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 x x x ( 2 0 1 6 ) 1 e1 2

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Quantification of temperature effect on batch

production of bio-hydrogen from rice crop wastes
in an anaerobic bio reactor

Asma Sattar a,1, Chaudhry Arslan a,b,1, Changying Ji a,*, Saba Sattar c,
Muhammad Umair d, Sumiyya Sattar e, Muhammad Zia Bakht f
a
College of Engineering, Nanjing Agricultural University, Nanjing, 210031, China
b
Department of Structures and Environmental Engineering, University of Agriculture, Faisalabad, Faisalabad,
38000, Pakistan
c
National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan
d
Department of Agricultural Engineering, PMAS Arid Agriculture University, Rawalpindi, Pakistan
e
Veterinary Research Institute, Lahore, Cantt, 54810, Pakistan
f
Institute of Food Science and Technology, University of Agriculture, Faisalabad, 38000, Faisalabad, Pakistan

article info abstract

Article history: Rice is an important cereal crop and different types of wastes are produced from it such as;
Received 11 February 2016 rice straw as a crop residue, rice husk and rice bran as agro-industrial waste and rice waste
Received in revised form as a food waste, which are needed to be tested for bio-hydrogen production under similar
6 April 2016 set of experimental conditions. The bio-hydrogen production potential of these wastes was
Accepted 13 April 2016 compared by co-digestion with heat shocked sludge under mesophilic thermophilic tem-
Available online xxx peratures by using an anaerobic bio reactor under no pH control conditions. It was
observed that the bio-hydrogen production potential increased with an increase in tem-
Keywords: perature from 37
C to 55
C for all wastes, except for rice waste. Although, the highest
Rice crop experimental yield of 40.04 mL/VSrem was produced from thermophilic rice straw, but the
Bio-hydrogen production maximum bio-hydrogen production rate of 97.08 mL/h was observed from rice bran after
Optimum pH 29.03 h under thermophilic conditions. The bio-hydrogen production period of rice bran
Kinetic parameters was 33e38% smaller than rice straw due to which the highest yield was observed from rice
Volatile fatty acids straw. The lignin content of rice husk was 19.34%, which was double than rice straw and
hence the rice husk produced the least experimental yield of 23.05 mL/VSrem under mes-
ophilic conditions. Increasing the temperature from mesophilic to thermophilic conditions
changed the final pH by 1.79%, 8.33%, 5.17% and 5.41% for rice straw, rice bran, rice husk
and rice waste, respectively. The optimum pH range of bio-hydrogen production from rice
straw, bran and husk was observed from pH 7 to pH 6. The average yield of wastes pro-
duced from rice crop was 30.36 and 33.16 mL/VSrem under mesophilic and thermophilic
conditions, respectively. The quadratic modeling found useful in order to explain more
than 90% variation caused by incubation time and temperature on cumulative bio-
hydrogen production, drop in pH and VFA production. The 3D and 2D plots developed on

* Corresponding author.
E-mail address: chyji@njau.edu.cn (C. Ji).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ijhydene.2016.04.087
0360-3199/© 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Sattar A, et al., Quantification of temperature effect on batch production of bio-hydrogen from rice
crop wastes in an anaerobic bio reactor, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/
j.ijhydene.2016.04.087
2 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 x x x ( 2 0 1 6 ) 1 e1 2

the basis of quadratic modeling explained the process parameter is in better way as
compared to conventional representation of experimental results. As a whole, increasing
temperature not only increased bio-hydrogen yield, but also decrease the volatile fatty acid
production.
© 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

