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Hydrogen production from acid hydrolyzed molassesby the hydrogen overproducing Escherichia coli strainHD701 and subsequent use of the waste bacterial biomassfor biosorption of Cd(II) and Zn(II)

Fatthy Mohamed Morsy*

Botany Department, Faculty of Science, Assiut University, Assiut 71516, Egypt

a r t i c l e i n f o

Article history:

Received 26 April 2011

Received in revised form

23 July 2011

Accepted 29 July 2011

Available online 3 September 2011

Keywords:

Hydrogen production

Biosorption

Cd2þ

Heavy metals

Zn2þ

Waste Escherichia coli biomass

* Tel.: þ20 0167714518; fax: þ20 088 234270E-mail address: fmorsy@aun.edu.eg.

0360-3199/$ e see front matter Copyright ªdoi:10.1016/j.ijhydene.2011.07.121

a b s t r a c t

This study was devoted to investigate production of hydrogen gas from acid hydrolyzed

molasses by Escherichia coli HD701 and to explore the possible use of the waste bacterial

biomass in biosorption technology. In variable substrate concentration experiments (1, 2.5,

5, 10 and 15 g L�1), the highest cumulative hydrogen gas (570 ml H2 L�1) and formation rate

(19 ml H2 h�1 L�1) were obtained from 10 g L�1 reducing sugars. However, the highest yield

(132 ml H2 g�1 reducing sugars) was obtained at a moderate hydrogen formation rate (11 ml

H2 h�1 L�1) from 2.5 g L�1 reducing sugars. Subsequent to H2 production, the waste E. coli

biomass was collected and its biosorption efficiency for Cd2þ and Zn2þ was investigated.

The biosorption kinetics of both heavy metals fitted well with the pseudo second-order

kinetic model. Based on the Langmuir biosorption isotherm, the maximum biosorption

capacities (qmax) of E. coli waste biomass for Cd2þ and Zn2þ were 162.1 and 137.9 (mg/g),

respectively. These qmax values are higher than those of many other previously studied

biosorbents and were around three times more than that of aerobically grown E. coli. The

FTIR spectra showed an appearance of strong peaks for the amine groups and an increase

in the intensity of many other functional groups in the waste biomass of E. coli after

hydrogen production in comparison to that of aerobically grown E. coli which explain the

higher biosorption capacity for Cd2þ or Zn2þ by the waste biomass of E. coli after hydrogen

production. These results indicate that E. coli waste biomass after hydrogen production can

be efficiently used in biosorption technology. Interlinking such biotechnologies is poten-

tially possible in future applications to reduce the cost of the biosorption technology and

duplicate the benefits of biological H2 production technology.

Copyright ª 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights

reserved.

1. Introduction succeed in bringing down costs [1e4]. Biological hydrogen

Hydrogen is currently more expensive than traditional

energy sources, so it is likely to play a major role in the

economy in the long run, if technology improvements

8.

2011, Hydrogen Energy P

production processes are found to be more environment

friendly and less energy intensive as compared to thermo-

chemical and electrochemical processes [5e7]. Biological

hydrogen production requires a waste and by-product

ublications, LLC. Published by Elsevier Ltd. All rights reserved.

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 6 ( 2 0 1 1 ) 1 4 3 8 1e1 4 3 9 014382

strategy to compete with other production technologies of

hydrogen gas. A way to reduce the cost of biological

hydrogen production is through improving the practical

bacterial hydrogen productivity from organic wastes [8e14].

Another way is to make use of its waste products in other

biotechnological applications. One possibility is to make use

of the waste bacterial biomass in heavy metals biosorption

technology for wastewater treatment. The biosorption of

heavy metals by certain types of microorganisms’ biomass

is a highly cost-effective technology for the decontamina-

tion of metal-containing effluents [15]. Both living and non-

living microorganisms biomass have been used in bio-

sorption of toxic metal ions [16e21]. The use of heterotro-

phic bacteria for biosorption of heavy metals [22e24] is

rather expensive in comparison to green algae and cyano-

bacteria [25e27] which grow autotrophically. Using the

waste bacterial biomass of hydrogen production in heavy

metals biosorption would make the biosorption technology

of heavy metals by heterotrophic bacteria inexpensive and

duplicates the benefits of biological hydrogen production

technology.

