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Technical Communication

Enhancing biohydrogen production through sewagesupplementation of composite vegetable based market waste

G. Mohanakrishna, R. Kannaiah Goud, S. Venkata Mohan*, P.N. Sarma

Bioengineering and Environmental Centre, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500 607, India

a r t i c l e i n f o

Article history:

Received 11 September 2009

Received in revised form

27 October 2009

Accepted 1 November 2009

Available online 18 November 2009

Keywords:

Anaerobic consortia

Volatile fatty acids

Treatment

Carbohydrates

Co-substrate

* Corresponding author. Tel.: þ91 40 2719166E-mail address: vmohan_s@yahoo.com (S

0360-3199/$ – see front matter ª 2009 Profesdoi:10.1016/j.ijhydene.2009.11.002

a b s t r a c t

The function of domestic sewage supplementation as co-substrate with composite vege-

table based market waste was studied during the process of fermentative hydrogen (H2)

production. Significant improvement in H2 production and substrate degradation were

noticed upon supplementing the waste with domestic sewage. Maximum H2 production

(cummulative) was observed at 5.2 kg COD/m3 with pulp operation and 4.8 kg COD/m3 with

non-pulp operation accounting for improvement of 51 and 55% respectively after sewage

upplementation. Substrate degradation was also found to improve with respect to both

carbohydrates [8% (with pulp); 5% (non-pulp)] and chemical oxygen demand [COD, 12%

(with pulp); 13% (non-pulp)] after adding domestic sewage. Specific H2 yield improved

especially at lower concentrations. Supplementation of waste with co-substrate helps to

maintain good buffering microenvironment supports fermentation process and in addition

provides micro-nutrients, organic matter and microbial biomass. Variation in the outlet pH

was less in supplementation experiments compared to normal operation.

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

1. Introduction supplementing the deficient nutrients [18] and also provides

Generation of waste is an integral part of society. Exploitation

of these wastes as substrate for H2 production with simulta-

neous treatment is one of the attractive and effective ways of

tapping clean energy from renewable resources in a sustain-

able approach [1–7]. This provides dual environmental bene-

fits like renewable energy generation and waste stabilization

[8–17]. Market based solid waste of vegetable origin is

considered to be one of the potential wastes with higher

organic composition and easily biodegradable nature which

can be used to harness energy.

Co-substrate supplementation usually improves the biogas

yields from anaerobic digester due to the positive synergy,

established in the digestion medium by means of

4.. Venkata Mohan).sor T. Nejat Veziroglu. Pu

proper C/N ratio [19]. A more stable fermentation process is

achieved because the buffering capacity is improved [20]. Co-

digestion was reported to increase biodegradable organic

fraction of substrate [21,22]. Application of co-substrates helps

to improve the overall economics of the plant [23]. Moreover,

anaerobic fermentation was reported to be more stable when

a variety of substrates were applied at the same time

[18,22,23]. In this communication, we made an attempt to

study the feasibility of supplementing domestic wastewater

as co-substrate to enhance H2 production from the fermen-

tation of composite vegetable based market waste. The effect

of co-substrate on H2 production process was compared with

already reported studies from our laboratory using composite

vegetable based wastewater as substrate without any

blished by Elsevier Ltd. All rights reserved.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 5 3 3 – 5 4 1534

co-substrate [25]. The experiment was also performed with

pulp (cellulosic) and non-pulp substrates to evaluate the role

of cellulosic content of the vegetable waste on the fermenta-

tion process.

2. Materials and methods

2.1. Composite vegetable waste

The vegetable based solid waste was collected from the waste

dumping yard at the market [25]. The waste was composite in

nature with different types of unused/residual vegetables like

tomato, potato, carrot, cabbage, brinjal, beet-root, okra, coc-

cinia, etc. After getting to laboratory, the waste was masti-

cated in an electrical mixer and the resulting slurry was used

in the experiment in two variations viz., extract with pulp and

extract without pulp (Table 1). Pulp was removed from the

extract through filtration. Extracted waste has high concen-

trations of COD and carbohydrates. Domestic sewage (COD,

420 mg/l; BOD5, 320 mg/l; carbohydrates, 235 mg/l; nitrates,

115 mg/l; chlorides, 350 mg/l; alkalinity 280 mg/l, pH 7.2,

microbial composition 1.8 � 104 CFU/ml) was used in diluting

the masticated vegetable waste which acts as co-substrate in

the reactor.