common pretreatment is comminution in which the ultra-

Introduction
Rice is one of the most popular staple food in the world and
Asia has 90% share in worldwide rice production out of which
28.7% is produced by China alone. The rice was planted over
29.8 million hectare area of China, which produced 196.7
million tons of paddy in the year 2009 [13,50]. The major res-
idues obtained upon the cultivation of rice crop are straw,
husk and bran. Rice straw is produced at the time of har-
vesting and termed as agricultural waste, whereas rice husk
and rice bran are obtained as a byproduct of the milling pro-
cess and termed as agro-industrial wastes [53]. In China, rice
straw has a share of 62% in total crop residues and 1.35 tons of
rice straw are produced for every ton of rice grain harvested
[21,55]. The straw is utilized in the paper making industry,
building construction, animal feed and home warming, but
still a lot of straw is left in the field. The leftover straw is
burned which causes serious environmental threats. On the
other hand, 0.2e0.33 tons of rice husk is produced per ton of
paddy during milling process, which is used as animal feed,
insulating material and fertilizer [22,27]. Apart this, many
power plants in China are producing electricity through gasi-
fication of rice husk [30]. Even after the multidirectional use of
rice husk, still lot of rice husk is burnt causing serious envi-
ronmental issues [22,23]. Along with husk, 0.1e0.12 tons of
rice bran is also produced per ton of paddy, which is used to
extract oil as well as in food products and animal feed.
Another type of waste is also produced from rice crop as part
of organic fraction of municipal solid waste and termed as rice
waste. The rice waste has 10e15% share in food waste pro-
duced at canteens [47].
Apart from the conventional utilization of wastes produced
from rice crop, converting the said wastes into more valuable
products like methane, ethanol and bio-hydrogen are better
options by which environmental concerns as well as energy
challenges can be addressed together. The conversion can be
done by gasification and pyrolysis, but it can cause a threat to
the environment. This issue can be resolved by opting bio-
logical means, such as; anaerobic fermentation, which is an
environmental friendly and accommodate wide range of
substrates [10,35,49]. In this regard, bio-hydrogen production
through anaerobic fermentation is more advantageous over
other fuel, as hydrogen producing microorganisms could uti-
lize a wide range of sugar hydrolysates as compared to other
microbes [19]. The sugar hydrolysates were available in the
form of holo-cellulose in rice straw and husk, whereas in case
of ice bran and rice waste, carbohydrates are the sources of
sugar hydrolysates. The holo-cellulose in bounded by lignin
and is not easily bio-degraded. To overcome this issue, the
structure of lignocellulosic biomass is changed. In this tech-
nique final particle size of 0.2e2 mm is achieved, which
increases the surface area and reduces the cellulose crystal-
linity for better biodegradability [28,34]. The mix consortia of
Clostridium are the common group of microorganism that can
be used for this purpose as they are easily available in the form
of sludge [12]. The sludge also contains hydrogen consumers
like methanogens, which can be deactivated by exposing
sludge to higher temperature, UV radiations, extremely high
or low pH and hazardous chemicals [39]. Among all these
methods, heat shock is the most suitable as it is an easy and
economical method [11]. In heat shocked sludge, metha-
nogens are unable to survive, but Clostridium can survive by
forming protective spores [52]. Apart Clostridium, sludge can
provide some nutrients to fulfill the nutritional requirement
for bacterial growth, which can make the process more
economical and simple [24,40].
The incubation temperature is an important parameter for
optimum bio-hydrogen production from wastes co-digested
with heat shocked sludge. In case of rice crop wastes, the ef-
fect of temperature was not intensively studied. Previously,
Alemahdi et al. [2] reported the bio-hydrogen yield of 14.7 mL/
VS under mesophilic condition, which was improved to
21 mL/VS by Kim et al. [24] by opting thermophilic condition.
In case of rice husk, few studies are conducted by using rice
husk or its hydrolysate for bio-hydrogen production under
mesophilic conditions only [31,40]. Similarly, bio-hydrogen
production was reported under mesophilic conditions from
rice bran [1,38]. Even if the bio-hydrogen is produced from said
wastes under both temperature conditions, but still it is not
comparable as the experimental conditions are different. The
effect of temperature on rice waste was studied by Fang et al.
[12] and Arslan et al. [9] under similar set of experimental
conditions, but still further work is needed to verify the
experimental results over larger scale.
Although intensive research is done on methane and
ethanol production from agricultural waste still there is a
gap for research on bio-hydrogen production from the
wastes produced from rice crop [5,6]. So, the following study
was designed to quantify and compare the bio-hydrogen
production potential of lignocellulosic wastes like rice
straw and rice husk along with carbohydrate rich wastes like
rice bran and rice waste produced from rice crop when they
were co-digested with treated sludge under mesophilic
thermophilic conditions. The volatile solids, volatile fatty
acids, soluble chemical oxygen demand and pH were also
measured to observe various aspects of the fermentation
process.

Please cite this article in press as: Sattar A, et al., Quantification of temperature effect on batch production of bio-hydrogen from rice
crop wastes in an anaerobic bio reactor, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/
j.ijhydene.2016.04.087
 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 x x x ( 2 0 1 6 ) 1 e1 2 3

Co., China) was also attached to the reactor to develop

Material and methods anaerobic conditions [9,44].

