Indian Journal of Chemical Technology
Vol. 13, November 2006, pp. 591-596
Studies on bacterial growth and lead(IV) biosorption using Bacillus subtilis
Sk Masud Hossaina* & N Anantharamanb
a
Department of Chemical Engineering, Mohamed Sathak Engineering College, Kilakarai 623 806, India
b
Department of Chemical Engineering, National Institute of Technology, Tiruchirapalli 620 015, India
Email: [email protected]
Received 11 November 2005; revised received 28 June 2006; accepted 11 July 2006
Gram-positive bacterium Bacillus subtilis biosorbs lead(IV) ion from its aqueous solution. The maximum biosorption
of lead is 97.68% (w/w) within 48 h of incubation time with optimum pH 4.5 and optimum temperature 40°C for 700 ppm
initial loading of lead in a shake flask (optimum rpm 60). 7 days old and 30% (v/v) of suspension inoculum culture is used in
the studies. Lead is measured by using atomic absorption spectrophotometer (AAS) into an air-acetylene flame and
absorbance is measured at 283.3 nm. The maximum bacterial growth is noticed as 4.90×108 cells/mL at optimum bioprocess
conditions.
Keywords: Bacteria, Biosorption, Incubation, Lead
IPC Code: C02F3/00, C12N1/02
Certain species of microbes have been found to
adsorb surprisingly large quantities of metals1-3. These
metals include the one involved in toxicity to humans
and of commercial economic value. The use of
microorganisms to treat aqueous streams for the
removal, concentration and recovery of toxic and
valuable heavy metals although receiving increased
attention in the last decades, the removal of heavy
metals from municipal and industrial wastes by
biological treatment systems has continued to be of
interest. Bacterial surfaces have great affinity to
adsorb and precipitate metals resulting in metal
concentration on bacterial surface. There have been
several reports1-14 on the uptake of toxic heavy metals
by bacteria, so that the metals are accumulated. Both
metabolically mediated and biosportive phenomena
can occur in such systems. Therefore, it is not
unexpected that biosorptive metal uptake is subject to
environmental conditions that affect the reaction
chemistry of both the receptive sites and the
metals1-12.
Bacillus subtilis has a well-studied gram-positive
wall. If this bacterium is grown in the presence of
phosphate, its wall has essentially two chemical
components, peptidoglycan (A1γ) and teichoic acid
(glycerol based). For polyelectrolytes such as teichoic
acid, which can make up approximately 50% (dry
weight) of B. subtilis walls, Mg2+ is the preferred
metal ions. Other metals (especially potassium) are
also present in the walls in much smaller amounts,
and hence are reduced but not teichoic acid
removal6-12. It is pointed out that metals are necessary
for the walls and indeed for both the outer membrane
and phospolipids and lipopolysaccharides of outer
membrane and even affect the bonding forces that
hold the two faces of the membrane together6-12. Their
replacement with other metals can seriously alter the
wettability of the wall surface. In fact, it is possible
that the hydrophilicity or hydrophobicity of this
membrane can be modulated by its outer leaflet of the
outer membrane, this lipid wall be the first to contact
exogenous metal. Lipopolysaccharide has an
abundance of phosphoryl group, when compared to
phospolipids, and these groups have been implicated
as the primary sites for metal interactions6-12. Yet
despite the broad surfaces on bacterial surface, it is
still difficult to explain absolutely the high sorption
capacity that results in metallic precipitates and
minerals. A variety of precipitates and minerals have
been found associated with bacterial surfaces10.
The studies have shown that most metal binding
occurs after initial metal complexation and
neutralization of the chemically active sites11-17. For
B.subtilis walls, Beveridge and Murrey10 have
predicted at least a two-step mechanism for the
development of metal precipitation. The first step is
the stoichiometric interaction of metal with relative
chemical groups, which reside primarily in the
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INDIAN J. CHEM. TECHNOL., NOVEMBER 2006
peptidoglycan. After, complexation these same sites
nucleate the deposition of more metal by chemical
precipitation. Those sites constrained within the
interstices of the wall can develop only small-grain
precipitates, since sites (especially those on the other
surface) have no such constraints, and with time and
enough metal, very large-grain precipitates can
develop.
