.65 (Table 1).
Water 2024, 16, x FOR PEER REVIEW 4 of 20
Water 2024, 16, 2025 4 of 19

Table 1. Physicochemical characteristics of dye wastewater and sludge inoculum.

Table 1. Physicochemical characteristics of dye wastewater and sludge inoculum.
Parameter Unit Value
Parameter Unit
Dye wastewater Value
pH -
Dye wastewater 11.35 ± 0.53
TotalpH COD mg/L
- 1810 ±
11.35 ± 131
0.53
Soluble
Total CODCOD mg/L 1180±± 131
1810 59
Soluble COD (TOC)
Total organic carbon mg/L
mg/L 1180 ± 59
2954 ± 135
Total organic carbon (TOC) mg/L 2954 ± 135
Ammonia nitrogen (NH4-N) mg/L 34.16 ± 0.56
Ammonia nitrogen (NH4 -N) mg/L 34.16 ± 0.56
Volatile
Volatile fatty acids(VFA)
fatty acids (VFA) mg/L
mg/L 378.26
378.26 ±± 17.39
17.39
Sludge inoculum
Sludge inoculum
Total solids g/L 21.35 ± 1.00
Total solids g/L 21.35 ± 1.00
Volatile solids
Volatile solids
g/L
g/L
16.95 ± 0.85
16.95 ± 0.85
Volatile suspended solids
Volatile suspended solids g/L
g/L 13.88 ± 0.71
13.88 ± 0.71
Moisture
Moisture content
content (w/w%)
(w/w%) 85.65 ± 3.50
85.65 ± 3.50
Ash content
Ash content (w/w%)
(w/w%) 17.50 ± 0.72
17.50 ± 0.72

2.2. Experiemental Set Up

The experimental procedures included the anaerobic co-digestion of DW and WS for
biogas recovery, followed by dry digestate pyrolysis
pyrolysis for
for biochar
biochar production
production (Figure
(Figure1).
1).

Figure 1.
Figure Experimental setup
1. Experimental setup of
of the
the anaerobic
anaerobic co-digestion
co-digestion of
of dye
dye wastewater
wastewater (DW)
(DW) and
and wheat
wheat straw
straw
(WS) for bio-CH recovery, followed by biomass pyrolysis for biochar production.
(WS) for bio-CH4 recovery, followed by biomass pyrolysis for biochar production.
4

The substrates
The substrates (DW
(DW and
and WS)
WS)were
weremixed
mixedaccording
accordingtotoa apreviously
previouslyreported
reported method-
meth-
ology, with a slight modification allowing for up to 50% WS in the mixtures [27].
odology, with a slight modification allowing for up to 50% WS in the mixtures [27]. WS was
WS
added
was to DW
added in the
to DW inproportions (%) of
the proportions (%)100/0, 90/10,
of 100/0, 80/20,
90/10, 65/35,
80/20, 65/35,and
and50/50
50/50(DW/WS
(DW/WS
v/v). The batch experiments were performed using 200 mL working volume in 250 mL
v/v). The batch experiments were performed using 200 mL working volume in 250 mL
serum bottles, allowing for 50 mL headspace. A certain amount of sludge inoculum
serum bottles, allowing for 50 mL headspace. A certain amount of sludge inoculum was
was added to each reactor, maintaining a SIR range of 0.28–0.50 g COD/g VSS. Both al-
Water 2024, 16, 2025 5 of 19

kaline (0.5 M NaOH) and acidic (0.5 M HCl) solutions were used to adjust the reactors’
pH to 7.0 ± 0.2, which is suitable for dominating methane-producing bacteria [24]. The
headspaces of batch reactors were initially purged with nitrogen gas for 3–5 min and sealed
with rubber stoppers and aluminum crimp caps to create anaerobic conditions. The batch
experimentation assays were performed in a temperature-controlled water bath (37 ◦ C) at
a 40-rpm mixing speed. The batch tests were conducted in triplicate to obtain mean ± stan-
dard deviation values. Methane-rich biogas was obtained by passing the gas through an
alkaline solution (0.5 M NaOH) and then quantified with an inverted measuring cylinder
using a water displacement technique following a previous study [12].
In the second experiment, anaerobic digestate was collected after terminating the
AD process and oven-dried at 105 ◦ C for 24 h. The dry biomass was pyrolyzed at 550 ◦ C
for 2 h under an oxygen-limited condition to obtain biochar, as described elsewhere [28].
The obtained carbon material was ground (<0.1 mm particle size) and further studied for
elemental compositions, crystallinity, surface morphology, and main functional groups.

2.3. Analytical Methods

Wastewater samples were analyzed for ammoniacal nitrogen (NH4 -N), volatile fatty
acids (VFAs), COD, and alkalinity following APHA standard methods [29]. COD was
measured using a multiparameter portable calorimeter HACH system (DR900, Loveland,
CO, USA). To measure the solution color, the samples were centrifuged at 5000 rpm
for 10 min and analyzed according to the procedures described by Bashiri et al. [30].
Decolorization was monitored with UV spectroscopy (Jasco V-630 spectrometer, Jasco
Inc., Tokyo, Japan) at the maximum visible absorbance wavelength of 664 nm for MB
detection. The sludge inoculum was analyzed for total solids (TSs) and volatile solids
(VSs), following the APHA methods [29]. A portable multimeter (Lutron, YK-2001PHA,
Taiwan) was used to monitor pH adjustments. The solid phase samples (AD sludge and
biochar) were characterized according to previously reported procedures [12]. For example,
the variation in surface functional groups was estimated using Fourier transform infrared
(FTIR) spectroscopy (Bruker Optics, ALPHA, Ettlingen, Germany) in the 4000–500 cm–1
wavenumber range. The surface morphology was determined using a scanning electron
microscope (JCM-6000PLUS Neoscope Benchtop SEM, Tokyo, Japan). The sample’s main
elemental compositions were detected using an energy-dispersive X-ray (EDX) spectroscopy
(JOEL JSM-6510LV, Tokyo, Japan). The X-ray diffraction (XRD) instrument (XRD-7000,
Shimadzu, Tokyo, Japan) was used to determine the mineral phases present in solid
substrates and the associated crystallographic structures. The crystallinity index (CrI) was
calculated with Equation (1) [31]:

I200 − Iam
CrI = × 100, (1)
I200

where I200 is the peak intensity of the 002 lattice plane (i.e., the maximum intensity of the
crystalline peak), and Iam is the peak intensity of the amorphous phase.