Feed stock preparation Analytical and assay methods

The rice straw was collected from Ba Bai Qiao field and air
dried after removing foreign materials. Later, it was cut up to
the length of 8e10 cm and ground in a grinder (LH-08B Speed
Grinder, CNC Instruments Inc., China). Finally, the grounded
straw was passed through a 2 mm sieve and used for bio-
hydrogen production. Rice Husk was obtained from a local
industry, ground and sieved in the same way as opted for rice
straw. Rice bran was also obtained from local supplier and
sieved in the similar way like husk and straw. The food waste
was collected from student dining, bones, potato, noodles,
meat, green vegetables and tofu were removed to obtained
rice waste. Later, the rice waste was ground in a food grinder
by adding an equal amount of water on weight basis and
resultant slurry was used for bio-hydrogen production [43].
The sludge was obtained from a settling channel in Pukou.
It was sieved and washed with tap water to remove dust and
foreign materials [36]. Later, it was placed in preheated oven at
100
C for 30 min in order to deactivate hydrogen consumers
[29,42]. The volatile solids, chemical oxygen demand, volatile
fatty acids, alkalinity and pH of the sludge were 2.87%, 50 g/L,
13,950 mg/L, 3700 mg/L and 7.1, respectively.
The total solids (TS), volatile solids (VS), chemical oxygen de-
mand (COD), volatile fatty acids (VFA) and alkalinity were
measured by standard methods [3]. The volume of hydrogen
gas was measured by 3% NaOH method as used in our previous
studies [4,9,44]. The compositional properties of straw and husk
were measured by the procedure opted by Ververis [54]. The
bio-hydrogen production was modeled by modified Gompertz
Equation for the determination of kinetic parameters [41]

 
Rm e
H ¼ Pexp  exp ðl  tÞ þ 1 (1)
P
where H, t, P, Rm, l and e represent cumulative bio-hydrogen
production (mL), incubation time (h), bio-hydrogen produc-
tion potential, maximum bio-hydrogen production rate (mL/
h), lag phase duration (h) and 2.71828 respectively. The values
of H, t, P, Rm were solved by using curve fitting tool in Matlab
(Ver. 2010 a).
In this study, the effects of temperature and incubation
time on cumulative bio-hydrogen production, drop in pH and
on VFA production were analyzed by full quadratic model as
shown below [20,26]

X
n X
n X
n X
n

Anaerobic bio-reactor (ABR) Y ¼ a0 þ ai Xi þ aii X2i þ aij Xi Xj (2)

i¼1 i¼1 i¼1 i < j¼2

In the present study, a 20 L stainless steel double jacket
anaerobic bio reactor was designed in collaboration with
Zhejiang Instruments Limited (Fig. 1). The reactor was
equipped with proportionaleintegralederivative (PID) con-
trollers (CAN-C700, Aivpen Instruments, China and PH900,
Acitek Instrument, China) for temperature and pH manage-
ment. Thorough mixing was done by a three stage stirrer
operated by DC motor (ZSD05A, Shanghai ShuDong Motor Co.,
Ltd, China). A vacuum pump (FY-1H-N, Zhejiang E &M Value
where Xi and Xj are the controlled parameters, which influ-
ence Y, a0, aii, aij, are the offset term, linear and quadratic
coefficients respectively.
Batch experiments
The reactor was operated at 10% TS by adding waste and
seeded sludge in equal proportion on TS basis and finally

Fig. 1 e Schematic Diagram for double jacket anaerobic bio-reactor.

Please cite this article in press as: Sattar A, et al., Quantification of temperature effect on batch production of bio-hydrogen from rice
crop wastes in an anaerobic bio reactor, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/
j.ijhydene.2016.04.087
4 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 x x x ( 2 0 1 6 ) 1 e1 2

