Acetate and butyrate as substrates for hydrogen production through photo-fermentation: Process...

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 ) 7 5 1 3 – 7 5 2 2

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Acetate and butyrate as substrates for hydrogen productionthrough photo-fermentation: Process optimizationand combined performance evaluation5

S. Srikanth, S. Venkata Mohan*, M. Prathima Devi, Dinakar Peri, P.N. Sarma

Bioengineering and Environmental Centre, Indian Institute of Chemical Technology, Tarnaka, Hyderabad, AP 500 007, India

a r t i c l e i n f o

Article history:

Received 11 July 2008

Received in revised form

22 May 2009

Accepted 22 May 2009

Available online 21 June 2009

Keywords:

Data enveloping analysis (DEA)

Anaerobic fermentation

Soluble acid metabolites

Wastewater treatment

Mixed photosynthetic culture

5 Oral presentation No. O41; International* Corresponding author. Tel./fax: þ91 40 271

E-mail addresses: [email protected]/$ – see front matter ª 2009 Interndoi:10.1016/j.ijhydene.2009.05.095

a b s t r a c t

Organic acids viz., acetate and butyrate were evaluated as primary substrates for the

production of biohydrogen (H2) through photo-fermentation process using mixed culture at

mesophilic temperature (34 �C). Experiments were performed by varying parameters like

operating pH, presence/absence of initiator substrate (glucose) and vitamin solution, type

of nitrogen source (mono sodium salt of glutamic acid and amino glutamic acid) and gas

(nitrogen/argon) used to create anaerobic microenvironment. Experimental data showed

the feasibility of H2 production along with substrate degradation utilizing organic acids as

metabolic substrate but was found to be dependent on the process parameters evaluated.

Maximum specific H2 production and substrate degradation were observed with acetic acid

[3.51 mol/Kg CODR-day; 1.22 Kg CODR/m3-day (92.96%)] compared to butyric acid [3.33 mol/

Kg CODR-day; 1.19 Kg CODR/m3-day (88%)]. Higher H2 yield was observed under acidophilic

microenvironment in the presence of glucose (co-substrate), mono sodium salt of glutamic

acid (nitrogen source) and vitamins. Argon induced microenvironment was observed to be

effective compared to nitrogen induced microenvironment. Combined process efficiency

viz., H2 production and substrate degradation was evaluated employing data enveloping

analysis (DEA) methodology based on the relative efficiency. Integration of dark fermen-

tation with photo-fermentation appears to be an economically viable route for sustainable

biohydrogen production if wastewater is used as substrate.

ª 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights

reserved.

1. Introduction alternate fuel which can replace the fossil fuels and mitigate

In recent times, the ever increasing usage of hydrocarbon

fuels is resulting in the emission of greenhouse gases and

other pollutants in alarming proportions leading to global

warming and various health disorders, pushing mankind to

the brink of extinction. Furthermore, the supply of hydro-

carbon resources in the world is limited, while the demand for

hydrocarbon fuels is increasing. Worldwide search is on for an

Workshop on Biohydroge63159., [email protected] (S.ational Association for H

all their harmful effects. In this context, hydrogen (H2) is

generating a lot of interest due to its clean, non-polluting and

eco-friendly nature thus being considered as an alternative

fuel [1–4]. At present, H2 is produced mainly from fossil fuels,

biomass and water. Most of these methods mainly use fossil

fuels as energy source, which are considered to be energy

intensive and not always environmental friendly [5–9]. Bio-

logical production of H2 is one of the feasible and alternative

n Technologies (IWBT2008).

Venkata Mohan).ydrogen Energy. Published by Elsevier Ltd. All rights reserved.

mailto:[email protected]

mailto:[email protected]

http://www.elsevier.com/locate/he


methods being pursued extensively by the research fraternity

recently. Biological H2 production processes can be broadly

classified as biophotolysis of water using algae and cyano-

bacteria, photodecomposition of organic compounds by

photosynthetic bacteria and fermentative H2 production from

organic compounds [2,3,10–16]. These processes mostly occur

at ambient temperatures and pressures, which are less energy

intensive, more environment friendly and further opening

new avenues for the utilization of renewable energy sources

which are inexhaustible [2,5,8–10].

