i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 7 ( 2 0 2 2 ) 4 0 6 7 2 e4 0 6 8 2

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High-solid dark fermentation of cassava pulp and

cassava processing wastewater for hydrogen
production

Noppamas Chantawan a,b, Ayyapruk Moungprayoon a,b,

Siriporn Lunprom a,b, Alissara Reungsang a,b,c, Apilak Salakkam a,b,*
a
Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen, 40002, Thailand
b
Research Group for Development of Microbial Hydrogen Production Process from Biomass Khon Kaen University,
Khon Kaen, 40002, Thailand
c
Academy of Science, Royal Society of Thailand, Bangkok, 10300, Thailand

highlights

Hydrogen production from cassava pulp (CP) and cassava processing wastewater (CPW).

Enzymatic hydrolysis of CP-CPW suspension was statistically optimized.

Proper mixing improved hydrogen production in high-solid dark fermentation.

Hydrogen yield (HY) equivalent to 83.1% of a stoichiometric maximum HY was attained.

article info abstract

Article history: Dark fermentation (DF) is a promising biological process for hydrogen production from
Received 28 March 2022 biomass. However, low hydrogen yield (HY) is a major hurdle impeding its use at large-
Received in revised form scale operation. A potential way to mitigate this problem is to increase the concentra-
22 July 2022 tion of substrate in the process to increase hydrogen production. The present study
Accepted 11 September 2022 investigated the possibility of using high-solid DF to produce hydrogen from cassava
Available online 6 October 2022 processing wastes, i.e., cassava pulp (CP) and cassava processing wastewater (CPW). CP was
suspended in CPW and hydrolyzed enzymatically under optimum conditions of 150 g-CP/L,
Keywords: 29 U/g of a-amylase, 47 U/g of glucoamylase, and 60 FPU/g of cellulase. The hydrolysis
Cassava wastes performed at 50  C for 24 h yielded a reducing sugar concentration of 117.7 ± 1.8 g/L,
Enzymatic hydrolysis equivalent to 0.78 g-reducing-sugar/g-CP. Subsequent DF of CP-CPW enzymatic slurry,
High-solid fermentation which contained 12.1% water insoluble solids, resulted in a cumulative production of
Biofuels 13.72 ± 0.22 L-H2, equivalent to 225.2 ± 3.7 mL-H2/g-VS. This was 83.1% of a maximum
Waste valorization stoichiometric HY, based on carbohydrate content of CP and soluble metabolites produc-
tion. The present study shows clearly the applicability of high-solid DF in the production of
hydrogen from cassava processing wastes.
© 2022 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