adding water to achieved desired experimental condition. The straw till 144 h of incubation because of which rice straw has
initial pH was maintained to 7.5 for straw and husk [32,37]. For the highest cumulative bio-hydrogen production (not shown
rice bran and rice waste, the initial pH was maintained to 7 in Fig. 2).
with the help of pH controller using 3 M HCl or 3 M NaOH Shifting the incubation temperature from mesophilic
[9,16]. The co-digestion was done under mesophilic (37
C) and (37
C) to thermophilic (55
C) increased the bio-hydrogen
thermophilic (55
C) conditions and all experiments were production potential for all wastes except rice waste. The
performed in duplicate [4]. The pH and volume of bio- time during which the bio-hydrogen was produced bio-
hydrogen was measured after 12 h interval and VFAs were hydrogen is termed as production time and for rice bran the
measured on daily basis. The reactor were closed when bio- production time was 96 h under both temperatures. The gas
hydrogen production was completely ceased, after which TS production was stopped in rice bran due to consumption of
and VS were analyzed [24]. carbohydrates. It is also observed that the co-digestion with
sludge have increased the bio-hydrogen production time to
96 h from 50 h as reported by Noike and Mizuno [38]. It might
Results and discussion be due to presence of Clostridium Saccharoper Butylacetonicum
N1-4 as reported by Al-shograni [1].
Cumulative bio-hydrogen production and kinetic parameters The maximum cumulative bio-hydrogen production was
observed in the thermophilic rice straw reactor as the bio-
The bio-hydrogen production ceased for all wastes within five hydrogen production continued till 156 h (not shown in
days of incubation except for rice straw. The cumulative Fig. 2). On the other end, the increase in temperature caused
production is shown in Fig. 2, where five-day data is given for negative effect on bio-hydrogen production from rice waste,
better representation. Rice bran represented a higher initial which was in agreement with the findings of Fang [12] and
bio-hydrogen production within 12 h of incubation under Arslan [4].
mesophilic condition, whereas rice husk showed a late start The change in kinetic parameters with temperature were
between 12 and 24 h of incubation. During 12e60 h, the bio- not the same in all waste types (Table 1). With the shift in
hydrogen production in rice bran and rice waste reactor was temperature from 37
C to 55
C, the Rm value for rice bran
almost same (Fig. 2), after which production in rice bran was became 97.08 mL/h after an increase of 68.40%, which is the
slightly decreased till 96 h under mesophilic condition. After highest value observed and also higher than that achieved by
96 h, bio-hydrogen production was ceased in the rice bran and Al Shorgani [1]. On the other hand, increase in a P value
rice waste reactor under mesophilic temperature and at that (45.33%) due to an increase in temperature for rice bran was
time, cumulative bio-hydrogen production was the highest in not the same. It is because of the bio-hydrogen production
rice bran reactor. Although, mesophilic bio-hydrogen pro- from rice bran was higher under mesophilic conditions as
duction in rice husk reactor ceased at 108 h, still the cumu- compared to thermophilic conditions after 60 h of incubation.
lative bio-hydrogen production was the least as compared to Secondly, the duration for bio-hydrogen production was
other wastes under same temperature. The bio-hydrogen almost the same under both temperatures and there was only
production as started after 12 h of incubation in mesophilic about one-hour difference between l values (Table 1). The rice
rice husk reactor whereas in other reactors it was started straw represented quite different trend, the increase in Rm
before 12 h of incubation. The late start and lower bio- value (5.22%) was much smaller as compared to increase in P
hydrogen production during every 12 h intervals were the value (26.87%) due to increase in temperature. It is because of
reasons for the least cumulative bio-hydrogen production smaller thermophilic l value, which cause an early start in
from rice husk under mesophilic conditions. On the other bio-hydrogen production and longer thermophilic production
hand, production remained continued in mesophilic rice period i.e., 154.73 and 134.74 h under thermophilic and

6,000
Cumulative Bio-hydrogen Production (mL)

5,000

4,000

3,000

2,000

1,000

0
0 12 24 36 48 60 72 84 96 108 120
Time (h)

Fig. 2 e Cumulative bio-hydrogen production from tested wastes.

Please cite this article in press as: Sattar A, et al., Quantification of temperature effect on batch production of bio-hydrogen from rice
crop wastes in an anaerobic bio reactor, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/
j.ijhydene.2016.04.087
 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 x x x ( 2 0 1 6 ) 1 e1 2 5

Table 1 e Effect of temperature on kinetic parameters.