Hydrogen production by Escherichia coli is mediated by the

formate hydrogenlyase (FHL) complex [28e34]. The mutant E.

coli HD701 constructed previously [35] cannot synthesize the

(Hyc A) repressor of the FHL complex. Consequently, this

mutant has an up-regulated FHL system and can, therefore,

evolve hydrogen at a greater rate than its parental wild type, E.

coli MC4100 [36]. The main objectives of this work were to

investigate the hydrogen production from acid hydrolyzed

molasses by the hydrogen overproducing E. coli HD701 and to

examine the biosorption efficiency of the waste E. coli biomass

for Cd2þ and Zn2þ.

2. Materials and methods

2.1. Culture conditions for E. coli

E. coli was maintained aerobically at 30 �C on nutrient agar

[containing (g L�1): peptone, 5; beef extract, 5; NaCl, 3; agar,

15]. Prior to hydrogen production, E. coli was grown aerobi-

cally overnight on nutrient broth [containing (g L�1):

peptone, 5; beef extract, 5; NaCl, 3] with shaking (100 rpm) at

30 �C.

2.2. Acid hydrolysis of molasses

Several preliminary experiments were conducted to optimize

the conditions of acid hydrolysis of molasses. The optimum

conditions used for acid hydrolysis of molasses were as

fellow: Molasses was diluted two times by distilled water and

adjusted to 2 M HCl. Subsequently, the acid hydrolysis was

conducted at 120 �C for 20 min.

2.3. Determination of reducing sugars concentration

Samples of acid hydrolyzed molasses were neutralized by 1 M

NaOH. Subsequently, the reducing sugars concentration was

determined by Nelson reagent [37].

2.4. Hydrogen production from acid hydrolyzedmolasses by E. coli

Batch experiments for hydrogen production by E. coli were

conducted as described by Penfold et al. [36] with modifica-

tions using the reducing sugars of acid hydrolyzed molasses

as a carbon source. The experiments were conducted at 30 �Cin 1 L fermentor with a working volume of 970 ml. The

fermentor contained 80% (v/v) of 10mMpotassium phosphate

buffer (pH 7.3) and 10% (v/v) of acid hydrolyzed molasses

(previously neutralized to pH 7.3 by 1 M NaOH) in a final

concentration of 1, 2.5, 5, 10 and 15 (g/L) reducing sugars. After

autoclaving, 10% (v/v) inoculum of E. coli cells, pre-grown in

nutrient broth (OD600 equal 0.25), was added. The fermentor

was fitted with rubber stopper and purged with N2 gas for 1 h

to ensure an anaerobic environment. Fermentation tempera-

ture was controlled at 30 �C in a water bath. The fermentor

was set in dark and the evolved H2 gas was collected and

measured by displacement of 1 M NaOH in an inverted

cylinder. NaOH was used to absorb CO2 [36]. All gas volumes

are reported at 1 atm and 25 �C.

2.5. Preparation of the waste E. coli biomass forbiosorption

Subsequent to the hydrogen production experiments, cells of

E. coli in the spent medium of the fermentor were collected by

centrifugation at 10,000 �g for 20 min at 4 �C. The biomass

pellets of E. coli were washed three times with distilled water.

Thewashed biomasswas freeze-dried and stored at�80 �C for

subsequent heavy metals biosorption experiments.

2.6. Metal solution for biosorption

Stock solutions of Cd2þ and Zn2þ (1000mg/L) were prepared by

dissolving 1.63, and 4.39 g of CdCl2 and ZnSO4$7H2O, respec-

tively in a 1000 ml of distilled water.

2.7. Biosorption experiments

Biosorption experiments were conducted at pH 6. The freeze-

dried E. coli waste biomass (20 mg) was suspended in 20 ml of

Cd2þ and Zn2þ solutions at various initial concentrations (Ci)

ranging from 0 to 300 mg L�1. Shaking was conducted at

200 rpm for 25 min till equilibrium attained at room temper-

ature (25 � 2 �C). Subsequent to centrifugation at 10,000 �g for

20 min, the heavy metal equilibrium (final) concentration (Ce)

in the supernatant was measured by atomic absorption

spectroscopy (AAS) (Model 210 VGP Buck Scientific). The

quantity of metal ion (mg) biosorbed per gram (dry weight) of

E. coli biomass was calculated using the following equation:

qeðmg=gÞ ¼�Ci�Ce

M

�� V (1)

where, qe ¼ equilibrium (final) quantity of metal ions bio-

sorbed (mg metal ions/g bacterial biomass); Ci ¼ initial metal

ions concentration (mg/L); Ce ¼ equilibrium (final) metal ion

concentration (mg/L); M ¼ dry weight of E. coli biomass (g);

V ¼ the volume of metal ions solution (L).