2.4. Determination of the System Performance

The removal efficiency R(%) of each pollutant was calculated with Equation (2) [21]:

CO − Ct
R(%) = , (2)
CO

where CO and Ct are the initial pollutant concentration and the corresponding value at
time t, respectively (mg/L).
The modified Gompertz model (Equation (3)) was used to fit the variation of methane
productivity from different reactors following a previous study [24]:
  
R.e
P = P(t)·exp −exp (λ − t) + 1 , (3)
P(t)
Water 2024, 16, 2025 6 of 19

where P is the cumulative methane production (mL), P(t) is the CH4 potential (mL) at an
operating time (t in day), R is the maximum CH4 production rate (mL/d), e is Euler’s
number (2.71828), and λ is the lag phase to initiate bacterial growth (day) estimated using
an optimization algorithm with MATLAB mathematical software (R2019b).
The COD mass balance model was performed to validate the accuracy of data obtained
from the biodegradation of organic matter in the bioreactors according to Equation (4), as
reported earlier [20]:

CODsoluble + CODbiomass + CODCH4

CODbalance = , (4)
tCODinitial

where CODsoluble is a soluble COD fraction in the final effluent (g), CODbiomass is the COD
equivalent to biomass growth (in g based on 1.42 g COD/g VSS), CODCH4 is COD-to-CH4
formation (in g based on 1 g COD/350 mL CH4 ), and tCODinitial is the influent total COD.

2.5. Economic Estimation

The economic feasibility of the AD/pyrolysis scheme was computed based on the
data derived from the COD mass balance model. Public sources, supplier quotations,
and manufacturer specifications were consulted to obtain the prices for materials and
equipment as capital input. Capital and operational costs were computed based on a
medium-scale textile industry discharging 35 m3 of wastewater per day (equivalent to
106.75 kg COD/day). The profits related to pollution reduction (environmental benefits)
were estimated with Equation (5) [32].

ENVprofit = CODremoved × PCOD , (5)

where ENVprofit is the profit related to the prevention of environmental damages (USD/y),
CODremoved is the mass of COD removed (kg/y), and PCOD is the shadow price per kg of
COD removed (USD/kg).
The revenues from biogas and biochar selling were computed with Equation (6) and
Equation (7), respectively [6].

Biogasprofit = CODin × Ybiogas × Pbiogas , (6)

Biocharprofit = Md × Ychar × Pchar , (7)

where Biogasprofit and Biocharprofit are the incomes derived from biogas and biochar selling
(USD/y), CODin is the influent COD (kg/y), Ybiogas is the biogas yield (m3 /kg COD), Md is
the annual dry sludge for biochar production (kg/y), Ychar is the biochar yield (kg/kg), and
Pbiogas (USD/m3 ) and Pchar (USD/kg) are the biogas and biochar selling prices, respectively.
The biogenic carbon footprint was computed for biogas (Equation (8)) and biochar
(Equation (9)), as reported elsewhere [20]:

CCbiogas = CH4 × GWP × methane density × CO2,trading , (8)

CCbiochar = Charmass × Carboncontent × Ec × CO2,trading , (9)

where CCbiogas and CCbiochar represent the carbon credit related to the biogenic CO2 of
biogas and biochar (USD/y), respectively, CH4 is the annual methane production (m3 /y),
GWP is the global warming potential for CH4 (i.e., the 100 year GWP for CH4 is 25),
CH4 density = 0.717 kg/m3 , CO2,trading is the global carbon trading price (USD/kg CO2 ),
Charmass is the annual biochar production (kg/y), Carboncontent is the carbon fraction in
biochar (w/w%), and Ec is the carbon emission coefficient (CO2 /C = 44/12).
The time taken to recover the initial investment was calculated with Equation (10) [33]:

Capital cost
PBP = , (10)
Net Profit
Water 2024, 16, 2025
Water 2024, 16, x FOR PEER REVIEW 77 of
of 19
20

where PBP is the payback period (years).

3. Results and Discussion
3. Results
3.1. and Discussion
Performance of Anaerobic Batch Reactor toward Pollutant Removal and Bio-CH4 Production
3.1.
fromPerformance
Dye Wastewater of Anaerobic
(ResultsBatch
of the Reactor toward Pollutant Removal and Bio-CH4 Production
First Experiment)
from Dye Wastewater (Results of the First Experiment)
3.1.1. Effect of Mixing Ratio on Color and COD Removal Efficiencies
3.1.1. Effect of Mixing Ratio on Color and COD Removal Efficiencies
Increasing the co-substrate fraction in the feed wastewater from 10% to 35% v/v pos-
Increasing the co-substrate fraction in the feed wastewater from 10% to 35% v/v
itively impacted color R(%) from 65.30 ± 3.13% to 83.70 ± 4.12%, respectively. A similar
positively impacted color R(%) from 65.30 ± 3.13% to 83.70 ± 4.12%, respectively. A similar
correlation between color removal and co-substrate supplementation was also observed
correlation between color removal and co-substrate supplementation was also observed
for the treatment of real alkaline textile wastewater using pineapple wastewater as a co-
for the treatment of real alkaline textile wastewater using pineapple wastewater as a co-
substrate [19]. Their study revealed that elevating the co-substrate fraction was associated
substrate [19]. Their study revealed that elevating the co-substrate fraction was associated
with the supply of more electron donors for catalyzing dye bond degradation [19]. A fur-
with the supply of more electron donors for catalyzing dye bond degradation [19]. A
ther increase in co-substrate fraction to 50% v/v insignificantly (p > 0.05) improved color
further increase in co-substrate fraction to 50% v/v insignificantly (p > 0.05) improved color
R(%) to 89.46 ± 4.47% (see Figure 2a). It is suggested that a co-substrate fraction between
R(%) to 89.46 ± 4.47% (see Figure 2a). It is suggested that a co-substrate fraction between
35% and
35% and 50%
50% v/v
v/v could
could be be suitable
suitable forfor supplying
supplying sufficient
sufficient electron
electron donors
donors to to enhance
enhance thethe
dye decolorization
dye decolorizationefficiency.
efficiency.InInaacomparable
comparablestudy, study, glucose
glucose was
was used
used as as
anan electron
electron do-
donor
nor to improve MB biodegradation was initiated through the
to improve MB biodegradation was initiated through the demethylation cleavage by an demethylation cleavage by
an isolated bacterium [4]. However, this microbe’s capacity to break
isolated bacterium [4]. However, this microbe’s capacity to break down the heterocyclic anddown the heterocyclic
and aromatic
aromatic structure
structure of MBofdropped
MB dropped significantly
significantly (p < to
(p < 0.05) 0.05) to ±
28.31 28.31
1.73%± 1.73% at 100/0
at 100/0 (%
(% v/v)
v/v) DW/WS mixing ratio. This pattern could be because of the limited
DW/WS mixing ratio. This pattern could be because of the limited substrate available to substrate available
to generate
generate thethe required
required electron
electron donors
donors andand
thethe recalcitrant
recalcitrant nature
nature of the
of the MBMB phenothia-
phenothiazine
zine ring [5]. This inadequate electron transfer pattern suggests the
ring [5]. This inadequate electron transfer pattern suggests the occurrence of adsorption occurrence of adsorp- as
tion as another pathway for color removal. As such, some functional
another pathway for color removal. As such, some functional groups (e.g., —OH, —COOH, groups (e.g., —OH,
—COOH,
and —NH2and ) on—NH 2) on cell
microbial microbial
surfaces cellcould
surfaces could be in
be involved involved
makingincomplexations
making complexa- with
the dye molecules [34]. This theory has been demonstrated previously previously
tions with the dye molecules [34]. This theory has been demonstrated [35], where[35],
the
where thegroups
functional functional
(e.g.,groups
C=O, —NH (e.g.,2 , C=O, —NH2,on
and P—OH) andtheP—OH)
Klebsiellaon the Klebsiella
oxytoca oxytoca
strains provided
strains provided
binding sites withbinding
MB cationssites to
with MB cations
facilitate to facilitate color removal.
color removal.

