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Continuous biohydrogen production from

coagulation-pretreated textile desizing wastewater

Chiu-Yue Lin a,b,*, Chih-Cheng Chiang c, Thi Mai Linh Nguyen a,b,
Chi-How Lay d
a
Green Energy Technology Research Group, Ton Duc Thang University, Ho Chi Minh City, Viet Nam
b
Faculty of Environment and Labor Safety, Ton Duc Thang University, Ho Chi Minh City, Viet Nam
c
Department of Environmental Engineering and Science, Feng Chia University, Taiwan
d
Green Energy Development Center, Feng Chia University, Taiwan

article info abstract

Article history: A real textile desizing wastewater (TDW) was coagulation-pretreated and used as the
Received 10 July 2017 substrate to investigate its potential of continuous biohydrogen production. The experi-
Received in revised form ments used a completely stirred tank reactor digester with operating conditions of pH 6.8,
4 September 2017 35
C, and hydraulic retention times (HRTs) 8 h and 4 h. Biohydrogen production perfor-
Accepted 3 October 2017 mance at HRT 8 h was detailed and effectives of doubled organic loading rate (OLR) were
Available online xxx elucidated at HRT 4 h. The experimental results indicate that coagulated TDW at an OLR of
30 g total sugar/L gave 3.8 L/L-d as the peak hydrogen production rate (HPR), which value is
Keywords: comparable to feedstock like sugarcane vinasse, palm oil mill effluent and cassava
Biohydrogen wastewater. TDW is a good source for biohydrogen production. Moreover, an OLR man-
Coagulation agement is an efficient strategy to enhance HPR. Methane production occurred in a long
Dark fermentation run H2 fermenter and its prevention methods were discussed. Acetate and butyrate were
Desizing wastewater the main soluble metabolic products. Future works for using the experimental data in
Organic loading rate industry applications are proposed with (1) HPR increment, (2) H2 fermenter effluent
Textile industry treatment, and (3) re-confirming the application feasibility of the experimental data.
© 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

environment-friendly characteristics are getting more

Introduction
Hydrogen has positive combustion characteristics of high
heating value and no pollutant emission when comparing
with methane and is known as a clean energy carrier. Many
thermochemical and biological methods have been proposed
and used for hydrogen production in practical applications or
researches but green ones with low energy-consumption and
attraction. Dark fermentation or anaerobic digestion of
organic wastes/wastewaters is the most promising mode of
biohydrogen generation [1,2]. This green biohydrogen pro-
duction technology is currently reported to have the potential
to become a major hydrogen-generating method in green
economy [3].
Exploring new feedstock sources is important in bioenergy
industry. Textile industry wastewaters contain high strength

Abbreviations: BPR, Biogas production rate; COD, Biochemical oxygen demand; CSTR, Completely stirred tank reactor; HPR, Hydrogen
production rate; HRT, Hydraulic retention time; HY, Hydrogen yield; OLR, Organic loading rate; ORP, Oxidation-reduction potential;
POME, Palm oil mill effluent; SMP, Soluble metabolic products; TDW, Textile desizing wastewater; VFAs, Volatile fatty acids; VSS, Volatile
suspended solids.
* Corresponding author. Green Energy Technology Research Group, Ton Duc Thang University, Ho Chi Minh City, Viet Nam.
E-mail address: chiu-yuelin@tdt.edu.vn (C.-Y. Lin).
https://doi.org/10.1016/j.ijhydene.2017.10.012
0360-3199/© 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

