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Research Article
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Effect of Salts Addition on Hydrogen Production by C. acetobutylicum |
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H. Alshiyab,
M.S. Kalil,
A.A. Hamid
and
W.M. Wan Yusoff
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ABSTRACT
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The objective of this study is to investigate the effect
of salts addition to fermentation medium on hydrogen production, under
anaerobic batch culture system. In this study, batch experiments were
conducted to investigate the inhibitory effect of both NaCl and sodium
acetate on hydrogen production. The optimum pH and temperature for hydrogen
production were at initial pH of 7.0 and 30°C. Enhanced production
of hydrogen, using glucose as substrate was achieved. In the absence of
Sodium Chloride and Sodium Acetate enhanced hydrogen yield (YP/S)
from 350 mL g-1 glucose utilized to 391 mL g-1 glucose
utilized with maximum hydrogen productivity of 77.5 ml/L/h. Results also
show that sodium chloride and sodium acetate in the medium adversely affect
growth. Hydrogen yield per biomass (YP/X) of 254 ml/L/g, biomass
per substrate utilized (YX/S) of 0.268 and (YH2/S)
of 0.0349. The results suggested that Sodium at any concentration resulted
to inhibit the bacterial productivity of hydrogen.
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INTRODUCTION
The modern economy is largely built on combustion of fossil fuels such
as oil, coal and natural gas. This will eventually lead to the depletion
of limited fossil energy resource. Furthermore, the combustion of fossil
energy source contributed to heavy air pollution and the greenhouse effect.
To reduce air pollution and greenhouse gas emissions, H2 has
been considered to be an ideal energy alternative for the future. Compared
with conventional H2 generation processes, biological H2
production using fermentative and photosynthetic bacteria is not only
environmental friendly, but also energy saving (Das and Veziroglu, 2001).
Biohydrogen fermentation by anaerobic fermentation by anaerobic process
is greatly influenced by many factors, such as pH, temperature, toxic
substance and ionic strength (Zheng et al., 2005).
The effects of the different factors, such as substrate, bacteria strain,
reactors used and conditions on anaerobic fermentation for hydrogen production
have been investigated. However few study investigated the impact of metal
ion concentration on bio-hydrogen production. Lee et al. (2001)
only studied the effect of normal metal ion iron concentration in the
external environment on hydrogen fermentation using FeC12,
treating sucrose solution by the mixed microorganisms from a soybean-meal
silo and found out the relationship between iron concentration and VFAs
and solvents. Subsequently, Lin and Lay (2005) reported that magnesium,
sodium, zinc and iron were important trace metals affecting hydrogen production
magnesium being the most significant. Metal ions are easily found in wastewaters,
whereas in practice some amount of sodium salts is frequently found in
various wastewaters. The presence of high sodium ion concentration is
considered to have an inhibitory effect on the anaerobic wastewater treatment
and activated sludge treatment (Xiaolong et al., 2006).
Earlier investigations on sodium inhibition were mainly concentrated
on undomesticated methanogenic bacteria in anaerobic digestion process;
and under different conditions, different bacteria (microbes) have different
tolerances to sodium toxicity. However, few study have been done on the
effect of sodium salt on hydrogen-producing bacteria (pure bacteria).
However, ionic strength is one of the important factors influencing H2
production because the growth of fermentative bacteria requires a number
of ions such as sodium and potassium. The ionic strength in the solution
is determined by both ion species and its concentrations and it can be
either stimulatory or inhibitory and even toxic to H2 production.
A low level of ionic strength may have no or even stimulatory effect on
H2 production. However, a high level of ionic strength can
result in cell lysis (Niel et al., 2003). Therefore, a proper ionic
strength is necessary to optimize anaerobic hydrogen production process.
Inhibitory effect of ionic strength on hydrogen fermentation is available
in the literature. Therefore this study was conducted to investigate the
influence of ionic strength on glucose degradation, hydrogen production.
The ionic strength in the solution was provided in the form of sodium
chloride and sodium acetate. New medium we formulated in the lab for hydrogen
production using C. acetobutylicum NCIMB 13357 and glucose is the
sole carbon source.