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 6 ( 2 0 1 1 ) 1 4 3 8 1e1 4 3 9 0 14383

2.8. Biosorption isotherms

Several isothermmodels are available to describe equilibrium

distribution of metal ions between the aqueous and solid

phases. The two most common types for describing bio-

sorption isotherms are the Langmuir and Freundlich models

[16,17,38,39].

2.8.1. Langmuir isotherm modelThe mathematical formula of Langmuir equation can be

expressed as:

qe ¼ qmaxbCe

1þ bCe(2)

The linear form of the equation is:

Ce=qe ¼ 1=qmaxbþ Ce=qmax (3)

where, qmax (mg/g) is the maximum biosorption capacity of

the metal ions (mg) per unit weight (g) of bacterial biomass

to make monolayer coverage by the metal ions on the

bacterial surface. The Langmuir constant b (L/mg) represents

the affinity between biosorbent and biosorbate. The

maximum biosorption capacity qmax, and b can be deter-

mined from the linear form of Langmuir equation Eq. (3) by

plotting Ce/qe versus Ce. The plot of Ce/qe versus Ce gives

a straight line. The slope equals 1/qmax while intercept

equals 1/(qmaxb).

2.8.2. Freundlich isotherm modelThe general form of the Freundlich isotherm equation is:

qe ¼ KfC1=ne (4)

The linear form of the equation is:

ln qe ¼ ln Kf þ 1=n ln Ce (5)

where, Kf and n are the biosorption capacity and intensity,

respectively. If the value of n is greater than unity, this is an

indication of a favorable adsorption. A plot of ln qe versus ln Ce

gives a straight line. The slope equals 1/n while the intercept

equals ln Kf.

2.9. Biosorption rate kinetics

Experiments for determining the kinetics of the biosorption

process were performed at room temperature (25 � 2 �C). Thefreeze-dried E. coli waste biomass (100 mg) was suspended in

100 ml of 50 mg L�1 Cd2þ or Zn2þ solutions (pH 6) with shaking

at 200 rpm. Aliquots (2 ml) of metal solution were taken at

desired time intervals and were immediately centrifuged at

15,000 �g for 5 min. Subsequently, the heavy metal concen-

tration in the supernatant wasmeasured by AAS. The changes

in Cd2þ and Zn2þ biosorption (mg/g) with time were deter-

mined and the data were analyzed using pseudo first-order

Lagergren and pseudo second-order kinetic models.

2.9.1. The pseudo first-order modelThe first-order rate equation [40] is one of the most widely

used for the sorption of solute from a liquid solution. The

pseudo first-order Lagergren model is generally expressed as:

dqt

dt¼ K1

�qe � qt

�(6)

where qt is the amount of metal ions biosorbed (mg/g) at any

given time t (min), qe is the amount of metal ion biosorbed

(mg/g) at equilibrium and K1 is the Lagergren rate constant of

pseudo first-order biosorption (min�1).

Integrating Eq. (6) between the limits, t¼ 0 to t¼ t and qt¼ 0

to qt ¼ qe, it becomes

log�qe � qt

� ¼ logqe � K1

2:303t (7)

The plot of log (qe � qt) vs. t should give a linear relationship

from which k1 and qe can be determined from the slope and

intercept of the plot, respectively.

2.9.2. The pseudo second-order modelThe pseudo second-order model [40] can be expressed as:

dqt

dt¼ K2

�qe � qt

�2(8)

where qt is the amount ofmetal ions biosorbed by freeze-dried

waste E. coli biomass (mg/g) at any given time t (min), qe is the

amount of metal ion biosorbed (mg/g) at equilibrium and k2 is

the rate constant of the pseudo second-order biosorption

(g mg�1 min�1).

For the boundary conditions t¼ 0 to t¼ t and qt¼ 0 to qt¼ qe,

the integrated form of Eq. (8)

tqt

¼ 1k2q2

e

þ 1qe

t (9)

The plot of (t/qt) vs. t of Eq. (9) should give a linear rela-

tionship from which qe and k2 can be determined from the

slope and intercept of the plot, respectively.

