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 x x x ( 2 0 1 6 ) 1 e1 1

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Biofilm formation on granular activated carbon in

xylose and glucose mixture for thermophilic
biohydrogen production

Nur Syakina Jamali a,c, Jamaliah Md Jahim a,b,*,

Wan Nor Roslam Wan Isahak a
a
Department of Chemical and Process Engineering, Universiti Kebangsaan Malaysia, UKM, 43600 Bangi, Selangor,
Malaysia
b
Research Centre for Sustainable Process Technology, Universiti Kebangsaan Malaysia, UKM, 43600 Bangi,
Selangor, Malaysia
c
Department of Chemical and Environmental Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400
Serdang, Selangor Darul Ehsan, Malaysia

article info abstract

Article history: Granular activated carbon (GAC) was used as a support carrier to develop biofilm of
Received 21 January 2016 Thermophilic biohydrogen producer in an immobilized system of dark fermentation. The
Received in revised form optimum ratio of the sludge to GAC loading was investigated in batch fermentation using
7 May 2016 glucose and xylose mixture as a carbon source. It was found that the highest hydrogen
Accepted 10 May 2016 yield of 1.77 mol H2/mol substrate consumed and hydrogen production rate (HPR) of
Available online xxx 2.0 mmol H2/l.h, were achieved at a sludge-GAC ratio of 1:2. On the other hand, the ex-
periments with suspended culture as a control gave poor performance of hydrogen yield
Keywords: (0.86 mol H2/mol of substrate consumed) and HPR (0.5 mmol H2/l.h). The sludge-GAC bio-
Biofilms film was further developed in a sequencing batch feeding mode through controlled accli-
Fermentation matization condition. Stable hydrogen production was achieved after day 40th with
Biohydrogen consistent HPR of 2.4 mmol H2/l.h and yield of 1.17 mol H2/mol substrate consumed with
Thermophiles 44.2% of hydrogen. Acetic and butyric acids dominate volatile fatty acids (TVFAs) as a major
Batch processing by-product while ethanol being the only alcohol produced but in a minor amount. This
Granular activated carbon (GAC) work has proven the possible future of GAC attached biofilm sludge as promising attach-
ment system to achieve consistent hydrogen production even at thermophilic conditions.
2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

from renewable resources contributes the decrease in CO2

Introduction emissions. With the current proliferation of global economy,
production of biohydrogen fuel has emerged as a platform to
Hydrogen gas is well-known as a green, clean, high energy fuel utilize the abundant biological wastes [2,3]. A thermophilic,
without harming the environment [1]. Biohydrogen derived dark fermentation (45e65
C) process has gained attention due

