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Biohydrogen production by dark fermentation of wheat


powder solution: Effects of C/N and C/P ratio on hydrogen
yield and formation rate

Hidayet Argun, Fikret Kargi, Ilgi K. Kapdan, Rukiye Oztekin


Department of Environmental Engineering, Dokuz Eylul University, Buca, Izmir, Turkey

art i cle info ab st rac t

Article history: The effects of C/N and C/P ratio on hydrogen yield and specific production rate were
Received 28 November 2007 investigated in dark fermentation of wheat powder solution (WPS) by external addition of N
Received in revised form and P since the wheat powder was N and P deficient. A BoxWilson statistical experiment
30 January 2008 design approach was used. C/N ratio was varied between 20 and 200 while C/P ratio was
Accepted 31 January 2008 between 50 and 1000. A quadratic response function was used to correlate the objective
Available online 12 March 2008 functions with the independent variables and the response function coefficients were
determined by regression analysis. Hydrogen yield increased with increasing C/N and C/P
Keywords:
ratios. Low nitrogen concentrations required low phosphorous contents for high hydrogen
Biohydrogen
yields. A C/N ratio of 200 and C/P ratio of 1000 yielded the highest H2 yield of
BoxWilson experiment design
281 ml H2 g1 starch at STP. SHPR also increased with increasing C/N and C/P ratios. Again
Dark fermentation
the highest SHPR (98 ml H2 g1 biomass h1 at STP) was obtained at C/N and C/P ratios of 200
Wheat powder solution (WPS)
and 1000, respectively. The C/N/P ratio maximizing the yield and formation rate of
C/N and C/P ratio
hydrogen was 100/0.5/0.1 w w1 w1 .
& 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights
reserved.

1. Introduction annually worldwide with a growth rate of nearly 10% per


year [1]. Based on the reports of the national hydrogen
The need for alternative energy sources has increased in program of the United States, the contribution of hydrogen to
recent years, due to rapid depletion of fossil fuels. Direct total energy market will be nearly 10% by 2025 [2].
utilization of fossil fuels results in considerable environmen- Hydrogen may be produced by different chemical processes
tal problems due to CO2 , SOX , NOX emissions causing air which are all energy intensive requiring high temperatures
pollution and global warming. Hydrogen gas is a clean fuel 4850  C [3]. Biomass and water can be used as renewable
with no CO2 emissions and can easily be used in fuel cells for resources for hydrogen gas production. Hydrogen production
electricity generation. Besides, hydrogen has a high energy from water is realized by electrolysis and photolysis. Biomass
content of 122 kJ g1 , which is 2.75 times greater than can also be processed chemically (pyrolysis, gasification) or
hydrocarbon fuels. Hydrogen is considered as the major biologically (hydrolysis, fermentation) for production of
energy carrier of the future. The major problem in utilization hydrogen.
of hydrogen gas as a fuel is its unavailability in nature and Bioprocesses have been given special attention for hydro-
the need for inexpensive production methods. It has been gen production in the last thirty years due to operation under
reported that 50 million tones of hydrogen are traded mild conditions (2535 1C, 1 atm). However, biohydrogen

Corresponding author. Tel.: +90 232 4127109.


E-mail address: fikret.kargi@deu.edu.tr (F. Kargi).
0360-3199/$ - see front matter & 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijhydene.2008.01.038
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1814 I N T E R N AT 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 33 (2008) 1813 1819