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2 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 x x x ( 2 0 1 7 ) 1 e7

organic and inorganic compounds such as starch, dye and
some chemicals. Recently, circular economy of efficiently
reusing industrial waste, sustainable textile production and
reusing and recycling textile wastewater are attracting at-
tentions [4e6]. Desizing operation is a main process in textile
industry and produces many pollutants including starch [7].
Individually treating this textile desizing wastewater (TDW) is
attractive in having simple technology and economic advan-
tages over a centralized-treatment [8]. Starch-containing TDW
has chemical oxygen demand (COD) concentrations of
20e40 g/L, indicating the potential of generating renewable
energy biogas such as biohydrogen and biomethane via dark
fermentation [9]. However, most of the desizing operation
uses bacterial growth-inhibitive dyes that might negatively
affect biogas fermentation. Recently, coagulation-
pretreatment has shown its high potential of enhancing bio-
hydrogen production from starch-containing TDW with 120%
and 550% increments in hydrogen yield (HY) and hydrogen
production rate (HPR), respectively, in our previous batch test
[10]. This coagulation-pretreated TDW gave an HY of 1.52 mol/
mol glucose and an HPR of 3.9 L/L-d which values are com-
parable to some other feedstock like cassava and paperboard
mill wastewaters [10] indicating the importance of pretreat-
ment in using TDW as a feedstock for biohydrogen produc-
tion. The biohydrogen production process in a continuous
mode is important in industry applications [11,12].
Based on the above observations, this work aims to
conduct a continuous fermentation fed on the coagulation-
pretreated starch-containing real TDW to elucidate the
usage of TDW for biohydrogen production. The biohydrogen
production performance of the bioreactor was investigated by
long operation (more than 2 months) and then the hydraulic
retention time (HRT) was reduced to double the organic
loading rate (OLR) to elucidate the performance. This work has
the novelties of using a TDW as a biohydrogen production
resource, developing a new TDW treatment method and
proposing a stagey for increasing HPR from TDW via OLR
management.
Materials and methods
The experiences of batch hydrogen production from
coagulation-pretreatment TDW in coagulant dosage and
coagulation operation conditions obtained from our previous
studies [10] were used as the references for operating the
continuous-feeding biohydrogen fermentation system.
Seed, bioreactor and substrate TDW
Seed sludge
In biohydrogen production experiments, a granular sludge
was used as the seed. This granular sludge was collected from
a fructose factory's anaerobic digester and had characteristics
of pH 7.71, total COD 50 g/L, total sugar 5.3 g/L, NH3eN 45 mg/L
and volatile suspended solids (VSS, expressing the biomass
concentration) 28.8 g/L. Before being used as the seed micro-
flora, the collected granular sludge was treated via water bath
(95
C, 1 h) to activate hydrogen-producers and inactivate
methanogens.
Bioreactor
A lab-scale completely stirred tank reactor (CSTR) digester
was used to in the continuous-feeding H2 production experi-
ments. This glass-made circular digester (inter diameter
13 cm, height 18 cm) had a working volume of 2.3 L (Fig. 1). Hot
water jacket was used to control the bioreactor operation
temperature. A drum-type gas meter (Ritter TG05, Germany)
was used to measure biogas production. This bioreactor was
operated in an ambient atmosphere pressure.

Fig. 1 e A scheme of the CSTR biohydrogen production bioreactor for continuously feeding operation.