The removal of heavy metal ions from aqueous
solution by bacteria is effected not only by the surface
properties of the organism but also by various
other physico-chemical parameters of metal ion
solution. The present batch investigations were
undertaken to develop an effective bacterial treatment
of lead(IV) ion in aqueous solution using Bacillus
subtilis. Aerobic studies were conducted to optimize
bioprocess parameters18-21 such as biosorption time,
initial lead(IV) loading, pH, temperature and shaking
speed using aerobic batch suspension culture of the
acidophilic, mesophilic bacteria B. subtilis.
Experimental Procedure
Collection of bacteria and growth
The bacterium Bacillus subtilis (MTCC-1427), was
procured from Microbial Type Culture Collection and
Gene Bank, Institute of Microbial Technology
(CSIR), Chandigarh, India. The slant cultures
were prepared with prescribed growth medium
containing beef extract 1.0 g, yeast extract 2.0 g,
peptone 5.0 g, agar 15.0 g, sodium chloride 5.0 g, and
distilled water 1.0 L. It was sterilized in an autoclave
maintained at 15 psi for 15 min. The slant cultures
were maintained at constant temperature of 37°C in
an incubator. The bacterium was subcultured
regularly within 30 days and stored in freezer at 4°C.
The same slant cultures were used for suspension
culture preparation.
Suspension culture preparation
Whenever the suspension cultures were needed, the
freezed slant cultures were transferred out and kept in
an incubator at 25°C for 7 days for sufficient
sporulation. The slant cultures were prepared from the
freezed slant cultures. Spore crops were harvested in a
culture medium by washing the slant with sterile
distilled water. The resulting spore suspension was
filtered through several layers of sterile cheesecloths.
The spore densities are adjusted to 2×108 numbers per
mL (Luckey Drop method)22 in the inoculum
suspension.
Suspension culture media preparation
The following constituents were used for
suspension culture media preparation per liter:
KH2PO4 — 20.0 g, MgSO4 — 5 g, CaCl2 — 1.0 g,
CuSO4 — 0.1 g, ZnSO4.7H2O — 0.1 g CuSO4 —
0.1 g, AlK(SO4).12H2O — 0.01 g, H3PO3 — 0.01 g,
Na2MoO4.2H2O — 0.01 g, Glucose — 10 g, and
peptone 0.1 % (w/v).
General method
The biosorption of lead(IV) ion is studied in batch
system. Experiments are carried out in 1L Erlenmeyer
flasks (bioreactor) containing 250 mL of the medium.
250 mL of 500 ppm of lead (IV) ion solution and 30%
(v/v) suspension inoculum are taken to the reactors.
Inoculum is taken from a seven days old culture. The
maximum biomass (B. subtilis) concentration22 is
2.0×108 no. of cells/mL and weight of dry biomass is
3.6 g/L. The aerobic condition of the system is
maintained by putting nonabsorbent cotton to the
mouth of the reactors15-18. The dissolved oxygen (DO)
level is measured to 6.8 ppm in the reactors22. The
reactors are incubated in a constant temperature water
bath18-21 maintained at 30°C with constant shaking of
20 rpm. The initial pH of reactor medium is
maintained at 3.5 by using 0.1 N H2SO4 acid and/or 1
M CaCO3 slurry. To assess the extent of chemical
reaction, a set of experiment is carried out under
sterile conditions (without bacteria).
Effect of incubation time and initial lead loading
The general method is repeated for 600, 700 and
800 ppm of initial lead(IV) ion loading respectively.
Reactors are taken out on a regular interval basis18-21
i.e. after 12, 24, 36, 48, 60, and 72 h of incubation
respectively, followed by analysis of lead(IV) ion
presence in solution23. The bacterial growth is also
measured for each incubation time22. The lead(IV) ion
biosorption and bacterial growth with time are shown
in Figs 1 and 2, respectively.
