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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

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http://dx.doi.org/10.1016/j.ijhydene.2012.12.135



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




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]).


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




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.


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


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


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.


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)


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.


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




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.

r e f e r e n c e s

[1] National Biodiesel Board, www.biodiesel.org.[2] Fernando S, Adhikari S, Kota K, Bandi R. Glycerol based

automotive fuels from future biorefineries. Fuel 2007;86(17e18):2806e9.

[3] Heming M. Oleolone glycerine market Report No. 71;2005.[4] ZhangY, DubeMA,McLeanDD,KatesM. Biodiesel product ion

from waste cooking oil: 1. Process design and technologicalassessment. Bioresour Technol 2003;89(1):1e16.

[5] NBB, editor. The official site of the National Biodiesel Board.National Biodiesel Board; 2006.

[6] Ito T, Nakashimada Y, Senba K, Matsui T, Nishio N. Hydrogenand ethanol production from glycerol-containing wastesdischarged after biodiesel manufacturing process. J BiosciBioeng 2005;100(3):260e5.

[7] AlhanashA,KozhevnikovaEF,Kozhevnikov IV.Hydrogenolysisof glycerol to propanediol over Ru: polyoxometalatebifunctional catalyst. Catal Lett 2008;120(3e4):307e11.

[8] Lammers PJ, Kerr BJ, Weber TE, Dozier WA, Kidd MT,Bregendahl K, et al. Digestible and metabolizable energy ofcrude glycerol for growing pigs. J Anim Sci 2008;86(3):602e8.

[9] U.S. Census Bureau Foreign Trade Data Dissemination.Available from: http://www.asasea.com/download.doc.php[accessed 10.01.2012].

http://www.biodiesel.org

http://www.asasea.com/download.doc.php




[10] Rosa DS, Bardi MAG, Machado LDB, Dias DB, Silva LGA,Kodama Y. Starch plasticized with glycerol from biodieseland polypropylene blends: mechanical and thermalproperties. J Therm Anal Calorimetry 2010;102(1):181e6.

[11] Li C, Fang HHP. Fermentative hydrogen production fromwastewater and solid wastes by mixed cultures. Crit RevEnviron Sci Technol 2007;37(1):1e39.

[12] Selembo PA, Perez JM, Lloyd WA, Logan BE. Enhancedhydrogen and 1,3-propanediol production from glycerol byfermentation using mixed cultures. Biotechnol Bioeng 2009;104(6):1098e106.

[13] Sharma Y, Parnas R, Li B. Bioenergy production from glycerolin hydrogen producing bioreactors (HPBs) and microbial fuelcells (MFCs). Int J Hydrogen Energy 2011;36(6):3853e61.

[14] Kraus GA. Synthetic methods for the preparation of 1,3-propanediol. Clean 2008;36(8):648e51.

[15] Biebl H, Zeng AP, Menzel K, Deckwer WD. Fermentation ofglycerol to 1,3-propanediol and 2,3-butanediol by Klebsiellapneumoniae. Appl Microbiol Biotechnol 1998;50(1):24e9.

[16] Gonzalez-Pajuelo M, Andrade JC, Vasconcelos I. Productionof 1,3-propanediol by Clostridium butyricum VPI 3266 usinga synthetic medium and raw glycerol. J Ind MicrobiolBiotechnol 2004;31(9):442e6.

[17] Murarka A, Dharmadi Y, Yazdani SS, Gonzalez R.Fermentative utilization of glycerol by Escherichia coli and itsimplications for the production of fuels and chemicals. ApplEnviron Microbiol 2008;74(4):1124e35.

[18] Seifert K, Waligorska M, Wojtowski M, Laniecki M. Hydrogengeneration from glycerol in batch fermentation process. Int JHydrogen Energy 2009;34(9):3671e8.

[19] Sharma Y, Li B. Optimizing energy harvest in wastewatertreatment by combining anaerobic hydrogen producingbiofermentor (HPB) and microbial fuel cell (MFC). Int JHydrogen Energy 2010;35(8):3789e97.

[20] Van Ginkel S, Sung S, Lay JJ. Biohydrogen product ion asa function of pH and substrate concentration. Environ SciTechnol 2001;35(24):472. 6e30.

[21] Mu Y,Wang G, Yu HQ, Zheng JC. Response surfacemethodological analysis onbiohydrogenproduction by enrichedanaerobic cultures. EnzymeMicrob Technol 2005;38(7):905e13.

[22] Jo JH, Lee DS, Park D, Choe WS, Park JM. Optimization of keyprocess variables for enhanced hydrogen production byEnterobacter aerogenes using statistical methods. BioresourTechnol 2008;99(6):2061e6.

[23] Temudo Margarida F, Poldermans Rolf,Kleerebezem Robbert, van Loosdrecht Mark CM. Glycerolfermentation by (open) mixed cultures: a chemostat study.Biotechnol Bioeng 2008;100(6):1088e98.

[24] Logan BE, Oh SE, Kim IS, Van Ginkel S. Biological hydrogenproduction measured in batch anaerobic respirometers.Environ Sci Technol 2002;36(11):2530e5.

[25] “UConn Biofuel Consortium”. http://biodiesel.engr.uconn.edu/index.html.

[26] ChristiansenK. Separations Process Biodiesel-derivedGlycerolProduction 1,3-propanediol, undergraduate thesis; 2012.

[27] Hajek M, Skopal F. Treatment of glycerol phase formed bybiodiesel production. Bioresour Technol 2010;101(9):3242e5.

[28] Sharma Y, Li B. Optimizing hydrogen production fromorganic wastewater treatment in batch reactors throughexperimental and kinetic analysis. Int J Hydrogen Energy2009;34(15):6171e80.

[29] Shepherd MA, Harrison R, Webb J. Managing soil organicmatter e implications for soil structure on organic farms.Soil Use Manage 2002;18(s1):284e92.

[30] Li JZ, Ren NQ. The operational controlling strategy about theoptimal fermentation type of acidogenic phase. Chin EnvironSci 1998;18(5):398e402.

[31] Xiu ZL, Song BH, Wang ZT, Sun LH, Feng EM, Zeng AP.Optimization of dissimilation of glycerol to 1,3-propanediolby Klebsiella pneumoniae in one- and two-stage anaerobiccultures. Biochem Eng J 2004;19(3):189e97.

[32] Sun YQ, Qi WT, Teng H, Xiu ZL, Zeng AP. Mathematicalmodeling of glycerol fermentation by Klebsiella pneumoniae:concerning enzyme-catalytic reductive pathway andtransport of glycerol and 1,3-propanediol across cellmembrane. Biochem Eng J 2008;38(1):22e32.

[33] Varrone C, Giussani B, Izzo G, Massini G, Marone A,Signorini A, et al. Statistical optimization of biohydrogen andethanol production from crude glycerol by microbial mixedculture. Int J Hydrogen Energy 2012;37(21):16479e88.

http://biodiesel.engr.uconn.edu/index.html

http://biodiesel.engr.uconn.edu/index.html



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