A practical, efficient and economically feasible process of H2

generation are extremely critical [4,11]. Various strategies in

this direction were reported earlier [1–3]. H2 production through

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

fermentative conversions of organic substrates is of great

importance due to its dual function of substrate reduction and

H2 production [2,4,11]. The generation of H2 by fermentation is

accompanied by the formation of organic acids as metabolic

products but these microbes are not capable of breaking the

acids [1,15]. Generation and accumulation of these soluble acid

metabolites causes a sharp drop in the system pH and inhibit

the process of H2 production. Persistent acidophilic conditions

resulted in suppression of methanogenic process and account

for only 15% of the energy conversion from the organic source

in the form of H2 [10]. Usage of unutilized carbon sources in

wastewater for additional H2 production will sustain practical

applicability of the process. Further, soluble metabolites can be

utilized by photosynthetic bacteria to produce additional H2 or

CH4 through photo-fermentation [2–4,11]. Photo-fermentation

is the process of fermentative conversion of organic substrate

manifested by a diverse group of photosynthetic bacteria

through a series of biochemical reactions similar to anaerobic

fermentation. Photo-fermentation only proceeds in the pres-

ence of light unlike dark fermentation. Photosynthetic bacteria

can produce H2 by consuming organic acids which are abun-

dant in the effluents generated from acidogenic H2 fermenta-

tion processes [11,17–19]. Moreover, the economics related to

phototrophic H2 production are limited by poor H2 production

rate of photosynthetic bacteria due to slow growth rate and

inefficient utilization of light energy [2,3]. Improving produc-

tion rate by integrating dark and photo-fermentation could aid

in the development of sustainable H2 production process.

Combination of dark and photo-fermentation appears to be an

ideal route leading to near highest theoritically possible yield

[12,13]. The main scope of this communication is to evaluate

the feasibility of utilizing volatile fatty acids generated from

dark fermentation as primary substrate for subsequent H2

production through photo-fermentation. In this direction an

attempt was made to evaluate organic acids viz., acetate and

butyrate as primary substrates for H2 production using

enriched photosynthetic mixed culture.

2. Material and methods

A series of thirty-two batch experiments each with acetate

and butyrate were designed and performed at mesophilic

temperature (34 �C) employing adapted mixed photosynthetic

culture by varying parameters like operating pH (7/6), pres-

ence/absence of initiator substrate (glucose), vitamin solution,

variable nitrogen source [mono sodium salt of glutamic acid

(MSG) and amino glutamic acid (AG)] and gas (Nitrogen/Argon)

used to create anaerobic microenvironment.

2.1. Substrate composition

Acetic acid (HAc) and butyric acid (HBu) were the two volatile

fatty acids considered in this study for evaluating photo-bio-

logical H2 production. Experiments were performed both in the

presence and absence of carbon source (glucose, 1 g/l) to

enumerate the role of metabolic initiator/co-substrate. AG

(8 mM) and MSG (8 mM) were the two nitrogen sources used in

the feed composition. All experimental studies wereperformed

in the presence of trace elements (mg/l; 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 and

MnCl2 –0.015) supplemented at 50 ml/l. Feed composition

in the presence and absence of vitamins (mg/l; thiamine

mononitrate – 10.0; riboflavine – 10.0; nicotinic acid – 25.0;

niacinamide – 75.0; pyridoxine hydrochloride – 3.0; calcium

pantothenate – 50.0; folic acid – 1.5; vitamin B12 – 0.015;

vitamin C – 150.0 mg; biotin – 0.260) (2.5 ml/l) was studied.

Various feed compositions used are described in Table 1.

2.2. Mixed culture

The parent photosynthetic culture used as inoculum in the

experiments was acquired from a lake at Saroor Nagar in Hy-

derabad, India [4]. Culture was sampled from a depth of 2–3 feet

of the water bodies. Prior to usage, the inoculum was enriched

with the combination of dairy and domestic wastewater (1:1)

supplementedwith vitamin solution (2.5 ml/l) in the presence of

fluorescent light (4 Klux) at 34 �C for 48 h. As the culture was

collected from lake with diverse characteristics and enriched

with wastewater, there might be possible presence of anaerobic

mixed consortia along with the predominant photosynthetic

mixed consortia [4].