Please cite this article in press as: Lin C-Y, et al., Continuous biohydrogen production from coagulation-pretreated textile desizing
wastewater, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.10.012
 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 x x x ( 2 0 1 7 ) 1 e7
Substrate TDW
A real TDW was coagulation-pretreated and used as the sub-
strate. The real TDW was obtained from a textile factory and had
the characteristics (average values) of COD 47 g/L, total sugar
17.0 g/L, sulfate 0.39 g/L, suspended solids 2.0 g/L and pH 6.8. The
range values for these characteristics were detailed in our pre-
vious study [10]. Coagulation-pretreatment gave COD and total
sugar removal rates of 12% and 8.5%, respectively. Coagulant
GGEFloc-653 (concentration 1 g/L) was used in the coagulation-
pretreatment [10]. The coagulation operation conditions ob-
tained in our previous work [10] were used: pH 7.0, rapid mixing
(100 rpm) 3 min, slow mixing (30 rpm) 20 min and sludge settling
time 1 h. The coagulated TDW supernatant was diluted (with tap
water) to total sugar concentrations of 5 and 10 g/L. They were
used as the substrate (substrate TDW) for further H2 fermenta-
tion. This substrate TDW was stored at 4
C before usage to
minimize any biochemical reactions. The inorganic nutrients
for bacterial growth were detailed in our previous study [10].
Fermentative biohydrogen production
Continuous operation of H2 fermentations was at pH 6.8 and
35
C using the CSTR fermenter. In starting-up the bioreactor,
seed inoculum 0.5 L and substrate TDW 1.8 L (10 g total sugar/L
in concentration) were put in the reactor and then gassed with
argon for 3 min to remove oxygen from the head space for
maintaining anaerobic environment. This reactor used a
batch mode operation for 1 day and then the continuous
feeding started at HRT 8 h. At day 17, the tested TDW con-
centration was changed to 5 g/L (as total sugar) because the
collected real TDW concentration was low from factory
operation. For this stage (5 g/L, HRT 8 h ¼ OLR 15 g/L-d), the
performance of the H2 fermenter was observed as reactor re-
sponses to environmental changes such as feeding-failure.
After the desired data were collected, the HRT was then
reduced to 4 h to elucidate the hydrogen production perfor-
mance when the OLR was doubled (OLR ¼ 30 g/L-d). During all
the experiments, the volume and content of biogas and
metabolite concentrations such as volatile fatty acids (VFAs)
were determined at proper time intervals. H2 concentration
(%) and HPR were used as the main indicators to show the
performance of H2 fermentation.
Analysis
The concentrations of H2, CO2 and VFAs were determined with
Shimadzu GC-14A gas chromatographs equipped with ther-
mal conductivity and flame ionization detectors. The opera-
tion conditions of these detectors were similar to those of our
previous study [11]. The methods used for measuring
oxidation-reduction potential (ORP), pH, sugar concentration,
VSS and COD were similar to a previous work of us [10].
Results and discussion
Biohydrogen production at HRT 8 h (OLR 15 g total sugar/L-d)
The H2 fermentation performances of the coagulation-
pretreated TDW were shown in Fig. 2 (daily variations of H2
Please cite this article in press as: Lin C-Y, et al., Continuous biohydrogen production from coagulation-pretreated textile desizing
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3
concentration, HPR, biogas production rate (BPR), pH and ORP
at TDW 5 g total sugar/L). The reactor performances during
started-up stage of using TDW 10 g total sugar/L were not
shown.
ORP is an important monitoring indicator showing the
anaerobic environment of a bioreactor. Fig. 2 shows that at
HRT 8 h through the reactor operations, the ORP generally
fluctuated between 420 mV and 510 mV with average ORP
values being in the range of around 450~480 mV. In the
beginning, marked fluctuation ORP values were observed;
they might result from the adaption of the microbes to new
cultivation environments. These average ORP values were in
the range favoring biohydrogen production. Some fluctua-
tions in ORP were observed when feeding failures occurred. H2
concentration in a biogas is a common indicator to show the
performance of a biohydrogen fermenter. During the start-up
of the reactor, H2 concentration reached 30% at day 1 (1 day
after the reactor start-up) and reached 60% at day 2 (1 day after
the continuous feeding began) but decreased to near 20% at
day16 (data were not shown in Fig. 2). At day 17, the substrate
TDW concentration was changed to 5 g/L (from 10 g/L), which
made the H2 concentration increase markedly from 20% (at
day 16) to 45% at day 23 (6 days after TDW concentration
change, Fig. 1) and then some decreases were observed from
feeding or mixing failures. The reasons of feeding failures
included power failures, pipe blocks and feedstock shortages.
Fig. 2 also indicates that H2 concentrations reached a constant
value of 30% after day 40. Another biogas CH4 that was not an
expected component appeared at days 40e60 in a small level
of 0.2e2%. CH4 generation might result from the growth of
methanogens in the reactor wall. We inactivated the metha-
nogens via heating the reactor content at 95
C for 1 h at day
60. After that, there was no methane until day 72; days 72e78
had 0.3e4% of methane. At day 78, the reactor content was
heat-treated again (95
C for 1 h) to inactivate the metha-
nogens. A similar operation strategy had been reported
recently. Bundhoo et al. [13] indicate that in the long run of
biohydrogen production, some seed pre-treatment methods
are not effective and repeated treatments for the fermenter
content are necessary for continuously suppressing H2-
consuming bacteria.
HPR is a well-used parameter in monitoring or evaluating
biohydrogen production performance. HPR indicates the
capability of a biohydrogen-producing reactor with higher
value favoring practical applications. A HPR value correlates
with feedstock source and concentration, reactor configura-
tion and reactor operation conditions. Fig. 2 also shows that
the HPR value was dependent on the reactor operations and
its variation trend met the variation of H2 concentration but in
bigger fluctuation. The average HPR value during reactor's
steady-state condition at the TDW 5 g total sugar/L was
0.88 ± 0.30 L H2/L-d. These values are rather lower than those
obtained from our batch operation results (HPR 3.9 L H2/L-d at
TDW 15 g total sugar/L [10]). However, the HPR value is com-
parable to some other feedstock like cassava (0.39 L H2/L-
d [14]; 2.1 L H2/L-d [15]), sugarcane vinasse (1.12 L-H2/L-d [16])
and palm oil mill effluent (POME) (2.64 L H2/L-d [17]; 3.68 L H2/
L-d [18]).
BPR represents total biogas production rate. In this study,
after reactor's long run operation, the Ar gas added during the
4 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 x x x ( 2 0 1 7 ) 1 e7

