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Critical assessment of anaerobic processes for continuousbiohydrogen production from organic wastewater

Kuan-Yeow Show a,*, Zhen-Peng Zhang b, Joo-Hwa Tay c, David Tee Liang d, Duu-Jong Lee e,Nanqi Ren f, Aijie Wang f

a Faculty of Engineering and Green Technology, University Tunku Abdul Rahman, Jalan University, Bandar Barat, 31900 Kampar,

Perak, Malaysiab Beijing Enterprises Water Group Limited, BLK 25, No. 3 Minzhuang Road, Beijing 100195, Chinac School of Civil and Environmental Engineering, Nanyang Technological University, Nanyang Avenue, Singapore 639798d Institute of Environmental Science and Engineering, Nanyang Technological University, Singapore 637723e Department of Chemical Engineering, National Taiwan University, Taipei, Taiwan, ROCf State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China

a r t i c l e i n f o

Article history:

Received 8 November 2009

Accepted 18 November 2009

Available online 29 December 2009

Keywords:

Granule

Biofilm

Continuous stirred tank reactor

(CSTR)

Anaerobic fluidized bed reactor

(AFBR)

* Corresponding author. Tel.: þ60 605 466232E-mail addresses: kyshow2003@yahoo.co

dtl168@gmail.com (D.T. Liang), djlee@ntu.ed0360-3199/$ – see front matter ª 2009 Profesdoi:10.1016/j.ijhydene.2009.11.110

a b s t r a c t

Production of biohydrogen using dark fermentation has received much attention owing to

the fact that hydrogen can be generated from renewable organics including waste mate-

rials. The key to successful application of anaerobic fermentation is to uncouple the liquid

retention time and the biomass retention time in the reactor system. Various reactor

designs based on biomass retention within the reactor system have been developed. This

paper presents our research work on bioreactor designs and operation for biohydrogen

production. Comparisons between immobilized-cell systems and suspended-cell systems

based on biomass growth in the forms of granule, biofilm and flocs were made. Reactor

configurations including column- and tank-based reactors were also assessed. Experi-

mental results indicated that formation of granules or biofilms substantially enhanced

biomass retention which was found to be proportional to the hydrogen production rate.

Rapid hydrogen-producing culture growth and high organic loading rate might limit the

application of biofilm biohydrogen production, since excessive growth of fermentative

biomass would result in washout of support carrier. It follows that column-based granular

sludge process is a preferred choice of process for continuous biohydrogen production

from organic wastewater, indicating maximum hydrogen yield of 1.7 mol-H2/mol-glucose

and hydrogen production rate of 6.8 L-H2/L-reactor h.

ª 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.

1. Introduction the mixed liquor and less suffered from the mass transfer

A continuous stirred tank reactor (CSTR) is frequently used for

continuous hydrogen production [1–3]. In such a conventional

system, hydrogen-producing bacteria are well suspended in

3; fax: þ60 605 4667449.m.sg (K.-Y. Show), zpzh

u.tw (D.-J. Lee).sor T. Nejat Veziroglu. Pu

resistance, but biomass retention is substantially influenced

by the culture hydraulic retention time (HRT) and washout of

biomass may occur at a shorter HRT. Hydrogen production

rates are thus restricted considerably by a low CSTR biomass

ang@yahoo.com.cn (Z.-P. Zhang), cjhtay@ntu.edu.sg (J.-H. Tay),

blished by Elsevier Ltd. All rights reserved.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 3 3 5 0 – 1 3 3 5 5 13351

retention and a low hydraulic loading [4,5]. Immobilized-cell

systems have become popular alternatives to suspended-cell

systems in continuous operations since they are more capable

of maintaining higher biomass concentration and could be

operated at high dilution rates without biomass washout.

Biomass immobilization can be achieved through forming

granules [6], biofilms [7] or gel-entrapped bioparticles [8].

Previous studies suggested that biofilms and granules showed

advantages compared to the cell-entrapped bioparticles in the

hydrogen production [8,9]. The inferior performance of gel-

immobilized sludge processes is mainly attributed to the low

mass transfer efficiency. Furthermore, stability and durability

of gel-entrapped bioparticles are generally questionable.

