i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 4 ( 2 0 0 9 ) 1 7 7 1 – 1 7 7 9

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Effluents with soluble metabolites generated from acidogenicand methanogenic processes as substrate for additionalhydrogen production through photo-biological process

S. Srikanth, S. Venkata Mohan*, M. Prathima Devi, M. Lenin Babu, P.N. Sarma

Bioengineering and Environmental Centre, Indian Institute of Chemical Technology, Tarnka, Hyderabad 500 007, Andhra pradesh, India

a r t i c l e i n f o

Article history:

Received 16 October 2008

Received in revised form

17 November 2008

Accepted 18 November 2008

Available online 21 January 2009

Keywords:

Mixed culture

Acidophilic

Dark fermentation

Wastewater treatment

Total volatile fatty acids

* Corresponding author.E-mail address: vmohan_s@yahoo.com (S

0360-3199/$ – see front matter ª 2008 Interndoi:10.1016/j.ijhydene.2008.11.060

a b s t r a c t

The feasibility of utilizing effluents generated from acidogenic [producing biohydrogen

(H2)] and methanogenic [producing methane] processes was studied for additional H2

production by terminally integrating with photo-biological process employing enriched

mixed culture. Experimental data has depicted enhanced process efficiency with respect to

additional H2 production and substrate degradation through photo-biological process.

However, the efficiency was found to depend on the process used in the first stage along

with nature and composition of the substrate. Acidogenic process in the first stage had

more positive influence on photo-biological H2 production [synthetic wastewater –

14.40 mol/Kg CODR and 15.16 mol/Kg CODR (with vitamins); dairy wastewater – 13.29 mol/

Kg CODR and 13.70 mol/Kg CODR (with vitamins)] over the corresponding methanogenic

process. Effluent generated from acidogenic treatment of dairy wastewater yielded high

substrate degradation rate (SDR) [1.20 Kg COD/m3 day and 1.34 Kg COD/m3 day (vitamins)]

followed by synthetic wastewater [0.92 Kg COD/m3 day and 1.05 Kg COD/m3 day (vitamins)].

Among the studied experimental variations chemical wastewater evidenced poor H2

production and SDR. Vitamin solution showed positive influence on both H2 production

and wastewater treatment irrespective of the experimental variations studied.

ª 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights

reserved.

1. Introduction cyanobacteria, photodecomposition by photosynthetic bacteria

Increasing demand for energy and the resulting crisis in asso-

ciation with the green house pollution from the combustion of

fossil fuels led for search towards alternative and eco-friendly

energy sources. In this direction, a great deal of attention is

being focused on the usage of hydrogen (H2) as an alternative

energy source which may play a pivotal role in future energy

supply. H2 production by biological route is considered to be

advantageous compared to traditional methods due to its

environmental friendly nature. Biological H2 production

processes can be classified as biophotolysis by algae/

. Venkata Mohan).ational Association for H

and dark fermentation by anaerobic bacteria [1–6]. Most of these

processes occur at ambient temperatures and pressures, are

less energy intensive and more environmental friendly and

thus opening a new avenue for the utilization of renewable

energy sources such as wastewater which are inexhaustible

[1,6–11].

A practical and efficient H2 generation process is a growing

concern among the research fraternity [9] and various strat-

egies in this direction were reported [3–6,12–17]. Light-

dependent (photo-biological) or light independent (dark)

fermentative conversions of organic substrates manifest

ydrogen Energy. Published by Elsevier Ltd. All rights reserved.