Fig. 2 e Daily variations of hydrogen production rate (HPR), H2 concentrations, biogas production rate (BPR), pH and ORP at
HRTs of 8 h and 4 h.

reactor starting-up stage was replaced by the produced biogas.
During these long run operations, the biogas contained only
H2 and CO2. Therefore, the daily BPR variation trend was
similar to that of HPR. The average BPR value during the re-
actor's steady-state stage was 5 L/L-d.
Biohydrogen production performance at HRT 4 h (OLR 30 g
total sugar/L-d)
When the HRT was decreased from 8 h to 4 h for TDW 5 g total
sugar/L, the reactor OLR was accordingly doubled as 30 g total
sugar/L-d. Fig. 2 shows that when the HRT was decreased to
4 h, H2 concentration, HPR and BPR elevated promptly. These
parameters reached constant values and did not decrease
except at the times of feeding failures. Constant H2 concen-
tration, HPR and BPR values reached 35%, 3.8 L H2/L-d and 12 L/
L-d, respectively. Moreover, very low level of methane
(0.2e0.5%) appeared again at day 104 and after day 109.
Liquid metabolic products during fermentation
Soluble metabolic products (SMP) such as alcohols and VFAs
are produced during fermentative biohydrogen production.
Fig. 3 depicts the daily variations of SMP concentrations dur-
ing reactor operations. An observation on these SMP data of
days 0e27 indicates that the major liquid metabolites were
ethanol, acetate, propionate and butyrate when the reactor
was in varied environments raised from reactor starting-up.
During the reactor start-up period of days 1e17, there were
batch feeding, continuous feeding and substrate concentra-
tion changing, which operations made environmental
changes of the digester contents. However, ethanol (around
4000 mg/L) and propionate (around 2000 mg/L) concentrations
markedly decreased to lower than 200 mg/L after the starting-
up period. In contrast, acetate (around 2700 mg/L) and buty-
rate (around 3000 mg/L) concentrations had no such marked
decrease with values of 1000 mg/L (acetate) to 1500 mg/L

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 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 x x x ( 2 0 1 7 ) 1 e7 5

Fig. 3 e Daily concentration variations of soluble metabolic products at HRTs of 8 h and 4 h.