Feed stock Temperature (
C) P (mL) Rm (mL/h) l (h) tmax (h) R2 Temperature effect (%)
P Rm l tmax
Rice straw 37 4258 42.51 10.26 47.22 0.9973 26.87 5.22 87.61 2.93
55 5402 44.73 1.271 45.84 0.9955
Rice husk 37 1855 24.29 22.89 51.07 0.9989 50.57 70.11 46.13 27.02
55 2793 41.32 12.33 37.27 0.9978
Rice bran 37 3473 57.65 10.87 33.10 0.9975 45.35 68.40 9.48 12.30
55 5048 97.08 9.84 29.03 0.9992
Rice waste 37 3085 54.73 16.76 37.56 0.9989 44.15 24.70 82.52 22.52
55 1723 41.21 30.59 46.02 0.9988

mesophilic conditions, respectively. The rice husk repre-
sented almost same trend observed for rice bran, whereas rice
waste represented totally opposite trend as the bio-hydrogen
production decreased with an increase in temperature
within experimental range.
The time to attain the maximum Rm (tmax) can be calculated
by taking the derivative of Equation (2) with respect to time
and later comparing the results with zero [7]. Following
equation will be achieved;
P
tmax ¼ l þ
e$Rm
The resultant tmax values are shown in Table 1. The in-
crease in temperature decreased the tmax for all wastes except
for rice waste. The experimental results showed the
maximum bio-hydrogen production during 24e48 h for all
waste except for mesophilic rice husk for which it was
observed during 36e60 h. The above tmax is in agreement with
the experimental results. On the other end, the tmax can be
used as hydraulic retention time (HRT) in continuous pro-
duction of bio-hydrogen. Among all wastes tested, the rice
bran has the minimum value of tmax, which make it more
suitable for continuous bio-hydrogen process.
The coefficients obtained through quadratic modeling of
cumulative bio-hydrogen production are listed in Table 2. The
coded values were used for incubation time x1 ranged from 2
to þ2 to represent 24e120 h of incubation for all wastes except
rice straw for which the coded values ranged from 3 to þ3 to
represent 24e168 h of incubation. The x2 represented the
temperature with coded value ranged from 1 to þ1 to
represent 37
C to 55
C temperature, respectively. The linear
terms represented positive effect on bio-hydrogen production
with an increase in time and temperature for all wastes except
for rice waste (Table 2), which is in agreement with the
experimental values. Similarly, the effect of time and tem-
perature on bio-hydrogen production was the highest for rice
bran as observed experimentally. On the other end, the R2
were represented that the quadratic model explained more
than 90% of variation in the bio-hydrogen production with
time and temperature for all wastes. On the basis of the
model, the 3D and 2D plots were developed as shown in Fig. 3.
(3) It can be observed that the rice husk (Fig. 3b) represented the
least effect of temperature and time as compared to other
wastes and the rice waste showed a negative effect of tem-
perature (Fig. 3d). The effect of temperature on bio-hydrogen
production from rice straw and rice bran became more
dominated after 120 h and 72 h of incubation (Fig. 3a and c).
The rice straw represented more variation in bio-hydrogen
production with time till 132 h of incubation, whereas the
rice bran represented the similar trend till 84 h of incubation.
On the other hand, the variation of bio-hydrogen production
was not so high throughout the incubation in rice husk and
rice waste. As a whole, the 3D and 2D plots represented a
better picture regarding the effect of temperature and incu-
bation time.
Bio-hydrogen yield
The bio-hydrogen yield was calculated by dividing the P with
volatile solids removed during incubation (VSrem). Although, it
can be calculated on the basis of initial volatile solids, but in
that case the effect of temperature would be the same as

Table 2 e Quadratic equation coefficient.

Parameter Feed stock x1 x2 x21 x22 x1x2 F R2
Cumulative bio-hydrogen Rice straw 3401.33 609.63 361.12 80.25 22.05 39.63 1210.45 0.9902
Rice bran 3473.03 770.00 565.00 218.75 1.97 112.50 381.96 0.9795
Rice husk 1326.82 453.13 331.25 23.33 7.76 73.13 157.11 0.9515
Rice waste 1726.93 515.00 460.00 98.33 6.40 110.00 264.80 0.9707
Drop in pH Rice straw 6.07 0.17 0.23 0.02 0.05 0.02 103.94 0.8965
Rice bran 5.95 0.18 0.20 0.01 0.15 0.02 381.96 0.9795
Rice husk 5.99 0.09 0.13 0.03 0.05 0.01 161.80 0.9529
Rice waste 4.15 0.33 0.10 0.08 0.01 0.03 191.01 0.9598
VFA production Rice straw 1384.95 378.43 405.60 11.81 1270.65 59.68 402.98 0.9711
Rice bran 2080.51 365.62 318.75 28.13 0.74 9.38 138.81 0.9455
Rice husk 1259.45 243.75 75.00 37.50 15.55 18.75 138.13 0.9453
Rice waste 1716.83 462.74 110.58 9.38 22.00 28.37 458.62 0.9829