* Corresponding author. Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti
Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia. Tel.: 603 8911 6427; fax: 603 8911 8345.
E-mail address: jamal@ukm.edu.my (J. Md Jahim).
http://dx.doi.org/10.1016/j.ijhydene.2016.05.092
0360-3199/ 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Jamali NS, et al., Biofilm formation on granular activated carbon in xylose and glucose mixture for
thermophilic biohydrogen production, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.05.092
2 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 x x x ( 2 0 1 6 ) 1 e1 1
to its ability of higher yield that could be due to its thermo-
dynamic advantages, better pathogenic destruction, as well as
the thermophilic process limits the growth of hydrogen con-
sumers like methanogens and homoacetogens [3e6]. Howev-
er, besides the above advantages, lower cell density remains
the drawback of fermentation at the thermophilic tempera-
ture [7e14].
Good transfer of nutrient into microorganisms can be
achieved via free suspended cell culture. However, at high
hydraulic pressure and low hydraulic retention time (HRT),
the microbial population in the bioreactor is hard to main-
tain as washout of the cells is often experienced [15,16].
Therefore, many research efforts have been made to increase
the cell density by using physical and biological immobili-
zation approaches onto support matric as an alternative
approach to the suspended cell systems [16e26]. There are
three types of immobilization cell systems in biohydrogen
production being introduced so far including surface
attachment [16,18,19,27,28], self-flocculation [22,29e33] and
gel entrapment [20,21,24,25] approaches. Of all the methods,
surface attachment approach becomes the most popular and
frequently sought by researchers in dark hydrogen fermen-
tation [34]. Attached cell immobilization is more advanta-
geous than suspended cell since the system is more
tolerance towards environment perturbation, process sta-
bility, reusable, higher biological activity. Moreover, this
system can operate at higher dilution rates without biomass
washout from the reactor [35,36].
In many types of support matric used in developing the
attached-biofilm, granular activated carbon (GAC) has been
well documented as a support matric in thermophilic
fermentation. GAC has high surface area, inertness, low
toxicity, and good mechanical properties, which are suitable
for high-temperature fermentation [37e40]. Its character of
highly porous structure also helps to sustain cell viability
which served excellently in microbial colonization, where the
fermentative bacteria can grow freely inside the porous
structure and on the surface of the carrier material, forming a
biofilm [41].
In our preliminary work, the focus on implementing the
advantages of GAC as support material in thermophilic
fermentation were done on glucose and xylose mixture, as the
mixture of carbon source that mostly composed in lignocel-
lulosic residue. As the first attempt in investigating the ability
of the microbial culture to self-attach and retain on the sur-
face of GAC, the efforts were made to create high cell density
that able to grow and produce good hydrogen production. The
studied parameters were subjected to the best GAC-sludge
ratio obtained in batch fermentation to form the attached-
biofilm, that in the end be used in a continuous hydrogen
production.
Material and methods
Microorganism, the GAC carrier and medium conditions
The source of microorganism was obtained from the sludge
pit of palm oil mill effluent (POME) located at Sime Darby
Plantation, West Oill Mill, Pulau Carey, Selangor, Malaysia.
Please cite this article in press as: Jamali NS, et al., Biofilm formation on granular activated carbon in xylose and glucose mixture for
thermophilic biohydrogen production, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.05.092
The sludge containing mixed culture was subjected to heat
treatment process at 80
C for 60 min to inhibit the growth of
methanogenic population. The granular activated carbon
(GAC), made from coconut shell, was purchased from Con-
cepts Ecotech Sdn Bhd, Malaysia. Initially, the GAC were
sieved to obtain a particle size of 2e3 mm as shown in Fig. 1.
The characteristics of the GAC were summarized in Table 1.
The prepared synthetic nutrient contains a mixture of
glucose and xylose (as carbon source); and other components
that prepared in deionized water such as NH4Cl 1 g l1, NaCl
2 g l1, MgCl2.6H2O 0.5 g l1, CaCl2.2H2O 0.05 g l1,
K2HPO4.3H2O 1.5 g l1, KH2PO4 0.75 g l1, NaHCO3 2.6 g l1 and
yeast extract 2 g l1. The medium composition was taken
from Wu et al. [42] with slight modification. The initial pH of
the culture medium was pH 6.0 and cultivated in a water bath
shaker at 60
C and 200 rpm for 48 h.
Selection of ratio of microbial sludge to GAC loading
The sludge added to the medium was chosen at constant
amount of 10% (v/v), which 800 ml of final culture volume
were prepared in a 1 l bioreactor. The amount of sludge sus-
pension (in ml) to clean GAC loading (in g) was varied as
shown in Table 2.
Biofilm development through cell acclimatization on GAC
The microbial sludge was acclimatized in a system operated at
sequencing batch and the experiments were run at HRT of 2
days in 800 ml working volume. The feed medium initial pH
was set at pH 6. The acclimatization were conducted in a
water bath shaker (model SW22, 230 V/50e60 Hz) at 200 rpm
and 60
C. Sequencing batch operation was carried out by the
removal of 50% of culture medium and replacing with new
medium after each cycle. The process cycle was continued
until hydrogen production rate achieved consistently. The
samples of fermentation products were taken at each cycle
and sent for analysis.
Characterization of the biofilm
The cell biomass adhered on the GAC were measured to
quantify volatile suspended solid (VSS) according to the
standard protocol by APHA [43]. The density of the GAC was
Fig. 1 e Granular activated carbon with particle size of
2e3 mm as microbial support carrier.
 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 x x x ( 2 0 1 6 ) 1 e1 1
Table 1 e Characteristics of GAC.
Surface area (BET-m2/g) 1000e1150
Pore Volume (cm3/g) 0.44
Moisture Content (%) <5%
Ash Content (%) <3%
Table 2 e Ratio of mixed culture sludge to GAC used in
fermentation.
Samples labeling Ratio (sludge: GAC) v:w
A (control) 1: 0.0 (without GAC)
B 1: 0.5
C 1: 1.0
D 1: 2.0
E 1: 3.0
F 1: 4.0
calculated by measuring the weight of the GAC (g) over the
occupied volume (l). Before the weight of clean GAC (g) was
measured, the GAC was subjected to determination of VSS to
ensure all bacteria grown on the surface was removed. The
ratio of the attached biomass present on GAC over clean GAC
was calculated as shown in Equation (1).
Weight of attached  biofilm g cell=g GAC
VSS of attached biomass on the GAC surfaceg=l
Density of clean on the GAC g=l
Analysis of H2 production
The biogas evolved from the fermentation was analyzed for
CO2 and H2 using gas chromatography (GC, model SRI 8600C,
USA). The system consisted of two detectors, helium ioniza-
tion detector (HID) and a thermal conductivity detector (TCD).
High purity helium gas (MOX 99.99%) was used as carrier gas
at flow rate 25 ml/min. The gas sample with the volume of
0.5 ml was injected using a gas-tight syringe into the GC at the
oven temperature of 43
C and 2.7 psi, followed by a ramping
of 30
C per minute and held for another 10 min once the
temperature has reached 220
C.
A modified Gompertz equation was used to correlate
experiment data in order to quantify the hydrogen production
and the correlation fitting was done using Matlab 7.9.0
(R2009b). The modified Gompertz equation is given in the
equation below [44]:
  
Rm $e
Ht Hm $exp  exp l  t 1
Hm
where, Ht is the cumulative hydrogen production (ml), Hm is
the maximum hydrogen production (mL), Rm is the maximum
hydrogen production rate (ml/h), e is Euler number, l is the lag
phase time (h) and t is the incubation time (h).
Analysis of sugar concentration
The filtered fermentation samples were analyzed for sugar
concentration using the HPLC system model Agilent 1200
HPLC (California, USA) with a REZEX ROA column
Please cite this article in press as: Jamali NS, et al., Biofilm formation on granular activated carbon in xylose and glucose mixture for
thermophilic biohydrogen production, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.05.092
3
(Phenomenex, USA) and equipped with a refractive index
detector (RID). The mobile phase of 5 mM H2SO4 was set at a
constant 0.6 ml/min flow rate at room temperature. The
glucose and xylose concentration were determine using
standard curves of pure glucose and xylose.
Yield and productivity
Hydrogen yield was expressed as moles of hydrogen per moles
of sugar consumed. While, for HPR, it was calculated based on
the percentage of hydrogen gas obtained in the biogas at every
feeding cycle and calculated in mmol H2 over the working
volume used (l) over the feeding time (h). In the case of batch
fermentation, the HPR was calculated based on the highest
hydrogen gas obtained in the exponential phase over
fermentation duration (h) in the microbial growth profile.
Microbial morphology
The biofilm formation on the GAC was observed using Field
Emission Scanning Electron Microscope (FESEM). The samples
were prepared using a Critical Point Dryer (Model Leica EM
CPD 300, Leica Microsystems, Germany), held for 1 h and
30 min before visualization with the FESEM was made. The
GAC attached biofilm was fixed with 4% glutaraldehyde for
12e24 h at 4
C. The samples were then washed with 0.1 M
phosphate buffer solution three times per 10 min each. The
samples were dehydrated with a series of alcohol dilutions
(1)
through 30, 50, 70, 80, 90, and 100% (w/w) alcohol. The dehy-
drated samples were then transferred to the CPD for 1 h and
were sputter-coated with platinum. The samples were then
being analyzed with FESEM under 5000 and 10,000 magni-
fication for comparisons.
Results and discussions
Effect of GAC-sludge support carriers on biohydrogen
production
The experiment was performed to find optimal ratio of added
mixed culture sludge (ml) to clean GAC (g) on biohydrogen
production. The results of hydrogen gas produced in each run
were plotted against time and were fitted using the modified
Gompertz [44] to determine the highest hydrogen production
(mL H2) and HPR (mmol H2/l.h), as shown in Fig. 2.
The results obtained from modified Gompertz equation
is given in Table 3. The highest hydrogen production was
(2) found in run D that representing the ratio of 1:2, which
corresponding to the Gompertz constants of Hm 1790 ml,
Rm 64.1 ml/h and l 13.3 h. The hydrogen production
rate (HPR) was 1.77 mmol H2/l.h, the highest hydrogen yield
was 2.0 mol H2/mol substrate consumed. The cells density
(in VSS) of the GAC attached-biofilm produced at the highest
value of 85.41 g/l, as compared to the density of the control
of suspended cell was only 4.92 g/l.
Meanwhile, the trends of hydrogen yield and the HPR ob-
tained (Table 3) were comparable among other runs that
contained GAC at different ratio of B, C, E and F. There is not
much different in term of hydrogen yield under different
4 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 x x x ( 2 0 1 6 ) 1 e1 1

