applied

sciences
Article
Effect of pH on the Economic Potential of Dark Fermentation
Products from Used Disposable Nappies and
Expired Food Products
Dimitris Zagklis 1 , Marina Papadionysiou 2 , Konstantina Tsigkou 2 , Panagiota Tsafrakidou 1 ,
Constantina Zafiri 1 and Michael Kornaros 2, *

1 Green Technologies Ltd., 5 Ellinos Stratiotou Str., 26223 Patras, Greece; dimitris_2502@yahoo.gr (D.Z.);
panag.tsafrak@gmail.com (P.T.); nzafeiri@tee.gr (C.Z.)
2 Laboratory of Biochemical Engineering & Environmental Technology (LBEET),
Department of Chemical Engineering, University of Patras, 26504 Patras, Greece;
marinapap@chemeng.upatras.gr (M.P.); ktsigkou@chemeng.upatras.gr (K.T.)
* Correspondence: kornaros@chemeng.upatras.gr

Abstract: Used disposable nappies constitute a waste stream that has no established treatment
method. The purpose of this study was the assessment of the dark fermentation of used disposable
nappies and expired food products under different pH values. The biodegradable part of the used
disposable nappies was recovered and co-fermented with expired food products originating from



supermarkets. The recoverable economic potential of the process was examined for different volatile


fatty acids exploitation schemes and process pH values. The process pH strongly affected the
Citation: Zagklis, D.;
products, with optimum hydrogen production at pH 6 (4.05 NLH2 /Lreactor), while the amount of
Papadionysiou, M.; Tsigkou, K.;
produced volatile fatty acids was maximized at pH 7 (13.44 g/L). Hydrogen production was observed
Tsafrakidou, P.; Zafiri, C.;
at pH as low as pH 4.5 (2.66 NLH2 /Lreactor). The recoverable economic potential was maximized at
Kornaros, M. Effect of pH on the
Economic Potential of Dark
two different pH values, with the first being pH 4.5 with minimum NaOH addition requirements
Fermentation Products from Used (181, 138, and 296 EUR/ton VS of substrate for valorization of volatile fatty acids through microbial
Disposable Nappies and Expired fuel cell, biodiesel production, and anaerobic digestion, respectively) and the second being pH 6,
Food Products. Appl. Sci. 2021, 11, where the hydrogen production was maximized with the simultaneous production of high amounts
4099. https://doi.org/10.3390/ of volatile fatty acids (191, 142, and 339 EUR/ton VS of substrate respectively).
app11094099
Keywords: disposable nappies; expired food products; hydrogen production; dark fermentation;
Academic Editor: Francisco volatile fatty acids
Jesus Fernandez-Morales

Received: 2 April 2021

Accepted: 27 April 2021
1. Introduction
Published: 30 April 2021
Used disposable hygiene products make up 1.5–6.3% of the municipal organic wastes
Publisher’s Note: MDPI stays neutral
stream [1,2]. Disposable nappies (DNs) typically consist of an absorbent core, with its
with regard to jurisdictional claims in
main components being cellulosic fibers and a super absorbent polymer (SAP), usually
published maps and institutional affil- sodium polyacrylate, while the exterior is made from various types of plastics. The
iations. composite nature of DNs is detrimental for their treatment and valorization after their
use, being part biodegradable material (cellulosic fibers, excreta, and urine up to 88% [1])
and part recyclable plastics and absorbent. As nappies are usually led for incineration or
landfilling due to their complex structure, many environmental issues arise, such as plastics
Copyright: © 2021 by the authors.
accumulation in landfills, methane release and potential pollution due to virus-infected
Licensee MDPI, Basel, Switzerland.
excreta [3]. A treatment method has been employed by the authors that facilitates the
This article is an open access article
separation of the different DN fractions and their valorization [4–6], according to which the
distributed under the terms and biodegradable fraction is co-digested with expired food products (EFPs). EFPs consisting
conditions of the Creative Commons of fruits and vegetables have already been tested by the authors with very promising
Attribution (CC BY) license (https:// results [5]. The simultaneous food waste and used DNs treatment is suggested, as in most
creativecommons.org/licenses/by/ European countries these are the main waste streams consisting of organic matter (partially
4.0/). or entirely) and no specific regulations or strategies of waste management are pursued.