(a) (b)
Figure 2. Effect of DW/WS mixing ratios on the removal efficiencies of (a) MB color and (b) COD.
Figure 2. Effect of DW/WS mixing ratios on the removal efficiencies of (a) MB color and (b) COD. The
The statistical significance between DW/WS mixing ratios was derived by one-way ANOVA fol-
statistical significance between DW/WS mixing ratios was derived by one-way ANOVA followed by
lowed by Tukey’s post hoc test estimated using MATLAB mathematical software. Groups sharing
Tukey’s
the commonpost hoc test
letter doestimated using MATLAB
not statistically mathematical
differ from software.
each other at Groups
a significant sharing
level the=common
of alpha 0.05.
letter do not statistically differ from each other at a significant level of alpha = 0.05.
The reduction in COD value followed a similar pattern for the degradation of MB dye
The reduction in COD value followed a similar pattern for the degradation of MB
molecules, where the COD R(%) remained almost stable between 65.41 ± 3.27% and 68.52
dye molecules, where the COD R(%) remained almost stable between 65.41 ± 3.27% and
± 3.40% by adapting the co-substrate fractions in the 20–35% v/v range (Figure 2b). The
68.52 ± 3.40% by adapting the co-substrate fractions in the 20–35% v/v range (Figure 2b).
enzymatic hydrolysis of feedstock straw could produce monosaccharides, like xylose, glu-
The enzymatic hydrolysis of feedstock straw could produce monosaccharides, like xylose,
cose, and galactose, utilized by microorganisms as electron donors to form colorless aro-
glucose, and galactose, utilized by microorganisms as electron donors to form colorless aro-
matic amines [17]. Moreover, some microbial species could utilize dye molecules and their
matic amines [17]. Moreover, some microbial species could utilize dye molecules and their
metabolites as C/N sources [4]. However, the microbial’s ability to convert COD dropped
Water 2024, 16, 2025 8 of 19

metabolites as C/N sources [4]. However, the microbial’s ability to convert COD dropped
to 58.38 ± 2.92% (see Figure 2b) when the co-substrate fraction in the feed wastewater
increased beyond 35% (v/v). Although the 50/50% (v/v) contained the largest fraction of
readily biodegradable organics, microbial inhibition could occur due to altered nutrient bal-
ance in the culture. This condition showed a C/N ratio of 33/1, which was beyond the ratio
of 30/1 reported during the treatment of textile wastewater [36]. Similarly, Malik et al. [15]
noted that excessive carbon supplementation would disrupt the bacterial activity, e.g.,
phylum Halobacteriota, Euryacrhaeota, Desulfobacteriota, and Actinobacteriota, under anaerobic
conditions to degrade dye Remazol blue because of nutrient imbalance in the mixtures. The
lowest R(%) of 27.35 ± 1.37% was recorded for the batch run with no co-substrate addition
(i.e., 100/0 v/v), thus elucidating microbial inhibition to degrade textile compounds at
high dye concentrations. The COD removal pattern by this group reflects the microbial’s
ability to utilize organic matter from decaying biomass as a substrate via endogenous
metabolism, a similar pathway reported previously [18]. It has also been reported that
long-term exposure to textile-dyeing wastewater stress would encourage microorganisms
to secrete extracellular polymeric substances (EPSs), e.g., exopolysaccharides, for soluble
COD utilization [25]. In particular, using 35% v/v of WS in the DW/WS digestion process
could provide a suitable environment for enabling microbial consortia to degrade the MB
recalcitrant compound.

