optimizing the production of hydrogen and 1,3-propanediol in anaerobic fermentation of biodiesel...
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i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 8 ( 2 0 1 3 ) 3 1 9 6e3 2 0 5
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Optimizing the production of hydrogen and 1,3-propanediol in anaerobic fermentation of biodieselglycerol
Bingchuan Liu a, Kyle Christiansen b, Richard Parnas b, Zhiheng Xu a, Baikun Li a,*aDepartment of Civil and Environmental Engineering, University of Connecticut, Storrs, CT 06269, USAbDepartment of Chemical, Materials and Biomolecular Engineering, University of Connecticut, Storrs, CT 06269, USA
a r t i c l e i n f o
Article history:
Received 2 July 2012
Received in revised form
28 December 2012
Accepted 29 December 2012
Available online 30 January 2013
Keywords:
Anaerobic fermentation
Biodiesel glycerol
Hydrogen
1,3-propanediol (1,3-PD)
Metabolic pathway
Hydrogen retention time (HyRT)
* Corresponding author.E-mail address: [email protected]
0360-3199/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.ijhydene.2012.12.1
a b s t r a c t
The conversion of glycerol in biodiesel waste streams to valuable products (e.g. hydrogen
and 1,3-propanediol (1,3-PD)) was studied through batch-mode anaerobic fermentation
with organic soil as inoculum. The production of hydrogen in headspace and 1,3-PD in
liquid phase was examined at different hydrogen retention times (HyRTs), which were
controlled by gas-collection intervals (GCIs) and initial gas-collection time points (IGCTs).
Two purification stages of biodiesel glycerol (P2 and P3) were tested at three concentrations
(3, 5 and 7 g/L). Longer HyRT (longer GCI and longer IGCT) led to lower hydrogen yield but
higher 1,3-PD yield. The P3 glycerol at the concentration of 7 g/L had the highest 1,3-PD
yield (0.65 mol/mol glycerolconsumed) at the GCI/IGCT of 20 h/65 h and the highest hydro-
gen yield (0.75 mol/mol glycerolconsumed) at the GCI/IGCT of 2.5 h/20 h), respectively. A
mixed-order kinetic model was developed to simulate the effects of GCI/IGCT on the
production of hydrogen and 1,3-PD. The results showed that the production of hydrogen
and 1,3-PD can be optimized by adjusting HyRT in anaerobic fermentation of glycerol.
Copyright ª 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
reserved.
1. Introduction process also exist in biodiesel waste stream [4], which make
The production of biodiesel has increased substantially over
the past decade as the alternative energy resource to solve the
shortage of fossil fuels and the release of greenhouse gases.
According to national biodiesel board, the production capacity
of biodiesel has reached 700 million gallons/year in 2008 [1].
For every gallon biodiesel produced, 1 pound glycerol is pro-
duced as one of the major by-products in biodiesel waste
streams [2]. This overproduction of glycerol lowered the price
of purified glycerol from $1.00/lb in 1995 to $0.38/lb in 2005 [3].
In addition, short-chain aliphatic alcohols (e.g. methanol) and
catalysts (e.g. NaOH or KOH) used in the biodiesel production
(B. Li).2013, Hydrogen Energy P35
the glycerol in biodiesel waste streams difficult to use.
Although most of the impurities can be removed by vacuum
distillation and carbon treatment (a method for removing
organic contaminants using carbon as absorbent), these pro-
cesses are energy intensive [5] and pose the obstacles for the
real-world application of the crude glycerol generated from
biodiesel waste streams.
Several energy efficient processes (e.g. combustion, com-
posting, anaerobic digestion, and thermo-chemical or bio-
logical conversions to value-added products) for utilizing
glycerol have been investigated [6e10], among which anae-
robic fermentation has gained most intention, since it
ublications, LLC. Published by Elsevier Ltd. All rights reserved.
Nomenclature
1,3-PD 1,3-Propanediol
GCI Gas-collection time interval
HPP Hydrogen partial pressure
HyRT Hydrogen retention time
IGCT Initial gas-collection time point
P0 Biodiesel glycerol of purification phase 0
P1 Biodiesel glycerol of purification phase 1
P2 Biodiesel glycerol of purification phase 2
P3 Biodiesel glycerol of purification phase 3
SSE Sum of squares errors
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 en e r g y 3 8 ( 2 0 1 3 ) 3 1 9 6e3 2 0 5 3197
produces hydrogen in biogas as a clean energy and ethanol,
butanol, and 1,3-propanediol (1,3-PD) as valuable liquid
products [11e15]. Anaerobic fermentation of crude glycerol
from biodiesel waste streams using either pure cell cultures
(e.g. Clostridium butyricum, Escherichia coli) [6,16,17] or mixed
cultures (e.g. wheat soil, compost, and wastewater sludge)
[12,13,18] have been performed. However, most of these
studies were conducted in the batch-mode bioreactors, in
which hydrogen produced accumulated in the headspace of
the bioreactors. The gas-collection approach or the gas-col-
lection interval (GCI) was not clearly described in literature.