* Corresponding author. Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen, 40002, Thailand.
E-mail addresses: noppamas_ch@kkumail.com (N. Chantawan), m.ayyapruk@kkumail.com (A. Moungprayoon), sirilun@kku.ac.th
(S. Lunprom), alissara@kku.ac.th (A. Reungsang), apilsa@kku.ac.th (A. Salakkam).
https://doi.org/10.1016/j.ijhydene.2022.09.106
0360-3199/© 2022 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 7 ( 2 0 2 2 ) 4 0 6 7 2 e4 0 6 8 2
Introduction
Hydrogen is a gas that has a very wide range of applications,
including the use as a propellant in aerospace sector [1], as a
reactant in ammonia production [2], as a therapeutic gas [3],
and as an extractant of rare-earth metals [4]. In recent years,
however, it has gained much attention as an alternative bio-
fuel [5], owing to its high energy content (120 MJ/kg), which is
much higher than that of other conventional fuels, e.g., gas-
oline (44 MJ/kg). Besides, hydrogen is clean energy, giving only
water as a by-product during its combustion [6]. For these
reasons, hydrogen is widely regarded as a potential solution to
issues related to both increasing global energy demand and
global warming. However, one major concern about hydrogen
utilization is that the majority of it is presently produced via
steam reforming of non-renewable hydrocarbons, which
leaves enormous greenhouse-gas footprint [7]. Hydrogen can
be alternatively produced through more environmentally
processes, e.g., photo-fermentation (PF) and dark fermenta-
tion (DF). PF has an advantage of high substrate conversion
rate, i.e., 12 mol-H2/mol-glucose, and can convert volatile fatty
acids, e.g., acetate, malate, butyrate, lactate, formate, succi-
nate, and propionate, into hydrogen [8]. However, it has a
major drawback of light dependence [9]. DF, on the other
hand, gives lower hydrogen yield (4 mol-H2/mol-glucose) [10],
but does not require light. DF operation is also simpler, and
this process can be used with various biomasses [11],
including lignocellulosic biomass [12] and starchy wastes [13],
making it a highly promising process for environmentally
friendly hydrogen production.
Lignocellulosic biomass has been widely investigated as
hydrogen feedstock. However, its use is hampered by the
presence of lignin and the complexity of its structure, which
underlies the need of complicated pretreatment and
saccharification processes. This not only increases the pro-
duction costs, but also generates microbial inhibitors [14] that
adversely affect the subsequent fermentation. Wasted starch-
rich biomass, e.g., cassava pulp (CP), is considered a potential
alternative to lignocellulosic biomass. This kind of biomass
has advantages over lignocellulose in that it has less lignin
content and comprises mainly starch that is easily hydrolyzed
[15]. Cassava (Manihot esculenta Crantz) pulp is starch-rich
fibrous residues generated as a by-product of a starch
extraction process from cassava roots. Around 10%e15% of
cassava roots entering the process ends up as CP [16]. Starch
content in CP can be very high, ranging from 54.4% [17] to
88.0% [18], which is higher than cellulose content of typical
lignocellulosic biomasses [19]. Despite this, CP is scarcely
utilized as fermentation feedstock, and is instead used as
animal feed with a low economic value [15]. It is only recently
that CP has gained increasing attention as a feedstock for
industrially valuable products, e.g. lactic acid [20], ethanol
[15,16], and hydrogen [21]. However, a survey of recent liter-
ature showed only a handful of reports on hydrogen produc-
tion from CP, and to the best of our knowledge, there has been
no reports on hydrogen production from CP under high-solid
conditions.
A fermentation process in which at least 10% (w/v) of solid
substrate is present in the system can be referred to as high-
40673
solid fermentation [22e25]. This process is known to be ad-
vantageous in many aspects, including reducing water con-
sumption [26], bioreactor volume [27], and specific energy
consumption [28], as well as increasing product titer [29],
which can lead to lower capital and operating costs. In fact,
when complex biomass, e.g. lignocellulose biomass, is used,
high-solid fermentation is considered a requisite for large-
scale economic process [30]. However, one major drawback
of high-solid systems is the high viscosity of the medium [31]
that impedes efficient mass and heat transfers, thus
hampering substrate consumption and product formation,
and resulting in low hydrogen yield and a delay of active
hydrogen production phase [32]. This effect is generally
referred to as “high-solid effect” [8]. Furthermore, pH control is
difficult under high-solid conditions, and this can substan-
tially affect some physiological functions of the hydrogen
producers, e.g., the internal pH membrane potential and pro-
ton motive force [33]. pH also plays a key role in controlling of
metabolic pathways for hydrogen production. For instance,
acetate-type pathway is favored at neutral pH, while acidic pH
conditions favor the butyrate pathway [34]. However, at low
pH in the range 4.0e5.0, the fermentation pathway could shift
to ethanol-type fermentation, which yields ethanol and acetic
acid as the soluble metabolite products [35]. Other undesirable
products, e.g., lactic acid, could also be produced at low pH
[28]. Besides, hydrogenase, which is responsible for hydrogen
production [36], has optimum pH in the range 5.0e7.0 [37].
Therefore, deviation of pH outside this range could result in
reduced hydrogen production. Fed-batch fermentations, in
which solid substrate is fed intermittently or periodically into
the bioreactor, can be employed to avoid this effect [14,26].
Alternatively, hydrolysis of the substrate can be conducted
preceding the fermentation to reduce the viscosity of the
medium, and the resulting hydrolysis slurry is used directly in
the fermentation process without solids separation. This
approach has an advantage that the entire substrate can be
added into the bioreactor at once. Mixing is also considered an
important factor that needs to be controlled properly to
ensure homogeneity of the fermentation medium [38].
With the advantages of high-solid fermentation, and the
limited information on hydrogen production from CP, the
present study aimed to develop a high-solid DF process to
produce hydrogen from CP. Cassava processing wastewater
(CPW) was used to prepare CP suspensions to reduce the use
of clean water. Conditions for CP-CPW hydrolysis, i.e., CP
loading and enzymes (a-amylase, glucoamylase, cellulase)
loading, were optimized. The CP-CPW enzymatic slurry was
subsequently fermented in 120-mL serum bottles and a 2-L
bioreactor to investigate the effect of mixing on substrate
consumption and hydrogen production. Based on the results
of the 2-L bioreactor experiment, applicability of the high-
solid DF was assessed.
Material and methods
Inoculum
Anaerobic granules collected from an anaerobic digester of a
wastewater treatment plant of Khon Kaen Brewery Co., Ltd.,
40674 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 7 ( 2 0 2 2 ) 4 0 6 7 2 e4 0 6 8 2