Appl. Sci. 2021, 11, 4099. https://doi.org/10.3390/app11094099 https://www.mdpi.com/journal/applsci

Appl. Sci. 2021, 11, 4099 2 of 12

Dark fermentation is the bioconversion of an organic substrate to hydrogen and other

metabolic products, like volatile fatty acids (VFAs) and ethanol. Biohydrogen can be used
to produce heat and electricity, either through its combustion, or through electrochemical
processes like fuel cells. The increasing energy demand of human activities and the
deterioration of the environment has led to an increasing need for alternatives to fossil
fuels, with hydrogen produced through dark fermentation of wastes being an excellent
candidate [7].
The metabolic products that are co-produced during dark fermentation can also be
employed as substrate for processes that produce valuable materials and energy [8]. VFAs
can be directly converted to electricity through microbial fuel cells (MFC), where acetic
acid is anaerobically converted to CO2 (anode), with simultaneous production of protons
and electrons. Through a proton exchange membrane, the protons react with oxygen in
an aerobic chamber (cathode), while the electrons are transported from the anode to the
cathode through an external circuit producing electricity [9]. Biodiesel can also be produced
from VFAs by utilizing bacteria, yeasts, or microalgae [10], with studies demonstrating an
accumulation of 20% w/w in the culture biomass as lipids [11]. Finally, the simplest method
for the conversion of VFAs is their anaerobic digestion, with a typical methanogenic reactor
treating the effluent of the dark fermentation process, in a two-step anaerobic digestion
system. This kind of systems have been tested by the authors with similar substrates as the
one examined in this study, proving the synergy of the two processes [4].
The literature regarding the valorization of used DNs for the recovery of materials and
energy is limited [1,12–14]. Colon et al. [1] examined the co-composting of the biodegrad-
able fraction of used DNs with separately collected biodegradable municipal waste, proving
that the occurring compost had very small differences compared to compost produced
solely from separately collected biodegradable municipal waste. Torrijos et al. [13] ex-
amined the co-digestion of the biodegradable part of used DNs with waste activated
sludge, with 3.6 MJ being produced from 1 kg of VS of the biodegradable part of used
DNs. Khoo et al. in their review paper [14] present different treatment methods for used
DNs, including the biodegradation of polymers and cellulosic materials, as well as the
implementation of pyrolysis, concluding that there are innovative technologies for the
treatment of used DNs but at very early stages, with no established treatment method.
A number of companies operating all over the world use disposable nappies for the
production of valuable materials [15–18], mostly focused on their non-biodegradable part,
but these technologies are not widely used. Though this study, the recovery of energy from
the biodegradable part of DNs and EFPs is examined through dark fermentation, a process
that can complement existing technologies and boost used DN valorization. Separate
collection of both waste streams is necessary.

2. Materials and Methods

In the present study, the co-digestion of the biodegradable fraction of the used DNs,
with a mixture of expired fruits, vegetables, bakery products, and meat, originating from
Spanish supermarkets was tested under different pH conditions, and the effect of pH on
the distribution of the energy potential of the substrate to hydrogen, VFAs, and ethanol
was examined. Data from supermarkets were used for the calculation of the ratio of the
different EFPs in the feed mixture that corresponded to the ratios of the different waste
streams being produced. The EFPs mixture used as feed consisted of 51% w/w bakery
products, 27.5% w/w fruits and vegetables and 21.5% w/w meat. The used DNs originated
form an elderly home located in Zamudio, Spain. The biodegradable fraction of the used
DNs was separated in the form of a hydrolysate [4,5]. The first step to produce the DN
hydrolysate was the shredding of the DNs and deswelling of SAP with the use of calcium
and magnesium salts, dissolved in 10 L of water per adult DN. The plastics were removed
from the surface of the mixture and then the cellulosic fibers were separated from SAP
under different stirring conditions. The specifics of the DN separation are currently under
review for a patent acquisition.
 and magnesium salts, dissolved in 10 L of water per adult DN. The plastics were removed
from the surface of the mixture and then the cellulosic fibers were separated from SAP
Appl. Sci. 2021, 11, 4099 under different stirring conditions. The specifics of the DN separation are currently 3 of 12under

review for a patent acquisition.

The optimum mixture for the co-digestion had been previously identified as 9.7:1
w/wThe DNoptimum
hydrolysate to EFPs
mixture [19].
for the Prior to the
co-digestion haddark
beenfermentation experiments,
previously identified as 9.7:1partial
w/w hy-
drolysis
DN of the to
hydrolysate substrate
EFPs [19].mixture
Prior towas carried
the dark out, with experiments,
fermentation the addition partial
of 0.5%hydrolysis
H3PO4, at 100
of°C,
thefor 1 h. The
substrate dark was
mixture fermentation
carried out,experiments wereofcarried
with the addition 0.5% H3out
PO4in 100 ◦ C,
, atbatch reactors
for 1 h. with
controlled
The pH through
dark fermentation NaOH addition,
experiments at mesophilic
were carried out in batch conditions (37 °C),
reactors with with an
controlled pHactive
through NaOH addition, at mesophilic conditions (37 ◦ C), with an active reactor volume
reactor volume of 0.85 L, while the pH examined ranged from 4.5 to 7, with 0.5 increment,
of
as0.85 L, whilealso
described the in
pHFigure
examined ranged
1. The fromoxygen
chemical 4.5 to 7, demand
with 0.5 increment, as described total
(COD), carbohydrates,
also in Figure
suspended 1. The
solids chemical
(TSS), oxygen
volatile demand
suspended (COD),
solids carbohydrates,
(VSS), total suspended
biogas composition, and the con-
solids (TSS), volatile suspended solids (VSS), biogas composition, and the
centrations of intermediary products (VFAs and ethanol) were monitored during the pro- concentrations
ofcess.
intermediary products (VFAs and ethanol) were monitored during the process.