MATERIALS AND METHODS
Microorganism and culture conditions: C. acetobutylicum NCIMB
13357 was purchased from a British culture collection, NCIMB Ltd. Scotland,
UK. This study was conducted from 2006 to 2007 in Department of Chemical
Engineering; Universiti Kebangsaan Malaysia. The bacterium was cultivated
in anaerobic condition in Reinforced Clostridial Medium (RCM) for 24 h
at 30 °C. Liquid medium of RCM was used for inoculum preparation.
The growth of culture in RCM was monitored by measuring an optical density
at 600 nm using a spectrophotometer. Only inoculum with optical density
(OD) values greater than 0.4 after 18 h cultivation was used as inoculum.
An inoculum of 10% (v/v) was used throughout this study.
Cultivation medium: New medium was formulated in the lab to be
used for hydrogen production have the following composition in (g L-1):
glucose (5), yeast extract (5), L-cystine. HCl (1.0) and bacteriological
agar (0.5). The initial anaerobic condition in the reactor after inoculation
inside the anaerobic glove box was established by replacing the gaseous
phase with nitrogen at start of cultivation. Then incubated at 30°C
in temperature controlled water bath without shaking. The evolved gas
was monitored and collected in a gas collection cylinder and the volume
of evolved gas was measured at room temperature by the water displacement
method (Morimoto et al., 2004) in a graduated cylinder (inverted),
that had been filled with water of pH 3 or less in order to prevent dissolution
of the gas components.
Analytical methods: The gas composition was determined by gas
chromatography (Shimadzu Co., Kyoto, GC-8A) under the following conditions:
column: Porapack-Q, carrier gas: Nitrogen, flow rate: 33 mL min-1;
column temperature: 50°C; injection temperature: 100°C; detector
temperature: 50°C, detector: Thermal Conductivity Detector (TCD).
The soluble glucose concentration was measured at the end of each batch
experiment for the calculation of the amount of glucose consumed by DNS
method modified by Miller, (1959) using spectrophotometer (UV 1601IPC,
Shimadzu corporation- Japan) optical density (OD550 nm). Individual
batch experiments were observed until the hydrogen production from each
bottle stopped. Final medium pH was measured by pH meter (Mettler Toledo)
and final biomass was measured by Spectrophotometer (UV 1601IPC, Shimadzu
corporation- Japan) at optical density (OD600 nm). All of these
data were the average (mean) of three trials.
Experimental design: Batch experiments were conducted with 500-mL
Scotch bottle with working volume of 100 mL (90% medium and 10% inoculum).
Different concentrations of NaCl and NaCH3COOH were used to
find out its effect on fermentative bacteria we used in this study, ranging
from 0.0 to 5000 mg L-1 for NaCl and from 0.0 to 3000 mg L-1
for NaCH3COOH.
RESULTS AND DISCUSSION
The results shown in Fig. 1 and 5
indicated that at 0.0 g L-1 of NaCl or NaCH3COOH,
the maximum H2 yield was obtained at 0.0 g L-1 of
NaCl was 391 mL g-1 glucose utilized and for NaCH3COOH
was 355 mL g-1 glucose utilized. The increase of hydrogen yield
was reversed to bacterial productivity of hydrogen which resulted to increase
as shown in Fig. 3 and 6 which indicated
that hydrogen productivity was dropped from 77.5 ml/L/h at 0.0 g L-1
for NaCl to 63.5 ml/L/h at 5 g L-1 and for NaCH3COOH
from 72.5 ml/L/h at 0.0 g L-1 to 66.5 ml/L/h at 3 g L-1.
The maximum productivity of 77.5 ml/L/h obtained by using this medium
was lower than 137 ml/L/h which reported by Morimoto et al. (2004)
at 50°C using POME sludge and the other reported values was for continuous
process not batch culture.
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Fig. 1: |
Effect of NaCl addition on H2 yield (mL
g-1) utilized |
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Fig. 2: |
Effect of NaCl addition on glucose consumption (%) |
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Fig. 3: |
Effect of NaCl addition on Hydrogen productivity H2P
(ml/L/h) |
The glucose consumption was influenced by the presence of NaCl or NaCH3COOH
or both in fermentation medium. The results shown in Fig.