production is realized with a lower rate as compared to


the chemical processes [36]. Bio-photolysis of water by 2. Materials and methods
algae is rather slow, inhibited by the oxygen in the air,
and also is dependent on the availability of the sun- 2.1. Experimental setup and procedure
light [711]. Therefore, production of biohydrogen from
renewable sources such as biomass has considerable advan- Batch dark fermentation experiments were carried out in 2 L
tages over other alternatives [3,6]. Biohydrogen production serum bottles (Isolab-Germany Boro 3.3). Silicone rubber
can be realized by anaerobic (dark fermentation) and photo- stoppers and screw caps were used to avoid gas leakage from
heterotrophic (light fermentation) microorganisms using the bottles. The wheat particles were ground to 200 mesh
carbohydrate rich biomass as a renewable resource [3]. size to obtain the wheat powder (WP). The bottles were filled
The first step is the acid or enzymatic hydrolysis of with 1 L water containing 20 g L1 WP of 200 mesh. The WP
biomass to highly concentrated sugar solution which is contained approximately 97% w w1 starch, 3:4 mg g1 total
further fermented by anaerobic organisms to produce volatile nitrogen and 1:72 mg g1 phosphate-P. The nitrogen and
fatty acids (VFA), hydrogen and CO2 [1218]. The organic phosphorous contents of the wheat were not considered in
acids are further fermented by the photo-heterothrophic C/N and C/P ratios since the nature and availability of N and P
bacteria (Rhodobacter sp) to produce CO2 and H2 which compounds were not known. Only the externally added N and
is known as the light fermentation [1925]. Combined P were considered in C/N and C/P ratios. The WPS was boiled
utilization of dark and photo fermentations was reported to for 1.5 h for partial hydrolysis of starch before placing into the
improve the yield of hydrogen formation from carbohydrates serum bottles. Initial pH of the medium was adjusted to 7.
[2628]. The oxidation reduction potential (ORP) was adjusted to
Starch containing biomass such as waste agricultural nearly 200 mV by addition of 200 mg L1 Na-thioglycolate.
products offer special advantages for biohydrogen production C/N and C/P ratio were adjusted by adding required amount of
since those raw materials are readily available, inexpensive urea CON2 H4 as nitrogen source and KH2 PO4 as P-source to
and starch can easily be hydrolyzed to carbohydrates. A every bottle yielding the desired ratio. The bottles containing
number of studies is reported in literature for production of 20 g L1 WP were inoculated with the heat treated anaerobic
hydrogen from starch and cellulose containing biomass by sludge to yield 1 L total volume with an initial biomass
dark and light fermentations [1225]. Usually anaerobic concentration of 0:22 g dw biomass L1 and were placed in an
bacteria such as Clostridia sp, Enterobacter sp. and anaerobic incubator at a constant temperature of 37 1C. The bottles were
sludge were used for dark fermentation of carbohydrates to mixed manually several times a day.
produce hydrogen and VFA [1218]. Rhodobacter species have
been the most widely used bacteria for photo-fermentation of 2.2. Organisms
VFA to CO2 and H2 [1925]. The reported hydrogen yields and
formation rates are rather low in dark fermentations since Anaerobic sludges obtained from acidogenic phase of anae-
part of hydrogen ends up in VFA [1215]. robic wastewater treatment plant of PAK MAYA Bakers yeast
Starch containing biomass such as waste wheat is rich company in Izmir, Turkey was used after heat treatment by
in terms of carbohydrates and deficient in other nutrients boiling in water. The concentrated sludge was boiled for 5 h in
such as nitrogen (N), phosphorous (P) and minerals (MN). order to eliminate hydrogen consuming methanogens and to
Therefore, when used as a substrate for biohydrogen produc- select spore forming and hydrogen producing acidogenic
tion, the wheat powder (WP) may not provide all required bacteria. The heat treated anaerobic sludge was cooled and
nutrients for microbial metabolism and hydrogen production. cultivated in a synthetic media containing glucose 60 g L1 ,
External addition of N and P may improve microbial peptone 10 g L1 , yeast extract 0:6 g L1 , MgSO4 :7H2 O
activities (enzyme synthesis, growth) and enhance biohydro- 0:25 g L1 , K2 HPO4 1 g L1 , KH2 PO4 1 g L1 , L-cysteine-
gen production from starch since starch is N and P deficient. HCl:H2 O 0:1 g L1 at 37 1C and pH 6:8 in an incubator.
Medium optimization studies were reported in literature Argon gas was passed through the cultivation media before
for biohydrogen production by dark fermentation of incubation and the cultivation flasks were closed with gas
different substrates [2932]. However, the effects of N and P tight rubber stoppers. The cultivated organisms were used for
supplementations or C/N and C/P ratios on the yield and inoculation of experimental bottles after three days of
the rate of hydrogen formation from starch were not incubation.
reported in literature. As an attempt to improve the rate
and yield of biohydrogen formation from wheat starch by 2.3. Analytical methods
dark fermentation, the effects of external supplementation of
nitrogen and phosphorous or C/N and C/P ratios were Samples were removed from the liquid phase everyday for
investigated in this study. The wheat powder solution (WPS) analysis of starch (STR), total sugar (TSG), and total volatile
was fermented at 37 1C and pH 627 for hydrogen gas fatty acids (TVFA). For starch analysis, the samples were
production using heat treated acidogenic anaerobic sludge. acidified and boiled for 1.5 h for complete hydrolysis of
Variations of hydrogen yield and formation rate with the starch to sugar and the resulting total sugar concentration
C/N and C/P ratio were investigated by using a BoxWilson was determined by using the acid-phenol method [33].
statistical experiment design approach and the C/N and Starch concentration was determined by dividing total sugar
C/P ratios yielding the highest yield and the rate were concentration by 1.05 since one water molecule is added
determined. to a disaccharide to produce two monosacharides during
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I N T E R N AT 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 33 (2008) 1813 1819 1815