1800 between the HPR, H2 yield and SMP in the different of GAC
1600 A (1: 0 - control) loading on hydrogen production were also compared in Fig. 3.
B (1:0.5) Two major acids of acetic acids (HAc) and butyric acids (HBu)
1400
C (1:1) were produced and ethanol was found in minor amount from
Hydrogen production (ml)

1200
D (1:2) the fermentation by-products. Highest TVFAs obtained in run
1000 E (1:3) D with 39.72 mM which consists mainly of 18.57 mM HAc and
800 F (1:4) 21.15 mM HBu with 0.89 ratio of TVFAs to SMP (Table 4). The
600 ratio in the range 0.83 0.89 suggests that the pathway of the
thermophilic hydrogen production from mixture of glucose
400
and xylose was of acetate type.
200
0 Development of self-attached cells on GAC for stable
0 10 20 30 40 50 60
Fermentation time (h)
biohydrogen production

Fig. 2 e Hydrogen production (ml H2) at different ratio of
GAC to sludge in an 800 mL of a 1 L modified lab scale
bioreactor in batch fermentation.
loading of GAC, that is when results of run B and C is
compared to D. It can be said that the porous structure and
irregular space present on the surface of the carrier provided
an adequate space for the sludge to grow well as in agreement
in the results obtained by Lutpi et al. [45]. The trends, however,
decrease when further increase of added GAC in run E and F
which shows that the microbial population of the sludge have
some limitation to grow in the surrounding of large amount of
support carrier in a batch fermentation.
In the control run using suspended culture, poor biomass
2.55 g/l VSS was obtained that corresponds to the lowest
hydrogen yield of 0.86 mol H2/mol sugar consumed and HPR of
0.5 mmol H2/l.h (Table 3). The work by Seol et al. [35], had
reported a fermentation of immobilization by cell attachment
onto carriers over free suspended cell culture was successfully
increased their biomass cultural density in the fermentation
medium and hence the study was in the agreement with this
results. The optimum ratio of 1:2 sludge to GAC, otherwise in
detail, remarks the use of GAC to occupy optimal porous space
by the microbes in order to promote a stable biological activity
for biohydrogen production.
Amounts of soluble microbial products (SMP), total volatile
fatty acids (TVFAs), ethanol (EtOH) solvents and the ratio of
TVFAs/SMP, associated to the fermentation at different
sludge:GAC values, are shown in Table 4. The correlation
The development of biofilm on the surface of GAC was con-
ducted to investigate the adherence of mixed culture sludge
towards the porosity and irregular surface of the GAC carriers.
The capability of the cells to self-attach on the surface of GAC
has been thoroughly evaluated. Optimum result obtained
from Section 3.1 at run D of sludge: GAC of 1:2 was used as
fixed parameter in this experiment which subjected to 20% w/
v of GAC as microbial support carrier and 10% v/v sludge
suspension in 800 mL working volume. The results of
hydrogen productions (mL H2), HPR (mmol H2/l.h), and
hydrogen yield (mol H2/mol sugars consumed) was plotted
over fermentation time (day) as shown in Fig. 4(a, b).
It can be clearly seen at the first day of the cycle, 51.4% of
hydrogen gas evolved from 4000 ml total biogas which
equivalent to 2056 ml H2 with almost five-fold the working
volume of fermentation. The HPR obtained was highest at
4.4 mmol H2/l.h. The biogas production however was fluctu-
ated over the fermentation period and the hydrogen gas began
to appear consistent at day 40 towards the end of 60 days
operation (Fig. 4a, 4b). The stable hydrogen production
reached an average of 44.2 1.7% H2 gas with HPR of
2.4 0.1 mmol H2/l.h (equivalent to1.4 0.1 l H2/l.d) and H2
yield of 1.17 mol H2/mol of total sugar consumed through the
acclimatization of sequencing feeding system with 2 days of
HRT (Fig. 4a, 4b).
Soluble metabolites formation and COD balance
It was found that major soluble metabolites that have been
generated were HAc, HBu and EtOH. However, propionate

Table 3 e Hydrogen productivity obtained from GAC loading to sludge ratio in batch fermentation.
Samples (sludge: GAC) v/w H2 Modified Gomertz equation
parameter values for H2
production (per 800 ml
working volume)
Yield HPR Hm Rm l
mol H2/mol sugar consumed mmol H2/l.h ml ml/h h
A (1:0) 0.86 0.5 419 13.6 12.8
B (1:0.5) 1.63 1.5 1513 43.0 10.4
C (1:1) 1.59 1.6 1628 46.0 12.4
D (1:2) 1.77 2.0 1790 64.1 13.3
E (1:3) 1.30 1.9 1457 58.1 9.9
F (1:4) 0.90 1.4 1140 41.0 8.9

Please cite this article in press as: Jamali NS, et al., Biofilm formation on granular activated carbon in xylose and glucose mixture for
thermophilic biohydrogen production, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.05.092
 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 x x x ( 2 0 1 6 ) 1 e1 1 5

Table 4 e Summary of liquid metabolites at various GAC loading.