Fig. 1 – Experimental setup used for photo-biological

hydrogen production.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 4 ( 2 0 0 9 ) 1 7 7 1 – 1 7 7 91772

diverse metabolic pathways and substrate utilization pattern

during H2 production [1,10,18–22]. In the case of dark

fermentation, the organic substrate was converted to H2 or

CH4 along with organic acids as metabolic (soluble) by-prod-

ucts mainly by acidogenic bacteria [1,5,6,23]. Generation and

accumulation of soluble acid metabolites causes sharp drop in

the system pH and inhibit the H2 production process. Persis-

tent acidophilic conditions result in suppression of meth-

anogenic process and accounts only for 15% of the available

energy conversion from the organic source in the form of H2

[6]. Usage of unutilized carbon associated with soluble

metabolites present in the effluents for additional H2

production will sustain the practical applicability of the

process. Further, photosynthetic organisms are capable of

utilizing organic acids as substrate besides the original

substrates to produce additional H2 [1,5,6,12,13,22,23]. There-

fore, integration of photo-biological process after dark

fermentation could result in additional H2 production and

substrate degradation efficiency. This route appears to be an

ideal one which might lead to highest possible theoretical

yield [6,8,20]. An attempt was therefore made in this

communication to study the feasibility of utilizing soluble

metabolites along with residual carbon source present in

effluents generated from dark fermentation processes

(acidogenic and methanogenic) as primary substrates for

additional H2 production and substrate degradation through

integration of photo-biological process.

2. Experimental procedure

2.1. Mixed culture

Photosynthetic mixed culture acquired from Saroor nagar

lake, Hyderabad was used after screening and testing its

viability in different wastewaters [6]. The inoculum (pH, 8.36;

total volatile fatty acids (TVFA), 754 mg/l) was preserved at

4 �C for further use. Prior to use, the inoculum was enriched in

a mixture of dairy wastewater and domestic sewage (1:1)

along with vitamin solution [2.5 ml/l; thiamine mononitrate,

10 mg/l; riboflavin, 10 mg/l; nicotinic acid, 25 mg/l; niacin-

amide, 75 mg/l; pyridoxine hydrochloride, 3 mg/l; calcium

pantothenate, 50 mg/l; folic acid, 1.5 mg/l; vitamin B12,

15 mcg/l; vitamin C, 150 mg/l; biotin, 260 mcg/l] and incubated

in the presence of fluorescent light (4 Klux) at 34 �C [6] for 24 h

(Fig. 1)

2.2. Substrates

Initially, H2 producing acidogenic bioreactor (HBR) and CH4

producing methanogenic bioreactor (MBR) were operated with

three substrates [synthetic wastewater ((SW); g/l, glucose-3.0/

6.0, NH4Cl-0.5, KH2PO4-0.25, K2HPO4-0.25, MgCl2$6H2O-0.3,

FeCl3-0.025, NiSO4-0.016, CoCl2-0.025, ZnCl2-0.0115, CuCl2-

0.0105, CaCl2-0.005, MnCl2-0.015) [COD: 3200 mg/l, pH: 6.8,

TDS: 1420 mg/l, ORP: 2.6], chemical wastewater (CW) [COD:

9200 mg/l, pH: 7.8, TDS: 18,400 mg/l, ORP: �24.6] and dairy

wastewater (DW) [COD: 4800 mg/l, pH: 7.2, TDS: 4200 mg/l,

ORP: 10.4]] in the first stage. Feed characteristics varied from

simple to complex depending upon the source obtained. The

effluents generated from first stage mostly constitute of

residual carbon associated with total volatile fatty acids

(TVFA)/soluble acid metabolites which were used as

substrates for subsequent H2 production by photo-biological

process as depicted in Table 1.

2.3. Experimental design

In the first stage, HBR was operated under acidophilic condi-

tion (pH 6) using selectively enriched mixed consortia and

MBR was operated under neutral condition (pH 7) using

anaerobic mixed inoculum. Both the processes were operated

in biofilm configured systems employing batch mode opera-

tion at ambient room temperature with three types of

wastewaters for a total cycle period of 48 h (retention time).