Effect of initial pH on lead biosorption
The general method is repeated for various initial
pH values such as 2.5, 3.5, 4.5, and 5.5, respectively
with 700 ppm of initial lead(IV) ion loading. The
reactors are taken out after 48 h of incubation
(optimum time) and are analyzed for lead(IV) ion
presence in solution. The bacterial growth is also
measured for every pH value22. The lead(IV) ion
biosorption and bacterial growth with pH are shown
in Figs 3 and 4, respectively.
HOSSAIN & ANANTHARAMAN: BIOSORPTION OF Pb(IV) USING BACILLUS SUBTILIS
Effect of temperature on lead biosorption
The general method is repeated for various reactor
temperatures such as 30, 35, 40 and 45°C,
respectively with 700 ppm of initial lead(IV) ion
loading. The initial pH value is maintained at 4.5
(optimum). The reactors are taken out after 48 h of
incubation (optimum biosorption time) and were
analyzed for lead(IV) ion presence in solution. The
bacterial growth is also measured for every
temperature value22. The lead(IV) ion bioremediation
and bacterial growth with temperatures are shown in
Figs 5 and 6, respectively.
Effect of shaking speed on lead biosorption
The general method is repeated for different
shaking speeds of shaker such as 20, 40, 60 and 80
rpm, respectively with 700 ppm of initial lead ion
loading. The initial pH value is maintained at 4.5
(optimum). The temperature is maintained at 40°C
(optimum). The reactors are taken out after 48 h of
incubation (optimum time) and are analyzed for
lead(IV) ion in solution. The bacterial growth is also
measured for every shaking value22. The lead(IV) ion
biosorption and bacterial growth with shaking speed
are shown in Figs 7 and 8, respectively.
Lackey’s drop method of bacterial count
Exactly 0.1 mL volume of the sample was put by
using a calibrated medicinal dropper onto a glass
slide22. A cover slip of known area was placed,
avoiding any air bubble. The slide was put under a
microscope and the width of the high power
microscopic field was measured. Suppose the area
visible at one time is one micro transect. Now, the
slide is moved from one corner to another, counting
bacteria in each visible microscopic field. It was
counted in several fields by moving the slide in
horizontal and vertical directions. Counting must be
quick to avoid drying of the sample. Calculation of
the bacteria count is as follows:
Number of bacteria per mL = (No. of organisms
counted in all fields × Area of cover slip, mm2)/(Area
of one macroscopic field, mm2 × No. of field counted
× volume of sample in the cover slip)
Determination of
spectrophotometer
lead(IV)
using
atomic
593
biomass and unsolubilized materials and the solution
was filtered18-21. A lead hollow cathode lamp was
placed in the operating position, the current was
adjusted to 2-3 mA, and the lead line was selected at
283.3 nm23 using the appropriate monochromator slit
width. The appropriate acetylene gas was supplied to
the burner, following the instructions detailed for the
instrument, and adjusted the operating conditions to
give a fuel- lean air- acetylene flame. The chelate was
extracted with ammonium pyroline dithiocarbamate
into methyl isobutyl ketone for low levels of lead. The
organic layer was aspirated into the air-acetylene
flame and the absorbance of three readings for each
sample was recorded. Between each experiment, deionized water was aspirated in the burner. Finally, the
absorbance of the test sample was determined. A
calibration curve is plotted by aspirating samples of
solutions containing known solutions of the lead(IV),
into the flame measuring the absorption of each
solution and then constructing a graph in which the
measured absorption is plotted against the
concentration of solutions. Using calibration curve the
concentration of the relevant lead(IV) ion in the
solution23 is interpolated from the measured
absorbance of the test solution.
Results and Discussion
Effect of incubation time and initial lead (IV) loading
The effect of initial lead(IV) loading and incubation
time with bacteria B. subtilis are shown in Fig. 1. The
biosorption of lead is measured after 12 h of
incubation with bacteria. This incubation period is
necessary for avoidance of lag-phase of bacterial
growth and adaptation to the environment of solid
substrate i.e. lead(IV) ion18-21. The biosorption by the
bacteria B. subtilis, is maximum after 48 h of
absorption
At the end of the specific biosorption, 100 mL of
the solution was taken out of the reactors and
centrifuged at 1000 rpm for 10 min in Sorval RC-5
super speed centrifuge at room temperature to remove
Fig. 1 — Effect of initial lead loading and time on biosorption
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INDIAN J. CHEM. TECHNOL., NOVEMBER 2006
incubation as equilibrium reaches nearly at this
contact time. The biosorptions are 98.66, 90.85, 76.83
and 34.57% (w/w) for 500, 600, 700 and 800 ppm of
initial lead ion loading after 48 h of incubation,
respectively (Fig. 1). It can be concluded that the
tolerance limit for the bacteria is 800 ppm of initial
lead ion loading, as the biosoption is very less at this
initial lead ion loading. The growth of bacteria is very
less for 800 ppm of initial lead ion loading (Fig. 2).