2.3. Experimental design

A series of thirty-two batch experiments were performed with

each of the two organic acids (HAc/HBu) as metabolic

substrates in the presence or absence of glucose and vitamins,

variation in the type of nitrogen source and the gas used to

create anaerobic microenvironment to evaluate the photo-

biological H2 production (Table 1). The experimental varia-

tions were studied for each acid in a series of 250 ml conical

flasks (with a total working volume of 160 ml). A 20 ml of

previously developed mixed photosynthetic culture (24 h;

Saroor Nagar lake) was inoculated into the flask under aseptic

anaerobic conditions [4]. Subsequently 150 ml of feed (with

a final concentration of COD 5900� 100 mg/l) was loaded and

the reaction mixture was adjusted to the desired pH (6/7)

using concentrated orthophosphoric acid and/or 3 N NaOH

solutions. The flasks were then sparged with argon or nitrogen

for a period of 30 s to remove dissolved oxygen and to create

an oxygen free microenvironment prior to closing with rubber

septum (butyl rubber). All the flasks were placed in a temper-

ature controlled shaking incubator (100 rpm) in the presence

of a fluorescent light (4 Klux), at 34 �C.

Table 1 – Details of experimental variations studied.

Ex. No. Volatile fatty acid (HAc, 0.07 M;HBu, 0.05 M concentration)

Operating pH Co-substrate(1 g glucose/l)

Nitrogensource

Vitamins Sparinggas

1 HAc/HBu Acidophilic Yes AG Yes Argon

2 HAc/HBu Acidophilic Yes AG Yes Nitrogen

3 HAc/HBu Acidophilic Yes MSG Yes Argon

4 HAc/HBu Acidophilic Yes MSG Yes Nitrogen

5 HAc/HBu Acidophilic Yes AG No Argon

6 HAc/HBu Acidophilic Yes AG No Nitrogen

7 HAc/HBu Acidophilic Yes MSG No Argon

8 HAc/HBu Acidophilic Yes MSG No Nitrogen

9 HAc/HBu Acidophilic No AG Yes Argon

10 HAc/HBu Acidophilic No AG Yes Nitrogen

11 HAc/HBu Acidophilic No MSG Yes Argon

12 HAc/HBu Acidophilic No MSG Yes Nitrogen

13 HAc/HBu Acidophilic No AG No Argon

14 HAc/HBu Acidophilic No AG No Nitrogen

15 HAc/HBu Acidophilic No MSG No Argon

16 HAc/HBu Acidophilic No MSG No Nitrogen

17 HAc/HBu Neutral Yes AG Yes Argon

18 HAc/HBu Neutral Yes AG Yes Nitrogen

19 HAc/HBu Neutral Yes MSG Yes Argon

20 HAc/HBu Neutral Yes MSG Yes Nitrogen

21 HAc/HBu Neutral Yes AG No Argon

22 HAc/HBu Neutral Yes AG No Nitrogen

23 HAc/HBu Neutral Yes MSG No Argon

24 HAc/HBu Neutral Yes MSG No Nitrogen

25 HAc/HBu Neutral No AG Yes Argon

26 HAc/HBu Neutral No AG Yes Nitrogen

27 HAc/HBu Neutral No MSG Yes Agron

28 HAc/HBu Neutral No MSG Yes Nitrogen

29 HAc/HBu Neutral No AG No Agron

30 HAc/HBu Neutral No AG No Nitrogen

31 HAc/HBu Neutral No MSG No Agron

32 HAc/HBu Neutral No MSG No Nitrogen

HAc: acetic acid; HBu: butyric acid; Acidophilic: pH 6; Neutral: pH 7; AG: glutamic acid ammonium salt; MSG: mono sodium salt of glutamic acid.

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 ) 7 5 1 3 – 7 5 2 2 7515

2.4. Analytical methods

H2 gas generated during photo-biological process was esti-

mated using a microprocessor based electrochemical gas

sensor (ATMI GmBH Inc., Germany). Volatile fatty acids (VFA),

oxidation reduction potential (ORP), pH and COD (soluble,

dichromate closed refluxing method) were determined as per

the procedures outlined in standard methods [20]. The sepa-

ration and quantification estimation of soluble metabolites

were carried out by High performance liquid chromatography

(HPLC; Shimadzu LC10A) operated under optimized condi-

tions [UV–vis detector; C18 column – reverse phase column

–250� 4.6 mm and 5 m 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 injection –20 ml].