Khon Kaen, Thailand, were used as hydrogen inoculum. The
granules were heated at 105  C for 3 h to inactivate metha-
nogens [36], and stored in screw-cap glass bottles at room
temperature (35 ± 2  C) until use. Total solids (TS) and volatile
solids (VS) of the granules were 75.29 ± 0.04% and
87.23 ± 0.01% of TS, respectively.
Substrates
Dry CP, commercially available as animal feed, was purchased
from a local vendor. It was sieved to obtain particles of
0.5e1 mm, and stored in an air-tight plastic container at room
temperature until use. Composition analysis revealed that CP
contained 47.7% starch, 20.32% cellulose, 18.32% hemicellu-
lose, and 3.36% lignin. CPW was provided by Kaensiri Starch
Co., Ltd., Phra Yuen, Khon Kaen, Thailand. It was filtered
through muslin cloth to remove suspended solids (e.g., dirt
and sand), then stored in screw-cap plastic bottles at 20  C
until use. CPW contained 5.71 g/L of starch, and several min-
erals as listed in Table 1.
Enzymes
iKnowZyme HTAA (a-amylase), iKnowZyme GA (glucoamy-
lase), and Cellic® CTec2 (cellulase), were used in the present
study. iKnowzyme HTAA and iKnowZyme GA contained 135
U/mL of a-amylase and 1300 U/mL of glucoamylase, respec-
tively, and were purchased from Reach Biotechnology Co.,
Ltd., Bangkok, Thailand. Cellic® CTec2, with an activity of 100
filter paper unit (FP) per mL, was purchased from Novozyme,
Denmark. All the enzymes were stored in screw-cap glass
bottles at 4  C until use.
Enzymatic hydrolysis of CP-CPW suspension
glucoamylase, and cellulase, each at 20 U/g-CP. Then, pH of
the suspensions was adjusted to 6.0 ± 0.1 before these were
incubated at 50  C, 120 rpm, for 24 h. The optimum CP con-
centration was selected based on hydrolysis efficiency (Eq. (1))
and reducing sugar production.
RS
HEð%Þ ¼  100 (1)
½ðCP  SCP Þ þ ðCP  CCP Þ  1:11
where HE is the hydrolysis efficiency (%), RS is the reducing
sugar concentration (g/L), CP is the concentration of CP (g/L),
SCP is the starch content of CP (%), CCP is the cellulose content
of CP (%), and 1.11 is the conversion factor for starch or cel-
lulose to glucose [15].
Enzymes loadings were optimized using RSM with CCD.
Each factor was varied in 5 levels, i.e., extreme low (a), low
(1), center (0), high (þ1), and extreme high (þa). The design
matrix consisted of 19 runs, 5 of which were conducted at the
center point. CP was suspended in CPW at the optimum
concentration (150 g/L) before a-amylase (10e30 U/g-CP), glu-
coamylase (20e60 U/g-CP), and cellulase (20e60 FPU/g-CP)
were added at the designated loadings (Table 2). The range
of each enzyme was selected based on our preliminary study
(unpublished data). pH of the mixtures was adjusted to
6.0 ± 0.1 with 5 M NaOH before incubation at 50  C, 120 rpm, for
24 h. HE and reducing sugar production were used as the re-
sponses. The results obtained were used to generate quadratic
models to describe the relationship between the variables and
the responses, and to optimize the conditions. The optimum
conditions obtained from this experiment were later used to
hydrolyze CP-CPW suspension in 2-L bottles (1.5-L working
volume) to produce CP-CPW enzymatic slurry.
High-solid DF of CP-CPW enzymatic slurry in 120-mL serum
bottles

Enzymatic hydrolysis of CP-CPW suspension was conducted CP-CPW enzymatic slurry obtained after the hydrolysis under
in 250-mL Erlenmeyer flasks, with a working volume of the optimum conditions was supplemented with basic
100 mL. The conditions for CP-CPW hydrolysis, i.e., CP con- anaerobic (BA) nutrients following the formula of Angelidaki
centration and enzymes loadings, were optimized using one- and Sanders [39], but without resazurin and a vitamin solu-
factor-at-a-time method, and response surface methodology tion. After adjusting pH of the slurry to 6.0 using 5 M NaOH and
(RSM) with central composite design (CCD), respectively. In mixing well, 70 mL of the slurry was transferred into 120-mL
optimizing CP concentration, CP was suspended in CPW at serum bottles and inoculated with 10% (w/v) heat-treated
50e225 g/L, and the suspensions were added with a-amylase, anaerobic granules. The bottles were then tightly capped
and flushed with nitrogen gas for 5 min to create anaerobic
conditions. Then, the bottles were incubated at room tem-
Table 1 e Mineral composition of cassava processing perature (35 ± 2  C) for 120 h on an orbital shaker set at
wastewater. 100 rpm. During the fermentation, liquid samples were
Component Concentration (mg/L) collected to follow changes in volatile fatty acids (VFAs) and
Calcium 47.8 reducing sugars concentrations, while gas samples were
Copper 0.06 collected for hydrogen production determination.
Chloride 365.0
Iron 0.92 High-solid DF of CP-CPW enzymatic slurry in 2-L bioreactor
Magnesium 62.0
Manganese 1.44
The experiment was conducted in a 2-L bioreactor (B.E. Mar-
Nitrate as N 0.92
ubishi, Thailand), with a working volume of 0.5 L. The biore-
Phosphorus 61.8
Potassium 585.0 actor was equipped with 4 baffles and a six-blade Rushton
Sodium 172.0 impeller. After transferring CP-CPW enzymatic slurry into the
Sulfur 34.1 bioreactor and supplementing with BA nutrients, the inoc-
Total Kjeldahl Nitrogen 156.0 ulum (10%, w/v) was added, and the bioreactor was flushed
Zinc 0.5 thoroughly with nitrogen gas for 10 min with an agitation rate
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 7 ( 2 0 2 2 ) 4 0 6 7 2 e4 0 6 8 2 40675

