Journal of Environmental Chemical Engineering 11 (2023) 111252

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Journal of Environmental Chemical Engineering

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Optimization of fermentative parameters to improve hydrogen production:

Is the co-fermentation of waste from the citrus agroindustrial an interesting
alternative for energy recovery?
Danilo Henrique Donato Rocha *, Isabel Kimiko Sakamoto , Maria Bernadete Amâncio Varesche *
Department of Hydraulics and Sanitation, São Carlos School of Engineering, University of São Paulo, Av Trabalhador São Carlense, 400, São Carlos, SP 13566-590,
Brazil

A R T I C L E I N F O A B S T R A C T

Editor: Yujie Men The co-fermentation of peel and wastewater from citrus processing is a proposal to promote the complementation
of the individual characteristics of these substrates, making it possible to obtain higher efficiency in hydrogen
Keywords: (H2) production, in addition to equating problems related to the logistics of destination and treatment of these
Calcium carbonate waste. To build a model for optimizing hydrogen production, based on the variables of citrus peel waste con­
Citrus peel waste
centration (9.9 – 35.1 gTVS.L-1), citrus processing wastewater concentration (0.3 – 8.7 gCOD.L-1), and calcium
Citrus wastewater
carbonate (CaCO3) concentration (0.3 – 7.3 g.L-1), the Rotational Central Composite Design and Response Sur­
Clostridium
Energy potential face Methodology (RSM) were applied, using a mesophilic granular sludge as inoculum for batch assays subjected
Hydrogen to thermal treatment. Hydrogen production from 199.2 to 1241.9 mLH2.L-1 was obtained in the assays, with the
Organic acid lowest production obtained with 22.5 gTVS.L-1 of citrus peel waste, 4.5 gCOD.L-1 of citrus processing wastewater,
and 0.3 g.L-1 of calcium carbonate, for a gCaCO3/gCarbohydrate ratio of 0.03. In the optimized condition with
21.4 gTVS.L-1 of citrus peel waste, 3.4 gCOD.L-1 of citrus wastewater and 4.5 g.L-1 of calcium carbonate, which
resulted in a gCaCO3/gCarbohydrate ratio of 0.53, 1249.0 mLH2.L-1 were obtained. In the hydrogen optimized
condition assay a relative abundance of 95% was observed for Clostridium Sensu Stricto 1, and therefore the
Clostridial metabolic pathway was inferred as predominant, with a maximum butyric acid and acetic acid
production of 2230.8 mg.L-1 and 1213.6 mg.L-1, respectively. For the lowest hydrogen production assay, there
was a significant reduction in the Clostridium sensu stricto 1 (51.2%) abundance, and an increase in the Para­
clostridium (20.0%) and Lactobacillus (21.9%) abundances, inferring lactic acid fermentation (6367.8 mg.L-1) as
the predominant pathway. An energy potential of 523.2 MJ.t-1 of citrus waste was estimated based on the
hydrogen production for the optimized condition assay.

1. Introduction products [2].

Agroindustrial waste, which includes peel, seeds, pits, pulp, press
cakes, leaves, stems, and others, resulting from various processing sec­
tors, are still deposited in landfills or just discarded in inappropriate
places. These by-products can be used to obtain value-added products
such as biofuels, and other biotechnological products [1]. The
Waste-to-energy (WTE) supply chain is an alternative to simultaneously
address the need to increase energy supply and improve waste man­
agement, thus achieving a circular economy model. Using WTE tech­
niques, different types of biomass, such as agricultural, forestry,
domestic, and animal waste, can be converted into several bioenergetic
The agroindustrial sector of citrus processing for juice production is
an important source of lignocellulosic solid waste and wastewater,
considering the significant production of these fruits. The annual global
orange production is estimated at around 75 million tons. Brazil is the
most important country in the global market of oranges and their de­
rivatives, with an annual production of 17 million tons of fruit, repre­
senting almost a quarter of world production [3,4]. A large amount of
production is directed towards processing, which makes Brazil the main
global orange juice producer and exporter, in which the European Union
and the United States are the most relevant consumer markets [5,6].
The estimate is that from 1000 kg of oranges, an average of 553 kg of

* Corresponding authors.
E-mail addresses: danilorocha21@usp.br (D.H.D. Rocha), varesche@sc.usp.br (M.B.A. Varesche).

https://doi.org/10.1016/j.jece.2023.111252
Received 13 June 2023; Received in revised form 4 September 2023; Accepted 11 October 2023
Available online 13 October 2023
2213-3437/© 2023 Elsevier Ltd. All rights reserved.
D.H.D. Rocha et al.
juice is extracted in its natural condition, as well as 30.0 kg of pulp, 3.0
kg of peel oil, and a high amount of solid waste from the peel and
bagasse: altogether more than 400 kg [7]. In addition to the peel and
bagasse waste generated directly from the fruit after juice extraction,
there is also wastewater generation resulting from various processing
steps, which include fruit washing, evaporation of the citrus juice,
washing of the pulp, cleaning operations, effluents from production of
pectin, citric acid, molasses and essential oils, estimated from 1 m3 to 3
m3 of wastewater per ton of processed fruit, reaching up to 17 m3 ,
depending on the water use and reuse practices in the industry, and in
function of the adopted processing technology [8–12].
Conventional alternatives for CPW disposal are treated as insufficient
and problematic in terms of environmental impacts and energy effi­
ciency [13], and therefore routes for this waste valuation have been
proposed to establish sustainable disposal logistics [14]. Alternatives
such as the production of fertilizers, pectin, ethanol, essential oils,
chemical products, cattle feed, and absorbent material, have been
evaluated from a technical and economic point of view [15]. Applica­
tions such as animal feed or organic fertilizer are the most common and
simple alternatives to process this waste because they require little
infrastructure and investment [16], however, due to the high waste
amount and the low value added of the recovered material, they are not
capable of absorbing all the waste generation [17]. Another important
aspect is the seasonality in the generation of these waste, with high
generation concentrated in the harvest period, as highlighted by Calabrò
et al. [18]. These authors evaluated the anaerobic digestion of orange
peel waste after ensiling as an alternative for better waste management.
Energy recovery with hydrogen production through fermentation
using agroindustrial waste has been widely evaluated [19–23]; however,
some factors are still obstacles to conducting a viable process with high
H2 production. Adjusting the operating conditions, such as pH, tem­
perature, substrate concentration, hydraulic retention time (HRT), and
supplementation with buffering agents and nutrients, among others, is
fundamental for the fermentation success [24,25], and the peculiarities
of each substrate show the need to investigate conditions that are suit­
able for each case.
The co-fermentation of peel and wastewater from citrus processing is
an alternative to facilitating the logistics of treatment and disposal of
these waste in the industry itself [26]. The co-fermentation of two or
more by-products is interesting because it can complement the indi­
vidual substrate characteristics to improve hydrogen production effi­
ciency [27]. Co-fermentation makes it possible to improve nutrient
balance, toxic or recalcitrant compounds dilution, and increase the
available organic load, in addition to promoting synergistic effects be­
tween microorganisms [28,29]. The lignocellulosic characteristic of
citrus waste is an aspect that makes its biodegradation more difficult,
imposing limitations on the use of this carbon source as the hydrolysis
step for direct conversion to a readily assimilable substrate is hampered,
leading to a slower conversion of polymeric carbohydrates into simple
sugars [30–32], therefore the provision of a soluble substrate by
wastewater promotes synergism between substrates.
As the co-digestion of these substrates has not been explored so far,
there is little information related to the process parameters, so a deep
investigation of variables with a significant effect on the process is
necessary, aiming to reach a high hydrogen production. For processes in
which many variables can influence the responses, the factorial designs
can minimize the difficulties of achieving the best process optimization
[33]. Through factorial experimental designs, the effects of many vari­
ables can be optimized as the statistical model can establish relation­
ships between responses of two or more experimental variables that are
simultaneously analyzed [34]. In general, this methodological approach
obtains the best response and even reduces the number of assays, which
implies a reduction in time and costs [33].
Experimental design methodologies and response surface models
(RSM) to optimize hydrogen production are applied in many studies for
different independent variables that affect the fermentation process,
2
Journal of Environmental Chemical Engineering 11 (2023) 111252
using various waste from agroindustrial processes as substrates. Citrus
peel waste was evaluated by Camargo et al. [35], whose maximum H2
production was 1085.0 mLH2. L-1, obtained by optimizing the inoculum
concentration, initial pH, and solid substrate concentration. Villa Mon­
toya et al. [36] used waste from coffee processing to produce hydrogen,
and optimizing the initial pH, substrate concentration, and headspace
percentage, obtained a maximum production of 686.0 mLH2. L-1. Rabelo
et al. [34] and Soares et al. [25] sought to increase hydrogen production
using sugarcane bagasse as a substrate, for which variables such as yeast
extract concentration, substrate concentration, and initial pH were
optimized, whose maximum productions were 517.0 mLH2. L-1 and 36.0
mlH2. L-1, respectively. Banana waste as a substrate for hydrogen pro­
duction was evaluated by Mazareli et al. [37], and optimizing the initial
pH and temperature variables, obtained a maximum production of 468.0
mLH2. L-1.
In the present study, the potential of co-digestion of solid waste and
wastewater from the citrus processing agroindustry was investigated,
which until now has not been explored despite the inherent advantages
of using by-products from the same productive chain. For this purpose, a
Rotational Central Composite Design (RCCD) and Response Surface
Methodology (RSM) were carried out to obtain a model for optimizing
H2 production, based on the variables of citrus peel waste concentration
(9.9 – 35.1 gTVS.L-1), citrus processing wastewater concentration (0.3 –
8.7 gCOD.L-1) and calcium carbonate concentration (0.3 – 7.3 g.L-1). The
choice of these variables for optimization aims to establish a direction
for the feasibility of using these substrates together. In addition, the
buffering agent effect was explored as a strategy for better stabilization
of the H2 production, and to promote a significant increase in H2 yield.
Thus, based on the maximum production achieved, it can be evaluated if
the process is favorable compared to other studies in fermentative
conditions. The microbial community characterization was also carried
out for different assays, aiming to associate changes in the H2 production
and soluble metabolite concentrations with changes in the metabolic
pathways inferred for the process. An evaluation for energy recovery of
citrus waste was estimated to verify the H2 potential use as an alterna­
tive energy source in the citrus agroindustry, stimulating the circular
economy proposal.
Therefore, this study provides scientific information to support the
application of citrus waste anaerobic co-digestion as an alternative for
waste management in a circular economy model for agroindustry,
providing a better fit in the proportion between substrates, the specific
demand for buffering agents, and the potential for energy recovery.
2. Material and methods
2.1. Substrate
The juice and seeds of oranges var. Pera-Rio (Citrus sinensis L.
Osbeck) were removed by manual processing, and the peel and bagasse
from the process were used as substrates in the assays. Citrus peel waste
(CPW) composed of peel and bagasse was dehydrated in an oven at 50 ◦ C
for 48 h and then submitted to mechanical grinding pre-treatment
reducing particles < 0.60 mm (knife mill type Willye Model SL 32,
1750 rpm – Solab), and the analytical characterization of in natura and
dry waste is shown in Table 1SM.
The wastewater from citrus processing used in the co-fermentation
assays was supplied by a juice production agroindustry located in Ara­
raquara, São Paulo, Brazil, and stored at − 20 ◦ C, and its analytical
characterization is shown in Table 2SM.
2.2. Inoculum
The sludge used as inoculum in the batch reactors comes from a
mesophilic UASB reactor for wastewater from a poultry slaughterhouse
treatment (Avícola Dacar – Tietê, São Paulo, Brazil), whose character­
ization is presented in the Supplementary Material.
 D.H.D. Rocha et al. Journal of Environmental Chemical Engineering 11 (2023) 111252