The performance was evaluated in terms of H2 and CH4

production along with substrate removal efficiency. Effluent

from the reactors was collected separately after 48 h of oper-

ation. In the second stage, after separating biomass (by

settling; 30 min), 150 ml of supernatant (adjusted to pH 6 using

concentrated orthophosphoric acid and/or 3 N NaOH) along

with 20 ml of enriched photosynthetic mixed culture (24 h;

Saroor Nagar lake, Hyderabad) was loaded in the flask under

anaerobic conditions. After feeding, the flasks were sparged

with oxygen free nitrogen gas for a period of 30 s to remove

dissolved oxygen and to create an oxygen free microenvi-

ronment prior to closing with rubber septum (butyl rubber).

Batch experimental protocol was employed to evaluate the

experimental objectives using a series of 250 ml conical flasks

(with a total working volume of 160 ml) under aseptic anaer-

obic microenvironment. Twelve experiments were designed

and evaluated based on the differential combinations of

substrate (Table 2). All the experiments were performed in

presence of fluorescent light (4 Klux) in an incubator orbital

shaker (100 rpm; 34 �C) for 24 h. Experiments were performed

in the presence and absence of vitamins (3.2 ml/l) to

enumerate its role on process performance. Periodically

samples were collected to evaluate the process efficiency

along with the quantification of H2 gas produced.

Table 1 – Characteristics of wastewater used as feed in the first stage of treatment [acidogenic (HBR) and methanogenic(MB)] and resulting process efficiency.

Wastewaterused as feed

Reactormicroenvironment

pH TVFA (mg/l)a SDR (KgCOD/m3 day)

(mg/l)a

xCOD (%)a H2 yield (molH2/Kg CODR)a

CH4 yield (molCH4/Kg CODR)a

Inlet Outlet Inlet Outlet

SW Acidogenic 6.0 6.8 360.4 782.2 0.525 31.58 18.64 –

Methanogenic 7.0 8.1 246.2 804.6 1.137 68.42 – 21.98

CW Acidogenic 6.0 6.6 1440 422 0.263 12.76 8.26 –

Methanogenic 7.0 8.1 1260 462.8 0.831 40.42 – 16.45

DW Acidogenic 6.0 7.2 642.6 574.8 0.306 16.27 11.12 –

Methanogenic 7.0 8.4 726.4 242 0.875 46.51 – 18.75

a Based on mean value (n¼ 2) S.E varied between 0.3 and 3.4%.

Table 2 – Design criteria of experiments carried byintegrating with photo-biological process.

Experimentno.

Feed OLR (KgCOD/m3 day)

Operational conditions

1 H-SW 2.21 Acidogenic

2 H-SW 2.21 Acidogenic; with vitamin

3 M-SW 1.11 Methanogenic

4 M-SW 1.11 Methanogenic; with vitamin

5 H-DW 3.59 Acidogenic

6 H-DW 3.59 Acidogenic; with vitamin

7 M-DW 2.39 Methanogenic

8 M-DW 2.39 Methanogenic; with vitamin

9 H-CW 3.18 Acidogenic

10 H-CW 3.18 Acidogenic; with vitamin

11 M-CW 2.12 Methanogenic

12 M-CW 2.12 Methanogenic; with vitamin

All experiments were performed with adopted photosynthetic

mixed culture from Saroor Nagar pond in presence of incandescent

light source (4 Klux) (temperature 34 �C; pH 6.0); OLR, Organic

loading rate; SW, Designed synthetic wastewater effluent; DW,

Dairy wastewater effluent; CW, Industrial wastewater effluent; H,

Effluent from acidogenic bioreactor; M, Effluent from methano-

genic bioreactor;.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 4 ( 2 0 0 9 ) 1 7 7 1 – 1 7 7 9 1773