Table 2 e Design matrix for optimization of enzymes loading for CP-CPW hydrolysis.
Run a-amylase (U/g) Glucoamylase (U/g) Cellulase (FPU/g) Reducing sugar (g/L) HE (%)
1 20 40 40 107 89.6
2 20 6 40 88 73.9
3 20 40 6 90 75.1
4 37 40 40 107 89.6
5 30 20 60 109 91.8
6 20 40 40 103 86.1
7 20 40 40 103 86.4
8 30 20 20 96 80.3
9 20 40 40 101 84.9
10 10 20 20 89 74.4
11 20 40 40 101 84.3
12 20 74 40 101 84.9
13 20 40 74 112 94.2
14 30 60 20 96 80.3
15 3 40 40 91 75.9
16 10 60 20 94 78.2
17 30 60 60 113 95.3
18 10 60 60 106 89.0
19 10 20 60 98 82.1

of 300 rpm to create anaerobic conditions. The fermentation
was carried out at pH 6.0 ± 0.1, controlled using 1 M NaOH and
1 M HCl, an agitation rate of 150 rpm, and a temperature of
35 ± 2  C. Liquid samples were collected regularly to follow
VFAs and reducing sugar concentrations, and gas samples
were collected for determination of hydrogen production.
Analytical methods
Cellulose, hemicellulose, and lignin contents of CP were
determined using the method of Goering and Van Soest [40] at
the Animal Nutrition Laboratory, Kasetsart University,
Bangkok, Thailand. Starch content of CP was analyzed using a
Total Starch HK assay kit (K-TSHK, Megazyme, Ireland). Total
solids (TS) and volatile solids (VS) were determined according
to the standard methods of the American Public Health As-
sociation (APHA) [41]. Mineral compositions of CPW were
analyzed at the ALS Laboratory Group (Thailand), Co., Ltd.,
Bangkok, Thailand, using standard methods of the American
Public Health Association (APHA) [42] and the United States
Environmental Protection Agency (US EPA) [43]. Water insol-
uble solids (WIS) content in CP-CPW enzymatic slurry was
determined using a no-wash method reported by Sluiter et al.
[44]. Alpha-amylase activity was determined following the
method of Dey et al. [45]. One unit of a-amylase was defined as
an amount of enzyme that released 1 mmol of reducing sugar
(maltose equivalent) from starch under the assay conditions.
Glucoamylase activity was determined using the method of
Huo et al. [46]. One unit of glucoamylase was defined as an
amount of enzyme that released 1 mg of glucose from starch
under the assay conditions. Cellulase activity was determined
using the method of Adney and Baker [47] and reported as
FPU/mL. Reducing sugar concentration was determined using
the DNS method [48] with D-glucose as the standard. Glucose
and VFAs concentrations were determined using a high-
performance liquid chromatography (HPLC) using the
method of Phanduang et al. [36]. Volume of biogas was
measured using a 50-mL wetted glass syringe. Hydrogen
content was determined using gas chromatography (GC),
following the method of Phanduang et al. [36]. Cumulative
hydrogen production was calculated using Eq. (2), where V is
cumulative hydrogen production (mL), V0 is head space vol-
ume (mL), Vi is biogas volume at time i (mL), and gi is the
hydrogen content in the biogas at time i (%) [49].
X
V ¼ V0 gi þ Vi gi (2)
Hydrogen yield (HY) was calculated by dividing the cu-
mulative hydrogen production by the initial VS of substrate.
The modified Gompertz model (Eq. (3)) was used to estimate
the kinetic parameters for hydrogen production, i.e., the
maximum hydrogen production (Hm, mL/g-VS), the maximum
hydrogen production rate (Rm, mL/(g-VS$h)), and the lag time
(l, h), where e is the Euler's number (2.71828), and t is the
fermentation time (h).
  