2.2. Enriched acidogenic mixed consortia

Anaerobic mixed microflora from an operating lab-scale

upflow anaerobic sludge blanket (UASB) reactor, where

chemical based wastewater is being used as feed was

employed as parent inoculum. Prior to use the culture was

selectively enriched for three times in nutrient broth (32 �C;

120 rpm; 48 h) at pH 6 under anaerobic conditions [26].

Subsequently, the culture was subjected to repeated

pretreatment (three times) by changing between heat-shock

[100 �C; 2 h], acid [pH 3 adjusted with orthophosphoric acid

(88%); 24 h] and chemical [2-bromoethane sulphonic acid

sodium salt solution (BESA), 0.2 g/l; 24 h] methods to restrain

the growth of methanogenic bacteria (MB) and to selectively

Table 1 – Details of operational conditions used in batch ferme

Experiment No. SubstrateLoading

(kg COD/m3)

Weight of rawvegetablewaste (g)

Amoobtaine

masticat

With pulp

C1 5.2 32 16

C2 11 64 33

C3 20 128 66

C4 32 192 99

C5 40 256 132

Without pulp

C6 4.8 32 16

C7 9.7 64 32

C8 19 128 64

C9 30 192 96

C10 36 256 128

enrich the H2 producing acidogenic bacteria (AB) [2,26,27]. The

resulting enriched mixed culture was used as inoculum in the

batch fermentation experiments.

2.3. Experimental design

Batch experiments were performed to evaluate the feasibility

of biohydrogen production from vegetable based waste after

supplementing with domestic sewage as co-substrate. In

total, two experimental sets were designed (each set contains

5 experiments both with pulp and without pulp substrate)

(Table 1). All the batch experiments were performed in

specifically fabricated anaerobic flasks (250 ml) in duplicate.

Three separate flasks were used (two replicates and one blank)

for each set of experimental variation. The experiments were

studied separately for two types of substrates viz., wastewater

with pulp and wastewater without pulp. Both the substrates

were diluted with domestic sewage (co-substrate). All the

experiments were performed with un-sterilized substrate

therefore one blank was maintained parallely for each set of

experimental run. Each flask was inoculated with 20 ml of

selectively enriched consortia (VSS of 4.5 g/l) along with 160 ml

of feed under aseptic conditions in anaerobic chamber. The

flasks were tightly capped with rubber septum (butyl rubber)

and placed in temperature controlled orbital shaker (120 rpm;

32 �C). Prior to loading, feed pH was adjusted to 6.0 using

concentrated orthophosphoric acid or 3 N NaOH. Process

evaluation was observed at various substrate loading condi-

tions [with pulp, 5.2, 11.0, 20.0, 32.0 and 40.0 kg COD/m3;

without pulp, 4.8, 9.7, 19.0, 30.0 and 36.0 kg COD/m3] (Table 1).

Each set of experiments were studied for 6 cycles by changing

fresh feed once per cycle and biomass was retained in the

reactor. The results presented here were an average of all

the studied cycles, which correlated well (R2- 0.9011) with all

the experimental variations studied.

2.4. Process monitoring

H2 generated during the experiment was estimated using

a microprocessor based pre-calibrated H2 sensor (ATMI GmbH

ntative experiments.

untd afterion (ml)

Domesticwastewaterused for thedilution (ml)

Totalcarbohydrates

(g/l)

Fermentationperiod (h)

.5 148.5 2.1 144

132 4.5 168

99 7.9 216

66 9.5 216

33 12.1 240

149 1.7 144

133 3.8 168

101 7.1 216

69 8.6 216

37 11.3 240

0 40 80 120 160 200 240

0

5

10

15

20

25

0

5

10

15

20

25

4.8 kg COD/m3

9.7 kg COD/m3

19.0 kg COD/m3

30.0 kg COD/m3

36.0 kg COD/m3

Time (h)