2.4. Bio-analytical methods

Water displacement method was employed for quantitative

estimation of the biogas generated from the process. H2 gas

was estimated using a microprocessor based electrochemical

gas sensor (ATMI GmBH Inc., Germany). The output signal

displayed the percent volume of H2 in the reactor head space,

which was converted to mmol. Process performance was also

evaluated by estimating the soluble COD (closed refluxing

titrimetric method) and TVFA according to the procedures

outlined in the standard methods [24]. The separation and

quantitative determination of the composition of soluble

metabolites [acetic acid (HAc), butyric acid (HBu) and pro-

pionic (HPr) along with ethanol (EtOH)] were estimated by high

performance liquid chromatography [HPLC; UV–vis detector;

C18 reverse phase column – 250� 4.6 mm and 5 m particle size;

flow rate – 0.5 ml/h; wavelength – 210 nm; mobile phase – 40%

of acetonitrile in 1 mN H2SO4 (pH 2.5–3.0); sample injection –

20 ml]. COD removal efficiency (x) was calculated as shown in

Eq. (1), where, CSO represents the initial COD concentration

(mg/l) in the feed and CS denotes COD concentration (mg/l) in

the reactor outlet.

x ¼ ½ðCSO � CSÞ=CSO� � 100 (1)

Substrate degradation rate (SDR – Kg COD/m3 day) was

calculated to study the rate and pattern of COD removal

where, xt represent substrate removal efficiency (%) at time ‘t’

according to Eq. (2).

SDR ¼ ½OLR� xt=100� (2)

Organic loading rate (OLR – Kg COD/m3 day) was calculated

using Eq. (3), where F represents feed rate (l/day) and V

denotes volume of substrate (l ).

OLR ¼ f½CSO � F�=Vg (3)

3. Results and discussion

During initial stage of process operation considerable varia-

tion in biogas (H2 and CH4) production, substrate degradation

and TVFA generation was observed with the function of

wastewater used as substrate and the process used for

substrate metabolism (Table 1). Among the wastewaters

studied, SW showed highest biogas yield followed by DW and

CW irrespective of the metabolic process used. Higher

substrate degradation was observed in the methanogenic

process rather than acidogenic process. The higher substrate

degradation and biogas yields observed with SW might be

attributed to its simple composition which may facilitate easy

metabolism. Comparatively, CW and DW were complex in

nature and are les bio-degradable. Irrespective of the waste-

water used, effluents generated from acidogenic process

documented higher concentration of TVFA along with carbon

(residual) than the methanogenic process irrespective of the

wastewater used.

3.1. Biohydrogen production

Experimental data evidenced the feasibility of photo-biolog-

ical process in using effluents generated from dark fermen-

tation processes (both acidogenic and methanogenic) as

primary substrates for additional H2 production and substrate

degradation. Fig. 2 depicts the H2 production profile as

a function of the substrates used. H2 production was found to

depend mostly on the first stage of treatment adapted and

nature of wastewater used as substrate. Details of photo-

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 4 ( 2 0 0 9 ) 1 7 7 1 – 1 7 7 91774

biological H2 production with respect to experimental varia-

tion studied in the descending order of performance is as

shown below.

H�DW > H� SW > M�DW > M� SW > H� CW > M� CW

Higher H2 production was observed with the effluents

generated from acidogenic reactor (HBR) irrespective of the

nature of wastewater. Among effluents, DW documented

higher efficiency of H2 generation followed by SW and CW.

Effluents supplemented with vitamins showed marked

improvement in H2 production in all the experimental varia-

tions studied (Fig. 3a). Outlet of acidogenesis reactor (H-DW)

showed maximum H2 production [17.28 mmol/day and

19.87 mmol/day (vitamins)] followed by H-SW [14.40 mmol/

day and 17.28 mmol/day (vitamins)]. Similarly, methanogenic

outlet also showed higher H2 production in the case of M-DW

0

5

10

15

20

25

1 2 3 4 5 6

Experim

1 2 3 4 5 6

Experim

H2 P

ro

du

ctio

n/H

2 Y

ield

0

5

10

15

20

25

H2/C

H4 Y

ield

(m

ol/K

g C

OD

R)