hydrolysis. In other words 360 g monosachharide (glucose) is Table 1 Experimental conditions of the BoxWilson
obtained from 342 g of starch by hydrolysis. Total carbon statistical design
content of starch was 40% of the starch concentration.
The samples were centrifuged at 7000g to remove solids Axial X1 X2 Factorial X1 X2
from the liquid media and total sugar and TVFA analyses points points
were carried out in clear supernatants. Total sugar content
A1 20 525 F1 173.6 860.7
was determined by the acidphenol spectrometric method
A2 200 525 F2 173.6 189.31
[33]. TVFA analyses were carried out by using analytical kits A3 110 50 F3 46.4 860.69
(Spectroquant, 1.01763. 0001, Merck, Darmstadt, Germany) A4 110 1000 F4 46.4 189.31
and a PC spectrometer (WTW Photolab S12). Individual VFAs Center point 110 525
in the fermentation effluent were determined by using a high
performance liquid chromatograph (HPLC, Agilent 1100) with
a prevail organic acid column of 5 mm  150 mm  4:6 mm and
a UV detector of 220 nm. The carrying medium was 25 mM
1
KH2 PO4 at pH 2.5 with a flow rate of 1 ml min . (Y 1 , mg H2 g1 starch) and the specific rate of H2 formation
Hydrogen gas was sampled from the head space of the mg H2 g1 biomass h1 were considered as the objective
bottles by using gas-tight glass syringes and hydrogen functions. N and P are the externally added nutrients exclud-
concentration was determined by using a gas chromatograph ing the N and P content of the wheat. The C/N and C/P ratios
(Agilent 6890). The GC column was Alltech, Hayesep D 80/100 were varied between X1 202200 and X2 5021000, respec-
6 in  1=8 in  085 in. Nitrogen gas was used as carrier with a tively. The experiments consisted of four axial and four
flow rate of 30 ml min1 and the head pressure was 22 psi. factorial and a center point totaling nine experiments at
Temperatures of the oven, injection, detector, and filament different C/N and C/P ratios. The center point was repeated
were 35, 120, 120, 140 1C, respectively. The amount of total gas three times to test the reproducibility of the experiments.
produced was determined by water displacement method Table 1 summarizes the list of experiments conducted by using
everyday using sulfuric acid and NaCl containing solution. the BoxWilson statistical experiment design. A control
The cumulative hydrogen gas production was determined by experiment with no inoculation and another experiment with
using the following equation: no external N and P addition were performed along with the
VH2 ;i VH2 ;i1 VW CH2 ;i VG;i CH2 ;i  VG;i1 CH2 ;i1 , (1) BoxWilson design experiments.
The following quadratic response function was used to
where VH2 ;i and VH2 ;i1 are the volumes of cumulative correlate the independent variables, X1 and X2 with the
hydrogen (ml) calculated after the ith and the previous objective function, Y.
measurement; VW is the total gas volume measured by the
water displacement method (ml); CH2 ;i is the concentration of Y b0 b1 X1 b2 X2 b12 X1 X2 b11 X21 b22 X22 . (2)
H2 gas in the total gas measured by the water displacement The experimental data obtained from the experiments
method (%); VG;i and VG;i1 are the volumes of the gas in the presented in Table 1 was used for determination of the
head space of the bottle for the ith and the previous coefficients in Eq. (2) using a STATISTICA 5 program for
measurement (ml); CH2 ;i and CH2 ;i1 are the percent H2 in the regression analysis.
head space of the bottle for the ith and the previous
measurement. The amount of released hydrogen gas and in
the head space of the bottle were measured independently 3. Results and discussion
and added up to determine cumulative H2 formation for every
period of sampling. The results of the BoxWilson design experiments are
Biomass concentration in the inoculum was determined by presented in Table 2 along with the predicted values from
filtering 20 ml sample through a 0:45 mm milipore filter, drying the response functions. The coefficients of the response
at 105 1C and determining the constant dry weight [34]. pH functions determined by regression analysis are summarized
and ORP of the fermentation medium were monitored by in Table 3 and were used to predict the response function
using a pH meter and ORP meter with relevant probes (WTW, values presented in Table 2. Hydrogen yield predictions were
Germany). pH of the medium decreased from an initial value in good agreement with the experimental results R2 0:86
of 7.0 to nearly 4.5 in early stages of fermentation due to VFA as shown in Table 2. A control experiment was conducted
production. Medium pH was adjusted to 7.0 by addition of without any N and P addition. The results of this experiment
10 M NaOH twice a day. pH was maintained between 6 and 7 are also presented in Table 2 (No NP addition).
by manual pH control. ORP values varied between 100 and Fig. 1 depicts variation of cumulative hydrogen (ml), starch,
300 mV, in general. glucose and TVFA concentrations mg L1 with time for the
experiment F1 where C/N and C/P ratios were 173 and 860,
2.4. BoxWilson statistical experiment design respectively since the hydrogen yield and the rate were the
highest for this experiment. Starch concentration decreased
A BoxWilson statistical experiment design method was used from an initial value of nearly 18 to 2 g L1 after 150 h of
to determine the effects of C/N and C/P ratios on hydrogen fermentation indicating effective starch hydrolysis by the
yield and production rate. C/NX1 and C/PX2 ratios were anaerobic sludge bacteria. Cumulative hydrogen volume
considered as independent variables; the hydrogen yield increased rather fast within the first 100 h of fermentation
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Table 2 Experimental and predicted values of the yields and SHPR

