processes

Article
Effect of Hydraulic Retention Time on Anaerobic
Baffled Reactor Operation: Enhanced Biohydrogen
Production and Enrichment of
Hydrogen-producing Acetogens
Fan Jiang 1 , Zhiying Peng 2 , Huaibo Li 2 , Ji Li 2,3 and Shuo Wang 2,3,4, *
1 Nanjing Municipal Engineering Quality and Safety Supervision Station, Nanjing 210000, China;
jf_850613@163.com
2 Jiangsu Key Laboratory of Anaerobic Biotechnology, School of Environment and Civil Engineering, Jiangnan
University, Wuxi 214122, China; pengzhiying2021@163.com (Z.P.); lhbo1995@126.com (H.L.);
liji@jiangnan.edu.cn (J.L.)
3 Jiangsu College of Water Treatment Technology and Material Collaborative Innovation Center,
Suzhou 215009, China
4 Department of Civil Engineering, Schulich School of Engineering, University of Calgary, Calgary,
AB T2N 1N4, Canada
* Correspondence: shuowang@jiangnan.edu.cn; Tel.: +(86)-510-85197210




Received: 23 January 2020; Accepted: 9 March 2020; Published: 15 March 2020


Abstract: Biohydrogen production by fermentation has become a promising technology developed

in the world. In this study, diluted normal molasses wastewater is used as the raw material,
and biohydrogen production efficiency and operation characteristics of an anaerobic baffled reactor
(ABR) are studied. The effect of hydraulic retention time (HRT) on biohydrogen production
efficiency and operating characteristics of ABR is extensively discussed. Experimental results
showed that methanogen residuals were still observed in the last three compartments under HRT
of 24 h and COD(Chemical oxygen demand) concentration of 8000 mg/L. Meanwhile, the first
three compartments presented an ethanol fermentation type. The characteristics of butyric acid
fermentation in Compartment IV were also enhanced. The average removal efficiency of COD
was reduced to 15.4%. The average rates of biohydrogen production and specific biohydrogen
production were 12.85 and 360.22 L/kg COD, respectively. The extension of HRT was beneficial to
enrich hydrogen-producing acetogens and could increase the production rate of biohydrogen by
a factor of 1.65. However, with the decrease in the bioactivity of acidogenic fermentation bacteria,
the biohydrogen production efficiency of ABR was significantly reduced when HRT was longer than
30 h. The specific biogas production rate decreased from 191 to 92 L/(kg MLVSS·d). The specific
biohydrogen production rate also decreased from 24.34 to 2.7 L/(kg MLVSS·d).

Keywords: hydraulic retention time (HRT); biohydrogen production; normal molasses wastewater;
anaerobic baffled reactor (ABR); biohydrogen-producing acetogens (HPA)

1. Introduction
With the rapid development of the modern food industry, the wastewater produced with high
organic matter is also gradually increasing. Thus, a huge amount of organic wastewater is directly
discharged to receiving water. Normal molasses wastewater is a typical high-strength organic
wastewater, which is also the by-product of sugar refinement. Gokcek et al. found that COD removal
increased along with the increase of organic loading rate, while the biogas yield decreased [1]. In order
to reduce the environmental risks, enhanced biogas and lipid production are well developed. By the

Processes 2020, 8, 339; doi:10.3390/pr8030339 www.mdpi.com/journal/processes

Processes 2020, 8, 339 2 of 18

enrichment of biohydrogen-producing acetogens, the specific hydrogen and methane yields from COD
removal increased by factors of 1.54 and 1.63 in normal molasses wastewater treatment [2]. In addition,
lipid production through microalgae cultivation in normal molasses wastewater treatment presented
its economic feasibility when a lipid content of 28.9% and lipid productivity of 94.4 mg·m−3 ·d−1 were
finally obtained [3,4].
The use of fermentation to produce biohydrogen is a process in which hydrogen-producing
microorganisms metabolize organic matter to produce hydrogen under anaerobic conditions [5].
The mechanism of the fermentation to produce biohydrogen has three possible ways: pyruvate
decarboxylation in the EMP(Embden–Meyerhof–Parnas) pathway, the oxidation and reduction
balance adjustment of acetyl coenzyme I, and the bacteria producing hydrogen and acetic acid [6].
The Enterobacter aerogenes ho-39 and E. aerogenes hu-10l strains have been adopted to conduct
biohydrogen production by non-immobilized and immobilized experiments. The results showed
that the continuous and maximum biohydrogen rates of the immobilized model is higher than that
of the un-immobilized model [7,8]. Continuous flow fermentation for biohydrogen production on
anaerobic-activated sludge (AnAS) with self-flocculation ability broke through the limitation of pure
bacteria compartment immobilization and created a new field to enhance biohydrogen production
based on non-immobilized mixed strains (AnAS) [9,10].
An anaerobic baffled reactor (ABR) is the third generation of anaerobic bioreactors in which
the specific design is to set up a series of vertical baffles in the reactor. Thus, the wastewater can
be introduced up and down along the baffle in ABR and then passed through the sludge bed of
each compartment [11]. The operation flow path of ABR is similar to that of the plug flow process,
which presents the provision of upper and lower baffles to form a compartment [12]. Such processes
can connect and effectively separate microorganisms from methanogenic and acidogenic phases [13].
Therefore, two-phase anaerobic digestion can be established in a single reactor. The organic loading
rate, influent type, sludge reflux, and agitation, temperature, and mathematical models have been
studied comprehensively to achieve biohydrogen production through high-load organic wastewater
fermentation in ABR [14–17].
Methanogens have strict living conditions and slow metabolism and growth rate. The traditional
theory considers methanogens as the first rate-limiting step in the anaerobic wastewater treatment
process because biohydrogen production and acetic acid production stage is prior to methane production
stage [18,19]. High organic matter can lead to low pH value through fermentation [20]. Thus,
the inhibition of methanogens caused by relatively low pH is beneficial to increase the biohydrogen
production rate. Therefore, this study proposes the startup and operation of ABR to investigate
the biohydrogen production performance. In accordance with the regulation of hydraulic retention
time (HRT), the characteristics of each compartment are discussed comprehensively. Furthermore,
the enhanced biohydrogen production through HPA is discussed to provide a potential approach for
eliminating methane production and preserving biohydrogen resources.

2. Materials and Methods

2.1. ABR Configuration and Setup

A four-compartment ABR with a total volume of 28.75 L was adopted in the experiment (Figure 1).
Each compartment had an equal size and had downflow and upflow chambers of 2.5 and 11.5 cm
in width, respectively. Normal molasses wastewater (NMWW) was introduced to the bottom of the
upflow chamber and finally drained through a guide plate with an inclination of 45◦ at the downflow
chamber. The sampling ports were arranged at different heights of each compartment. An effluent pipe
was set at the top to connect with the water seal. The sealed water bottle and wet gas flowmeter were
filled with HCl (pH of 3) to prevent gas dissolution. The ABR temperature was maintained at 35 ± 1 ◦ C.
The operation control of ABR was conducted in stages, and Table 1 presents the specific control mode.
Processes 2020, 8, 339 3 of 18
Processes 2020, 8, x FOR PEER REVIEW 3 of 18

Figure 1. Schematic diagram of the experiment.