H2

)y

ad/l

om

m(e

ta

rn

oit

cu

do

rp

5 .2 kg COD/m3

11.0 kg COD/m3

20.0 kg COD/m3

32.0 kg COD/m3

40.0 kg COD/m3

With Pulp

Without Pulp

Fig. 1 – Hydrogen production with the function of

fermentation time at different organic loading conditions

in concurrence with substrate composition.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 5 3 3 – 5 4 1 535

Inc., Germany) where the output signal displayed the %

volume of H2. The separation and quantitative determination

of the soluble metabolites composition was performed by the

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]. The performance was also assessed by

monitoring chemical oxygen demand (COD- potassium

dichromate closed refluxing titrimetric method), volatile sus-

pended solids (VSS), nitrates, chlorides, pH and volatile fatty

acids (VFA) according to the standard methods [28]. Total

carbohydrates were analyzed by anthrone method [25]. After

monitoring H2 and collecting samples for analysis, the

contents of the flasks were discarded safely.

Table 2 – Influence of co-substrate supplementation on biohydmarket wastea.

Pulp operation

Maximumwithout

co-substrateaddition [25]

Improvementwith co-substrate

addition

Impro(

Cumulative H2

production (CHP)

26.8 mmol 13.7 mmol 51.

Specific H2 Yield (SHY) 12.2 mol/kg CODR 2.1 mol/kg CODR 16.

Carbohydrate removal 6.1 kg/m3 0.8 kg/m3 7.5

COD removal 16 kg/m3 4 kg/m3 12.

Outlet pH variation 4.6–5.5 4.3–6.9 –

VFA 8971 mg/l 1901 mg/l 123

a Compared with experimental data performed under same conditions w

3. Results and discussion

3.1. Biohydrogen production

Supplementation of domestic sewage as co-substrate to

vegetable waste showed positive influence on both H2

production and substrate degradation (Fig. 1). After sewage

supplementation, marked improvement in overall process

efficiency including system buffering capacity was noticed

compared to non-supplemented operation [25]. Comparative

assessment of supplemented and non-supplemented opera-

tions were depicted in Table 2 based on important output

parameters. Enhanced cumulative H2 production (CHP) was

observed by 13.7 mmol (5.2 kg COD/m3; with pulp operation)

and 18.53 mmol (4.8 kg COD/m3; non-pulp operation)

accounting for percent improvement of 51.1% and 55.3%

respectively. It is also evident from experimental data that the

substrate concentration (organic load) and composition

showed marked influence on the overall process performance.

Non-pulp based substrate had evidenced the higher H2

production compared to the pulp based substrate (Table 3). A

steady improvement in H2 production rate was observed with

pulp based operation from 5.2 kg COD/m3 (18.4 mmol/day;

48 h) to 20 kg COD/m3 (22.4 mmol/day; 48 h) which sustained

upto 32 kg COD/m3 (22.4 mmol/day; 48 h) followed by drop in

the performance at 40 kg COD/m3 (20.97 mmol/day, 48 h).

Cumulative H2 production (CHP) showed a gradual improve-

ment with increase in organic load from 5.2 kg COD/m3 to

40 kg COD/m3. Higher H2 production was observed during

operation in absence of pulp except at higher load conditions.

Inconsistent trend in H2 production was noticed from 4.8 kg

COD/m3 (20.2 mmol/day; 72 h) upto 19 kg COD/m3 (24.1 mmol/

day; 48 h) and showed a decreasing trend from 30 kg COD/m3

(23.9 mmol/day; 48 h) to 36 kg COD/m3 (19.8 mmol/day; 48 h).