Rm  e
H ¼ Hm  exp  exp ðl  tÞ þ 1 (3)
Hm
Results and discussion
Enzymatic hydrolysis of CP-CPW suspension
Fig. 1 shows that reducing sugar production increased with
increasing CP concentration. The production increased from
29.45 ± 0.39 g/L to 119.32 ± 5.28 g/L when the CP concentration
was increased from 50 to 225 g/L. However, the highest HE
(83.0 ± 1.9%) was observed at 75 g-CP/L, and this tended to
decrease with further increasing CP concentration, implying
adverse effects of increasing solids concentration on mixing
and mass transfer during the process. Similar effects were
reported in a study of Larnaudie et al. [50], where HE of liquid
hot water-pretreated switchgrass decreased with increasing
solids loading from 10% to 25%, using a cellulase (Cellic®
CTec2) loading of 10 mg-protein/g-glucan. Du et al. [31] also
reported the same phenomenon when the loading of
delignified corncob residues was increased from 2% to 20%.
The low HE at high solid loadings could be attributed to
40676 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 7 ( 2 0 2 2 ) 4 0 6 7 2 e4 0 6 8 2
Fig. 1 e Reducing sugar production and hydrolysis
efficiency at CP concentrations of 50e225 g/L.
feedback inhibition and mass transfer limitation. It is well
known that cellulase is susceptible to glucose [51], which is
the end product of cellulose and starch hydrolyses, while
amylases have long been shown to be inhibited by glucose
feedback inhibition [52]. Therefore, with high glucose pro-
duction at high solid loadings, higher degrees of feedback in-
hibition could be expected. The high solid loading could also
increase the viscosity of the mixture, impeding efficient
mixing and decreasing the contact frequency between the
substrate and enzymes [31]. It should, however, be noted that
despite high HE is desirable for the biomass saccharification,
high sugar production is also crucial for high product forma-
tion. Therefore, in any reaction, a solid loading should be
carefully selected to ensure high HE, while maintaining high
sugar production. In the present study, the CP concentration
of 75 g/L gave 83.0 ± 1.9% HE, with only 47.1 ± 1.1 g/L of
reducing sugar being produced. This was considered inade-
quate for high hydrogen productivity. Further inspection of
the results revealed that a slightly lower yet relatively high HE
(ca. 80%), and reasonably high reducing sugar production
(89.10 ± 1.67 g/L) were still achievable at 150 g-CP/L. Therefore,
150 g-CP/L was selected for the subsequent optimization step.
The use of higher substrate concentration in a hydrolysis re-
action could be beneficial in reducing the size of the reactor,
saving both the capital and operating costs.
Using the selected CP concentration of 150 g/L, a-amylase,
glucoamylase, and cellulase loadings were optimized through
RSM with CCD. Reducing sugar production and HE obtained in
each experimental run are given in Table 2. Analysis of vari-
ance (ANOVA) of the results (Table 3) showed that the data
sets were best described by quadratic equations (Eq. (4) and
(5)), with the p-values of both equations being less than 0.05
and lack of fit greater than 0.05. The coefficient of determi-
nation (R2) of Eqs. (4) and (5) were 0.9492 and 0.9495, respec-
tively. The ANOVA also revealed that, for both equations, the
mean square of the lack of fit was close to the mean square of
the pure error. These indicated that both equations fitted the
data well, and were adequate to predict the responses. The
ANOVA also showed that enzymes loadings had significant
effects on reducing sugar production and HE, despite their
interactions did not affect significantly the results. Numerical
optimizations were subsequently performed to maximize the
reducing sugar production and HE. It was found that both
reducing sugar production and HE increased with increasing
enzymes loadings, and the highest reducing sugar production
(113.6 g/L) and HE (95.6%) could be attained using 30 U/g-CP of
a-amylase, 47 U/g-CP of glucoamylase, and 60 FPU/g-CP of
cellulase (Fig. 2). A confirmation experiment conducted under
these predicted conditions gave 117.7 ± 1.8 g/L of reducing
sugar, and HE of 98.8 ± 1.6%. These fell within the 95% pre-
diction intervals (PI) of 107e120 g/L, and 90.0%e101.1%,
respectively, confirming the validity of the predicted condi-
tions. Compared with the results shown in Fig. 1, where each
enzyme was used at 20 U/g, it was found that more enzymes
were required to efficiently hydrolyze CP at 150 g/L. This was
consistent with a report of Larnaudie et al. [50] that high
enzyme loading could be used to compensate the effects of
feedback inhibition. The CP-CPW enzymatic slurry obtained
after the hydrolysis had 12.1% WIS, and a VS concentration of
121.9 ± 5.0 g/L.
Reducing sugar ðg=LÞ ¼ 65:28 þ 0:77A þ 0:67B þ 0:17C
 0:0056AB  0:0056AC þ 0:0021BC
 0:0094A2  0:0063B2  0:0006C2
(4)
Hydrolysis efficiency ð%Þ ¼ 54:9 þ 0:66A þ 0:53B þ 0:14C
 0:0045AB  0:005AC þ 0:002BC
 0:0084A2  0:005B2  0:0005C2
(5)
High-solid DF of CP-CPW enzymatic slurry in 120-mL serum
bottles
The CP-CPW enzymatic slurry obtained after the hydrolysis
was used in high-solid DF in 120-mL serum bottles. The
fermentation showed a lag period followed by a production
period that started at around 12 h and lasted until 72 h,
yielding 22.5 ± 0.6 mL-H2/g-VS (Fig. 3A). Eq. (3) estimated the
lag time, maximum hydrogen production rate (Rm), and
maximum hydrogen yield (Hm) to be 10.3 h, 0.69 mL/(g-VS$h)
and 22.7 mL/g-VS, respectively. The low HY correlated well
with the low acetic and butyric acids production, where only
6.2 ± 0.9 g/L of acetic acid, and 7.4 ± 1.1 g/L of butyric acid were
detected at the end of the process. However, a vigorous pro-
duction of lactic acid and ethanol were observed after 48 h,
reaching 18.8 ± 1.7 and 10.4 ± 0.6 g/L at 120 h, respectively
(Fig. 3B). Reducing sugar was consumed steadily during the
course of the fermentation, though it was not depleted. The
volumetric reducing sugar consumption rate was 0.35 ± 0.03 g/
(L$h). With a relatively high WIS content of 12.1%, and the
presence of heat-treated anaerobic granules at 10% (w/v),
orbital shaking at 100 rpm was thought to be inadequate to
efficiently mix the fermentation medium. Since the insoluble
solid concentration of ca. 12%e15% has been reported to
represent the upper limit for efficient mixing [30], it was
therefore possible that both the substrate and microbial cells
were not uniformly mixed, leading to low substrate con-
sumption and thus low HY.
The inadequate mixing could also result in a decrease of
local pH of the medium, leading to a metabolic shift of the
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 7 ( 2 0 2 2 ) 4 0 6 7 2 e4 0 6 8 2 40677