The performance of batch experiments was evaluated by

estimating the substrate (COD) removal efficiency (x) during

operation using Eq. (1). 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)

Organic loading rate based on COD (OLR – Kg COD/m3-day)

was calculated using Eq. (2), where F represents feed rate

(l/day) and V denotes the volume of substrate (l).

OLR ¼ f½CSo�F�=Vg (2)

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

calculated to study the rate and pattern of COD removal

during the cycle operation according to the Eq. (3), where, xt

represent substrate removal efficiency (%) at time ‘t’.

SDR ¼ ½OLR� xt=100� (3)

3. Results and discussion

3.1. Biohydrogen production

The experimental data illustrated the feasibility of utilizing

both HAc and HBu as metabolic substrates for H2 production by

photo-fermentation process apart from substrate degradation

(Fig. 1a). Among the two substrates studied, HAc showed

comparatively higher H2 production than HBu. Experiments

performed in the presence of glucose as co-substrate, vitamins

and MSG as nitrogen source when sparged with argon gas at

acidophilic pH registered higher H2 production with both HAc

(3.51 mol/kg CODR-day) and HBu (3.33 mol/kg CODR-day). The

experimental variations studied with HBu in presence of

glucose as co-substrate showed H2 production irrespective of

the nitrogen source used and gas sparged at both acidophilic

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

3231302928272625242322212019181716151413121110987654321Experiment Number

Sp

ecific H

2 p

ro

du

ctio

n (m

ol/kg

C

OD

R-d

ay)

Acetic acid Butyric acid

0

10

20

30

40

50

60

70

80

90

100

312927252321191715131197531

CO

D rem

oval efficien

cy (%

)

Experiment Number


0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40


SD

R (kg

C

OD

R/m

3-d

ay)


a

b

c

Fig. 1 – (a) Specific H2 production; (b) COD removal efficiency; (c) Substrate degradation rate (SDR) with the function of

experimental variations studied.


and neutral microenvironment. However, in the absence of

vitamins, acidophilic microenvironment showed lower H2

production. On the contrary, HAc as metabolic substrate

showed higher H2 production irrespective of the presence or

absence of glucose (co-substrate), vitamins, nitrogen source

used and gas sparged at both acidophilic and neutral micro-

environment. HAc evidenced good H2 yield among the two

acids studied which is due to its simple structure and easy


degradability [2]. Among the 32 experimental variations

studied with HAc, 20 experimental variations showed H2

production while in the case of HBu only 11 experimental

variations showed H2 production. This might be attributed due

to the prevalence of acetogenic bacteria present in the mixed

culture.

The experiments under acidophilic pH showed higher H2

production compared to the experiments operated at neutral

pH with same combination [HAc: 2.83 mol/kg CODR-day (pH 6)

and 2.23 mol/kg CODR-day (pH 7); HBu: 2.6 mol/kg CODR-day

(pH 6) and 1.54 mol/kg CODR-day (pH 7)]. The experiments

carried out at acidophilic pH without vitamin solution also

evidenced H2 production with both the acids. The presence of

vitamins enhanced the H2 production but did not showed any

significant effect on substrate removal [4]. Higher yields of H2

observed in the flasks sparged with nitrogen were irrespective

of the nitrogen source used (HAc: 3.1 mol/kg CODR-day; HBu:

2.71 mol/kg CODR-day). However, irrespective of the nitrogen

source [2.47 mol/Kg CODR-day(AG); 1.21 mol/Kg CODR-day

(MSG)] used HAc when sparged with nitrogen evidenced H2

production even at neutral pH. The experiments with HBu did

not showed any H2 production in the absence of glucose, but

HAc evidenced H2 production in the flasks sparged with argon

irrespective of the nitrogen source used at acidophilic pH

[1.98 mol/kg CODR-day (AG); 1.44 mol/kg CODR-day (MSG)].