Samples (sludge:GAC) v/w HAc HBu EtOH SMP TVFAs TVFAs/SMP
mM mM mM mM mM
A (1:0) 3.08 3.61 1.41 8.10 6.69 0.83
B (1:0.5) 11.57 11.21 4.22 27.00 22.78 0.84
C (1:1) 13.24 15.27 4.53 33.03 28.50 0.86
D (1:2) 18.57 21.15 4.78 44.51 39.72 0.89
E (1:3) 10.99 22.19 5.17 38.35 33.17 0.87
F (1:4) 19.06 23.10 5.10 47.25 42.16 0.89

TVFAs (total volatile fatty acids) HAc HBu; SMP (soluble microbial products) TVFAs EtOH.

Yield mmol H2/l.h TVFAs/SMP

2.5 1
0.9

Yield (mol H2 / mol sugars)

2.0 0.8
HPR (mmol H2 /l.h)

0.7
1.5 0.6
0.5
1.0 0.4
0.3
0.5 0.2
0.1
0.0 0
A B C D E F
GAC loading

Fig. 3 e Correlation of hydrogen production rate (HPR, mmol H2/l.h), yield (mol H2/mol substrate consumed) and soluble
microbials products (SMP) in different GAC loading.

(HPr) contributed in negligible amounts. The acclimatization
results were dominated with 23.1 2.8 mM of HAc and
22.5 3.5 mM of HBu in 1:1 ratio. TVFAs obtained were
45.5 mM and SMPs (TVFAs EtOH) were 49.8 mM as
4.3 0.7 mM of EtOH as the main alcohol was produced in
minor amount. As reported by Mohan et al. [46], high TVFAs
value was attributed to the desirable microenvironment for
acidogenic bacteria, hence high hydrogen productivity. The
close value 0.51 HAc/TVFAs and 0.49 HBu/TVFAs indicated the
dominance of one-to-one production of acetate to butyrate as
the fermentation by-product. Meanwhile the TVFAs/SMP ob-
tained was 0.91, owing to both acids as the main SMP and
suggests that the pathway of the thermophilic hydrogen
production from the mixed sugar solution of glucose and
xylose was of an acetate-butyrate type. The performance of
hydrogen production is usually monitored collectively with
the ratio of HAc/HBu and TVFAs. The HAc/HBu ratio of 1.03
has been applied for dark fermentative hydrogen production.
The concentration of TVFAs and their relative proportions
were successfully used as indicators of this anaerobic
hydrogen production [47].
The performance of the GAC attached-biofilm in terms of
chemical oxygen demand (COD) removal efficiency has been
analyzed which initially the substrate contain 55.03 0.29 g
COD/l in synthetic media fermentation. The COD reading of
the effluent was consistent 11.90 1.16 g COD/l from day 40
towards the end with COD removal efficiency of 78.4% with
32.3 ml H2/g of COD removal. Percentage of hydrogen gas
present in the biogas to the percentage of COD removal was
0.56 which was almost similar when compared in term of per
percentage of sugar consumption of 0.53.
Biomass of biofilm formation
The cells biomass suspended in the liquid samples at the
effluent and the attached cells as biofilm on the surface of the
GAC were examined after consistent HPR was obtained. The
microbial cell weight (VSS) on the GAC analyzed at day 40
towards the end was 133.85 0.25 g GAC attached-biofilm/l,
with 5.74 0.09 g cell/l suspended in the medium which
represented the total biomass accumulated in the system of
139.59 0.54 g cell/l. The weight ratio of biofilm attached cell
presented on the GAC over the clean GAC as the blank was
examined consistent at 1.31 0.01 g of GAC attached-biofilm/g
of clean GAC.
From the above result, it shows that the formation of bio-
film on the surface of the GAC had successfully developed.
The attachment formed biofilm helped sustaining cell
viability and which would prevent the cells washout from the
system, thereby enhanced culture density. The surface
porosity of the GAC eased the sludge to attach on the carrier
surface and forming a stable biofilm [45], likewise, a consistent
biohydrogen production was also obtained in the system. The
results also illustrated that the acclimatization process has

Please cite this article in press as: Jamali NS, et al., Biofilm formation on granular activated carbon in xylose and glucose mixture for
thermophilic biohydrogen production, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.05.092
6 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 x x x ( 2 0 1 6 ) 1 e1 1

a)
4500
ml biogas

Biogas and H2 gas production (ml)

4000
ml hydrogen
3500
3000
2500
2000
1500
1000
500
0
0 10 20 30 40 50 60
Fermentation time (day)

b)
5.0 mmol H2/l.h
4.5 l H2 / l.d
mol H2 / mol total sugar
H2 yield (mol H2/mol sugar)

4.0
HPR (mmol H2/l.h ; l H2/l.d),

3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0 10 20 30 40 50 60
Fermentation time (day)

Fig. 4 e (a)- Biogas and hydrogen gas production (ml); and (b) e HPR (mmol H2/l.h and l H2/l.d) together with hydrogen yield
(mol H2/mol of total sugar consumed), over fermentation time (day) by thermophilic mixed culture sludge with 2 days HRT
in sequencing batch feeding system.