Specifically, GCI was the parameter indicating the gas-col-
lection frequency and the time duration between two con-
tiguous gas collections from the headspace. Unlike
continuous stirred tank reactors (CSTR) where hydrogen
generated from anaerobic reactions can be continuously col-
lected (or released) from headspace [19], hydrogen was accu-
mulated in the headspace of batch-mode reactors. It was
found that the accumulation of hydrogen inhibited anaerobic
fermentation and decreased the specific hydrogen yields, due
to the buildup of hydrogen partial pressure (HPP) in the
headspace [19e22]. Additionally, higher HPP could increase
hydrogen absorption to the fermentation solution, thus pos-
sibly affecting the metabolic fermentative pathways. Hydro-
gen retention time (HyRT) indicates the average retention
time that hydrogen gas stays in the headspace of reactors. The
HyRT of the batch reactors mainly depends on the duration of
GCI (gas collection frequencies). Longer GCI (or lower fre-
quency of gas collection) leads to longer HyRT and increases
the contact time of hydrogen and fermentation solution,
which is expected to accelerate the absorption of hydrogen to
the solution and lower the pH of the solution. In addition, the
initial gas collection time point (IGCT, a parameter indicating
the gas collection/sampling starting time from the headspace
of reactors) also affects HyRT in the batch-mode fermentation.
Because the lag time for gas production in anaerobic fer-
mentation of glycerol was around 18 h [7], an IGCT longer than
20 hwas expected to prolongHyRT in batch-mode fermentors.
Fermentation is carried out through different metabolic
pathways depending on the bacterial types and the opera-
tional conditions. In anaerobic fermentation of glycerol,
1,3-PD is produced in a reductive pathway (Fig. 1, adapted
from [23]) and competes with the oxidative pathways of
hydrogen production and other liquid products (e.g. ethanol,
acetate and butyrate). Compared with the other liquid
products (e.g. ethanol, butynol), 1,3-PD has much higher
market values and is an important component of industrial
polyesters which are widely used in thermoplastics, textiles,
carpets, and upholstery [14]. Additionally, 1,3-PD is only pro-
duced from glycerol fermentation, not from glucose fermen-
tation [13,23]. Thereby, anaerobic fermentation of glycerol has
a unique advantage of harvesting 1,3-PD as a valuable liquid
product. Hydrogen accumulation caused by longerHyRT could
increase hydrogen concentration in liquid phase, lower the pH
of fermentation solution, affect the metabolic pathways of
acidogenic bacteria and hydrogenic bacteria, and ultimately
affect hydrogen yields and 1,3-PD yields. Therefore, it is crit-
ical to effectively solve hydrogen accumulation in batch-mode
fermentation treatment of glycerol in order to achieve high
1,3-PD production and hydrogen production.
In terms of inocula for anaerobic fermentation of glycerol,
organic soil containing diverse anaerobicmicroorganisms and
rich in nutrients was used previously [12,13]. Because the
metabolic activities of microorganisms and the nutrient levels
could decrease over time after the organic soil was collected,
fresh organic soil was normally stored in sealed bags at 4 �Crefrigerators [24]. It is important to understand whether the
soil freshness affected the anaerobic fermentation efficiency.
Different soil sources (e.g. compost, potato soil, and soybean
soil) were compared in anaerobic hydrogen-producing re-
actors [20]. The impact of the freshness of tomato soil on
hydrogen yields using different substrates (e.g. glucose, su-
crose, lactate, and potato starch) was studied, and the results
showed that the soils collected over 1 month had no clear
effect on gas production [24]. Until now, it was unclear
whether the long-term stored organic soil (e.g. 2e6 months)
could have less hydrogen yields or less liquid yields than the
fresh collected organic soil.
Researchers at the University of Connecticut have suc-
cessfully produced biodiesel fuel from vegetable oil [25]. The
biodiesel waste stream contains (by mass): 61.6% methanol,
26.5% glycerol, 5.8% potassium hydroxide (KOH), and 6.1%
biodiesel [26]. Separation steps were developed to purify
glycerol in the biodiesel waste streams [27], in which four
purification levels of glycerol, namely, phase 0 (P0), phase
1(P1), phase 2 (P2) and phase 3 (P3), were achieved. Specifically,
KOH in P0, methanol in P1, and the non-polar layer (biodiesel
leftover) in P2 were removed from each purification step to
produce P1, P2 and P3 glycerol [26,27], respectively. In this
study, these four purification levels (P0, P1, P2, and P3) of
glycerol from biodiesel waste streams were examined in
anaerobic fermentation tests for high production of hydrogen
and 1,3-PD.