Figure 1. Schematic diagram of the experiment.
Table 1. The parameters of an anaerobic baffled reactor (ABR).
Table 1. The parameters of an anaerobic baffled reactor (ABR).
Running Temperature Influent COD Organic Loading
Running
Phase HRTHRT (h)
Temperature(◦ C)
Influent pH
Influent Influent COD (mg/L) Organic
Concentration (kgCOD·mloading
−3 ·d−1 )
-3 -1
phase
First stage (h) (℃) pH concentration（mg/L） （kgCOD·m ·d ）
24 35 ± 1 6.4–7.6 7730–8490 7.73–8.49
First(1–10
staged)
24 35 ± 1 6.4–7.6 7730–8490 7.73–8.49
(1–10
Second d)stage 30 35 ± 1 7.0–7.6 7273–8776 7.34–8.71
(11–70 d)
Second stage
Third stage 30 40 35 ± 1 35 ± 1 7.0–7.6 7.0–7.5 7273–8776 7.34–8.71
(11–70
(71–87d)d) 7309–8287 7.21–8.32
Third stage
40 35 ± 1 7.0–7.5 7309–8287 7.21–8.32
(71–87 d)
2.2. Inoculated Sludge and NMWW
2.2. Inoculated
The NMWW sludge and NMWW
(Table 1), with pH ranging from 5.3 to 5.8, was obtained from a sugar beet factory.
The total nitrogen(Table
The NMWW of NMWW 1), withwaspH35ranging
mg/L; thus,
from the corresponding
5.3 to 5.8, was obtainedC/N from
was 230. To maintain
a sugar the
beet factory.
bioactivity of the anaerobic activated sludge, NH
The total nitrogen of NMWW was 35 mg/L; thus,4 the corresponding Cl and K 2 HPO 4 were added at a COD:N:P
C/N was 230. To maintain the ratio of
200:5:1. NaHCO
bioactivity was used activated
of the3anaerobic to adjust the initial
sludge, NH pH value of the wastewater to 7.5–8.0. In addition, a
4Cl and K2HPO4 were added at a COD:N:P ratio of
large amount of particulate matter is generated
200:5:1. NaHCO3 was used to adjust the initial pH value at the bottom
of theofwastewater
the influenttotank afterInstorage
7.5–8.0. for aa
addition,
long period of time, which can significantly reduce the bioactivity of activated
large amount of particulate matter is generated at the bottom of the influent tank after storage forsludge. Therefore, a newa
type of NMWW with litter particulate matter was provided after 32 days of ABR
long period of time, which can significantly reduce the bioactivity of activated sludge. Therefore, a operation to ensure
the stable
new operation
type of NMWWofwith the ABR
littersystem and investigate
particulate matter wasthe effect ofafter
provided HRT32ondaysbiohydrogen production.
of ABR operation to
The inoculated aerobic activated sludge was obtained from Wenchang
ensure the stable operation of the ABR system and investigate the effect of HRT on biohydrogen Wastewater Treatment Plant
(Harbin, China),
production. The and the anaerobic-activated
inoculated aerobic activated sludge
sludge(AnAS) was collected
was obtained frominWenchang
a beer brewhouse.
WastewaterThe
AnAS was initially filtered and washed to remove inorganic particles. In order
Treatment Plant (Harbin, China), and the anaerobic-activated sludge (AnAS) was collected in a beer to enhance biohydrogen
production efficiency
brewhouse. The AnASinwas each compartment
initially of ABR,
filtered and washeddifferent inoculation
to remove strategies
inorganic were
particles. adopted
In order to
to maintain biomass retention and increase the ratio of the functional
enhance biohydrogen production efficiency in each compartment of ABR, different inoculation genus. The first and fourth
compartments
strategies were inoculated
were adopted to maintainwithbiomass
aerobicretention
and anaerobic sludges,
and increase therespectively. Compartments
ratio of the functional genus.2
and first
The 3 were
andinoculated with mixedwere
fourth compartments aerobic activated
inoculated withsludge
aerobicandandAnAS in thesludges,
anaerobic ratios ofrespectively.
5:2 and 2:5
(v/v), respectively.
Compartments 2 and 3 were inoculated with mixed aerobic activated sludge and AnAS in the ratios
of 5:2 and 2:5 (v/v), respectively.
2.3. Analytical Methods
COD, pH,methods
2.3. Analytical alkalinity (ALK, in terms of CaCO3 ), and mixed liquor volatile suspended solids (MLVSS)
were regularly determined by the standard method (APHA, 1995) [21]. The biogas production of ABR
COD, pH, alkalinity (ALK, in terms of CaCO3), and mixed liquor volatile suspended solids
(MLVSS) were regularly determined by the standard method (APHA, 1995) [21]. The biogas
production of ABR compartments was measured with a wet biogas flowmeter. The morphology of
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Processes 2020, 8, x FOR PEER REVIEW 4 of 18

AnAS was was

compartments observed
measured with with
a scanning electrical
a wet biogas microscope
flowmeter. The(JSM-5610).
morphology The ofcomponents
AnAS was observed and contents
withof fermentation
a scanning biogas,
electrical volatile (JSM-5610).
microscope fatty acids (VFAs) in wastewater,
The components and ethanol
and contents were determined
of fermentation biogas, by
volatile fatty acids (VFAs) in wastewater, and ethanol were determined by gas chromatography [22]. gas
gas chromatography [22]. The liquid terminal product was determined with a GC-122
chromatograph,
The liquid terminal product whichwas wasdetermined
determinedwith under the following
a GC-122 conditions: supporter
gas chromatograph, which wasGDX-103
determined (60–80
under mesh) + 2% H3PO
the following 4; size of packed
conditions: supporter column of 2 m
GDX-103 × Ф 5mesh)
(60–80 mm; and+ 2%carrier
H3 POgas4 ; rates
size of of N
packed2 , H , and
column
2 air of
of 2 50,
m ×50, Φ and
5 mm; 500andmL/min,
carrierrespectively.
gas rates of The N2 , highest
H2 , andtemperature
air of 50, 50,ofand the500
columnmL/min,box respectively.
was 250 °C, the
temperature
The highest of the sampler
temperature was 200
of the column °C,was
box and250the◦ C,
temperature of theofion
the temperature thechamber
samplerwas was240 200 °C.◦ C, The

and column temperature

the temperature of the was
ion190 °C, andwas
chamber the240 ◦ C. The
sample volume
columnwastemperature
2 mL. Before wasthe190 ◦ C, and the the
determination,
samplesample should
volume wasbe pretreated
2 mL. Before theas follows: after centrifugation
determination, the sample shouldor filtration of the water
be pretreated sample after
as follows: from the
continuous
centrifugation or flow reactor,
filtration of the5 water
mL was taken,
sample from and
the2continuous
drops of 6flow mol/L HCl5were
reactor, mL was added.
taken,The andbiogas
2
components in fermentation gas were determined with gas chromatography
drops of 6 mol/L HCl were added. The biogas components in fermentation gas were determined with (Lunan SC-7, Tengzhou,
China) under the
gas chromatography following
(Lunan SC-7,conditions: oven temperature
Tengzhou, China) of 60 °C,
under the following injection temperature
conditions: oven temperature of 80 °C,
◦ C, injection
of 60detection temperature
temperature of 200
of 80°C,◦ C,
N2detection
= 28 mL/min, and sample
temperature of 200 ◦ C, N of
volume 2 = 200
28 mL.
mL/min, and sample
volume of 200 mL.
3. Results
3. Results
3.1. Biomass and morphology of AnAS
3.1. Biomass and Morphology of AnAS
Figure 2 shows the variations of biomass concentration from each compartment of ABR, in which
Figure
the 2 shows
initial MLVSS thewere
variations
7.0, 6.0,of 5.8,
biomass concentration
and 11.5 from each The
mg/L, respectively. compartment
biomass in of Compartments
ABR, in which I to
the initial MLVSS were
III presented 7.0, 6.0,
a decrease 5.8,and
first andthen11.5an
mg/L, respectively.
increase along with Thethe
biomass in Compartments
regulation of HRT (fromI Stage to III I to
presented a decrease first and then an increase along with the regulation of HRT
Stage II), indicating that the microbial activity and stable operation of ABR can be well maintained (from Stage I to Stage
II), indicating
when suitable that the microbial
extension HRT activity and stable
is provided. operation
However, of ABR can
the biomass be well maintained
concentration in Compartment when IV
suitable extension
notably decreased HRT dueis provided.
to sludgeHowever,
floatationtheandbiomass
loss whenconcentration
HRT was in Compartment
extended to 40 h,IV notably that
implying
decreased due to sludge floatation and loss when
relatively long HRT is beneficial for the growth of methanogens. HRT was extended to 40 h, implying that relatively
long HRT is beneficial for the growth of methanogens.

Figure 2. Biomass concentration trend over time in each compartment of ABR.

Figure 2. Biomass concentration trend over time in each compartment of ABR.
Figure 3 illustrates the morphology of AnAS in each compartment during the stable phase of
Figure
Stage III. The 3 illustrates
morphology the morphology
in each compartmentofwasAnAS in each compartment
significantly different. The during the stable phase
first compartment was of
Stage III. The morphology in each compartment was significantly different. The
dominated by Bacillus, and the population of short Bacillus in Compartment II increased. The dominant first compartment
wasindominated
bacteria Compartmentby Bacillus, andBacillus,
III was still the population of short
and the long Bacillus
Bacillus in Compartment
increased. However, largeII increased.
amounts The
dominant
of cocci bacteria
were found in Compartment
in Compartment IV,III
andwas
thestill Bacillus,
number and thestrains
of plump long Bacillus
decreasedincreased. However,
significantly.
large amounts of cocci were found in Compartment IV, and the number of plump strains
The reason could be due to the lack of substrate, which further led to a decrease in microbial activity. decreased
significantly. The reason could be due to the lack of substrate, which further led to a decrease in
microbial activity.
Processes 2020, 8, 339 5 of 18

Processes 2020, 8, x FOR PEER REVIEW 5 of 18

(a)

(b)

(c)

(d)

Figure 3. (a) Morphology of anaerobic-activated sludge (AnAS) in Compartment I at Stage III;

(b) morphology of AnAs in Compartment II at Stage III; (c) morphology of AnAs in Compartment III
at Stage III; (d) morphology of AnAs in Compartment IV at Stage III.
Processes 2020, 8, x FOR PEER REVIEW 6 of 18

Figure 3. (a) Morphology of anaerobic-activated sludge (AnAS) in Compartment I at Stage III; (b)
Processes 2020, 8, 339 of AnAs in Compartment II at Stage III; (c) morphology of AnAs in Compartment III at6 of 18
morphology
Stage III; (d) morphology of AnAs in Compartment IV at Stage III.

