ARTICLE IN PRESS

WAT E R R E S E A R C H 41 (2007) 2652– 2664

Available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/watres

Biohydrogen production from chemical wastewater

treatment in biofilm configured reactor operated in periodic
discontinuous batch mode by selectively enriched
anaerobic mixed consortia

S. Venkata Mohan, Y. Vijaya Bhaskar, P.N. Sarma

Indian Institute of Chemical Technology, Bioengineering and Environmental Centre, Tarnaka, Hyderabad 500007, India

ar t ic l e i n f o abs tra ct

Article history: Molecular hydrogen (H2) production with simultaneous wastewater treatment was studied
Received 1 August 2006 in biofilm configured periodic discontinuous/sequencing batch reactor using chemical
Received in revised form wastewater as substrate. Anaerobic mixed consortia was sequentially pretreated with
5 February 2007 repeated heat-shock (100 1C; 2 h) and acid (pH—3.0; 24 h) treatment procedures to
Accepted 12 February 2007 selectively enrich the H2 producing mixed consortia prior to inoculation of the reactor.
Available online 5 April 2007 The bioreactor was operated at mesophilic (room) temperature (2872 1C) under acidophilic
Keywords: conditions with a total cycle period of 24 h consisting of FILL (15 min), REACT (23 h), SETTLE
Biohydrogen (30 min) and DECANT (15 min) phases. Reactor was initially operated with synthetic
Mixed consortia wastewater (SW) at OLR of 4.8 kg COD/m3-day and subsequently operated using composite
Anaerobic fermentation chemical wastewater (CW) at OLR of 5.6 kg COD/m3-day by adjusting pH to 6.0 prior to
Selectively enrichment feeding to inhibit the methanogenic activity. H2 evolution rate differed significantly with
Biofilm reactor the nature of wastewater used as substrate [SW—volumetric H2 production rate—12.89 -
Periodic discontinuous batch mmol H2/m3-min and specific H2 production rate—0.0084 mmol H2/min-g CODL
process (0.026 mmol H2/min-g CODR); CW—volumetric H2 production rate—6.076 mmol H2/m3-min
Volatile fatty acids and specific H2 production rate—0.0089 mmol H2/min-g CODL (0.033 mmol H2/min-g CODR)].
Scanning electron microscopy (SEM) Relatively rapid progress towards higher H2 yield (2 h) was observed with SW compared to
Transmission electron microscopy the CW (10 h). Substrate (COD) reduction of 32.4% (substrate degradation rate
(TEM) (SDR)—1.55 kg COD/m3-day) and 26.7% (SDR-1.49 kg COD/m3-day) was observed with SW
and CW, respectively. The system showed rapid stabilization tendency (SW—37 days;
CW—40 days) with respect to H2 generation and COD reduction. H2 evolution showed
relatively good correlation with VFA concentration in the case of SW (R2-0.961) compared to
CW (R2-0.912). A surge in pH values from 5.87 to 4.23 (SW) and 5.93 to 4.62 (CW) was
observed during the cycle operation. Integration of biofilm configuration with periodic
discontinuous batch operation under the defined operating conditions showed potential to
influence the microbial system by selectively enriching the specific group of microflora
capable of producing H2.
& 2007 Elsevier Ltd. All rights reserved.

Corresponding author. Tel./fax: +91 40 27015744.

E-mail address: vmohan_s@yahoo.com (S. Venkata Mohan).
0043-1354/$ - see front matter & 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2007.02.015
 ARTICLE IN PRESS
WAT E R R E S E A R C H 4 1 (200 7) 265 2 – 266 4 2653