2 and 6 indicated that with increase in NaCl from
0.0 to 5000 mg L-1 and NaCH3COOH from 0.0 to 3000
mg L-1, the glucose consumption decreased gradually. For the
control, both NaCl (5 g L-1) was 73% and NaCH3COOH
(3 g L-1) was 80%. Compared with the control, at 0.0 mg L-1
of NaCl and NaCH3COOH, the glucose consumption was enhanced
and reached to 87 and 90%, respectively, but in the presence of a low
level of NaCl or NaCH3COOH, glucose consumption was decreased
as the concentration of each was increased. This demonstrates that glucose
consumption by C. acetobutylicum NCIMB13357 was influenced by the
presence of any amount of NaCl or NaCH3COOH in the fermentation
medium and that might be the inhibitory effect of sodium at any concentration.
The results showed that, NaCl and NaCH3COOH affect in negative
way on glucose degradation.
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Fig. 4: |
Effect of NaCl addition on Biomass concentration (g
L-1) Glucose 0.5 g L-1, inoculum size 10% (v/v),
I pH 7.0, Temp. 30°C |
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Fig. 5: |
Effect of NaCH3COOH addition H2
yield (mL g-1) Utilized |
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Fig. 6: |
Effect of NaCH3COOH addition on glucose consumption
(%) |
Table 1: |
Results the effect of NaCl on glucose consumption and
H2P (ml/L/h) by C. acetobutylicum NCIMB13357 |
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Glucose: 5 g L-1, inoculum size 10% (v/v)
I pH 7.0. Temp. 30 °C |
Table 2: |
Results the effect of NaCl on hydrogen production by
C. acetobutylicum NCIMB13357 |
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Y1P/S (H2 mL g-1
glucose supplied) (mL g-1)), Y2P/S
(mL g-1) (Utilized): (H2 mL g-1 glucose
utilized), [Biomass] (g L-1). Biomass production g L-1
culture, YP/X (mL/g/L): (H2 mL g-1
Biomass L-1), YX/S: (Biomass production per
g glucose supplied), YH2/s: (conversion of H2
mL to H2 (g) per g glucose utilized) Glucose: 5 g L-1,
inoculum size 10% (v/v), I pH 7.0, Temp. 30 °C |
Obtained data shown in Table 1 and 2
demonstrated that hydrogen production depends on the presence of NaCl
and its concentration and as NaCl concentration increased the produced
hydrogen was decreased. Niel et al. (2003) reasoned that inhibition
of decreased hydrogen production to increase in concentration of the NaCl
in the fermentation medium and suggested that due to the increase of the
ionic strength in the fermentation medium concluded that the high ionic
strength affect the bacterium metabolic pathway.
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Fig. 7: |
Effect of NaCH3COOH addition on hydrogen
productivity H2P (ml/L/h) |
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Fig. 8: |
Effect of NaCH3COOH addition on biomass concentration
(g L-1) glucose 0.5 g L-1, inoculum size 10%
(v/v), I pH 7.0, Temp. 30°C |
The effect of NaCl it was also shown on the growth of bacteria. It appeared
that the concentration of inhibitor affects the bacterial growth and as
concentration increased, the final biomass concentration was lower. The
results shown in Fig. 4 and 8 shown
that the presence of NaCl or NaCH3COOH in fermentation medium
affect on bacterial growth with maximum biomass concentration was enhanced
from 1.22 at 5 to 1.31 at 0.0 g L-1 whereas for NaCH3COOH
enhanced from 1.18 at 3 to 1.34 at 0.0 g L-1, suggested that
both components were inhibited the bacterial growth and that resulted
to reduce the produced hydrogen. Niel et al. (2003) suggested that
high salt concentration affect the bacterial growth and resulted to cell
lyses.