3.1.2. Effect of Mixing Ratio on Methane Production from Dye Wastewater and
WS Substrate
The cumulative amount of bio-CH4 for about 40 d was successfully fitted with the
modified Gompertz model (Figure 3a), with R2 values between 0.97 and 0.99. The onset
of CH4 production experienced an initial delay lasting 10–12 d (lag phase) required for
microbial acclimatization to the dye-related environment. Following this adaptation phase,
there was a gradual rise in CH4 production, indicating an augmentation in microbial activity
to facilitate substrate conversion into CH4 . The maximum cumulative CH4 production of
113.08 ± 7.35 mL was achieved at the 65/35% v/v (DW/WS) mixing ratio. This finding
was also consistent with the highest organic matter degradation (given as COD removal
68.52 ± 3.40%, as discussed in Section 3.1.1; see Figure 2b; Supplementary Table S1).
This condition was also accompanied by exhibiting the maximum CH4 production rate
(R) of 5.20 ± 0.26 mL/d, with a lag period (λ) of 10.1 ± 0.1 days, indicating favorable
physiological methanogenic activity for readily converting the substrate into CH4 . An
extended λ was observed for the 100/0 v/v group, also corresponding to the lowest R
of 1.30 ± 0.07 mL/d. This finding demonstrated that microbes needed a more extended
period to acclimatize to the toxicity induced by textile-dyeing effluents.
Cumulative CH4 production significantly increased from 65.64 ± 4.25 to 113.08 ± 7.33 mL
by increasing the WS co-substrate fraction from 10% to 35%, respectively (Figure 3b). Rais-
ing the WS fraction in the digesters could offer more carbon sources to offset the nutrient
limitation in DW, making the entire digestion process simpler for microbes. Digestion
at 65/35 (v/v) DW/WS maintained a C/N ratio of 24.12, complying with the preferable
range (C/N of 21–25) for maximizing biogas from textile wastewater with food waste and
dairy effluent [37]. Similarly, the addition of 40% of food waste to garden waste and tofu
waste digestion enhanced CH4 productivity (370.54 mL/g VS) under a balanced C/N ratio
(16.68–22.04), favoring the enrichment of genus Methanosarcina archaea species to utilize
H2 /CO2 to produce CH4 [38]. Further, increasing the WS fraction beyond 35% showed
a slight drop in P to 107.16 ± 7.25 mL observed at a 50/50 (v/v) DW/WS mixing ratio,
indicating substrate underutilization (i.e., the amount of biodegradable organics could
exceed the microbe utilization capacity). It is also suggested that elevating feeding substrate
concentration over a certain threshold could lead to a shift in SIR to an unfavorable value,
dominating hydrogenotrophic methanogenic and syntrophic bacteria [39]. Their study
also demonstrated that the unpreferable SIR value caused a shift in the methanogenesis
 tivity to facilitate substrate conversion into CH4. The maximum cumulative CH4 produc-
tion of 113.08 ± 7.35 mL was achieved at the 65/35% v/v (DW/WS) mixing ratio. This find-
ing was also consistent with the highest organic matter degradation (given as COD re-
moval 68.52 ± 3.40%, as discussed in Section 3.1.1; see Figure 2b; Supplementary Table S1).
Water 2024, 16, 2025
This condition was also accompanied by exhibiting the maximum CH4 production rate (R)
9 of 19
of 5.20 ± 0.26 mL/d, with a lag period (λ) of 10.1 ± 0.1 days, indicating favorable physio-
logical methanogenic activity for readily converting the substrate into CH4. An extended
λ was observed for the 100/0 v/v group, also corresponding to the lowest R of 1.30 ± 0.07
pathway (i.e., from acetoclastic methanogenesis to syntrophic pathways) with an about
mL/d. This finding demonstrated that microbes needed a more extended period to accli-
60% reduction in the genus Aminobacterium species responsible for CH4 production [39].
matize to the toxicity induced by textile-dyeing effluents.

Figure 3. Cumulative bio-CH4 patterns at various DW/WS mixing ratios of 100/0, 90/10, 80/20, 65/35, and
Figure 3. Cumulative bio-CH4 patterns at various DW/WS mixing ratios of 100/0, 90/10, 80/20,
50/50 (v/v), (a) experimental data “Exp.” compared with fitting the Gompertz model to data “Fit”, and
65/35, and 50/50
(b) Gompertz (v/v),
model (a) experimental
parameters, data “Exp.”
where Maximum Compared
P is the with
cumulative fittingproduction
methane the Gompertz
at themodel
end of
to data “Fit”, and (b) Gompertz model parameters, where Maximum P is the cumulative
the anaerobic digestion period (mL), and R is the maximum CH4 production rate (mL/d). methane
production at the end of the anaerobic digestion period (mL), and R is the maximum CH4 production
rate (mL/d).

3.1.3. COD Mass Balance for Validation of Bio-CH4 Production Data

The conversion of total influent COD into CH4 and other end products showed a mass
balance of over 85% (Table 2). Some portions of COD mass, such as non-degradable organic
matter, COD stored in bacterial cells, and dissolved CH4 , were undetected in this mass
balance model [12].

Table 2. COD mass balance at varying DW/WS mixing ratios of 100/0, 90/10, 80/20, 65/35, and
50/50 (v/v), showing the transformation of CODinitial to CH4 and biomass accumulation.

Mixing Ratios 100/0 90/10 80/20 65/35 50/50

SIR 0.50 ± 0.03 0.31 ± 0.02 0.30 ± 0.02 0.29 ± 0.01 0.28 ± 0.01
C/N 7.96 ± 0.54 11.6 ± 0.83 17.9 ± 1.22 24.12 ± 1.67 33.44 ± 2.39
Final pH 5.19 ± 0.23 6.38 ± 0.25 6.77 ± 0.29 6.85 ± 0.29 6.82 ± 0.30
CODinitial total (mg/L) 1810 ± 131 2240 ± 155 2610 ± 180 3050 ± 218 3530 ± 252
CODfinal total (mg/L) 1320 ± 94 1040 ± 76 902 ± 60 960 ± 69 1470 ± 109
CODsoluble final (mg/L) 987 ± 71 865 ± 64 850 ± 63 848 ± 63 1260 ± 91
VFA/alkalinity 0.32 ± 0.02 0.27 ± 0.02 0.20 ± 0.01 0.19 ± 0.01 0.27 ± 0.02
CH4 yield (mL/g COD) 75.90 ± 5.42 146.52 ± 10.37 176.73 ± 12.44 185.37 ± 12.58 151.78 ± 10.22
CH4 yield (mL/g
280.36 ± 19.03 273.50 ± 19.25 270.06 ± 19.20 270.52 ± 19.14 260.09 ± 18.81
CODremoved )
∆VSS (mg/L) 125.44 ± 8.82 191.04 ± 13.07 252.21 ± 17.35 358.53 ± 25.80 369.15 ± 26.24
CODbiomass /CODinitial (%) 9.84 ± 0.72 12.11 ± 0.82 13.72 ± 1.04 16.69 ± 1.37 14.85 ± 1.14
CODCH4 /CODinitial (%) 21.69 ± 1.58 41.86 ± 3.03 50.49 ± 3.31 52.96 ± 3.61 43.37 ± 2.84
CODsoluble /CODinitial (%) 54.53 ± 3.95 38.62 ± 2.52 32.57 ± 2.19 27.80 ± 2.11 35.69 ± 2.71
COD mass balance (%) 86.06 ± 5.72 92.59 ± 6.82 96.78 ± 7.07 97.46 ± 7.22 93.91 ± 6.87