3.2. Biogas Production Characteristics of ABR

3.2. Biogas production characteristics of ABR
3.2.1. Biogas Production in Compartment I
3.2.1. Biogas production in Compartment I
In the startup phase of Stage I, the biogas production rate in Compartment I was stable at 13 L/d,
In the startup phase of Stage I, the biogas production rate in Compartment I was stable at 13 L/d,
the biohydrogen content fluctuated between 44.4% and 58.1%, and the methane content was always
the biohydrogen content fluctuated between 44.4% and 58.1%, and the methane content was always
below 0.5% (Figure 4). On day 7, the biogas production rate was approximately 13.4 L/d, and the
below 0.5% (Figure 4). On day 7, the biogas production rate was approximately 13.4 L/d, and the
biohydrogen content increased to 68.9%. However, a decrease in biohydrogen content occurred from
biohydrogen content increased to 68.9%. However, a decrease in biohydrogen content occurred from
day 21, and the methane content was always below 0.5%.
day 21, and the methane content was always below 0.5%.
40 80
Gas production rate
35 hydrogen 70
methane
Gas production rate（L/d）

30 60

percent gas volume(%)

25 50

20 40

15 30

10 20

5 10

0 0
0 10 20 30 40 50 60 70 80 90
Time（d）
Figure 4. Biogas
Figure 4. Biogas yield
yield and
and content
content in
in Compartment
Compartment I.I.

After HRT was extended to 30 h (Stage II), the biogas production rate in Compartment I increased
After HRT was extended to 30 h (Stage II), the biogas production rate in Compartment I
rapidly and reached a peak of 26 L/d on day 15. However, the biohydrogen production rate decreased
increased rapidly and reached a peak of 26 L/d on day 15. However, the biohydrogen production rate
to 1.7 L/d on day 32. The biohydrogen content did not change immediately with the variation in
decreased to 1.7 L/d on day 32. The biohydrogen content did not change immediately with the
pH. The content increased to approximately 50% on day 17 and remained at nearly 45% on day 32.
variation in pH. The content increased to approximately 50% on day 17 and remained at nearly 45%
By introducing a new type of NMWW to ABR, the biogas production started to recover from day 36
on day 32. By introducing a new type of NMWW to ABR, the biogas production started to recover
and continued to increase to the highest value of 19.9 L/d on day 49. The biohydrogen production
from day 36 and continued to increase to the highest value of 19.9 L/d on day 49. The biohydrogen
rate dropped to 7 L/d on day 62 and further reduced to less than 1 L/d. The biohydrogen content
production rate dropped to 7 L/d on day 62 and further reduced to less than 1 L/d. The biohydrogen
suddenly decreased to the lowest value of 33.4% on day 34, gradually recovered to 54.3% on day 36,
content suddenly decreased to the lowest value of 33.4% on day 34, gradually recovered to 54.3% on
and remained stable until the end of Stage II. The average biohydrogen content in the stable period
day 36, and remained stable until the end of Stage II. The average biohydrogen content in the stable
was approximately 50%, and the methane content was always below 0.5%.
period was approximately 50%, and the methane content was always below 0.5%.
In Stage III, with the increase in HRT (HRT = 40 h), the biogas production rate increased rapidly
In Stage III, with the increase in HRT (HRT = 40 h), the biogas production rate increased rapidly
from less than 1 to 4.3 L/d on day 71 and reached 5.6 L/d on day 72. On days 73 to 87, the ABR system
from less than 1 to 4.3 L/d on day 71 and reached 5.6 L/d on day 72. On days 73 to 87, the ABR system
was still unable to recover to the previous level. In other words, the microbial activity was severely
was still unable to recover to the previous level. In other words, the microbial activity was severely
inhibited, and the biogas production continued to decline. The biohydrogen content decreased to
inhibited, and the biogas production continued to decline. The biohydrogen content decreased to
42.5% and finally stabilized at approximately 49%.
42.5% and finally stabilized at approximately 49%.
3.2.2. Biogas Production in Compartment II
The variation in biogas production in Compartment II was different from that in Compartment I.
The biogas production rate reached a maximum value of 26.0 L/d on day 4 and then dropped to 15.7 L/d
on day 8 (Figure 5). The biohydrogen content gradually increased to 51% and suddenly decreased
Processes 2020, 8, x FOR PEER REVIEW 7 of 18

3.2.2. Biogas production in Compartment II

The
Processes variation
2020, in biogas production in Compartment II was different from that in Compartment
8, 339 7 of 18
I. The biogas production rate reached a maximum value of 26.0 L/d on day 4 and then dropped to
15.7 L/d on day 8 (Figure 5). The biohydrogen content gradually increased to 51% and suddenly
to 28.45% on day 8 but was recovered immediately on day 9. At the end of Stage I, the biohydrogen
decreased to 28.45% on day 8 but was recovered immediately on day 9. At the end of Stage I, the
content was approximately 50%.
biohydrogen content was approximately 50%.
40 80
Gas production rate
35 hydrogen 70
methane
Gas production rate（L/d）

30 60

percent gas volume(%)

25 50

20 40

15 30

10 20

5 10

0 0
0 10 20 30 40 50 60 70 80 90
Time（d）
Figure 5. Biogas yield and content in Compartment II.
Figure 5. Biogas yield and content in Compartment II.
In Stage II, the biogas production rate achieved a peak value of 31.6 L/d on day 28. As HRT was
In Stage
regulated to 30II,h,thethebiogas production
biohydrogen ratedecreased
content achieved arapidly
peak value of 31.6toL/d
and began riseon onday
day28.15.AsTheHRT was
content
regulated
reached 63.7%to 30 h, onthe biohydrogen
day 32. However, content
by decreased
introducing rapidly
a new and began
type to rise onto
of NMWW day 15. The
ABR, thecontent
biogas
production rate significantly reduced to 1.7 L/d and fluctuated between 14.3 and 17.7 L/d from biogas
reached 63.7% on day 32. However, by introducing a new type of NMWW to ABR, the day 36
production
to day 47. The raterate
significantly
suddenly reduced
droppedto to1.7
theL/d and value
lowest fluctuated
of 3.4between
L/d. The14.3biogas andproduction
17.7 L/d from day
started
36 to day 47. The rate suddenly dropped to the lowest value of 3.4 L/d. The
to increase slowly from day 56 and stabilized at approximately 8.1 L/d at the end of this stage (day biogas production started
to
70).increase slowly from
The biohydrogen day 56decreased
content and stabilized
slightly at approximately
and fluctuated 8.1 L/d at40%
between the end
and of60%thisinstage (day
this stage,
70). The
while thebiohydrogen
methane content contentwasdecreased
always belowslightly and fluctuated between 40% and 60% in this stage,
0.5%.
whileInthe methane content was always below 0.5%.
the third stage, the biogas production did not change immediately after HRT was extended.
In the the
However, thirdratestage, the biogas
suddenly production
decreased to 4.0did L/dnot
on change
day 73 immediately
and then slowly after increased
HRT was to extended.
7.9 L/d
However, the rate suddenly decreased to 4.0 L/d on day 73 and then slowly
after 84 days. At the end of this stage, no significant change in biogas production was observed. increased to 7.9 L/d after
The biohydrogen content slightly decreased and stabilized at approximately 46% at the end of The
84 days. At the end of this stage, no significant change in biogas production was observed. this
biohydrogen
stage. Methane content slightly
production decreased
occurred fromand day stabilized at approximately
72, but the content was always 46% at the end
below 2%.of this stage.
Methane production occurred from day 72, but the content was always below 2%.
3.2.3. Biogas Production in Compartment III
3.2.3. Biogas production in Compartment III
The biohydrogen production rate slightly changed at the beginning of Stage I. Specifically,
The biohydrogen
the value fluctuated between production 10.9rate
andslightly
13.8 L/d changed
(Figureat6).theMeanwhile,
beginning ofthe Stage I. Specifically,
biohydrogen the
content
valuealways
was fluctuated
belowbetween
5%. The10.9 and 13.8
methane L/d (Figure
content 6). Meanwhile,
drastically dropped andthe wasbiohydrogen
28% at the end content was
of Stage
always
I. Frombelowday 7,5%.theThe methane content
biohydrogen contentdrastically
increased dropped
rapidly and and reached
was 28%40.5%at the on enddayof Stage
10. InI. Stage
From
day 7, the
II, the biohydrogen
biogas production content
rate increased rapidly
to 19.6 and L/d reached 40.5% on day
at the maximum. After10. In
HRTStagewasII, the biogas
increased,
production
the biohydrogen rate increased to 19.6 L/d
content decreased at the
rapidly maximum.
to 7.1% AfteraHRT
but reached peakwas increased,
of 68.3% on daythe34. biohydrogen
The methane
content increased
decreased to rapidly
35.8% to and7.1%
thenbut reached
rapidly a peaktoofbelow
dropped 68.3%0.5%on day
on day34. The
32. Bymethane contenta
introducing
increased
new type of NMWW to ABR, the biogas production rate suddenly decreased to 6.2 L/d due to type
to 35.8% and then rapidly dropped to below 0.5% on day 32. By introducing a new sludgeof
NMWW
floatationtoand ABR,loss. theThe
biogas production
biogas production raterate
suddenly
graduallydecreased
recoveredto 6.2
and L/d due to7.4
reached sludge
L/d on floatation
day 70.
The biohydrogen content decreased slowly to the lowest value of 19.8% on day 61, which further
remained at approximately 40% until the end of this stage. On day 39, the content of methane suddenly
Processes 2020, 8, x FOR PEER REVIEW 8 of 18