Hence, gel immobilization techniques may not be the tech-

nology of choice for fermentative hydrogen production. Those

immobilized hydrogen-producing cultures have been

employed in different reactor systems, i.e. CSTR, fixed- or

packed-bed reactor, anaerobic fluidized bed reactor (AFBR),

upflow anaerobic sludge blanket (UASB) reactor, and trickling

biofilter. From the viewpoint of reactor configuration, those

reactor systems can be divided into two types: tank-shaped

reactor and column-shaped reactor.

Hydrogen production performances of granule system and

biofilm system, however, are inconsistent. For example,

hydrogen production rate varies from 0.20 L/L h with granules

in a UASB reactor [10] to 15.09 L/L h with silicone-immobilized

and self-flocculated granular sludge in a CSTR [8]. It seems to

indicate that microbial cell immobilization methods and

reactor configuration play a crucial role in hydrogen produc-

tion rate. Nevertheless, it is stressed that those systems con-

tained different microbial cultures and were operated under

different conditions. This should be taken into consideration

when making comparison in the system performance. To

realize a valid comparison, it is of importance to maintain

a homogenous microbial culture in the reactors while oper-

ating under the same conditions. However, comparative

studies between biofilm and granular sludge or between tank

reactor and column reactor for hydrogen production remain

limited so far. The present study was conducted to fill up the

knowledge gap by summarizing the experimental results

obtained.

The objective of the present study was to evaluate the

system performance in terms of hydrogen production. These

systems included suspended sludge CSTR system, granular

sludge CSTR system, granular sludge AFBR system and biofilm

AFBR system.

Table 1 – Biomass concentration in a CSTR under differentHRT conditions.

HRT (h) D (h�1) Biomassconcentration

(g-VSS/L)

Residual glucoseconcentration

(g/L)

YX/S (�)

12 0.08 1.01 0.06 0.10

10 0.10 0.84 0.38 0.09

8 0.13 0.90 0.84 0.10

6 0.17 0.85 2.22 0.11

3 0.33 0.33 7.05 0.11

2 0.50 Washout

2. Materials and methods

2.1. Experimental approaches

A CSTR (6 L, working volume) and two AFBRs (1.4 L, working

volume) were used in the present work. All reactors were

operated at a constant temperature of 37 �C and fed with

10 g-glucose/L glucose-based synthetic wastewater containing

nutrients as described previously [11,12]. Seed sludge was

obtained from a local wastewater treatment plant, which was

acclimated with glucose (10 g-glucose/L) in an anaerobic CSTR

at pH 5.5 for more than 2 months as described in a previous

study [11]. Granular sludge and biofilm sludge were cultivated

from the acclimated hydrogen-producing culture through the

acid incubation method developed by Zhang et al. [13,14].

Characteristics of granular sludge used in CSTR and AFBR

systems were as follows: diameter 1.7� 0.9 mm (CSTR),

1.5� 0.3 mm (AFBR); roundness 2.6� 1.4 m/h (CSTR),

1.3� 0.5 m/h (AFBR); and extracellulous polymers 180� 14 mg/

g-VSS (CSTR), 187� 13 mg/g-VSS (AFBR). Biofilm particles in

AFBR had a diameter of 1.8� 0.4 mm, a roundness value of

1.6� 0.6 m/h, and an extracellulous polymer content of

181� 18 mg/g-VSS. The CSTR was operated in an HRT short-

ening mode from 12 h to 2 h for suspended sludge, and from 2 h

to 0.5 h for granular sludge. The HRT of AFBRs was shortened

from 2 h to 0.25 h.

2.2. Analytical methods

Biogas composition in H2, CO2 and CH4 was analyzed by gas

chromatography. Detailed procedures for measurement of

biogas amount and composition had been described in

previous works [11,13]. Glucose concentration was deter-

mined following the phenol–sulfuric acid method reported by

Dubois et al. [15]. Measurements of suspended solids (SS) and

volatile suspended solids (VSS) were performed according to

the standard methods [16].