Figure1.1. Experimental
Figure Experimental procedure
procedure and
and set-up
set-up of
of the
the DNs
DNs hydrolysate
hydrolysate and
and EFPs
EFPs mixture
mixture dark
dark fer-
mentation process.
fermentation process.

2.1.
2.1.Inoculum
Inoculum
The
Thebatch
batchexperiments
experiments were
werecarried outout
carried using anaerobic
using sludge
anaerobic fromfrom
sludge a municipal
a municipal
wastewater treatment plant (Attiki, Greece) as inoculum. The anaerobic sludge
wastewater treatment plant (Attiki, Greece) as inoculum. The anaerobic sludge was pre-
was pre-
treated ◦ for 20 min, aiming at the hydrogen-consuming bacteria in-
treatedby
byboiling
boilingatat100
100 C
°C for 20 min, aiming at the hydrogen-consuming bacteria inac-
activation andhydrogen-producing
tivation and hydrogen-producing bacteria
bacteria enrichment
enrichment [20].
[20]. The
The inoculum
inoculum constituted
constituted 15%
15% v/v of the reactor working volume and was characterized by a TSS content of
v/v of the reactor working volume and was characterized by a TSS content of 56.6 ± 3.8
56.6 ± 3.8 g/L and a VSS content of 35.3 ± 5.3 g/L.
g/L and a VSS content of 35.3 ± 5.3 g/L.
2.2. Substrates
2.2. Bakery
Substrates
products, fruit and vegetables and meat in a ratio of 51:27.5:21.5 were collected
from aBakery products,
Super Market fruit and(Biscay,
in Zamudio vegetables and
Spain). meat
These in a ratio
products did of
not51:27.5:21.5 were col-
meet the supply
lected from a Super Market in Zamudio (Biscay, Spain). These products did not
chain quality standards as their expiration date had passed or their quality had been inade- meet the
quate
supplyforchain
consumption. Hard pieces
quality standards as like
theirstalks or bones
expiration were
date hadremoved,
passed while grinding,
or their quality had
mixing and homogenization
been inadequate followed.Hard pieces like stalks or bones were removed, while
for consumption.
Various
grinding, sizes of
mixing andused DNs were obtained
homogenization from a nursing home in Zamudio (Biscay,
followed.
Spain).Various
The DNs sizes of used DNs were obtainedand
were cut manually with scissors frommixed with water
a nursing homeand reagents [12],
in Zamudio (Biscay,
preparing a hydrolysate, which contained cellulosic fibers and excreta,
Spain). The DNs were cut manually with scissors and mixed with water and while the plastic [12],
reagents
parts and SAP were separated.
preparing a hydrolysate, which contained cellulosic fibers and excreta, while the plastic
EFPs and used DNs hydrolysate were initially mixed with a ratio of 1:9.7 w/w and
parts and SAP were separated.
afterwards acid hydrolysis with 0.5% H3 PO4 at 100 ◦ C for 60 min was implemented. Acid
hydrolysis serves two purposes, as it promotes the organic matter solubilization [19,21],
while pasteurization of the substrate is achieved. According to the European Commission
Regulation No 142/2011 [22], animal by-products and derived products not intended for
Appl. Sci. 2021, 11, 4099 4 of 12

human consumption should be pasteurized before any biological treatment. The physico-
chemical characteristics of used DN hydrolysate, EFPs and their hydrolyzed mixture are
presented in Table 1, including pH, carbohydrates, COD, total solids (TS), volatile solids
(VS), TSS, VSS, total Kjeldahl nitrogen (TKN), oil and grease, and phosphorus (P). The
prefixes t- and d- refer to total and dissolved content, respectively.

Table 1. Physicochemical characteristics of used DNs hydrolysate, EFPs, and their mixture after hydrolysis (mean value ±
standard deviation of three measurements).