advocated the cell density to adhere on the GAC surface, carrier for the sludge bacteria has been observed before and
thereby provided consistent and stable hydrogen production after acclimatization. Fig. 5(a, b) represents clean GAC; 5 (c, d)
at thermophilic condition as in agreement with Zhang et al., are the FESEM images of attached cell on GAC after 60-day
[46]. This is due to the mechanical stability of the biofilms operation when stable hydrogen production was achieved;
produced on the activated carbon, which have a high ten- and Fig. 5(e, f) exhibits the images of suspended cell culture
dency in binding capacity which mostly for organic matter, under 5,000 and 10,000 magnification respectively.
and therefore provide an environment that is rich in nutrients A relatively uniform biofilm was successfully found
and hence promoting the microbial adhesion [47]. The attached onto GAC as illustrated in Fig. 5 (c, d). The bacteria
immobilization technologies are mainly based on granulation colonization with rod-shaped were dominated on the biofilm
process or biofilm attachment process [48], which in this and fully covered on the porous surface of GAC. A closer view
experiment, biofilm attachment method has been conducted. of 10,000 magnification in Fig. 5 (d) illustrates the attachment
Biofilm-based system has been widely explored in the of microbial sludge was successfully developed and filled the
hydrogen production as immobilization cell systems as they cavities of GAC. The same attachment behavior was also
enhance production rate and population dynamics [49]. observed by Lutpi et al. [45], in their work using synthetic
media containing sucrose. It is clearly noticeable that several
Microbial FESEM images observation layers of cell had adhered on the porous surface of GAC,
forming a stable biofilm that is believed as a result of the cell
Mixed cultures of POME were cultivated during acclimatiza- growth on glucose and xylose during the fermentation.
tion and observed under Field Emission Scanning Electron Among the reviewed studies by Barca et al., most of biofilm
Microscope (FESEM). Single GAC particle acted as support formation performed through the immobilization approach

Please cite this article in press as: Jamali NS, et al., Biofilm formation on granular activated carbon in xylose and glucose mixture for
thermophilic biohydrogen production, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.05.092
 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 x x x ( 2 0 1 6 ) 1 e1 1 7

Fig. 5 e FESEM images of (a,b) clean GAC, (c,d) attached cell on micro-pores GAC surface and (e,f) suspended cell in the
fermentation medium at 5000 magnification and 10,000 magnification respectively.

were using synthetic solutions [41] that was prepared with
distilled water and simple sugars (glucose and sucrose) as the
main sugar carbon source, with some addition of nutrients (P
and N) and micronutrients such as Fe, Ca, Cu, Mg, Mn, Zn and
others which are usually required for bacterial growth and
activity [48,49]. Therefore, it is evident from this study that
consistent HPR observed during repeated batch cultivation
with GAC could facilitate the attachment of more bacterial
cells on the GAC surface.
Microorganism characterization
The microbial culture obtained in this experiment was
subjected to isolation process and consequently analyzed
for bacterial species barcoding and polymerase chain
reaction (PCR). Two-isolated microorganisms were identified
dominated in the fermentation medium. The DNA of the
isolates were sequenced and analyzed through GenBank se-
quences using basic local alignment search tools (BLAST). It
was found that both isolates were predominant species of
Bacillus (Table 5; Fig. 6) where 99% were identical to Bacillus
coagulans and Bacillus smithii (Table 5).
Nakamura et al. [50], and Grady et al. [51], discovered in
their works that B. coagulans and B. smithii can work very well
in thermophilic condition at operating temperature of
50e60
C with optimum pH around 5.5e6.5, and had cultivated
the hydrogen producer from waste. Their work was also
supported by Patel et al., [52] when his group showed that B.
coagulans 36D1 can work at optimum pH of 5.5 at temperature
50
C [52]. Works done by Kotay and Das had also determined
that the soluble microbial products of acetic acid, butyric acid
and ethanol as the primary metabolites were produced by the
B. coagulans IIT-BT SI, which coincides with the fermentation
products found in this work [53].

Please cite this article in press as: Jamali NS, et al., Biofilm formation on granular activated carbon in xylose and glucose mixture for
thermophilic biohydrogen production, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.05.092
8 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 x x x ( 2 0 1 6 ) 1 e1 1

Table 5 e Sequences producing significant alignments from isolated sample culture by BLAST.
Samples Description Max score Total score Identical
1 Bacillus coagulans 36D1, complete genome 2665 26438 99%
Bacillus shackletonii strain LMG 18435 super19, whole genome shotgun 2353 9561 96%
sequence
Bacillus shackletonii strain LMG 18435 scaffold8, whole genome shotgun 2348 2348 96%
sequence
Bacillus shackletonii strain LMG 18435 super11, whole genome shotgun 2342 7028 96%
sequence
Bacillus methanolicus MGA3, complete genome 2259 20253 95%
2 Bacillus smithii strain DSM 4216, complete genome 2612 28735 99%
Bacillus shackletonii strain LMG 18435 super11, whole genome shotgun 2322 6944 96%
sequence
Bacillus shackletonii strain LMG 18435 super19, whole genome shotgun 2316 9439 96%
sequence
Bacillus shackletonii strain LMG 18435 scaffold8, whole genome shotgun 2316 2316 96%
sequence
Bacillus thermotolerans strain SGZ-8 Contig10, whole genome shotgun 2266 2266 95%
sequence

Fig. 6 e Phylogenetic Tree diagram of isolated samples, yellow highlighted of a) sample 1; b) sample 2, shows Bacillus is the
predominant species in the mixed culture sludge.(For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)

Table 6 summarizes the comparison of similar study on et al., [54]. As shown in Table 6, the highest H2 yield obtained
hydrogen production from other researchers using various by this group was found to be 2.7 mol H2/mol hexose, which
type of support carriers at thermophilic fermentation. Close was slightly comparable to our study (at 1.8 mol H2/mol total
related comparison to the results was obtained from Lutpi sugar consumed). Other study that used support carrier done

Please cite this article in press as: Jamali NS, et al., Biofilm formation on granular activated carbon in xylose and glucose mixture for
thermophilic biohydrogen production, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.05.092
 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 x x x ( 2 0 1 6 ) 1 e1 1 9