3. Results and discussion

3.1. Microbial kinetics

The anaerobic seed sludge was cultivated in a CSTR with HRT

reducing from 48 h to 2 h. As a completely mixed reactor,

biomass retention of suspended bacteria was related to the

operating HRT. A relatively consistent biomass concentration

of 0.84–1.01 g-VSS/L was observed as the reactor was operated

at HRT in a range of 6–12 h (Table 1). However, reducing HRT

further caused a substantial decrease in biomass retention,

and complete washout of hydrogen-producing bacteria took

place at an HRT of 2 h, corresponding to a dilution rate (D) of

0.5 h�1. This suggests that the peak specific growth rate (mmax)

of hydrogen-producing culture used in the present study was

close to 0.5 h�1. The mmax value was similar to that obtained by

Ghosh which was referred in a previous study [17] and was

higher compared with those (0.14–0.33 h�1) reported by other

researchers [18,19]. It was noted that mixed cultures of

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 3 3 5 0 – 1 3 3 5 513352

hydrogen-producing bacteria were used and cultured at

different conditions in the studies, and it was likely that they

exhibit inconsistent growth rates due to diversed microbial

population.

Microbial kinetic parameters of suspended culture could be

determined by employing the following equation [2,20]:

D ¼ YDðS0 � SÞ

X� kd ¼ Y,V � kd (1)

where, V is specific substrate utilization rate (g-glucose/g-

VSS h); D is dilatation rate (¼HRT�1, h�1); Y is true yield coef-

ficient expressing the cell mass produced per unit substrate

(g-VSS/g-glucose); S0 and S are influent and effluent concen-

trations of glucose (g/L), respectively; X is the biomass

concentration (g-VSS/L); kd is the endogenous decay coeffi-

cient (h�1). By dint of plotting D vs. V as indicated in Eq. (1), the

kinetic constants of Y, kd can be determined by using the

experimental data presented in Table 1.

The kinetic parameters were estimated to be kd 0.016 h�1

and Y 0.12 g-VSS/g-glucose. The kd value was extremely low,

and Y value was quite close to specific growth yield of

biomass, YX/S indicating that operation conditions are suitable

for the growth of hydrogen-producing bacteria. At pH 5.5,

almost all the energy obtained by catabolism was mainly

utilized to maintain cell growth, rather than cell activity. The

Y value is in the range of 0.08–0.16 g-VSS/g-COD reported in

other mixed hydrogen-producing cultures [6,19,21], but much

lower compared to 0.33 g-VSS/g-COD reported by Yu and Mu

[10] in a UASB reactor fed with sucrose.

It was noted that microbial composition of suspended

sludge became simple as the HRT was reduced [11]. Only one

microbial strain was detectable at an HRT of 6 h at steady-

state conditions. The strain was identified at an HRT of 2 h,

which was very close to species Clostridium pasteurianum [22].

It has been known that Clostridia are most popular microbial

species involved in anaerobic hydrogen fermentation. The

following evaluation on culture type and reactor type was

based on the systems containing such microbial species.

3.2. Evaluation on culture type

3.2.1. Suspended sludge vs. granular sludgeEvaluation on suspended sludge and granular sludge was con-

ducted with a CSTR. In contrast to the system upset occurring at

an HRT of 2 h, steady hydrogen production was observed in the

suspended-cell CSTR with HRT ranging from 3 h to 12 h. Fig. 1a

illustrates the effect of HRT on hydrogen yield, hydrogen

2 4 6 8 10 120.0

0.5

1.0

1.5

2.0

2.5

0.0

0.1

0.2

0.3

0.4

8

10

12

14

16

H2

)lom/lo

m(dleiy

HRT (h)

HY HPR SHPR

H2

)h.L/

L(etar

noitcudorp

Hcificep

S2

noitcudorp

)h.SS

V-g/lom

m(etar

a

Fig. 1 – Hydrogen production by suspended slu

production rate and specific hydrogen production rate in the

suspended-cell CSTR. Hydrogen yield stabilized at 1.92 mol-H2/

mol-glucose at HRTs of 6–12 h, but it decreased substantially to

0.97 mol-H2/mol-glucose as the HRT was shortened to 3 h.