Processes
and loss. 8, 339biogas
2020,The production rate gradually recovered and reached 7.4 L/d on day 70.8 ofThe18
biohydrogen content decreased slowly to the lowest value of 19.8% on day 61, which further
remained at approximately 40% until the end of this stage. On day 39, the content of methane
increased to 12.4% and remained at 13.7% to 16.6%, although it reduced to 3% on day 47. In Stage
suddenly increased to 12.4% and remained at 13.7% to 16.6%, although it reduced to 3% on day 47.
III, the biohydrogen content suddenly dropped to 25.4% and then fluctuated at approximately 27.3%.
In Stage III, the biohydrogen content suddenly dropped to 25.4% and then fluctuated at
The methane content suddenly increased to 21.8% on day 79.
approximately 27.3%. The methane content suddenly increased to 21.8% on day 79.
40 80
Gas production rate
35 hydrogen 70
methane
Gas production rate（L/d）

30 60

percent gas volume(%)

25 50

20 40

15 30

10 20

5 10

0 0
0 10 20 30 40 50 60 70 80 90
Time（d）
Figure 6. Biogas yield and content in Compartment III.
Figure 6. Biogas yield and content in Compartment III.
3.2.4. Biogas Production in Compartment IV
3.2.4. Biogas production in Compartment IV
The average biogas production in compartment IV slightly changed and remained at approximately
2 L/d The average
(Figure 7). Thebiogas
methane production
content wasin higher
compartment
than 40% IVat slightly
Stage I. In changed
Stage II,and remained at
the biohydrogen
approximately 2 L/d (Figure 7). The methane content was higher than 40% at
content increased slowly and reached 11.7% on day 32, and the methane content reached 29.6%. Stage I. In StageOnII,day
the
biohydrogen
33, content
the biohydrogen increased
content slowly
suddenly and reached
increased to 29.0%11.7% on day 32,
by introducing and
a new theofmethane
type NMWW to content
ABR.
reached 29.6%.
However, On day
the content 33, thetobiohydrogen
dropped content
the lowest value suddenly
of 3.4% on dayincreased to 29.0%
56 and reached by introducing
approximately 18%a
new type of NMWW to ABR. However, the content dropped to the lowest value
on day 70. The methane content drastically dropped to below 0.5% and recovered to approximately of 3.4% on day 56
and reached
11%. In Stage approximately
III, although the18% on day
volume 70.was
load Thereduced,
methane content
the drastically
biohydrogen dropped
content to below 0.5%
in Compartment IV
and recovered to approximately 11%. In Stage III, although the volume
dropped to below 2% on day 72. Meanwhile, the methane content increased to approximately 23%.load was reduced, the
biohydrogen content in Compartment IV dropped to below 2% on day 72. Meanwhile, the methane
content increased to approximately 23%.
Processes 2020, 8, 339 9 of 18
Processes 2020, 8, x FOR PEER REVIEW 9 of 18

30 60
Gas production rate
hydrogen
Gas production rate（L/d） 25 methane 50

percent gas volume(%)

20 40

15 30

10 20

5 10

0 0
0 10 20 30 40 50 60 70 80 90
Time（d）
Figure 7. Biogas yield and content in Compartment IV.
Figure 7. Biogas yield and content in Compartment IV.
3.2.5. Comparison of Biogas Production Rate in Each Compartment
3.2.5. Comparison of biogas production rate in each compartment
Figures 4–7 shows that, in the early phase of Stage I, the biogas production rate in Compartments
Figures 4–7 shows
I and III slightly changed. that,
Atinthethesame
earlytime,
phasethe of biogas
Stage I,production
the biogas production rate in Compartments
rate in Compartment II gradually
Idecreased,
and III slightly changed.
and that At the same time,
in Compartment IV was thealways
biogas production
at a low level.rate in Compartment
With the increaseII in gradually
HRT to
decreased,
30 h on day and11,that
thein Compartment
biogas production IV rate
was in always at a three
the first low level. With the increase
compartments rapidly in HRT to 30
increased andh
on day 11, the biogas production rate in the first three compartments
reached the highest values on days 15, 28, and 23. However, the biogas production rate decreased rapidly increased and reached
the highestThe
thereafter. values on days 15,content
biohydrogen 28, andin23.theHowever, the biogasincreased
first compartment production rate after
to 32% decreased thereafter.
HRT promotion,
The biohydrogen content in the first compartment increased to 32%
but it showed a downward trend in Compartment I. Therefore, the microbial activity in Compartment after HRT promotion, but it
showed
I was low.a downward
The content trend in Compartment
of biohydrogen I. Therefore, the
in Compartments microbial
II and activity in
III decreased Compartment
rapidly, while theI
was low.ofThe
contents content ofand
biohydrogen biohydrogen
methane ininCompartment
Compartments IVIIincreased
and III decreased
slowly but rapidly,
remained while the
at a low
contents of biohydrogen and methane in Compartment IV increased
level. By introducing a new type of NMWW to ABR, the biogas production rate of the first three slowly but remained at a low
level. By introducing
compartments a new
drastically type of NMWW
decreased. The biogas to ABR, the biogas
production rate production
in Compartments rate of IItheand first
III three
were
compartments
maintained at adrastically
low level due decreased.
to sludgeThe biogas and
floatation production
loss. Therate in Compartments
biohydrogen content inIICompartment
and III were
maintained
I decreased at a low level
rapidly on day due33 toand
sludge
thenfloatation and loss. The
slowly recovered biohydrogen content
to approximately 30%, and in Compartment
the methane
Icontent
decreased
was rapidly on day
below 0.5%. The33 and then
content slowly recovered
of methane in Compartments to approximately 30%, and
III and IV greatly the methane
changed, both of
content was below 0.5%. The content of methane in Compartments
which experienced a sudden increase and then a rapid decrease. Moreover, the content of methaneIII and IV greatly changed, both
of
in which experienced
Compartment a sudden
IV even dropped increase and then
to below 0.5%a rapid
on days decrease.
33 andMoreover,
44. This resultthe content
indicates of methane
that the
in Compartment
bioactivity IV even dropped
of methanogens to below
is difficult 0.5% on
to improve. Thedays 33 and
average 44. This result
biohydrogen indicatescapacities
production that the
bioactivity of methanogens
of ABR gradually decreased is difficult
with valuesto improve.
of 0.46, The 1.87,average
and 1.32 biohydrogen production capacities
L/d in Compartments of
I, II, and III,
ABR gradually
respectively. decreased
After with in
the increase values
HRT to of 400.46, 1.87,biogas
h, the and 1.32 L/d in Compartments
production rate in Compartments I, II, and III,
II and
respectively. After the while
III slowly decreased, increase thatin in
HRT to 40 h, the biogas
Compartment production
I increased rapidly.rateHowever,
in Compartments II and III
the biohydrogen
slowly
contentdecreased,
decreased while
to 24.5%that in Compartment
and continued to decline.I increased
Methanerapidly.
wasHowever,
detected inthe biohydrogenIIcontent
Compartment during
decreased
this stage, to
and 24.5% and continued
its content to decline.IIIMethane
in Compartments was detected
and IV increased. in Compartment
However, the microbial II during
activity this
of
stage, and its content in Compartments III and IV increased.
methanogens was ineffectively enhanced due to the low rate of total biogas production. However, the microbial activity of
methanogens was ineffectively enhanced due to the low rate of total biogas production.
Processes 2020, 8, 339 10 of 18

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3.3. Characteristics of Fermentation Products of ABR