Experiment Yieldexp Yieldpred SHPRexp SHPRpred


mg H2 g1 starch mg H2 g1 starch mg H2 g1 bm h1 mg H2 g1 bm h1

A1 7.82 8.73 2.27 2.46


A2 14.25 14.89 5.53 5.20
A3 9.41 8.46 3.19 2.16
A4 6.42 8.92 1.77 2.67
F1 17.87 15.96 5.62 5.19
F2 8.37 8.89 2.06 2.99
F3 6.95 4.85 2.21 1.41
F4 10.94 11.28 2.32 2.89
C (ave) 6.74 6.74 1.84 1.84
No NP 6.09 2.36

Table 3 Coefficients of the response functions for different C/N and C/P ratios

b0 b1 b2 b12 b11 b22 R2

Y 1 , Yield 21.806 0.186 0.026 1:58  104 6:26  104 9:0  106 0.86
Y 2 , SHPR 6.034 0.061 0.006 4:30  10 5
2:45  10 4
2:5  106 0.78

4500 22500 Fermentation of starch to VFA and H2 has the following


reaction scheme
4000 20000
Cumulative Hydrogen Gas (ml)

3500 17500 Starch!n Glucose maltose!VFA H2 . (3)


Concentration (mg.l-1)

3000 15000 The first step is the hydrolysis of starch to carbohydrates and
2500 12500 the second step is fermentation of carbohydrates to VFA and
H2 . Glucose and maltose constitute total sugars as inter-
2000 10000
mediate fermentation products and are converted to VFA and
1500 7500 H2 simultaneously. Since no sugar accumulation was ob-
1000 5000 served during fermentation, microbial hydrolysis of starch
was the slowest or the rate limiting step in this conversion
500 2500
scheme. Sugars were converted to VFA and H2 much faster
0 0 than they were produced.
0 50 100 150 200 250 300 350
The response functions with determined coefficients were
Time (h)
used to predict the variations of hydrogen yield and formation
Fig. 1 Variation of cumulative hydrogen E, starch (), rate with the C/N and C/P ratio. Variations of hydrogen yield
TVFA m, and total sugar concentrations with time for mg H2 g1 starch with C/P ratio at different C/N ratios are
the experiment F1 (C=N 173, C=P 860). depicted in Fig. 2. At low C/N ratios o46 indicating excess
nitrogen (high N/C ratio), hydrogen yield increased with
decreasing C/P or increasing P/C ratio indicating high
phosphorous requirements at high nitrogen concentrations.
and reached nearly 3650 ml (30 1C, 1 atm) at the end of 200 h of At high C/N ratios 4110 indicating low nitrogen contents
incubation which is equivalent to 3590 ml at STP (25 1C, 1 atm). (low N/C ratios), hydrogen yield increased with increasing C/P
Sugar was an intermediary product in starch fermentation to or decreasing P/C ratio indicating low phosphorous require-
organic acids and hydrogen gas. Therefore, total sugar ments at low nitrogen contents. At a high C/P ratio of 950 (low
concentration increased from an initial level of 2 g L1 to P content) hydrogen yield increased with increasing C/N or
nearly 9 g L1 within 50 h and then decreased to less than decreasing N/C ratio indicating low nitrogen requirements at
1 g L1 due to sugar conversion to VFA and hydrogen gas. low phosphorous contents. N and P requirements were
Sugar concentration remained almost constant at 0:4 g L1 for proportional with each other and also with the carbon or
the rest of the fermentation period. The TVFA concentration starch content of the wheat due to prevailing stoichiometry of
increased steadily from 2 to 11 g L1 at the end of 300 h of the anaerobic metabolism. The highest hydrogen yield
fermentation. HPLC analysis of VFA indicated that the major (23 mg H2 g1 starch or 281 ml H2 g1 starch at STP) was ob-
organic acids were acetic, butyric, propionic and lactic acids. tained at the highest C/N and C/P ratios of 200 and 1000
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30 low P requirements. For a constant carbon content of the


medium (e.g., 20 g starch L1 or 8 g C L1 ) the nitrogen and
25 phosphorous contents should change in the same direction
Yield (mgH2.g-1starch)

200 requiring low P for low N contents for high hydrogen yield due
20
to prevailing stoichiometry of anaerobic metabolism. Within
173 the range of independent variables the highest hydrogen yield
15 20
46 (23 mg H2 g1 starch or 281 ml H2 g1 starch at STP) was ob-
10 tained with the highest C/N and C/P ratios of 200 and 1000 or
110 the lowest N/C and P/C ratios of 0.005 and 0.001. N/P ratio
5 maximizing the hydrogen yield was 5. The hydrogen yield
C/N obtained with no nitrogen and phosphorous addition was
0 6:09 mg H2 g1 starch or 74:4 ml H2 g1 starch at STP which was
50 200 350 500 650 800 950 considerably lower than the maximum yield obtained at
C/P ratio C=N 200 and C=P 1000. The N and P content of the WP was
Fig. 2 Variation of hydrogen yield with the C/P ratio at not sufficient and external addition of N and P was required
different C/N ratios. for effective hydrogen production by dark fermentation of
WPS.
The specific hydrogen production rate (SHPR) is another
30 important parameter indicating effectiveness of the fermen-
tation. SHPR was calculated as the mg or ml hydrogen
25
produced per gram dry weight of initial biomass per unit
Yield (mgH2.g-1starch)