periodic discontinuous batch reactor (PDBR) using selectively

1. Introduction enriched mixed anaerobic consortia under acidophilic
conditions.
Nowadays, global energy requirements are mostly dependent
on fossil fuels, which eventually lead to foreseeable depletion
due to limited fossil energy resources (Aman, 1996; Das and
Veziroglu, 2001). In recent times a great deal of attention is 2. Experimental
being paid to the utilization of hydrogen (H2) as alternative
and eco-friendly fuel throughout the world. H2 is produced 2.1. Selective enrichment of H2 producing mixed
mainly from fossil fuels, biomass and water. About half of all microflora
the H2 gas currently produced is obtained from thermocata-
lytic and gasification processes using natural gas as a starting Anaerobic mixed microflora acquired from an operating
material, heavy oils and naphtha make up the next largest laboratory scale upflow anaerobic sludge blanket (UASB)
source, followed by coal, and only 4% is generated from water reactor treating CW for the past three years was used as
using electricity (Logan, 2004). These methods mainly con- parent inoculum. Prior to inoculation, dewatered sludge
sume fossil fuels as energy source and are considered to be acquired from UASB reactor was subjected to cyclic pretreat-
energy intensive and not always environmental friendly. ment sequences (four times) changing between heat-shock
Present utilization of H2 is equivalent to 3% of the energy (100 1C; 2 h) and acid [pH 3 adjusted with ortho-phosphoric
consumption and with a growth rate estimated at 5–10% acid (88%); 24 h] treatment to restrain the growth of metha-
year1 (Das and Veziroglu, 2001; Logan, 2004). nogenic bacteria (MB), at the same time to selectively enrich
Sustainably generating H2 by a method that is more the H2 producing acidogenic bacteria (AB). The resulting
economical and environmentally friendly than the natural enriched mixed culture was used as inoculum to startup the
gas requires advances in direct H2 generation (Logan, 2004). biofilm reactor.
Biological production of H2 is one of the alternative methods
where processes can be operated at ambient temperatures
and pressures and are less energy intensive and more 2.2. Wastewater composition
environmental friendly. Broadly, biological H2 production
processes can be classified as biophotolysis of water using Designed synthetic wastewater (SW) [(g/l) glucose—3.0,
algae and cyanobacteria, photodecomposition of organic NH4Cl—0.5, KH2PO4—0.25, K2HPO4—0.25, MgCl2.6H2O—0.3,
compounds by photosynthetic bacteria and fermentative H2 FeCl3—0.025, NiSO4—0.016, CoCl2—0.025, ZnCl2—0.0115,
production from organic compounds (Das and Veziroglu, CuCl2—0.0105, CaCl2—0.005 and MnCl2—0.015] and composite
2001; Hawkes et al., 2002; Logan, 2004). So far H2 production CW were used as substrates for H2 production (Table 1). The
by photosynthetic microorganisms was extensively studied CW was composite/combined CW collected from a common
(Fascetti et al., 1998; Das and Veziroglu, 2001), while H2 effluent treatment plant (CETP) in Hyderabad, India where
evolution by fermentation was treated with little attention wastewater received from about 100 chemical processing
(Logan, 2004). Comparatively, the fermentative evolution is industries was being treated. The wastewater was aggregated
more advantageous than photochemical evolution for mass from bulk drugs, chemical intermediates, dye and dye
production of H2 where various wastewaters can be used as intermediates, pharmaceuticals, pesticides and various che-
substrates. Of late, H2 production through anaerobic fermen- mical process units. Characteristically, the wastewater is
tation using wastewater as substrate has been attracting complex in nature due to its composite nature, low-biode-
considerable attention (Yu et al., 2002; Logan, 2004; Lin and gradability (biological oxygen demand (BOD)/chemical oxy-
Lay, 2004; Atif et al., 2005; Ginkel et al., 2005; Ginkel and gen demand (COD)  0.3) and high sulfate concentration
Logan, 2005; Fan et al., 2006; Gavala et al., 2006; Venkata (1750 mg/l).
Mohan et al., 2006a; Vijayaraghavan et al., 2006; Yang et al.,
2006). Exploitation of wastewater as substrate for H2 produc-
tion with simultaneous wastewater treatment is an attractive Table 1 – Characteristics of wastewaters used as feed
and effective way of tapping clean energy from renewable
resource in a sustainable approach. This provides dual Parameters SW CW
environmental benefits in the direction of wastewater reduc-
tion from raw material and reduction of pollution from pH 7.64 7.80
product (H2) utilization. However, the microbial conversion TDS (mg/l) 1350 11,000
Suspended solids (mg/l) — 920
of substrate to H2 by anaerobic fermentation is a complex
Oil and grease (mg/l) — 14
series of biochemical reactions manifested by diverse
COD (mg/l) 5840 9840
group of selective bacteria, which requires considerable BOD5 (mg/l) 3910 2950
optimization. Chlorides (mg/l) 184 5096
The study was therefore, an evaluation of anaerobic Sulfates (mg/l) 12 1750
biohydrogen production process using composite Phosphates (mg/l) 230 360
chemical wastewater (CW) as substrate. The investigation is Total nitrogen (TKN) (mg/l) 140 125

focused to study on the feasibility of H2 production in

SW, synthetic wastewater; CW, composite chemical wastewater.
conjugation with wastewater treatment in biofilm configured
 ARTICLE IN PRESS
2654 WAT E R R E S E A R C H 41 (2007) 2652– 2664

2.3. Reactor design and operation
Bench scale biofilm configuration anaerobic reactor was
designed and fabricated in the laboratory using ‘perplex’
material to have a designed working volume of 4 l, liquid
volume of 2 l and gas holding capacity of 0.35 l. Schematic
details of the bioreactor used in this study along with
photograph of the experimental setup are depicted in Fig. 1.
The reactor was fabricated using leak proof sealing along with
proper inlet and outlet arrangements. The bioreactor was
designed to operate in upflow mode (L/D ratio6) and was
filled with inert stone chips (0.02 cm  0.05 cm; void ratio 
0.54) as fixed bed packing material to support the growth of H2
producing mixed microflora. The reactor was operated in
periodic discontinuous batch (PDBR)/sequencing batch (SBR)
mode with a total cycle period (hydraulic retention time) of