2.10. Comparison between waste E. coli biomass afterhydrogen production and aerobically grown E. coli forbiosorption of Cd2þ and Zn2þ

Biosorption of Cd2þ and Zn2þ by freeze-dried biomass of aero-

bically grown E. coli in nutrient broth were also investigated for

comparison with the biosorption capacity of the waste E. coli

biomass after hydrogen production. E. coli HD701 was grown

aerobically onnutrient broth as described above. Subsequently,

E. coli cells were collected by centrifugation at 10,000 �g for

20min at 4 �C. The biomass pellets of E. coliwere washed three

times with distilled water. The washed biomass was freeze-

dried and stored at �80 �C for subsequent heavy metals

biosorption experiments. Biosorption experiments were con-

ducted at pH 6. The freeze-dried biomass of aerobically grown

E. coli (20mg)was suspended in20ml ofCd2þ andZn2þ solutions

at various initial concentrations (Ci) ranging from 0 to

300 mg L�1. Shaking was conducted at 200 rpm for 25 min till

equilibrium attained at room temperature (25 � 2 �C). Subse-quent to centrifugation at 10,000�g for 20min, the heavymetal

equilibrium concentration (Ce) in the supernatant was

measured by AAS. The data were analyzed as described above.

2.11. Fourier transform infra-red (FTIR) analysis

The freeze-dried cells of aerobically grown E. coli and the

freeze-dried waste biomass of E. coli after hydrogen

f H

form

atio

n (m

l hL

)

d (

ml)

/g r

educ

ing

suga

rs

10

15

20

60

80

100

120

140

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 6 ( 2 0 1 1 ) 1 4 3 8 1e1 4 3 9 014384

production from acid hydrolyzed molasses (metal free

biomass) as will as the waste E. coli biomasses after bio-

sorption of Cd2þ or Zn2þ (metal ions loaded) were analyzed

using an Fourier transform infra-red (FTIR) spectrometer

(Model: Nicolet 6700; Thermo-Electron Corporation). The FTIR

analysis was performed to give a qualitative characterization

of the main chemical groups present on the cell wall that are

responsible for heavy metal biosorption.

Reducing sugars (g L )

Rat

e o

Hyi

el

0

5

0

20

40

0 2 4 6 8 10 12 14 16

Fig. 2 e Average rate of hydrogen formation and the

hydrogen yield by E. coli HD701 from acid hydrolyzed

molasses. Mean values were estimated from three

independent experiments.

3. Results and discussion

3.1. Hydrogen production by E. coli from acidhydrolyzed molasses

Dark fermentation batch experiments were conducted for

hydrogen production by E. coli using the reducing sugars of the

acid hydrolyzed molasses as a carbon source. Hydrolysis of

molasses was substantial for hydrogen production by E. coli.

The major component of molasses was sucrose (data not

shown). However, E. coli cannot normally utilize sucrose

[36,41]. Thus, it could not produce hydrogen from non-

hydrolyzed molasses. E. coli HD701 was previously reported

to produce only H2 and CO2 [36]. Hydrogen evolution began

after two hours from the fermentation start time and

continued for a period of 30 h followed by a plateau in the

hydrogen evolution pattern (Fig. 1). The highest cumulative

hydrogen (570 ml H2 L�1) (Fig. 1) and formation rate (19 ml H2

h�1 L�1) (Fig. 2) were obtained from 10 g L�1 reducing sugars.

However, the highest yield (132 ml g�1 reducing sugars) was

obtained from 2.5 g L�1 reducing sugars (Fig. 2). The rate of

hydrogen evolution increased by increasing the substrate

concentration up to 10 g L�1 but it declined at 15 g L�1 (Fig. 2).

The accumulation of fermentation end-products (e.g. ethanol

and organic acids) and fall in pH can exert sufficient ‘stress’ to

halt mesophilic H2 production by E. coli in an excess of

substrate [42,43]. It is more advantageous to operate the

fermentation by E. coli at low reducing sugars concentration

(2.5 g L�1) to obtain the highest hydrogen yield at a moderate

hydrogen formation rate (11 ml H2 h�1 L�1) (Fig. 2).

Time (h)

Cum

ulat

ive

hydr

ogen

gas

(m

l)

0

100

200

300

400

500

600

700

0 10 20 30 40

012.551015

Reducing Sugars (g L )

Fig. 1 e Cumulative hydrogen production from acid

hydrolyzed molasses by E. coli HD701 at various reducing

sugars concentrations. Mean values and standard errors

from three independent experiments are shown.

3.2. Biosorption of Cd2þ and Zn2þ by the waste E. colibiomass

Subsequent to hydrogen production by E. coli from the acid

hydrolyzed molasses, the waste bacterial biomass was

collected and its biosorption efficiency for Cd2þ and Zn2þ was

investigated. The optimum pH for biosorption of Cd2þ and

Zn2þ by freeze-dried waste biomass of E. coli was 6 and the

optimum contact time was 20 min at room temperature (data

not shown).

