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Biofuels
ISSN: 1759-7269 (Print) 1759-7277 (Online) Journal homepage: http://www.tandfonline.com/loi/tbfu20
Effect of operational parameters on biohydrogen
production from dairy wastewater in batch and
continuous reactors
Panga Kirankumar, S. Vijaya Krishna, N. Chaitanya, D. Bhagawan, V.
Himabindu & M. Lakshmi Narasu
To cite this article: Panga Kirankumar, S. Vijaya Krishna, N. Chaitanya, D. Bhagawan, V.
Himabindu & M. Lakshmi Narasu (2016): Effect of operational parameters on biohydrogen
production from dairy wastewater in batch and continuous reactors, Biofuels, DOI:
10.1080/17597269.2016.1196327
To link to this article: http://dx.doi.org/10.1080/17597269.2016.1196327
Published online: 04 Jul 2016.
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Effect of operational parameters on biohydrogen production from dairy
wastewater in batch and continuous reactors
Panga Kirankumar
a
, S. Vijaya Krishna
a
, N. Chaitanya
a
, D. Bhagawan
a
, V. Himabindu
a
and M. Lakshmi Narasu
b
a
Centre for Environment, Institute of Science and Technology, Jawaharlal Nehru Technological University Hyderabad, Kukatpally,
Hyderabad 500085, Telangana, India;
b
Centre for Biotechnology, Institute of Science and Technology, Jawaharlal Nehru Technological
University Hyderabad, Kukatpally, Hyderabad 500085, Telangana, India
ARTICLE HISTORY
Received 15 March 2016
Accepted 18 May 2016
ABSTRACT
Hydrogen (H
2
) is a sustainable and variable form of green and alternative energy sources. H
2
is
one of the most promising alternative fuel sources for satisfying future energy demands.
Current research on H
2
production is being conducted on carbohydrate-rich wastewater which
has the ability to produce renewable energy outputs like hydrogen. Dairy wastewater is one
such carbohydrate-rich high-volume industrial wastewater which is suitable for H
2
production.
The biohydrogen production using dairy wastewater influencing factors such as initial pH of
the influent, temperature, and hydraulic retention time (HRT) were investigated in this paper.
Optimized operational conditions for biohydrogen production were found to be 6.5 pH, 55C
temperature and 6 h HRT.
KEYWORDS
Biohydrogen; dairy
wastewater; chemical oxygen
demand; hydraulic retention
time; anaerobic sludge
Introduction
Hydrogen is an alternative energy source, a clean
energy, possessing a high energy yield up to 142 kJ/g
(Boboescu et al. [1], Shi et al. [2], Cardoso et al. [3]),
which does not contribute to the greenhouse effect.
Moreover, hydrogen is an odorless, colorless, tasteless,
and non-poisonous gas. There are a lot of advantages
of hydrogen utilization, such as its high conversion effi-
ciency, its ability to be recycled, and its combustion
releases only water, thus reducing carbon dioxide
emission. From these motives, hydrogen has become
an unrealized ‘fuel of the future’(Poontaweegeratigarn
et al. [4]). Hydrogen, with several unique characteris-
tics, such as high energy yield and non-polluting com-
bustion, should be considered as one of the most
promising energy carriers for the future.
Capturing of energy as a molecular biohydrogen
especially from wastewater treatment process is gain-
ing prominence. Various biological routes are there for
H
2
production including biophotolysis, photo fermen-
tation, dark fermentation process, or a combination of
these processes. Among them, dark fermentation is
considered a variable method on the practical front
thus leading to avenues for the utilization of renewable
energy sources.[5]
Biohydrogen production using industrial wastewa-
ter results in bioenergy recovery and environmental
cleanup.[6,7] Several industrial wastewaters, like palm
oil mill effluent, rice slurry, condensed molasses, distill-
ery wastewater, dairy wastewater, and alcohol industry
wastewater, are employed for biohydrogen production
by dark fermentation in both batch and continuous
processes.[8] Among these, dairy wastewater, due to
its immense carbohydrate content, is an attractive can-
didate for H
2
production. Dairy processing is a high-
volume water-consuming industry with water use
throughout the process steps including cleaning, sani-
tization, heating, cooling, and floor washing; wastewa-
ter volumes generally range between two- and three-
fold of the volume of processed milk. Dairy plants gen-
erate wastewater flows with characteristics that are
heavily dependent on the raw materials used, the proc-
essing technology, and the recovery rate of effluent
wastewater.[9]
The dairy products industry produces wastewaters
with different polluting characteristics depending on
the plant and production type so that fats, proteins,
and carbohydrates constitute different percentages
of the organic matter. Physicochemical treatment
processes are less favorable than biological pro-
cesses owing to the high cost of chemicals, high
energy consumption, large amounts of waste sludge
production, organic loading limitation, and sludge
bulking problems thus making the aerobic processes
a less practical treatment than anaerobic digestion.