Parameter Used DN Hydrolysate (g/L) EFPs (g/kg) Mixture after Hydrolysis (g/L)
pH 7.40 ±0.01 N/A 4.21 ±0.03
t-Carbohydrates 3.50 ±1.70 183.80 ±37.20 31.59 ±8.42
d-Carbohydrates 0.06 ±0.01 N/A 12.24 ±0.18
t-COD 5.01 ±1.68 663.27 ±86.59 49.95 ±6.41
d-COD 0.40 ±0.01 N/A 20.42 ±1.45
TS 8.17 ±0.52 554.51 ±58.60 42.37 ±0.85
VS 6.51 ±0.55 509.20 ±7.13 32.95 ±0.95
TSS 6.70 ±0.01 N/A 22.03 ±0.35
VSS 6.16 ±0.31 N/A 22.00 ±0.30
TKN 0.07 ±0.02 3.92 ±0.42 0.91 ±0.06
NH3 -N 0.07 ±0.01 2.65 ±0.06 0.25 ±0.02
Oil & Grease 0.02 ±0.01 47.00 ±1.38 3.14 ±0.14
t-P 0.05 ±0.01 1.24 ±0.25 2.16 ±0.07
d-P 0.02 ±0.01 N/A 0.87 ±0.01

The used DNs hydrolysate used in this study had similar characteristics as the one
used in a previous work of the authors [19], with slightly alkaline pH probably caused by
the presence of urine and urea decomposition [23]. The COD content of the hydrolysate
was low, because of the large amounts of water needed to be added for the separation of
the nappies’ constituents in other steps of the process, with only 8% being soluble COD.
Most of the COD appears to originate from non-soluble carbohydrates probably in the form
of cellulosic fibers. All the measured TKN appears to be in the form of NH3 -N, probably
originating form urine.
The food waste mixture contained high concentrations of carbohydrates, proteins, and
lipids. Due to the low moisture of the samples, measurement of the dissolved part was not
possible. Carbohydrates mostly originated from the bakery products and the fruits and
vegetables used, while the proteins and lipids originated from the animal by-products and
partly from bakery products [24].
The mixture of used DNs hydrolysate and EFPs was analyzed after its partial hydroly-
sis and the addition of water for the adjustment of the organic load that would be fed to the
fermenters. As it was to be expected, the mixture had acidic pH, but within an acceptable
range, as fermentation has been recorded in the literature at lower pH [25]. Around 40%
of the total carbohydrates and COD was in soluble form. It has been reported that by
using 0.8% H3 PO4 at 150–200 ◦ C for 1 h, the dissolved biomass can reach 30–45% [26].
All the suspended matter appears to be biomass, while the use of H3 PO4 led to increased
phosphorus concentrations.

2.3. Experimental Set-Up and Procedure

All experiments were performed in 1 L double wall, stainless steel (INOX 316) bioreac-
tors at mesophilic conditions, as described in detail by Tsigkou et al. [5]. Physicochemical
characterization of the substrates and the batch tests effluents was performed. During the
experimentation period samples were taken every 4–6 h depending on the progress of
the experiment.
TS, VS, TSS, VSS, t-COD, d-COD, TKN, ammonium N, t-P, and d-P were measured
according to Standard Methods [27]. Total and dissolved carbohydrates were determined
according to Joseffson [28], while off-line pH values were measured by Orion ROSS Ultra
 Appl. Sci. 2021, 11, 4099 5 of 12

Refillable pH/ATC Triode, Thermo Scientific. Lactic acid was measured with a DIONEX
IC300 ion chromatography as described by Tsigkou et al. [5] while ethanol and VFAs were
analyzed through gas chromatography (Agilent Technologies, Sant Clara, CA, USA, 7890A)
equipped with a flame ionization detector, as described by Dareioti et al. [29]. Oil and
grease were determined using hexane and a Soxhlet extractor (Velp Scientifica, Usmate
Velate MB, Italy, SER 148). The biogas volume was measured by custom-made equipment
and converted to standard conditions (STP = 0 ◦ C and 1 atm) while the biogas analysis was
performed by gas chromatography, according to Tsigkou et al. [4].
Concerning the calculation of normalized cumulative H2 production per L of reactor,
the produced hydrogen was converted to STP conditions and divided by the working
volume of the reactor. The calculation for the H2 production yield was carried out from the
produced H2 (NL) converted to mols (22.4 NL/mol H2 ) divided by the mol of consumed
carbohydrates in glucose equivalents.

3. Results and Discussion

3.1. Effect of pH
The main metabolic products were in, all cases, acetic and butyric acid, with the
former always preceding the latter. Using just fruits and vegetables and DNs hydrolysate
in similar experiments [5], the same main metabolic products were observed at the range
of pH examined. The substrate carbohydrates were cellulosic fibers (cellulose and hemicel-
lulose) [30] from the nappies, cellulose, and soluble sugars from fruit and vegetables [4,31],
and starch [31], due to the presence of bakery products in the mixture. According to
Chatellard et al. [32], when using sugars and disaccharides such as glucose, fructose, and
maltose, the production of butyric acid is favored, followed by acetic acid. Depending on
the inoculum used, the presence of starch can lead to different metabolic pathways [32].
In the case of cellulose, the products are mostly acetic and butyric acid, followed by low
concentrations of propionic acid, exhibiting a behavior similar to the results recorded in
this study for the mixture of substrates.
As it can be observed in Figures 2 and 3, acetic acid concentration increased inde-
pendently of the hydrogen production rate for pH 5 and 7 (Figures 2a and 3b,f), while
butyric acid production followed the hydrogen production pattern (Figures 2 and 3a–f).
Similar behavior for acetic acid production during the dark fermentation of food waste
has been reported in literature, with butyric acid production diminishing with increasing
pH [33]. Some metabolic products, like caproic and propionic acid, where detected towards
the end of the experiment, and not as intermediary products that were converted during
11, x FOR PEER REVIEW the process. Small concentrations of ethanol were also detected (pH 5.5 6and of 6),
13 as well as
valeric acid (pH 4.5 and 5), in agreement with literature [33].