20 time mg H2 g1 biomass h1 at the end of 240 h where


1000
860 cumulative hydrogen production was leveled off. The follow-
15 50 ing equation was used in SHPR calculations,
190 525
10 mg or ml H2 produced
SHPR , (4)
XT0 :t
5
C/P where mg or ml H2 produced is the amount of cumulative
0 hydrogen produced in 240 h, XT0 is the initial amount of
20 45 70 95 120 145 170 195 biomass (g, X0 V0 ) and t is the fermentation period (240 h).
C/N ratio Variation of SHPR with C/P ratio at different C/N ratios is
Fig. 3 Variation of hydrogen yield with the C/N ratio at depicted in Fig. 4. At low C/N ratios o46, or high nitrogen
different C/P ratios. contents, the SHPR decreased with increasing C/P ratio due to
limitations caused by low phosphorous content when nitro-
gen is in excess. However, at high C/N ratios 446 where
corresponding the lowest N/C and P/C ratios of 0.005 and nitrogen was the rate limiting substrate, the SHPR increased
0.001. The C/N/P ratio maximizing hydrogen yield was 100/ with increasing C/P ratio indicating low phosphorous require-
0.5/0.1 corresponding to a starch/N/P ratio of 100/0.2/0.04 ments when nitrogen concentration was low. N and P
w w1 w1 . The results indicated that the external nitrogen requirements were proportional as dictated by the stoichio-
and phosphorous requirements of the anaerobic sludge metry of anaerobic metabolism. The highest SHPR
organisms for the highest hydrogen yield was 0.2% and (8:0 mg H2 g1 biomass h1 or 98 ml H2 g1 biomass h1 at STP)
0.04% of dry weight starch or 0.5% and 0.1% of total organic was obtained at C=N 200 and C=P 1000 indicating low
carbon available. Low external N and P requirements
are partly due to presence of N and P in the WP and partly
due to low growth yields of the anaerobic organisms
10
(0:06 g biomass g1 sugar).
9
Fig. 3 depicts variation of hydrogen yield mg H2 g1 starch
SHPR (mgH2.g-1cells.h-1)

8
with C/N ratio at different C/P ratios. At low C/P ratios (o525,
7
excess P), hydrogen yield decreased with increasing C/N ratio
(decreasing N/C ratio) indicating limitations by the nitrogen 6 200
source at high phosphorous contents. However, at high C/P 5
173
ratios 4525 or low P contents hydrogen yield increased with 4
increasing C/N ratio (decreasing N/C ratio) steadily indicating 3 20
46 110
low nitrogen requirements at low phosphorous concentra- 2
1 C/N
tions. Similarly, at a low C/N ratio of 45 indicating presence of
excess nitrogen, hydrogen yield increased with decreasing 0
50 200 350 500 650 800 950
C/P ratio or increasing P/C ratio indicating the requirement for
C/P ratio
high phosphorous contents. At high C/N ratios of 195 and
above indicating low nitrogen content, hydrogen yield in- Fig. 4 Variation of SHPR with the C/P ratio at different C/N
creased with increasing C/P or decreasing P/C ratio indicating ratios.
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1818 I N T E R N AT 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 33 (2008) 1813 1819