In spite of the variations observed in H2 production rate, CHP

showed a steady improvement in concurrence with the

organic load especially from 4.4 to 19 kg COD/m3 and

decreased thereafter. After supplementing, the outlet pH

varied between 4.3 and 6.9 (with pulp) and 4.6 and 6.8

(without pulp). pH showed specific shift towards acidic

rogen process during fermentation of composite vegetable

Non-pulp operation

vement%)

Maximumwithout

co-substrateaddition [25]

Improvement withco-substrate

addition

Improvement(%)

1% 33.5 mmol 18.5 mmol 55.3%

8% 13.4 mol/kg CODR 1.5 mol/kg CODR 10.5%

% 7.6 kg/m3 0.5 kg/m3 4.7%

5% 14 kg/m3 4 kg/m3 13.3%

4.5–4.9 4.6–6.9 –

.4% 4556 mg/l 2039 mg/l 75.8%

ith same waste without domestic sewage supplementation [25].

micro

en

viro

nm

en

t[v

arie

db

etw

een

4.5

an

d4.9

(with

ou

tp

ulp

);

4.6

an

d5.5

(with

pu

lp)]in

the

ab

sen

ceo

fsu

pp

lem

en

tatio

n[2

5].

Th

ed

ilutin

gp

rop

ortio

ns

of

vegeta

ble

wa

steex

tract

to

do

mestic

sew

age

va

ried

betw

een

0.1

1to

4.0

an

d0.1

1to

3.4

6in

the

case

of

pu

lpa

nd

no

n-p

ulp

op

era

tion

sre

spectiv

ely

wa

s

use

din

this

stud

y.A

mo

ng

these

con

ditio

ns,0

.11

ratio

(C1

an

d

C6)

registe

red

ma

xim

um

imp

rov

emen

tin

H2

pro

du

ction

.

Imp

rov

ed

bu

fferin

gen

viro

nm

en

tw

as

ob

serv

edb

yth

e

ad

ditio

no

fd

om

estic

wa

stea

sco

-sub

strate

.Bu

fferin

gn

atu

reis

Table 3 – Consolidated data of batch fermentation experim nts performed vegetable waste when supplemented with sewage as the co-substrate.

Experiment No. Substrateloading

(kg of COD/m3)

Max H2

production(mmol/day)at time (h)

Cum lative H2

pro uction(CH ) (mmol)

Specific H2

Yield (SHY)(mol/kg CODR)

Carbohydrateremoval(kg/m3)

Carbohydrateremoval (%)

CODremoval

(kg/m3)/%COD removal

VFAproduction

(mg/l)

Outlet pH

With pulp

C1 5.2 18.4 � 1.2 (48) 40 4 � 3.0 21.3 � 0.4 1.5 � 0.1 70.9 � 5.8 1.9/36.5 578 � 26 6.9 � 0.1

C2 11 18.9 � 1.3 (48) 51 7 � 3.9 10.4 � 0.5 2.9 � 0.2 66.1 � 3.9 4.9/45.3 3172 � 113 6.8 � 0.1

C3 20 22.5 � 1.1 (48) 66 5 � 3.9 6.7 � 0.4 5.2 � 0.2 66.3 � 2.8 10.0/50.0 3442 � 138 5.9 � 0.2

C4 32 22.5 � 0.7 (48) 70 2 � 2.4 3.5 � 0.2 6.9 � 0.4 72.9 � 4.0 20.0/62.5 7272 � 342 4.3 � 0.2

C5 40 20.9 � 0.8 (48) 85 9 � 2.7 3.2 � 0.3 9.6 � 0.4 79.4 � 2.9 26.8/67.0 6581 � 365 6.8 � 0.3

Without pulp

C6 4.8 20.2 � 1.3 (48) 52 0 � 4.2 25.8 � 0.6 1.2 � 0.1 68.6 � 8.1 2.0/42.1 548 � 39 6.8 � 0.1

C7 9.7 19.7 � 1.0 (72) 54 1 � 4.6 12.7 � 0.6 2.6 � 0.2 69.5 � 5.0 4.3/43.9 1161 � 210 6.7 � 0.2

C8 19 24.2 � 2.1 (48) 69 5 � 4.7 8.1 � 0.5 4.7 � 0.2 65.9 � 2.8 8.6/45.3 4730 � 270 4.6 � 0.2

C9 30 23.9 � 0.6 (48) 63 7 � 3.3 3.5 � 0.3 5.9 � 0.3 69.2 � 3.5 18.0/60.0 4628 � 239 4.8 � 0.2

C10 36 19.8 � 1.4 (48) 51 5 � 4.8 2.1 � 0.4 8.1 � 0.4 71.7 � 3.6 25.0/69.4 4428 � 168 6.9 � 0.2

5.2

11

20

32

40

0 2 4 6 8

10

5.2

11

20

32

40

50

60

70

80

Org

an

ic

lo

ad

(k

gC

OD

/m

)