5 1.6 100
(a) pH 4.5 pH 5 pH 5.5 H2 production yield
(b)
H 2 production yield (mol H 2/mol eq. Glucose)

pH 6 pH 6.5 pH 7 Carbohydrates removal

4
1.2 90
Carbohydrates removal (%)

3
H2 (NL/Lr)

0.8 80

0.4 70
1

0 0.0 60
0 20 40 60 80 100 120 140 160 180 4.5 5.0 5.5 6.0 6.5 7.0
Time (h) pH

(a) H production per L of reactor, (b) H production

Figure 2. (a) Normalized cumulative (b)yield and carbohydrates removal
2 2
duringFigure
the batch dark
2. (a) fermentation
Normalized of used DNs
cumulative Hhydrolysate and
2 production EFPs
per L ofatreactor,
different(b)
pHHvalues.
2 production yield and

carbohydrates removal during the batch dark fermentation of used DNs hydrolysate and EFPs at
different pH values.

The components typically consumed during dark fermentation for hydrogen produc-
tion are carbohydrates. Around 52% of the total substrate COD originated from carbohy-
Appl. Sci. 2021, 11, 4099 6 of 12
Appl. Sci. 2021, 11, x FOR PEER REVIEW 7 of 13

9000 9000
(a) (b)
VFAs and ethanol concentrations (mg/L)

VFAs and ethanol concentrations (mg/L)

8000 8000

7000 7000

6000 6000

5000 5000

4000 4000

3000 3000

2000 2000

1000 1000

0 0
0 20 40 60 80 100 120 140 160 180 0 20 40 60 80 100 120 140 160 180
Time (h) Time (h)
9000 9000

(c) (d)
VFAs and ethanol concentrations (mg/L)
VFAs and ethanol concentrations (mg/L)

8000 8000

7000 7000

6000 6000

5000 5000

4000 4000

3000 3000

2000 2000

1000 1000

0 0
0 20 40 60 80 100 120 140 160 180 0 20 40 60 80 100 120 140 160 180
Time (h) Time (h)
9000 9000
(e) (f)
VFAs and ethanol concentrations (mg/L)

VFAs and ethanol concentrations (mg/L)

8000 8000

7000 7000

6000 6000

5000 5000

4000 4000

3000 3000

2000 2000

1000 1000

0 0
0 20 40 60 80 100 120 140 160 180 0 20 40 60 80 100 120 140 160 180
Time (h) Time (h)
Acetic Propionic Isobutyric Butyric Isovaleric Valeric Caproic Ethanol Lactic

Figure 3.
Figure 3. Concentrations
Concentrations ofof VFAs
VFAsand
andethanol
ethanolduring
during the
the batch
batch dark
darkfermentation
fermentation of
of used
used adult
adult diapers
diapers hydrolysate
hydrolysate and
and
expired food products, at (a) pH 4.5, (b) pH 5, (c) pH 5.5, (d) pH 6, (e) pH 6.5, and (f) pH
expired food products, at (a) pH 4.5, (b) pH 5, (c) pH 5.5, (d) pH 6, (e) pH 6.5, and (f) pH 7.7.
Appl. Sci. 2021, 11, 4099 7 of 12

The components typically consumed during dark fermentation for hydrogen pro-
duction are carbohydrates. Around 52% of the total substrate COD originated from
carbohydrates, with 1.7–5.6% of total COD being converted to hydrogen and 25–36% to
VFAs and ethanol at the pH range examined. Around 10% of the carbohydrates were not
consumed during the process, probably because they were present in a form difficult to
biodegrade, like cellulose. The 89–92% consumption of carbohydrates (Figure 3b) is in
good agreement with relevant literature [33].
The pH of the process appears to strongly affect the products, with hydrogen pro-
duction peaking near pH 6 (Figure 2). The production of hydrogen at pH 4.5 (0.97 mol
H2 /mol equivalent glucose) is also worth mentioning, as it is not typically observed [34].
Concerning the efficiency of the process, the maximum yield (1.33 mol H2 /mol equivalent
glucose) was observed at pH 6.0. In a previously published work by the authors [5], where
fruit and vegetables and DNs hydrolysate were used as substrate, the maximum yield
was 1.12 mol H2 /mol equivalent glucose at pH 7.5. It is also worth mentioning that the
hydrogen yield of EFPs and DNs hydrolysate at pH 5.5 was estimated at 1.12 mol H2 /mol
equivalent glucose while the hydrogen yield of the same substrate mixture at identical
pH and temperature conditions in CSTR reactors remained unchanged (1.17 mol H2 /mol
equivalent glucose) [19]. Regarding the total VFAs concentration, the maximum amounts
were observed at pH 6.5 and 7, at 13.44 and 13.37 g/L, respectively.