Table 6 e Comparisons study on the hydrogen production from various types of microbial support carriers at thermophilic
fermentation.
Inoculum Support carrier Operational condition H2 yield Ref
Substrate Temp pH (mol H2/mol
glucose)
Mixed cultures sludge in CSTR Methanogenic granule Glucose 55 5 1.2 [56]
Mixed cultures sludge in CSTR Methanogenic granule Starch 55 4.8 1.68 [56]
Mixed culture sludge H2 granule Glucose 70 7.2 2.47 [57]
Mixed culture sludge H2 granule Glucose 70 7.1 1.14 [57]
Mixed culture sludge H2 granule Glucose 70 7.15 1.87 [57]
Household solid waste sludge Plastic carrier Glucose 70 NA 0.69 [58]
Heat pretreated sludge GAC Sucrose 60 5.5 2.7 [54]
Heat pretreated sludge GAC Sucrose 60 5.5 2.8 [45]
Thermoanaerobacterium species Calcium alginate Glucose 60 7 1.9 [59]
Thermoanaerobacterium Methanogenic granule Sucrose 60 5.5 1.76 [10]
thermosaccharolyticum PSU-2
Mixed cultures sludge in CSTR Fibrous polymeric material Glucose 55 5.5 1.21 [38]
Clostridium species Ceramic ring Sucrose 55 6.4 2.5 [60]
Clostridium species Pumice stone Sucrose 55 6.4 2 [60]
Heat and acid pretreated sludge Stainless steel scrubber Acid hydrolyzed wheat starch 55 5.5e6 1.15 [55]
Heat and acid pretreated sludge Volcanic stone Acid hydrolyzed wheat starch 55 5.5e6 1.44 [55]
Heat and acid pretreated sludge Tulle Acid hydrolyzed wheat starch 55 5.5e6 1.64 [55]
Heat and acid pretreated sludge Polyester fiber Acid hydrolyzed wheat starch 55 5.5e6 1.96 [55]
Heat and acid pretreated sludge Loofah sponge Acid hydrolyzed wheat starch 55 5.5e6 1.32 [55]
Heat and acid pretreated sludge Plastic bath sponge Acid hydrolyzed wheat starch 55 5.5e6 1.53 [55]
Heat and acid pretreated sludge Sea sponge Acid hydrolyzed wheat starch 55 5.5e6 0.85 [55]
Heat and acid pretreated sludge Aquarium biological sponge Acid hydrolyzed wheat starch 55 5.5e6 1.72 [55]
Heat pretreated sludge GAC Mixture of glucose and xylose 60 6 1.77 This study

by Gokfiliz and Karapinar [55] on thermophilic hydrogen Zero Waste Technology, Trust Area Biohydrogen (KK-2015-
fermentation at 55
C from acid hydrolyzed wheat starch, 002). This project also received funding from Research Uni-
achieved maximum of 1.97 mol H2/mol hexose using polyester versity Grant (DIP-2014-002).
fiber [55]. This study had contributed additional information
in developing the attached-biofilm on GAC for thermophilic
references
biohydrogen.

[1] Ozmihci S, Kargi F, Cakir A. Thermophilic dark fermentation

Conclusion
This work had successfully developed GAC attached-biofilm
fermentation system from glucose and xylose fermentation
using thermophilic mixed cultures. It was found that GAC
loading of 20% gave the highest amount of the cell attached on
the surface of GAC. The attached-biofilm was obtained by
culture acclimatization in sequencing batch mode on 2 day
HRT until stable biohydrogen production was obtained. The
results shows that the attached-biofilm enabled stability in
hydrogen production after 40 days with consistent HPR of
2.4 mmol H2/l.h, and H2 yield of 1.17 mol H2/mol sugar
consumed. Taken together, all key findings emphasized that
the acclimatization of cell with GAC as the carrier material
reassures to obtain consistent HPR and enhance cultural
density when dealing with synthetic wastewater at thermo-
philic hydrogen production.
Acknowledgment
The authors wish to acknowledge the profound financial
support from Sime Darby Plantation, Sdn Bhd under project
of acid hydrolyzed waste ground wheat for hydrogen gas
production. Int J Hydrogen Energy 2011;36:2111e7.
[2] Kapdan IK, Kargi F, Oztekin R, Argun H. Bio-hydrogen
production from acid hydrolyzed wheat starch by photo-
fermentation using different Rhodobacter sp. Int J Hydrogen
Energy 2009;34:2201e7.
[3] Das D, Vezirolu TN. Hydrogen production by biological
processes: a survey of literature. Int J Hydrogen Energy
2001;26:13e28.
C. Progress in bioethanol processing.
[4] Balat M, Balat H, Oz
Prog Energy Combust Sci 2008;34:551e73.
[5] Nielsen AT, Amandusson H, Bjorklund R, Dannetun H,
Ejlertsson J, Ekedahl L-G, et al. Hydrogen production from
organic waste. Int J Hydrogen Energy 2001;26:547e50.
[6] Zinder SH. Conversion of acetic acid to methane by
thermophiles. FEMS Microbiol Lett 1990;75:125e37.
[7] O-Thong S, Prasertsan P, Intrasungkha N, Dhamwichukorn S,
Birkeland N-K. Improvement of biohydrogen production and
treatment efficiency on palm oil mill effluent with nutrient
supplementation at thermophilic condition using an
anaerobic sequencing batch reactor. Enzyme Microb Technol
2007;41:583e90.
[8] O-Thong S, Prasertsan P, Intrasungkha N, Dhamwichukorn S,
Birkeland N-K. Optimization of simultaneous thermophilic
fermentative hydrogen production and COD reduction from
palm oil mill effluent by Thermoanaerobacterium-rich
sludge. Int J Hydrogen Energy 2008;33:1221e31.