Please cite this article in press as: Sattar A, et al., Quantification of temperature effect on batch production of bio-hydrogen from rice
crop wastes in an anaerobic bio reactor, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/
j.ijhydene.2016.04.087
6 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 x x x ( 2 0 1 6 ) 1 e1 2

Fig. 3 e 3D and 2D plots for Cumulative bio-hydrogen production (a) Rice straw, (b) Rice husk, (c) Rice bran, (d) Rice waste.

observed in case of cumulative bio-hydrogen production. So
the process efficiency could be better represented if the bio-
hydrogen yield is calculated on the basis of volatile solids
removed during incubation. The variation in bio-hydrogen
yield and specific hydrogen production rate (SHPR) with
temperature and waste type is shown in Fig. 4. Increasing the
temperature from 37
C to 55
C found useful in order to in-
crease the bio-hydrogen yield from all wastes except rice
waste as 9.34%, 31.31% and 23.51% increase in yield was
observed from rice straw, husk and bran, respectively. The

Please cite this article in press as: Sattar A, et al., Quantification of temperature effect on batch production of bio-hydrogen from rice
crop wastes in an anaerobic bio reactor, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/
j.ijhydene.2016.04.087
 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 x x x ( 2 0 1 6 ) 1 e1 2 7

45 Bio-Hydrogen Yield (mL/VS rem)

be due to the growth of Clostridium aceticum [48]. As a whole,
40 Specific Hydrogen Production Rate (mL/day/VS rem) rice straw had the highest bio-hydrogen yield of 40.04 mL/
35 VSrem under thermophilic condition, which was close to
30 thermophilic rice bran yield 38.6 mL/VSrem. On the other end,
25 rice husk and rice straw are lignocellulosic part of rice crop
20 and the grinding has no effect on the compositional properties
15 but it reduced the crystalline nature of the cellulose, increased
10 active surface area as well as increased the degree of poly-
5 merization [18]. As the lignin content in the rice husk is almost
0 double than rice straw due to which less cellulose was avail-
able in rice husk due to lignin binding and hence produced the
Rice Straw Rice Husk Rice Bran Rice Waste least yield as compare to other wastes [33]. The bio-hydrogen
Fig. 4 e Effect of temperature and waste type on bio- yield could be increased by selecting the most efficient pre-
hydrogen yield. treatment method as well as providing nutrient solution
[17,24,40]. The reduction in bio-hydrogen production and
decrease in intensity of drop in pH indicated the possibility of
60 P VS rem Bio-hydrogen yield SHPR activation of methanogenic bacteria, which were deactivated
initially by heat treatment of sludge [41,45].
40
The effect of increasing temperature from 37
C to 55
C
Percentage Change

on cumulative bio-hydrogen production, VSrem, bio-

20
0 straw, the bio-hydrogen production increased more as
Rice Straw Rice Husk
-20
-40
-60
Fig. 5 e Effect of increasing temperature from 37
C to 55
C.
increase in yield might be due to the presence of Thermoa-
naerobacterium thermosaccharolyticum that grow at higher
temperature and produce more hydrogen [46].
Under mesophilic conditions, the bio-hydrogen yield of
rice bran and rice waste was close to each other, as carbohy-
drates were the main source for bio-hydrogen, but rice waste
has higher carbohydrates which causes negative effect of
temperature on yield as reported by Fang [12]. The decrease in
the hydrogen production at thermophilic temperature might
hydrogen yield and SHPR is shown in Fig. 5. In case of rice
Rice Bran Rice Waste compared to VS removed and bio-hydrogen yield, which
represents that the conversion of VS to bio-hydrogen under
mesophilic condition was more efficient than thermophilic.
The decrease in SHPR also at higher temperature also in-
dicates the same results for rice straw. Similarly, increase in
P and bio-hydrogen yield due to increase in temperature was
higher than the VS removed during incubation. The rela-
tionship between VSrem and bio-hydrogen yield along with
increase in SHPR due to temperature indicate that the VS
conversion efficiency for rice husk and rice bran increased
with an increase in temperature within experimental range.
In case of rice waste, the increase in temperature decreased
the value of P more than the VSrem and bio-hydrogen yield
decreased slightly less than VSrem, which ultimately caused
little increase in SHPR (Fig. 5). Apparently, it seems that the
rice straw is converted most efficiently into bio-hydrogen
but detailed analysis in the light of VSrem represents that it
is the least efficient in term of VS conversion to bio-
hydrogen at elevated temperature (55
C).