Table 1 (100%).
Independent variables evaluated at the factorial (+1 and − 1) and axial (1.68 and Table 1 presents the independent variables with the values corre­
− 1.68) levels and for the central points (0) in the rotational central composite sponding to the delimited factorial and axial levels, and central point.
design (RCCD). The range of values defined for each variable was delimited to ensure
Variables Coded -1.68 -1 0 1 1.68 that an experimental condition for optimizing hydrogen production
Citrus Peel Waste X1 9.9 15.0 22.5 30.0 35.1 could be identified, considering the three investigated variables. For
Concentration (CPW) CPWW, the concentration variation was taken close to the possible limits
(gTVS.L-1) due to the raw effluent characteristics, according to the physical-
Citrus Processing X2 0.3 2.0 4.5 7.0 8.7 chemical characterization (Table 2SM).
Wastewater
Concentration (CPWW)
Table 2 presents the experimental design matrix of the Rotational
(gCOD.L-1) Central Composite Design used to establish the experimental conditions
Calcium Carbonate X3 0.3 1.7 3.8 5.9 7.3 of each assay.
Concentration (CaCO3) (g. A quadratic polynomial equation (Eq. 1) describes the maximum
L-1)
hydrogen production potential (P) based on the independent variables
(X1, X2, X3). The individual and interaction of variable effects on the
experimental response were evaluated at a 10% confidence level.
Table 2
Matrix of the experimental design (RCCD) with the coded levels and the Y = β0 + β1 X1 + β2 X2 + β3 X3 + β11 X 21 + β22 X 22 + β33 X 23 + X1 X2 + X2 X3 + X1 X3
respective values for each variable. (1)
Assay X1 X2 X3 CPW CPWW CaCO3
(gTVS.L-1) (gCOD.L-1) (g.L-1) Where Y is the experimental response (maximum H2 production po­
tential), β0 , β1 , β2 andβ3 are the linear coefficients, β11 , β22 andβ33 are the
E-1 -1 -1 -1 15.0 2.0 1.7
E-2 1 -1 -1 30.0 2.0 1.7
quadratic coefficients, and X1 , X2 andX3 are the independent variables.
E-3 -1 1 -1 15.0 7.0 1.7
E-4 1 1 -1 30.0 7.0 1.7 2.4. Validation
E-5 -1 -1 1 15.0 2.0 5.9
E-6 1 -1 1 30.0 2.0 5.9
E-7 -1 1 1 15.0 7.0 5.9 Based on the results obtained through the response surface, a model
E-8 1 1 1 30.0 7.0 5.9 validation assay was carried out, allowing verification of its accuracy by
E-9 -1.68 0 0 9.9 4.5 3.8 comparing the predicted and experimental H2 production. Therefore,
E-10 1.68 0 0 35.1 4.5 3.8
the validation assay was performed in an experimental condition where
E-11 0 -1.68 0 22.5 0.3 3.8
E-12 0 1.68 0 22.5 8.7 3.8
maximum hydrogen production is obtained, with 21.4 gTVS.L-1 of CPW,
E-13 0 0 -1.68 22.5 4.5 0.3 4.5 gCOD.L-1 of CPWW, and 3.4 g.L-1 of CaCO3.
E-14 0 0 1.68 22.5 4.5 7.3
E-15 0 0 0 22.5 4.5 3.8
2.5. Physicochemical and chromatographic analysis
E-16 0 0 0 22.5 4.5 3.8
E-17 0 0 0 22.5 4.5 3.8
For monitoring and data collection during the fermentation assays,
the liquid fraction analyses of the batch reactors were carried out, such
In order to select endospore-forming bacteria, commonly associated as total soluble carbohydrates [41], chemical oxygen demand - COD
with high H2 yields, and mainly to inactivate hydrogen-consuming mi­ [42], total phenols [43], total volatile solids (TVS), total suspended
croorganisms, a heat treatment was applied to the anaerobic granular solids (TSS) and volatile suspended solids (VSS), organic acids [44].
sludge, according to the methodology applied by Kim et al. [38] and Carbohydrates, phenols, and COD analyses were performed for the
Menezes and Silva [39]. sample filtered through a 0.45 µm filter. The quantification of total
soluble carbohydrates, pH, and organic acids was performed on the
2.3. Factorial Design initial and final samples, and also on samples collected during the assay.
The D-limonene quantification in the samples from the reactors was
A set of fermentation assays was carried out using different con­ performed using the standard addition method, through gas chroma­
centrations of citrus peel waste - CPW (gTVS.L-1), citrus processing tography coupled to mass spectrometry (GC-MS Shimadzu, Column SH-
wastewater - CPWW (gCOD.L-1), and calcium carbonate - CaCO3 (g.L-1). Rtx-5MS 30 m x 0.25 mm × 0.25 mm). The sample temperature was
The experimental design of the assays was defined based on the Rota­ raised to 240 ◦ C in the oven, at a rate of 30 ◦ C, with a retention time of
tional Central Composite Design (RCCD) methodology, evaluating a 5 min. Injection of 250 µL of headspace sample was performed, with an
range of values for the independent variables, and identifying an opti­ injection temperature of 200 ◦ C, Split 10:1, using helium as carrier gas.
mized experimental condition for accumulated H2 production through For mass spectrometry, the full scan method was used for the mass range
the Response Surface Methodology (RSM); the factorial design has been from 50 to 200.
frequently applied to assess and relate significant independent variables The biogas production monitoring was carried out from 500 µL
to responses of interest from diverse research fields. [40]. The factorial headspace samples, using a syringe equipped with a pressure lock. Gas
design was carried out with three factors (k = 3), and, therefore, ± α = chromatography was used to analyze the biogas composition (Shimadzu
1.68, and according to the methodology, there are 2k factorial points, GC- 2014, equipped with a thermal conductivity detector (TCD), Car­
totaling eight assays; 2 *k axial points, totaling six assays, and three boxenTM 1010 PLOT capillary column 30 m x 0.53 mm, Supelco), with
central point assays. Thus, the experimental design consisted of 17 as­ a temperature of 220 ◦ C in the injector, 130 ◦ C for the column, and
says, carried out in triplicate, totaling 51 batch reactors. 230 ◦ C for the detector, using argon as carrier gas (12 mL.min-1). A
The assays were carried out in borosilicate flasks (250 mL) with a periodic measurement of the reactor headspace pressure (mbar) was
plastic screw cap and equipped with a set of valves coupled to the butyl carried out before and after gas sampling.
cap to collect liquid and gas samples, and incubated in an Informs HT –
Multitron Pro shaker, kept stirred at 130 rpm and a mesophilic tem­ 2.6. Data Analysis
perature of 37 ◦ C. In addition, fixed conditions for all assays were 3.5
gTVS.L-1 of inoculum, and 50% headspace (v/v), under N2 atmosphere The accumulated hydrogen production (Hi ) was obtained by sum

3
­
D.H.D. Rocha et al.
ming the H2 produced volume between the monitoring measurements (i)
carried out at intervals of 2 – 6 h throughout the assay, calculated ac­
cording to Eq. (2), based on a hydrogen produced volume (VH,i ) between
the previous (i-1) and current (i) sampling points. Moreover, the
hydrogen volume (VH,i ) was obtained based on the Clapeyron equation
principles, according to Eq. (3).
∑n
Hi = i=1
VH,i (2)
VH,i =
ΔPi ∗ V ∗ CH,i
(3)
Pi
Where CH,i is the hydrogen percentage in the biogas at the sampling
point (i); Pi is the reactor headspace pressure; ΔPi the headspace pres­
sure variation between the previous (i - 1) and current (i) sampling
points; and V the headspace volume.
The hydrogen percentage in the biogas (%H2) for each assay was
given by the arithmetic mean of the H2 percentage values from the
sample points for the period in which biogas composition stabilization
was identified; this stabilization was verified by a coefficient of variation
(CV) < 5%.
The accumulated H2 production – H (mL.L-1) was adjusted to the
modified Gompertz kinetic model (Eq. 4) using the OriginPro 8.5®
software, obtaining the kinetic parameters of maximum H2 production
potential – P (mL.L-1), maximum H2 production rate – Rm (mL.L-1.h-1),
and time to start H2 production - λ (h).
{ [ ]}
Rm ∗ e
H(t) = P ∗ exp − exp (λ − t) + 1 (4)
P
Where e = 2.718281828.
The experimental data from the factorial design and the response
surface model were adjusted and statistically analyzed using Minitab®
19.1 and STATISTICA 10 software, at a confidence level of 10%
(p < 0.10) [24,45,46].
2.7. Energy balance Assessment
The hydrogen energy potential - PEH2 (MJ.kg-1CPW) was calculated
by Eq. (5) based on maximum hydrogen production potential – P (mL.L-
1
), on the lower heating value of H2 – LHVH2 (10.8 MJ.m-3) [47], and the
CPW concentration added to the reactor – CPWC (g.L-1).
EPH2 =
PH2 ∗LHV H2
(5)
1000 ∗ CPW C
EPH2 ∗ η
EPPH2 = (6)
3600
Based on the PEH2, the electricity production potential was estimated
- PPEH2 (MWh.kg-1CPW) through Eq. (6), considering 60% efficiency (η)
for electrical conversion into fuel cells [48,49]. To estimate the process
scale-up, the PPEH2 was extrapolated based on the estimated citrus
waste generation – CPWG, based on data from a processing industry
[50].
2.8. Microbial Community Analysis
Extraction and sequencing of the 16 S rRNA gene were performed for
samples collected at the end of the validation assay for the optimized H2
production and for the E-10 assay. The samples were centrifuged
(6000 rpm - 6 min), and the biomass pellet was washed with PBSX1
solution (NaCl 8.2 g.L-1, Na2HPO4 1.05 g.L-1, NaH2PO4 0.35 g.L-1), and
then stored at − 20 ◦ C. Genomic DNA from the samples was extracted
using the FastDNA™ SPIN Kit for Soil DNA Extraction (MP Biomedicals).
The integrity of the extracted DNA was checked on 0.8% agarose gel,
and the quantification (ng/µL) and purity analysis (260/280 nm ratio)
were performed on the Nanodrop 2000 Spectrophotometers
4
Journal of Environmental Chemical Engineering 11 (2023) 111252
(ThermoFisher Scientific). DNA sequencing was performed by the
company NGS genomic solutions (Piracicaba-SP, Brazil), and the 16 S
rRNA genes in the V3-V4 region were amplified adopting the polymer­
ase chain reaction (PCR) using the primer set 341 F - 806 R [51], per­
formed with Phusion® High-Fidelity PCR Master Mix (New England
Biolabs). Sequencing libraries were generated using NEBNext Ultra DNA
Library Pre ® Kit for Illumina and following the manufacturer’s rec­
ommendations. The DADA2 package [52] was used to transform the
sequencer fastq files into inferred, dismembered, chimera-free sample
sequences. Fastq file filtering was performed to cut PCR primer se­
quences and filter out the 3’-ends of the reads due to quality decay
(Q<30). Taxonomies were assigned to each ASV (Amplicon Sequencing
Variants) using a DADA2 program implementation of the naive Bayesian
classifier method. The Silva 138 database was used as a reference. The
taxonomic classifications generated by DADA2, and their quantifica­
tions, were imported into the phyloseq program [53], and implemented
in the R language. The sequences were submitted to the National Center
for Biotechnology Information (NCBI) database under accession number
PRJNA909571.
The BRENDA [54] and KEGG [55] enzyme databases were used to
infer the predominant metabolic pathways and enzymes that catalyze
conversion reactions, based on the microorganism genera identified in
abundance > 1% in the assays OC-H and E-10.
3. Results and discussion
3.1. H2 production and optimization by RCCD
The kinetic parameters of the fermentation assays are shown in
Table 3. Maximum H2 production potential (P) values were observed
between 199.21 mLH2. L-1 (E-13) and 1241.91 mLH2. L-1 (E-17); the
maximum H2 production rate (Rm) was observed between 7.35 mLH2. L-
1 -1
.h (A-13) and 106.58 mLH2. L-1.h-1 (A-3); and the time to start H2
production (λ) observed between 7.75 h (A-13) and 14.83 h (A-17). As
verified, an important variation was observed for P, Rm, and λ under
different experimental conditions (Fig. 1), which reflects the importance
of adjusting the concentrations of solid waste, wastewater, and calcium
carbonate, to obtain a better response to H2 production.
According to the analysis of variance (ANOVA), the linear term X2
(CPWW concentration, p = 0.093) and the quadratic terms X21 (CPW
concentration, p = 0.015), X22 (CPWW concentration, p = 0.037), and X23
(CaCO3 concentration, p = 0.019) of the model were statistically sig­
nificant (p < 0.10), and resulted in individual effects on hydrogen pro­
duction (Table 4). The linear terms X2 and X3, and the interaction terms
X1X2 (p = 0.912), X1X3 (p = 0.308) and X2X3 (p = 0.166) were not
significant (p > 0.10), probably requiring individual interpretation of
the independent variables.
Based on the data regression analysis, a second-order polynomial
function given by the three coded variables was adjusted, resulting in a
quadratic model of maximum hydrogen production potential. Based on
the significant terms (p < 0.10) for hydrogen production, the model can
be represented by Eq. (7).
( )
Y mL.L− 1 = − 1241 + 254.0X2 − 3.49X 21 − 25.13X 22 − 42.2X 23 (7)
The model significance was evaluated based on the ANOVA test and
was statistically significant based on the F values; the F calculated by
regression (3.23) was higher than the tabulated F value (2.72) for a
confidence interval of 10%, and 7 degrees of freedom for the residuals,
and 9 degrees of freedom for the regression. Thus, the null hypothesis H0
may be rejected, implying that at least one of the variables X1, X2, and X3
contributes significantly to the model. The model has a coefficient of
determination (R2) of 0.81, that is, the model explains 81.0% of the
variability in H2 production, indicating the aptness of the model to
establish an adequate correlation between predicted and observed
values for the response. R2 values between 0.75 and 1.00 indicate a good
D.H.D. Rocha et al. Journal of Environmental Chemical Engineering 11 (2023) 111252

Table 3
Kinetic parameters, yield, and pH, for RCCD assays.
Assay P mL.L-1 Rm mLL-1.h-1 λh %H2 Y1 mLH2. g-1CHO Y2 molH2.mol-1 hexose gCaCO3.g-1 CHO Initial pH Final pH

E-1 823.7 58.5 11.5 47.4% 214.36 2.06 0.32 6.64 4.95
E-2 541.0 33.6 10.0 30.2% 112.23 1.05 0.21 6.31 4.90
E-3 898.8 106.6 12.3 51.0% 182.65 1.87 0.24 6.41 4.56
E-4 578.6 38.9 12.7 44.8% 75.47 0.68 0.14 6.28 4.04
E-5 869.7 49.8 10.8 42.9% 169.53 1.62 0.87 6.67 5.94
E-6 835.5 55.8 10.4 37.3% 123.55 1.17 0.60 6.51 5.67
E-7 423.4 17.2 9.1 31.9% 69.73 0.61 0.77 6.56 5.79
E-8 493.8 46.4 11.4 30.5% 53.51 0.53 0.46 6.46 5.37
E-9 892.0 71.2 9.5 47.8% 209.30 2.13 0.66 6.82 5.64
E-10 522.7 53.3 11.2 33.2% 62.74 0.61 0.28 6.28 4.27
E-11 1056.6 77.4 10.5 42.2% 181.46 1.83 0.46 6.65 5.63
E-12 578.8 56.3 10.5 39.5% 73.88 0.70 0.35 6.39 4.32
E-13 199.2 7.4 7.8 48.3% 75.87 0.65 0.03 5.86 3.52
E-14 1224.5 88.5 13.4 43.5% 144.40 1.46 0.67 6.60 5.72
E-15 1211.4 97.4 12.9 45.8% 151.46 1.65 0.36 6.58 5.33
E-16 1235.1 98.9 13.8 45.2% 172.67 1.69 0.37 6.48 5.38
E-17 1241.9 89.9 14.8 43.4% 180.87 1.64 0.40 6.57 5.47

CHO: Carbohydrate.