b

a

Fig. 2 – (a) H2 production and yield during photo-biological oper

stages of the experimental study (acidogenic/methanogenic/ph

[8.93 mmol/day; 9.79 mmol/day (vitamins)] followed by M-SW

[8.35 mmol/day; 8.93 mmol/day (vitamins)]. Among the efflu-

ents, CW documented lowest H2 production [H-CW –

5.76 mmol/day; 7.49 mmol/day (vitamins); M-CW – 2.30 mmol/

day; 2.59 mmol/day (vitamins)] irrespective of the preliminary

treatment processes adapted. However, specific H2 production

showed marked deviation with the production observed as

depicted below.

H� SW > H�DW > M� SW > M�DW > H� CW > M� CW

Acidogenic reactor outlet treating H-SW showed higher

specific H2 yield [14.40 mol/Kg CODR and 15.16 mol/Kg CODR

(vitamins)] followed by H-DW [13.29 mol/Kg CODR and

13.70 mol/Kg CODR (vitamins)]. Similarly the methanogenic

outlets evidenced higher specific H2 production in the case of

M-SW [11.60 mol/Kg CODR; 11.75 mol/Kg CODR (vitamins)]

7 8 9 10 11 12

ent Number

7 8 9 10 11 12

ent Number

mmol/day

mol/Kg COD R

Acidophilic/AnaerobicPhotosynthetic

ation (b) Biogas (H2 and CH4) yield observed during two

otosynthetic).

0

2

4

6

8

10

12

14

16

2.21 1.11 3.59 2.39 3.18 2.12OLR (Kg COD/m

3-day)

H2 Y

ield

(m

ol/kg

C

OD

R)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

2.21 1.11 3.59 2.39 3.18 2.12OLR (Kg COD/m

3-day)

SD

R (K

g C

OD

/m

3-d

ay)

b

a

Without Vitamin

With Vitamin

Without Vitamin

With Vitamin

Fig. 3 – Variation in (a) Hydrogen yield and (b) substrate degradation rate (SDR) in presence and absence of vitamin

supplementation with the function of organic loading rate.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 4 ( 2 0 0 9 ) 1 7 7 1 – 1 7 7 9 1775

followed by M-DW [9.62 mol/Kg CODR; 10.20 mol/Kg CODR

(vitamins)]. The CW effluent from both the processes evi-

denced lower H2 yield among the experimental variations

studied [H-CW – 6.70 mol/Kg CODR; 8.23 mol/Kg CODR (vita-

mins); M-CW – 5.24 mol/Kg CODR; 5.71 mol/Kg CODR (vita-

mins)]. It was evident from the experimental results that, the

initial treatment and characteristics of wastewater (before

and after first stage of treatment) appeared to have significant

influence on H2 production by photo-biological process. Initial

placement of acidogenic process appears to have positive

influence on subsequent integration with photo-biological H2

production over the integration of corresponding methano-

genic process. This may be attributed to the presence of higher

concentrations of TVFA associated with residual carbon after

acidogenic process in the effluents.