7.5

6.5

6
pH

5.5

4.5

3.5
0 12 24 36 48 60 72 84 96 108 120
Time (h)

Fig. 6 e Drop in pH with incubation time.

Please cite this article in press as: Sattar A, et al., Quantification of temperature effect on batch production of bio-hydrogen from rice
crop wastes in an anaerobic bio reactor, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/
j.ijhydene.2016.04.087
8 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 x x x ( 2 0 1 6 ) 1 e1 2

Fig. 7 e 3D and 2D plots representing drop in pH (a) Rice straw, (b) Rice husk, (c) Rice bran, (d) Rice waste.

Change in pH After 12 h of incubation, the intensity of drop in pH reduced

The observed mesophilic pH drop was higher than ther-
mophilic drop in case of rice straw, rice husk and rice waste
whereas, rice bran gives an opposite trend as shown in Fig. 6
[44]. It can be seen that there was a sudden drop in pH
during 12 h of incubation in all wastes except rice bran.
in all reactors except for rice waste, as observed in our
previous studies [8,9]. The final pH for mesophilic bio-
hydrogen production were observed as 5.6, 5.8, 6.1 and 3.8
for rice straw, rice husk, rice bran and rice waste, respec-
tively. Increasing the temperature to thermophilic condi-
tions changed the final pH for rice straw, rice husk, rice bran

Please cite this article in press as: Sattar A, et al., Quantification of temperature effect on batch production of bio-hydrogen from rice
crop wastes in an anaerobic bio reactor, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/
j.ijhydene.2016.04.087
 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 x x x ( 2 0 1 6 ) 1 e1 2
4000
3500
3000
VFA (mg/L) 2500
2000
1500
1000
500
24
Fig. 8 e Change in VFA content during incubation.
and rice waste as 5.7, 6.2, 5.7, and 4, respectively. Experi-
mentally, increase in bio-hydrogen production from rice
straw was observed from pH 6.7 to pH 6.1 and pH 7.1 to pH
6.6 under mesophilic as well as thermophilic temperatures,
respectively. After which, the bio-hydrogen production
started to decrease till the terminal pH was achieved. As a
whole, pH 7 to pH 6 was observed as an optimum pH range
for bio-hydrogen production from rice straw under both
tested temperatures as shown in Figs. 2 and 5. The same pH
range could be opted for optimum bio-hydrogen production
from rice husk and rice bran but in case of rice waste, the
optimum pH drop was observed from pH 5.5 to pH 4 [12].
Another important aspect is that the increase in tempera-
ture from 37
C to 55
C shorten the pH range in which bio-
hydrogen could be produced for all tested wastes except rice
bran.
The results of quadratic modeling on pH are also listed in
Table 2, using the same coded values, which were previously
used for cumulative bio-hydrogen production. It can be
observed the effect of time (x1) is negative on all waste and
the temperature effect is negative only for rice bran as
observed experimentally. The R2 values represented a good
fit that could explain about 90% of variation. The plots
developed by using quadratic modeling are shown in Fig. 7.
As the incubation time increased, the intensity of drop in pH
decreased in all wastes. The effect of temperature became
more dominated at higher incubation and the intensity was
much higher in rice bran (Fig. 7c). In case of rice straw, the
drop in pH was higher initially but after 72 h of incubation
the intensity of the drop was reduced. The similar pattern
was observed for rice waste till 84 h of incubation. The drop
in pH was the least in rice bran at elevated temperature after
60 h of incubation, whereas mesophilic conditions repre-
sented an opposite situation. As a whole the rice husk rep-
resented a uniform drop in pH throughout, but the intensity
of drop in pH under mesophilic conditions was high as
compared to thermophilic conditions. Apart this, the drop in
pH trend in rice bran was quite opposite to other wastes as
observed experimentally.
Please cite this article in press as: Sattar A, et al., Quantification of temperature effect on batch production of bio-hydrogen from rice
crop wastes in an anaerobic bio reactor, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/
j.ijhydene.2016.04.087
9
48 72 96 120
Time (h)
VFA production
The VFA contents increased with time, because the wastes
were co-digested with heat shocked sludge and it is in
agreement with the finding of Kim [25]. The effect of tem-
perature and time on VFA is represented in Fig. 8. The
highest experimental VFA at the end of incubation was
observed in mesophilic rice straw reactor and the increase in
temperature decreased the VFA content in all wastes as
previously [9,14]. As whole, at the end of incubation, the
decreased in VFA content due to temperature increase was
18.23%, 22.22%, 15.79% and 22.62% for rice straw, rice bran,
rice husk and rice waste, respectively. During 72 h of incu-
bation, the VFA and bio-hydrogen production was higher in
all reactors after which VFA and bio-hydrogen production
rate both started to decrease. As a whole, during the same
duration the bio-hydrogen production was higher under
thermophilic conditions in all wastes except rice waste. The
possible reason of increase in bio-hydrogen production is the
change in physical state of the bacterial cell at high VFA
contents and need higher energy to pump ions through cell,
which could be inhibitory to hydrogen production bacteria
[9]. Such high energy was readily available under thermo-
philic conditions and ultimately bio-hydrogen production
was increased [15,51,56].
The quadratic coefficient in Table 2 for VFA production
represented the same trend as observed experimentally. The
plot developed on the basis of the modeling are shown in
Fig. 9. The rice straw represented the curve type pattern with
temperature (Fig. 9a), which is explained by the square term of
temperature (x22 ) having the most high value (Table 2). On the
other end, rice husk has the least effect of time and temper-
ature (x1&x2), which was also observed experimentally. Simi-
larly, rice straw has the highest effect of the said parameters
as compared to other wastes, by which the VFA production in
rice straw reactor was higher under mesophilic conditions
and the effect of increasing temperature on VFA production
was also high for rice straw. With time, the VFA production
10 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 x x x ( 2 0 1 6 ) 1 e1 2