However, irrespective of the nitrogen source used, H2 produc-

tion was observed at neutral pH when the solution was sparged

with nitrogen [0.504 mol/kg CODR-day (AG); 0.925 mol/kg

CODR-day (MSG)]. The experiments without both co-substrate

and vitamins also did not showed any H2 production with HBu

as primary substrate. But with HAc, H2 production was

observed in all the cases except with MSG and nitrogen

(sparged)at acidophilic pH [2.218 mol/kgCODR-day (argon; AG);

0.564 mol/kg CODR-day (nitrogen; AG); 1.934 mol/kg CODR-day

(argon; MSG)]. However, at neutral pH, H2 production was

observed only in the experiment with AG when sparged with

nitrogen (2.44 mol/kg CODR-day). The presence of MSG as

nitrogen source supports higher performance at neutral pH,

while AG contains one extra amino group in its structure,

which may be toxic to the organism and its lower activity

towards the degradation of substrate and H2 production [7].

3.2. Substrate degradation

Substrate degradation is a very important factor when treat-

ment of wastewater is considered. In the experimental varia-

tions studied, both higher (with MSG 92.96%; 1.22 kg CODR/

m3-day) and lower (with AG 69.07%; 0.90 kg CODR/m3-day) COD

removal efficiency was observed with HAc in the experiments

with only variation in nitrogen source while other experi-

mental conditions were same. In case of HBu, higher COD

removal efficiency (88%; 1.19 kg CODR/m3-day) was docu-

mented under similar experimental conditions as with HAc

and lower efficiency (74.33%; 1.00 kg CODR/m3-day) was

observed in the experiment with glucose in presence of vita-

mins, nitrogen source AG and sparged with nitrogen under

acidophilic condition (Fig. 1b & c). However, HAc showed

higher SDR at acidophilic pH while HBu showed higher SDR

compared to HAc at neutral pH. Comparatively neutral pH

seems to be favorable for higher substrate removal irrespective

of the experimental variations studied [(HAc: 1.22 kg CODR/

m3-day/92.96% (neutral pH) and 1.17 kg CODR/m3-day/89.35%

(acidophilic pH); HBu: 1.19 kg CODR/m3-day/88% (neutral pH)

and 1.14 kg CODR/m3-day/84.33% (acidophilic pH)]. Presence of

co-substrate showed enhanced SDR in all the cases HAc:

1.22 kg CODR/m3-day (92.96%) and 1.21 kg CODR/m3-day

(92.10%) – without glucose; HBu: 1.19 kg CODR/m3-day (88%)

and 1.04 kg CODR/m3-day (76.93%)-without glucose). The effect

of nitrogen source was clearly observed in the case of HAc. MSG

showed higher SDR compared to AG as nitrogen source irre-

spective of the other experimental variations (neutral: 0.90 kg

CODR/m3-day (AG) and 1.22 kg CODR/m3-day (MSG); acido-

philic: 1.10 kg CODR/m3-day (AG) and 1.17 kg CODR/m3-day

(MSG)). However, with HBu greater variation was not observed

among the two nitrogen sources used [neutral: 1.11 kg CODR/

m3-day (AG) and 1.19 kg CODR/m3-day (MSG); acidophilic:

1.03 kg CODR/m3-day (AG) and 1.14 kg CODR/m3-day (MSG)].

The presence of vitamins and the gas used to create anaerobic

microenvironment did not show any marked influence on

substrate degradation.

3.3. Bioprocess monitoring

The system pH is considered to be one of the crucial operating

parameters having significant influence on biological H2

production. Marked variation in pH was observed during the

study (Fig. 2a&b). After 24 h of operation, pH showed shift

towards acidophilic range. In the case of experiments per-

formed at pH 6, acidophilic phase was observed upto 48 h prior

to reversing to neutral condition. While in case of neutral

operations, after 24 h the acidophilic condition slowly turned

towardsbasic pHleading tosolventogenesis.Thevariation inpH

values strongly support higher H2 production rate in acidophilic

conditions.Acidogenicbacteriapresent asco-culture with inthe

photosynthetic mixed culture might be responsible for the

initial acidophilic condition observed in the experiments. Shift

towards neutral/basic microenvironments from acidophilic

condition suggests the consumption of soluble organic acid

metabolites. This further corroborated well with the variation in

total VFA concentration observed during the study.