b
T
P T
pH/ORP meter T
H2 monitoring facility

T
T H3PO4 /NaOH

P T

P T
Outlet Storage Tank Influent storage tank

Recirculation loop

Fig. 1 – (a) Photograph depicting the total experimental set up (biofilm reactor, peristaltic pump and H2 monitoring system);
(b) schematic details of the experimental set up (T, timer; P, peristaltic pump).
 ARTICLE IN PRESS
WAT E R R E S E A R C H
Table 2 – Details of reactor operation
Mode of reactor operation Periodic discontinuous
batch mode/sequencing
batch reactor
Reactor microenvironment Anaerobic
Hydraulic retention time (HRT) 24 h
Total (single) cycle period FILL—15 min
REACT—23 h
SETTLE—30 min
DECANT—15 min
Operating temperature 2872 1C
Feeding pH (influent) 6.0
Feed volume 1.5 l
Organic loading rate (OLR) 4.8 kg COD/m3-day (SW)
5.6 kg COD/m3-day (CW)
Recirculation rate (feed to 1:3
recirculation)
24 h consisting of 15 min of FILL, 23 h of REACT (anaerobic),
30 min of SETTLE and 15 min of DECANT phases (Table 2).
At the beginning of each cycle, immediately after withdrawal
(earlier sequence), a pre-defined volume (1.5 l) was fed
to the reactor during FILL phase and the reactor volume
was circulated with reactor outlet in closed loop at recircula-
tion rate (recirculation volume to feed volume ratio) of 3
during the REACT phase to achieve a homogeneous distribu-
tion of the substrate as well as uniform distribution of
requisite consortia along the reactor depth. Peristaltic
pump controlled by preprogrammed electronic timer (ETTS,
Germany) was used to regulate the FEED, recirculation, and
DECANT operations. The controller was programmed to
operate on a repeating 24 h cycle with a sub-program and
output dedicated to the operation of each controllable
element.
After inoculation, the reactor was initially operated with
synthetic feed (SW) as substrate to support the biofilm
formation on the packing medium and facilitate adaptation
at lower organic loading rate (OLR; 2.4 kg COD/m3-day) by
adjusting feed pH to 6. Constant COD removal and gas
production (75% variation) were considered as indicators
for successful formation of biofilm and subsequently the
reactor was operated at higher OLR (4.8 kg COD/m3-day) with
SW till stable performance was attained. The molecular H2
evolution was evaluated at operating OLRs of 4.8 kg of COD/
m3-day [SW (100%); COD—6.0 g/l] and 5.6 kg of COD/m3-day
[CW (25%) and SW (75%); COD—7.0 g/l]. The influent pH was
adjusted to 6.0, before feeding the wastewater using concen-
trated ortho-phosphoric acid (88%). Reactor was operated at
mesophilic (room) temperature (2872 1C).
2.4. Analysis
H2 gas generated during the bioreactor operation was
estimated using a microprocessor based pre-calibrated H2
sensor (electrochemical 3 electrode H2 sensor, FMK satellite
4 1 (200 7) 265 2 – 266 4 2655
4–20 mA version, ATMI GmbH Inc., Germany). The output
signal displays the percentage volume of H2 in the head space
of the bioreactor and the system was calibrated once in two
days using calibration cap provided with the instrument.
Sensor has a measuring range of 0.01–10% H2 with 5 s
response time in a temperature range of 20–80 1C. H2
monitoring was done under closed conditions to avoid
external environmental contamination. Alkalinity (total),
volatile suspended solids (VSS), pH, ORP, volatile fatty
acids (VFA), COD (closed refluxing method) and BOD5
were determined according to the standard methods (APHA,
1998). Oxidation–reduction potential (ORP) and pH values
were determined by a pH meter (Model 20, Denver
instruments Ltd.). The separation and quantitative determi-
nation of VFA was carried out by high performance liquid
chromatography (HPLC; Shimadzu LC10A) employing
optimized conditions (UV–VIS detector; C18 column—reverse
phase column—250  4.6 mm and 5 mm particle size; flow
rate—0.5 ml/h; wave length—210 nm; mobile phase—40% of
acetonitrile in 1 mN H2SO4 (pH 2.5—3.0); sample injec-
tion—20 ml).
The biofilm formed on the stones and dominant colonies
was subjected to scanning electron microscopy (SEM). Prior to
SEM (JOEL-JSM 5600) imaging the samples were transferred to
vials and fixed in glutaraldehyde (2.5%) in 0.05 M phosphate
buffer (pH 7.2) for 24 h at 4 1C and post fixed in aqueous
osmium tetroxide (2%) in same buffer for 2 h. After the post
fixation samples were dehydrated in series of graded alcohol
and dried. Then samples were mounted over the stubs with
double sided conductivity tape and a thin layer of platinum
metal was applied over the sample using an automated
sputter coater for about 2 min and then scanned in SEM at
various magnifications. For transmission electron microscopy
(TEM; Hitachi, H-7500) dehydrated samples were infiltrated
and embedded in Spurr’s resin. Both semithin (200–300 nm
thick) and ultra thin sections (50–70 nm thick) were cut with a
glass knife on a Leica Ultra cut (UCT-GA-D/E-1/00) microtome
and stained with toludine blue. Ultra thin sections were
mounted on grids and the sections were stained with
saturated aqueous uranyl acetate and counter stained with
lead citrate (4%) and then samples were observed under a
TEM at various magnifications.
H2 production rate (HPR) was calculated by estimating H2
evolution in the reactor between specified time intervals
using the following equation:
HPR ðmmol H2 =minÞ ¼ ½ðHX  HY Þ=ðtx  ty Þ, (1)
where, HX and HY represents, the H2 concentration (mmol) at
time X and Y, respectively, and tX and tY denotes time (min) at
X and Y, respectively. Substrate degradation rate
(SDR—kg COD/m3-day) was calculated to study the rate and
pattern of COD removal during the cycle operation according
to Eq. (2).
SDR ¼ f½ðCODO  CODT ÞXFR =Rv g, (2)
where, CODO and CODT represent COD (mg/l) at ‘O’ and ‘T’
times, respectively, FR represents feed rate (m3/day) and Rv
denotes reactor volume (m3).
 ARTICLE IN PRESS
2656 WAT E R R E S E A R C H 41 (2007) 2652– 2664

volumetric HPR of 6.08 mmol H2/m3-min and specific HPR of

3. Results 0.0089 mmol H2/min-g CODL (0.033 mmol H2/min-g CODR). H2
yield showed maximum value (0.0181 mmol H2/min) after
3.1. Biohydrogen production 600 min of the cycle operation and thereafter gradually
approached 0.009 mmol H2/min (0.0019 mmol H2/g CODL) at
After inoculation with the selectively enriched mixed con- the end of the cycle period. With CW the reactor recorded a
sortia, the biofilm reactor was initially operated with SW as cumulative yield of 13.125 mmol H2/day at the end of cycle
feed at OLR of 2.4 kg COD/m3-day by adjusting the influent period. It is apparent from the experimental data that, SW
feed pH to 6 for a period of 35 days. Subsequently, the reactor (2 h) showed comparatively high and rapid H2 yield compared
was shifted to higher OLR of 4.8 kg COD/m3-day with same to CW (10 h). This may be attributed to the complex and
wastewater and operated for 37 days. After achieving stable composite nature of the CW having low-biodegradability
performance the reactor was shifted to CW and operated at (BOD/COD0.3) which manifest slow down in the substrate
an OLR of 5.6 kg COD/m3-day until stable performance (40 metabolism leading to lower H2 yield. Relatively, substrate
days) was attained. The experimental data depicted the metabolism was easy in the case of SW, which is easily
feasibility of H2 production from the two types of substrates biodegradable (BOD/COD 0.67).
studied (Fig. 2). Fig. 3 shows the H2 production rate during
single cycle operation at stabilized performance for the
studied wastewaters. The H2 evolution rate differed signifi- 3.2. Wastewater treatment
cantly with the nature of wastewater used as substrate.
Experimental data (Figs. 2 and 3) illustrate the influence of Substrate degradation (as COD reduction) during the bior-
nature and composition of the substrate/feed used on the H2 eactor operation and cycle period are depicted in Fig. 4. A
production. In the case of simple substrate (SW) as feed, the steady decrease in the COD concentration was observed
reactor showed a maximum H2 yield of 0.042 mmol/min after with the function of cycle period irrespective of the substrate
2 h of cycle operation through the stable operation phase studied. Both SW and CW had participated as primary
(Fig. 3) and registered cumulative H2 yield of 27.89 mmol carbon source in the metabolic reactions involving molecular
H2/day at the end of the cycle period. This accounts for a H2 generation and this was evident from the reduction
volumetric HPR of 12.89 mmol H2/m3-min and specific HPR of in substrate (COD) with concomitant H2 evolution. The
0.0084 mmol H2/min-g CODL (0.026 mmol H2/min-g CODR). H2 substrate reduction started with 12.5% (SW) and 15.6%
production showed a gradual rise up to 120 min of the cycle (CW) during the initial stages and gradually approached
operation and thereafter the production reduced gradually a steady state condition after 30 days (SW) and 32 days
and approached 0.007 mmol H2/min (0.0014 mmol H2/g CODL) (CW) of the reactor operation (Fig. 4a). Reactor operation with
at the end of the cycle period. While in the case of complex SW registered 32.4% of COD removal efficiency at the end of
substrate (CW), maximum H2 yield of 0.0181 mmol/min was the cycle period accounting for SDR of 1.55 kg COD/m3-day.
observed after 10 h of the cycle operation accounting for a While in the case of CW, the substrate degradation was