3.3. Characteristics of fermentation products of ABR
3.3.1. Fermentation Products in Compartment I
3.3.1. Fermentation products in Compartment I
In the early phase of Stage I, the content of VFAs in Compartment I was relatively stable, and the
In the
ethanol early gradually
content phase of Stage I, the content
decreased. In Stage of II,
VFAsthe in Compartment
content of ethanol I was relatively
continued stable, and
to decrease to
the
473 ethanol
mg/L atcontent
18 days,gradually decreased.
and the total amount Inof
Stage
VFAs II,also
the decreased
content of ethanol continued
to its lowest value.toFrom
decrease to
day 19,
473 mg/L atcontent
the ethanol 18 days, and the increased
gradually total amount of VFAs
to 1314 mg/L,also and decreased to its lowest
the total amount of VFAs value. From
reached day
2129 19,
mg/L
the ethanol content gradually increased to 1314 mg/L, and the total amount
at day 23 (Figure 8). Subsequently, the content of ethanol continued to decrease, and the total amount of VFAs reached 2129
mg/L
of VFAs at day 23 (Figure
decreased 8).mg/L.
to 805 Subsequently, the content
By introducing a new of ethanol
type of NMWW continued to decrease,
to ABR, andofthe
the content total
ethanol
amount of VFAs decreased to 805 mg/L. By introducing a new type of NMWW
reached 818.8 mg/L at day 38, and the total amount of VFAs was as high as 2086.7 mg/L. From days 39 to to ABR, the content
of
56,ethanol reached
the content 818.8 mg/L
of ethanol at day
gradually 38, and during
increased, the total amount
which of VFAsofwas
the content as acid
acetic highslowly
as 2086.7 mg/L.
increased
From days 39the
and reached tohighest
56, the value
content
of of ethanol
1356.8 mg/L.gradually
The totalincreased,
amount ofduringVFAs was which up the content
to 2412.2 of acetic
mg/L on 56
acid slowly increased and reached the highest value of 1356.8 mg/L. The
days. From day 57, the content of ethanol and acetic acid decreased, and the total amount of VFAs total amount of VFAs was
up to 2412.2
decreased to mg/L
1258.2on 56 days.
mg/L. From
In Stage III,day
the57, the content
content of ethanolof ethanol
changed and aceticand
greatly acidshowed
decreased, and the
a decreasing
total amount of VFAs decreased to 1258.2 mg/L. In Stage III, the content
trend. Meanwhile, the other components of volatile acid changed slightly, and the total amount of ethanol changed greatly
of
and showed a decreasing trend. Meanwhile, the other components of volatile
VFAs also showed a decreasing trend. The ethanol content also exceeded 50% of the total volatile acid, acid changed slightly,
and the acetic
and the total amount of VFAs
acid content was also showed a 26.3%.
approximately decreasing trend. The
The ethanol andethanol content
acetic acid alsoaccounted
contents exceeded
50% of the total volatile acid, and the acetic acid content was approximately
to approximately 84.8% of the total VFA content. Thus, the ethanol fermentation type in Compartment 26.3%. The ethanol and
acetic acid contents accounted to approximately 84.8% of the total VFA content.
I was a typical one. In Stages II and III, although the content of ethanol was reduced, the contents Thus, the ethanol
fermentation
of ethanol and type in Compartment
acetic I was afor
acid still accounted typical
moreone. thanIn80%
Stages II and
of the III,content
total although of the
VFAs content of
content.
ethanol was reduced, the contents of ethanol and acetic
Therefore, the fermentation maintained the ethanol fermentation type. acid still accounted for more than 80% of the
total content of VFAs content. Therefore, the fermentation maintained the ethanol fermentation type.
4000
Total VFAs
Liquid terminal fermentation product（mg/L）

Ethanol
Acetic acid
Propionic acid
3000
Butyrate
Valerate

2000

1000

0
0 15 30 45 60 75 90
Time（d）
Figure 8.
Figure Ethanol and
8. Ethanol and volatile
volatile fatty
fatty acids
acids (VFAs)
(VFAs) yields
yields in
in Compartment
Compartment I.
I.

3.3.2. Fermentation Products in Compartment II

3.3.2. Fermentation products in Compartment II
The VFA content was stable at Stage I. On day 10, the ethanol content rapidly increased to
The
1217.6 VFAwhile
mg/L, content
thewas stable
content ofatother
Stage I. On day 10,
components thestable.
was ethanol content
The rapidly increased
total amount to 1217.6
of VFAs decreased
mg/L, while the content of other components was stable. The total amount of VFAs decreased
rapidly to 2114.3 mg/L (Figure 9). In Stage II, the ethanol content continued to increase to 2651.4 rapidly
mg/L
to 2114.3 mg/L (Figure 9). In Stage II, the ethanol content continued to increase to 2651.4
at the highest level on the 15th day. At this time, the contents of acetic and butyric acids increasedmg/L at the
highest level on the 15th day. At this time, the contents of acetic and butyric acids increased
to 1505.4 and 356.5 mg/L, respectively, and the total amount of VFAs reached 4867.0 mg/L. From day to 1505.4
and 356.5 mg/L, respectively, and the total amount of VFAs reached 4867.0 mg/L. From day 16, the
content of VFAs decreased, and the total amount of VFAs decreased to 2794.6 mg/L with the decrease
in the content of ethanol. With the operation of ABR, the content of ethanol increased to 2404.4 mg/L,
Processes 2020, 8, 339 11 of 18

16, the content of VFAs decreased, and the total amount of VFAs decreased to 2794.6 mg/L with the
Processes
decrease 2020,
in 8,the
x FOR PEER REVIEW
content of ethanol. With the operation of ABR, the content of ethanol increased 11 of 18
to
2404.4 mg/L, and the total amount of VFAs increased to 3926.7 mg/L in day 23. At this time, the acetic
and
acidthe total was
content amount of VFAs
1217.2 mg/L, increased to 3926.7and
and the propionic mg/L in dayacid
butyric 23. contents
At this time,
werethe acetic
268.5 andacid content
118.0 mg/L,
was 1217.2 mg/L, and the propionic and butyric acid contents were
respectively, which presented a typical ethanol fermentation type. By introducing a new type 268.5 and 118.0 mg/L, of
respectively, which presented a typical ethanol fermentation type. By introducing
NMWW to ABR, the VFA content significantly decreased. On day 34, the ethanol content drastically a new type of
NMWW
decreasedtofrom
ABR, theto
1340 VFA
140.4content
mg/L. significantly decreased.
The total amount of VFAsOn day 34, the
decreased ethanol
to 1173.9 content
mg/L with drastically
the decline
decreased from 1340 to 140.4 mg/L. The total amount of VFAs decreased to 1173.9
in acetic acid. The content of ethanol recovered to 1154.3 mg/L from day 39 and slightly fluctuated mg/L withwith
the
decline in acetic
the operation acid. The
of ABR. Theacetic
contentacidofcontent
ethanolgradually
recovered to 1154.3
recovered to mg/L
1194.7from
mg/Lday
and39 and
then slightly
decreased
fluctuated with the operation of ABR. The acetic acid content gradually recovered
slowly. The propionic and butyric acid contents remained at approximately 219.4 and 261.4 mg/L. to 1194.7 mg/L and
then decreased
The total amountslowly.
of VFAsThewas
propionic and butyric
approximately acid
2000 contents
mg/L, whichremained
denotedat approximately
ethanol 219.4type.
fermentation and
261.4 mg/L. The total amount of VFAs was approximately 2000 mg/L, which
In Stage III, the ethanol content began to decrease from day 76. However, the other components denoted ethanol
fermentation type.and
changed slightly, In Stage III, the
the total ethanol
amount ofcontent
VFAs was began to decrease from
approximately 1916day 76. However,
mg/L, which stillthe other
showed
components changed type.
ethanol fermentation slightly, and the total amount of VFAs was approximately 1916 mg/L, which
still showed ethanol fermentation type.
5000
Total VFAs
Liquid terminal fermentation product（mg/L）

Ethanol
Acetic acid
4000 Propionic acid
Butyrate
Valerate
3000

2000

1000

0
0 15 30 45 60 75 90
Time（d）
Figure 9. Ethanol and VFAs yields in Compartment II.
Figure 9. Ethanol and VFAs yields in Compartment II.
3.3.3. Fermentation Products in Compartment III
3.3.3. Fermentation products in Compartment III
In the early phase of Stage I, the content of ethanol and the total amount of VFAs in Compartment
In the early
III drastically phaseHowever,
changed. of StagetheI, VFAs
the content
graduallyofbecame
ethanolstable
and with
the the
total amount
ABR of VFAs
operation. in
In Stage
Compartment III drastically
II, the butyric acid changed.
content increased, and However, thecontent
the ethanol VFAs began
gradually becameand
to decrease stable with the
remained ABR
stable at
operation. In Stage II, the butyric acid content increased, and the ethanol content
approximately 769.3 mg/L when HRT was prolonged to 40 h (Figure 10). The total amount of VFAs began to decrease
and
was remained stable2553.8
approximately at approximately
mg/L in the769.3
stablemg/L when
period. TheHRT waspropionic,
acetic, prolongedbutyric,
to 40 h and
(Figure 10). acid
valeric The
total amount
contents wereofapproximately
VFAs was approximately
1000, 280, 530,2553.8
and 70mg/L in respectively,
mg/L, the stable period.
which The acetic, mixed
presented propionic,
acid
butyric, and valeric
fermentation type. acid contents were approximately 1000, 280, 530, and 70 mg/L, respectively,
which presented mixed acid fermentation type.
Processes 2020, 8, 339 12 of 18
Processes 2020, 8, x FOR PEER REVIEW 12 of 18

6000
Total VFAs

Liquid terminal fermentation product（mg/L）

Ethanol
5000
Acetic acid
Propionic acid
Butyrate
4000 Valerate

3000

2000

1000

0
0 15 30 45 60 75 90
Time（d）
Figure 10.
Figure Ethanoland
10. Ethanol andVFAs
VFAs yields
yields in
in Compartment
Compartment III.
III.