3.2. Uses of Produced VFAs

The produced VFAs are a valuable resource that can be utilized though several dif-
ferent processes. This includes the production of electricity through MFC, biodiesel, and
methane through anaerobic digestion. The distribution and amount of VFAs produced per
examined pH is expected to affect the optimum pH for each process. It should be noted
that this is not a full technoeconomic analysis of each process. The operational cost can
vary greatly per process and can lead to very different results.

3.2.1. Microbial Fuel Cells

The maximum energy that can be recovered though an MFC can be calculated from
the change in the Gibbs free energy from the oxidation of the VFAs examined (Table 2).

Table 2. Oxidation reactions of VFAs produced by dark fermentation and their ∆G◦ calculated from
∆Gf ◦ of pure substances.

VFA Reaction ∆G◦ (MJ/Mole)

Acetic Acid C2 H4 O2 + 2 O2 → 2 CO2 + 2 H2 O −0.873
Propionic Acid C3 H6 O2 + 3.5 O2 → 3 CO2 + 3 H2 O −1.603
Butyric Acid C4 H8 O2 + 5 O2 → 4 CO2 + 4 H2 O −2.243
Valeric Acid C5 H10 O2 + 6.5 O2 → 5 CO2 + 5 H2 O −2.883
Caproic Acid C6 H12 O2 + 8 O2 → 6 CO2 + 6 H2 O −3.523

The analysis is based on the energy that can be released from the oxidation of VFAs
produced from 1 kg VS of substrate used in the dark fermentation process Equation (1),
h i
g
◦
h
MJ
i miLreactor

MJ
 ∑ ∆Gi mole ∗ g
[ ] MWi mole
E = (1)
kgVSsubstrate kgVSsubstrate /Lreactor

where E is the energy released from the oxidation of VFAs, ∆G◦ i the change in the Gibbs
free energy from VFA i, mi , the g of VFA i produced per L of reactor, and MWi its
molecular weight.
The percentage of electrons produced in an MFC that can be harvested for the pro-
duction of electricity is called coulombic efficiency (CE), with the rest of the electrons
being lost due to biomass production, or oxygen diffusion through the proton exchange
Appl. Sci. 2021, 11, 4099 8 of 12

membrane [35,36]. Teng et al. [35] have reported how different mixtures of VFAs lead to
different coulombic efficiencies and developed an empirical formula for its calculation from
the distribution of chemical energy in the form of COD fractions of acetic (X1 ), propionic
(X2 ) and butyric acid (X3 ) Equation (2).

CE = 29.7X1 + 20.4X2 − 22.6X3 + 0.2X1 X2 + 95.3X1 X3 − 100.0X2 X3 + 475.9X1 X2 X3 (2)

As it can be observed from Equation (2) [35], the VFA that favors higher CE is acetic
acid, while the presence of butyric acid has a negative effect. The presence of acetic acid is
beneficial to the process probably because longer-chain VFAs have first to be converted to
acetic acid before releasing most of their chemical energy through its oxidation [35]. By
multiplying the chemical energy released from the oxidation of the different VFAs mixtures
that were produced per kg of substrate through dark fermentation with the corresponding
CE (Table 3), the recoverable electric energy can be estimated (Table 4).

Table 3. Coulombic efficiency and chemical energy from the oxidation of different mixtures of VFAs
produced under different pH values, per kg of VS of substrate used in the dark fermentation process.

pH CE (%) Chemical Energy from VFAs Oxidation (MJ/kg VS)

4.5 26.20 8.51
5.0 33.44 7.22
5.5 22.64 8.04
6.0 26.84 10.65
6.5 30.12 10.01
7.0 34.31 10.55

Table 4. Consumed NaOH, produced H2 and VFAs followed by the equivalents of VFAs conversion through MFC, biodiesel
production and anaerobic digestion, for the pH values examined in dark fermentation, per kg of VS of substrate used in the
dark fermentation step.