)%(lavomeretardyhobraC

Org

an

iclo

ad

(k

gC

OD

/m

3

)

m/gk(lavomeretardyhobraC3

)

a

4.8

9.7

19

30

36

0 2 4 6 8

Org

an

iclo

ad

(k

gC

OD

/m3)

m/gk(lavomeretardyhobraC3)

b

0 4 6 8

10

12

05

10

15

20

25

30

35

40

45

Org

an

ic

lo

ad

(kg

CO

Dm

3/d

ay)

m/gk(lavomeretardybarC3

)

With

pu

lp

With

ou

tp

ulp

c

Fig

.2

–C

arb

oh

yd

rate

rem

ov

al

with

the

fun

ction

of

org

an

ic

loa

d(a

)w

ithp

ulp

(b)

with

ou

tp

ulp

(inse

rted

fig

ure

s

ind

icatin

gca

rbo

hy

dra

tere

mo

va

leffi

cien

cyfo

rre

spectiv

e

typ

eo

ffe

ed

);(c)

Co

rrela

tion

co-e

fficie

ncy

of

carb

oh

yd

rate

rem

ov

al

an

do

rga

nic

loa

dfo

rb

oth

feed

com

po

sition

s.

in

te

rn

at

io

na

ljo

ur

na

lo

fh

yd

ro

ge

ne

ne

rg

y3

5(2

01

0)

53

3–

54

15

36

the

cap

ab

ilityo

fa

na

qu

eo

us

med

ium

tom

ain

tain

stab

leio

nic

e

ud

P

.

.

.

.

.

.

.

.

.

.

2

ho

0 24 48 72 96 120 144 168 192 216 240

0

5

10

15

20

25

0

5

10

15

20

25

5.2 kg COD/m3

11.0 kg COD/m3

20.0 kg COD/m3

32.0 kg COD/m3

40.0 kg COD/m3

Time (h)

m/g

k(

la

vo

me

rD

OC

3

)

4 .8 kg COD/m3

9.7 kg COD/m3

19.0 kg COD/m3

30.0 kg COD/m3

36.0 kg COD/m3

y = 0.7207x - 2.8604 [R2

= 0.9904]

y = 0.7241x - 2.8342 [R2

= 0.9701]

0

5

10

15

20

25

30

0 5 10 15 20 25 30 35 40 45

Organic load (kg COD m3

/day)

m/

DO

Cg

k(

la

vo

me

rD

OC

3

)

With pulp

Without pulp

a

b

With Pulp

Without Pulp

Fig. 3 – (a) COD removal with the function of fermentation time at different organic loading conditions in concurrence with

substrate composition; (b) correlation co-efficiency of organic load and COD removal for both feed compositions.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 5 3 3 – 5 4 1 537

strength by resisting the changes in the system pH against

acidic and basic constituents [18]. Alkalinity is generally

considered as an agent that provides buffering to the system

microenvironment in the anaerobic bioreactor. Experiments

operated with vegetable waste showed to have lower amounts

of alkalinity (52–280 mg/l) [25] compared to the experiments

operated with domestic waste as co-substrate (320–465 mg/l).

H2 production is associated with the generation of VFA which

in turn results in pH drop. Lower pH, below 4.5 inhibits the

activity of the enzymes (hydrogenase) involved in H2 produc-

tion. Maintaining high alkalinity conditions favors H2 produc-

tion. After adding co-substrate, the pH has maintained

between 5.1 and 6.5 which is considered to be favorable for H2

production (Table 2). While in the case of non-

supplementation operation the pH was maintained between

4.5 and 5.5. The positive shift in pH values against extreme

acidic condition observed after co-substrate supplementation

helped to increase the duration of H2 production period due to

feasible buffering microenvironment (Table 2). Bicarbonates

present in the system, increases the duration of H2 production

by maintaining its pH due to the buffering capacity of the

system [28]. Mild acidic microenvironment facilitates the

optimum system’s buffering capacity required for a good H2

production. Generally, the extract of vegetable based waste

contains relatively less concentration of micro-nutrients [18].