gTVS.L-1 of CPW, 4.5 gCOD.L-1 of CPWW, and 3.8 g.L-1 of CaCO3, for an
initial carbohydrate concentration of 10.12 ± 0.48 g.L-1, and a gCaCO3/
gCHO ratio of 0.38 ± 0.01. For assay E-14, performed at the same level
for the substrate variables (22.5 gTVS.L-1 of CPW, 4.5 gCOD.L-1 of
CPWW) as those central point assays, very close H2 production was
observed, 1224.50 mLH2. L-1. However, it should be noted that for E-14,
a higher calcium carbonate concentration was applied (7.33 g.L-1),
resulting in a CaCO3/CHO ratio of 0.67.
Based on these considerations, two important points can be
observed: the first is related to the buffering concentration; that is,
values above 3.8 g.L-1 did not result in higher P for the same substrate
concentration, but also did not cause significant negative effects on H2
production. The second point refers to the perception that intermediate
substrate concentrations (22.5 gTVS.L-1 of CPW, 4.5 gCOD.L-1) resulted
in a better process adjustment, considering the higher H2 production in
Fig. 1. Accumulated H2 production fitted to the modified Gompertz model, for the central point assays. Similar to what was observed in the present
factorial design assays. study, Pu et al. [59] highlighted that low substrate concentrations do not
provide enough organic matter for hydrogen fermentation, while very
high concentrations imply changes in metabolic pathways and lactic
Table 4 fermentation.
ANOVA for the effects of CaCO3, CPW, and CPWW at maximum production Harmful effects to the process directly associated with the high cal­
potential (P) for a 90% confidence interval. cium carbonate concentration (7.33 g.L-1) were not observed, this fact is
Source SQ DF QM F Value P Value probably related to the low solubility and slow dissolution of this buff­
Model 1230063 9 136674 3.23 0.068 ering agent in the liquid medium, and therefore high CaCO3 concen­
X1 (L) 103317 1 103317 2.44 0.162 trations do not result in too much increase in pH, but in a buffering
X2 (L) 160165 1 160165 3.78 0.093 capacity increase [60]. Otherwise, some calcium carbonate concentra­
X3 (L) 113194 1 113194 2.67 0.146 tions would lead to the process establishment above the upper limit of
X1 (Q) 433292 1 433292 10.24 0.015
X2 (Q) 278015 1 278015 6.57 0.037
the ideal pH range for hydrogen production (pH from 4.5 to 6.5) [61].
X3 (Q) 391187 1 391187 9.24 0.019 Consistent with this behavior described for carbonate, in the present
X1X2 561 1 561 0.01 0.912 study, despite the wide range of calcium carbonate concentration
X1X3 51061 1 51061 1.21 0.308 applied to the reactors (0.3–7.3 g.L-1), no impact was observed on the
X2X3 101387 1 101387 2.40 0.166
initial pH of the assays, which were close to 6.5 (Table 3). The percep­
Erro 296226 7 42318
Total SQ 1526289 16 tion of a reduction in the stability of maintaining the H2 production in
the assays conducted at the upper level for the substrate variables is
associated with the imbalance in the kinetics of the calcium carbonate
fit, as discussed by Reungsang et al. [56], Niladevi et al. [57], and dissolution processes and organic acid production; therefore, a high
Kainthola et al. [58]. organic acid production imposes the need for high calcium carbonate
The interaction and optimal level of independent variables in dissolution [62], which sometimes cannot be achieved.
hydrogen production can be observed on the 3D response surface The satisfactory adjustment of the variables for the assays in which
(Fig. 2). The surfaces approximate the characteristic of an elliptical the highest P values were obtained is confirmed by the response surface,
hyperboloid, and in this case, a maximum point can be identified within so that the point of H2 production optimization was verified in the
the range of values evaluated for each variable, which represent the x central region of the surface, for the condition with 21.4 gTVS.L-1 of
and y axes in the three-dimensional plane. Therefore, an optimized CPW, 3.4 gCOD.L-1 of CPWW, and 4.50 g.L-1 of CaCO3; while for points
condition for H2 production can be identified. located towards the surface peripheries, experimental conditions with
The highest H2 production was verified for the central point assays gradual reduction for maximum production potential were observed. As
(E-15/E-16/E-17), of 1229.45 ± 14.1 mLH2. L-1, performed with 22.5 obtained in the present study of co-fermentation of citrus waste and

5
D.H.D. Rocha et al. Journal of Environmental Chemical Engineering 11 (2023) 111252

Fig. 2. Response surface for H2 production model with (a) fixed CaCO3 concentration 3.8 g.L-1; (b) fixed CPWW concentration 4.5 gCOD.L-1; (c) fixed CPW 22.5
gTVS.L-1.