3.3.4. Fermentation Products in Compartment IV

3.3.4. Fermentation products in Compartment IV
The variation in fermentation products in Compartment IV was different from those in
The variation in fermentation products in Compartment IV was different from those in
Compartments I, II, and III. The content of acetic acid was higher than that of ethanol in Stage
Compartments I, II, and III. The content of acetic acid was higher than that of ethanol in Stage I, which
I, which denoted mixed acid fermentation type. From Stage III, the content of acetic acid suddenly
denoted mixed acid fermentation type. From Stage III, the content of acetic acid suddenly decreased
decreased due to the extension of HRT, while the content of ethanol kept increasing, and ethanol
due to the extension of HRT, while the content of ethanol kept increasing, and ethanol fermentation
fermentation type finally formed. Moreover, the contents of ethanol, acetic acid, propionic acid,
type finally formed. Moreover, the contents of ethanol, acetic acid, propionic acid, and valeric acid
and valeric acid tended to be stable. The butyric acid content suddenly increased from 334.5 to
tended to be stable. The butyric acid content suddenly increased from 334.5 to 990.2 mg/L at day 56.
990.2 mg/L at day 56. At this time, the contents of ethanol, acetic acid, propionic acid, and valeric acid
At this time, the contents of ethanol, acetic acid, propionic acid, and valeric acid were 960.1, 1146.3,
were 960.1, 1146.3, 246.3, and 58.8 mg/L (Figure 11), respectively, with a total amount of 3401.7 mg/L,
246.3, and 58.8 mg/L (Figure 11), respectively, with a total amount of 3401.7 mg/L, which showed
which showed mixed acid fermentation type.
mixed acid fermentation type.
3.3.5. Comparison of Fermentation Products in Each Compartment
3.3.5. Comparison of fermentation products in each compartment
The variations in fermentation products of each compartment showed similar performance to
The
biogas variations(Figures
production in fermentation
8–11). Beforeproducts of each compartment
the promotion of HRT, stable showed
ethanolsimilar performance
fermentation type wasto
biogas production (Figures 8–11). Before the promotion of HRT, stable ethanol
presented in Compartments I, II, and III, and Compartment IV denoted butyric acid fermentation type. fermentation type was
presented in Compartments
After the extension of HRT to 30 I, II,
h, and III, andcontent
the ethanol Compartment IV denoted
in Compartment butyric significantly
I decreased acid fermentation
with a
type. After the extension of HRT to 30 h, the ethanol content in Compartment
value of 473 mg/L on day 18 and a value of 180 mg/L on day 32. Furthermore, the total amount of VFAsI decreased significantly
with a value
decreased to of
805473 mg/L
mg/L. on day 18
However, theand a value of 180
fermentation mg/L
types onfirst
in the daythree
32. Furthermore,
compartments thedid
total
notamount
change.
of
TheVFAs decreased
content to 805
of acetic acidmg/L. However, theIV
in Compartment fermentation types in theand
suddenly decreased, firstthat
threeofcompartments did
ethanol increased
not change. The content of acetic acid in Compartment IV suddenly decreased,
rapidly, and thus indicated an ethanol fermentation type. By introducing a new type of NMWW to and that of ethanol
increased rapidly,ofand
ABR, the content thus acid
butyric indicated an ethanol IV
in Compartment fermentation
increased to type. By introducing
990 mg/L on day 60.aHowever,
new typethe of
NMWW to ABR,
composition the content
of other of butyric
liquid terminal acid inchanged
products Compartment IVThe
slightly. increased to 990 mg/L
fermentation on day 60.
characteristics of
However, the composition of other liquid terminal products changed
AnAS in Compartment IV were restored to butyric acid fermentation type. With the extension of slightly. The fermentation
characteristics
HRT to 40 h, the of ethanol
AnAS incontent
Compartment IV were
in the first three restored to butyric
compartments acid fermentation
decreased. However, thetype. With the
contents of
extension of HRT to 40 h, the ethanol content in the first three compartments
ethanol and acetic acid still accounted for more than 60% of the total amount of VFAs. Therefore, the decreased. However,
the contents oftype
fermentation ethanol
was andstill acetic
ethanol acid still accounted
fermentation, andfor
thatmore than 60% of the
in Compartment IV total amount
denoted of VFAs.
a butyric acid
Therefore,
fermentationthetype.
fermentation type was still ethanol fermentation, and that in Compartment IV denoted
a butyric acid fermentation type.
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7000
Total VFAs

Liquid terminal fermentation product（mg/L）

7000 Ethanol
6000 Total VFAs
Acetic acid

Liquid terminal fermentation product（mg/L）

Ethanol
Propionic acid
6000 Acetic acid
Butyrate
5000 Propionic acid
Valerate
Butyrate
5000
4000 Valerate

4000
3000

3000
2000

2000
1000

1000
0
0 15 30 45 60 75 90
0
0 15 30 Time（d）
45 60 75 90

Figure 11. Ethanol andTime（d）

VFAs yields in Compartment IV.
Figure 11. Ethanol and VFAs yields in Compartment IV.
Figure 11.and
3.4. Variations in pH, alkalinity, Ethanol
CODand VFAs yields in Compartment IV.
3.4. Variations in pH, Alkalinity, and COD
3.4.3.4.1.
Variations in pH,in
Variations alkalinity,
pH and and COD
alkalinity
3.4.1. Variations in pH and Alkalinity
Figures 12
3.4.1. Variations andand
in pH 13 demonstrate
alkalinity the variation in pH value and alkalinity in the ABR system. In
Figures
the early 12 andof
phase 13Stage
demonstrate
I, the pHthe variation
value of theininfluent
pH value wasand alkalinity
nearly 6.6, butinthe
thepH
ABR system.
values of the
In theFigures
early 12 andof13
phase demonstrate
Stage I, the pH the variation
value of the in pH value
influent was and alkalinity
nearly 6.6, butinthe
thepHABR system.
values of In
the
compartments decreased to below 4.7, 5.3, 5.3, and 5.0. The alkalinity of each compartment greatly
the early phasedecreased
compartments of Stage toI, the
below pH4.7,
value
5.3,of the
5.3, andinfluent was nearly 6.6, but compartment
the pH valuesgreatly
of the
changed due to sludge loss in Compartments I5.0.
andThe IV.alkalinity of each
The alkalinity of each compartment was
compartments
changed due decreased
to sludge to
loss below
in 4.7, 5.3,
Compartments 5.3, and
I and5.0.
IV. The
Thealkalinity of
alkalinity each
of eachcompartment
compartment greatly
was
oscillatory. Specifically, the alkalinity in Compartment I increased from 510 to 640 mg/L, and that in
changed due
oscillatory. to sludgethe
Specifically, lossalkalinity
in Compartments
in CompartmentI and IV. The alkalinity of each compartment was
Compartment IV decreased from 1780 to 860 mg/L. I increased from 510 to 640 mg/L, and that in
oscillatory. Specifically, the alkalinity
Compartment IV decreased from 1780 to 860 mg/L. in Compartment I increased from 510 to 640 mg/L, and that in
Compartment IV decreased from 1780 to 860 mg/L.

Figure 12. Variation of pH values in each compartment.

Figure 12. Variation of pH values in each compartment.

Figure 12. Variation of pH values in each compartment.

 Processes 2020, 8, x FOR PEER REVIEW 14 of 18
Processes 2020, 8, 339 14 of 18

Figure 13. Variation of alkalinity in each compartment.