Hence anaerobic treatment has gained popularity
because of its applicability to variant characters of
dairy wastewater.
The objective of this work is to optimize the hydro-
gen fermentative process from dairy wastewater using
batch and continuous reactors, inoculated with pre-
treated anaerobic sewage sludge.
CONTACT V. Himabindu drvhimabindu@jntuh.ac.in; kirankumarjntuh11@gmail.com
© 2016 Informa UK Limited, trading as Taylor & Francis Group
BIOFUELS, 2016
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Materials and methods
Dairy wastewater
The dairy wastewater which was used as substrate for
biohydrogen production in the present study was col-
lected from a local dairy plant. Its initial characteriza-
tion was done according to standard methods for
water and wastewater characterization (APHA, [10])
and given in Table 1.
Inoculum (anaerobic sewage sludge)
The inoculum was obtained from the secondary set-
tling tank of a local municipal wastewater treatment
plant located at HMWWS (Hyderabad Metropolitan
Water Supply & Sewerage Board, Amberpet). The initial
characteristics of the anaerobic sewage sludge are
given in Table 2.
Pretreatment
The anaerobic sludge was heat treated at 102C for 1 h
to inhibit the methanogens and the sludge was sieved
to remove stones, sand and other coarse materials. The
sludge was acid treated by maintaining the pH at 2.0
for 24 h.
Experimental setup and conditions
Batch and continuous experiments were carried out in
order to determine the biological hydrogen production
with dairy wastewater as a substrate.
Batch fermentative hydrogen production in lab scale
The hydrogen production experiments were con-
ducted in 100 ml vials with working volumes of 80 ml.
Initially 10% seed sludge, 10% nutrient solution, and
80% substrate were taken in the batch reactor. To
ensure anaerobic conditions, nitrogen was purged for
10 min. The medium circulation in the vials was main-
tained at 100 rpm using an orbital shaker or magnetic
stirrer.
Further studies were carried out in a scale up 6L
batch reactor with 5L working volume at optimum
conditions as shown in Figure 1; 10% of the working
volume of inoculum was introduced into the batch
reactor. The reactor was equipped with a gas
Table 1. Initial characterization of dairy effluent.
S.No Parameter Concentration (mg/l)
1 pH 5.9
2 Chemical oxygen demand (COD) 1760
3 Biological oxygen demand (BOD) 810
4 Alkalinity 500
5 Total solids (TS) 2572
6 Total dissolved solids (TDS) 2492
7 Total suspended solids (TSS) 80
8 Volatile solids (VS) 2130
9 Volatile dissolved solids (VDS) 1060
10 Volatile suspended solids (VSS) 700
11 Volatile fatty acids (VFA) 540
12 Nitrates as NO
3
¡
7.837
13 Sulfates as SO
4
¡
101.39
14 Phosphates as PO
4
¡
0.737
15 Fluorides as F
¡
1.588
16 Total hardness 800
17 Color White
Note: except pH, all are represented in mg/l
Table 2. Initial characteristics of anaerobic sewage sludge.
Parameters Concentrations (mg/l)
pH 7.84
Chemical oxygen demand (COD) 6400
Alkalinity 600
Total solids (TS) 10036
Total dissolved solids (TDS) 968
Total suspended solids (TSS) 9068
Volatile suspended solids (VSS) 1190
Volatile fatty acids (VFA) 148
Nitrates as NO
3
¡
37.16
Sulfates as SO
4
¡
65.81
Phosphates as PO
4
¡
56.81
Note: except pH, all are represented in mg/l
Figure 1. Schematic diagram of the batch study.
2P. KIRANKUMAR ET AL.
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measuring cylinder filled with 40% KOH solution to
absorb the CO
2
from the biogas. Nitrogen purging via
bubbling was provided. An outlet tube fitted with a
stop cork was fitted to the reactor for removing efflu-
ent and venting biogas. A trapper was fixed between
the reactor and CO
2
absorber for biogas collection and
the reactor was placed on a magnetic stirrer provided
with a heating mantle for continuously mixing and
maintaining a constant temperature. The gas outlet
was connected through a rubber cork to the water dis-
placement system. The entire system was checked for
gas leaks and protected by a black cover to avoid the
growth of photosynthetic bacteria. Nitrogen gas was
purged into the reactor for 35 min to create strict
anaerobic conditions prior to the seeding of the active
anaerobic sludge. The pH was adjusted by using 2N
HCl and 2N NaOH solutions. The batch reactor was rou-
tinely monitored for pH, gas production, gas composi-
tion, VFA composition, and volatile suspended solids
(VSS). Each series was repeated and carried out in
triplicate.