Therewere three objects in this study. First, hydrogen yield
and 1,3-PD yield of these biodiesel glycerol products were
examined in the batch-mode fermentors. The effects of glyc-
erol purification steps on anaerobic fermentation were eluci-
dated. Second, the impacts of HyRT on hydrogen yields and
liquid product yields were investigated to explore the rela-
tionship between GCI, IGCT, and anaerobic fermentation of
glycerol. The correlations of the hydrogen yield, 1,3-PD yield
and other liquid product yield (e.g. ethanol, acetate) were
established. A kinetic model was developed to simulate the
trends of hydrogen/1,3-PD production at different GCIs and
IGCTs. Third, the effects of soil freshness on anaerobic
H2O
NADH
1, 3 Propanediol
ADP
ATP
DHAP
Glycerol
Glyceroldehyde-3-P
ADP
2ATP
NADH
Pyruvate
HsCoA
CO2
Fdox
Fdred
2H+
H2
Acetyl-CoA
2NADH
HsCoA
2NADH
Butyryl-CoA
HsCoA
Pi
ADP
ATP
Ethanol
Butyrate
Acetyl-phosphate
Pi
HsCoA
NADH
Acetate
CoA transfer
ADP
2ATP
Fig. 1 e Important metabolic pathways during anaerobic fermentation of glycerol by mixed cultures (revised from [23]).
i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 8 ( 2 0 1 3 ) 3 1 9 6e3 2 0 53198
fermentation were examined by comparing the performances
of the six-month-old organic soil and fresh organic soil.
2. Material and methods
2.1. Batch fermentation reactor setup and operationalconditions
Serum bottles (volume: 160 mL) (Wheaton Scientific, Millville,
NJ) were used in this study as batch-modeanaerobic bioreactors
[28]. A nutrient solution of 120 mL (consisting of per liter of
water: 2.0 g NH4HCO3, 1.0 g KH2PO4, 100mgMgSO4$7H2O, 10mg
NaCl, 10 mg Na2MoO4$2H2O, 10 mg CaCl2$2H2O, 15 mg
MnSO4$7H2O, and 2.78 mg FeCl2) was used as the inorganic
medium for anaerobic bacteria to grow [24]. Organic soil col-
lected from an organic farm near the University of Connecticut
was used as inocula [28]. The soil was stored at 4 �C and then
pre-heated at 105 �C for 2h to eliminatemethanogensand select
for hydrogen-producing bacteria [15]. The soil was then added
into the bioreactors along with the nutrient solution. The soil
concentration was kept as 15 g/L in all the tests.
The fermentative solutionwaswellmixed in thebioreactors
and then buffered with 2-(N-morpholino) ethanesulfonic acid
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 en e r g y 3 8 ( 2 0 1 3 ) 3 1 9 6e3 2 0 5 3199
and hydrate 4-morpholineethanesulfonic acid (MES hydrate,
Sigma-Aldrich) [28]. The pH of the solution was adjusted to 6.0
with MES in all tests and was further adjusted to 5.5 using hy-
drochloric acid (HCl) [28]. The final volume of the fermentation
solutionwas 120mL, whichmade the volume of the headspace
as 40 mL. The volume ratio of the headspace to the bioreactor
volume was designed as 25% in order to shorten the lag period
of gas pressure accumulation [24]. All the bioreactors were
constantly stirred at 350 rpmona stirrer (Variomag poly stirrer,
Thermo Corporation) at 30 �C during the test period [28].
Before the tests, the fermentative solutionwas spargedwith
nitrogen for 10 min to reduce oxygen concentration [28]. The
bioreactors were then sealed with rubber septum (Wheaton
Scientific, Millville, NJ). The oxygen left in the headspace was
expelled by injecting nitrogen to the headspace. This procedure
guaranteed an anaerobic environment in the batch-mode bio-
reactors. Pure glycerol and biodiesel glycerol were used as the
organic substrates, and the glycerol concentrations were
examined at three levels (3, 5 and 7 g/L). In these batch-mode
tests, a metabolic cycle was considered complete after hydro-
gen production stopped in fermentation solution.
2.2. Control test with soil
Organic substances (e.g. roots, manure, organic acids, carbo-
hydrates, humic acids, etc. [29].) originally contained in
organic soil could be utilized/degraded by anaerobic bacteria,
so a control test with soil only (without the addition of glyc-
erol) was conducted under each operational condition in order
to identify the contribution of these organic substances to the
production of hydrogen and liquid products [28]. The actual
generation of hydrogen and liquid products (e.g. 1,3-PD) from
the degradation of glycerol (pure glycerol or biodiesel glycerol)
equals the hydrogen and liquid products generated from
glycerol as the substrates minus the hydrogen and liquid
products generated from soil in the control tests [28].
2.3. Hydrogen gas measurement and analytical methods
The hydrogen concentration in the headspace of anaerobic
bioreactors was analyzed by collecting the biogas generated in
the headspace using a 50 mL gas tight syringe (Gastight #1705,
Hamilton Co. Reno, Nevada) and then analyzed using a gas
chromatograph (Agilent 6890N) equipped with a packed col-
umn (60/80 Carboxen-1000, Supelco, PA) and a thermal con-
ductivity detector. The total volume of the biogas generated in
the headspace was measured by inserting a 160 mL gas-tight
syringe into the headspace and collecting the biogas. Due to
the high gas pressure in the headspace, the biogas will push
the syringe plunger against the atmospheric pressure until the
equilibrium was reached. The gas volume was then recorded.
Different levels of GCIs (gas collection interval, 2.5, 5.5, 8.5,
13.0 and 20.0 h) and IGCTs (initial gas collection time point, 15,
45 and 65 h) were examined to determine the effects of HPP on
fermentation performance.