40

OLR - 4.8 kg COD/m3-day (SW)

Hydrogen generation (mmol/day)

30

OLR - 5.6 kg COD/m3-day (CW)

20

10

0
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77
Time (days)

Fig. 2 – Variation in hydrogen production during the run of bioreactor.

 ARTICLE IN PRESS
WAT E R R E S E A R C H 4 1 (200 7) 265 2 – 266 4 2657

OLR 4.8

Hydrogen production rate (mmol/min)

0.04 OLR 5.6

0.03

0.02

0.01

0
1 2 4 6 8 10 12 16 20 24
Cycle period (h)

0.03
mmol/g COD/min
0.025 mmol/g COD reduced/min
Hydrogen (mmol)

0.02

0.015

0.01

0.005

0
1 2 4 6 8 10 12 16 20 24
Cycle period (hrs)

0.04
mmol/g COD/min
0.035
mmol/g COD reduced/min
0.03
Hydrogen (mmol)

0.025

0.02

0.015

0.01

0.005

0
1 2 4 6 8 10 12 16 20 24
Cycle period (hrs)

Fig. 3 – (a) Variation in hydrogen production during single cycle operation [H2 production rate; (b) specific H2 production rate
(4.8 kg COD/m3-day); (c) specific H2 production rate (5.6 kg COD/m3-day)].

comparatively on the lower side (COD removal effi-
ciency—26.7%) accounting for SDR of 1.49 kg COD/m3-day.
Comparatively good correlation was observed when substrate
degradation data was correlated with H2 production (R2-0.953)
in the case of SW compared to CW (R2-0.782). This is
indicative of the fact that the metabolic activity is critical
for effective H2 yield.
3.3. Bioprocess monitoring
Along with COD, parameters such as VFA (represented as the
total of all acids generated during acidogenic fermentation
step), pH, ORP and alkalinity (total) were also monitored
during the reactor operation to evaluate the bioprocess
mechanism during H2 production. Under acidophilic micro-
 ARTICLE IN PRESS
2658 WAT E R R E S E A R C H 41 (2007) 2652– 2664

OLR 4.8 kg COD/m3-day (SW) OLR 5.6 kg COD/m3-day (CW)

30
COD Removal Efficiency (%)

20

10

0
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77
Time (days)

40

30
COD Removal Efficiency (%)

20

10

0
1 4 8 12 16 20 24
Cycle period (h)

Fig. 4 – Variation of substrate (COD) removal [(a) during reactor operation; (b) during single cycle operation at OLR 4.8 kg COD/
m3-day (n) and 5.6 kg COD/m3-day (J)].

environment, H2 production is generally accompanied by acid
and solvent production due to acidogenic metabolism. Acidic
intermediates generation reflects changes in the metabolic
pathway of the microorganisms involved and provides
information, which can be used to improve the conditions
favorable for H2 production. VFA production was always
associated with conversion of organic fraction to acid
intermediates in the anaerobic microenvironment with the
help of specific group of bacteria (Cha and Noike, 1997;
Dinopoulou et al., 1998). Fig. 5 illustrates VFA produced during
the reactor operation. It is interesting to note that VFA
production varied consistently with the substrate composi-
tion. Relatively higher VFA concentrations were recorded with
SW as substrate. VFA concentration showed a steady increase
from 2993 to 8464 mg/l at the end of the cycle period. In the
case of CW, VFA production was slightly on the lower side
which approached 7470 mg/l at the end of the cycle period
from 4914 mg/l. With CW as substrate comparatively low yield
of VFA was observed which might be attributed to the
substrate complex conditions leading to substrate metabo-
lism inhibition. H2 evolution showed relatively good correla-
tion with VFA concentration in the case of SW (R2-0.961)
 ARTICLE IN PRESS
WAT E R R E S E A R C H 4 1 (200 7) 265 2 – 266 4 2659

8800 1150
VFA
Aklalinity
8400

Total alkalinity (mg/l)

1050
Total VFA (mg/l)

8000

OLR 4.8 kg COD/m3–day (SW)

7600 OLR 5.6 kg COD/m3–day (CW)
950

7200

6800 850
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77
Time (days)

9000 2500

8000

7000 2000

Total alkalinity (mg/l)

6000
Total VFA (mg/l)

1500
5000

4000
1000
3000

2000 500
1000 VFA
Alkalinity
0 0
0 1 2 4 6 8 10 12 16 20 24
Cycle period (h)

8000 2000

1600
6000
Total alkalinity (mg/l)
Total VFA (mg/l)

1200
4000

800

2000
400
VFA
Alkalinity
0 0
0 1 2 4 6 8 10 12 16 20 24
Cycle period (h)