Increasing co-substrate concentration in the feed provided a favorable environment

for microbial growth, probably due to the availability of easily degradable organics in WS,
which also justifies the highest biomass growth (CODbiomass /CODinitial = 16.69 ± 1.37%)
at 65/35 mixing ratio. In particular, WS could be microbially consumed and fermented
into glucose, VFAs, reducing sugars, soluble proteins, and other low molecular weight
monomers to induce metabolic enzymes and co-enzymes [18]. Moreover, WS addition main-
tained a proper medium’s buffering capacity (VFA/alkalinity = 0.19) with a pH condition
of 6.85 ± 0.29, which is within a favorable range (pH 6.6–7.3) for acetoclastic methanogens
Water 2024, 16, 2025 10 of 19

growth [40]. Likely, methane production returned the highest yield (CODCH4 /CODinitial )
of 50–53% (Table 2) at 80/20 and 65/35 (v/v) DW/WS, probably due to the adaptation
of methanogenic consortia to the balanced SIR range. Similarly, the methanogens grown
under the optimum pH range (6.4–7.2) during garden waste (GW) digestion maintained
better CH4 potential, assigning to the quick adaptation of the genus Methanosaeta archaea
species to the balanced SIR range [38].
For the group with no co-substrate (100/0 v/v), most of the feed COD remained in
soluble form (CODsoluble /CODinitial = 54.53 ± 3.95%). This poor COD conversion efficiency
could be attributed to the high fraction of DW in the mixture and the lack of interaction
between C—O as electron donors to the aromatic ring of MB (electron acceptor) [41].
Some microorganisms are encouraged to activate physiological antioxidative defense
systems (release more metabolic secretions) during exposure to the toxic effects of textile
dyes, increasing the COD soluble fraction in the final effluent [42]. Moreover, higher dye
concentrations could be associated with faster cell lysis and death (i.e., biomass decay
that inhibits methanogenesis), giving the lowest CODbiomass /CODinitial of 9.84 ± 0.72%.
This condition was also associated with the smallest CODCH4 /CODinitial (21.69 ± 1.58%)
fraction, possibly due to substrate limitation to supply the required nutrients to microbial
consortia. The insufficient CODCH4 /CODinitial percentage could also be validated by a
pH drop to 5.19 ± 0.23, attributed to the accumulation of VFAs in the digester (i.e., a high
portion of CODfeed was acidified and not converted to CH4 ). Similarly, lower methane
conversion efficiency was observed under pH-stressed AD (pH< 6.0) due to the dominance
of acidogenesis over methanogenesis responsible for VFA accumulation and subsequently
raising CODsoluble in the final supernatant [43].

3.2. Biochar Production (Results of the Second Experiment)

3.2.1. Morphology and Elemental Composition of Raw Sludge, Exhausted Sludge,
and Biochar
The pyrolysis of textile sludge yielded 61.3% biochar (0.613 g/g sludge), complying
with the 0.40–0.73 g/g dry sludge range reported earlier [44]. Moreover, this value closely
aligns with the biochar yield (0.60 g/g sludge) obtained from the pyrolysis of petrochemical
digestate [12]. The percentage loss in weight could be associated with the dehydration
(release of moisture) and volatilization of some organic compounds [45]. The main elements
in the ash were Si, Al, Fe, P, Ca, Mg, and K, showing ash contents of 28.38%wt for sludge
and 47.78%wt for biochar. These values agree with the enrichment of inorganics (ash) in
the biochar by the order of 1.4–2.4 times the ash fraction in the raw sludge [44].
The SEM morphology of raw sludge was relatively compact, with well-arranged layers
and almost vacant pores (Figure 4a). Some bright white spots appeared on the exhausted
sludge surface, probably because various bacterial species were more metabolically active
within the surface layer to form granular structures [46]. In a comparable study, granular
sludge containing decolorization and degradation microbial communities was responsible
for dye mineralization, generating aromatic amines [47]. The folding structure on the
sludge surface subsequently disappeared in biochar, depending on the decomposition and
carbonization degree of organic matter at higher temperatures. The gradual increase in the
concentration of inorganic components (e.g., minerals and metals) during pyrolysis could
make the biochar pores smaller, further increasing the associated surface area [22]. This
pattern, in addition to the increased decomposition of organic substances after pyrolysis,
could increase the ash content and other residues occupying the pores initially present in
the digestate.
 gions of digestate were partially disintegrated, and the degree of carbonization and aro-
matization was enhanced, yielding a stable biochar material against the biological decom-
position in the soil environment [16]. This low O/C ratio could also be linked to the de-
composition of oxygen functional groups, e.g., –C=O, –COOH, and O–H, in the digestate
Water 2024, 16, 2025
during the decarbonylation and decarboxylation processes [49]. Based on the aforemen-
11 of 19
tioned properties, the prepared biochar possesses a high carbon sequestration potential
(O/C atomic ratio < 0.2), with an estimated half-life of over 1000 years [50].

(a)

Water 2024, 16, x FOR PEER REVIEW 12 of 20

(b)

(c)
Figure 4. SEM/EDX characterization of (a) raw sludge, (b) exhausted sludge, and (c) anaerobic
Figure 4. SEM/EDX characterization of (a) raw sludge, (b) exhausted sludge, and (c) anaerobic
sludge biochar.
sludge biochar.

3.2.2.The
Crystallographic Structure
raw sludge particles of Raw Sludge,
contained Exhausted
elements, Sludge,
e.g., carbon (C:and Biochar
53.82%), oxygen (O:
The and
14.10%), XRDnitrogen
of the raw(N:sludge (Figure
11.63%). 5a)
It also showed a broad
compressed non-crystalline
various peak at 2θ =
inorganic microelements,
20.93°, corresponding to the (002) plane. Other diffraction peaks appeared at 2θ≈ 26.67°
(101), 29.39° for calcite [51], and 42.65° for quartz [52], representing the sludge major com-
ponent. However, more peaks appeared in the exhausted sludge, possibly explaining the
reduction in the amorphous cellulose content [53]. Incrementing the crystalline cellulose
was further validated by having a greater CrI value of 44.5% for exhausted sludge com-
pared with 32.5% for raw sludge. As such, some microbes tended to cleave the amorphous
Water 2024, 16, 2025 12 of 19
Water 2024, 16, x FOR PEER REVIEW 12 of 20