Continuous formative hydrogen production
Continuous mode biohydrogen production studies
were carried out in an up-flow anaerobic sludge
blanket reactor (UASB) with dairy wastewater as
substrate.
Up-flow anaerobic sludge blanket reactor
Figure 2 shows a schematic diagram of the UASB
reactor used in this study. Lab scale experiments were
conducted in the UASB reactor. The UASB reactor was
a circular column with a length of 90 cm, internal diam-
eter of 10 cm, and wall thickness of 2 mm. The reactor
was provided with a hopper bottom. Four sampling
ports are provided along its length at equal distance.
The inlet end opens towards the bottom of the reactor,
so the feed strikes at the bottom. An outlet was pro-
vided at the top, which was connected to the effluent
tank. On the top of the reactor, a gassolid separator
was provided to separate gas and solid raised due to
the upward movement of the feed. The gas outlet was
connected through rubber tubing to the liquid dis-
placement system to measure the gas production. The
amount of gas produced is directly proportional to the
amount of liquid displaced and hence gas produced
can be measured at regular intervals of time.
Analytical methods
Samples of bioreactor effluent were routinely collected
for COD determination according to the standard
methods.[10] Gas production was recorded daily by
the water displacement method. The hydrogen gas
percentage was calculated by comparing the sample
biogas with a standard of pure hydrogen using a gas
Figure 2. Schematic description of the up-flow anaerobic sludge blanket reactor.
BIOFUELS 3
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chromatograph (Amilnucon 5700, Italy) equipped with
thermal conductivity detector (TCD) and a six feet
stainless column packed with porapak Q (80/100
mesh). The operational temperatures of the injection
port, the oven, and the detector were 100, 80, and
100C respectively. Nitrogen was used as the carrier
gas at a flow rate of 30 ml/min. The concentrations of
the volatile fatty acids (VFAs) and the alcohol were ana-
lyzed using GC under the following conditions: column:
chromosorb 101, carrier gas: nitrogen, flow rate: 30 ml/
min, column temperature: 200C, injector temperature:
220C, detector temperature 220C, and detector:
flame ionization detector (FID). The pH values inside
the digesters were measured by pH meter.
Results and discussion
Effect of pH on hydrogen production
The pH is an important factor which affects the biohy-
drogen production by fermentation process and thus
needs to be optimized in order to achieve maximum
hydrogen yield. In the present study the initial pH of
the effluent was varied from 4 to 7 at (room) tempera-
ture 29 §2
◦
C. A maximum of 43.3% hydrogen was
noted at pH 6.5 along with COD reduction of 80%. The
percentage of hydrogen increased from 8 to 43.3%
with increasing pH from 4 to 6.5. Similarly, the COD%
reduction potential improved from 28 to 80% with
increasing pH from 4.5 to 6.5. At pH above 6.5, the
hydrogen percentage was decreased. These findings
were in accordance with those reported by Wongtha-
nate et al.[11]
An acidic pH inhibits the growth of microbes thus
lowering the production of hydrogen. Therefore, maxi-
mum hydrogen production of 6.8 ml/l/h and hydrogen
yield of 4.3 mol/g COD were achieved at the initial pH
of 6.5, but slightly decreased when the initial pH was
increased to 7.0 (see Figure 3). Nevertheless, pH control
could stimulate microorganisms to achieve maximum
hydrogen production ability because the activity of
hydrogenase was inhibited by low or high pH in fer-
mentation. Hence, the initial pH 6.5 was found to be
optimum for hydrogen production due to the fermen-
tative conversion of substrate to hydrogen which was
increased by maintaining an operating pH of 6.0 com-
pared to neutral pH. The same trend was reported by
(Venkata Mohan et al.[5]
Effect of temperature on hydrogen production
Temperature plays an important role in determining the
metabolic activity of bacteria and influencing the essen-
tial enzymes such as hydrogenase for fermentative
hydrogen production. In this study the effect of temper-
ature on the production of hydrogen was carried out at
3060C in intervals of 5C under anaerobic condition
at pH 6.5. From Figure 4 it was observed that maximum
hydrogen production was achieved at 55C that is 50%
and COD% reduction of 90% was achieved simulta-
neously. With increasing temperature from 30 to 55C,
hydrogen production increased from 44 to 50%. This
might be due to the fact that at low temperatures,
hydrolysis rates are slower and increasing temperature
could increase the ability of hydrogen-producing bacte-
ria to produce hydrogen.[12] At temperatures above
55C the yield was observed to be decreased which
might be due to the destruction of the metabolic activi-
ties with increasing temperature levels.[12] Further
experiments were conducted at the optimum pH and
temperature of 6.5 and 55C respectively.