6
D.H.D. Rocha et al.
wastewater, Liu et al. [63], who evaluated the co-digestion of food waste
and cattle manure, observed a significant reduction in the H2 production
when the substrate concentration differed from the concentration values
of the optimized condition.
The lowest H2 production (199.21 mLH2. L-1) was observed in assay
E-13 (22.5 gTVS.L-1 of CPW, 4.5 gCOD.L-1 of CPWW, and 0.3 g.L-1 of
CaCO3). The concentrations of CPW and CPWW in this assay are the
same as those for assays E-14 and the central point (E-15/E-16/E-17), in
which the highest P values were observed. However, in E- 13, only 0.3 g.
L-1 of CaCO3 was added, consequently resulting in a CaCO3/CHO ratio of
0.03. From the antagonistic behavior to the H2 production in this assay,
the fundamental importance of alkalinization for maintaining good
fermentation stability is highlighted. Similar results were verified by
Zhang et al. [64], who evaluated the alkalinity effect using lime mud
(1.0–4.0 g.L-1), for fermentation in batch reactors with food waste, and
identified that the decrease in buffering agent concentration resulted in
a significant impact on hydrogen production. As observed in the present
study, lower stability was observed due to a more acidic pH (4.0–5.0).
Through the response surfaces (Figs. 2b and 2c), there was a marked
P reduction, mainly for calcium carbonate concentrations in the range
from 0.3 to 1.7 g.L-1, combined with substrate concentrations ≥ 30.0 g.
L-1 of CPW, and ≥ 7.0 gCOD.L-1 of CPWW. For the condition with a low
calcium carbonate concentration (1.7 g.L-1), however, with intermedi­
ate values for initial carbohydrate concentration (5.0–7.0 gCHO.L-1,
Fig. 3), there was no sharp reduction in H2 production as the combi­
nation of variables at these levels did not result in a sufficiently low
gCaCO3/gCHO ratio. The importance of the buffer:substrate ratio for
fermentation was also verified by Sangyoka et al. [65], using sodium
bicarbonate as a buffering agent in a study with sugarcane bagasse in
batch reactors. The authors identified a reduction in the hydrogen po­
tential in assays with low values for the buffer:substrate ratio (<0.15),
similar to the present study, and identified an optimized condition with
a buffer: substrate ratio of approximately 0.25.
The effect of this adjustment between concentrations of CaCO3, CPW,
and CPWW could be seen in assays E-1 and E-3, which were performed
with a low CaCO3 concentration (1.7 g.L-1), however with a low initial
substrate concentration in comparison to other assays (5.25 gCHO.L-1
and 7.12 gCHO.L-1, respectively), resulting in a CaCO3/CHO ratio of
0.30. Therefore, there was no marked reduction in hydrogen production
(823.72 mLH2. L-1 for E-1, and 898.80 mLH2. L-1 for E-3) when
compared to the central point assays, in which it obtained the highest
production.
The process stability in the assays with the best responses to the H2
Fig. 3. Carbohydrate concentration at initial and final, and carbohydrate
removal from experimental design assays.
7
Journal of Environmental Chemical Engineering 11 (2023) 111252
production was associated with the behavior observed for the opera­
tional pH, which after a reduction in the first hours of the process (10 –
15 h) starting from an initial pH of 6.5 and reaching values close to at
5.95, a slight reduction was observed, reaching values close to 5.5 at the
end of the assay (~40 h).
As can be seen on the response surface (Fig. 2a), hydrogen produc­
tion was significantly increased when a better fit was established be­
tween substrate variables X1 and X2 (CPW and CPWW). In the peripheral
areas of the surface, referring to conditions that simultaneously have
high concentrations of CPW (≥ 30.0 gTVS.L-1) and CPWW (≥ 7.0 gCOD.
L-1) or even conditions with low concentrations for one of these sub­
strates, did not result in a favorable response to hydrogen production.
Under conditions with addition of 30.0 gTVS.L-1 of CPW and 7.0 gCOD.
L-1 of CPWW, such as E-4 and E-8, the H2 production was 578.6 mLH2. L-
1
and 493.8 mLH2. L-1, respectively.
Regarding the effect of adjusting substrate proportions in co-
digestion for hydrogen production, Li et al. [66] evaluated the
co-digestion of food waste with sewage sludge and observed that the H2
production was negatively impacted as the proportion between these
substrate concentrations was altered. This was similar to the results of
the present study, in which it was verified that the simultaneous increase
in the concentration of both substrates (CPW and CPWW) resulted in
negative effects on H2 production.
It is observed from these results that there are no adverse effects in
the co-use of these substrates, since for duly adjusted concentrations
were obtained high H2 production. The negative results observed in
some assays were associated with the lack of adjustment in their pro­
portions, for example, for the assay E-7 performed at the upper level for
CPWW concentration (7.0 gCOD.L-1) but at the lower level for CPW
concentration (7.0 gTVS.L-1), the production was of only 423.4 mLH2. L-
1
. Another important aspect of the co-digestion is that the optimized
condition obtained ensures the use of significant concentrations for both
substrates (21.4 gTVS.L-1 of CPW, 3.4 gCOD.L-1 of CPWW), postulating
how a concrete alternative for the treatment of both. Considering the
specific generation data of CPW and CPWW in the industry per ton of
fruit processed (400 kg of CPW in natura [7], and 1 – 3 m3 of CPWW [8,
12]), it is estimated that for every 1 kg of CPW (dry basis, moisture
<20%), there is a generation of 10–30 liters of CPWW. For the optimized
experimental conditions obtained, for each 1 kg of CPW (dry basis)
applied in the reactor, there is a demand of 18.7 liters of CPWW (based
on the COD of the wastewater of the study). Thus, it is verified that the
process of co-digestion of these substrates is also feasible from a quan­
titative point of view, consistent with the average of CPW and CPWW
generation.
The H2 percentage (%H2) in the biogas composition between 30.2%
and 51.0% was identified, which was an important variation, making it
possible to infer that different experimental conditions significantly
influenced the biogas quality. The variables of CPW concentration (X1)
and CaCO3 concentration (X2) were identified as statistically significant
(p = 0.001 and p = 0.008, respectively) to %H2. Assays E-2 (30.0 gTVS.
L-1 of CPW, 2.0 gCOD.L-1 of CPWW, and 1.7 gTVS.L-1 of CaCO3) and E-8
(30.0 gTVS.L-1 of CPW, 7.0 gCOD.L-1 of CPWW, and 5.9 g.L-1 of CaCO3)
were those in which the lowest H2 percentage was found (30.2% and
30.5%, respectively), while in E-3 (15.0 gTVS.L-1 of CPW, 7.0 gCOD.L-1
of CPWW, and 1.7 g.L-1 of CaCO3) and E-13 (22.5 gTVS.L-1 of CPW, 4.5
gCOD.L-1 of CPWW, and 0.3 g.L-1 of CaCO3) were those with the highest
%H2 (51.0% and 48.3%).
For the two assays with the highest %H2, distinct patterns were
identified, the first with the higher volumetric production, as verified in
E-3, and therefore adequate fermentation stability with the predomi­
nance of H2 production pathways. While, for assay E-13, the high %H2 in
the biogas despite the low P occurred due to the lower CaCO3 addition
(0.3 g.L-1) compared to the other assays, resulting in a reduction in the
total CO2 release through the carbonate system. The neutralization re­
action of the acids formed in fermentation, through the carbonate sys­
tem, results in CO2 release to the gaseous phase of the reactor, as can be
D.H.D. Rocha et al.
seen in Eq. (8), taking as an example the neutralization reaction of acetic
acid [62], so that higher calcium carbonate availability in the liquid
medium enables higher CO2 release. Lin and Lay [67] point out that
carbonate correlates with CO2, therefore the increase in carbonate
concentration tends to provide a higher CO2 percentage in biogas.
2CH 3 COOH + CaCO3 ⇆Ca2+ + 2CH 3 COO− + H2 O + CO2 (8)
3.2. Substrate utilization, limonene removal, and hydrogen yield
Carbohydrate removal in the assays ranged from 29.25% (E-13) to
88.31% (E-7). As can be seen in Fig. 3, a significant difference in this
parameter indirectly allows us to evaluate the stability and efficiency of
the fermentation process through the substrate utilization. However, the
low conversion observed in E-13 was an exception among the experi­
mental conditions (Fig. 3), and except for this condition, the average
carbohydrate removal of the other assays was 81.4% ± 5.7%. High
carbohydrate removal (88.3% for E-7% and 81.5% for E-8) was observed
even for conditions with reduced H2 yield (0.61 mol H2.mol-1 hexose for
E-7 and 0.53 mol H2.mol-1 hexose for E-8).
The positive response to the carbohydrate removal, even for assays
with high initial substrate concentration, was directly associated with
CaCO3, since its gradual solubilization throughout the assay ensured the
pH maintenance in a range that did not limit the continuation of the
process. In some hydrogen production studies that used only the initial
pH correction as a process parameter, they obtained carbohydrate
removal lower than those obtained in the present study, even starting
from an initial carbohydrate concentration in a lower range than that
evaluated for citrus waste. For example, Villa Montoya et al. [36], in a
study on co-digestion of coffee waste in batch reactors, verified carbo­
hydrate removal of 60.4% ± 11.3%, in an initial pH range from 4.8 to
8.5. Camargo et al. [35] evaluated the citrus waste fermentation in batch
reactors and verified a conversion of only 46.8% ± 12.2%, with an
initial pH variation between 4.5 and 8.5. In both cases, initial pH values
in the neutral or alkaline range were insufficient to ensure high carbo­
hydrate removal.
The H2 yield (Y1) for the assays was calculated indirectly based on
substrate concentration and cumulative H2 production and ranged from
53.5 mLH2.g-1CHO to 214.4 mLH2.g-1CHO. In the assays with the
highest P, intermediate yields were obtained, whose values were 168.3
mLH2.g-1CHO (E-15/E-16/E-17) and 144.4 mLH2.g-1CHO for E-14.
For assays E-1 (15.0 gTVS.L-1 of CPW, 2.0 gCOD.L-1 of CPWW, and
1.7 g.L-1 of CaCO3) and E-9 (9.9 gTVS.L-1 of CPW, 4.5 gCOD.L-1 of
CPWW, and 3.8 g.L-1 of CaCO3) yields of 214.4 mLH2.g-1CHO and 209.3
mLH2.g-1CHO, respectively, were observed. These assays have the
lowest initial substrate concentrations in common among all assays
evaluated (5.25 and 5.74 gCHO.L-1, respectively). Therefore, it was
verified that lower substrate concentrations combined with the CaCO3/
CHO ratio > 0.3 were favorable to obtain the highest H2 yields.
The lowest yields found were 53.5 mLH2.g-1CHO for E-8 (30.0 gTVS.
L of CPW, 7.0 gCOD.L-1 of CPWW, and 5.9 g.L-1 of CaCO3), and 62.7
-1
mLH2.g-1CHO for E-10 (35.1 gTVS.L-1 of CPW, 4.5 gCOD.L-1 of CPWW,
and 3.8 g.L-1 of CaCO3), assays which have in common the highest initial
soluble carbohydrate concentrations among all assays (12.8 and 13.5
gCHO.L-1, respectively). The high substrate concentration is likely to
have favored a higher organic acid production rate leading to a modi­
fication in the predominant metabolic pathways, which is unfavorable
for hydrogen production. In other studies, an increase in the H2 yield has
also been reported as the substrate concentration was adjusted, with a
reduction in this yield for substrate concentrations that resulted in the
process overload, causing organic acid accumulation and consequently a
marked pH reduction [68,69].
Considering the hypothesis that the initial soluble carbohydrates
were composed only of hexoses, H2 yields (Y2) with values between 0.53
and 2.13 molH2.mol-1hexose were obtained representing 13.3–53.2% of
the theoretical maximum yield, which is 4 molH2.mol-1hexose obtained
8
Journal of Environmental Chemical Engineering 11 (2023) 111252
in the metabolic reactions of the glucose to acetic acid conversion
pathway [70]. The yields of some assays were high, such as E-1 (2.06
molH2.mol-1hexose) and E-9 (2.13 molH2.mol-1hexose), considering the
practical limitations to reaching the theoretical maximum, as presented
by Thauer et al. [70]. Lee et al. [71] infer that the maximum yields
observed in fermentation studies have been up to 2.8 molH2.mol-1hex­
ose, which is about 70% of the theoretical yield, similar to the highest
yields obtained in the present study. As observed, it is highlighted that a
significant variation in Y2 is often verified due to different experimental
conditions evaluated, showing evidence of the importance of carrying
out an adequate adjustment of significant variables. Akhlaghi et al. [72],
for example, obtained H2 yields from 0.36 to 1.45 mol H2.mol-1hexose,
and Gioannis et al. [73] obtained Y2 from 0.04 to 2.6 mol H2.
mol-1lactose.
The limonene concentration was determined for initial and final
samples of the fermentation assays in batch reactors. As can be seen in
Fig. 4, the initial limonene concentration was generally less than 15 mg.
L-1. A significant difference was observed between the initial and final
limonene concentration in the assays, with a maximum removal of
96.7% in assay E-8 and a minimum of 9.9% in E-1, which represented a
removal of 9.0 mg.L-1 and 0.13 mg.L-1, respectively. Considering the
limonene concentrations reported by Camargo et al. [74], which resul­
ted in negative effects for P and λ, it is unlikely that there was inhibition
or negative effects on P related to the presence of this compound at the
concentrations observed in the present study, lower than 15 mg.L-1.
Camargo et al. [74] evaluated the effect of different limonene concen­
trations on the kinetic parameters of H2 production in batch reactors and
found that for concentrations of up to 100 mg.L-1, there was no signifi­
cant inhibitory effect for P. However, a statistical difference was
observed in the values of λ for assays with limonene concentration above
50.0 mg.L-1.
In the batch assays of the present study, the limonene removal likely
did not occur via degradation, considering the short duration of the
assays. Calabrò et al. [17] identified the D-limonene degradation in
batch reactors only after the 15th day of digestion, detecting the pres­
ence of degradation by-products such as p-cymene. The limonene
adsorption in the sludge was not evaluated as this mechanism has not
been observed as significant in the removal of this compound in
anaerobic reactors [74]. The adsorption process in the solid phase of
citrus waste is a likely mechanism that may have contributed to the
limonene removal from the liquid medium. Calabrò et al. [17] evaluated
the limonene content in anaerobic digestion in batch reactors and
identified adsorption of up to 76% in the solid phase of citrus waste,
pointing out that this mechanism plays an important role in reducing the
limonene content in the liquid phase of the reactors.
Fig. 4. Limonene concentration at initial and final, and limonene removal.
D.H.D. Rocha et al.
3.3. Volatile fatty acids
The analysis of the distribution of soluble metabolites in the assays
was carried out for complementary interpretation with the hydrogen
production data and possible understanding of the predominant meta­
bolic pathways in the assays of the experimental design. Acetic (HAc),
butyric (HBu), propionic (HPr), lactic (HLa), and formic (HFo) acids
were observed in at least one of the assays (Fig. 5).
As can be seen in Fig. 5 concerning the predominant metabolites at
the end of the fermentation process, lactic acid (HLa) was observed as
the main metabolite in assays A-4 with 4302.1 mg.L-1 (representing
64.1% of total), A-6 with 3333.5 mg.L-1 (60.3%), A-7 with 3926.3 mg.L-
1 2Lactate + H +
(70.5%), A-8 with 7312.2 mg.L-1 (79.6%), A-10 with 6367.8 mg.L-1
(81.9%), A-12 with 6247.5 mg.L-1 (78.0%) and A-13 with 3992.6 mg.L-1
(91.5%). However, for assay A-1, a predominance of acetic acid was
identified with 1897.3 mg.L-1 (63.6%); and for assays A-2, A-3, A-5, A-9,
A-11, A-14, and central points, acetic and butyric acids were observed as
the main metabolites, each one representing from 40% to 50% of the
total (Fig. 5).
For assays with HLa predominance, a correspondence pattern with
another important process response, which is hydrogen production, was
observed, except for E-6. The H2 yield for assays A-4, A-7, A-8, A-10, A-
12 and A-13 was only 0.63 ± 0.06 molH2.mol-1hexose, consistent with
what was expected considering the biochemical reactions of lactic acid
production, in which the carbohydrate molecules catabolism does not
involve the direct H2 production [75].
Among these assays with HLa predominance, for E-8, E-10, and E-12,
higher concentrations of this metabolite were observed, of 7.3 g.L-1,
6.4 g.L-1, and 6.3 g.L-1, respectively. In these assays, the combination of
high concentrations of both substrate (12.8 gCHO.L-1, 13.5 gCHO.L-1,
and 10.8 gCHO.L-1, respectively) and CaCO3 (5.9 g.L-1, 3.8 g.L-1, and
3.8 g.L-1, respectively) favored lactic bacteria populations impacting
hydrogen production under these conditions. However, for E-13, despite
the high HLa proportion to the total metabolites (91.5%), its concen­
tration was not observed as high (4.0 g.L-1) as in E-8, E- 10, and E-12.
The low CaCO3 concentration added to E-13 (0.3 g.L-1) was insufficient
to buffer the process, resulting in a significant pH reduction and
consequent inhibitory effects on fermentation as the pH final was only
3.52, the lowest of all assays. As observed in the present study, Tang
et al. [76] identified that the prevention of a sharp reduction in the
operational pH in the assays made it possible to obtain higher HLa
concentrations, avoiding pH values < 4.0 that were inhibitory to the
growth and activity of microorganisms.
For assay E-6, unlike what was identified for the other assays whose
HLa production was high, there was no correspondence with low H2
production as an intermediate H2 yield was obtained (1.17 molH2. mol-
1
hexose) for this assay. It is likely that the intermediate H2 yield ob­
tained, despite the predominance of HLa production, is related to H2
production via lactic acid oxidation pathways. The acid profile
Fig. 5. Organic acid distribution at the end of the experimental design assays.
9
Journal of Environmental Chemical Engineering 11 (2023) 111252
throughout this assay infers that the production of HBu and H2 occurred
from the HLa consumption (Eq. 9), highlighting that this metabolic
pathway is thermodynamically favorable (ΔG◦ =− 64.1) [77,78]. Meta­
bolic pathways of lactic acid utilization during the fermentation were
investigated by Baghchehsaraee et al. [79] in batch reactors using starch
as substrate. The authors observed that the HLa availability in the
fermentation medium resulted in a significant increase in hydrogen
production, associated with an increase in the butyric acid concentra­
tion. These results are similar to those observed in assay E-6, in which
partial consumption of the HLa produced occurred concomitantly with
the increase in HBu concentration, maintaining the H2 production.
butyrate + 2H2 + 2CO2 (9)
The metabolite profile of the assays with the highest hydrogen pro­
duction (E-14 and central point) was characterized by the prevalence of
acetic and butyric acids, totaling around 95% of the metabolites at the
process end. Substrate conversion probably occurred mainly through the
Clostridial pathway, which represents an advantage for higher H2 pro­
duction per mole of carbohydrate consumed [80].
In the central point assays (E-15/E-16/E-17), the concentrations of
HAc and HBu at the process end were 1590.1 ± 11.3 mg.L-1 and 2153.7
± 156.0 mg.L-1, respectively, and represented 40.1% (HAc) and 54.2%
(HBu) of the final composition of soluble metabolites. This characteristic
was similar to that observed for assay E-14 (1684.1 mg.L-1 of HAc and
2199.8 mg.L-1 of HBu), in which the H2 production was similar to the
central point assay. However, for E-1 (1897.3 mg.L-1) and E-4
(1785.2 mg.L-1), the HAc concentrations identified were similar or even
higher than the assays of higher hydrogen production (E-14, E-15, E-16,
and E-17). This aspect was not observed for butyric acid, whose con­
centration was notably higher in E-14, E-15, E-16, and E-17.
For assays E-3, E-5, E-9 and E-11, the HBu concentration (1305.6 mg.
L-1, 1728.8 mg.L-1, 1776.4 mg.L-1 and 1240.0 mg.L-1, respectively) was
intermediate, as was observed for hydrogen production in these assays,
close to 900 mLH2. L-1. Therefore, a higher correlation between H2
production and HBu predominance was identified.
Matyakubov et al. [81] verified that the HBu/HAc ratio has an
important correlation with the H2 yield. A relationship between 0.3 and
1.5 was observed in the present study for citrus waste. Thus, the assays
with the highest values for HBu/HAc (>0.80) were those with the
highest P. For example, an HBu/HAc ratio of 1.35 was obtained for
central point assays, while for E-4, with P of 578 mLH2. L-1, this ratio was
0.30.
3.4. Validation of the hydrogen production model
The adjustment provided by the experimental design and the
response surface model led to an optimized P of 1249.04 mLH2. L-1, and
a maximum production rate (Rm) of 89.60 mLH2. L-1.h-1, with time to
start production of 8.56 h (Fig. 6), in an experimental condition with
21.4 gTVS.L-1 of CPW, 3.4 gCOD.L-1 of CPWW, and 4.5 g.L-1 of CaCO3
(Table 5). The hydrogen production obtained is 5.3 times higher (530%)
than the H2 production verified in assay E-13 (199.21 mLH2. L-1), for
example, in which the lowest H2 production was obtained.
In the experimental condition of the optimized assay (OC-H) was
obtained a gCaCO3/gCHO ratio of 0.53; this value is an important
guideline about alkalinization for fermentation focused on the H2 pro­
duction from citrus waste. A sharp decrease in pH (from 6.62 to 5.65)
between the start and the exponential phase of H2 production
(~10–12 h) was verified (Fig. 6). However, until the process ended
(~42 h), there was a low pH variation (from 5.65 to 5.51), even with
high production of H2 and organic acids. Therefore, adjusting the con­
ditions allowed sufficient buffering for the process to occur stably, even
with a high initial substrate concentration (8.4 gCHO.L-1). For OC-H was
obtained 86.6% of carbohydrate removal and H2 yield of 195.07 mLH2.
g-1CHO or 2.07 molH2.mol-1hexose.
The H2 percentage in the biogas composition of the optimized
D.H.D. Rocha et al. Journal of Environmental Chemical Engineering 11 (2023) 111252

its use, but also for transport and storage [82,83].

Fig. 6. Accumulated H2 production fitted to the modified Gompertz model, for
validation assay.
Based on the metabolite distribution profile throughout the process,
it was found that acetic and butyric acids were the metabolites at the
highest concentration throughout the entire assay; however, changes to
their proportions in the total metabolite composition were observed
(Fig. 7). It is noteworthy that after 30 h, an increase in the HBu con­
centration was observed at a higher proportion than that observed for
HAc, in which butyric acid was the soluble metabolite at the highest
concentration at the assay end (2230.8 mg.L-1), while the acetic acid
concentration was 1213.6 mg.L-1, representing proportions of 60.8%
and 33.1% of the total soluble metabolites, respectively.
For OC-H, an HBu/HAc ratio of 1.87 was obtained. This value is
higher than that verified for all assays previously performed in the
factorial design, and consistent with what is described in the literature
[81]; that is, a higher HBu/HAc ratio is associated with a higher H2
production.
As can be seen in Table 6, H2 production from citrus waste is higher
than that obtained by other studies that evaluated co-digestion, for
example, compared to Villa Montoya et al. [36] when evaluating the
co-digestion of solid waste and wastewater from coffee processing. The

Table 5 Table 6
Experimental condition and main parameters for the validation assay of the Comparison of optimized H2 production from citrus waste with H2 production by
optimized condition of H2 production. other lignocellulosic substrates.

Experimental Condition Substrate Optimized Conditions H2 Reference

production*
-1
CPW 21.4 gTVS.L Predict Value
CPWW 4.5 gCOD.L-1 P (mLH2. L-1) Solid waste and 21.4 gTVS.L-1 of CPW, 4.5 1249.1 mL.L- This study
Calcium Carbonate 3.4 g.L-1 1287.5 wastewater from gCOD.L-1 of CPWW, and 1

Parameters of the validation assay citrus processing 3.4 g.L-1 of CaCO3

P (mL.L-1) 1249.04 ± 40.1 Pulp/Husk and 7.0 g.L-1 of coffe pulp and 240 mL [36]
Rm (mL.L-1.h-1) 89.60 wastewater from husk, 30% headspace, (686 mL.L-1)
λ (h) 8.56 coffe processing initial pH of 7.0
%H2 47.5% Citrus Peel Waste 4 gTVS.L-1 of sludge, 29.8 48.47 mmol. [35]
Y1 (mLH2.g-1CHO) 195.07 gTVS.L-1 of CPW, and L-1
Y2 (molH2.mol-1 hexose) 2.07 initial pH of 8.98 (1085 mL.L-
1
Final pH 5.51 )
gCaCO3.g-1 CHO 0.53 Sugarcane bagasse 2.77 g.L-1 of yeast extract 1.5 mmol.L-1 [25]
Carbohydrate Removal (%) 86.6% and 5.84 g.L-1 of (36 mL.L-1)
Predominant Organic Acid HBu sugarcane bagasse
HBu/HAc 1.84 Sugarcane bagasse 7.0 g.L-1 of sugarcane 23.1 mmol.L- [34]
1
bagasse, initial pH of 7.2
-1
(517 mL.L )
Banana waste 5.0 g.L-1 of substrate, 70.2 mL [37]
condition was 47.5%. This H2 percentage is important for equating the
temperature of 37 ◦ C, (468 mL.L-1)
technical and economic viability of energy recovery from the biological initial pH of 7.0
H2 production as the biogas obtained in the fermentation is not feasible *
values in parentheses were approximately based on data provided by the
for its direct use, due to the presence of other gases. Moreover, hydrogen
authors and using the ideal gas conversion of 1 mol = 22.4 liters.
separation/concentration processes are fundamental not only to enable

Fig. 7. Temporal profile for organic acid distribution in the validation assay.