(butyrate). Higher butyrate concentration shows that there
was a butyrate-type fermentation that giving good bio-
hydrogen production [19]. Moreover, the concentrations of
acetate and ethanol were both around 200 mg/L showing their
concentration ratio was one, which value indicating a good
hydrogen production [20].
Significances of the experimental results
Starch-containing TDW as substrate for biohydrogen
production
Our studies using real TDW as the substrate for producing
biohydrogen via dark fermentation in batch [10] and contin-
uous (this work) modes resulted in peak HPR values of
3.8e3.9 L H2/L-d. Since industry applications prefer a contin-
uous operation mode, the results obtained from the contin-
uous operation were used to compare with other substrates to
show the applicable characteristics of TDW for biohydrogen
production. The peak HPR value 3.8 L H2/L-d was obtained in
this continuous H2 fermentation at the operation conditions
of HRT 4 h and 5 g total sugar/L-d (equal to an OLR of 30 g total
sugar/L-d). A comparison of continuous biohydrogen pro-
duction from various feedstock/wastewaters is shown in
Table 1. It indicates that a coagulation-pretreated starch-
containing TDW has biohydrogen production potential with a
HRP comparable to some wastewaters such as POME and
cassava wastewater.
Biohydrogen production performance in the long run of the
reactor
In biohydrogen production operation, it is designed to maxi-
mize the H2 fraction with lower CO2 fraction and even no CH4
composition in the biogas. However, this work shows that CH4
appeared in the biogas when the reactor had long operations
both at HRT 8 h and 4 h. In this situation, we had heated the
reactor contents for inactivating methanogens and then pro-
moting biohydrogen production. The cases of long reactor
operation making the slow development of methanogenic
activity have also been reported in running biohydrogen fer-
menters [13,22]. The appearance of CH4 indicates the

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L-d). Of course, a HRT decrease to 3 or 2 h or even lower values