pH NaOH (kg/kgVS ) H2 (MJ/kgVS ) VFAs (kg/kgVS ) Eq. Electric Energy Eq. Biodiesel Eq. CH4 (MJ/kgVS )
(MJ/kgVS ) (kg/kgVS )
4.5 0.022 1.41 0.41 2.23 0.124 7.26
5 0.085 1.30 0.34 2.41 0.102 6.16
5.5 0.169 1.83 0.41 1.82 0.123 7.27
6 0.129 2.15 0.55 2.86 0.165 9.32
6.5 0.175 0.95 0.54 3.02 0.162 8.86
7 0.192 0.66 0.56 3.62 0.169 9.34

3.2.2. Biodiesel Production

Biodiesel can be produced from waste biomass through the transesterification of lipids
by oleaginous microorganisms, with VFAs being a possible substrate [10,11]. A conversion
of Ylipids/VFAs = 0.3 g/g has been reported by Park et al. [10]. Fey et al. [37] examined the
effect of different VFA ratios for acetic propionic and butyric acid, with higher amounts of
acetic acid once again leading to higher yields of biodiesel and being completely consumed
by the microorganisms. The ratio that was closest to the results of this study demonstrated
a yield of Ylipids/VFAs = 0.159 g/g. Karmee et al. [38] studied the production of lipids from
food derived VFAs and their conversion to biodiesel that can reach 100%. The amount of
biodiesel that can be produced from the VFAs produced under the different pH values
examined was calculated by multiplying the total mass of VFAs produced per kg of VS
of substrate treated with dark fermentation, with a yield of 0.159 g biodiesel per g VFAs
(Table 4).

3.2.3. Anaerobic Digestion Process

A two-stage anaerobic digestion process is a process that consists of two reactors. The
first acidogenic reactor is identical to the system described in this work, where hydrogen
and VFAs are produced, followed by a methanogenic reactor, where the produced VFAs
Appl. Sci. 2021, 11, 4099 9 of 12

are converted to methane and carbon dioxide. In the second stage, apart from the VFAs,
most of the substrate not converted in the acidogenic reactor is typically also consumed.
For comparison of the different VFAs utilization processes, only the methane that can
be produced from the VFAs produced in the first stage will be calculated, but it should
be mentioned that the methane that would be produced from the rest of the substrate is
expected to be much higher because of the lipid and protein content that remains mostly
unaffected at the first stage of the process. The maximum methane that can be produced
can be calculated through a COD balance, where the COD present in the form of VFAs
will be equal to the COD of the methane produced, with YCH4/VFAs = 0.35 NL/g COD.
55.5 KJ/gCH4 heat of combustion was used for the calculations. Through the ideal gas
assumption, the conversion can be calculated as YCH4/VFAs = 13.9 KJ/g CODVFAs . The
COD of each VFA mixture per pH examined can be calculated through the stoichiometry
of its oxidation. For a compound with chemical formula: Cn Ha Ob , COD can be calculated
from Equation (3).  
n + 4a − b2 32
!
gO2
COD = (3)
gcompound 12n + a + 16b

The biomethanization efficiency of VFAs, especially acetic acid, is almost 100%, be-
cause of the low growth yield of acetoclastic methanogens that does not allow significant
conversion of chemical energy to biomass [39]. The expected energy content of the pro-
duced methane per kg of VS of substrate used in the dark fermentation process is presented
in Table 4.

3.3. Comparison of Different VFAs Exploitation Methods

The results of the dark fermentation process under different pH, along with the final
products from VFAs utilization are summarized in Table 4. The amount of NaOH used for
pH adjustment during dark fermentation is also presented, as it may have a strong impact
in the feasibility of the process. As it can be observed, in order to adjust the pH to seven,
the amount of NaOH increases almost tenfold compared to the amount used for pH 4.5.
Hydrogen production is presented in MJ equivalents, with 142 MJ/gH2 . The results of the
VFAs exploitation methods correspond to the equivalent products from the VFAs reported,
with the methods discussed thus far. Everything is expressed per kg of VS of substrate
used in the dark fermentation process.
Even though pH 7 seems optimal for the examined processes from the amount of
equivalent products, a basic economic balance can be carried out with the data presented
in Table 4, in order to compare the cost of NaOH with the value of the products. The
prices used were 0.52 EUR/kg of NaOH [40], 0.8 EUR/kg of biodiesel [41], 0.0637 EUR/MJ
of electric energy [42], and 0.0198 EUR/MJ of thermal energy [43]. For hydrogen and
methane, 40% was calculated as electric energy, 50% as thermal energy and 10% losses
during combustion. Here it must be noted that this is a simplified economic balance
that does not take into account the operational costs of the VFAs exploitation process
that can be vastly different, especially for emerging technologies when compared to well
established technologies, as well as the cost of initial investment. The results refer only to
the conversion of the produced VFAs from the substrate and does not take into account
the amount of substrate that was not converted but could be utilized, like the lipids and
proteins for the production of methane with anaerobic digestion. From the result of each
process per pH, the cost of NaOH has been subtracted and the profit from hydrogen gas
has been added. The results are presented in Figure 4.
Appl. Sci. 2021, 11, x FOR PEER REVIEW
Appl. Sci. 2021, 11, 4099 10 of 12