At the beginning of StageFigureII,13.the

Variation
pH and of alkalinity
alkalinityin of
each compartment.
each compartment slightly changed.
By introducing a new type of NMWW to ABR, the pH value in Compartments I and II began to
At while
decrease, the beginning
those in of Stage II, the pH
Compartments and IV
III and alkalinity
began to of increase.
each compartment
On day 39, slightly
the pH changed.
value inBy
introducing a new type of NMWW to ABR, the pH value in Compartments I and
Compartment I was the highest, and the pH values in the last three compartments were close. The II began to decrease,
pH
while those in Compartments III and IV began to increase. On day 39, the pH
value in Compartment IV exceeded that in Compartment I and reached approximately 5.2 on day 45. value in Compartment
I was
After the highest,
operation andthan
for more the 50
pHdays,
values theinpH
the last three
values compartments
in each compartmentwere were close.
similar. The
ThepH value
effect on in
Compartment IV exceeded that in Compartment I and reached
pH value and alkalinity was only slight when HRT was prolonged to 40 h. approximately 5.2 on day 45. After
operation for more than 50 days, the pH values in each compartment were similar. The effect on pH
value
3.4.2. and alkalinity
Relationship was only
between CODslight
Removal whenandHRT was prolonged
Biohydrogen to 40 h.Efficiency
Production
COD removal by anaerobic treatment system was mainly realized by methanogenic bacteria.
3.4.2. Relationship between COD removal and biohydrogen production efficiency
The ABR system, which was characterized by the metabolism of acidogenic fermentation bacteria,
COD removal
could remove COD mainlyby anaerobic
through the treatment system
release of CO2 andwasHmainly
2 because realized
the pHby methanogenic
value bacteria.
was not conducive
to The ABR system,
the relatively highwhich was characterized
bioactivity of methanogens. by the metabolism
However, mostof acidogenic
COD fermentation
was converted bacteria,
into volatile
could
acids, removeacetic,
including CODpropionic,
mainly through the acids.
and butyric releaseThus,
of CO₂ and H₂ rate
the removal because
of CODthe was
pH relatively
value was low.not
conducive to the relatively high bioactivity of methanogens. However, most
Figure 14 shows the variation in COD from the effluent of each compartment during the operation of COD was converted into
volatile
ABR. In theacids,
early including
phase of Stageacetic, propionic,
I, the influent and butyric COD
and effluent acids.ofThus,
each the removal rate
compartment wereofrelatively
COD was
relatively low. Figure 14 shows the variation in COD from the effluent
stable, and the COD removal efficiency showed a slow upward trend (Figure 14). Subsequently, of each compartment during
the operation of ABR. In the early phase of Stage I, the influent and
the COD concentration in each compartment also changed with the fluctuation of influent due to the effluent COD of each
compartment
fluctuation wereHowever,
of COD. relativelythestable,
CODand the COD
removal removal
efficiency efficiency
of COD changedshowed a slow
slightly and upward
maintained trend
at approximately 18%. In Stage II, the influent COD concentration still fluctuated, but the removalthe
(Figure 14). Subsequently, the COD concentration in each compartment also changed with
fluctuation
efficiency of influent
of COD due to
increased to 27.3%
the fluctuation
and thenof COD. However,
stabilized at 18.0%.the COD
With theremoval efficiency
continuous of COD
fluctuation
of changed slightly andfrom
COD concentration maintained
influent,atthe
approximately
content of VFAs 18%.inIn Stage
each II, the influent
compartment COD accordingly,
changed concentration
andstill
thefluctuated, but the removal
removal efficiency efficiency to
of COD decreased of approximately
COD increased11.4% to 27.3%
at theandendthen stabilized
of this stage. InatStage
18.0%.
III,With the continuous
the COD fluctuation
concentration of eachofcompartment
COD concentration from influent,improved
was insignificantly the content of VFAs
with in each
the further
compartment changed accordingly, and the removal efficiency of COD decreased
extension of HRT. Meanwhile, the COD removal efficiency gradually increased and finally stabilized at to approximately
11.4% at the15.5%.
approximately end of this stage. In Stage III, the COD concentration of each compartment was
insignificantly improved with the further extension of HRT. Meanwhile, the COD removal efficiency
gradually increased and finally stabilized at approximately 15.5%.
Processes 2020, 8, x FOR PEER REVIEW 15 of 18

Processes 2020, 8, 339 15 of 18

Figure 14. Variation of COD in each compartment.

4. Discussion Figure 14. Variation of COD in each compartment.

4.4.1.
Discussion
Effect of Biomass on Biohydrogen Production Efficiency
When
4.1. Effect HRT was
of biomass extended toproduction
on biohydrogen 30 h, the biomass
efficiency of each compartment firstly began to decrease,
and that in Compartment IV decreased to less than 20% of the original biomass (Figure 2). At the same
time,When
biogasHRTandwas extended production
biohydrogen to 30 h, therates biomassshowed of each
a slowcompartment firstly4–7),
decline (Figures beganwhichto decrease,
indicated
and that in Compartment IV decreased to less than 20% of the
that the microorganisms with poor adaptability and low microbial activity in the reactionoriginal biomass (Figure 2). At the
system
same
were time, biogasdischarged
gradually and biohydrogen
from theproduction
ABR. Afterrates showed
further a slowofdecline
extension HRT, the (Figures
sludge4–7), which
bioactivity
indicated that the microorganisms with poor adaptability and low microbial
continued to decrease. In Compartment II, the total specific biogas production rate decreased from activity in the reaction
system were
285 to 191 gradually discharged
L/(kg·MLVSS·d). However, from the ABR.
the biogas After further
production capacity extension of HRT, IV
in Compartment theincreased,
sludge
bioactivity continued
while the sludge to decrease.
bioactivity in the In
threeCompartment
other compartments II, the total specific biogas
significantly decreased.production rate
The specific
decreased from 285 to 191 L/(kg·MLVSS·d). However, the biogas
biogas and biohydrogen production rates of AnAS in Compartment I were 76 and 55 L/(kg·MLVSS·d), production capacity in
Compartment IV increased, while the sludge bioactivity in the three other
those in Compartment II were 81 and 73 L/(kg·MLVSS·d), and those in Compartment III were 53 and compartments significantly
decreased. The specific biogas and biohydrogen production rates of AnAS in Compartment I were 76
41 L/(kg·MLVSS·d).
and 55 L/(kg·MLVSS·d),
The microbial community those instructure
Compartment in ABRIIwas were no81longer
and 73 L/(kg·MLVSS·d),
suitable for the current and operating
those in
Compartment
conditions [6,17] III were
when53 andwas
HRT 41 L/(kg·MLVSS·d).
extended to 40 h; the possible reason could be due to the continuous
The microbial community
loss of biomass. The biogas and biohydrogenstructure in ABR was nocapacities
production longer suitable
of eachfor the currentsignificantly
compartment operating
conditions [6,17] when HRT was extended to 40 h; the possible reason could
decreased, with the total specific biogas production rate reduced to 92 L/(kg·MLVSS·d). The specific be due to the continuous
loss of biomass.production
biohydrogen The biogas and rate biohydrogen productiondecreased
of each compartment capacities ofbyeach
more compartment
than 30%. significantly
The specific
decreased, with the total specific biogas production rate reduced
biogas production rate of AnAS in Compartment I decreased to 29 L/(kg·MLVSS·d), and to 92 L/(kg·MLVSS·d). The specific
those in
biohydrogen
Compartments production
II, III, andrate
IVof each43,
were compartment
18, and 1.6 L/(kg decreased by more
MLVSS·d), than 30%. In
respectively. The specific biogas
Compartment IV,
production rate of AnAS in Compartment I decreased to 29 L/(kg·MLVSS·d),
the biogas production rate decreased the least; the reason was attributed to the further enrichment of and those in
Compartments II, III, and
methanogens (Figures 4–7)IVinwere
Stage43,II.18,
Theand 1.6 L/(kg
presence andMLVSS·d),
metabolism respectively.
of methanogens In Compartment
were conducive IV,
the biogas
to the production
enrichment rate decreased
of HPA the least;
[2,6]. However, the reason was
the biohydrogen attributedefficiency
production to the further enrichment of
in Compartment IV
methanogens (Figures 4–7) in Stage II. The presence and metabolism
was low due to the hydrogen consumption [23,24] and still showed a decreasing trend. At of methanogens were conducive
this time,
tothe
theABR
enrichment
operationofperformance
HPA [2,6]. However,
was poorthe biohydrogen
because production
the microbial efficiency
community in Compartment
structure of AnAS tended IV
was
to below duealong
stable to thewith
hydrogen consumption
the operation of ABR, [23,24]
and theandmetabolic
still showed a decreasing
speed became slow. trend. At this time,
the ABR operation performance was poor because the microbial community structure of AnAS
tended to be stable
4.2. Enrichment along
of HPA andwith the operation
Biohydrogen of ABR,
Production and theofmetabolic
Efficiency ABR speed became slow.
The COD concentration reached 8000 mg/L and was stable for 10 days. The biogas production rate,
biohydrogen, and methane content in Compartments III and IV remained at a low level. The enrichment
Processes 2020, 8, 339 16 of 18

effect of HPA was not obvious (Figures 4–7). After HRT was extended to 30 h, the decrease in OLR
reduced its effect on the system. As a result, the overall pH value of the system improved for
biohydrogen production [25]. Figure 5 shows that, after HRT was extended to 30 h, the ethanol
concentration in Compartment I and other formats continued to decrease. The concentration of acetic
acid in Compartments III and IV decreased evidently, while the contents of methane and biohydrogen
in biogas increased correspondingly. Therefore, the production rates of biohydrogen, acetic acid,
and methane in the reaction system were gradually increasing, and the corresponding bacteria groups
were gradually enriched [6]. The biohydrogen production efficiency of the system increased from
14.78 L/d in Stage I to 20.46 L/d (Figures 6 and 7). After HRT was extended for 40 h, the ethanol and
butyric acid contents in the first three compartments continued to decrease and led to a significantly
high metabolic transformation effect [6,26]. However, the contents of ethanol and acetic acid still
accounted for more than 60% of the total amount of VFAs, which was still manifested as an ethanol
fermentation type, and the fermentation type in Compartment IV was maintained. Compared with the
performance in Stage II (HRT 30 h), the contents of biohydrogen and methane in biogas of Compartment
III were significantly increased. HPA gradually become prevalent because HPA microflora has a
transitional role in anaerobic wastewater treatment [17,27]. For Compartment IV, the content of
biohydrogen in biogas increased significantly in the previous stage and then decreased, while the
content of methane significantly increased. Therefore, the methanogens in Compartment IV were
further enriched and presented dominant position [2,6,14] although the biomass was relatively low.
The presence and metabolism of methanogens were conducive to the enrichment of HPA. However,
the biohydrogen production efficiency in Compartment IV was only 0.1 L/d due to the biohydrogen
consumption (Figure 7).