e.g., Al, Si, Na, Cl, Ca, Mg, P, K, and Fe (Figure 4a). The elemental contents of C and
N reduced by 30% and 33%, respectively, after the AD process (see exhausted sludge in
Figure 4b). These elements could have been utilized by the methanogenic microorganisms.
Pyrolysis temperatures enriched most of the inorganic elements present in the sludge (see
biochar in Figure 4c), possibly due to the gradual loss of volatile organic contents (C, H,
N, and O) during the sludge thermal treatment. These features are comparable with the
characteristics of biochar prepared from the fermenter digestate, showing enrichment of
the Ca, Mg, Na, Si, S, P, K, and Fe elements [48]. The increased amount of cationic elements
(e.g., Na+ , Mg2+ , Ca2+ , and K+ ) supports the use of biochar for heavy metal adsorption
from wastewater via the ion exchange mechanism [12]. The presence of K, Mg, Ca, S, and
P elemental composition makes biochar suitable for soil amendment applications. The
reduction in N and S contents suggests their volatilization, releasing gaseous products
as NOX , NH3 , and SO2 [45]. The C content presented a significant increase of 26%, and
the O/C ratio reached ≈0.2 after pyrolysis (Figure 4c). It is supposed that the amorphous
regions of digestate were partially disintegrated, and the degree of carbonization and
aromatization was enhanced, yielding a stable biochar material against the biological
decomposition in the soil environment [16]. This low O/C ratio could also be linked to
the decomposition of oxygen (c) functional groups, e.g., –C=O, –COOH, and O–H, in the
digestate during the decarbonylation and decarboxylation processes [49]. Based on the
Figure 4. SEM/EDX characterization of (a) raw sludge, (b) exhausted sludge, and (c) anaerobic
aforementioned properties, the prepared biochar possesses a high carbon sequestration
sludge biochar.
potential (O/C atomic ratio < 0.2), with an estimated half-life of over 1000 years [50].
3.2.2.
3.2.2.Crystallographic
CrystallographicStructure
StructureofofRaw
RawSludge,
Sludge,Exhausted
ExhaustedSludge,
Sludge,and andBiochar
Biochar
The
The XRD of the raw sludge (Figure 5a) showed a broad non-crystalline peak peak
XRD of the raw sludge (Figure 5a) showed a broad non-crystalline at 2θ =at20.93
2θ =◦ ,
20.93°, corresponding to the (002) plane. Other diffraction peaks appeared at
corresponding to the (002) plane. Other diffraction peaks appeared at 2θ ≈ 26.67 (101), 29.39◦
◦ 2θ≈ 26.67°
(101), 29.39°[51],
for calcite for calcite [51], ◦and
and 42.65 for42.65° for[52],
quartz quartz [52], representing
representing the sludge
the sludge major major com-
component.
ponent.
However,However,
more peaksmoreappeared
peaks appeared in the exhausted
in the exhausted sludge, possibly
sludge, possibly explaining explaining the
the reduction
reduction in the amorphous cellulose content [53]. Incrementing the crystalline
in the amorphous cellulose content [53]. Incrementing the crystalline cellulose was further cellulose
was furtherby
validated validated
having abygreater
havingCrI a greater
value ofCrI44.5%
valuefor
of 44.5% for exhausted
exhausted sludge com-
sludge compared with
pared with 32.5% for raw sludge. As such, some microbes tended
32.5% for raw sludge. As such, some microbes tended to cleave the amorphous to cleave the amorphous
fraction
fraction of cellulose
of cellulose duringduring
the AD theprocess.
AD process. Disrupting
Disrupting the amorphous
the amorphous cellulose
cellulose through
through the
the enzymatic
enzymatic hydrolysis
hydrolysis ofof waterhyacinth
water hyacinthwaswasalso
also accompanied
accompanied by increasing
increasing CrI
CrIfrom
from
50.13%
50.13%toto60.21%
60.21%[3].[3].

(a) (b)
Figure 5. Characterization of raw sludge, exhausted sludge, and anaerobic sludge biochar for (a)
Figure 5. Characterization of raw sludge, exhausted sludge, and anaerobic sludge biochar for
crystallinity and (b) main functional groups.
(a) crystallinity and (b) main functional groups.

The crystalline phases of the digestate denoted the presence of silicates, aluminosilicates,
and carbonates, e.g., wollastonite (CaSiO3, PDF#00-001-0720), sillimanite (Al2SiO5), and
Water 2024, 16, 2025 13 of 19

The crystalline phases of the digestate denoted the presence of silicates, aluminosili-
cates, and carbonates, e.g., wollastonite (CaSiO3 , PDF#00-001-0720), sillimanite (Al2 SiO5 ),
and calcium carbonate as calcite (CaCO3 , PDF#01-072-1650) [54], due to sufficient Si, Ca,
and Al contents of WS. The intensity of these crystalline peaks decreased in biochar, indi-
cating a shift toward a more amorphous structure due to the decomposition of crystalline
domains within the solid digestate under pyrolysis temperatures [55]. The CrI reduced from
44.51% to 35.65% after pyrolysis, elucidating the formation of more amorphous carbon [56].
The mineral phases of the biochar could be monitored by the diffraction peaks at
2θ = 26.61◦ for SiO2 (silica quartz structure) [57], confirming the high ash content of biochar.
Other peaks at 2θ = 31.65◦ (dellaite) and 39.47◦ (crystalline components of sylvine, KCl) [58]
were noticed for the biochar XRD pattern. This pattern also comprised other inorganic
minerals related to magnetite (Fe3 O4 crystalline, PDF#01-076-0955) and aluminum oxide
II (Al2 O3 , PDF#01-075-0278). In a comparable study [59], sludge-derived biochar also
contained quartz and kaolinite as the major crystalline minerals and major elements of Ca,
Al, Fe, and Si.