10
D.H.D. Rocha et al.
results of H2 production obtained in the present study are also higher
than those of studies that used the mono-digestion of agro-industrial
waste as an alternative, such as Mazareli et al. [37] with banana
waste, and Soares et al. [25] and Rabelo et al. [34] with sugarcane
bagasse.
The technical viability of the co-digestion of wastewater and solid
waste from citrus processing can be inferred considering its H2 pro­
duction compared with mono-digestion assays of citrus waste. For
example, Camargo et al. [35] obtained an H2 production of 1085 mLH2.
L-1; that is, 15.0% lower than that obtained in the present co-digestion
study. Therefore, from the point of view of H2 production, and also
based on other process parameters, such as carbohydrate removal, and
%H2 in biogas, the co-digestion proposal is technically feasible as it
enables important energy content recovery using two residual flows
from the same productive chain. Another disadvantage to the
mono-digestion of CPW is the water demand, which in some situations
can be considered a bottleneck for the process viability. Among the H2
production routes, dark fermentation is one of the most water-intensive,
with a high water footprint [84]. Co-digestion with processing waste­
water allows for a change in the approach to water footprint analysis for
this H2 production route, with a reduction in the water footprint. Under
the optimized conditions of the present study, wastewater meets about
40% of the process water demand; considering organic matter concen­
tration of the CPWW and OC-H assay concentration of 3.4 gCOD.L-1.
3.5. Microbial community characterization
Microbial community characterization was carried out to identify the
predominant microorganism groups in the reactors of the selected as­
says, helping to infer the metabolic profile of the process. For this pur­
pose, samples from two reactors with antagonistic results for the main
responses of the process, which are the production of H2 and soluble
metabolites, were analyzed.
The first sample selected was from the optimized condition valida­
tion assay reactor - OC-H (21.4 gTVS.L-1 of CPW, 4.5 gCOD.L-1 of CPWW,
and 3.4 g.L-1 of CaCO3), with the highest P, and with HAc and HBu as the
predominant soluble metabolites. The second sample was from the E-10
assay reactor (35.1 gTVS.L-1 of CPW, 4.5 gCOD.L-1 of CPWW, and 3.8 g.
L-1 of CaCO3), with lower P and Rm, and absolute HLa predominance
among the soluble metabolites.
For the OC-H and E-10 assays samples, 45,813 and 37,906 16 S rRNA
gene sequences were obtained, grouped into 19 ASV and 33 ASV
(Amplicon Sequencing Variants), respectively. The similarity obtained
between the samples was only 26.8%, and probably the different
nutritional conditions resulted in a significant difference in the bacterial
population’s distribution in the batch reactors.
The microbial diversity in the samples was evaluated based on the
Shannon-Wiener and Simpson ecological indices. Significantly higher
diversity (1.30) and lower dominance (0.34) were observed at E-10;
while in the optimization assay (OC-H), were 0.30 and 0.90, respec­
tively. It is likely that the statistical adjustment of the variables in the
assay OC-H to establish a condition with maximum hydrogen production
potential with operational pH maintenance in a stable range from 5.5 to
6.5 throughout the fermentation, provide a favorable environment to
the development and establishment of specific populations, without
creating transitory environments to favor other bacteria groups, thus
resulting in lower diversity and higher dominance. Based on the
Shannon-Wiener index, Camargo et al. [35] also identified a reduction
in diversity for the sample of an optimized condition reactor for H2
production from citrus waste, attributing this fact to the population’s
selection due to the process variable optimization to favor the H2
production.
For both samples, from the OC-H and E-10 assays, bacteria belonging
to the Firmicutes phylum were identified as the ones with the highest
relative abundance, respectively 96.9% and 93.7%. For assay OC-H, the
other phyla identified were Proteobacteria (3.0%), Halobacterota, and
11
Journal of Environmental Chemical Engineering 11 (2023) 111252
Chloroflexi with abundance < 0.01%. Regarding E-10 Proteobacteria
(5.9%), Halobacterota (0.2%), Actinobacteriota (~0.1%), Desulfo­
bacterota (~0.1%), and Chloroflexi, Spirochaetota, and Synergistota
with relative abundance < 0.01%. The abundance observed for the
Firmicutes phylum for the assays OC-H and E-10 is in line with what has
been reported in several studies applied to the various substrates
fermentation [85–87], characterized by bacteria with a wide physio­
logical variety in the organic matter fermentation [88].
For the OC-H assay, bacteria belonging to the Clostridiaceae family
were identified in high abundance, with a relative abundance of
96.15%, and Enterobacteriaceae (3.0%) and Peptostreptococcaceae
(0.6%) were identified with lower abundance. However, for E-10, bac­
teria belonging to three families of the Firmicutes phylum were identi­
fied, such as Clostridiaceae (51.6%), Lactobacillaceae (21.9%), and
Peptostreptococcaceae (20.0%); in addition to Enterobacteriaceae
(5.58%), Moraxellaceae (~0.2%) and Methanosaetaceae (~0.2%), in
lower relative abundance. A significant reduction in the relative abun­
dance of the Clostridiaceae family in assay E-10 was observed, compared
to the assay OC-H (Fig. 8), from 96% to 50%, respectively. Combined
with this abundance reduction, an increase in the relative abundance of
the Peptostreptococcaceae family was observed in E-10.
Regarding the genera, for the optimized condition assay, only two of
them were found with relative abundance > 1%, Clostridium sensu stricto
1 (95.0%) and Enterobacter (3.0%), and other genera with relative
abundance < 1%, such as Paraclostridium (0.54%), Clostridium sensu
stricto 2 (0.34%), Clostridium sensu stricto 10 (0.10%), and Hathewaya
(0.14%). For assay E-10, Clostridium sensu stricto 1 was also identified as
the one with the highest relative abundance (51.2%), in addition to
Paraclostridium (20.0%) and Lactobacillus (21.9%). This change in these
genera abundance can be pointed out as the most important change in
the structure of the E-10 microbial community compared to assay OC-H.
Other genera such as Enterobacter (4.7%), Kosakonia (0.6%), Clostridium
sensu stricto 18 (0.4%), Klebsiella (~0.2%), and Acinetobacter (~0.2%)
were also identified, however at low relative abundances in both assays
(E-10 and OC-H) (Fig. 8).
The granular sludge from a mesophilic UASB reactor for wastewater
from a poultry slaughterhouse treatment used as inoculum for the assays
is characterized by a mixed culture with a diversity of bacteria and
Fig. 8. Taxonomic groups for bacteria Genus, for assays Optimized condition
(OC-H) and E-10.
D.H.D. Rocha et al.
archaea [89], and therefore, the strategy of application of the heat
treatment was adopted as a way to establish a selective pressure, fa­
voring endospore-forming microorganisms in response to environ­
mental stress, such as the genus Clostridium sp. [90]. However, even
starting from the same inoculum, the different nutritional conditions
applied to the assays OC-H and E-10 resulted in the establishment of
microbial communities with important differences in the relative
abundance of the main genera identified in the reactors.
Bacteria groups similar to Paraclostridium and Clostridium sp. are
characterized by endospore formation [91]. This characteristic was
likely important so that under favorable conditions to their develop­
ment, as was assay E-10, these genera could play a fundamental role in
the microbial community structure, which was identified with high
abundance. Another microorganism group with important relative
abundance in E-10 was Lactobacillus, which is endospore negative [92].
The origin of these bacteria is likely from autochthonous sources of solid
waste [93] and wastewater from citrus processing [94].
Clostridium sensu stricto 1 mainly uses carbohydrates and amino acids
as a substrate, with butyric acid as its main metabolite product [95];
furthermore, this genus is widely reported as a notable hydrogen pro­
ducer, capable of using other types of more complex substrates such as
food waste, starch, cellulose [96]. These metabolic characteristics are
consistent with the responses observed for the H2 optimization assay
(OC-H), with a P of 1249.1 mL.L-1, and 2230.8 mg.L-1 of HBu. Lower
process pH stability in E-10 due to a lower gCaCO3/gCHO ratio
compared to OC-H, given the high CPW concentration (35.1 gTVS.L-1)
for 3.8 g.L-1 of CaCO3, resulted in a reduction of Clostridium sensu stricto
1 abundance in this assay, which is likely to be associated with lower
production of H2 (507.1 mL.L-1), acetic and butyric acids (867.1 mg.L-1
and 393.5 mg.L-1, respectively).
Luo et al. [95] point out that the H2 production, and HBu and HAc
concentrations, were positively correlated with Clostridium sensu stricto 1
in batch reactors with food waste. Similarly, Mazareli et al. [97]
observed an important abundance of Clostridium sp. (~40%) in
fermentation assays in batch reactors using banana waste for producing
H2, acetic and butyric acids. Soares et al. [25] and Rabelo et al. [34] also
identified Clostridium sp. in the batch reactors with sugarcane bagasse,
associated with acetic and butyric acids as the main soluble metabolites
of the process.
Lactobacillus, identified in high abundance in assay E-10, performs
carbohydrate fermentation, in which lactic acid is the main by-product,
in addition to acetic acid, ethanol, formic acid, and CO2 [92]. High lactic
acid production is often associated with lactic acid bacteria (LAB)
belonging to the genus Lactobacillus, classified into homofermentative
and heterofermentative [98]. The significant lactic acid production
(6367.8 mg.L-1) in assay E-10 and the high proportion (81.9%) of this
among the soluble metabolites, such as HAc (867.1 mg.L-1), allow us to
infer that this genus’ abundance was associated with mostly homo­
fermentative LAB [99]. The significant reduction in H2 production in
assay E-10, associated with the higher Lactobacillus abundance, may also
be related to bacteriocins secreted by LAB, which have deleterious ef­
fects on other bacteria [100]. Furthermore, Liu et al. [100] point out that
Lactobacillus sp. produces high amounts of CO2, which is an adverse
condition for stabilizing the H2 concentration in biogas. This fact may be
associated with the low H2 percentage (33.2%) in the biogas composi­
tion for assay E-10 compared to OC-H (47.5%).
Similar to the results observed in the present study, other authors
have observed a reduction and failures in H2 production associated with
an increase in the Lactobacillus abundance in fermentation reactors. For
example, Jo et al. [101] evaluated the H2 production from food waste in
a continuously stirred reactor and observed that the Lactobacillus spp.
abundance increase was associated with reduced H2 production and
changes in the soluble metabolite distribution, with an increased lactic
acid concentration in the effluent. Park et al. [102] observed changes in
the microbial community of the reactor throughout the operation with
an increase in the relative abundance of Lactobacillus sp., which
12
Journal of Environmental Chemical Engineering 11 (2023) 111252
negatively affected the H2 production, and resulted in lactic acid
accumulation.
Paraclostridium identified in assay E-10 has been commonly identi­
fied in hydrogen production reactors [103] and is capable of using a
wide variety of organic substrates [104] in addition to its hydrolytic
metabolism [105].
It is also noteworthy that for both assays, Enterobacter was identified
with relative abundance between 3% and 5%. Bacteria similar to this
genus are characterized by rapid growth using various substrates [106],
and robustness to pH variation in fermentation reactors [107]. The
Enterobacter metabolism is also characterized by lactic acid production.
For example, Thapa et al. [108] verified high levorotatory lactic acid
production (L-lactic acid) (46.0 g.L-1) by Enterobacter sp. in batch re­
actors with synthetic medium and various carbohydrates (glucose,
mannitol, sucrose, fructose).
Paraclostridium and Enterobacter have metabolic characteristics
similar to those of other genera identified with high abundance in E-10
and OC-H assays, such as Clostridium sensu stricto 1 and Lactobacillus,
resulting in a soluble metabolite distribution with a restricted diversity
of organic acids, basically with HAc and HBu in OC-H, and HLa in E-10.
The assessment that there was not a wide metabolic diversity is an