3.2. Substrate degradation

Experiments also evidenced the possibility of additional

substrate degradation due to the terminal integration of

photo-biological process. Residual carbon source remaining in

the outlets generated from acidogenic and methanogenic

processes was observed to be utilized by subsequent integra-

tion with photo-biological process (Fig. 4; Table 1). However,

the rate of substrate degradation was found to depend on the

characteristics/nature of the wastewater used as substrate

and the process used in the first stage. Visible improvement in

the substrate degradation efficiency was also observed after

supplementing with vitamins (Fig. 3b). COD removal efficiency

showed a decreasing trend with increasing complexity of the

substrated. SW which contained simple carbon source in the

form of glucose yielded high COD removal efficiency [H-SW –

41.67% and 47.50% (vitamins); M-SW – 60.00% and 63.33%

(vitamins)] compared to the other two substrates studied. DW

effluents showed lower COD removal efficiency compared to

SW [H-DW – 33.33% and 37.18% (vitamins); M-DW – 35.69%

and 36.92% (vitamins)]. Among all the substrates studied CW

effluent evidenced poor COD removal efficiency [H-CW –

24.93% and 26.38% (vitamins); M-CW – 19.13% and 19.74%

(vitamins)] among all the substrates studied. Complex nature

of the CW might be the reason for the poor substrate degra-

dation along with lower H2 production observed during the

experimental study. Among all the experimental variations

studied, higher SDR was observed with the effluent generated

0

1

2

3

4

10987654321 11 12Experiment Number

10987654321 11 12Experiment Number

OL

R/S

DR

(K

g C

OD

/m

3-d

ay)

0

10

20

30

40

50

60

70

CO

D rem

oval efficien

cy (%

)

a

b

OLRSDR

Acidophilic/AnaerobicPhotosynthetic

Fig. 4 – (a) Variation in SDR in comparison with OLR during photo-biological process (b) COD removal pattern observed

during two stages of experimental studies (acidogenic/methanogenic/photosynthetic).

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 4 ( 2 0 0 9 ) 1 7 7 1 – 1 7 7 91776

from the acidogenic process rather than the methanogenic

process effluents as shown below.

H�DW > H� SW > M�DW > H� CW > M� SW > M� CW

Among the effluents generated from the acidogenic process,

DW showed higher SDR [H-DW – 1.20 Kg COD/m3 day and

1.34 Kg COD/m3 day (vitamins)] followed by SW [H-SW –

0.92 Kg COD/m3 day and 1.05 Kg COD/m3 day (vitamins)].

Likewise in the methanogenic effluents also DW evidenced

higher SDR [M-DW – 0.85 Kg COD/m3 day and 0.88 Kg COD/

m3 day (vitamins)] compared to SW [M-SW- 0.67 Kg COD/

m3 day and 0.79 Kg COD/m3 day (vitamins)]. CW documented

lower SDR irrespective of the initial process [H-CW – 0.79 Kg

COD/m3 day and 0.84 Kg COD/m3 day (vitamins); M-CW –

0.41 Kg COD/m3 day and 0.42 Kg COD/m3 day (vitamins)].

Higher substrate degradation observed in the acidogenic

outlets might be attributed to the presence of higher carbon

source (residual) even after the first stage of treatment. While,

in the case of methanogenic outlets the carbon source might

be exhausted in the first stage of treatment and the remaining

carbon source might be more complex in nature which cannot

be degraded easily. Higher SDR with DW might be attributed to

the presence of higher concentration of TVFA during the first

stage of treatment. TVFA concentration was relatively low in

the case of SW which might be the causative for lower

substrate degradation observed.

3.3. Soluble metabolites

3.3.1. Acidogenic/methanogenic processesComposition and distribution of soluble metabolites during H2

generation were often considered as a crucial factor in

understanding the metabolic pathway of H2 production

process [1,6,9,10]. Therefore, composition of soluble metabo-

lites viz., acetic acid (HAc), butyric acid (HBu) and propionic

(HPr) along with ethanol (EtOH) was determined using HPLC

during the experimental study (Table 3). Concentration of

soluble metabolites and its utilization showed good correla-

tion with H2 production and substrate degradation. Presence of

soluble metabolites viz., HAc, HBu, HPr and EtOH was observed

Table 3 – Variation pattern of soluble metabolites composition during photo-biological H2 production.