nitrogen and phosphorous requirements for maximum rate was found to be 200. The external nitrogen requirement in
of hydrogen formation. The C/N/P ratio yielding the highest our case was lower making the process economically more
SHPR was 100/0.5/0.1. The SHPR obtained with no nitrogen attractive.
and phosphorous addition was 2:36 mg H2 g1 biomass h1 or Theoretical, hydrogen yields are 4 mol H2 mol1 glucose and
28:8 ml H2 g1 biomass h1 which was considerably lower than 2 mol of H2 mol1 glucose when acetic (2 mol) or butyric
that of the maximum SHPR indicating the need for external (1 mol) acids were the only end products from one mole of
nitrogen and phosphorous addition to yield C=N=P glucose, respectively. Formation of propionic acid (2 mol)
100=0:5=0:1 in the fermentation medium. consumes 2 moles of hydrogen per mole of glucose. That is
Fig. 5 depicts variation of SHPR with the C/N ratio at propionic acid formation is a hydrogen consuming reaction
different C/P ratios. At low C/P ratios (o525, high P content), and should be avoided. Lactic acid and ethanol formations do
SHPR decreased with increasing C/N ratio up to C=N 95 not yield hydrogen gas.
indicating limitations by the available nitrogen when phos- Since a mixture of acetic, butyric, propionic and lactic acids
phorous was in excess. However, at high C/N ratios above 95, were produced in this study, the highest hydrogen yield was
the SHPR increased steadily with increasing C/N ratio estimated to be 2 mol H2 mol1 sugar. At the experimental
indicating low requirement for nitrogen source. Similarly, at point of F1 with the highest hydrogen yield, 9072 mg L1 TVFA
high C/N ratios above 95 (low nitrogen content), the SHPR was produced at the end of the fermentation which is nearly
increased with increasing C/P ratio due to low phosphorous 50% of the initial starch. TVFAS consisted of acetic, butyric,
requirements by the organisms. The highest SHPR lactic and propionic acids with the concentrations of
(8:0 mg H2 g1 biomass h1 or 98 ml H2 g1 biomass h1 at STP) 3755 mg L1 (41%), 1627 mg L1 (18%), 1130 mg L1 (12.5%) and
was obtained at the highest C/N and C/P ratios of 200 and 1520 mg L1 (17%), respectively.
1000, respectively. The optimal C/N/P ratio maximizing the
SHPR was found to be 100/0.5/0.1. Low nitrogen and phos-
phorous requirements are due to low growth yields in
anaerobic organisms. 4. Conclusions
The highest hydrogen yield obtained in this study
(281 ml H2 g1 starch at STP) corresponds to a hydrogen yield Hydrogen production by dark fermentation of WPS using heat
of nearly 2 mol H2 mol1 glucose which is comparable with the treated anaerobic acidogenic sludge was investigated at
literature studies reporting hydrogen production from starch different initial C/N and C/P ratios in order to determine the
by dark fermentation [12,14,17]. Liu and Shen [13] studied N and P requirements of the organisms for effective hydrogen
hydrogen production from starch by dark fermentation and production.
obtained a maximum hydrogen yield of 194 ml H2 g1 starch A BoxWilson statistical experiment design was used by