interesting aspect of the H2 optimization assay (OC-H) considering the
objective of directing the substrate utilization towards pathways of
higher H2 yield, as was observed.
Prediction of the metabolic pathways involved in the citrus waste
fermentation was performed for assays OC-H (4.5 of gCaCO3. L-1, 21.4
gTVS.L-1 of CPW, 3.4 gCOD.L-1 of CPWW) and E-10 (3.8 of gCaCO3. L-1,
35.1 gTVS.L-1 of CPW, 4.5 gCOD.L-1 of CPWW) (Fig. 9). The metabolic
prediction was carried out based on the main genera (Clostridium,
Paraclostridium, Lactobacillus, and Enterobacter) and the main meta­
bolic products identified (acetic, butyric, lactic, propionic acids, and
hydrogen).
For assay OC-H, the hydrogen production was associated with in­
termediate reactions of the pathways for the formation of acetic and
butyric acids, with no inference for the occurrence of lactic acid pro­
duction pathways. The conversion of pyruvic acid to acetyl-CoA, and
subsequently to other metabolites such as HAc and HBu, has been
identified as a key reaction for OC-H. This reaction, characteristic of
clostridial fermentation [109], is catalyzed by the enzyme
pyruvate-ferredoxin oxidoreductase [1.2.7.1] (PFOR), in which reduced
ferredoxin (Fdred) is also formed, which is subsequently reoxidized
resulting in hydrogen formation by the action of hydrogenases [110,
111]. According to the inference, the acetic acid formation occurred
predominantly through the clostridial pathway, for which the role of the
enzymes phosphate acetyltransferase [2.3.1.8] and acetate kinase
[2.7.2.1] stands out. Regarding the butyric acid, the main catalytic en­
zymes of the reactions for this metabolic pathway were acetyl-CoA
acetyltransferase [2.3.1.9], butyrate kinase [2.7.2.7], and
hydroxybutyryl-CoA dehydrogenase [1.1.1.157], attributed mainly to
the metabolism of microorganisms of the genus Clostridium.
For assay E-10, a fundamental role of the homofermentative meta­
bolism of lactic acid bacteria was inferred, highlighting the importance
of the enzyme L-lactate dehydrogenase [1.1.1.27] (Fig. 9), which cata­
lyzes the conversion reaction of pyruvic acid into HLa, with the ability to
produce this enzyme in fermentative processes attributed mainly to
microorganisms of the genus Lactobacillus [112]; but some microor­
ganisms of the genera Clostridium and Paraclostridium are also associ­
ated with the production of L-lactate dehydrogenase [55].
3.6. Energy balance assessment
The energy balance of the H2 optimization assay (OC-H) was calcu­
lated and taken as a reference scenario (SCN-R), comparing it to alter­
native scenarios represented by assays E-7 (SCN-A1) and E- 13 (SCN-
A2), whose conditions resulted in maximum and minimum buffer de­
mands per unit of energy produced, respectively.
D.H.D. Rocha et al.
Fig. 9. Simplified metabolic map, with the probable enzymes (EC) in the conversion of citrus waste in H2 and organic acids, in the optimized condition for H2
production (OC-H), and assay E-10.
For the reference scenario (SCN-R), the EPH2 was 523.2 MJ.t-1CPW,
while for the alternative scenarios, it was only 253.0 MJ.t-1CPW for SCN-
A1 and 79.4 MJ.t-1CPW for SCN-A2 (Table 7). It can be observed by the
EPH2 values that the H2 production optimization resulted in a significant
gain in the energy recovery potential. However, it should be noted that
despite this, the H2 energy potential still represents a tiny portion of the
13
Journal of Environmental Chemical Engineering 11 (2023) 111252
total energy content of the citrus waste, around 3%, considering the
lower heating value of 16.97 MJ.t-1CPW [113].
Regarding the electricity production potential (EPPH2) from the
hydrogen utilization in a fuel cell, values of 0.872 MWh.t-1CPW for SCN-
R, 0.422 and 0.132 MWh.t-1CPW for SCN-A1 and SCN-A2 were obtained,
respectively. Despite not representing a high electricity production,
D.H.D. Rocha et al.
Table 7
Energy Potential (EPH2), Electricity Production Potential (EPPH2), and buffering
agent demand for different experimental conditions of hydrogen production.
Scenario Assays MJ.t-1 MWh. t-1 tCaCO3. US$.MWh-
CPW CPW MWh-1 1
SCN-R OC-H 523.2 0.087 2.00 941.45
SCN-A1 E-7 253.0 0.042 7.74 3641.33
SCN-A2 E-13 79.4 0.013 0.75 351.85
EPPH2 can meet a portion of the energy demand of the processing in­
dustry itself. Galvagno et al. [50] performed an energy integration
analysis for a citrus juice production plant localized in Sicily - Italy. In
this evaluation to process 72,000 tons of citrus per year, there was a
demand close to 9000 MWh and the generation of close to 8600 tons of
dry waste (residual moisture < 20%). Taking into account this electricity
demand, waste generation, and the EPPH2 obtained in the present study,
it can be inferred that the energy recovery based on the H2 production
for SCN-R could supply 8.3% of the electricity demand of an industry
with characteristics similar to the one that was the object of the study by
Galvagno et al. [50], while SCN-A1 would supply 4.0%, and SCN-A2
only 1.3%.
An important comparison between the evaluated scenarios is related
to the buffering agent demand, representing an important drawback for
the economic viability of energy recovery from hydrogen fermentation
since the average cost per ton of calcium carbonate is US$ 470.35 [114].
For SCN-R relative to the optimized condition of H2 production, a de­
mand of 2.0 tCaCO3. MWh-1is identified, which results in an associated
cost of 941.45 US$. MWh-1, higher than the demand and the cost asso­
ciated obtained for SCN-A2 which was 0.75 tCaCO3. MWh-1, resulting in
a cost of 351.85 US$. MWh-1. However, despite the lower cost associated
with alkalinization in SCN-A2, EPPH2 in this condition was incipient,
almost 7 times lower than that obtained in the reference scenario
(SCN-R). By observing the demand for the buffering agent (7.74 tCaCO3.
MWh-1) and the associated cost (3641.33 US$. MWh-1) for SCN-A1, it
can be seen how a non-optimized condition can result in a distancing
from the process viability. Therefore, economic aspects related to pro­
cess inputs should be evaluated as variables in studies that aim to
evaluate the energy recovery potential from H2 fermentation, aiming at
process scale-up.
To the process scale-up, an alternative would be the incorporation of
an additional co-substrate in the proposed co-digestion of citrus agro-
industry waste, which can effectively contribute to the system’s alka­
linity, reducing the demand for buffering agents supplementation. As
discussed by Neshat et al. [115], animal manure is a feasible substrate
for anaerobic digestion due to its high buffering capacity, contributing
to process stability. Li et al. [116] verified an alkalinity of 4.2 gCaCO3.
L-1 for cattle manure, indicating that this material could increase the
buffering capacity of the digesters, preventing failures in the process
related to acidification. Yin et al. [117] reported a high alkalinity of 5.09
gCaCO3. L-1 for swine manure, highlighting the high ammonia release
contributing to the VFAs neutralization [118].
4. Conclusions
The application of optimization methodology made it possible to
obtain a maximum hydrogen production of 1249.0 mL.L-1 from the
citrus waste co-digestion (21.4 gTVS.L-1 of CPW, 3.4 gCOD.L-1 of
wastewater, and 4.5 g.L-1 of CaCO3), such production was up to 5.3
times higher than the production obtained in the non-optimized con­
ditions. The optimized condition for hydrogen fermentation was asso­
ciated with the Clostridial metabolic pathway, with a high abundance of
the genus Clostridium sensu stricto 1 (95.0%), with predominance of
acetic and butyric acids. For the opposite condition, with low H2 pro­
duction, there was a predominance of lactic acid fermentation with
increased abundance for Lactobacillus (21.9%) and Paraclostridium
14
Journal of Environmental Chemical Engineering 11 (2023) 111252
(20.0%).
A maximum energy potential of 523.2 MJ.t-1CPW was estimated,
based on the optimized H2 production, representing an electricity pro­
duction potential of 0.872 MWh.t-1CPW. In this condition, the buffering
agent demand was 2.0 tCaCO3. MWh-1. It is concluded, from the results
obtained that the co-fermentation of citrus waste is a technically feasible
alternative for hydrogen fermentation, considering the H2 production
achieved; however, alternatives must be evaluated to meet the demand
for alkalinity in the system. These results are important to guide the
investigation of the process in continuous and semi-continuous reactors,
allowing the process scale-up to reach the industrial application.
CRediT authorship contribution statement
Danilo Henrique Donato Rocha: Conceptualization, Methodology,
Formal analysis, Investigation (reactor’s monitoring, data acquisition
and interpretation), Data curation, Writing – original draft and Writing –
review & editing, Visualization. Isabel Kimiko Sakamoto: Molecular
Biology, Formal analysis (metabolic pathway analysis). Maria Berna­
dete A. Varesche: Conceptualization, Methodology, Resources, Writing
– review & editing, Supervision, Project administration, Funding
acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data Availability
Data will be made available on request.
Acknowledgements
The authors gratefully acknowledge the São Paulo Research Foun­
dation (FAPESP Processes 2015/02640-2), and the Coordination for the
Improvement of Higher Education Personnel (CAPES – Finance Code
001).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.jece.2023.111252.
References
[1] L.C. Freitas, J.R. Barbosa, A.L.C. Costa, F.W.F. Bezerra, R.H.H.J. Pinto, R.
N. Carvalho, From waste to sustainable industry: How can agro-industrial wastes
help in the development of new products ? Resour. Conserv Recycl 169 (2021),
105466 https://doi.org/10.1016/j.resconrec.2021.105466.
[2] S. Pan, M. Alex, I. Huang, I. Liu, E. Chang, P. Chiang, Strategies on
implementation of waste-to-energy ( WTE) supply chain for circular economy
system: a review, J. Clean. Prod. 108 (2015) 409–421, https://doi.org/10.1016/j.
jclepro.2015.06.124.
[3] B.B. Kist, C. Carvalho, M. Treichel, C.E. Santos, Anuário brasileiro da fruticultura,
Gaz. St Cruz (2018) 88.
[4] FAO. Food and agriculture data 2019. 〈http://www.fao.org/faostat/en/#home〉
(accessed July 15, 2020).
[5] CITURSBR. Total Brazilian Orange Juice Exports. Brazilian Assoc Citrus Export
2020. 〈http://www.citrusbr.com/en/statistics/?ins=03〉.
[6] BRASIL. Grupos de Produtos: Exportação Brasileira. Ministério Da Econ - Secr
Comércio Exter 2020. 〈http://www.mdic.gov.br/index.php/comercio-exterior/
estatisticas-de-comercio-exterior/series-historicas〉 (accessed July 15, 2020).
[7] Tetra Pak. The Orange Book. Lund, Sweden: 2004.
[8] E.S.R. Mendoza, J.M.M. Contreras, A.M. Sibaja, N.A.V. Cantú, A.L. Alvarado,
Anaerobic digestion of citrus industry effluents using an Anaerobic Hybrid
Reactor, Clean. Technol. Environ. Policy (2018) 1–11, https://doi.org/10.1007/
s10098-017-1483-1.
[9] A. Koppar, P. Pullammanappallil, Anaerobic digestion of peel waste and
wastewater for on site energy generation in a citrus processing facility, Energy 60
(2013) 62–68, https://doi.org/10.1016/j.energy.2013.08.007.
D.H.D. Rocha et al.
[10] P.S. Calabro, A. Folino, V. Tamburino, G. Zappia, D.A. Zema, S.M. Zimbone,
Valorisation of citrus processing waste: a review. Sixt. Int. Waste Manag. Landfill
Symp, CISA Publisher,, Cagliari, Italy, 2017.
[11] E.S. Rosas-Mendoza, J.M. Mendez-Contreras, A.A. Aguilar-Lasserre, N.A. Vallejo-
Cantú, A. Alvarado-Lassman, Evaluation of bioenergy potential from citrus
effluents through anaerobic digestion, J. Clean. Prod. 254 (2020) 1–11, https://
doi.org/10.1016/j.jclepro.2020.120128.
[12] D.A. Zema, P.S. Calabro, A. Folino, V. Tamburino, G. Zappia, S.M. Zimbone,
Wastewater management in citrus processing industries: an overview of
advantages and limits, Water 11 (2019) 1–23, https://doi.org/10.3390/
w11122481.
[13] D.A. Zema, P.S. Calabrò, A. Folino, V. Tamburino, G. Zappia, S.M. Zimbone,
Valorisation of citrus processing waste: A review, Waste Manag. 80 (2018)
252–273, https://doi.org/10.1016/j.wasman.2018.09.024.
[14] Z. Berk. Citrus Fruit Processing, 1st ed., Elsevier, 2016 doi:books/citrus-fruit-
processing/berk/978-0-12-803133-9.
[15] P.S. Calabró, E. Paone, D. Komilis, Strategies for the sustainable management of
orange peel waste through anaerobic digestion, J. Environ. Manag. 212 (2018)
462–468, https://doi.org/10.1016/j.jenvman.2018.02.039.
[16] J.A. López, Q. Li, I.P. Thompson, Biorefinery of waste orange peel, Crit. Rev.
Biotechnol. 30 (2010) 63–69, https://doi.org/10.3109/07388550903425201.
[17] P.S. Calabrò, L. Pontoni, I. Porqueddu, R. Greco, F. Pirozzi, F. Malpei, Effect of the
concentration of essential oil on orange peel waste biomethanization: preliminary