Table 3 e Analysis of variance for response surface quadratic models (Eq. (4) and (5)).
Source Sum of Squares df Mean Square F value p-value
Eq. (4) 1010.85 9 112.32 18.44 <0.0001
A-a-amylase 213.18 1 213.18 34.99 0.0002
B-Glucoamylase 110.94 1 110.94 18.21 0.0021
C-Cellulase 567.09 1 567.09 93.08 <0.0001
AB 10.12 1 10.12 1.66 0.2295
AC 10.13 1 10.13 1.66 0.2295
BC 6.13 1 6.13 1.01 0.3422
A2 12.58 1 12.58 2.06 0.1846
B2 88.17 1 88.17 14.47 0.0042
C2 0.9007 1 0.9007 0.1478 0.7095
Residual 54.83 9 6.09
Lack of Fit 30.83 5 6.17 1.03 0.5035
Pure Error 24 4 6
Eq. (5) 757.41 9 84.16 19.74 <0.0001
A-a-amylase 162.29 1 162.29 38.06 0.0002
B-Glucoamylase 78.55 1 78.55 18.42 0.002
C-Cellulase 435.53 1 435.53 102.15 <0.0001
AB 6.48 1 6.48 1.52 0.2489
AC 8 1 8 1.88 0.204
BC 5.45 1 5.45 1.28 0.2877
A2 10.02 1 10.02 2.35 0.1597
B2 56.49 1 56.49 13.25 0.0054
C2 0.4872 1 0.4872 0.1143 0.7431
Residual 38.37 9 4.26
Lack of Fit 21.48 5 4.3 1.02 0.5075
Pure Error 16.89 4 4.22