The hydrogen generation was calculated based on the
following equation (Eqs. (1)e(3)):
VH2 ;1 ¼ ð40þ V1Þ � C1 (1)
VH2 ;2 ¼ ð40þ V2Þ � C2 � 40 � C1 (2)
/VH2 ;n ¼ ð40þ VnÞ � Cn � 40 � Cn�1 (3)
where 40 is the headspace volume (mL) of bioreactors; VH2 ;1,
VH2 ;2 and VH2 ;n are the hydrogen volume calculated based on
the 1st, 2nd and nth times of gas collection; V1, V2 and Vn are
the gas volumemeasured on the 1st, 2nd and nth times of gas
collection; C1, C2, Cn�1 and Cn are the hydrogen concentrations
(by volume) measured on the 1st, 2nd, (n � 1)th and nth times
of gas collection, respectively. The total hydrogen volume
generated was calculated by adding all the hydrogen volumes
calculated from each gas collection (Eq. (4)):
VH2 ;total ¼Xn
i
VH2 ;i (4)
Specific hydrogen yield (mol/mol glycerolconsumed) was
defined as hydrogen generated (mole) per mole glycerol
consumed.
2.4. Liquid production measurement and analyticalmethods
The liquid products including 1,3-PD, acetate, butyrate, meth-
anol, and ethanol were measured with a gas chromatograph
(GC) (Agilent 6890N) equipped with a fused silica capillary (007
series bonded phase fused silica capillary) and flame ionization
detector (FID). Prior to analysis, all liquid samples were filtered
through filter membrane filters with 0.2 mm pore size to
remove all dissolved solid substances [19]. After each test, the
residual glycerol in the fermentative solution was measured
with an enzyme based kit (free glycerol reagent, Sigmae
Aldrich) and a spectrophotometer (Cary 50, Varian, CA) [19]. The
pH of the fermentative solution was measured at the end of
each test using anAg/AgCl reference electrode (Accumet AP72).
Liquid products yield (mol/mol glycerolconsumed) was defined as
liquid products generated (mole) per mole glycerol consumed.
3. Results and discussion
3.1. Effects of glycerol concentrations, GCI and IGCT onhydrogen and liquid products yields
The anaerobic fermentation of P2 and P3 glycerol using fresh
soil as inocula was conducted at three glycerol concentrations
(3, 5, and 7 g/L). The results showed that the glycerol con-
centrations (3e7 g/L) did not clearly affect hydrogen yields and
liquid products yields at various GCIs and IGCTs tested (Figs.
2e6), which indicated that glycerol concentration was not
the critical factor for anaerobic fermentation of glycerol.
However, other studies [18,28] with glucose as the substrate
and broader concentrations (1e30 g/L) had found that glycerol
concentrations affected the hydrogen yields, especially at
high glycerol concentrations. It is possible that low glycerol
concentrations (e.g. lower than 10 g/L) did not substantially
affect hydrogen yields and liquid products yield, but would
affect themat high concentrations. In addition, therewas only
little difference in hydrogen yields and liquid products yields
Fig. 2 e The hydrogen and liquid product yields of glycerol
at the GCI of 5.5 h and the IGCT of 15 h.Fig. 4 e Hydrogen and liquid product yields of glycerol at
the GCI of 8.5 h and the IGCT of 15 h.
i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 8 ( 2 0 1 3 ) 3 1 9 6e3 2 0 53200
between P2 and P3 glycerol (Fig. 2), even though P3 glycerol
had a higher purification level than P2 glycerol. This indicates
that the impurities (biodiesel leftover) in P2 glycerol had no
significant influence on anaerobic fermentation. When P0 and
P1 glycerol were used as the substrates, hydrogen yields were
comparable with P2 and P3 glycerol under the same GCI/IGCT,
but liquid products yields (including 1,3-PD yield) were low (all
products yields were less than 0.05 mol/mol glycerolconsumed).
Highmethanol contents (61.56% and 62.49% bymass [26]) in P0
and P1 glycerol inhibited 1,3-PD yields.
Longer GCI and IGCTwere expected to increase HyRT in the
headspace of reactors. To study the effects of GCI and IGCT on
hydrogen yields and liquid products yields, five sets of GCI and
IGCT were examined with GCI ranging from 2.5 h to 20 h and
IGCT from 15 h to 65 h. The tests started at the lower end of GCI
5.5 h and IGCT 15 h (Fig. 2), in which HyRT was short. The
hydrogen yields reached 0.47e0.71 mol/mol glycerolconsumed,
whichwerehigher than the reportedhydrogen yieldsunder the
same experimental condition [13]. Liquid products only con-
tained ethanol and 1,3-PD with high ethanol yields
(0.35e0.53 mol/mol glycerolconsumed) and low 1,3-PD yields
(0e0.20 mol/mol glycerolconsumed). Previous study did not find
the ethanol production at the same condition [13].Moreover, P3
glycerol had stable hydrogen and ethanol yields among dif-
ferent glycerol concentrations (3e7 g/L), while the yields from
P2 glycerol varied substantially with glycerol concentrations.