3.2.3. Main Functional Groups of Raw Sludge, Exhausted Sludge, and Biochar
FTIR spectra of the raw sludge exhibited dominant peaks at 3445 cm–1 (–OH) for the
hydroxyl group and 2970–2850 cm–1 (C–H) in the aliphatic CH2 and CH groups (Figure 5b).
The intensity of the sludge FTIR peaks showed a slight shift after AD, assigned to the
microbial degradation of the substrate structure into CH4 , CO2 , and other metabolites. For
instance, the shift in band for the O–H group around 3425 cm–1 could be assigned to the
binding of MB with the sludge biomass due to N–H stretching [60]. The hydrogen atoms on
the adsorbent hydroxyl groups could form hydrogen bonds with nitrogen atoms in MB. A
previous article also demonstrated that a shift in transmittance peak from 3407 to 3411 cm−1
could describe the occurrence of hydrogen bonding between the nitrogen atoms (N + ) in the
MB molecule and OH− (as negatively charged hydroxyl groups) [3]. The absorption peaks
between 2850 and 2972 cm–1 for the aliphatic C–H stretching bonds were slightly widened
and shifted, probably due to the activity of bacterial species able to decompose aliphatic
fraction contents accompanied by the conversion of VFAs into CH4 and CO2 [61]. The peak
at ≈2900 cm−1 could represent the C–H stretching vibration in the methylene group [62],
suggesting that MB biodegradation initiated through the demethylation cleavage by some
bacterial species [4]. Some peaks appeared in the treated sample, as compared with the
raw sludge, suggesting the dye degradation scheme (e.g., the C=N and C—S bonds in the
MB molecular structure could be oxidized to generate small molecular substances [9]). The
appearance of a new vibration band at 1503 cm–1 agrees with the bending vibrations of MB
molecule adsorbed into sludge, while narrowing the band characteristic of OH vibration
suggests MB molecule fixation [63]. In a similar study, the adsorption of MB onto the
bacterial cell wall of sludge could be supported by C=O electrostatic attraction, –NH2 ,
P-OH hydrogen bonding, and van der Waals forces [35]. Similarly, the appearance of peaks
in the 820–557 cm–1 region, describing the C-Cl stretching [64], could justify the interaction
of MB molecules with the sludge matrix.
Some peaks at 2972–2850 cm–1 and 1503 cm–1 disappeared after sludge pyrolysis,
indicating the thermal decomposition of the organic compounds into gaseous products.
The decrease in the intensity of multiple peaks at 1383–1051 cm–1 could also indicate the
transformation of the remaining organic fatty hydrocarbons of sludge into volatiles under
the thermal treatment process. The peaks in the 1633–1503 cm–1 range could represent
the conjugated aromatic ring stretching, where the C=C bonds in biochar suggested the
formation of stable aromatic ring systems [65]. The bands at the 1163–900 cm–1 region could
be related to the polysaccharide components [60]. The weak peaks between 810−526 cm–1
could reveal the existence of Si–O–Si and Si–C, consistent with the presence of quartz
in the as-prepared biochar (see Figure 5a). These functional groups would promote the
cation-exchange capacity of the soil and improve crop nutrition during the land application
of biochar [49].
Water 2024, 16, 2025 14 of 19

3.3. Economic Analysis for the Anaerobic Co-Digestion System Treating Dye Wastewater
The economic feasibility of the biogas and biogas/biochar scenarios was performed
based on the optimized mixing ratio of 65/35 (v/v) DW/WS for a medium-sized scheme
treating 35 m3 /day of DW (Table 3). The operational parameters of sludge retention time
(SRT) = 14.6–16.2 d and food-to-microorganism (F/M) ratio of 0.68–0.71 g COD/g VSS/d
complied with a previous study [66], avoiding VFA accumulation and maintaining balance
between the faster-growing bacteria and the slower-growing groups. The computation of
capital costs was based on elements like construction facilities, mechanical and electrical
apparatus, piping and fittings, and site footprint, following the previous study [32]. The
total capital cost of scenario#2 was estimated at USD 28,500 and reduced by ≈22% due
to the exclusion of the pyrolysis unit. Contingencies maintained USD 2400, which was
estimated at ≈9% of the capital costs. The prices of construction materials for digesters
with a volume of 15 m3 (hydraulic retention time of 10.29 h) were based on manufacturer
specifications, local contractors, and supplier quotes.
Electrical energy demands varied among the two scenarios and dominated most of
the operational costs, accounting for 34.02% and 40.84% for scenario#1 and scenario#2,
respectively. This cost comprised the energy consumed by electrical appliances and a
source of heat to maintain mesophilic conditions for the effective digestion of DW. It also
considered the power required to operate a screw press for sludge dewatering, further
facilitating the pyrolysis of dry sludge [67]. The energy consumed by drying sludge
(assuming 105 ◦ C for 24 h) was computed based on the previous study [68]. Energy
demand for pyrolysis at 550 ◦ C was estimated from 150 kWh/t total solids [68], assuming
4 h of daily operation. Elevating the electrical energy costs for scenario#2 was due to
additional energy demands for maintaining pyrolysis temperatures.
In both scenarios, the methanogenic microbes could convert 52.96 ± 3.61% of the feed
COD into CH4 (see COD mass balance in Table 2), giving a daily biogas production of
19.79 m3 . CH4 production would positively reduce the carbon footprint from the uncon-
trolled decomposition of waste organics. Thus, the carbon credit earned from biogenic
CH4 was estimated at 355 kg CO2 /d, contributing 1277 USD/y in environmental benefits.
This system was also responsible for reducing COD pollution up to 68.52 ± 3.40% to
give a yearly benefit of USD 2107, expressed as a profit of 0.08 USD/kg CODremoved [12].
This shadow profit reflects the benefit of avoiding environmental damages from the un-
controlled disposal of untreated DW. Although installing a pyrolysis unit raised capital
and operating costs of scenario#2, biochar production could add extra revenues to the
cash flow. Sludge accumulation was determined from the COD conversion efficiency
(CODbiomass = 16.69 ± 1.37% of CODinitial ; see Table 2), which could yield 3.76 kg/d of
solid digestate (estimated from 1.42 g COD/g biomass conversion ratio). The pyrolysis
scheme would increase the profitability of scenario#2, where the accumulated sludge
would produce 2.31 kg biochar/d estimated from 3.76 kg solid digestate/d multiplied by
a biochar yield of 0.163 kg/kg dry sludge. The GHG offset benefit was estimated from
the amount of carbon stored in biochar, giving 1.11 kg/d (using the C fraction of 48% in
EDX; Figure 4c). With the carbon trading price of 10 USD/ton CO2 and the biochar price of
1.5 USD/kg, the pyrolysis scheme could deliver an extra 2616 USD/y to the cash flow of
scenario#2. The biochar price is within the comparable range of the global biochar price
(0.09–8.85 USD/kg), as reported elsewhere [69]. Conversely, the net profits of scenario#1
suffered from sludge disposal expenses compared with scenario#2.
Incorporating a pyrolysis unit would recover the initial investment capital in 6.5 years
compared with 8.4 years for the AD alone scenario. Moreover, the profits of scenario#2
would maintain a positive net present value (NPV) with an internal rate of return greater
than the project discount rate (8%). These findings demonstrated that the study outputs
could pave the way toward the circular economy of recalcitrant textile dyes and residual
sludge, meeting several sustainable development goal (SDG) targets (see Supplementary
Figure S1). Further studies are required to consider the liquid (bio-oil) and gases (e.g.,
Water 2024, 16, 2025 15 of 19

non-condensable gases with a high-calorie value) generated from the co-pyrolysis of sludge
with wood biomass and estimate the profitability criteria with the NPV.