Please cite this article in press as: Jamali NS, et al., Biofilm formation on granular activated carbon in xylose and glucose mixture for
thermophilic biohydrogen production, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.05.092
10 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 x x x ( 2 0 1 6 ) 1 e1 1
[9] Luo G, Xie L, Zou Z, Wang W, Zhou Q. Evaluation of
pretreatment methods on mixed inoculum for both batch
and continuous thermophilic biohydrogen production from
cassava stillage. Bioresour Technol 2010;101:959e64.
[10] O-Thong S, Prasertsan P, Karakashev D, Angelidaki I. High-
rate continuous hydrogen production by
thermoanaerobacterium thermosaccharolyticum PSU-2
immobilized on heat-pretreated methanogenic granules. Int
J Hydrogen Energy 2008;33:6498e508.
[11] Koskinen PEP, Lay C-H, Beck SR, Tolvanen KES,
Kaksonen AH, Orlygsson J, et al. Bioprospecting thermophilic
microorganisms from icelandic hot springs for hydrogen and
ethanol production. Energy Fuels 2008;22:134e40.
[12] van Niel EW, Claassen PA, Stams AJ. Substrate and product
inhibition of hydrogen production by the extreme
thermophile, caldicellulosiruptor saccharolyticus.
Biotechnol Bioeng 2003;81:255e62.
[13] Liu D, Zeng RJ, Angelidaki I. Effects of pH and hydraulic
retention time on hydrogen production versus
methanogenesis during anaerobic fermentation of organic
household solid waste under extreme-thermophilic
temperature (70
C). Biotechnol Bioeng 2008;100:1108e14.
[14] Yokoyama H, Moriya N, Ohmori H, Waki M, Ogino A,
Tanaka Y. Community analysis of hydrogen-producing
extreme thermophilic anaerobic microflora enriched from
cow manure with five substrates. Appl Microbiol Biotechnol
2007;77:213e22.
[15] Chen C, Lin C, Chang J. Kinetics of hydrogen production with
continuous anaerobic cultures utilizing sucrose as the
limiting substrate. Appl Microbiol Biotechnol 2001;57:56e64.
[16] Oh YK, Kim SH, Kim MS, Park S. Thermophilic biohydrogen
production from glucose with trickling biofilter. Biotechnol
Bioeng 2004;88:690e8.
[17] Hawkes F, Dinsdale R, Hawkes D, Hussy I. Sustainable
fermentative hydrogen production: challenges for process
optimisation. Int J Hydrogen Energy 2002;27:1339e47.
[18] Chang J-S, Lee K-S, Lin P-J. Biohydrogen production with
fixed-bed bioreactors. Int J Hydrogen Energy
2002;27:1167e74.
[19] Kumar N, Das D. Continuous hydrogen production by
immobilized Enterobacter cloacae IIT-BT 08 using
lignocellulosic materials as solid matrices. Enzyme Microb
Technol 2001;29:280e7.
[20] YokoiH OT, Hirose J. Hydrogen production from tofu
wastewater by rhodbactersphaeroides Immobilized in agar
gels. Int J Ferment Bioeng 1995;80:571e4.
[21] Wu SY, Lin CN, Chang JS, Lee KS, Lin PJ. Microbial hydrogen
production with immobilized sewage sludge. Biotechnol Prog
2002;18:921e6.
[22] Rachman M, Nakashimada Y, Kakizono T, Nishio N.
Hydrogen production with high yield and high evolution rate
by self-flocculated cells of enterobacter aerogenes in a
packed-bed reactor. Appl Microbiol Biotechnol
1998;49:450e4.
[23] Palazzi E, Fabiano B, Perego P. Process development of
continuous hydrogen production by enterobacter
aerogenes in a packed column reactor. Bioprocess Eng
2000;22:205e13.
[24] Wu SY, Lin CN, Chang JS. Hydrogen production with
immobilized sewage sludge in three-phase fluidized-bed
bioreactors. Biotechnol Prog 2003;19:828e32.
[25] Wu S-Y, Lin C-N, Chang J-S, Chang J-S. Biohydrogen
production with anaerobic sludge immobilized by ethylene-
vinyl acetate copolymer. Int J Hydrogen Energy
2005;30:1375e81.
[26] Singh L, Wahid ZA, Siddiqui MF, Ahmad A, Rahim MHA,
Sakinah M. Biohydrogen production from palm oil mill
Please cite this article in press as: Jamali NS, et al., Biofilm formation on granular activated carbon in xylose and glucose mixture for
thermophilic biohydrogen production, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.05.092
effluent using immobilized clostridium butyricum EB6 in
polyethylene glycol. Process Biochem 2013;48:294e8.
[27] Lee K-S, Lo Y-S, Lo Y-C, Lin P-J, Chang J-S. H2 production with
anaerobic sludge using activated-carbon supported packed-
bed bioreactors. Biotechnol Lett 2003;25:133e8.
[28] Tanisho S, Ishiwata Y. Continuous hydrogen production
from molasses by fermentation using urethane foam
as a support of flocks. Int J Hydrogen Energy
1995;20:541e5.
[29] Kim JO, Kim YH, Ryu JY, Song BK, Kim IH, Yeom SH.
Immobilization methods for continuous hydrogen gas
production biofilm formation versus granulation. Process
Biochem 2005;40:1331e7.
[30] Fang HH, Liu H, Zhang T. Characterization of a hydrogen-
producing granular sludge. Biotechnol Bioeng 2002;78:44e52.
[31] Liu H, Fang H. Hydrogen production from wastewater by
acidogenic granular sludge. Water Sci Technol
2003;47:153e8.
[32] Lee K-S, Lo Y-S, Lo Y-C, Lin P-J, Chang J-S. Operation
strategies for biohydrogen production with a high-rate
anaerobic granular sludge bed bioreactor. Enzyme Microb
Technol 2004;35:605e12.
[33] Lee KS, Wu JF, Lo YS, Lo YC, Lin PJ, Chang JS. Anaerobic
hydrogen production with an efficient carrier-induced
granular sludge bed bioreactor. Biotechnol Bioeng
2004;87:648e57.
[34] Lin C-N, Wu S-Y, Chang J-S. Fermentative hydrogen
production with a draft tube fluidized bed reactor containing
silicone-gel-immobilized anaerobic sludge. Int J Hydrogen