Expt. No. Inlet composition (%)a,b Outlet composition (%)b

Acetate Butyrate Propionate Ethanol Acetate Butyrate Propionate Ethanol

1 68.21 7.28 1.26 23.23 26.28 25.16 2.26 46.28

2 71.54 1.55 1.86 25.05 31.55 29.35 2.86 36.24

3 28.66 15.78 0.94 54.61 28.78 24.67 2.94 43.61

4 21.41 10.30 1.06 67.23 15.30 31.58 2.06 51.06

5 14.62 16.50 21.63 47.25 3.50 15.57 47.64 33.29

6 17.77 12.08 14.89 55.26 2.08 8.89 56.88 36.15

7 15.93 18.47 17.27 38.31 2.47 24.04 41.67 31.82

8 10.72 16.01 33.24 40.02 2.01 5.94 58.04 34.01

9 12.10 17.92 44.64 25.34 14.02 5.36 34.54 46.08

10 16.89 16.09 48.38 18.64 12.05 9.56 27.30 51.09

11 18.42 17.91 41.45 22.22 18.91 9.38 36.42 35.29

12 19.21 22.84 39.31 18.64 24.64 8.68 24.51 42.16

a Effluent generated from acidogenic/methanogenic process.

b Based on mean value (n¼ 2) S.E varied between 0.2 and 2.9%.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 4 ( 2 0 0 9 ) 1 7 7 1 – 1 7 7 9 1777

in the effluents generated from both the acidogenic and

methanogenic processes. The distribution of metabolites

varied in accordance with the nature of wastewater and initial

process adapted. In the first stage of treatment, H-SW showed

a profile with higher HAc concentration (71.54%) followed by

the EtOH (25.05%), HBu (7.28%) and HPr (1.86%) which are

feasible for effective H2 production. Presence of higher

concentrations of HAc indicated feasible environment for

acidogenesis. In the case of M-SW, the concentration of EtOH

(67.23%) was higher followed by HAc (28.66%), HBu (15.78%)

and HPr (0.96%) supporting the possibility of solventogenesis.

In the case of DW also the concentration of EtOH was higher

(40–55%) followed by HAc, HBu and HPr concentrations almost

similar in the range of 14–21% irrespective of the experimental

variations studied. Lower concentration of HPr observed in all

the experimental variations studied with SW also indicated

positive microenvironment towards acidogenic H2 generation.

M-DW showed comparatively higher concentration of HPr

which is a negative sign with respect to H2 generation. CW

evidenced higher concentration of HPr (40–48%) irrespective of

the experimental variations which might be attributed to the

inhibition of H2 production. While the concentration of other

metabolites viz., HAc, HBu and EtOH were more or less similar

and varied within a narrow range (14–22%). However, the

concentration of EtOH was relatively higher (18–25%). More-

over, higher H2 yields could be observed in association with

a mixture of HAc and HBu where as HPr generation will result

in consumption or inhibition of H2 production [1,2]. While the

production of EtOH strongly supports the metabolic process

towards solventogenesis as observed in the case of DW efflu-

ents from both the processes along with the methanogenic

effluents of SW. Interestingly, in the case of M-CW also,

effluents generated from methanogenic process contributed to

higher concentration of HAc over the corresponding effluent

generated from acidogenic process. However, the concentra-

tion of HPr was higher in CW effluents from both the processes

compared to SW and DW which supports the least chances of

H2 production. Relatively higher concentration of HAc was

observed in the effluents generated from acidogenic process

compared to the corresponding methanogenic process.

3.3.2. Photo-biological processUtilization of TVFA was observed in most of the experimental

variations studied during photo-biological H2 generation and

this enumerated their participation as a substrate in the

subsequent metabolic process (Fig. 5a). H2 production was

observed in all the experimental variations studied except CW

and DW effluents generated from methanogenic process.

Acidogenic effluents showed relatively good TVFA utilization

with all the three feeds studied and this also corroborated well

with the corresponding H2 production rate. Among the

wastewaters, SW showed higher utilization of soluble TVFA.

Relatively low utilization of TVFA was observed with CW.