Fig. 5 – Variation in VFA and alkalinity concentration [(a) during reactor operation; (b) OLR 4.8 kg COD/m3-day (single cycle);
(c) OLR 5.6 kg COD/m3-day (single cycle)].

compared to CW (R2-0.912). When substrate (COD) conversion
was correlated with VFA generated, a reasonably good
correlation was observed with both SW (R2-0.914) and CW
(R2-0.912). Conversion of organic substrate to fatty acids was
observed at relatively shorter cycle period (o12 h) with both
the types of wastewaters studied.
Samples during the course of reactor operation were also
analyzed for the composition of VFA in terms of acetic acid
(HAc), butyric acid (HBu), propionic acid (HPa) and ethanol
(HEt) by chromatography to have a better understanding H2
producing metabolic pathway. The distribution of metabolites
formed during H2 fermentation was often crucial in assessing
 ARTICLE IN PRESS
2660 WAT E R R E S E A R C H 41 (2007) 2652– 2664

the efficiency of H2-producing cultures (Cha and Noike, 1997;
Dinopoulou et al., 1998; Lee et al., 1999). Chromatography data
showed the presence of butyric acid only in the case of SW.
While in the case of CW, higher concentrations of acetic acid
along with relatively lower concentrations of butyric acid
were observed. Formation of propionate and ethanol were not
observed during reactor operation with both the wastewaters
studied. HAc was a major metabolite in the H2-producing
bacterial population with CW as substrate. The butyrate
concentration was observed after 4 h of cycle operation and
the HAc/HBu ratio showed a gradual decline signifying an
increasing concentration of butyrate. The VFA composition

5.2 160
pH
ORP

5
140

4.8

ORP (mV)
pH

OLR 4.8 kg COD/m3-day (SW) 120

4.6

100
4.4

OLR 5.6 kg COD/m3–day (CW)

4.2 80
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77
Time (days)

7 200

6
160

ORP (mV)
5
120
pH

4
80
3

40
2 pH
ORP
1 0
0 1 2 4 6 8 10 12 16 20 24
Cycle period (h)

7 160

6
120
ORP (mV)

5
pH

4 80

3
40
2 pH
ORP
1 0
0 1 2 4 6 8 10 12 16 20 24
Cycle period (h)