Energy 2006;31:2200e10.
[35] Seol E, Manimaran A, Jang Y, Kim S, Oh Y-K, Park S.
Sustained hydrogen production from formate using
immobilized recombinant Escherichia coli SH5. Int J
Hydrogen Energy 2011;36:8681e6.
[36] Singh L, Siddiqui MF, Ahmad A, Rahim MHA, Sakinah M,
Wahid ZA. Biohydrogen production from palm oil mill
effluent using immobilized mixed culture. J Industrial Eng
Chem 2013;19:659e64.
[37] Leenen EJTM, Dos Santos VAP, Grolle KCF, Tramper J,
Wijffels R. Characteristics of and selection criteria for
support materials for cell immobilization in wastewater
treatment. Water Res 1996;30:2985e96.
[38] Ahn Y, Park E-J, Oh Y-K, Park S, Webster G, Weightman AJ.
Biofilm microbial community of a thermophilic trickling
biofilter used for continuous biohydrogen production. FEMS
Microbiol Lett 2005;249:31e8.
[39] Moteleb MA, Suidan MT, Kim J, Maloney SW. Pertubated
loading of a formaldehyde waste in an anaerobic granular
activated carbon fluidized bed reactor. Water Res
2002;36:3775e85.
[40] Maloney SW, Adrian NR, Hickey RF, Heine RL. Anaerobic
treatment of pinkwater in a fluidized bed reactor containing
GAC. J Hazard Mater 2002;92:77e88.
[41] Barca C, Soric A, Ranava D, Giudici-Orticoni M-T, Ferrasse J-
H. Anaerobic biofilm reactors for dark fermentative
hydrogen production from wastewater: a review. Bioresour
Technol 2015;185:386e98.
[42] Angelidaki I, Sanders W. Assessment of the anaerobic
biodegradability of macropollutants. Re/Views Environ Sci
Bio/Technology 2004;3:117e29.
[43] Association APH, Association AWW, Federation WPC,
Federation WE. Standard methods for the examination of
water and wastewater. American Public Health Association.;
1915.
[44] Chen W-H, Chen S-Y, Kumar Khanal S, Sung S. Kinetic study
of biological hydrogen production by anaerobic
fermentation. Int J Hydrogen Energy 2006;31:2170e8.
 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 x x x ( 2 0 1 6 ) 1 e1 1
[45] Lutpi NA, Jahim JM, Mumtaz T, Abdul PM, Nor MTM.
Physicochemical characteristics of attached biofilm on
granular activated carbon for thermophilic biohydrogen
production. RSC Adv 2015;5:19382e92.
[46] Mohan SV, Mohanakrishna G, Goud RK, Sarma PN.
Acidogenic fermentation of vegetable based market waste to
harness biohydrogen with simultaneous stabilization.
Bioresour Technol 2009;100:3061e8.
[47] Chu Y, Wei Y, Yuan X, Shi X. Bioconversion of wheat stalk to
hydrogen by dark fermentation: effect of different mixed
microflora on hydrogen yield and cellulose solubilisation.
Bioresour Technol 2011;102:3805e9.
[48] Hawkes FR, Hussy I, Kyazze G, Dinsdale R, Hawkes DL.
Continuous dark fermentative hydrogen production by
mesophilic microflora: principles and progress. Int J
Hydrogen Energy 2007;32:172e84.
[49] Li C, Fang HH. Fermentative hydrogen production from
wastewater and solid wastes by mixed cultures. Crit Rev
Environ Sci Technol 2007;37:1e39.
[50] Nakamura Lawrence K, B I, Claus Dieter. Taxonomic study of
bacillus coagulans hammer 1915 with a proposal for bacillus
smithii sp. nov. Int J Syst Evol Microbiol 1988;38:63e73.
[51] Grady JLCG. Bioconversion of waste biomass to useful
products. US Patent. 1998. US5821111A.
[52] Patel MA, Ou MS, Ingram LO, Shanmugam K. Simultaneous
saccharification and co-fermentation of crystalline cellulose
and sugar cane bagasse hemicellulose hydrolysate to lactate
by a thermotolerant acidophilic bacillus sp. Biotechnol Prog
2005;21:1453e60.
[53] Kotay SM, Das D. Microbial hydrogen production with
Bacillus coagulans IIT-BT S1 isolated from anaerobic sewage
sludge. Bioresour Technol 2007;98:1183e90.
Please cite this article in press as: Jamali NS, et al., Biofilm formation on granular activated carbon in xylose and glucose mixture for
thermophilic biohydrogen production, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.05.092
11
[54] Lutpi NA, Jahim JM, Mumtaz T, Harun S, Abdul PM. Batch and
continuous thermophilic hydrogen fermentation of sucrose
using anaerobic sludge from palm oil mill effluent via
immobilisation technique. Process Biochem
2016;51:297e307.
[55] Gokfiliz P, Karapinar I. The effect of support particle type on
thermophilic hydrogen production by immobilized batch
dark fermentation. Int J Hydrogen Energy 2016. http://
dx.doi.org/10.1016/j.ijhydene.2016.03.041.
[56] Akutsu Y, Lee D-Y, Chi Y-Z, Li Y-Y, Harada H, Yu H-Q.
Thermophilic fermentative hydrogen production from
starch-wastewater with bio-granules. Int J Hydrogen Energy
2009;34:5061e71.
[57] Kotsopoulos TA, Zeng RJ, Angelidaki I. Biohydrogen
production in granular up-flow anaerobic sludge blanket
(UASB) reactors with mixed cultures under hyper-
thermophilic temperature (70
C). Biotechnol Bioeng
2006;94:296e302.
[58] Zheng H, Zeng RJ, Angelidaki I. Biohydrogen production from
glucose in upflow biofilm reactors with plastic carriers under
extreme thermophilic conditions (70
C). Biotechnol Bioeng
2008;100:1034e8.
[59] Basile MA, Dipasquale L, Gambacorta A, Vella MF, Calarco A,
Cerruti P, et al. The effect of the surface charge of hydrogel
supports on thermophilic biohydrogen production. Bioresour
Technol 2010;101:4386e94.
[60] Keskin T, Akso yek E, Azbar N. Comparative analysis of
thermophilic immobilized biohydrogen production using
packed materials of ceramic ring and pumice stone. Int J
Hydrogen Energy 2011;36:15160e7.