Fig. 9 e 3D and 2D plots for VFA production (a) Rice straw, (b) Rice husk, (c) Rice bran, (d) Rice waste.

was also higher under mesophilic conditions as compared to almost uniform with time for rice husk and rice waste, but the
thermophilic conditions in rice straw, whereas the variation effect of temperature on rice husk was more dominated as
in VFA production became more dominating after 48 h of in- compared to rice waste. Keeping in view the VFA plots, it is
cubation in rice bran. The variation of VFA with time was clear that the effect of time and temperature can be better

Please cite this article in press as: Sattar A, et al., Quantification of temperature effect on batch production of bio-hydrogen from rice
crop wastes in an anaerobic bio reactor, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/
j.ijhydene.2016.04.087
 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 x x x ( 2 0 1 6 ) 1 e1 2
explain by such plots as compared to simple plots developed
only on experimental data (Figs. 8 and 9).
Conclusion
The effect of temperature on bio-hydrogen production po-
tential of wastes produced from rice crop i.e., straw, husk,
bran and rice waste (derived from food waste) co-digested
with heat shocked sludge was studied by using 20L anaer-
obic bio reactor. The increase in temperature increased the
bio-hydrogen yield from all wastes except rice waste and the
highest experimental increase of 31.31% in bio-hydrogen yield
was observed from rice husk. As a whole the rice straw has the
highest experimental yield of 40.04 mL/VSrem under thermo-
philic conditions. At the same time, increase in temperature
increased the bio-hydrogen production rate of rice bran by
68.40%, which results into the highest experimental rate of
97.08 mL/h. As a whole, the wastes produced from rice crop
could provide average bio-hydrogen yield of 30.37 mL/VSrem
under mesophilic conditions, which was increased by 9.19%
under thermophilic conditions. The optimum pH range of pH
7 to pH 6 was found suitable for all tested wastes under both
temperatures, except rice waste for which the range observed
as pH 5.5 to pH 4. As a whole, the increase in temperature not
only increased specific hydrogen production rate but also
reduced VFA content.
Acknowledgments
We thank K. Chen for providing lab facilities; H. Fang, Y. P.
Huang and J. B. Kang for their help during lab work. We extend
our thanks to Higher Education Commission, Pakistan, China
Scholarship council and the College of Engineering, Nanjing
Agricultural University, Nanjing for supporting and providing
research facilities for this study.
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