3.3. Biosorption isotherms

Zinc and cadmiumbiosorption performance by freeze-dried E.

coli waste biomass was achieved by the biosorption equilib-

rium measurements at initial concentrations ranging from

0 to 300 mg/L at pH 6. Fig. 3 shows the relationship between

the amount (mg/g) of Zn2þ and Cd2þ biosorbed by E. coli waste

biomass against the concentration of Zn2þ and Cd2þ ions

remaining in the solution (mg/L). For a given initial metal ion

concentration, the amount of Cd2þ biosorbed by E. coli biomass

was greater than that of Zn2þ ions (Fig. 3). The biosorption

equilibrium data were analyzed with Langmuir and Freund-

lich isotherms. Through the modeling of the biosorption

equilibrium data it is possible to characterize biosorbents

under various operational conditions and this point is essen-

tial for future industrial applications [44].

3.3.1. The Langmuir isotherm modelThe Langmuir isotherm model was chosen to estimate the

maximum biosorption capacity corresponding to complete

monolayer coverage by the metal ions on the biosorbent (E.

coli) surface. The biosorption data was analyzed using the

Langmuir model in its linear form Eq. (3). The data fitted quite

well with the Langmuir model (Fig. 4) with very high regres-

sion correlation coefficients (R2 > 0.99) for both Cd2þ and Zn2þ

(Table 1). The validity of these data through the Langmuir

model indicates that the biosorption of Zn2þ and Cd2þ in the

present study can be characterized by a monolayer formation

C (mg L )

Am

ount

ads

orbe

d q

(mg)

/ g d

ry m

ass

0

50

100

150

200

0 50 100 150 200

Zn

Cd

Fig. 3 e Biosorption of Cd2D and Zn2D by freeze-dried E. coli

waste biomass at various initial metal ion concentrations.

Table 1 e The Langmuir and Freundlich isothermconstants for Cd2D and Zn2D biosorption by freeze-driedwaste E. coli biomass.

Heavy metalions

Langmuir Freundlich

qmax (mg/g) b (L/mg) R2 Kf n R2

Cd2þ 162.1 0.192 0.999 1.41 2.57 0.921

Zn2þ 137.9 0.069 0.995 1.33 2.37 0.958

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of the metal ions on the surface of E. coli. The maximum bio-

sorption capacities (qmax) of E. coli for Cd2þ and Zn2þwere 162.1

and 137.9 mg/g, respectively (Table 1). Taking into account the

atomic weight of both heavy metals, the maximum bio-

sorption capacities (qmax) of E. coli for Cd2þ and Zn2þ would be

3.377 and 4.597 mmol g�1, respectively. Thus, the E. coli

maximum biosorption capacity for Zn2þ would be higher than

Cd2þ. This differential type of Cd2þ and Zn2þ biosorption can

be ascribed to the difference in their ionic radii [45]. The

smaller-ionic radii metal ions appear to adsorb more on the

biomass, which is consistent with surface adsorption mech-

anism, since smaller-radius metal ions have greater adsorp-

tion tendency than larger ones as the element with smaller-

ionic radius will compete faster for exchange sites than those

of lager ionic radius [45]. This may explain the greater bio-

sorption ability of Zn2þ on the E. coli biomass than Cd2þ as the

ionic radius of Zn2þ (0.74 A�) is smaller than that of Cd2þ

(0.95 A�).

3.3.2. The Freundlich isothermThe Freundlich isotherm model was chosen to estimate the

biosorption intensity of Cd2þ and Zn2þ toward the E. coli

biomass. This model is usually used in its linear form Eq. (5)

C (mg L )

C/q

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 50 100 150 200

Zn

Cd

Fig. 4 e The linear plot of Langmuir isotherm for Cd2D and

Zn2D biosorption by freeze-dried E. coli waste biomass.

for analyzing the biosorption equilibrium data [17,23,24]. The

linear plot of Freundlich equation for biosorption of Cd2þ and

Zn2þ by freeze-dried E. coli waste biomass is shown in Fig. 5.