Table 1 e Biohydrogen production performance of
to give higher OLR may be feasible because some reports show
various wastewaters.
that at these low HRT values high biohydrogen could be ach-
Substrate OLR Reactor HPR Reference
ieved in dark fermentation of some wastewaters [21,26,27].
(g COD/L-d) (L/L-d)
Moreover, an observation on the relationships between the
Cassava wastewater 161 AFBR 96 [21] reactor type, OLR and HPR in Table 1, it is known that to have
Cassava wastewater 25 UASB 0.39 [14]
high HPR, granular microflora or immobilized-cells systems
Cassava processing 14 AFBR 2.1 [15]
wastewater
such as up-flow sludge blanket bed and anaerobic packed bed
De-oiled jatropha e CSTR 0.15 [12] reactors can be applied and operated at high OLR for obtaining
waste high HPR values.
POMEa 94.4b Batch 2.64 [17]
POME 75 UASB 3.68 [18] Future studies on applying the process for industrial usage
Sugarcane vinasse 84.2 APBR 1.12 [16]
Continuous systems are necessary for using various biomass
TDW 60 CSTR 3.8 This study
sources to produce biohydrogen for industry applications. The
a
POME, Palm oil mill effluent; TDW, textile desizing wastewater; present work has successfully shown the feasibility and
OLR, organic loading rate; AFBR, anaerobic fluidized bed reactor;
possible strategy for conducting a continuous biohydrogen
APBR, up-flow anaerobic packed bed reactor; UASB, up-flow
production from a coagulated TDW. However, there are some
anaerobic sludge bed; CSTR, completely stirred tank reactor;
HPR, hydrogen production rate. further studies needed to do if a starch-containing TDW is to
b
g COD/L. be used as a substrate for bioenergy production in industry
applications. These issues include (1) how to increase the HPR,
(2) how to treat the H2 fermenter effluent, and (3) to re-confirm
presences of methanogens that might consume H2 to produce the application feasibility of the experimental data. High HPR
CH4. This might result from the fact that in a long run oper- favors industrial application of H2. As mentioned above, HPR
ation, seed pre-treatment methods are not effective and, could be increased by lowing the HRT to 3 h, 2 h or lower
therefore, it is necessary to find a suitable seed pre-treatment values [21,26,27]. Moreover, a direct H2 fermentation and in-
technology for industrial application [13]. In the present wok, direct bio-H2 production via methane-reforming might be
heat-pretreatment was applied to treat the seed sludge to used to increase HPR. It has been reported that the HPR was
obtain highly-active hydrogen-producers and to inhibit increased by 10% via direct and indirect bio-H2 production
methanogenic activity. Note that during heat-treatment the methods, which used a two-stage anaerobic digestion to
methanogens are inhibited but not killed and this fact leads to produce biogas and simultaneously to reduce the effluent
the slow development of methanogenic activities in a long- organic strength [28]. Some tests are needed to evaluate the
operating H2 fermenter. potential of continuous two-stage anaerobic digestion for a
TDW [29e31]. To re-confirm the industrial application feasi-
OLR management for enhancing HPR bility of the proposed total-solution process and experimental
To have high HPR is important for a biohydrogen production data, a techno-economic evaluation is suggested [32,33].
system in its industry applications. Recently, Arimi et al. [1]
used OLR management as a strategy to improve biohydrogen
production from a wastewater. In the present work, Conclusions
comparing the biohydrogen production performances ob-
tained at HRT 8 h and 4 h could elucidate the OLR effect. It is This work aims to elucidate the usage of TDW for producing
observed that a small increase in H2 concentration (30% vs. biohydrogen as a biofuel. Based on the continuous-feeding
35%), a big increase in BPR (5 vs. 12 L/L-d) and a very big in- operation results, the following conclusions are drawn.
crease in HPR (0.9 vs. 3.8 L H2/L-d) were obtained when the OLR Starch-containing TDW is a potential resource for fermenta-
was doubled. Increments of 140% [¼ (125)÷5] and 320% tive biohydrogen production if it is coagulation-pretreated. A
[(3.8e0.9)÷0.9] were obtained for BPR and HPR, respectively, continuous H2 fermenter can use an OLR management strat-
though only 100% increase in the OLR. The enhancement egy to enhance HPR but methane production might occur in
capability of increasing OLR was obtained. the long run operation. A continuous CSTR operation at HRT
There are many reports showing that OLR affects bio- 4 h (OLR 30 g total sugar/L) could give peak HPR 3.8 L H2/L-d,
hydrogen production in dark fermentation of various feed- which value is comparable to other feedstock as sugarcane
stock but not higher OLR gives higher HPR [16,23]. Moreover, vinasse, POME and cassava wastewaters. HPR increment, H2
the OLR values studied in literature are in a range of 2e480 g fermenter effluent treatment and re-confirming the applica-
COD/L-d and the optimal values to give peak HPR are 20e80 g tion feasibility are elucidated as the main future works for
COD/L-d [16,23e25]. In the present study, the tested OLR using the experimental data in industry applications.
values were 15 and 30 g total sugar/L-d (nearly equaling to 30
and 60 g COD/L-d, respectively) which are in the optimal value
range for biohydrogen production. These facts indicate that Acknowledgements
controlling a proper OLR is an important strategy to have a
high HPR for industry applications. The proper OLR for the This research is funded by Foundation for Science and Tech-
pretreated-TDW is suggested as 30 g total sugar/L-d (60 g COD/ nology Development of Ton Duc Thang University (FOSTECT)

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 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 x x x ( 2 0 1 7 ) 1 e7
(website: http://fostect.tdt.edu.vn) under Grant FOS-
TECT.2015.BR.17, and Ministry of Science and Technology of
Taiwan (MOST 104-2221-E-035 -006 -MY3).
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wastewater, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.10.012
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