batch results, Waste Manag 48 (2016) 440–447, https://doi.org/10.1016/j.
wasman.2015.10.032.
[18] P.S. Calabrò, F. Fazzino, R. Sidari, A.D. Zema, Optimization of orange peel waste
ensiling for sustainable anaerobic digestion, Renew. Energy 154 (2020) 849–862,
https://doi.org/10.1016/j.renene.2020.03.047.
[19] A. Tenca, A. Schievano, F. Perazzolo, F. Adani, R. Oberti, Bioresource Technology
Biohydrogen from thermophilic co-fermentation of swine manure with fruit and
vegetable waste: Maximizing stable production without pH control, Bioresour.
Technol. 102 (2011) 8582–8588, https://doi.org/10.1016/j.
biortech.2011.03.102.
[20] D.E. Algapani, W. Qiao, M. Ricci, M. Bianchi D, S. Wandera, F. Adani, et al., Bio-
hydrogen and bio-methane production from food waste in a two-stage anaerobic
digestion process with digestate recirculation, Renew. Energy 130 (2019)
1108–1115, https://doi.org/10.1016/j.renene.2018.08.079.
[21] S.F. Fu, R. Liu, W.X. Sun, R. Zhu, H. Zou, Y. Zheng, et al., Enhancing energy
recovery from corn straw via two-stage anaerobic digestion with stepwise
microaerobic hydrogen fermentation and methanogenesis, J. Clean. Prod. 247
(2020), 119651, https://doi.org/10.1016/j.jclepro.2019.119651.
[22] A.C. Villa Montoya, R. Cristina da Silva Mazareli, T.P. Delforno, V.B. Centurion, I.
K. Sakamoto, V. Maia de Oliveira, et al., Hydrogen, alcohols and volatile fatty
acids from the co-digestion of coffee waste (coffee pulp, husk, and processing
wastewater) by applying autochthonous microorganisms, Int J. Hydrog. Energy
44 (2019) 21434–21450, https://doi.org/10.1016/j.ijhydene.2019.06.115.
[23] F.P. Camargo, I. Kimiko, A. Bize, I. Cristina, S. Duarte, E. Luiz, Screening design of
nutritional and physicochemical parameters on bio-hydrogen and volatile fatty
acids production from Citrus Peel Waste in batch reactors, Int J. Hydrog. Energy
46 (2020) 7794–7809, https://doi.org/10.1016/j.ijhydene.2020.06.084.
[24] A.G.L. Moura, C.A.B.S. Rabelo, C.H. Okino, S.I. Maintinguer, E.L. Silva, M.B.
A. Varesche, Enhancement of Clostridium butyricum hydrogen production by
iron and nickel nanoparticles: Effects on hydA expression, 28477–28461, Int J.
Hydrog. Energy 45 (2020), https://doi.org/10.1016/j.ijhydene.2020.07.161.
[25] L.A. Soares, C.A. Borges Silva Rabelo, I.K. Sakamoto, T.P. Delforno, E.L. Silva, M.
B.A. Varesche, Metagenomic analysis and optimization of hydrogen production
from sugarcane bagasse, Biomass-.-. Bioenergy 117 (2018) 78–85, https://doi.
org/10.1016/j.biombioe.2018.07.018.
[26] D.H.D. Rocha, I.K. Sakamoto, M.B.A. Varesche, Evaluation of significant factors in
hydrogen production and volatile fatty acids in co-fermentation of citrus peel
waste and processing wastewater, Fuel 354 (2023), 129306, https://doi.org/
10.1016/j.fuel.2023.129306.
[27] L. Sillero, R. Solera, M. Perez, Anaerobic co-digestion of sewage sludge, wine
vinasse and poultry manure for bio-hydrogen production, Int J. Hydrog. Energy
47 (2022) 3667–3678, https://doi.org/10.1016/j.ijhydene.2021.11.032.
[28] A. Rabii, S. Aldin, Y. Dahman, E. Elbeshbishy, A Review on Anaerobic Co-
Digestion with a Focus on the Microbial Populations and the Effect os Multi-Stage
Digester Configuration, Energies 12 (2019) 1–25, https://doi.org/10.3390/
en12061106.
[29] G. Esposito, L. Frunzo, A. Giordano, F. Pirozzi, A. Liotta, F. Panico, Anaerobic co-
digestion of organic wastes, Environ. Sci. Biotechnol. (2012) 325–341, https://
doi.org/10.1007/s11157-012-9277-8.
[30] M.A. De la Rubia, V. Fernández-Cegrí, F. Raposo, R. Borja, Influence of particle
size and chemical composition on the performance and kinetics of anaerobic
digestion process of sunflower oil cake in batch mode, Biochem Eng. J. 58–59
(2011) 162–167, https://doi.org/10.1016/j.bej.2011.09.010.
[31] L. Pontoni, G. D’Antonio, G. Esposito, M. Fabbricino, L. Frunzo, F. Pirozzi,
Thermal pretreatment of olive mill wastewater for efficient methane production:
Control of aromatic substances degradation by monitoring cyclohexane
carboxylic acid, Environ. Technol. (U. Kingd. ) 36 (2015) 1785–1794, https://doi.
org/10.1080/09593330.2015.1012179.
[32] A.C. dos Santos, E. Ximenes, Y. Kim, M.R. Ladisch, Lignin–enzyme interactions in
the hydrolysis of lignocellulosic biomass, Trends Biotechnol. 37 (2019) 518–531,
https://doi.org/10.1016/j.tibtech.2018.10.010.
[33] A. Elhalil, H. Tounsadi, R. Elmoubarki, F.Z. Mahjoubi, M. Farnane, Factorial
experimental design for the optimization of catalytic degradation of malachite
15
Journal of Environmental Chemical Engineering 11 (2023) 111252
green dye in aqueous solution by Fenton process, Water Resour. Ind. 15 (2016)
41–48, https://doi.org/10.1016/j.wri.2016.07.002.
[34] C.A.B.S. Rabelo, L.A. Soares, I.K. Sakamoto, E.L. Silva, M.B.A. Varesche,
Optimization of hydrogen and organic acids productions with autochthonous and
allochthonous bacteria from sugarcane bagasse in batch reactors, J. Environ.
Manag. 223 (2018) 952–963, https://doi.org/10.1016/j.jenvman.2018.07.015.
[35] F.P. Camargo, I.K. Sakamoto, T.P. Delforno, M. Mariadassou, V. Loux, I.C.
S. Duarte, et al., Microbial and functional characterization of an allochthonous
consortium applied to hydrogen production from Citrus Peel Waste in batch
reactor in optimized conditions, J. Environ. Manag. 291 (2021), 112631, https://
doi.org/10.1016/j.jenvman.2021.112631.
[36] A.C. Villa Montoya, R.C. da Silva Mazareli, T.P. Delforno, V.B. Centurion, V.M. de
Oliveira, E.L. Silva, et al., Optimization of key factors affecting hydrogen
production from coffee waste using factorial design and metagenomic analysis of
the microbial community, Int J. Hydrog. Energy 45 (2020) 4205–4222, https://
doi.org/10.1016/j.ijhydene.2019.12.062.
[37] R.C. da S. Mazareli, A.C.V. Montoya, T.P. Delforno, V.B. Centurion, V.M.
de Oliveira, E.L. Silva, et al., Enzymatic routes to hydrogen and organic acids
production from banana waste fermentation by autochthonous bacteria:
Optimization of pH and temperature, Int J. Hydrog. Energy 6 (2021) 8454–8468,
https://doi.org/10.1016/j.ijhydene.2020.12.063.
[38] D. Kim, S. Han, S. Kim, H. Shin, Effect of gas sparging on continuous fermentative
hydrogen production, Int J. Hydrog. Energy 31 (2006) 2158–2169, https://doi.
org/10.1016/j.ijhydene.2006.02.012.
[39] C.A. Menezes, E.L. Silva, Hydrogen production from sugarcane juice in expanded
granular sludge bed reactors under mesophilic conditions: The role of
homoacetogenesis and lactic acid production, Ind. Crop Prod. 138 (2019),
111586, https://doi.org/10.1016/j.indcrop.2019.111586.
[40] Z. Cui, J.P. Kerekes, Impact of Wavelength Shift in Relative Spectral Response at
High Angles of Incidence in Future Landsat Design Concepts, IEEE Trans. Geosci.
Remote Sens 56 (2018) 5873–5883, https://doi.org/10.1109/
TGRS.2018.2827394.
[41] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric Method
for Determination of Sugars and Related Substances, Anal. Chem. 28 (1956)
350–356, https://doi.org/10.1021/ac60111a017.
[42] APHA/AWWA/WEF. Standard methods for the examination of water and
wastewater. 22nd ed. Washington, DC: Études Rabelaisiennes; 2012. doi:〈
https://doi.org/10.1021/ed076p1069.2〉.
[43] I.D. Buchanan, J.A. Nicell, Model development for horseradish peroxidase
catalyzed removal of aqueous phenol, Biotechnol. Bioeng. 54 (1997) 251–261,
https://doi.org/10.1002/(SICI)1097-0290(19970505)54:3<251::AID-BIT6>3.0.
CO;2-E.
[44] C.Z. Lazaro, D.V. Vich, J.S. Hirasawa, M.B.A. Varesche, Hydrogen production and
consumption of organic acids by a phototrophic microbial consortium, Int J.
Hydrog. Energy 37 (2012) 11691–11700, https://doi.org/10.1016/j.
ijhydene.2012.05.088.
[45] M.I. Rodrigues, A.F. Iemma. Experimental Design and Process Optimization, 1st
ed..,, CRC Press,, 2014, https://doi.org/10.1201/b17848.
[46] M.M. Gomes, I.K. Sakamoto, C.A. Rabelo, E.L. Silva, M.B.A. Varesche, Statistical
optimization of methane production from brewery spent grain: Interaction effects
of temperature and substrate concentration, J. Environ. Manag. 288 (2021),
112363.
[47] Goldmeer J. POWER TO GAS: HYDROGEN FOR POWER GENERATION Fuel
Flexible Gas Turbines as Enablers for a Low or Reduced Carbon Energy
Ecosystem. Gen Electr Co 2019.
[48] DOE Energy Efficiency And Renewable Energy Information Center. Hydrogen
Fuel Cells 2006.
[49] B. Shabani, J. Andrews, Hydrogen and Fuel Cells, Green. Energy Technol. (2015)
453–491, https://doi.org/10.1007/978-81-322-2337-5.
[50] A. Galvagno, M. Prestipino, S. Maisano, F. Urbani, Integration into a citrus juice
factory of air-steam gasi fi cation and CHP system: Energy sustainability
assessment, Energy Convers. Manag 193 (2019) 74–85, https://doi.org/10.1016/
j.enconman.2019.04.067.
[51] Y. Yu, C. Lee, J. Kim, S. Hwang, Group-Specific Primer and Probe Sets to Detect
Methanogenic Communities Using Quantitative Real-Time Polymerase Chain
Reaction, Biotechnol. Bioeng. (2005) 670–679, https://doi.org/10.1002/
bit.20347.
[52] B. Callahan, P. McMurdie, M. Rosen, A. Han, A. Johnson, S. Holmes, DADA2:
High-resolution sample inference from illumina amplicon data, Nat. Methods 13
(2016) 581–583, https://doi.org/10.1038/nmeth.3869.
[53] P. McMurdie, S. Holmes, Phyloseq: An r package for reproducible interactive
analysis and graphics of microbiome census data, PLoS One 8 (2013) 61217,
https://doi.org/10.1371/journal.pone.0061217.
[54] BRENDA Enzyme Database. The Comprehensive Enzyme Information System
2022. 〈https://www.brenda-enzymes.org/〉.
[55] KEGG Pathway Database. Wiring diagrams of molecular interactions, reactions
and relations 2022. 〈https://www.genome.jp/kegg/pathway.html〉.
[56] A. Reungsang, S. Pattra, S. Sittijunda, Optimization of key factors affecting
methane production from acidic effluent coming from the sugarcane juice
hydrogen fermentation process, Energies (2012) 4746–4757, https://doi.org/
10.3390/en5114746.
[57] K.N. Niladevi, R.K. Sukumaran, N. Jacob, G.S. Anisha, P.Ã. Prema, Optimization
of laccase production from a novel strain — Streptomyces psammoticus using
response surface methodology, Microbiol Res 164 (2009) 105–113, https://doi.
org/10.1016/j.micres.2006.10.006.
D.H.D. Rocha et al.
[58] J. Kainthola, A.S. Kalamdhad, V.V. Goud, Optimization of methane production
during anaerobic co-digestion of rice straw and Hydrilla verticillata using
response surface methodology, Fuel 235 (2019) 92–99, https://doi.org/10.1016/
j.fuel.2018.07.094.
[59] Y. Pu, J. Tang, X.C. Wang, Y. Hu, Hydrogen production from acidogenic food
waste fermentation using untreated inoculum: Effect of substrate concentrations,
Int. J. Hydrog. Energy 44 (2019) 27272–27284, https://doi.org/10.1016/j.
ijhydene.2019.08.230.
[60] B. Ai, J. Li, X. Chi, J. Meng, A.K. Jha, C. Liu, et al., Effect of pH and Buffer on
Butyric Acid Production and Microbial Community Characteristics in
Bioconversion of Rice Straw with Undefined Mixed Culture, Biotechnol.
Bioprocess Eng. 19 (2014) 676–686, https://doi.org/10.1007/s12257-013-0655-
z.
[61] H. Lee, W.F.J. Vermaas, B.E. Rittmann, Biological hydrogen production: prospects
and challenges, Trends Biotechnol. 28 (2010) 262–271, https://doi.org/10.1016/
j.tibtech.2010.01.007.
[62] S.S. Salek, A.G.Van Turnhout, R. Kleerebezem, M.C.M.Van Loosdrecht, pH
Control in Biological Systems Using Calcium Carbonate, Biotechnol. Bioeng.
(2015) 112, https://doi.org/10.1002/bit.25506.
[63] S. Liu, W. Li, G. Zheng, H. Yang, L. Li, Optimization of cattle manure and food
waste co-digestion for biohydrogen production in a mesophilic semi-continuous
process, Energies 13 (2020) 3848.
[64] J. Zhang, Q. Wang, J. Jiang, Lime mud from paper-making process addition to
food waste synergistically enhances hydrogen fermentation performance, Int. J.
Hydrog. Energy 38 (2013) 2738–2745, https://doi.org/10.1016/j.
ijhydene.2012.12.048.
[65] S. Sangyoka, A. Reungsang, C. Lin, Optimization of biohydrogen production from
sugarcane bagasse by mixed cultures using a statistical method, Sustain. Environ.
Res. 26 (2016) 235–242, https://doi.org/10.1016/j.serj.2016.05.001.
[66] Z. Li, Z. Chen, H. Ye, Y. Wang, W. Luo, J. Chang, et al., Anaerobic co-digestion of
sewage sludge and food waste for hydrogen and VFA production with microbial
community analysis, Waste Manag 78 (2018) 789–799, https://doi.org/10.1016/
j.wasman.2018.06.046.
[67] C. Lin, C.H. Lay, Effects of carbonate and phosphate concentrations on hydrogen
production using anaerobic sewage sludge microflora, Int J. Hydrog. Energy 29
(2004) 275–281, https://doi.org/10.1016/j.ijhydene.2003.07.002.
[68] G. Yang, J. Wang, Biohydrogen production by co-fermentation of sewage sludge
and grass residue: E ff ect of various substrate concentrations, Fuel 237 (2019)
1203–1208, https://doi.org/10.1016/j.fuel.2018.10.026.
[69] W. Jianlong, W. Wei, The effect of substrate concentration on biohydrogen