Fig. 6 – Variation in pH and ORP [(a) during reactor operation; (b) OLR 4.8 kg COD/m3-day (single cycle); (c) OLR 5.6 kg COD/m3-
day (single cycle)].
 ARTICLE IN PRESS
WAT E R R E S E A R C H
data suggests dominance of acid-forming metabolic flow
associated with the acidogenesis instead of solventogenesis,
which was considered as optimum environment for effective
H2 generation. It was observed that high H2 yields were
associated with a mixture of acetate and butyrate fermenta-
tion products, and low H2 yields were with propionate and
reduced end products (alcohols, lactic acid) (Hawkes et al.,
2002).
Generally pH drop in anaerobic microenvironment was
considered as an index of volatile acid generation in alliance
with the existing buffering capacity (total alkalinity) of the
system. Drop in pH showed a distinct trend towards
acidification with both the wastewaters studied (Fig. 6).
During reactor operation, due to acid production a gradual
reduction in the buffering capacity (total alkalinity) was
observed which resulted in a concomitant decline in the
system pH. With the exhaustion of cycle period a steady
decrease in pH values was observed from 5.87 to 4.23 (SW)
and 5.93 to 4.62 (CW). Higher pH drop visualized rapid H2
production with concomitant increase in VFA production.
Comparatively low pH surge along with low concentration of
VFA observed with CW might be reasoned to the metabolic
inhibition. When H2 yield was correlated with pH drop, SW
(R2-0.898) showed reasonably good correlation than the CW
(R2-0.5528). pH values showed relatively good correlation with
VFA concentration [SW (R2-0.920); CW (R2-0.739)] than the
buffering capacity i.e. alkalinity [SW (R2-0.884); CW (R2-0.727)].
Fig. 7 – SEM images of: (a) biofilm (  2500); (b) isolated strain (  1800); (c) isolated strain (  2200).
4 1 (200 7) 265 2 – 266 4 2661
It is reported that, optimum pH for the growth of MB was
between 6.0 and 7.5, while AB functions well below 6 pH (Liu
et al., 1985; Boopathy and Daniels, 1991; Ginkel et al., 2001;
Fang and Liu, 2002). Also by maintaining the operating pH
around 6 (optimum being pH 5.5–6.0) compared to a near
neutral pH the conversion efficiency (of H2 production) was
increased (Ginkel et al., 2001; Ginkel and Logan, 2005, Venkata
Mohan et al., 2006a, b). The pH range of 5.5–6 was considered
to be ideal to avoid both methanogenesis and solventogenesis
(Rogers, 1984; Fang and Liu, 2002) and could be considered
optimum pH range for effective H2 generation. The observed
pH drop during H2 production was considered to be favorable
microenvironment for effective H2 yield by inhibiting the
methanogenic group of bacteria. The pH drop from 6 to 5 was
considered to be the congenial pH range for the functioning of
the AB and at the same time for inhibiting the activity of MB.
Concomitant values of ORP with pH were observed in all the
experimental variations studied.
3.4. Microscopic studies
SEM (  2500; Fig. 7a) of biofilm acquired from the stone chips
(72nd day after startup) of bioreactor showed images of
slightly bent, scattered and short chain rods (predominant;
streptobacillus; 10 mm in length) along with relatively low
frequency of cocci-shaped bacteria. Fluorescence was also
visualized from the images. SEM images of isolated bacteria
 ARTICLE IN PRESS
2662 WAT E R R E S E A R C H
Fig. 8 – TEM images of isolated strain (a) 1.7 lm; (b) 333 nm.
strains which were profusely/dominantly grown in selective
medium (Wilkins-chalgren anaerobic broth; Himedia) (Fig. 7b
and c) visualized slightly bent, rod shaped, thick fluorescent
capsid bacteria with (10 mm in length). Images of both the
single strain and mixed consortia showed comparatively
similar morphology demonstrating the presence of related
group of bacteria proliferated in the bioreactor producing H2.
TEM image (Fig. 8a) showed oval centralized spore formation
with sub-terminal endospore development in rod-shaped
bacteria (1–7 mm in length). Terminal bulging with granulose
accumulation was not observed. Flagellum attached subapi-
cally to the bacterium (two times length of the cell body) was
observed. Vegetative cell surrounded by thick membrane
(peptidoglycan layer) with two layers (inner and outer
forming fibrillar capsule structure) on the cell surface was
also visualized (Fig. 8b).
4. Discussion
It was evident that typical anaerobic mixed cultures could not
produce H2 as it is an intermediate for methane formation,
and was rapidly consumed by methane-producing bacteria
(Sparling et al., 1997; Nandi and Sengupta, 1998). Most
effective ways to enhance H2 production from the anaerobic
culture is to restrict or terminate the methanogenesis process
41 (2007) 2652– 2664
by allowing H2 to become an end product in the metabolic
flow. In this study two pretreatment techniques were adopted
to limit methanogenic group in the mixed cultures. The
experimental data illustrated the efficacy of the pretreatment
methods [heat-shock (100 1C; 2 h) and acid (pH—3.0; 24 h)]
applied on parent anaerobic mixed culture for H2 generation.
Both heat-shock and acid treatments facilitates removal of
non-spore forming MB groups from parent inoculum. It is also
evident from the microscopic studies that the selective
enrichment procedure used in this study resulted in enu-
meration of specific group of morphologically similar group of
bacteria. In literature, studies were reported pertaining to
different pretreatment methods employed on diverse type of
inoculum (Lin et al. 2000; Ginkel et al., 2001; Logan et al., 2002;
Oh et al., 2003; Ferchichi et al., 2005; Venkata Mohan et al.,
2006a, b). In addition, to sustain acidophilic microenviron-
ment during reactor operation, reactor was fed with substrate
adjusted to pH 6.0 to suppress the methanogenic activity.
Methane concentration below detectable limit was observed
during the operation, whereas the H2 concentration showed a
gradual build-up with time. Hence, it can be concluded that
methanogenic population in the anaerobic inoculum may be
inhibited/killed due to the pretreatments (heat-shock and
acid) preliminary applied on the inoculum and the persistent
acidophilic microenvironment maintained during the reactor
operation. Moreover, using mixed microbial cultures is
considered to be a practical, cost-effective and promising
approach to achieve H2 production in large scale.
Reactor configuration and its operation conditions are
important factors, which govern the performance of any
biological system and may also have considerable influence
on the H2 evolution. Biofilm configured systems are reported
to be well suited for the treatment of wastewater containing
poorly degradable compounds (Wilderer et al., 1993). Immo-
bilization of microflora on support medium as biofilm results
in high biomass hold up, which enables the process to be
operated significantly at higher liquid throughputs and OLR
(Chandrasekhara Rao et al., 2005; Venkata Mohan et al., 2005a,