The hydrogen yield of the 3 g/L P2 glycerol was higher than
those of the 5 g/L and 7 g/L P2 glycerol (Fig. 2). Although it is
unclear about the mechanism causing the distinct yield vari-
ations in P2 glycerol, the dark non-polar layer “biodiesel left-
over” in P2 glycerol could affect the fermentative performance
Fig. 3 e The hydrogen and liquid product yields of glycerol
at the GCI of 2.5 h and the IGCT of 15 h.
of the anaerobic bacteria or block the contact between the
bacteria and glycerol substrates in batch-mode biofermentors.
The high yields of hydrogen at the short GCI and IGCT
verified our hypothesis that shorter HyRT alleviated the HPP in
the batch-mode bioreactors, decreased the hydrogen absorp-
tion activity in the fermentation broth, and thus proceeding
anaerobic reactionswell. Based on the fermentation pathways,
pyruvate is produced as the intermediate in the oxidation
pathways of glycerol anaerobic fermentation (Fig. 1 [23],). The
electrons (e�) generated later combine with protons (Hþ) to
formhydrogen gas (H2). The higher yields of hydrogen enhance
the production of Acetyl coenzyme A (Acetyl-CoA) and other
liquid products (e.g. ethanol, butyrate and acetate) [23], which
well explains the high yields of hydrogen and ethanol at short
GCIs and IGCTs in this study. On theother hand, theproduction
of 1,3-PD occurs in the reductive metabolic pathway, which is
different from the formation pathways of ethanol and hydro-
gen. The low yields of 1,3-PD at short GCIs and IGCTs indicated
that the production of 1,3-PD was suppressed by the high
production of ethanol and hydrogen.
To further decrease HyRT, GCI was shortened to 2.5 h without
changing IGCT (15 h). The hydrogen yields reached at
0.53e0.75mol/mol glycerolconsumed (Fig. 3), and the average values
forbothP2andP3glycerolwerehigherthanthoseattheGCIof5.5h
(Fig. 2). However, since the hydrogen yields for P2 glycerol varied
substantially (0.47e0.71mol/mol glycerolconsumed) at threeglycerol
concentrations, the increase in hydrogen yields (from Fig. 2 to
Fig. 3) was not significant for P2 glycerol. As indicated previously,
the dark non-polar layer “biodiesel leftover” in P2 glycerol could
causethisunstablehydrogenyield.Ethanolwasstill theonly liquid
Fig. 5 e The hydrogen and liquid product yields of glycerol
at the GCI of 13.0 h and the IGCT of 45 h.
Fig. 6 e The hydrogen and liquid product yields of P2 and
P3 glycerol at the GCI of 20 h and the IGCT of 65 h.
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 8 ( 2 0 1 3 ) 3 1 9 6e3 2 0 5 3201
product with the yields of 0.33e0.71mol/mol glycerolconsumed, the
maximumyield ofwhichwasmuchhigher than that at theGCI of
5.5h(Fig.2).Theresultsclearly indicatedthatshorterHyRTatshort
GCI and IGCT enhanced the yields of hydrogen and ethanol, and
the positive correlation of the two yields proved that ethanol and
H2 productions share the samemetabolic pathway. However, 1,3-
PDwas not detected in any liquid products, which confirmed that
that the enhancement in the oxidation pathways (the production
of hydrogen and ethanol) at short HyRT suppressed the reduction
pathways (the production of 1,3-PD) [23].
HyRT was then prolonged by increasing GCI (8.5 h) without
changing IGCT (15 h) (Fig. 4). Twomajor changes in the products
were observed. First, hydrogen and ethanol yields substantially
decreased comparedwith thoseat shortGCIs (Figs. 2 and 3). The
average hydrogen and ethanol yields were 0.26 and 0.17 mol/
mol glycerolconsumed. Second, 1,3-PD, acetic acid, and butyric
acid started to exist in the liquid products (Fig. 4). The average
yields for 1,3-PD, acetic acid, and butyric acid were 0.42, 0.12,
and 0.05 mol/mol glycerolconsumed, respectively. The 1,3-PD
yield found in this study was comparable with the highest 1,3-
PD yield (0.46 mol/mol glycerolconsumed) previously reported
under the same conditions [13]. Longer HyRT at longer GCI and
IGCT was supposed to increase the hydrogen absorption to the
fermentative solution and further affect the metabolic path-
ways and fermentative products yields. With the enhanced
absorption of hydrogen to the solution at longer HyRT, the
fermentation reactions slowed down, which caused the sub-
stantial decrease in the production of hydrogen and ethanol.
However, the reductive formation of 1,3-PD was enhanced at
longerHyRT. Based on the anaerobicmetabolic pathways (Fig. 1
[23],), the formation pathways of acetate and butyrate are dif-
ferent from those of ethanol, but they all come from the same
intermediate (Acetyl-CoA). The inhibition of hydrogen produc-
tion would increase the formation of Acetyl-CoA and further
increase the formation of acetate and butyrate (Fig. 4). Based on
the results of high yields of hydrogen and ethanol at short HyRT
(Figs. 2 and 3) and low yields of hydrogen and ethanol at long
HyRT (Fig. 4), it is possible that the hydrogenase would benefit
the formation of ethanologenic enzyme, and hydrogen pro-
duction would benefit the ethanol yield.