VFA concentrations also evidenced a good correlation with

the H2 production and substrate degradation (Fig. 3a&b).

Initially lower concentration of VFA was observed upto 24 h of

operation and reached a peak followed by a surge prior to

stabilization with both the substrates. The sudden increase in

VFA (from �400 mg/l to �1600 mg/l) resulted sharp drop in

corresponding pH values which favored H2 production. Gener-

ally the photosynthetic organisms utilize VFA (organic acids) as

substrates besides theoriginal substrates in photo-biological H2

production process [4,11,13,16]. The experiments performed

with organic acids as the only carbon source (without initiator

substrate) also showed increase in VFA concentration before

dropped. This may be attributed to substrate used (combination

of dairy and domestic wastewater) for the enrichment of

culture which might possess the residual substrate [4]. The

observed VFA utilization in most of the experimental variations

studied during photo-biological H2 generation enumerates its

participation in the process. The increase in hydraulic retention

time (96 h) showed visible variations in pH and VFA during the

present experimental study.

4

5

6

7

8

9

3231302928272625242322212019181716151413121110987654321

Experiment Number

pH

Inlet 24 hrs 48 hrs 72 hrs 96 hrsAcetic acid

4

5

6

7

8

9

3231302928272625242322212019181716151413121110987654321

Experiment Number

pH

Inlet 24 hrs 48 hrs 72 hrs 96 hrs

Butyric acid

a

b

Fig. 2 – pH variations observed during photo-fermentative H2 production (a. acetic acid; b. butyric acid).


The composition of soluble acid metabolites viz., HAc,

HBu and propionic (HPr) along with ethanol (EtOH) was also

determined. The concentration of soluble metabolites and

its utilization during the period of operation showed a good

correlation with both H2 production and substrate degrada-

tion. The presence of HAc, HBu and HPr along with EtOH was

observed in the outlets generated from photo-biological

process irrespective of the substrate used. However, the

distribution of metabolites varied in accordance with the

nature of substrate. The outlets from both HAc and HBu

showed a major amount of HAc and HBu with significant low

concentration of EtOH at acidophilic operation. On the

contrary, the outlets of HAc operated at neutral pH evi-

denced a greater amount of EtOH and HPr along with lower

amount of HAc and HBu. H2 production through a combina-

tion of dark and light fermentations is of great importance

due to its dual function in removing additional substrate

present in wastewater along with additional H2 produc-

tion [2,11]. The photo-fermentative bacteria therefore

utilizes the soluble acid intermediates generated from the

primary acidogenic process resulting in the generation of

additional hydrogen along with higher treatment efficiency

[2–4,11].

3.4. Combined process evaluation

Data envelopment analysis (DEA) is a frontier analysis tech-

nique to measure the performance by evaluating the relative

efficiency adapting graphical approach [4,10,21–24]. DEA

methodology helps to enumerate the combined effect of the

process by integrating the two outputs. DEA procedure was

used to analyze the relative performance of 64 experimental

variations studied, considering VFA as input and specific H2

production and SDR as output parameters for model analysis

(Fig. 4). The relative efficiency of the system can be calculated

as shown in Eq. (4) by comparing the current performance of

the system to the best possible performance that the system

could reasonably be expected to achieve.

Relative efficiency of the system ¼ ½ðX=YÞ � 100� (4)

0

200

400

600

800

1000

1200

1400

1600

1800


VF

A (m

g/l)

Inlet 24 hrs 48 hrs 72 hrs 96 hrsAcetic acid

0

200

400

600

800

1000

1200

1400

1600

1800

2000


VF

A (m

g/l)

Inlet 24 hrs 48 hrs 72 hrs 96 hrsButyric acid

a

b

Fig. 3 – Variation in VFA observed during photo-biological H2 production (a. acetic acid; b. butyric acid).