microbes towards undesirable metabolic pathways that do
not yield hydrogen [53], e.g. lactic acid fermentation [28].
Considering that no pH control was performed in this exper-
iment, the local pH of the medium might decrease, as a result
of VFAs accumulation, to an unfavorable level for hydrogen
production, e.g. pH 4.0e4.5 [54,55], but facilitative for lactic
acid production. As a consequence, lactic acid was produced
vigorously after 48 h (Fig. 3B). Furthermore, a slight increase in
HY after 48 h together with ethanol production suggested that
the metabolic pathway could possibly shift to ethanol-type
fermentation. This pathway was reported to be active at low
pH, e.g., pH 4.0e5.0 [35], and is used to reduce acidic terminal
products [56] to maintain the acidogenic fermentation. A close
inspection of the results (Fig. 3A and B) revealed that only
12.2 ± 2.6 g/L of reducing sugar (20.7 ± 3.5% of the initial
concentration) was used for hydrogen, acetic acid, and butyric
acid production during the first 48 h, while a larger portion of
the reducing sugar (29.5 ± 0.7 g/L, equivalent to 50.4 ± 2.0% of
the initial value) was used for lactic acid and ethanol forma-
tion after 48 h. The consumption of reducing sugar for lactic
acid and ethanol production not only lowers the substrate for
hydrogen production, but lactic acid also acidifies the medium
to the pH level that might be unfavorable for hydrogen syn-
thesis. Ethanol can also be inhibitory to cells at certain levels
[57]. These are considered other possible reasons for the low
hydrogen production observed in this experiment. These re-
sults were in accordance with studies of Ghimire et al. [58] and
Motte et al. [28]. Using food waste as the substrate, Ghimire
et al. [58] reported a simultaneous decrease in hydrogen pro-
duction and increase in lactic acid production at the substrate
concentration greater than 10%, while Motte et al. [28]
observed this phenomenon at the substrate (organic fraction
of municipal solid waste) concentrations greater than 14%.
High-solid DF of CP-CPW enzymatic slurry in 2-L bioreactor
Since it was difficult to achieve adequate mixing and pH
control in the serum bottle experiment, fermentation in a
bioreactor with mechanical agitation was conducted. The
presence of baffles in the vessel, as well as the use of me-
chanical agitation, could improve mixing [59], facilitating pH
control and enhancing hydrogen production. Comparing with
the serum bottles experiment, HY obtained in the bioreactor
was much higher. Hydrogen production was observed shortly
after the commencement of the process, and the rapid
hydrogen production period continued until around 54 h.
During this period, the reducing sugar was depleted, with a
consumption rate of ca. 1.0 ± 0.0 g/(L$h) (Fig. 4A). This revealed
the importance of mixing in improving substrate utilization
and product formation in high-solid fermentation process.
After 54 h, HY still increased, but at a lower rate, reaching
225.2 ± 3.7 mL/g-VS at 132 h. This was around 10.2 times that
obtained in the serum bottle experiment. Determinations of
acids and ethanol concentration showed that acetic acid and
ethanol were produced until 30 h, whereas lactic acid was
produced until 42 h, and the production of butyric acid
continued until the end of the process (Fig. 4B). During the first
42 h, despite the use of baffles and mechanical agitation,
mixing might not be very efficient as solid substrate was
present at a high concentration. Therefore, pH of the medium
might not be constantly controlled, resulting in the decrease
in local pH, which led to lactic acid and ethanol production as
40678 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 7 ( 2 0 2 2 ) 4 0 6 7 2 e4 0 6 8 2
Fig. 2 e Interaction effects between glucoamylase and a-amylase (A), cellulase and a-amylase (B), and cellulase and
glucoamylase (C) on reducing sugar production; and glucoamylase and a-amylase (D), cellulase and a-amylase (E), and
cellulase and glucoamylase (F) on hydrolysis efficiency.
discussed above. Nevertheless, with the progression of the
fermentation, substrate would be continually degraded,
reducing the viscosity of the medium. This facilitated better
mixing that helped to control pH at the designated level.
Consequently, lactic acid and ethanol were not further pro-
duced. Interestingly, unlike the concentration of ethanol that
stayed relatively constant, lactic acid concentration
decreased after 54 h, reaching zero at 108 h (Fig. 4B). The lactic
acid profile was found to differ from that observed in the
serum bottle experiment, suggesting that the metabolic
pathway in the two experiments might be different. Close
inspection of the results revealed a possibility that the
fermentation in the 2-L bioreactor consisted of two phases,
i.e., Phase I: hydrogen production from reducing sugar, and
Phase II: hydrogen production from lactic acid. Phase I
covered the period of the first 54 h, during which reducing
sugar was consumed for the production of hydrogen, acetic
acid, butyric acid, lactic acid, and ethanol. At the end of this
phase, HY and concentrations of acetic, butyric, and lactic
acids, and ethanol were 176.5 ± 11.6 mL/g-VS, 9.9 ± 0.2 g/L,
18.0 ± 0.0 g/L, 8.3 ± 0.2 g/L, and 2.9 ± 0.3 g/L, respectively
(Fig. 4A and B). Despite the simultaneous occurrence of
ethanol-type and butyrate-type pathways, the high produc-
tion of butyric acid observed in this experiment indicated that
the latter was the main pathway for hydrogen production
[28,60]. These results agreed well with a report of Ma et al. [61]
that starch is the main feedstock for butyrate formation
during acidogenic fermentation. The kinetic parameters for
hydrogen production during Phase I were a lag time of 12.9 h,
Rm of 8.5 mL/(g-VS$h), and Hm of 167.1 mL/g-VS.
Phase II started at 54 h, as indicated by the reduction of
lactic acid concentration (Fig. 4B). In this phase, HY increased
more or less linearly with time at a rate of 0.63 mL/(g-VS$h),
while lactic acid decreased at a rate of 0.15 g/(L$h). It is
noteworthy that although some Clostridia, e.g. Clostridium
butyricum and C. beijerinckii, were reported to be able to utilize
lactic acid and acetic acid for hydrogen production (Eq. (6))
[62], the relatively constant concentration of acetic acid
during Phase II suggested that this reaction did not likely
occur. Direct conversion of lactic acid into hydrogen was also
difficult under the conditions used in the present study. This