production by using kinetic models, Sci. China Ser. B Chem. 51 (2008)
1110–1117, https://doi.org/10.1007/s11426-008-0104-6.
[70] R.K. Thauer, K. Jungermann, K. Decker, Energy conservation in chemotrophic
anaerobic bacteria, Bacteriol. Rev. 41 (1977) 100–180, doi:https://doi.org/
10.1128%2Fbr.41.1.100-180.1977.
[71] H. Lee, R. Krajmalinik-brown, H. Zhang, B.E. Rittmann, An electron-flow model
can predict complex redox reactions in mixed-culture fermentative BioH 2:
microbial ecology evidence, Biotechnol. Bioeng. 104 (2009) 687–697, https://
doi.org/10.1002/bit.22442.
[72] M. Akhlaghi, M.R. Boni, A. Polettini, R. Pomi, A. Rossi, G. De Gioannis, et al.,
Fermentative H2 production from food waste: Parametric analysis of factor
effects, Bioresour. Technol. 276 (2019) 349–360, https://doi.org/10.1016/j.
biortech.2019.01.012.
[73] G. De Gioannis, M. Friargiu, E. Massi, A. Muntoni, A. Polettini, Biohydrogen
production from dark fermentation of cheese whey: Influence of pH, Int J.
Hydrog. Energy (2014) 1–12, https://doi.org/10.1016/j.ijhydene.2014.10.046.
[74] F.P. Camargo, A. Sarti, A.C. Alécio, C.A. Sabatini, M.A.T. Adorno, I.C.S. Duarte, et
al., Limonene qunatification by gas chromatography with mass spectometry (GC-
MS) and its effects on hydrogen and volatile fatty acids production in anaerobic
reactors, Quim. Nova (2020) 1–7, https://doi.org/10.21577/0100-
4042.20170557.
[75] X.M. Guo, E. Latrille, H. Carrère, J.-P. Steyer, E. Trably, Hydrogen production
from agricultural waste by dark fermentation: A review, Int J. Hydrog. Energy 35
(2010) 10660–10673, https://doi.org/10.1016/j.ijhydene.2010.03.008.
[76] J. Tang, X.C. Wang, Y. Hu, Y. Zhang, Y. Li, Effect of pH on lactic acid production
from acidogenic fermentation of food waste with different types of inocula,
Bioresour. Technol. 224 (2017) 544–552, https://doi.org/10.1016/j.
biortech.2016.11.111.
[77] Z. Lee, S. Li, P. Kuo, I. Chen, Y. Tien, Y. Huang, et al., Thermophilic bio-energy
process study on hydrogen fermentation with vegetable kitchen waste, Int J.
Hydrog. Energy 35 (2010) 13458–13466, https://doi.org/10.1016/j.
ijhydene.2009.11.126.
[78] G. Grause, M. Igarashi, T. Kameda, T. Yoshioka, Lactic acid as a substrate for
fermentative hydrogen production, Int J. Hydrog. Energy 37 (2012)
16967–16973, https://doi.org/10.1016/j.ijhydene.2012.08.096.
[79] B. Baghchehsaraee, G. Nakhla, D. Karamanev, A. Margaritis, Effect of extrinsic
lactic acid on fermentative hydrogen production, Int J. Hydrog. Energy 34 (2009)
2573–2579, https://doi.org/10.1016/j.ijhydene.2009.01.010.
[80] A. Sikora, M. Błaszczyk, M. Jurkowski, U. Zielenkiewicz, Lactic Acid Bacteria in
Hydrogen-Producing Consortia: On Purpose or by Coincidence ? Lact. Acid Bact. -
R D Food. Heal. Livest. Purp, InTech,, 2013, https://doi.org/10.5772/50364.
[81] B. Matyakubov, Y. Hwang, T. Lee, Evaluating interactive toxic impact of heavy
metals and variations of microbial community during fermentative hydrogen
production, Int J. Hydrog. Energy 47 (2022) 31223–31240, https://doi.org/
10.1016/j.ijhydene.2022.07.038.
16
Journal of Environmental Chemical Engineering 11 (2023) 111252
[82] K. Bélafi-bakó, D. Búcsú, Z. Pientka, B. Bálint, Z. Herbel, Integration of
biohydrogen fermentation and gas separation processes to recover and enrich
hydrogen, Int J. Hydrog. Energy 31 (2006) 1490–1495, https://doi.org/10.1016/
j.ijhydene.2006.06.022.
[83] J. Baeyens, H. Zhang, J. Nie, L. Appels, R. Dewil, R. Ansart, et al., Reviewing the
potential of bio-hydrogen production by fermentation, Renew. Sustain Energy
Rev. 131 (2020), 110023, https://doi.org/10.1016/j.rser.2020.110023.
[84] T.A.Z.De Souza, D.H.D. Rocha, A.A.V. Julio, C.J.R. Coronado, J.L. Silveira, R.
J. Silva, et al., Exergoenvironmental assessment of hydrogen water footprint via
steam reforming in Brazil, J. Clean. Prod. 311 (2021), 127577, https://doi.org/
10.1016/j.jclepro.2021.127577.
[85] J. Park, S. Lee, J. Yoon, S. Kim, H. Park, Predominance of cluster I Clostridium in
hydrogen fermentation of galactose seeded with various heat-treated anaerobic
sludges, Bioresour. Technol. 157 (2014) 98–106, https://doi.org/10.1016/j.
biortech.2014.01.081.
[86] J.C. Ribeiro, V.T. Mota, V.M. Oliveira, M. Zaiat, Hydrogen and organic acid
production from dark fermentation of cheese whey without buffers under
mesophilic condition, J. Environ. Manag. 304 (2022), 114253, https://doi.org/
10.1016/j.jenvman.2021.114253.
[87] M.A. Mazorra-manzano, G.R. Robles-porchas, M. Mart, J.C. Ram, C.O. Garc, M.
J. Torres-llanez, et al., Bacterial Diversity and Dynamics during Spontaneous
Cheese Whey Fermentation at Different Temperatures, Fermentation 8 (2022)
1–11, https://doi.org/10.3390/fermentation8070342.
[88] S.I. Maintinguer, I.K. Sakamoto, M.A.T. Adorno, M.B.A. Varesche, Bacterial
diversity from environmental sample applied to bio-hydrogen production, Int J.
Hydrog. Energy 40 (2015) 3180–3190, https://doi.org/10.1016/j.
ijhydene.2014.12.118.
[89] T. Palladino, D. Gileno, V. Lacerda, J. Melline, I.K. Sakamoto, M. Bernadete, et al.,
Microbial diversity of a full- - scale UASB reactor applied to poultry
slaughterhouse wastewater treatment: integration of 16S rRNA gene amplicon
and shotgun metagenomic sequencing, Microbiol Open (2017) 1–12, https://doi.
org/10.1002/mbo3.443.
[90] M. Diallo, S.W.M. Kengen, A.M. López-Contreras, Sporulation in solventogenic
and acetogenic clostridia, Appl. Microbiol Biotechnol. 105 (2021) 3533–3557,
https://doi.org/10.1007/s00253-021-11289-9.
[91] G. Yang, Y. Yin, J. Wang, Microbial community diversity during fermentative
hydrogen production inoculating various pretreated cultures, Int J. Hydrog.
Energy 4 (2019) 13147–13156, https://doi.org/10.1016/j.ijhydene.2019.03.216.
[92] E. Salvetti, S. Torriani, G.E. Felis, The genus lactobacillus: a taxonomic update,
Probiotics Antimicrob. Proteins (2012) 217–226, https://doi.org/10.1007/
s12602-012-9117-8.
[93] X. Hu, J. Zeng, F. Shen, X. Xia, X. Tian, Z. Wu, Citrus pomace fermentation with
autochthonous probiotics improves its nutrient composition and antioxidant
activities, LWT - Food Sci. Technol. (2022) 157, https://doi.org/10.1016/j.
lwt.2022.113076.
[94] I. Zerva, N. Remmas, P. Melidis, G. Tsiamis, S. Ntougias, Microbial succession and
identification of effective indigenous pectinolytic yeasts from orange juice
processing wastewater, Waste Biomass-.-. Valoriz. 12 (2021) 4885–4899, https://
doi.org/10.1007/s12649-021-01364-7.
[95] L. Luo, S. Sriram, D. Johnravindar, T. Louis, P. Martin, J.W.C. Wong, et al., Effect
of inoculum pretreatment on the microbial and metabolic dynamics of food waste
dark fermentation, Bioresour. Technol. 358 (2022), 127404, https://doi.org/
10.1016/j.biortech.2022.127404.
[96] G. Yang, J. Wang, Biohydrogen production by co-fermentation of antibiotic
fermentation residue and fallen leaves: Insights into the microbial community and
functional genes, Bioresour. Technol. 337 (2021), 125380, https://doi.org/
10.1016/j.biortech.2021.125380.
[97] R.C. d S. Mazareli, A.C. Villa-Montoya, T.P. Delforno, V.B. Centurion, V. Maia de
Oliveira, E.L. Silva, et al., Metagenomic analysis of autochthonous microbial
biomass from banana waste: Screening design of factors that affect hydrogen
production, Biomass-.-. Bioenergy 138 (2020), 105573, https://doi.org/10.1016/
j.biombioe.2020.105573.
[98] D. Cizeikiene, G. Juodeikiene, J. Damasius, Biocatalysis and Agricultural
Biotechnology Use of wheat straw biomass in production of L-lactic acid applying
biocatalysis and combined lactic acid bacteria strains belonging to the genus
Lactobacillus, Biocatal. Agric. Biotechnol. 15 (2018) 185–191, https://doi.org/
10.1016/j.bcab.2018.06.015.
[99] O. Kandler, Carbohydrate metabolism in lactic acid bacteria, Antonie Van.
Leeuwenhoek 49 (1983) 209–224, https://doi.org/10.1007/bf00399499.
[100] H. Liu, G. Wang, D. Zhu, G. Pan, Enrichment of the hydrogen-producing microbial
community from marine intertidal sludge by different pretreatment methods, Int
J. Hydrog. Energy 34 (2009) 9696–9701, https://doi.org/10.1016/j.
ijhydene.2009.10.025.
[101] J.H. Jo, C.O. Jeon, D.S. Lee, J.M. Park, Process stability and microbial community
structure in anaerobic hydrogen-producing microflora from food waste
containing kimchi, J. Biotechnol. 131 (2007) 300–308, https://doi.org/10.1016/
j.jbiotec.2007.07.492.
[102] J. Park, G. Kumar, J. Park, H. Park, S. Kim, Changes in performance and bacterial
communities in response to various process disturbances in a high-rate
biohydrogen reactor fed with galactose, Bioresour. Technol. 188 (2015) 109–116,
https://doi.org/10.1016/j.biortech.2015.01.107.
[103] Y. Yin, J. Wang, Enhanced Sewage Sludge Disintegration and Hydrogen
Production by Ionizing Radiation Pretreatment in the Presence of Fe 2+, ACS
Sustain Chem. Eng. 7 (2019) 15548–15557, https://doi.org/10.1021/
acssuschemeng.9b03362.
D.H.D. Rocha et al.
[104] T.S.S. Jyothsna, L. Tushar, C. Sasikala, C.V. Ramana, Paraclostridium
benzoelyticum gen. nov., sp. nov.,isolated from marine sediment and
reclassification of Clostridium bifermentans as Paraclostridium bifermentans
comb. nov. Proposal of a new genus Paeniclostridium gen. nov. to accommodate
Clostridium s, Int J. Syst. Evol. Microbiol (2016) 1268–1274, https://doi.org/
10.1099/ijsem.0.000874.
[105] C.A.B.S. Rabelo, C.H. Okino, I.K. Sakamoto, M.B.A. Varesche, Isolation of
Paraclostridium CR4 from sugarcane bagasse and its evaluation in the
bioconversion of lignocellulosic feedstock into hydrogen by monitoring cellulase
gene expression, Sci. Total Environ. 715 (2020), 136868, https://doi.org/
10.1016/j.scitotenv.2020.136868.
[106] S.G. Santiago, E. Trably, E. Latrille, The hydraulic retention time influences the
abundance of Enterobacter, Clostridium and Lactobacillus during the hydrogen
production from food waste, Lett. Appl. Microbiol. 69 (2019) 138–147, https://
doi.org/10.1111/lam.13191.
[107] L. Zhuang, S. Zhou, Y. Yuan, T. Liu, Z. Wu, J. Cheng, Development of Enterobacter
aerogenes fuel cells: From in situ biohydrogen oxidization to direct electroactive
biofilm, Bioresour. Technol. 102 (2011) 284–289, https://doi.org/10.1016/j.
biortech.2010.06.038.
[108] L.P. Thapa, S.J. Lee, C. Park, S.W. Kim, Enzyme and Microbial Technology
Production of L-lactic acid from metabolically engineered strain of Enterobacter
aerogenes ATCC 29007, Enzym. Micro Technol. 102 (2017) 1–8, https://doi.org/
10.1016/j.enzmictec.2017.03.003.
[109] P.C. Hallenbeck, Fundamentals of the fermentative production of hydrogen,
Water Sci. Technol. 52 (2005) 21–29, https://doi.org/10.2166/wst.2005.0494.
[110] I. Karube, T. Matsunaga, S. Tsuru, S. Suzuki, Continuous Hydrogen Production By
Immobilized Whole Cells Of Clostridium Butyricum, Biochim Biophys. Acta - Gen.
Subj. 444 (1976) 338–343, https://doi.org/10.1016/0304-4165(76)90376-7.
17
Journal of Environmental Chemical Engineering 11 (2023) 111252
[111] S. Feng, H. Hao, W. Guo, S. Woong, D. Duc, Y. Liu, et al., Wastewater-derived
biohydrogen: critical analysis of related enzymatic processes at the research and
large scales, Sci. Total Environ. 851 (2022), 158112, https://doi.org/10.1016/j.
scitotenv.2022.158112.
[112] S.K. Rhee, Y. Pack, Effect of environmental pH on fernentation balance of
lactobacillus bulgaricus, J. Bacteriol. 144 (1980) 217–221, https://doi.org/
10.1128/jb.144.1.217-221.1980.
[113] B. Tamelová, J. Malat’ák, J. Velebil, Energy valorisation of citrus peel waste by
torrefaction treatment, Agron. Res 16 (2018) 276–285, https://doi.org/
10.15159/AR.18.029.
[114] Intratec Primary Commodity Prices. Calcium Carbonate Prices. Calcium
Carbonate Prices 2018. 〈https://www.intratec.us/chemical-markets/calcium-ca
rbonate-price〉 (accessed February 2, 2023).
[115] S.A. Neshat, M. Mohammadi, G.D. Najafpour, P. Lahijani, Anaerobic co-digestion
of animal manures and lignocellulosic residues as a potent approach for
sustainable biogas production, Renew. Sustain Energy Rev. 79 (2017) 308–322,
https://doi.org/10.1016/j.rser.2017.05.137.
[116] X. Li, L. Li, M. Zheng, G. Fu, J.S. Lar, Anaerobic co-digestion of cattle manure with
corn stover pretreated by sodium hydroxide for efficient biogas production,
Energy Fuels 74 (2009) 4635–4639, https://doi.org/10.1021/ef900384p.
[117] D. Yin, W. Liu, N. Zhai, G. Yang, X. Wang, Y. Feng, Anaerobic digestion of pig and
dairy manure under photo-dark fermentation condition, Bioresour. Technol. 166
(2014) 373–380, https://doi.org/10.1016/j.biortech.2014.05.037.
[118] X. Meng, D. Yu, Y. Wei, Y. Zhang, Q. Zhang, Endogenous ternary pH buffer system
with ammonia-carbonates-VFAs in high solid anaerobic digestion of swine
manure: An alternative for alleviating ammonia inhibition ? Process Biochem. 69
(2018) 144–152, https://doi.org/10.1016/j.procbio.2018.03.015.