Both GCI and IGCT were then further extended to 13 h and
45 h, respectively, to examine the fermentation of glycerol at
even longer HyRT. It was expected that longer GCI/IGCT sup-
pressed the hydrogen production by increased hydrogen ab-
sorption in the solution at longer HyRT. The average yields for
hydrogen, ethanol, acetic acid, butyric acid were 0.20, 0.05, 0.06,
0.02 mol/mol glycerolconsumed, respectively (Fig. 5), all of which
were much lower than those acquired under shorter GCI and
IGCT (Figs. 2e4). However, the yields of 1,3-PDwere substantially
increased, with the average yields 0.55 mol/mol glycerolconsumed
(Fig. 5). These results confirmed the hypothesis that longer HyRT
increased the hydrogen absorption in fermentation solution,
which inhibited hydrogen production, but enhanced the 1,3-PD
production. In addition, it should be noted that the lag time for
hydrogen productionwas 20� 5 h at the GCI of 2.5e8.5 h and the
ICGT of 15 h, which was consistent with previous study [12].
When GCI and IGCT were further increased to 20 h and 65 h,
respectively, except hydrogen yield and 1,3-PDyield, other liquid
products yields were below 0.1 mol/mol glycerolconsumed (Fig. 6),
which were lower than those at the GCI of 13 h and IGCT of 45 h
(Fig. 5). The average hydrogen yield of P2 glycerol remained the
same, while the average hydrogen yield of P3 glycerol
(0.22 mol/mol glycerolconsumed, Fig. 6) was a bit higher than in
previous condition (0.19 mol/mol glycerolconsumed, Fig. 5). This
indicated that GCI/IGCT longer than 13/45 h would not have
significant influence on hydrogen yields. The average 1,3-PD
yield for P2 glycerol increased to 0.52 mol/mol glycerolconsumed
at the GCI 20 h and IGCT 65 h (Fig. 6), which was slightly higher
thanthat (Fig. 5).However, theaverage1,3-PDyield forP3glycerol
became lower (0.57 mol/mol glycerolconsumed, Fig. 6) than that at
the GCI 13 h and IGCT 45 h (0.60mol/mol glycerolconsumed, Fig. 5).
The highest 1,3-PD yield was 0.65 mol/mol glycerolconsumed pro-
duced by 7 g/L P3 glycerol. This is the highest 1,3-PD yield ever
reported in anaerobic fermentation of glycerol.
To conclusively demonstrate the changing trends of 1,3-PD
and H2 yields when increasing GCI/IGCTs, the p values were
acquired (to define the value differences between groups as
Boolean type) based on the yields of 1,3-PD and H2 from each
GCI/IGCT group, regardless of the glycerol types and concen-
trations. The p values for describing the increasing trend of
1,3-PD yields and the decreasing trend of H2 yields were 0.05
and 0.12, respectively, which showed that both trends were
significant, even though the latter was slightly less consid-
erable but still to an adequate extent.
3.2. Effects of glycerol concentrations, GCI and IGCT onglycerol degradation efficiency
The anaerobic fermentation had high removal efficiencies for
P2 (Fig. 7a) and P3 glycerol (Fig. 7b). More than 90% (bymass) P2
and P3 glycerol was consumed in the batch tests at all the
ranges of GCI (2.5e20 h), IGCT (15e65 h) and glycerol con-
centrations (3e7 g/L). However, one exception occurred at the
P3 glycerol concentration of 7 g/L with the longest GCI and
IGCT (GCI: 20 h and IGCT: 65 h) (Fig. 7b), which only 85%
glycerol was degraded at the end of the operation. This
exception might be caused by the low fermentation efficiency
under long HyRT that may slow down the overall metabolic
activities of anaerobic microorganisms. Moreover, higher
removal efficiencies achieved at lower glycerol concentra-
tions, and the GCI and IGCT had no obvious effect on glycerol
degradation efficiency. P2 and P3 glycerol exhibited the similar
removal efficiencies (90e100%). The results indicated that the
anaerobic fermentation of glycerol from biodiesel waste
stream had high removal efficiency.
(a)
(b)
Fig. 7 e The variations of glycerol degradation efficiencies
at different glycerol concentrations, GCI and IGCT (a: P2
glycerol, b: P3 glycerol).
(a)
(b)
Fig. 9 e The pH variation of glycerol fermentation at
different glycerol concentrations, GCI and IGCT.(a: P2
glycerol, b: P3 glycerol)
i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 8 ( 2 0 1 3 ) 3 1 9 6e3 2 0 53202
The maximum total fermentation time (h) for different
glycerol types (P2 and P3) and different concentrations at each
GCI/IGCT was examined (Fig. 8). It indicated that shorter
GCI/IGCT generally had shorter fermentation duration. With
prolonging the GCI/IGCT, the maximum total fermentation
time increased and the longer HyRT slowed down the fer-
mentation, whichwas correspondedwith the lower production
of hydrogen and liquid products (except 1,3-PD) at longer HyRT.