was because large positive free energy is required to drive the
reaction (Eq. (7)) [63]. To produce hydrogen from lactic acid,
typically, energy is supplied in the form of light to purple
non-sulfur bacteria to ferment lactic acid to hydrogen
through PF [64]. Alternatively, as recently reported by Park
et al. [65], a proper mixing could have enhanced the expres-
sion of some genes involving in the metabolism of hydrogen
producers, including LDH which encodes lactate dehydroge-
nase. Therefore, it was rather that lactic acid was converted
into pyruvate and used in the butyric acid pathway to pro-
duce hydrogen during Phase II [66], thus decreasing lactic
acid concentration and increasing butyric acid concentration
after 54 h.
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 7 ( 2 0 2 2 ) 4 0 6 7 2 e4 0 6 8 2
Fig. 3 e Hydrogen production and reducing sugar
consumption (A), and acids and ethanol production (B)
during DF of CP-CPW enzymatic slurry in 120-mL serum
bottles. The broken line in (A) represents the data
estimated using Eq. (3).
CH3 COOH þ 2CH3 CHðOHÞCOOH/H2 þ 2CO2
þ 1:5CH3 CH3 CH2 COOH þ H2 O
 0
CH3 CHOO þ 6H2 O/3HCO þ
3 þ2H þ6H2 DG0 ¼ þ 100:4 kJ
Applicability of high-solid DF of CP-CPW enzymatic slurry
for hydrogen production
With the use of 150 g-CP/L, the starch and cellulose contents of
47.7% and 20.32%, respectively, and the production of acetic,
butyric, and lactic acids, as well as ethanol, as the main sol-
uble metabolites, a stoichiometrically maximum hydrogen
production from CP was estimated to be 0.64 mol (Eq. (8)),
equivalent to 14.1 L of hydrogen at standard temperature and
pressure (STP, 273.15 K and 1 atm).
0:32C6 H12 O6 þ 0:16H2 O / 0:64H2 þ 0:58CO2
þ 0:097C2 H4 O2 þ 0:16C4 H8 O2 þ 0:097C3 H6 O3
The high-solid DF in the 2-L bioreactor gave a cumulative
hydrogen production of 13.73 ± 0.22 L at the fermentation
temperature. This was equivalent to 0.54 mol-H2 (12.17 L) at
STP and a HY on glucose of 1.7 mol-H2/mol-glucose, which
40679
Fig. 4 e Hydrogen production and reducing sugar
consumption (A), and acids and ethanol production (B)
during DF of CP-CPW enzymatic slurry in 2-L bioreactor.
The broken line in (A) represents the data estimated using
Eq. (3).
was 83.1% of the stoichiometric maximum. The fermentation
(6) also produced 10.1 ± 0.1 g/L of acetic acid (0.084 mol), and
23.1 ± 1.0 g/L of butyric acid (0.129 mol). The ratio between
 butyric and acetic acids (B/A ratio) was 1.53 mol/mol, which
was close to the stoichiometric ratio 1.5 mol/mol used to
(7)
quantitatively indicate substrate metabolism and hydrogen
production [37,67]. Calculation of chemical oxygen demand
(COD) balance revealed that the majority of substrate was
consumed for butyric acid formation (34.2%), followed by
hydrogen (14.4%), acetic acid (8.9%), lactic acid (7.3%), and
ethanol (5.6%). The COD of biomass was not included in the
calculation as it was not determined. Nevertheless, it could be
assumed that ca. 10% of COD is converted into biomass [68].
The rest (19.7%) could be attributed to the production of other
metabolites, e.g., propionic, citric, and succinic acids [69].
With the high HY obtained in the present study, it is clear
that the high-solid DF of CP-CPW enzymatic slurry is highly
applicable for hydrogen production, provided that proper
(8) agitation and pH control are performed during the process. The
hydrogenic effluent containing acetic and butyric acids can be
further used in a photo-fermentation to produce additional
hydrogen. Stoichiometrically, 0.084 mol-acetate and 0.129 mol-
butyrate can be converted into 0.34 and 1.29 mol-H2, respec-
tively [64]. These, coupling with the production of 0.54 mol-H2
40680 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 7 ( 2 0 2 2 ) 4 0 6 7 2 e4 0 6 8 2
through the high-solid DF, lead to a total hydrogen production
of 2.17 mol-H2, which was equivalent to HY of 6.8 mol-H2/mol-
glucose, being close to a desirable range of 7.2e9.6 mol-H2/mol-
glucose (60%e80% of the theoretical HY on glucose) recom-
mended for economically sustainable hydrogen production
[70]. However, further study, particularly process optimization
and upscaling, should be carried out to further assess the en-
ergy and economic feasibilities of this process.
Conclusions
Hydrogen production from cassava pulp (CP) and cassava
processing wastewater (CPW) under high-solid dark fermen-
tation (DF) process was investigated. CP was suspended in
CPW and hydrolyzed enzymatically under optimum condi-
tions, yielding 117.71 ± 1.84 g/L of reducing sugar, with a
hydrolysis efficiency of 98.8 ± 1.6%. Subsequent DF of CP-
CPW enzymatic slurry in 120-mL serum bottles, shaken on
an orbital shaker at 100 rpm, gave 22.5 ± 0.6 mL-H2/g-VS. A
fermentation in a 2-L bioreactor, with an agitation rate of
150 rpm, dramatically improved the hydrogen production to
225.2 ± 3.7 mL/g-VS. The experimental HY obtained in this
study was 83.1% of the stoichiometric maximum, based on
carbohydrate content of CP and soluble metabolites produc-
tion. These results demonstrate the applicability of high-solid
DF for hydrogen production from CP and CPW. Further opti-
mization of this process could make it a promising approach
for valorization of cassava processing wastes for bioenergy
production.
CRediT authorship contribution statement
Noppamas Chantawan: Investigation, Writing e Original draft.
Ayyapruk Moungprayoon: Investigation. Siriporn Lunprom:
Methodology, Writing e review & editing. Alissara Reungsang:
Resources, Funding acquisition. Apilak Salakkam: Conceptu-
alization, Methodology, Writing e review & editing, Resources,
Funding acquisition, Supervision. All authors discussed the
results and contributed to the final manuscript.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationship that could have
appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by the Thailand Research Fund (TRF)
Senior Research Scholar (Grant no. RTA6280001), and the
Graduate School Khon Kaen University, Khon Kaen, Thailand
(Grant no. 631T220). The authors would also like to thank the
Fermentation Research Center for Value Added Agricultural
Products (FerVAAP), Faculty of Technology, Khon Kaen Uni-
versity, Khon Kaen, Thailand, for the provision of chemicals
and equipment, and Kaensiri Starch Co. Ltd., Phra Yuen, Khon
Kaen, Thailand, for the provision of cassava processing
wastewater used in the present study.
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