and hydrogen formation rate of 9:8 ml H2 g1 biomass h1 at considering C/N (20200) and C/P (501000) ratio as independent
STP with a starch concentration of 15 g L1 . Our results were variables while hydrogen formation yield and the rate
considerably higher than those reported by Liu and Shen due were the objective functions. The experimental data was
to external addition of nitrogen and phosphorous and using correlated with a quadratic response function and the coeffi-
the most suitable C/N and C/P ratios to meet nutritional cients were determined by regression analysis. The response
requirements of the organisms. In a study by Lin and Lay [29] function predictions were in good agreement with the
the optimal C/N ratio was reported 47 for biohydrogen experimental data for hydrogen yield. The hydrogen yield
formation from sucrose solution. Since part of the nitrogen increased with increasing C/N and C/P ratio yielding the
was provided by the wheat in our study, the optimal C/N ratio maximum yield (23 mg H2 g1 starch or 281 ml H2 g1 starch at
STP) at C=N 200 and C=P 1000. The SHPR also increased
with increasing C/N and C/P ratios yielding the highest SHPR
(8:0 mg H2 g1 biomass h1 or 98 ml H2 g1 biomass h1 at STP)
at C=N 200 and C=P 1000. Low nitrogen contents required
10 low phosphorous for maximum hydrogen yields and formation
9 rates due to prevailing stoichiometry of the anaerobic
metabolism. High nitrogen and phosphorous concentrations
SHPR (mgH2.g-1cells.h-1)

8
inhibited hydrogen formation by dark fermentation probably
7 1000
by shifting the metabolic pathway. The C/N/P ratio yielding
6 860
the highest hydrogen yield 23 mg H2 g1 starch and formation
5
525 rate 8:0 mg H2 g1 biomass h1 was C=N=P 100=0:5=0:1.
4 50 The hydrogen yield and formation rate with no nitrogen
3 190
and phosphorous addition were 6:09 mg H2 g1 starch
2 (74:4 ml H2 g1 starch at STP) and 2:36 mg H2 g1 biomass h1
1 C/P
28:8 ml H2 g1 biomass h1 which were considerably lower
0 than the highest yield and the rate. The results indicated low
20 45 70 95 120 145 170 195
nitrogen and phosphorous requirements (N=P=starch
C/N ratio
0:2=0:04=100, w w1 w1 ) for high hydrogen yields and rates by
Fig. 5 Variation of SHPR with the C/N ratio at different C/P dark fermentation of WPS since part of the N and P present in
ratios. WP was also used for fermentation.
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I N T E R N AT 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 33 (2008) 1813 1819 1819

[19] Federov AS, Tsygankov AA, Rao KK, Hall DO. Hydrogen
Acknowledgment photoproduction by Rhodobacter sphaeroides immobilized on
polyurethane foam. Biotechnol Lett 1998;20:10079.
This study was supported by the Scientific and Technological [20] Eroglu I, Aslan K, Gunduz U, Yucel M, Turker L. Substrate
consumption rate for hydrogen production by Rhodobacter
Research Council of Turkey by a Grant number 105M296.
sphaeroides in a column photobioreactor. J Biotechnol
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