Where, X represents length of the line from the origin to the

point obtained by plotting two ratios of the system (H2/VFA

and SDR/VFA) and Y denotes length of the line from the origin

through the point obtained by the system to the efficient

frontier. Efficient frontier line was drawn as a horizontal line

from the Y-axis enveloping experiment nos. A2, A20 and B18

finally joining the X-axis (Fig. 2; Table 2). This line represents

a standard of performance from the experimental variations

studied. The best possible performance that a system could be

expected to achieve was given by the point where the line

from the origin through the point of that system meets the

efficient frontier line. Combined performance of experiments

with respect to H2 production and wastewater treatment was

visualized by assessing relative efficiency from DEA with

respect to the sixty-four experiments with HAc and HBu. Of

the experiments studied, A2, A20 and B18 are 100% efficient,

A22 and A28 are above 90% and A1, A3, A6, A7, A26, B1 and B3

are above 80%. The final output showed combined process

efficiency of 100% with three systems (A2, A20 and B18),

among the sixty-four studied.

Three experiments showed 100% efficiency among which

two experiments used HAc. The experiments with glucose in

the presence of vitamins and nitrogen source (AG) showed

100% process efficiency at acidophilic pH in the case of HAc

(A2) and at neutral pH in the case of HBu (B18). The experi-

ments with the same conditions having different nitrogen

source (MSG instead of AG) at neutral pH also evidenced 100%

efficiency in the case of HAc (A20). Only two experiments

0.000

0.010

0.020

0.030

0.040

0.00350.00300.00250.0020SDR/VFA

H2/V

FA

B2

A3B1

B7

B4A4

A9

B3A1 A22A6

B8A8

A18

B20A24

B6

A30A7

A2

A20

B18

A13

A26

B17

A14B5

A28A5

A15

A11

Fig. 4 – Data enveloping analysis (DEA) profile.


showed process efficiency between 90 and 100% among the

sixty-four studied with HAc. The experiments performed with

glucose, nitrogen source (AG) and sparged with nitrogen gas in

absence of vitamins at neutral pH depicted combined process

Table 2 – Relative and combined process efficiency (H2

production and wastewater treatment) of theexperiments variation studied based on DEAmethodology.

Experiment number Relative efficiency (%)

A1 82.76

A2 100

A3 88.57

A4 57.34

A5 69.33

A6 82.43

A7 82.76

A8 75.94

A9 54.05

A11 71.12

A13 72.41

A14 54.90

A15 78.32

A18 74.67

A20 100

A22 93.19

A24 68.96

A26 86.79

A28 92.72

A30 79.31

B1 89.28

B2 64.52

B3 86.62

B4 49.65

B5 65.33

B6 62.24

B7 50.67

B8 69.39

B17 51.85

B18 100

B20 51.72

A: acetic acid; B: butyric acid.

efficiency of 93.19% while the one without glucose in the

presence of vitamins, nitrogen source (MSG), sparged with

nitrogen at neutral pH evidenced an efficiency of 92.72%.

Among the sixty-four experiments performed seven systems

showed a relative efficiency in the range of 80–90%, among

which five experiments are with HAc. The experiments in

presence of glucose and vitamins at acidophilic pH sparged

with argon showed efficiency of about 80–90% irrespective of

the nitrogen source and substrate used [82.76% (AG); 88.57%

(MSG) HBu: 89.28% (AG); 86.62% (MSG)]. The experiment per-

formed without glucose in the presence of vitamins, nitrogen

source AG and sparged with nitrogen at neutral pH also evi-

denced an efficiency of 86.79%. The experiment without

vitamins but in presence of glucose and nitrogen source MSG

and sparged with argon at acidophilic conditions evidenced

an efficiency of 82.76% while experiments with the same

conditions except nitrogen source (AG) and sparging (nitrogen

instead) evidenced a process efficiency of 82.43%.

Experimental data documented the feasibility of utilizing

organic acids as primary substrates in the process of photo-

biological H2 production. H2 production can be improved by

integrating dark fermentation process with photo-fermenta-

tion process [11]. Photosynthetic culture is generally capable

of producing H2 by utilizing organic acids generated from

acidogenic H2 fermentation (dark) process [4,11,14,17,18].

Photosynthetic bacteria can use small chain organic acids as

electron donors for the production of H2 at the expense of light

energy and have an advantage over the acidogenic bacteria in

terms of high theoretical conversion [4,7,11,16].

Butyrate: C4H8O2þ 6H2O / 10H2þ 4CO2 (5)

Acetate: C2H4O2þ 2H2O / 4H2þ 2CO2 (6)

In addition to H2 production, photo-biological process also

showed substrate degradation and combined process effi-

ciency which was important when wastewater is used as

substrate. This study indicates the integration feasibility of

dark fermentative process with photo-biological H2 production


resulting in higher H2 production as well as substrate removal.