Supplemented domestic sewage can provide additional micro-

nutrients, organic matter and microbial consortia which might

have positive synergism with the resident mixed consortia on

5

10

15

20

Sp

ec

ific

H

2 y

ie

ld (

mo

l/k

g C

OD

R)

CO

D re

mo

va

l (%

)

40.032.022.011.05.2

Specific H2 yield

% CODR

O rganic Loading (kg COD/m3)

30

40

50

60

70

0

5

10

15

20

25

4.8

30

40

50

60

70

Specific H2 yield

% CO DR

36.030.019.09.7

O rganic Loading (kg CO D/m3)

Sp

ec

ific

H2 y

ield

(m

ol/

kg

CO

DR)

CO

D re

mo

va

l (%

)

y = -0.4524x + 18.795 [R = 0.7663]

y = -0.6582x + 23.527 [R = 0.828]

0

5

10

15

20

25

30

0 5 10 15 20 25 30 35 40 45

Organic load (kg COD m3

/day)

SH

Y (m

ol/k

g C

OD

R)

With pulp

Without pulp

a

b

c

y = 3.2011x - 4.0249 [R = 0.9695]

y = 3.456x - 4.0384 [R = 0.9467]

0

5

10

15

20

25

30

0 2 4 6 8 10 12

Carbohydrate removal (kg/m3

)

CO

D re

mo

va

l (k

g C

OD

/m

3

)

With pulp

Without pulp

d

Fig. 4 – Specific hydrogen yield (SHY) versus organic load in concurrence with substrate composition [(a) with pulp; (b)

without pulp]; [c] Correlation co-efficiency between organic load vs carbohydrate removal; (d) Correlation co-efficiency

between carbohydrate removal vs COD removal.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 5 3 3 – 5 4 1538

overall process efficiency. Micro-nutrients along with alka-

linity improve the buffering nature of the system [18,21,24].

3.2. Substrate degradation

Substrate removal during H2 production was evaluated by

estimating the substrate (COD and carbohydrates) removal

efficiency (x) using Eq. (1), where CSO represents either COD or

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

COD/carbohydrate concentration (mg/l) in the outlet [14,15].

x ¼ ½ðCSO � CSÞ=CSO� (1)

3.2.1. Carbohydrates removalDuring metabolic process, anaerobic consortia hydrolyzes the

complex carbohydrates to simple carbohydrates. In acido-

genic step, simple carbohydrates are converted to VFAs

associated with H2 production. Higher concentration of

carbohydrates was especially observed with pulp operation.

Sewage supplementation also showed enhanced degradation

efficiency of carbohydrates compared to normal operation [25]

(Table 2). Carbohydrate removal improved by 0.8 kg/m3 (32 kg

COD/m3; with pulp operation) and 0.5 kg/m3 (30 kg COD/m3;

non-pulp operation) accounting for percent improvement of

7.5% and 4.5% (Table 2). Carbohydrate removal was observed

to be more or less same in both the feed compositions studied

(Fig. 2 a, b). Carbohydrate removal efficiency was relatively

higher at lower load conditions and as the load increased the

removal efficiency was found to decrease in both the feed

compositions studied. At 5.2 kg COD/m3 carbohydrate

removal efficiency of 70.9% was registered with pulp operation

accounting for the removal of 1.5 kg carbohydrates/m3. At

loading conditions of 11 and 20 kg COD/m3, the efficiency of

carbohydrate removal was observed to be almost equal (66.1%

and 66.3%) with marked variation in the total amount of

carbohydrate removal (2.9 kg carbohydrates/m3 and 5.2 kg

carbohydrates/m3) respectively. Maximum carbohydrate

removal (79.4%; 9.6 kg carbohydrates/m3) was registered at

40 kg COD/m3 followed by a decrease at 32 kg COD/m3 (72.9%,

6.9 kg carbohydrate/m3). In the case of non-pulp operation

also carbohydrate removal efficiency varied inconsistently

between 65.9% (19 kg COD/m3) and 71.7% (36 kg COD/m3)

(Fig. 2). Relatively good correlation was observed between

organic load and carbohydrate removal (R2-0.9838, with pulp;

0.9772, without pulp) (Fig. 2c).