Table 3: |
Results the effect of sodium Acetate (NaCH3COOH),
on glucose consumption and H2 P (ml/L/h) by C. acetobutylicum
NCIMB13357 |
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Glucose: 5 g L-1, inoculum size 10% (v/v)
I pH 7.0. Temp 30 °C |
Table 4: |
Results the effect of sodium acetate (NaCH3COOH),
on hydrogen production by C. acetobutylicum NCIMB13357 |
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Y1P/S(H2 mL g-1
glucose supplied) (mL g-1), Y2P/S
(mL g-1) (Utilized): (H2 mL/g glucose utilized),
[Biomass] (g L-1). Biomass production g per L culture,
YP/X (ml/g/L): (H2 ml per g Biomass per L),
YX/S: (Biomass production per g glucose supplied), YH2/s:
(conversion of H2 mL to H2 (g) per g glucose
utilized) Glucose: 0.5 g L-1, inoculum size 10% (v/v),
I pH. 7.0, Temp. 30°C |
The data shown in Table 2 and 4 were
used to Calculate different yields like YP/X (ml/g/L): (H2
mL per g Biomass per L), YX/S: (Biomass production per g glucose
supplied) and YH2/s (conversion of H2 (ml) to H2
(g) per g glucose utilized). These results showed that fermentation medium
without salts addition resulted to enhanced the bacterial growth and that
would enhance the bacterial productivity of hydrogen. Suggested that the
enhancement of hydrogen production due to the increased in bacterial biomass.
Mechanisms of sodium chloride inhibition on h2 production:
Das and Veziroglu (2001) they mentioned that many ions can be inhibitory
or toxic to H2 producing microorganisms, depending on their
concentration. The results obtained in Table 1 and 2
showed that both H2 productivity and yield influenced by addition
of NaCl to fermentation medium. Zheng et al. (2005) reasoned that
to the ionic strength of the medium. They reported that a trace level
of many ions is required for activation of function of many enzymes and
co-enzymes. Excessive amounts, however, can lead to inhibition or toxicity.
This is mostly due to chemical binding of some metals to the enzymes,
resulting in the disruption of enzyme structure and activities. However,
the inhibitory effect of NaCl on hydrogen production should not be related
to the disruption of structure and activities of enzymes responsible for
H2 production, as NaCl dose not bind to the enzyme. With high
concentration of NaCl, high osmolarity environments trigger rapid fluxes
of cell water, thus causing a reduction in turgor and dehydration of the
cytoplasm (Ahring and Westermann, 1983).
In earlier studies about the effect of NaCl on methanogenic microorganisms,
Zheng et al. (2005) stated that high osmolarity has been found
to be responsible for the inhibition of NaCl on bacterial bioactivities.
This was also likely to be the mechanism for NaCl inhibition of hydrogen
production in the present study. Lin and Lay (2005) reported that magnesium,
sodium, zinc and iron were important trace metals to enhanced hydrogen
production by mixed culture with magnesium being the most significant.
To find out the effect of sodium ions on the enzymes activity, Maris et
al. (1984) study the levels of enzymes involved in the formation of
acetate and butyrate in C. acetobutylicum. They found that in the
pathway to form acetate, this bacterium produces two enzymes which are
phosphate acetyltransferase and acetate kinase, they found that the activity
of phosphate acetyltransferase inhibited by monovalent cation like Na+.
It seems that the medium components was totally responsible of the inhibition
of hydrogen production and with relation to other study weather the addition
was to activate or to inhibit depend on the microorganism (s) used. Finally,
NaCl is one of yeast extract (Nitrogen source) composition so, it might
be that has another inhibitory effect and reduce the quantity of produced
gases. In present study the results show to the first time that NaCl with
any concentration in fermentation medium have negative effect on hydrogen
production by C. acetobutylicum NCIMB13357.
Inhibition by sodium acetate: The results shown in Table
3 and 4 demonstrated that hydrogen production by
C. acetobutylicum NCIMB13357, was depending on sodium acetate concentration
and as Sodium Acetate concentration increased the hydrogen production
yield and that connected to bacterium productivity of hydrogen were decreased.
As mentioned above for sodium chloride, Niel et al. (2003) reasoned
that inhibition of decreased hydrogen production to increase in concentration
of the acetate in the fermentation medium and that affect the bacterium
metabolic pathway. The effect of sodium acetate concentration it was also
shown on the growth of bacteria. Growth was checked by final biomass concentration
(bacterial dry weight). It appeared that concentration of inhibitor affect
the bacterial growth and as concentration increased, the biomass concentration
was decreased. Niel et al. (2003) suggested that due to cell lysis.