The values of Freundlich parameters are summarized in Table

1. The value of the biosorption intensity (n) of E. coli biomass

for both Cd2þ and Zn2þ is greater than unity (Table 1) indi-

cating that they are both favorably adsorbed by the waste

E. coli biomass.

The equilibriumdatawere found to fit reasonablywell with

the Freundlich model (Fig. 5) with regression correlation

coefficient (R2 ¼ 0.921) for Cd2þ and (R2 ¼ 0.958) for Zn2þ (Table

1). However, these values of regression correlation coefficients

in Freundlich model are lower than their corresponding

values in Langmuir model. Thus, Langmuir model fitted the

equilibrium data better than the Freundlich isotherm for

describing the biosorption equilibriumof Cd2þ and Zn2þ by the

freeze-dried waste E. coli biomass.

3.4. Kinetics of Cd2þ and Zn2þ biosorption by the freeze-dried waste E. coli biomass

Fig. 6A shows the changes in the amount (mg g�1) of Cd2þ and

Zn2þ biosorbed by the freeze-dried waste E. coli biomass with

time. The amount of metal biosorbed increased rapidly up to

a constant value ofmetal concentration after 20min. A further

increase in incubation time up to 400 min or even up to 3 days

had no significant effect on the biosorption of the heavy

metals. Thus, it can be concluded that the appropriate equi-

librium time for biosorption is reached at 20 min. This short

time required for biosorption is in accordance with the results

given by other authors [22,24,46,47]. In order to examine the

Ln C

Ln

q

0

1

2

3

4

5

6

0 1 2 3 4 5 6

Zn

Cd

Fig. 5 e The linear plot of the Freundlich isotherm for Cd2D

and Zn2D biosorption by freeze-dried E. coliwaste biomass.

A

B

C

0

2

4

6

8

10

12

0 100 200 300 400 500Time (min)

t/q

ZnCd

-1.5

-1

-0.5

0

0.5

1

1.5

2

0 10 20 30 40Time (min)

Log

(q

-q)

ZnCd

0

10

20

30

40

50

0 100 200 300 400 500Time (min)

q(m

g/g)

ZnCd

Fig. 6 e Kinetics of Cd2D and Zn2D biosorption by freeze-

dried E. coli waste biomass. Panel (A) shows the changes in

the amount (qt) of Cd2D and Zn2D biosorbed by the freeze-

dried waste E. coli biomass with time (t). Panel (B) and (C)

show the linearized pseudo first- and second-order kinetic

models respectively; for biosorption of Cd2D and Zn2D by

freeze-dried E. coli waste biomass.

Table 2 e Kinetic parameters obtained from pseudo first-order and pseudo second-order for Cd2D and Zn2D

biosorption by freeze-dried waste E. coli biomass.

HeavyMetal

Initialconc.

(mg L�1)

qexp(mg/g)

Pseudofirst-order

Pseudosecond-order

K1 qe R2 K2 qe R2

Cd2þ 50 44.5 0.139 29.76 0.998 0.0187 45.16 0.999

Zn2þ 50 43 0.135 35.78 0.994 0.0146 43.21 0.999

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 6 ( 2 0 1 1 ) 1 4 3 8 1e1 4 3 9 014386

controlling mechanism of the biosorption process, experi-

ment kinetic data in Fig. 6A were analyzed using two kinetic

models; the pseudo first-order and the pseudo second-order

model. Fig. 6B and C show linear plots of the linearized

forms of the pseudo first-ordermodel in Eq. (7) and the pseudo

second-order model in Eq. (9) respectively, for the biosorption

of Cd2þ and Zn2þ by the freeze-dried waste E. coli biomass.

Table 2, shows the theoretical qe values obtained from both

models in comparison with the experiment values (qexp). The

theoretical biosorption capacities (qe) for Cd2þ and Zn2þ esti-

mated from the pseudo first-order model showed a deviation

from the experiment values (qexp). In contrast, the pseudo

second-order results showed a theoretical qe values very close

to the experiment values (Table 2). Therefore, it can be

concluded that the pseudo second-order biosorption model is

more suitable to describe the biosorption kinetics of Cd2þ and

Zn2þ by the freeze-dried waste E. coli biomass.