3.3. The pH variation during anaerobic fermentation
The protons (Hþ) generated throughout anaerobic fermenta-
tion reactions lowered the solution pH for both P2 (Fig. 9a) and
P3 glycerol (Fig. 9b). Generally, higher glycerol concentrations
resulted in higher amounts of liquid fermentation products
(e.g. acetic acid, butyric acid) and the lower pH [30]. The results
well corresponded with this trend of lower pH at higher glyc-
erol concentrations, with two exceptions (GCI: 2.5 h and 20 h)
for P2 glycerol (Fig. 9a).With the initial pHof 5.50 in all tests, the
final pH ranged from4.88 to 5.20. It should benoted that the low
pH (below 5.20) (Fig. 9b) could also reduce the fermentation
activities, which explained the glycerol degradation efficiency
decreased at high glycerol concentrations (7 g/L P3) (Fig. 7b).
Fig. 8 e Maximum fermentation time at different GCI and
IGCT (The unit of GCI/ICGT on the X axis is h/h).
3.4. Effects of fresh soil and old soil on fermentation
Fresh soil (stored at 4 �C for less than 1 month) was used in all
the experiments. To examine the effects of soil freshness on
anaerobic fermentation, old soil (collected and stored at 4 �Cfor 6 months) was used as inocula and compared with fresh
soil. Pure glycerol was used as organic substrate in these tests.
The GCI of 10 h and IGCT of 25 h were applied. The results
showed that average hydrogen yields of the fresh soil was
0.38 mol/mol glycerolconsumed and dropped to 0.31 mol/
mol glycerolconsumed for old soil (Fig. 10). This trend was fol-
lowed for all three glycerol concentrations (3, 5 and 7 g/L). The
1,3-PD yield was 0.44 mol/mol glycerolconsumed for fresh soil
and 0.49 mol/mol glycerolconsumed for old soil. It should be
noted that old soil had an average acetate yield of
0.15 mol/mol glycerolconsumed without ethanol yield (except
5 g/L glycerol), while fresh soil had an average ethanol yield of
0.15 mol/mol glycerolconsumed (except 3 g/L glycerol) without
acetate production (Fig. 10). This might imply that the
microbial communities changed through the 6-month soil
storage period. Overall, the results indicated that fresh soil
had better performances on hydrogen yield but lower 1,3-PD
yield than old soil.
3.5. Effects of GCI/IGCT on the net change of hydrogenand 1,3-PD yields
To elucidate the relationship of hydrogen yield and 1,3-PD yield
for P2 (Fig. 11a) and P3 glycerol (Fig. 11b) at different GCIs and
IGCTs, the net changes of these two major products were cal-
culated with the yields at the shortest GCI and IGCT (GCI: 2.5 h
and IGCT 15 h) as the baseline. It was clearly shown that the 1,3-
PD yields enhanced at longer GCI/IGCT (h/h). For P2 glycerol, the
1,3-PD yield only had a small increase (net increase: 0.07 mol/
mol glycerolconsumed) when GCI was increased from 2.5 to 5.5 h.
Later, it substantially increased 0.32 mol/mol glycerolconsumed
when GCI was further increased from 5.5 to 8.5 h. The net
increase of 1,3-PD reached a plateau when GCI was increased to
13 and 20 h. P3 glycerol followed the same trend on the net
Fig. 10 e The hydrogen and liquid products yields with fresh soil and old soil as inoculums.
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 en e r g y 3 8 ( 2 0 1 3 ) 3 1 9 6e3 2 0 5 3203
changesof 1,3-PDatdifferentGCIs/IGCTs.On theotherhand, the
net changes of hydrogen yields exhibited the opposite pattern
comparedwith1,3-PD (Fig. 11).Hydrogenyieldwas thehighest at
the shortest GCI/IGCT, and then steadily decreased with the
increase in GCI/IGCT (h/h). For P2 glycerol, the hydrogen yield
slightly decreased (�0.03 mol/mol glycerolconsumed) when GCI
was increased to 5.5 h, then decreased considerably
(�0.35 mol/mol glycerolconsumed) when GCI was increased to
8.5 h, and further deceased slowlywhenGCI was increased to 13
and 20 h. P3 glycerol fermentation followed the similar pattern.
The net changes of hydrogen yields and 1,3-PD yields with GCI/
IGCT (h/h) demonstrated that adjusting the gas collection time
could change HyRTs in the anaerobic bioreactors and further
influence the metabolic pathways of the fermentation. The
optimization of hydrogen yield and 1,3-PD yield should be bal-
anced at different GCIs and IGCTs.
Fig. 11 e The net changes of hydrogen yield and 1,3-PD yield a
baseline of the net changes was based on the hydrogen yield an
IGCT: 15 h).) (a: P2 glycerol, b: P3 glycerol).
3.6. Kinetic models for hydrogen and 1,3-PD yields atdifferent GCI/IGCTs
There were only a few studies for modeling hydrogen and 1,3-
PD yields in anaerobic treatment of glycerol, andmost of them
were based on glycerol concentrations. The model of hydro-
gen and liquid production from glycerol batch fermentation
using activated municipal sludge as inoculum was developed
[18]. By using the modified Gompertz equation, the best fit-
tings of the obtained results were made for the evolved biogas
(H2 and CO2) (R2 > 0.9015), liquid products (including 1,3-PD)
(R2 > 0.9310) and glycerol (R2 > 0.9676). Xiu et al. (2004) mod-
eled the optimal conditions of batch and continuous glycerol
fermentations by Klebsiella pneumoniae by setting volumetric
productivity of 1,3-PD as the optimization target [31]. Sun et al.