Specific hydrogen production rate increased from 8.33 to

16.09 mmol-H2/g-VSS h with the decreased HRT from 12 h to

3 h, whereas hydrogen production rate increased from

0.21 L/L h at an HRT of 12 h–0.32 L/L h at 6 h HRT, but decreased

to 0.13 L/L h when HRT was shortened to 3 h.

In contrast to the suspended sludge, granular sludge

concentration increased with the reduction in HRT (Fig. 2).

The maximum concentration of granular sludge was esti-

mated to be at 16.0 g-VSS/L in the granule-based CSTR at an

HRT of 0.5 h, which was far higher than that obtained in

suspended-cell system. It was found that granules dis-

aggregated and eventually evolved into dispersed cells at an

HRT of 3 h, whose performance in hydrogen production was

found to be close to the suspended sludge system at the same

HRT, suggesting that HRT might play a crucial role in the

stability of microbial granules. An HRT equal to or shorter

than the critical washout point of suspended sludge is

required in a granular sludge CSTR. This is consistent with the

hypothesis proposed by Tijhuis et al. [23] that, in ideally mixed

reactors, formation of biofilm covered carriers only takes

place as the HRT is shorter than the inverse of the maximum

growth rate (the dilution rate is larger than the maximum

growth rate, D> mmax).

Hydrogen yield, hydrogen production rate and specific

hydrogen production rate increased significantly with the

reduction in HRT from 2 h to 0.5 h as shown in Fig. 1b. Their

respective maximum values of 1.81 mol-H2/mol-glucose,

3.20 L/L h and 8.31 mmol-H2/g-VSS h were recorded at an HRT

of 0.5 h. An HRT of 0.5 h was thus suggested as the optimum

operating condition for the granular sludge, which was much

shorter as compared with an HRT of 6 h required for the sus-

pended sludge in the same CSTR. The ability to retain a large

amount of biomass of granular sludge at a shorter HRT

condition allowed high-rate hydrogen production to be

accomplished in the granule-based CSTR system. As

a sequence of the enriched granular sludge, granular sludge

exhibited a tenfold increase in hydrogen production rate while

keeping a comparable hydrogen yield to the suspended sludge.

3.2.2. Granular sludge vs. biofilm sludgeGranular sludge and biofilm sludge hydrogen production

were evaluated with two AFBRs, designated as granule

reactor and biofilm reactor, respectively. The HRT varied

0.5 1.0 1.5 2.0

1.4

1.6

1.8

2.0

0

1

2

3

0

2

4

6

8

10

H2

)lom/lo

m(dleiy

HRT (h)

HY HPR SHPR

H2

)h.L/

L(etar

noitcudorp

Hcificep

S2

noitcudorp

)h.SSV- g/lo

mm(

etar

b

dge (a) and granular sludge (b) in a CSTR.

0 2 4 6 8 10 120

5

10

15B

iom

ass

conc

entra

tion

(g-V

SS/L

)

HRT (h)

Suspended sludge

Granular sludge

Fig. 2 – Biomass concentrations of suspended sludge and

granular sludge.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 3 3 5 0 – 1 3 3 5 5 13353

from 2 h to 0.25 h with a consistent glucose concentration of

10 g/L, corresponding to OLRs of 5–40 g-glucose/L h. Table 2

shows a comparable hydrogen production performance of

granule and biofilm in terms of hydrogen yield and specific

hydrogen production rate. Hydrogen yields increased in both

reactors as the HRTs were shortened from 2 h to 0.5 h, but

decreased slightly with the further reduction in HRT,

achieving the respective maximum values of 1.83 and

1.81 mol-H2/mol-glucose for the granular sludge and biofilm

sludge. Hydrogen production rates increased substantially

with decreasing HRT, and reached the respective peaks of

6.77 and 7.34 L-H2/L h at the shortest HRT of 0.25 h in

granule-based reactors and biofilm-based reactors. Short-

ening HRT to 0.25 h, however, caused a substantial decrease

in glucose conversion rate to a level of 76–80% which was

much lower in contrast to nearly 100% glucose conversion

rate at HRT ranging from 2 h to 0.5 h.