3.5. Comparison between waste E. coli biomass afterhydrogen production and aerobically grown E. coli forbiosorption of Cd2þ and Zn2þ

Biosorption of Cd2þ and Zn2þ by freeze-dried biomass of

aerobically grown E. coli (in nutrient broth) were also investi-

gated for comparison with the waste E. coli biomass after

hydrogen production. Zinc and cadmium biosorption perfor-

mance by freeze-dried cells of aerobically grown E. coli was

achieved by the biosorption equilibrium measurements at

initial concentrations ranging from 0 to 300 mg L�1 of Cd2þ or

Zn2þ (Supplementary Fig. 1). The qmax deduced from Langmuir

isotherm for biosorption of Cd2þ and Zn2þ by aerobically

grown E. coli (Supplementary Fig. 2) were 55.3 and 42.6 mg g�1

respectively. In contrast, themaximum biosorption capacities

(qmax) of waste E. coli (after hydrogen production) for Cd2þ and

Zn2þ were around three times more than that of aerobically

grown E. coli in nutrient broth (Table 3). Such maximum bio-

sorption capacity values of waste E. coli (after hydrogen

production) for Cd2þ (162.1 mg/g) and Zn2þ (137.9 mg/g) are

also higher than those ofmany previously studied biosorbents

(Table 3).

3.6. Fourier transform infra-red (FTIR) analysis

The FTIR spectra were taken, in the range of 500e4000 cm�1,

for the freeze-dried cells of aerobically grown E. coli (Fig. 7A)

and the freeze-dried waste biomass of E. coli after hydrogen

production from acid hydrolyzed molasses (metal free

biomass) (Fig. 7B) as will as the waste E. coli biomasses after

biosorption of Cd2þ (Fig. 7C) or Zn2þ (Fig. 7D) (metal ions

loaded). The spectra display a number of absorption peaks,

reflecting the complex nature of the biomass. The FTIR spectra

of the waste biomass of E. coli after hydrogen production

(Fig. 7B) differed from that of aerobically grown E. coli (Fig. 7A).

The absorption peaks at 2359e2342 cm�1, corresponded to the

amine (]NeH2þ) group [74,75], appeared in the spectrumof the

waste biomass of E. coli after hydrogen production (Fig. 7B) in

comparison to that of aerobically grown E. coli (Fig. 7A). In the

spectrum of aerobically grown E. coli (Fig. 7A), the peak at

861 cm�1 is attributed to the presence of aromatic eCH

stretching [76]. Such peak disappeared in the spectrum of the

waste biomass of E. coli after hydrogen production (Fig. 7B).

The absorption bands characterizing hydroxyl and amine

groups were assigned at 3500e3200 cm�1. A peak at

3500e3200 cm�1 region is due to the stretching of the NeH

bond of amino groups and indicative of bonded hydroxyl

group [44,77]. Such bandswere stronger in the spectrum of the

Table 3 e Comparison of maximum biosorption capacity(qmax) of waste E. coli biomass (after H2 production) forZn2D and Cd2D with some other biosorbents and withaerobically grown E. coli.

Metal Biosorbent qmax (mg/g) References

Zn2þ Aspergillus niger 405 4.7 [48]

Citrobacter strain MCMB-181 23.6 [49]

Fucus spiralis 54.2 [50]

Padina sp. 35.1 [51]

Penicillium chrysogenum 6.5 [52]

Penicillium simplicissimum 65.5 [53]

Pseudomonas aeruginosa AT18 77.5 [54]

Pseudomonas putida 17.7 [55]

Pseudomonas syringae 8 [56]

Rhizopus nigricans 14 [57]

Spirogyra insignis 21.1 [50]

Streptomyces rimosus 30 [58]

Thiobacillus ferrooxidans 82.6 [59]

Escherichia coli 65.9 [60]

E. coli HD701 (aerobically grown

in nutrient broth)

42.6 This study

Waste E. coli HD701 biomass

(after H2 production)

137.9 This study

Cd2þ Fucus spiralis 114.9 [50]

Oedogonium sp. 88.2 [61]

Penicillium chrysogenum 56 [62]

Penicillium simplicissimum 52.5 [53]

Penicillium digitatum 3.5 [63]

Phanerochaete chrysosporium 84.5 [64]

Pseudomonas fluorescens 66.25 [65]

Pseudomonas stutzeri 21.3 [23]

Pseudomonas veronii 2E 54 [66]

Rhizopus arrhizus 25 [67]

Rhizopus arrhizus 30 [62]

Sargassum natans 115 [67]

Spirogyra insignis 22.9 [50]

Streptomyces noursei 3.4 [68]

Wheat straw, Triticum aestivum 4.88 [69]

Enterobacter sp. J1 46.2 [70]

Escherichia coli WS11 45.37 [71]

E. coli JM109 63.26 [72]

Polyvinyl alcohol immobilized

E. coli (33.0 wt.% of biomass)

4.29 [73]

E. coli HD701 (aerobically grown

in nutrient broth)

55.3 This study

Waste E. coli HD701 biomass

(after H2 production)

162.1 This study

Fig. 7 e The FTIR spectra for the freeze-dried cells of

aerobically grown E. coli (panel A) and the freeze-dried

waste biomass of E. coli after hydrogen production from

acid hydrolyzed molasses (metal free biomass) (panel B) as

will as the (metal ions loaded) waste E. coli biomasses after

biosorption of Cd2D (panel C) or Zn2D (panel D).