(2008) developed a metabolic kinetic model for simulating the
t different GCI/IGCT (h/h) with glycerol as substrate (The
d 1,3-PD yield at the shortest GCI and IGCT (GCI: 2.5 h and
i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 8 ( 2 0 1 3 ) 3 1 9 6e3 2 0 53204
concentrations of substrate (glycerol), key enzymes glycerol
dehydratase (GDHt) and 1,3-PD oxidoreductase (PDOR), inter-
mediate 3-hydroxypropionaldehyde (3-HPA), and target
products (1,3-PD) [32]. By using Box-Behnken design and
Response Surface Method (RSM), Varrone et al. (2012) effec-
tively modeled the biohydrogen and ethanol production of
glycerol fermentation process based on the parameter opti-
mization of glycerol concentration, reaction temperature, and
initial pH [33]. However, there has been no model to simulate
the production of hydrogen and 1,3-PD at different gas col-
lection frequencies (GCI/IGCT). This paper was, for the first
time, to establish themathematic correlation of hydrogen and
1,3-PD yields and GCI/IGCT for anaerobic treatment of
glycerol.
Based on the experimental results of H2 and 1,3-PD yields
at different GCI/IGCT from P2 and P3 glycerol, two models
were developed in this study to simulate the production of H2
and 1,3-PD at different GCI/IGCT, respectively. The software
PYTHON (Python�, version 3.3.0) was used for the model
development. Because the experimental results between P2
and P3 glycerol were similar, the average values of H2 and
1,3-PD were calculated and used to develop the models. Sum
of squares errors (SSE) was applied to describe the preciseness
of the models, which was a statistic approach to quantify the
difference between the estimated implied values and the true
values of the quantity being estimated. SSE is defined in Eq. (5)
for a model with a single variable.
SSE ¼Xn
i¼1
�yi � fðxiÞ
�2(5)
where yi is the ith value of the variable to be predicted, xi is the
ith value of the variable, and f(xi) is the predicted value of yi.
Two models were developed for the 1,3-PD (Eq. (6)) and H2
yields (Eq. (7)), respectively, with GCI and IGCT as variables.
The model for the average 1,3-PD yields with GCI/IGCT is
Y1;3�PD ¼�0:062�GCIþ0:085� IGCTþ0:012�GCI2�0:002
� IGCT2�0:787(6)
The model for the average H2 yields with GCI/IGCT is
YH2¼0:064� GCI� 0:080� IGCT� 0:012� GCI2 � 0:002� IGCT2
� 0:787 ð7Þ
where Y1,3-PD and YH2(mol/mol glycerolconsumed) are the average
yields of 1,3-PD and H2 at different GCI/IGCT (h/h). The SSEs for
the two models are 0.0093 and 0.0054, respectively, which indi-
cated that these models precisely described the relationships
between GCI/IGCT and the average H2 and 1,3-PD yields. The
models clearly indicated that themixed-order (1st order and 2nd
order) GCI/IGCT directly influenced theH2 and 1,3-PD yields. The
model provides a good guideline to estimate the H2 and 1,3-PD
yields at different gas sampling frequencies (GCI/IGCT).
4. Conclusion
The effects of HyRT on the production of hydrogen and 1,3-PD
in anaerobic fermentation of glycerol were extensively con-
ducted in this study. Different concentrations of purified
glycerol from biodiesel waste stream were tested as organic
substrates. Different levels of CGI and IGCTwere examined for
the effects of different HPPs in headspace on anaerobic fer-
mentation. The correlation of hydrogen production and 1,3-PD
production was established. The effects of soil freshness on
anaerobic fermentation were also investigated.
There were five major conclusions drawn from this study.
First, P2 and P3 glycerol exhibited similar performances on
hydrogen and 1,3-PD yields. In addition, glycerol concentra-
tion was not a critical factor for hydrogen and 1,3-PD yields at
low concentrations (<7 g/L). Higher methanol concentrations
in P0 and P1 glycerol inhibited the growth of anaerobic bac-
teria and had low 1,3-PD yields.
Second, hydrogen yield increased at shorter HyRT (shorter
CGI and IGCT),while 1,3-PDyield decreased at shorterHyRT. The
P3 glycerol at the concentration of 7 g/L had the highest 1,3-PD
yield (0.65 mol/mol glycerolconsumed) at the GCI/IGCT (20 h/65 h)
and thehighest hydrogen yield (0.75mol/mol glycerolconsumed) at
the GCI/IGCT (2.5 h/20 h), respectively.
Third, hydrogen yields from the old organic soil (stored for
6 months) as inoculum was lower than those from fresh soil
(stored for 1month), but 1,3-PD yields of these two types of soil
were similar.
Fourth, the mixed-order relationship of GCI/IGCT and the
production of H2 and 1,3-PD was established in two models.
Fifth, the optimization of hydrogen production and 1,3-PD
production can be achieved by adjusting HyRT in anaerobic
fermentation of biodiesel glycerol.
Acknowledgement
The project was supported by the Environmental Graduate
Research Scholarship (CESE) at the University of Connecticut.
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