It was reported by Chia et al. (1982) the summary of earlier reports
of the effect of sodium acetate salt on yeast growth. They reported that
elevated levels of inorganic electrolytes in the liquid growth medium
have been found to influence several parameters of yeast activity. (i)
Cell growth and multiplication: (a) the number of viable yeast cells per
unit volume of liquid growth medium decreases as salt content increases,
(b) the biomass of the culture (i.e., the total weight of yeast cells
per unit volume of liquid growth medium) decreases as salt content increases
and (c) the length of the lag phase (i.e., the incubation period between
inoculation of the culture and detectable initiation of cell growth) lengthens
as salt concentration increases. (ii) Utilization of the primary carbon
and energy source is reduced. (iii) Change in concentration of metabolic
products: (a) there is a decrease in the production of ethanol as salt
content increases and (b) there is an increase in the concentration of
other fermentation products (such as glycerol, acetaldehyde, etc.) as
salt content increases. Another study regarding the effect of sodium acetate
on the enzyme level was conducted by Biggins and Dilworth (1968) they
reported that an increase in the concentration of acetate within the cell
to partially inhibit acetate kinase activity, resulting in elevated concentrations
of acetyl phosphate in C. pasteurianum. They found also that acetyl
phosphate acted as a product inhibitor of pyruvate phosphoroclastic activity
and apparently resulted in a slowdown of general metabolism. Regarding
the effect of sodium acetate on bacterial metabolism, Bechtle (1969) reported
that sodium acetate was shown to have a potentiating effect on some proteases
in hydrolysis of milk proteins at acid pH values and that effect was minimal
in neutral pH media and there was an inhibition of the protease activity
at alkaline pH values under some conditions. Furthermore, Lawrence and
Sanderson (1969) reported that sodium acetate in the Cheesman assay medium
was inhibitory to some of the microbial enzymes tested. This, as well
as observations reported here, would indicate that acetate ions may have
an inhibitory action on an enzyme system.
Inhibition of growth by acetate and other weak organic acids is quite
common, but the level of tolerance varies tremendously among species.
The undissociated form of these compounds can function as uncoupling agents
(Booth, 1985). However, Niel et al. (2003) reported that, the ionic
strength of sodium acetate was responsible for the inhibition of the hydrogen
production by C. saccharolyticus and Donnison et al. (1989)
observed that lysis in exponentially growing cultures of this organism
after addition of 20 mM sodium acetate. Similar phenomena have already
been described for, C. thermoaceticum (Wang and Wang, 1984) and
Bacillus subtilis (Joliffe et al., 1981). Joliffe et
al. (1981) reported that C. thermoaceticum release of autolysins
upon addition of high salt concentrations. Another study by Kemper et
al. (1993), they reported that inactive autolysins are distributed
in the relatively acidic cell wall of gram-positive bacteria during exponential
growth. Suggested that it is thought that autolysins become activated
once the pH of the cell wall is neutralized. Results obtained in this
study imply a similar scenario in C. saccharolyticus, which possesses
also a gram-positive-type cell wall. Present results shows to the first
time that sodium with any concentration in fermentation medium have negative
effect on hydrogen production by bacterium we used in this study.
Finally the final H2 yield obtained by using the new medium
was 340 mL g-1 glucose supplied (2.72 mol H2/mol
glucose supplied): (According to Wooshin et al. (2005): each 125
mL of H2 ∼ 1 mole H2) and that was higher than
reported value in the literature for mesophilic species of clostridia
and using glucose as substrate. The entire reported H2 yield
reported in the literature shown in Table 5. Through
the revision of the components of the medium they were used, it was shown
that some they used NaCl and different nitrogen source with different
concentration and that what it discovered to inhibit the H2
production by C. acetobutylicum NCIMB13357 in this study. For
above reasons it was clear that the medium composition had remarkable
effect on hydrogen production by Clostridium species.
Table 5: |
Some of reported value of H2 by Clostridium
species through the literature |
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H2 yield (molH2/mol glucose supplied) |
CONCLUSIONS
This study demonstrated fermentative hydrogen production by C. acetobutylicum
NCIMB 13357 in a batch bioreactor. The maximum hydrogen yield was 391
mL g-1 glucose utilized. It found that both NaCl and sodium
acetate were inhibitor to hydrogen production as well as bacterial growth
of bacterium used in this study.
ACKNOWLEDGMENT
The author would like the thank Universiti Kebangsaan Malaysia for financial
assistance under grant No UKM-OUP-BTK-14/2007.
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