3.2.2. COD removalCOD removal efficiency also registered visible improvement

due to co-substrate addition, compared to normal operation

[25]. COD removal improved by 4 kg COD/m3 both in pulp and

20

40

60

VF

A c

om

po

sitio

n (

%)

5.2 kg COD/m3

11.0 kg COD/m3

20.0 kg COD/m3

32.0 kg COD/m3

40.0 kg COD/m3

a

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 5 3 3 – 5 4 1 539

non-pulp operations. COD removal efficiencies were also

found to improve by 12.5% (32 kg COD/m3; with pulp) and

13.3% (30 kg COD/m3; non-pulp operation) (Table 2). COD

removal efficiency varied between 36.5% and 67% accounts for

substrate degradation of 1.9 kg CODR/m3 and 26.8 kg CODR/m3

in the case of the experiments performed with pulp (Fig. 3a, b).

Experiments performed without pulp showed relatively

higher COD removal efficiency ranging from 42.1% (2 kg

CODR/m3) to 69.4% (25 kg CODR/m3). Higher substrate degra-

dation observed in the case of non-pulpy waste might be

attributed to the relatively easy degradability of the feed due

to the absence of cellulosic material. Unlike H2 production,

substrate degradation showed a consistent improvement with

increase in the organic load in both the cases of substrate

composition. A good correlation was observed between

organic load and substrate degradation [R2-0.9904 (pulp); R2-

0.9701 (non-pulp)] (Fig. 3c).

Specific H2 yield (SHY) also improved by 2.1 mol/kg CODR

(5.2 kg COD/m3) in the case of pulp operation and 1.5 mol/kg

CODR (4.8 kg COD/m3) with non-pulp operation after co-

substrate supplementation when compared to normal opera-

tion [25] (Table 2). SHY registered higher values at lower organic

load in both the feed compositions (Fig. 4 a, b). With pulp,

maximum SHY was registered at 5.2 kg COD/m3 (21.3 mol/kg

CODR) followed by decrement with increase in waste concen-

tration A minimum SHY (3.2 mol/kg CODR) was observed at

maximum load studied (40 kg COD/m3). A similar trend was

observed in without pulp operation also, where comparatively

higher yield was registered at lower load of 4.8 kg COD/m3

(28.9 mol/kg CODR) and higher load of 36 kg COD/m3 docu-

mented lower yield (2.1 mol/kg CODR). COD removal and

carbohydrate removal were also visualized good correlation

[R2, 0.9695 (pulp); 0.9467 (non-pulp)] indicating the fact that

cellulosic material (complex carbohydrate) was an integral part

of COD (Fig. 4 c, d). When the process was evaluated with

respect to substrate degradation and diluting proportions of

vegetable waste extract to domestic sewage, 1.50 (C4, pulp) and

1.39 (C9, non-pulp) showed maximum substrate degradation

with respect to both carbohydrate and COD.

Acetic acid Propionic acid Butyric acid Malic acid Ethanol

0

Acetic acid Propionic acid Butyric acid Malic acid Ethanol

0

20

40

60

80 4.8 kg COD/m3

9.7 kg COD/m3

19.0 kg COD/m3

30.0 kg COD/m3

36.0 kg COD/m3

VF

A c

om

po

sit

ion

(%

)

b

Fig. 5 – Soluble acid intermediates composition as function

of (a) with pulp and (b) without pulp operations.