Table 3. Economic analysis for the AD and AD/pyrolysis scenarios treating dye wastewater.

Scenario#1 Scenario#2
Item Justification
AD AD/Pyrolysis
Capital costs (USD)
Included site footprint, screening unit, receiving tanks,
anaerobic unit (assumed as 15 m3 of lightweight structural
Construction 10,000 10,000
concrete with a foundation), piping, and fitting. Maintained
35–45% of the total capital costs
Included the cost of pumps for lifting wastewater and sludge, a
Mechanical equipment 9800 11,100 screw press for sludge dewatering, a heat exchanger, and a
mixer. Maintained 40–44% of the capital costs
Purchase price and shipping of a pyrolysis unit. The price
Pyrolysis unit 0 5000 included an additional condenser, furnace, and gas
cleaning equipment.
Contingency 2400 2400 About 9% of the capital costs
The extra cost of purchasing and installing a pyrolysis unit
Total 22,200 28,500 increases the capital costs of scenario#2 by about 30% compared
with scenario#1
Operating costs (USD/y)
Energy consumption for maintaining mesophilic conditions
Electrical energy (37 ◦ C), drying, pyrolysis, and operating equipment (motors,
1218 1819
consumption pumps, lighting) computed by adding up individual energy
demands assuming 0.05 USD/kWh electricity tariff
Chemicals and reagents, e.g., for pH adjustment, buffering,
Chemical utilization 962 1062 nutrients, for effective microbial degradation assuming a unit
cost of 0.03 USD/kg
Water for mixing substrates, cleaning assuming a unit cost of
Water utilization 485 533
water 0.02 USD/m3
Maintenance 380 420 About 10% of the operating costs
Overhead and Staff
535 620 Taxes, process control, legal fees, insurance, and office supplies
salaries
Total operating costs 3580 4454 Extra electrical energy consumption for scenario#2 for pyrolysis
Profits (USD/y)
Calculated from an organic load of 106.75 kg COD/d, COD
Pollution reduction removal efficiency of 68.52%, and 0.08 USD/kg COD removed.
2107 2107
(COD removal) These values correspond to the design criteria of an organic
loading rate of 7.12 kg/m3 /d and an F/M ratio of 0.69 d–1
Computed from the COD mass balance of
Biogas selling 2850 2850 CODCH4 /CODin = 52.96% and assuming a unit cost of
0.4 USD/m3 of biogas
The biogenic carbon emissions were calculated from CO2
Carbon credit (biogas) 1277 1277
trading of 10 USD/ton CO2
Derived from CODbiomass /CODinitial of 16.69%, biochar yield of
Biochar selling 0 1246
0.613 g/g, and 1.5 USD/kg biochar selling price
Calculated from carbon content in biochar = 47.54% (see EDX
Carbon credit (biochar) 0 15
characteristics in Figure 4c), and CO2 /C molar ratio of 44/12.
Denotes transportation costs and fees paid for sludge disposal
(without pyrolysis) in a landfill. The amount of sludge
generated (10.04 kg/d) was based on an SRT of 15.49 d, which
Sludge disposal 0 1355
is considered suitable to avoid VFAs accumulation and
maintain a balance between the faster-growing bacteria and the
slower-growing groups [66]
Additional profits for scenario#2 from biochar selling and
Total profits 6233 8849
carbon credit from biochar
Water 2024, 16, 2025 16 of 19

Table 3. Cont.

Scenario#1 Scenario#2
Item Justification
AD AD/Pyrolysis
Profitability criteria
Computed as the difference between the total annual revenues
Net profit (USD/y) 2653 4395
and the annual operating costs
Payback period (PBP in y) 8.4 6.5 This time should be compared with the project lifetime (10 y)
Net present value is estimated using an 8% discount rate and a
NPV (USD) −4396 989
project lifetime of 10 years
Internal rate of return should be greater than or equal to the
IRR (%) 3.4% 8.8%
project discount rate (8%)

4. Conclusions
This study successfully developed a sustainable strategy that could cost-effectively
manage recalcitrant dye wastewater (DW) based on biological and thermal treatment pro-
cesses. Adding extracts of wheat straw (WS) to DW under anaerobic conditions enhanced
the methylene blue (MB) degradation efficiency by ≈3-fold higher than the mono-digestion
assays (only DW). The 35% v/v WS supplementation showed a better synergistic interac-
tion with DW, in which 52.96 ± 3.61% of the influent COD was transformed to bio-CH4
production. Moreover, the thermal treatment of solid digestate depicted a promising route
for sludge management, getting biochar characterized by a high degree of aromaticity
(O/C ≈ 0.2). The profits related to pollution reduction, biogas, biochar, and carbon trading
showed that the integrated digestion/pyrolysis scenario would overcome capital costs
in a time less than the project lifetime (<10 y). The profitability scenario of the diges-
tion/pyrolysis project also revealed a positive net present value, with an internal rate
of return greater than the discount rate (8%). Further studies are required to determine
the best post-treatment phase that could achieve complete mineralization of the aromatic
compounds to meet the discharge standards. Moreover, some future studies should aim to
identify the microbial strains and enzymatic activities involved in the degradation processes
and carry out phytotoxicity analysis of the biodegradation products.

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

www.mdpi.com/article/10.3390/w16142025/s1, Table S1. Data of CH4 recovery from the anaerobic
digestion of dye wastewater compared with the current study; Figure S1: Correlating the study
output from the anaerobic digestion (scenario-1) and anaerobic digestion/pyrolysis (scenario-2)
treating dye wastewater to the sustainable development goals (SDGs).
Author Contributions: Conceptualization, M.N., M.F. and M.G.I.; methodology, A.T.; software, A.T.;
formal analysis, A.T.; writing—original draft preparation, A.T.; writing—review and editing, M.N.,
M.F. and M.G.I.; visualization, M.N., M.F. and M.G.I.; supervision, M.N., M.F. and M.G.I. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was funded by TICAD7, the Egypt-Japan University of Science and Technol-
ogy (E-JUST), and the Japanese International Corporation Agency (JICA).
Data Availability Statement: The data that support the findings of this study are within the article
(and or) its Supplementary Materials.
Acknowledgments: The author is very grateful to TICAD7 for providing financial support in the
form of an MSc. scholarship. Also, thanks to JICA—Japan International Corporation Agency for
providing all the facilities and equipment needed to conduct this study.
Conflicts of Interest: The authors declare no conflicts of interest.
Water 2024, 16, 2025 17 of 19

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