However, the performance was found to depend on the nature

of organic acid used as substrate, co-substrate, presence of

vitamins, nitrogen source and nature of gas used for creating

anaerobic microenvironment. Combined process efficiency

studies performed with DEA method depicted 12 experi-

mental sets which were in the relative efficiency range of

80–100% among the 64 experimental sets studied (Table 2).

Evidently most of these experimental sets used acetate as

carbon source indicating its positive role on overall process

performance. Glucose as initiator substrate (co-substrate)

showed marked influence on H2 production as well as on

substrate degradation. Almost all the systems in the range of

80–100% efficiency were observed to be associated with

glucose supplementation suggesting the influence of initiator

substrate. Dark fermentative effluents with higher residual

carbon source along with HAc as major component of soluble

metabolites reported to generate higher H2 production in

subsequent photo-biological process [11]. Presence of nitrogen

in effluents showed marked influence on H2 production.

Although the nitrogenase reaction is a highly energy-

consuming process [25] heterocystous N2 fixing cyanobacteria

have an unique ability to sustain H2 production with simple

nutritional requirements [26]. The overall energy-consuming

reaction as explained by Miyake et al., [27] is as follows

N2þ 8Hþ þ 8e� þ 16ATP / 2NH3þH2þ 16ADPþ 16Pi. (7)

The ability of N2 fixation by photo-heterotrophic bacteria or

cyanobacteria is catalyzed by the nitrogenase enzyme which

also catalyses the evolution of H2, particularly in the absence

of N2. This supports the poor H2 production observed in the

presence of N2 compared to the argon in the study. However

for combined process efficiency, presence of nitrogen showed

positive influence. Nitrogen source is also one of the essential

factors for the effective growth of organisms and substrate

utilization [7]. Among the two nitrogen sources used MSG

showed higher H2 production compared to AG irrespective of

the organic acid used as substrate. However, combined

process efficiency evidenced higher performance with AG as

nitrogen source both with HAc and HBu at acidophilic and

neutral pH respectively. MSG as nitrogen source evidenced

higher performance with HAc at neutral pH. Major variation in

the case of combined process efficiency was not observed with

the type of gas used to create anaerobic microenvironment

and the nitrogen source used. Presence of glucose as co-

substrate along with vitamins, AG as nitrogen source and

nitrogen sparging supported higher combined process effi-

ciency with respect to H2 production and substrate removal at

both acidophilic and neutral pH.

4. Conclusions

The experimental data documented H2 production utilizing

acetate and butyrate as primary metabolic substrate along

with wastewater treatment through photo-fermentation

process using mixed culture. Relatively higher H2 yields were

observed with acetic acid compared to butyric acid.

Acidophilic microenvironment seems to be favorable for

higher H2 production. Presence of carbon source as co-

substrate and vitamins also showed positive influence on H2

production. Mono sodium salt of glutamic acid (MSG) evi-

denced higher H2 production compared to the amino glutamic

acid (AG) as nitrogen source. Argon induced anaerobic

microenvironment showed higher H2 production compared to

corresponding nitrogen induced microenvironment. The

presence of co-substrate also showed positive influence on

substrate removal in all the cases however, vitamins presence

and nature of the gas used for creating anaerobic microenvi-

ronment did not show any marked influence on the substrate

degradation. Data enveloping analysis (DEA) was successfully

applied to assess the combined process efficiency with respect

to H2 production and wastewater treatment. Presence of

carbon source as initiator along with vitamins, amino gluta-

mic acid as nitrogen source and sparging with nitrogen

showed higher combined process efficiency at both the

acidophilic and neutral pH. Combination of dark and photo-

fermentation appears to be an ideal route which might lead to

the economic viability of biohydrogen production process

when wastewater is used as substrate.

Acknowledgments

Authors wish to express their deep sense of gratitude to Dr J.S.

Yadav, Director, Indian Institute of Chemical Technology for

his encouragement and support. SS duly acknowledges

Council of Scientific and Industrial Research (CSIR) for

providing Senior Research Fellowship.

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