Obviously, there was no apparent difference in hydrogen

production between granule reactor and biofilm reactor as

observed in the present study. This is largely owing to the

relatively consistent and comparable biomass concentration

maintained in both reactors. Biomass amount was main-

tained consistently within the ranges of 34.7–36.9 g-VSS/L and

34.3–37.6 g-VSS/L at HRTs between 0.25 h and 2 h for the

granule reactor and biofilm reactor, respectively. This indi-

cated that adding support media in biofilm reactor did not

reduce the biomass retention as compared with the granule-

based reactor.

Our previous study [24] indicated that biofilm sludge

decreased significantly over time at an OLR of 40 g-glucose/L h

due to the washout of support media, associating with an

Table 2 – Hydrogen production performance of granule reactor

HRT (h) Glucose conversion rate (%) H2

Granule Biofilm Granu

2 99.28� 0.27 96.13� 2.45 1.44� 0

1 99.00� 0.73 99.12� 0.54 1.64� 0

0.5 99.30� 0.31 99.16� 0.32 1.83� 0

0.25 76.30� 1.34 80.14� 1.91 1.63� 0

increase of granular sludge in biofilm reactor. It was observed

that with the evolution of biofilm, the thickness of biofilm

increased significantly and attachment of microorganisms to

the support media might become weaker. As a result, biofilm

was separated from support media due to particle–particle

collision, leaving fragmented biofilm in the reactor. This

fragmented biofilm eventually developed into granules which

had predominated over biofilm in biofilm reactor after being

operated for 50 days. As washout of support carriers is not

a concern of granular sludge systems, this poses as an

advantage feature of the systems.

These findings indicated that biofilm process might not be

suitable for anaerobic hydrogen fermentation due to the rapid

growth characteristics of hydrogen-producing bacteria. As

stated earlier, mmax (0.5 h�1) and yield coefficient of hydrogen-

producing bacteria (0.12 g-VSS/g-glucose) would result in the

production of a large amount of biomass (ca. 72–127 g-VSS per

day) under an organic loading rate (OLR, 40 g-glucose/L h). The

maximum specific growth rate and biomass growth yield were

also found in other studies, ranging from 0.17 to 0.5 h�1 and

from 0.08 to 0.33 g-VSS/g-COD, respectively. Furthermore,

higher organic loading rates, such as 40 g-glucose/L h in the

present study and up to 80 g-COD/L h in a study conducted by

Wu et al. [25], were generally employed in the anaerobic

hydrogen fermentation. In a recent review, Nicolella et al. [26]

had mentioned that biofilm reactors are not particularly

useful with fast-growing organisms (i.e. with a maximum

specific growth rate >0.1 h�1). Rapid buildup of hydrogen-

producing biofilms could result in a system upset due to the

mass transfer limitation. Working on a trickling biofilter, for

example, Oh et al. [27] noted that microbial growth of

hydrogen-producing bacteria was too excessive under a mes-

ophilic condition, which caused system upset after merely

one-week of operation.

3.3. Evaluation on reactor type

Influence of reactor configuration on hydrogen production

was evaluated by examining CSTR and AFBR systems with

granular sludge for hydrogen production. Maximum hydrogen

production rates of the suspended sludge, CSTR granular

sludge, AFBR granular sludge and biofilm sludge were essen-

tially correlated to the biomass concentration as shown in

Fig. 3, indicating that the system performance is largely

influenced by the reactor biomass retention. Compared to the

CSTR, much more biomass retention was retained in the AFBR

reactor (35 vs. 16 g-VSS/L), which might be attributed to the

reactor intrinsic structure. A larger height/section diameter

and biofilm reactor.