Distribution of soluble metabolites showed marked variation

after photo-biological process (Table 3). Irrespective of first

stage of treatment used, the pattern of metabolite consump-

tion was almost the same in the subsequent photo-biological

process. Utilization of HAc (15–25%) associated with EtOH (35–

50%) production was observed with both acidogenic (H-SW)

and methanogenic (M-SW) outlets after photo-fermentation.

Utilization of HAc along with formation of EtOH during H2

production indicated the metabolic flow associated with the

solventogenesis. Increase in HBu formation (25–30%) associ-

ated with lower HPr concentration (2–2.5%) was observed

irrespective of the experimental variation indicating a positive

sign for photo-biological process. In the case of DW effluent

from both processes, almost complete utilization of HAc

(2.5%) was noticed with a decreasing EtOH concentration (30–

36%). The formation of HPr (40–58%) was higher compared to

all other soluble metabolites in this case. However, HBu

concentration was observed to be higher (15–24%) in the

absence of vitamins. On the contrary, with CW outlet from

both the processes, increase in EtOH (35–50%) was observed

along with the stable concentration of HAc (12–24%). Stable

HAc concentration observed might be attributed to the

transformation of residual substrate (during initial treat-

ment). Consumption of HPr (25–35%) and HBu (5–10%) was

observed suggesting the efficacy of photosynthetic culture.

The system pH showed gradual increase with time in all the

experimental variations studied which might be attributed to

the strong reducing phase observed during substrate

0

100

200

300

400

500

600

700

800

900

1 2 3 4 5 6 7 8 9 10 11 12Experiment Number

1 2 3 4 5 6 7 8 9 10 11 12Experiment Number

1 2 3 4 5 6 7 8 9 10 11 12

Experiment Number

VF

A (m

g/l)

0

2

4

6

8

10

pH

-65

-40

-15

10

35

60

85

110

OR

P (m

V)

a

b

c

InletOutlet

InletOutlet

InletOutlet

Fig. 5 – Change in (a) TVFA, (b) pH and (c) ORP from inlet to outlet during photo-biological operation.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 4 ( 2 0 0 9 ) 1 7 7 1 – 1 7 7 91778

metabolism (Fig. 5b). pH microenvironment is crucial during

photo-biological H2 production because of nitrogenase activity

which is crucial and functions well between pH range of 6–7

[6,25]. Methanogenic effluents showed higher up shift in pH

towards basic microenvironment rather than the correspond-

ing acidogenic effluents. Generally, basic pH doesnot support H2

production and leads to solventogenesis. The generation of

EtOH during the process also corroborated with the observation.

4. Conclusions

Experimental results evidenced the possibility of additional H2

production associated with higher substrate degradation by

integrating acidogenic and methanogenic processes with

photo-biological process. The residual carbon source along

with soluble metabolites present in the effluents generated

from acidogenic and methanogenic processes were reutilized

as primary substrates through subsequent photo-biological

process integration (terminal) leading to overall process effi-

ciency based on H2 production and wastewater treatment.

However, H2 production and substrate degradation were

found to depend on the process used in the first stage and

nature and composition of the substrate along with the

presence of vitamins. Acidogenic process of treatment

appeared to have more positive influence over the corre-

sponding methanogenic process in photo-biological H2

production. This observation could be attributed to the

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 4 ( 2 0 0 9 ) 1 7 7 1 – 1 7 7 9 1779

presence of excessive concentration of soluble metabolites

along with higher concentration of residual carbon source.

The presence of vitamins showed a positive influence on both

H2 production and substrate degradation. The process

described in this study documented the possibility of higher

rates of H2 generation and substrate removal. This was made

possible by reusing the unutilized substrate from acidogenic

and methanogenic processes by integrating with photo-bio-

logical process.

Acknowledgments

The authors wish to thank the Department of Biotechnology

(DBT), Government of India, New Delhi for funding the pre-

sented research study (Project No. BT/PR/4405/BCE/08/312/

2003). The authors also wish to thank the Director, IICT, for his

encouragement in carrying out this work.

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