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 6 ( 2 0 1 1 ) 1 4 3 8 1e1 4 3 9 0 14387

waste biomass of E. coli after hydrogen production (Fig. 7B)

than the spectrum of aerobically grown E. coli (Fig. 7A). The

absorption peaks at 3000e2900 cm�1 are ascribed to the

asymmetric stretching of CeH bond of the eCH2 groups

combined with that of the CH3 groups [44,78]. Such bands

were also stronger in the spectrum of the waste biomass of E.

coli after hydrogen production (Fig. 7B) than the spectrum of

aerobically grown E. coli (Fig. 7A). The C]O of amide I was

assigned at 1650�1 while the NH/C]O combination of the

amide II bond was present at 1544 cm�1. These peaks were

stronger in the spectrum of the waste biomass of E. coli than

the spectrum of aerobically grown E. coli. The absorption

peaks at 1650 and 1544 cm�1 confirm also the presence of

carboxyl groups. The peaks in the range 1300e1067 cm�1 are

attributable to the presence of carboxyl and phosphate groups

[79] which have same intensity in both spectra (Fig. 7A and B).

It is observed from the spectra of Cd2þ or Zn2þ-loaded E. coli

waste biomass (Fig. 7C and D) that the intensity of almost all

functional groups had decreased and some characteristic

absorption peaks even disappeared. Pradhan et al. [80] and

Volesky [81] affirm that the main functional groups respon-

sible for a biosorption process are the hydroxyl, carbonyl,

carboxyl, sulfonate, amide, imidazole, phosphonate and

phosphodiester groups. Some of these groups are present on

the E. coli biomass and may interact with the metal ions

leading to such observed change in the FTIR spectra of Cd2þ or

Zn2þ-loaded E. coli waste biomass. The appearance of strong

peaks for the amine groups at 2359e2342 cm�1 and the

increase in the intensity of many other functional groups in

the FTIR spectrum of the waste biomass of E. coli after

hydrogen production in comparison to that of aerobically

grown E. colimight explain the higher biosorption capacity for

Cd2þ or Zn2þ by the waste biomass of E. coli after hydrogen

production than aerobically grown E. coli.

4. Conclusion

In this study, the highest hydrogen formation rate by E. coli

HD701 from acid hydrolyzed molasses was obtained from

10 g L�1 reducing sugars. However, it is more advantageous to

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 6 ( 2 0 1 1 ) 1 4 3 8 1e1 4 3 9 014388

operate the fermentation by E. coli at low reducing sugars

concentration (2.5 g L�1) to obtain the highest hydrogen yield

at a moderate hydrogen formation rate. The waste E. coli

biomass showed a high efficiency for biosorption of Cd2þ and

Zn2þ withmaximumbiosorption capacities (qmax) of 162.1 and

137.9 (mg/g), respectively. These qmax values were around

three times more than that of aerobically grown E. coli and are

higher than those of many previously studied biosorbents,

indicating a potential biosorption of both heavy metals by the

waste E. coli biomass. The FTIR spectrum of the waste biomass

of E. coli after hydrogen production showed an appearance of

strong peaks for the amine groups and an increase in the

intensity of many other functional groups in comparison to

that of aerobically grown E. coli which might explain the

higher biosorption capacity for Cd2þ or Zn2þ by the waste

biomass of E. coli after hydrogen production than aerobically

grown E. coli. The use of the waste bacterial biomass in heavy

metals biosorption would make the biotechnology of heavy

metals biosorption by heterotrophic bacteria inexpensive and

duplicates the benefits of biological hydrogen production

technology. Further future studies, for exploring the efficiency

of the waste biomass of other dark fermentative and also

photofermentative hydrogen producing bacteria in bio-

sorption technology, would be of interest to interlink such

biotechnologies.

Acknowledgments

Sincere thanks to Professor Dr. L.E. Macaskie and Dr. Mark

Redwood (School of Biosciences, University of Birmingham,

Edgbaston, Birmingham, UK) for kindly providing E. coli

HD701.

Appendix. Supplementary data

Supplementary data associated with this article can be found,

in the online version, at doi:10.1016/j.ijhydene.2011.07.121.

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