Figure 3. Effect of pH and COD reduction on biohydrogen production from dairy.
(Conditions: Working volume: 80 ml, Substrate: Dairy effluent, Inocula: anaerobic sewage sludge, Temperature: Room temperature)
4P. KIRANKUMAR ET AL.
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Hydrogen production in batch fermentation
Experiments were conducted in 6L batch reactors for
hydrogen production using anaerobic sewage sludge
as the inocula and dairy effluent as the substrate at the
above optimized pH and temperature conditions.
These studies were conducted up to 36 h of fermenta-
tion time, where a significant reduction in hydrogen
yield was observed. From Figure 5 it is observed that
the maximum hydrogen yield is 950 ml/l at 36 h with
45% of COD reduction. Hydrogen content in the biogas
was changed with time as the CO
2
production was
significant. The H
2
% in the total biogas increased from
5 to 61% during the fermentation time of 6 h to 24 h
and it slightly decreased after 24 h of fermentation.
Figure 6 shows maximum COD degradation of 62.5%
at 36 h.
Hydrogen production in continuous fermentation
This study deliberated the hydrogen production per-
formance of the UASB system and the effect of dairy
substrate concentration on the stability and yield of a
continuous formative process that produces hydrogen.
Figure 4. Effect of temperature and COD reduction on biohydrogen production from dairy processing wastewater.
(Conditions: Working volume: 80 ml, Substrate: Dairy effluent, Temperature: 3060C)
Figure 5. Hydrogen production with different HRT.
(Conditions: Inocula: anaerobic sewage sludge, Substrate: Dairy effluent, pH: 6.5, Temperature: 35C, Batch reactor volume: 6L)
BIOFUELS 5
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Effect of HRT on hydrogen production
HRT is a critical design parameter since it determines
the microbial substrate reaction time and thus the
removal efficiency of the substrate, where it plays a key
role in hydrogen production. Figure 7 shows the hydro-
genproductionrateatdifferenthydraulicretention
times as well as the substrate removal efficiency.
HRT DVolume of aeration tank
in fluent flowrate
In continuous hydrogen production process the HRT
was studied from 0 to 30 h. The maximum hydrogen
Figure 6. Hydrogen production and COD reduction.
(Conditions: Inocula: anaerobic sewage sludge, Substrate: Dairy effluent, pH: 6.5, Temperature: 35C, Batch reactor volume: 6L)
Figure 7. COD reduction pattern and hydrogen yield at different HRTs.
(Conditions: Inocula: Anaerobic sewage sludge, Substrate: Dairy effluent (COD of 6700 §80 mg/lit), pH: 6.5, Temperature: 35C, UASB reactor volume: 10L,
Working volume: 8L (Inocula-2L, Substrate-6L)
6P. KIRANKUMAR ET AL.
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yield (71%) was observed at 6 h HRT. The hydrogen
yield values improved in continuous hydrogen produc-
tion when the HRT was decreased from 30 to 6 h.
These results suggest that the hydrogen yield values
increased when the HRT decreased. The same trend
was reported by de Amorim et al.[13] At low HRTs the
hydrogen consumers get inhibited, which promotes
the washing-out of methanogens.[14]
Conclusions
The optimum pH, temperature and HRT for biohy-
drogen production using dairy wastewater as sub-
strate and anaerobic sewage sludge as inocula
were found to be 6.5, 55C and 6 h respectively.
The present study results demonstrated that
hydrogen yield increased with decreasing hydrau-
lic retention time. The maximum hydrogen yield
(71%) was achieved at 6 h HRT.
The maximum COD% reduction of 71% was
obtained at 24 h HRT.
Continuous mode is found to be favorable for
hydrogen production using dairy wastewater. In
batch mode, the hydrogen yield of 61% was
achieved which was comparatively less to contin-
uous mode yields.
Acknowledgement
This work was supported by the financial assistance from
Ministry of New and Renewable Energy (MNRE), India, under
National Mission mode project on ‘Hydrogen Production
Through Biological Routes’with a sanction order No. 103/
131/2008-NT.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the Ministry of New and Renew-
able Energy India [grant number 103/131/2008-NT].
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