2006c, 2007). Attached biofilm acts as buffer to reduce the
concentration of toxic chemicals during process operation
thereby providing advantage for the treatment low-biode-
gradable industrial wastewater containing recalcitrant com-
pounds (Bishop, 1997). Moreover, the biofilm configured
systems are more resistant to change in the process
parameters and are less energy intensive (Chaudhry and
Beg, 1998) and are particularly useful where high hydraulic
loading variations occur and where slowly growing organisms
with special metabolic capacities are to be protected from
washout (Wilderer et al., 1993; Woolard and Irvine, 1995).
Periodic discontinuous batch mode operation generally facil-
itates controlled unsteady-state conditions (exposure time,
frequency of exposure and substrate concentration can be set
independent of inflow condition) where microorganisms are
periodically exposed to defined process conditions and there-
fore select organisms that are generally more robust and
withstand shock loads (Chiesa et al., 1985; Irvine and Moe,
2001; Wilderer et al., 2001; Venkata Mohan et al., 2005a, b).
Under transient conditions, growth is unbalanced, and the
population’s physiological state adapts to the imposed
conditions and the organisms experience high growth rates
 ARTICLE IN PRESS
WAT E R R E S E A R C H
that increase cellular RNA content resulting in microflora
with high reactivity (Woolard and Irvine, 1995). Periodic
discontinuous process coupled with biofilm configuration
combines the operational advantages of biofilm reactor and
periodic discontinuous batch operation, which maintains
high biomass concentration, encourages the culture of slow
growing organisms and can obtain homogeneous biomass
distribution throughout the reactor (Chiesa et al., 1985; Irvine
and Ketchum, 1989; Zhang and Bishop, 1994; Kaballo et al.,
1995; Irvine et al., 1997; Pochana et al., 1999; Venkata Mohan
et al., 2006c, 2007). Further, the selection of biomass
particularly effective for degrading toxic and/or recalcitrant
compounds is possible along with the maintenance of
uniform biomass concentration along the whole height of
the bed. Biofilm configured system integrated with periodic
operation imposes regular variations in substrate concentra-
tion on biofilm organisms (Bouwer, 1989; Wilderer et al., 1993).
Therefore, organisms throughout the biofilm achieve max-
imum growth rates which results in improved reaction
potential leading to stable and robust system well suited for
treating highly variable wastes. Bacteria capable of decom-
posing xenobiotic compounds generally have a comparatively
low growth rate and are especially esteemed where slowly
growing organisms with special metabolic capacities are to be
protected from washout. Biofilm reactor configuration
coupled with periodic discontinuous batch operation, main-
tains high biomass concentration, encourages the enrich-
ment of slow growing organisms and can obtain
homogeneous biomass distribution throughout the reactor
(Kaballo et al., 1995; Venkata Mohan et al., 2006c, 2007). This
process integration seems to be easy to operate, cost efficient,
provides rapid startup and stabilization, highly flexible and
has potential to provide the possibilities of influencing the
microbial system.
5. Conclusions
The study demonstrated the feasibility of H2 generation
utilizing composite chemical wastewater as substrate in
addition to wastewater treatment. However, the process of
H2 evolution from wastewater was found to be dependent on
the nature and composition of wastewater used as substrate.
The pretreatment steps adopted for enumerating the H2
producing mixed consortia from anaerobic inoculum was
found to be effective. The microscopic images showed
selectively enriched spore forming bacteria. The study
showed selective enrichment of a group of morphologically
similar bacteria capable of proliferating in bioreactor on stone
chips producing H2 under anaerobic microenvironment. The
selected reactor operating conditions (pH—6; acidophilic
conditions) were found to be optimum for effective H2 yield.
Integration of biofilm configuration with periodic discontin-
uous batch operation seems to be highly effective, flexible
and has great potential to provide the possibilities of
influencing the microbial system by selectively enriching
the specific group of microflora showed rapid startup and
stabilization. Using mixed microbial cultures is considered to
be a practical, cost-effective and promising approach to
achieve H2 production in large scale. The described process
4 1 (200 7) 265 2 – 266 4 2663
has a dual benefit of H2 production with simultaneous
wastewater treatment in an economical, effective and
sustainable way.
Acknowledgment
The authors are indebted to Department of Biotechnology
(DBT), Government of India, New Delhi for providing financial
support (BT/PR/4405/BCE/08/312/2003) carrying out research
work reported herein.
R E F E R E N C E S
Aman, C.A., 1996. Alternative fuels and power systems in the long
term. Int. J. Vehicle Design 17, 510–517.
APHA, 1998. Standard methods for the examination of water and
wastewater. 20th, American Public Health Association, Amer-
ican Water Works Association, Water Pollution Control
Federation, Washington, DC.
Atif, A.A.Y., Razia, A.F., Ngan, M.A., Morimoto, M., Iyukec, S.E.,
Veziroglu, N.T., 2005. Fed batch production of hydrogen from
palm oil mill effluent using anaerobic microflora. Water Sci.
Technol. 53 (8), 271–279.
Bishop, P.L., 1997. Biofilm structure and kinetics. Water Sci.
Technol. 36, 287–294.
Boopathy, R., Daniels, L., 1991. Effect of pH on anaerobic mild steel
corrosion by methanogenic bacteria. Appl. Environ. Microbiol.
57, 2104–2108.
Bouwer, E.J., 1989. Transformation of xenobiotics in biofilms. In:
Charcklis, W.G., Wilderer, P.A. (Eds.), Structure and Function of
Biofilms. Wiley, New York, pp. 251–256.
Cha, G.C., Noike, T., 1997. Effect of rapid temperature and HRT on
anaerobic acidogenesis. Water Sci. Technol. 36 (6–7), 247–253.
Chandrasekhara Rao, N., Venkata Mohan, S., Muralikrishna, P.,
Sarma, P.N., 2005. Treatment of composite chemical waste-
water by GAC-Biofilm configured sequencing batch reactor
(SBGR) operated in aerobic environment. J Haz. Material. 124,
59–67.
Chaudhry, M.R.S., Beg, S.A., 1998. A review on the mathematical
modeling of biofilm processes: advances in fundamentals of
biofilm modeling. Chem. Eng. Technol. 21 (9), 701–710.
Chiesa, S.C., Irvine, R.L., Manning, J.F., 1985. Feast/Famine growth
environment and activated sludge population selection.
Biotechnol. Bioeng. 27, 562–569.
Das, D., Veziroglu, T.N., 2001. Hydrogen production by biological
process: a survey of literature. Int. J. Hydrogen Energy 26 (1),
13–28.
Dinopoulou, G., Rudd, T., Lester, J.N., 1998. Anaerobic acidogenesis
of complex wastewater. The influence of operational para-