yield (mol/mol) H2 production rate (L/L h)

le Biofilm Granule Biofilm

.11 1.50� 0.08 0.97� 0.07 0.99� 0.06

.08 1.73� 0.12 2.20� 0.10 2.32� 0.16

.09 1.81� 0.08 4.93� 0.25 4.87� 0.24

.07 1.69� 0.31 6.77� 0.32 7.34� 1.34

0 5 10 15 20 25 30 35 400

2

4

6

8 Rate Fit of Rate

Rat

e (L

/L.h

)

Biomass concentration (g-VSS/L)

Fig. 3 – Hydrogen production rate (R2 [ 1.00, p [ 0.0002) as

a function of biomass concentration.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 3 3 5 0 – 1 3 3 5 513354

ratio of column reactor, such as AFBR, might be the advan-

tages favoring solid/liquid separation and biomass retention,

and hence the hydrogen production. It should be stressed that

Wu et al. [25] reported the highest hydrogen production rate of

up to 15 L/L h which was achieved by a granule-based CSTR

system at an HRT of 0.5 h. In their study. The biomass

concentration of the CSTR culture maintained at a level of

32.5 g-VSS/L, which was comparable to that of the present

AFBR system, but was two times higher than that of the

present CSTR. A further study on CSTR design and manage-

ment (tank configuration, stirrer location and control, etc.)

should be conducted in order to better understand the factors

affecting reactor biomass retention.

Additionally, since the recirculation flow rate was kept

consistently at 200 mL/min, its effect on mass transfer and

reactor performance was not examined in the present study.

Nevertheless, it is possible that increasing liquid recirculation

ratio might improve mass transfer of immobilized culture and

substrate in a column reactor. Fixed- or packed-bed reactor is

operated under the conditions with a lesser extent of hydraulic

turbulence, thus its immobilized cultures usually encounter

mass transfer resistance which would result in inferior rates of

substrate conversion and hydrogen production. Rachman

et al. [28] found that high hydrogen molar yield could not be

maintained consistently in a packed-bed reactor, although the

pH in the effluent was controlled at more than 6.0. This was

owing to pH gradient distribution along the reactor column

resulting in a heterogeneous distribution of microbial activity.

In order to overcome the mass transfer resistance and pH

heterogeneous distribution, they recommended that fluidized

bed or expanded bed reactor system with recirculation flow are

more appropriate for further enhancing the hydrogen

production rate and yield. In a later study, Kumar and Das [29]

observed that hydrogen production and substrate conversion

rates of a packed-bed reactor both increased with the recycling

ratio, and obtained the maximum hydrogen production rate of

1.69 L/L h at a recirculation ratio of 6.4. This means that the

mass transfer resistance in a packed-bed reactor can be

reduced by increasing slurry recycle ratio.

Appropriate reactor configuration would help to increase

hydraulic turbulence of the column reactors, and result in an

increase in hydrogen production. Kumar and Das [29] inves-

tigated hydrogen production by Enterobacter cloacae attaching

on coir in packed-bed reactors with different configurations,

i.e. tubular, tapered or rhomboid shape at an HRT of 1.08 h.

The comparative study indicated that the rhomboid biore-

actor with convergent–divergent configuration has

a maximum hydrogen production rate of 1.60 L/L h as

compared with 1.46 L/L h in tapered reactor and 1.40 L/L h in

tubular reactor. The enhancement in hydrogen production

rate could be attributed to higher turbulence caused by reactor

geometry favoring mass transfer and reduced gas hold-up.

4. Conclusions

Formation of granules or biofilms substantially enhanced

biomass retention. Reactor volumetric hydrogen production

rate was highly related to the biomass retention. Rapid

hydrogen-producing culture growth and higher OLR conditions

might limit the application of biofilm anaerobic biohydrogen

processes, since excessive production of fermentative biomass

would result in washout of support carriers in a fluidized bed

reactor or system upset in a fixed-bed reactor. As no support

carrier is needed by the granule-based systems, this poses as

an advantage of the systems. It can be concluded that the

column-shaped reactor with granular sludge is a preferred

choice of continuous biohydrogen production systems from

organic wastewater. However, this system may not be suitable

for operation under longer HRTs in which granules may

disaggregate, and also not suitable for substrate containing

high solid content.

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