5. Conclusions
The increase in influent COD led to the transformation of fermentation type and metabolism
characteristics of AnAS. When influent COD increased to 8000 mg/L, methanogen residues were still
observed in the last three compartments. The first three compartments showed the characteristics of
an ethanol fermentation type, while the last compartment presented as a butyric acid fermentation
type. The average removal efficiency of COD in ABR reduced to 15.4%, and the average biohydrogen
production rate and biohydrogen production by COD removal capacity were 12.85 L/d and 360.22 L/kg
COD, respectively. However, the specific biohydrogen production rate was 48 L/(kg MLVSS·d).
Appropriately prolonging HRT was beneficial to the enrichment of hydrogen-producing acetogen
and could improve the efficiency of biohydrogen production. When HRT was extended to 30 h,
the biohydrogen production rate increased from 14.78 to 24.34 L/d. However, the bioactivity of
acidogenic fermentation bacteria decreased, and the biohydrogen production efficiency of ABR
drastically reduced when HRT increased to 40 h. The biogas production rate decreased to 92 L/(kg
MLVSS·d), and the specific biohydrogen production rate finally decreased to 2.7 L/(kg MLVSS·d).

Author Contributions: Formulated the idea of the study and wrote the manuscript, F.J. and Z.P.; carried out
the experiment and performed the analytical calculation, F.J. and H.L.; contributed the interpretation of the
experimental results and revised the manuscript, J.L.; provided critical feedback and proposed the experiment in
discussions, S.W. All authors have read and agreed to the published version of the manuscript.
Funding: This research was supported by the Major Science and Technology Program for Water Pollution Control
and Treatment (2015ZX07306-001, 2017ZX07201-001).
Acknowledgments: Special thanks to Tay Joo Hwa for the contribution to this work; may you rest in peace.
Additionally, the authors are grateful to the editors and anonymous reviewers for their constructive comments.
Conflicts of Interest: The authors declare no conflict of interest.
Processes 2020, 8, 339 17 of 18

References
1. Gokcek, O.B.; Sarioglu, M. Anaerobic treatment of the mixture of automotive industry and molasses
wastewater for different organic loading rates in an upflow anaerobic sludge blanket (UASB) reactor.
Desalin. Water Treat. 2018, 105, 83–91. [CrossRef]
2. Wang, S.; Li, J.; Zheng, G.; Du, G.; Li, J. Bioaugmentation with Mixed Hydrogen-Producing Acetogen
Cultures Enhances Methane Production in Molasses Wastewater Treatment. Archaea 2018, 2018, 4634898.
[CrossRef] [PubMed]
3. Dong, X.; Zhao, Y.; Li, T.; Huang, L.; Zhao, P.; Xu, J.; Ma, H.; Yu, X. Enhancement of lipid production and
nutrient removal of Monoraphidium sp. FXY-10 by combined melatonin and molasses wastewater treatment.
J. Taiwan Inst. Chem. Eng. 2019, 99, 123–131. [CrossRef]
4. Ma, C.; Wen, H.; Xing, D.; Pei, X.; Zhu, J.; Ren, N.; Liu, B. Molasses wastewater treatment and lipid production
at low temperature conditions by a microalgal mutant Scenedesmus sp Z-4. Biotechnol. Biofuels 2017, 10, 111.
[CrossRef] [PubMed]
5. Wang, J.; Yin, Y. Fermentative biohydrogen production using various biomass-based materials as feedstock.
Renew. Sustain. Energy Rev. 2018, 92, 284–306. [CrossRef]
6. Li, J.; Zheng, G.; He, J.; Chang, S.; Qin, Z. Biohydrogen-producing capability of anaerobic activated sludge
in three types of fermentations in a continuous stirred-tank reactor. Biotechnol. Adv. 2009, 27, 573–577.
[CrossRef]
7. Kurokawa, T.; Tanisho, S. Effects of formate on fermentative biohydrogen production by enterobacter
aerogenes. Mar. Biotechnol. 2005, 7, 112–118. [CrossRef]
8. Tsioptsias, C.; Lionta, G.; Samaras, P. Microalgae-activated sludge treatment of molasses wastewater in
sequencing batch photo-bioreactor. Environ. Technol. 2017, 38, 1120–1126. [CrossRef]
9. Ren, N.; Li, J.; Li, B.; Wang, Y.; Liu, S. Biohydrogen production from molasses by anaerobic fermentation
with a pilot-scale bioreactor system. Int. J. Hydrog. Energy 2006, 31, 2147–2157. [CrossRef]
10. Grobicki, A.; Stuckey, D.C. Hydrodynamic characteristics of the anaerobic baffled reactor. Water Res. 1992,
26, 371–378. [CrossRef]
11. Wei, W.; Zhang, Y.; Huang, Q.; Ni, B. Polyethylene terephthalate microplastics affect hydrogen production
from alkaline anaerobic fermentation of waste activated sludge through altering viability and activity of
anaerobic microorganisms. Water Res. 2019, 163, 114881. [CrossRef] [PubMed]
12. Zhu, G.; Li, J.; Liu, C. Fermentative Hydrogen Production from Soybean Protein Processing Wastewater in
an Anaerobic Baffled Reactor (ABR) Using Anaerobic Mixed Consortia. Appl. Biochem. Biotechnol. 2012, 168,
91–105. [CrossRef] [PubMed]
13. Mahdieh, S.; Mahmood, S.K.; Reza, R.M.; Babak, G.; Hossein, S.K. Preparation and characterization of
cellulose nanocrystals from bacterial cellulose produced in sugar beet molasses and cheese whey media.
Int. J. Biol. Macromol. 2019, 122, 280–288.
14. Zou, R.; Zhu, G.; Kumar, J.A.; Liu, C.; Huang, X.; Liu, L. Hydrogen and methane production in a
bio-electrochemical system assisted anaerobic baffled reactor. Int. J. Hydrog. Energy 2014, 39, 13498–13504.
15. Li, J.; Li, B.; Zhu, G.; Ren, N.; Bo, L.; He, J. Hydrogen production from diluted molasses by anaerobic
hydrogen producing bacteria in an anaerobic baffled reactor (ABR). Int. J. Hydrog. Energy 2007, 32, 3274–3283.
[CrossRef]
16. Li, S.; Nan, J.; Gao, F. Hydraulic characteristics and performance modeling of a modified anaerobic baffled
reactor (MABR). Chem. Eng. J. 2016, 284, 85–92. [CrossRef]
17. Zheng, G.; Li, J.; Zhao, F.; Zhang, L.; Wei, L.; Ban, Q.; Zhao, Y. Effect of illumination on the hydrogen-production
capability of anaerobic activated sludge. Front. Environ. Sci. Eng. 2012, 6, 125–130. [CrossRef]
18. Pan, J.; Ma, J.; Zhai, L.; Luo, T.; Mei, Z.; Liu, H. Achievements of biochar application for enhanced anaerobic
digestion: A review. Bioresour. Technol. 2019, 292, 122058. [CrossRef]
19. Vasily, A.V.; Sergey, V.R.; Ljudmila, Y.L.; Jukka, A.R.; Gerasimos, L. Simplified hydrolysis models for the
optimal design of two-stage anaerobic digestion. Water Res. 2001, 35, 4247–4251.
20. Noblecourt, A.; Christophe, G.; Larroche, C.; Fontanille, P. Biohydrogen production by dark fermentation
from pre-fermented depackaging food wastes. Bioresour. Technol. 2018, 247, 864–870. [CrossRef]
21. APHA. Standard Methods for the Examination of Water and Wastewater, 20th ed.; American Public Health
Association: Washington, DC, USA, 1998.
Processes 2020, 8, 339 18 of 18

22. Liu, C.; Li, J.; Wang, S.; Nies, L. A syntrophic propionate-oxidizing microflora and its bioaugmentation
on anaerobic wastewater treatment for enhancing methane production and COD removal. Front. Environ.
Sci. Eng. 2016, 10, 13. [CrossRef]
23. Öztürk, M. Conversion of acetate, propionate and butyrate to methane under thermophilic conditions in
batch reactors. Water Res. 1991, 25, 1509–1513. [CrossRef]
24. Nielsen, H.B.; Uellendahl, H.; Ahring, B.K. Regulation and optimization of the biogas process: Propionate as
a key parameter. Biomass Bioenergy 2007, 31, 820–830. [CrossRef]
25. Barredo, M.S.; Evison, L.M. Effect of propionate toxicity on methanogen-enriched sludge,
methanobrevibacter-smithii, and methanosprillum-hungatii at different pH values. Appl. Environ. Microbiol.
1991, 57, 1764–1769. [CrossRef] [PubMed]
26. Chaucheyras, F.; Fonty, G.; Bertin, G.; Gouet, P. In-vitro H2 Utilization by a ruminal acetogenic bacterium
cultivated alone or in association with an archaea methanogen is stimulated by a probiotic strain of
saccharomyces-cerevisiae. Appl. Environ. Microbiol. 1995, 61, 3466–3467. [CrossRef]
27. Rajhi, H.; Puyol, D.; Martinez, M.C.; Diaz, E.E.; Sanz, J.L. Vacuum promotes metabolic shifts and increases
biogenic hydrogen production in dark fermentation systems. Front. Environ. Sci. Eng. 2016, 10, 513–521.
[CrossRef]

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