400
Microbial fuel cell
350 Biodiesel

Economic potential [€/ton VS substrate]

Anaerobic digestion
300

250

200

150

100

50

0
4.5 5.0 5.5 6.0 6.5 7.0
pH
Figure 4.4.Economic
Figure Economicpotential per tonper
potential VS of
tonsubstrate, recoverable through
VS of substrate, dark fermentation
recoverable followed
through dark fermentatio
by different VFAs exploitation methods.
lowed by different VFAs exploitation methods.
4. Conclusions
4. Conclusions
Dark fermentation proved capable of producing valuable products from two solid
wasteDark
streams, used DNs and
fermentation EFPs. The
proved distribution
capable of the substrate
of producing valuablechemical energyfrom tw
products
was affected by the process pH, presenting the opportunity of favoring the most preferred
waste streams, used DNs and EFPs. The distribution of the substrate chemical ener
product through pH adjustment. While maximum hydrogen production was observed at
affected
pH 6 (4.05 by
NLH the process pH, presenting the opportunity of favoring the most pr
2 /Lreactor ) and VFAs at pH 7 (13.44 g/L), the cost of NaOH had a significant
product
impact onthrough pH adjustment.
the net economic potential, While maximum
with the best resultshydrogen
at pH 4.5, production
with minimum was obse
NaOH addition (181 EUR/ton VS of substrate, 138 EUR/ton VS of substrate,
pH 6 (4.05 NLH2/Lreactor) and VFAs at pH 7 (13.44 g/L), the cost of NaOH had a sig and 296 for
valorization of volatile fatty acids through microbial fuel cell, biodiesel production, and
impact on the net economic potential, with the best results at pH 4.5, with mi
anaerobic digestion respectively), and pH 6 with moderate NaOH addition and increased
NaOH
amountsaddition
of VFAs and (181 EUR/ton
hydrogen gasVS
(191ofEUR/ton
substrate,
VS of138 EUR/ton
substrate, 142VS of substrate,
EUR/ton VS of and
valorization of volatile
substrate, and 339 EUR/ton fatty acids through
VS of substrate microbial
for valorization fuel cell,
of volatile biodiesel
fatty acids producti
through
anaerobic digestion respectively), and pH 6 with moderate NaOH addition and in
microbial fuel cell, biodiesel production, and anaerobic digestion respectively).
amounts of VFAs and hydrogen gas (191 EUR/ton VS of substrate, 142 EUR/to
Author Contributions: Conceptualization, D.Z. and K.T.; methodology, D.Z., M.P. and K.T.; valida-
substrate, and
tion, D.Z., K.T. and339
P.T.; EUR/ton VS D.Z.
formal analysis, of substrate for valorization
and K.T.; investigation, D.Z., K.T.of volatile
and fatty acids t
P.T.; resources,
microbial fuel cell,
M.K.; data curation, D.Z.,biodiesel production,
M.P. and K.T.; and anaerobic
writing—original digestion
draft preparation, respectively).
D.Z., K.T. and P.T.;
writing—review and editing, D.Z., K.T. and M.K.; visualization, D.Z. and K.T.; supervision, C.Z. and
Author Contributions:
M.K.; project Conceptualization,
administration, D.Z.C.Z.
C.Z.; funding acquisition, andandK.T.;
M.K.methodology, D.Z.,
All authors have readM.P.,
and and K
dation,
agreed toD.Z., K.T., and
the published P.T.;offormal
version analysis, D.Z. and K.T.; investigation, D.Z., K.T., and
the manuscript.
sources,
Funding: M.K.; data
This work wascuration,
supportedD.Z.,
by theM.P., and Project
European K.T.; writing—original draft preparation,
WASTE4THINK (H2020-GA 688995) D
and P.T.;towards
“Moving writing—review and editing,
Life Cycle Thinking D.Z.,Advanced
by integrating K.T., andWaste
M.K.; visualization,
Management D.Z. and K.T.;
Systems”.
sion, C.Z. and M.K.; project administration, C.Z.; funding acquisition, C.Z. and M.K. All
Institutional Review Board Statement: Not applicable.
have read and agreed to the published version of the manuscript.
Informed Consent Statement: Not applicable.
Funding: This work was supported by the European Project WASTE4THINK (H2020-GA
Data Availability Statement: Data is contained within the article, in Section 3.
“Moving towards Life Cycle Thinking by integrating Advanced Waste Management System
Conflicts of Interest: The authors declare no conflict of interest.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable
Data Availability Statement: Data is contained within the article, in Section 3.
Conflicts of Interest: The authors declare no conflict of interest.

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Appl. Sci. 2021, 11, 4099 11 of 12

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