3.3. Soluble metabolites

Production of acidic intermediates (VFA) indicates the

changes in the metabolic process involved and provides

information to improve the conditions favorable for process

efficiency [6]. Marked variation in VFA production was

observed as a function of feed composition. In the case of

experiments performed with pulp, maximum VFA concen-

tration was documented at 32 kg COD/m3 (7272 mg/l) followed

by 40 kg COD/m3 (6581 mg/l), 20 kg COD/m3 (3442 mg/l), 11 kg

COD/m3 (3172 mg/l) and 5.2 kg COD/m3 (578 mg/l). A steady

increase in VFA generation was observed till 32 kg COD/m3

followed by slight decrease. Whereas in without pulp opera-

tion, maximum VFA production was registered at 19 kg COD/

m3 (4730 mg/l) followed by 30 kg COD/m3 (4628 mg/l), 36 kg

COD/m3 (4428 mg/l), 9.7 kg COD/m3 (1161 mg/l) and 4.8 kg

COD/m3 (548 mg/l). In this case, a gradual improvement in VFA

generation was observed up to 19 kg COD/m3 which remained

more or less same thereafter.

The distribution of soluble metabolites formed during H2

generation was considered as a crucial signal in under-

standing the metabolic pathway of H2 production during

acidogenesis [29,30]. The following equations (Eqs. 2–6)

depicts the possibility of various soluble metabolites forma-

tion during acidogenic fermentation [31,32].

Acetic acid: C6H12O6 þ 2H2O / 2CH3COOH þ 2CO2 þ 4H2 (2)

Propoinic acid: C6H12O6 þ 2H2 / 2CH3CH2COOH þ 2H2O (3)

Butyric acid: C6H12O6 / CH3CH2CH2COOH þ 2CO2 þ 2H2 (4)

Malic acid: C6H12O6 þ 2H2 / COOHCH2CH2COOH þ CO2 (5)

Ethanol: C6H12O6 / 2CH3CH2OH þ 2CO2 (6)

Composition of soluble metabolic acid intermediates

showed the presenceof acetic acid, butyric acid, propionic acid,

malic acid and ethanol (Fig. 5). Experiments with lower load

conditions evidenced presence of acetic acid as a major

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 5 3 3 – 5 4 1540

metabolite in both feed compositions studied. As the concen-

tration of waste increased, acetic acid concentration decreased

while propionic acid and butyric acid concentrations

increased. Acetic acid concentration varied among 17.7–61.1%

(pulp) and 1.1–71% (non-pulp). Propionic acid concentration

was especially higher in the experiments performed with pulp

[22.1–38.7%] rather than without pulp [0–22%]. On the contrary,

higher composition of butyric acid was observed in the

experiments operated with pulp [9.3–76%] than non-pulp [13.3–

35.7%] operation. Production of acetic acid and butyric acid

favors H2 generation, while the propionic acid consumes H2

[1,6,33]. Generation of higher concentrations of acetic acid and

butyric acid in the experiments performed without pulp

signifies favorable microenvironment for acidogenic activity.

Distribution of metabolites also suggested the dominance of

acid-forming (formation of acetic acid) metabolic flow associ-

ated with acidogenesis. Both substrate compositions have

shown the presence of malic acid in small amounts [2.5–8.7%

(with pulp); 0–10.3% (without pulp)] and ethanol in negligible

amounts [0.2–5.5% (with pulp); 0–2.30% (non-pulp)].

4. Conclusions

Experimental studies illustrated the positive influence of

supplementation of domestic wastewater as co-substrate to

composite vegetable based market waste to improve

fermentative H2 production along with substrate degradation

efficiency. Enhanced H2 yield was noticed at lower organic

load conditions for both pulp and non-pulp operations. Period

of H2 evolution was improved after sewage supplementation

and this might contributed positively on enhanced process.

Soluble metabolite composition depicts that higher H2 yield

was accompanied with the higher acetic acid concentrations.

Domestic sewage, along with diluting the vegetable waste,

provided organic matter and microbial biomass to enhance

the fermentation process and also helps in maintaining a good

buffering microenvironment to support the acidogenic

fermentation.

Acknowledgements

We are thankful to the Director, IICT for the support and

encouragement in carrying out this work. One of the authors

(GM) duly acknowledges Council of Scientific and Industrial

Research (CSIR), New Delhi for providing research fellowship.

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