meters on reactor performance. Biotechnol. Bioeng. 31,
958–968.
Fan, Y.T., Zhang, Y.H., Zhang, S.F., Hou, H.W., Ren, B.Z., 2006.
Efficient conversion of wheat straw wastes into biohydrogen
gas by cow dung compost. Bioresource Technol. 97, 500–505.
Fang, H.H.P., Liu, H., 2002. Effect of pH on hydrogen production
from glucose by a mixed culture. Bioresource Technol. 82 (2),
87–93.
Fascetti, E., Daddario, E., Todini, O., Robertiello, A., 1998. Photo-
synthetic hydrogen evolution with volatile organic acids
derived from the fermentation of source selected municipal
solid wastes. Int. J. Hydrogen Energy 23 (9), 753–760.
Ferchichi, M., Crabbe, E., Gil, G., Hintz, W., Almadidy, A., 2005.
Influence of initial pH on hydrogen production from cheese
whey. J. Biotechnol. 120, 402–409.
 ARTICLE IN PRESS
2664 WAT E R R E S E A R C H
Gavala, H.N., Skiadas, I.V., Ahring, B.K., Lyberatos, G., 2006.
Thermophilic anaerobic fermentation of olive pulp for hydro-
gen and methane production: modeling of the anaerobic
digestion process. Water Sci. Technol. 53 (8), 271–279.
Ginkel, S.V., Logan, B., 2005. Increased biological hydrogen
production with reduced organic loading. Water Res. 39,
3819–3826.
Ginkel, S.V., Lay, J.J., Sung, S., 2001. Biohydrogen production as a
function of pH and substrate concentration. Environ. Sci.
Technol. 35 (24), 4726–4730.
Ginkel, S.W.V., Oh, S.E., Logan, B.E., 2005. Biohydrogen gas
production from food processing and domestic wastewaters.
Int. J. Hydrogen Energy 30, 1535–1542.
Hawkes, F.R., Dinsdale, R., Hawkes, D.L., Hussy, I., 2002. Sustain-
able fermentative hydrogen production: challenges for
process optimisation. Int. J. Hydrogen Energy 27 (11–12),
1339–1347.
Irvine, R.L., Ketchum, H., 1989. Sequencing batch reactors for
biological wastewater treatment. Crit. Rev. Environ. Control 18,
255–294.
Irvine, R.L., Moe, W.M., 2001. Periodic biofilter operation for
enhance performance during unsteady state loading condi-
tion. Water Sci. Technol. 45 (3), 231–239.
Irvine, R.L., Wilderer, P.A., Flemming, H.C., 1997. Controlled
unsteady state processes and technologies—an overview.
Water Sci. Technol. 35, 1–10.
Kaballo, H.P., Zahao, Y., Wilderer, P.A., 1995. Elimination of
p-chlorophenol in biofilm reactor—a comparative study of
continuous flow and sequenced batch operation. Water Sci.
Technol. 33 (1), 51–60.
Lin, CY., Lay, Ch., 2004. Effects of carbonate and Phosphate
concentrations on hydrogen production using anaerobic
sewage sludge microflora. Int. J. Hydrogen Energy 29, 275–281.
Lin, C.Y., Chen, C.C., Lin, M.C., 2000. Hydrogen production in
anaerobic acidogenesis process—influences of thermal isola-
tion and acclimation environment. J. Chin. Inst. Environ. Eng.
10, 163–168.
Liu, Y., Boone, D.R., Sleat, R., Mah, R.A., 1985. Methanosarcina
mazei LYC—a new methanogenic isolate which produces a
disaggregating enzyme. Appl. Environ. Microbiol. 57,
2104–2108.
Logan, B.E., 2004. Biologically extracting energy from wastewater:
biohydrogen production and microbial fuel cells. Environ. Sci.
Technol. 38, 160A–167A.
Logan, B.E., Oh, S.E., Ginkel, S.V., Kim, I.S., 2002. Biological
hydrogen production measured in batch anaerobic respirom-
eters. Environ. Sci. Technol. 36 (11), 2530–2535.
Nandi, R., Sengupta, S., 1998. Microbial production of hydrogen:
an overview. Crit. Rev. Microbiol. 24 (1), 61–84.
Oh, S.E., Van Ginkel, S., Logan, B.E., 2003. The relative effective-
ness of pH control and heat treatment for enhancing
biohydrogen gas production. Environ. Sci. Technol. 37,
5186–5190.
Pochana, L., Kellen, J., Lant, P., 1999. Model development for
simultaneous nitrification and denitrification. Water Sci.
Technol. 39, 235–243.
Rogers, O., 1984. Genetics and biochemistry of clostridium
relevant to development of fermentation process. Appl.
Microbiol. 31, 1–60.
41 (2007) 2652– 2664
Sparling, R., Risbey, D., Poggi-Varaldo, H.M., 1997. Hydrogen
production from inhibited anaerobic composters. Int. J.
Hydrogen Energy. 22 (6), 563–566.
Venkata Mohan, S., Chandrasekhara Rao, N., Krishna Prasad, K.,
Sarma, P.N., 2005a. Bioaugmentation of an anaerobic sequen-
cing batch biofilm reactor (AnSBBR) with immobilized sul-
phate reducing bacteria (SRB) for the treatment of sulphate
bearing chemical wastewater. Process Biochem. 40 (8),
2849–2857.
Venkata Mohan, S., Chandrasekhara Rao, N., Prasad, K.K., Murali
Krishna, P., Rao, R.S., Sarma, P.N., 2005b. Anaerobic treatment
of complex chemical wastewater in a sequencing batch
biofilm reactor: process optimization and evaluation of factors
interaction using the Taguchi dynamic DOE methodology.
Biotechnol. Bioeng. 90 (6), 732–745.
Venkata Mohan, S., Lalit Babu, V., Sarma, P.N., 2006a. Effect of
various pretreatment methods on anaerobic mixed microflora
to enhance biohydrogen production utilizing dairy wastewater
as substrate. Bioresource Technol. doi:10.1016/j.biortech.
2006.12.004.
Venkata Mohan, S., Vijaya Bhaskar, Y., Murali Krishna, P.,
Chandrasekhara Rao, N., Lalit Babu, V., Sarma, P.N., 2006b.
Hydrogen production from chemical wastewater as substrate
by selectively enriched anaerobic mixed consortia: influence
of fermentation pH and substrate composition. Int. J. Hydro-
gen Energy, in press.
Venkata Mohan, S., Chandrasekhar Rao, N., Sarma, P.N., 2006c.
Composite chemical wastewater treatment by biofilm config-
ured periodic discontinuous batch reactor operated in anae-
robic metabolic function. Enzyme Microb. Technol.
doi:10.1016/j.enzmictec.2006.10.023.
Venkata Mohan, S., Lalit Babu, V., Vijaya Bhaskar, Y., Sarma, P.N.,
2007. Influence of recirculation on the performance of
anaerobic sequencing batch biofilm reactor (AnSBBR) treating
hypersaline composite chemical wastewater. Bioresource
Technol. 98 (7), 1373–1379.
Vijayaraghavan, K., Ahmad, D., Ibrahim, M.K.B., 2006. Biohydro-
gen generation from jackfruit peel using anaerobic contact
filter. Int. J. Hydrogen Energy. 31, 569–579.
Wilderer, P.A., Roske, I., Ueberschar, L.D., 1993. Continuous flow
and sequencing batch operation of biofilm reactors: a com-
parative study of shock loadings responses. Biofoulings 6,
295–304.
Wilderer, P.A., Irvine, R.L., Goronszy, M.C., 2001. Sequencing batch
reactor technology. Scientific and Technical Report. IWA
publishing.
Woolard, C.R., Irvine, R.L., 1995. Response of a periodically
operated halophilic biofilm reactor to changes in salt con-
centration. Water Sci. Technol. 31, 41–50.
Yang, H., Shao, P., Lub, T., Shena, J., Wang, D., Xub, Z., Yuan, X.,
2006. Continuous bio-hydrogen production from citric acid
wastewater via facultative anaerobic bacteria. Int. J. Hydrogen
Energy 31, 1306–1313.
Yu, H., Zhu, Z., Hu, W., Zhang, H., 2002. Hydrogen production from
rice winery wastewater in an upflow anaerobic reactor by
using mixed anaerobic cultures. Int. J. Hydrogen Energy 27,
1359–1365.
Zhang, T.C., Bishop, P.L., 1994. Evaluation of tortuosity factors and
effective diffusivities in biofilms. Water Res. 28, 2279–2287.