The lead metal ions binding with bacteria are more at
initial stages, which gradually increase and remain
almost constant after an optimum time. After 48 h of
incubation time, the growth of bacteria remains
constant with constant lead ion removal (Figs 1 & 2)
for all initial lead(IV) ions loading. Hence, 700 ppm
of lead ion is taken as optimum initial loading with 48
h as optimum incubation time (contact time) for
further biological process parameters optimization
studies respectively.
It is evident from Fig. 1 that as the initial
concentration of lead ions increases the percentage
removal efficiency by the bacteria decreases. This is
because of the fact that the bacterial populations in the
broth can effect lead ion removal. The bacterial
growth decreases with increase of initial lead ion
loading (Fig. 2). The maximum bacterial growths
were found as 4.2×108, 3.94×108, 3.65×108 and
2.67×108 nos of cells/ mL for 500, 600, 700 and 800
ppm of initial lead(IV) ion loading respectively after
48 h of incubation (Fig. 2). It is essential to point out
that application of lead ions at higher concentrations
not only increased the extent of lag-phase but also
decreased the lead ion removal.
There is a real danger that lead metal ions may
poison the environment, stopping biological
(metabolic) activity and bacterial growth24-26. Lead
Fig. 2 — Effect of initial lead concentration and time on
bacterial growth
ion has a poisonous effect on growth of bacteria B.
subtilis beyond a certain concentration. Lead metal
ions accumulate on bacterial surface that slows the
rate of growth and halts due to nutrient exhaustion
and toxified lead ion medium. Exact criteria are more
difficult to ascertain: growth of nutrient limited
populations does slow somewhat before total
exhaustion and the growth rate of a poisoned
population may become imperceptibly slow. It has
been inferred in several instances that the
accumulation of metal results from the lack of
specificity in a normal metal transport system and
that, at high concentrations, metals may act as
competitive substrates in a transport system24-26.
Effect of initial pH on lead biosorption
The effect of initial pH on biosorption of lead metal
ions with B. subtilis is shown in Fig. 3. The lead(IV)
ion biosorption is measured after 48 h of incubation
(optimum time). An increase in lead removal is
observed with increase in initial pH of the medium up
to 4.5. It is observed that the maximum 87.54% (w/w)
removal of lead ion occurred at pH of 4.5 with 700
ppm of initial lead ion loading. At initial pH values of
2.5, 3.5 and 5.5, the biosorption of lead ion was
noticed as 76.83, 80.55 and 70.47% (w/w) with 700
ppm of initial lead ion loading, respectively. With
increase in initial pH beyond 4.5, the biosorptions of
lead(IV) ion sharply declines. An optimum initial pH
of 4.5 value for the biosorption of lead ions is found
in the studies Therefore, initial pH value of 4.5 as the
optimum is taken for further bioprocess parameters
optimization studies. At lower initial pH, the cell
surface becomes more positively charged, reducing
Fig. 3 — Effect of pH on lead biosorption
HOSSAIN & ANANTHARAMAN: BIOSORPTION OF Pb(IV) USING BACILLUS SUBTILIS
attraction between biomass and lead metal ions1-10. In
contrast, higher pH results in facilitation of lead metal
uptake, since the cell surface is more negatively
charged. At initial pH of 4.5 value, neutralization of
positive and negative ions occurred.
The bacterial growth at different initial pH values
with 700 ppm of initial lead(IV) ion loading is shown
in Fig. 4. The bacterial growth reaches its maximum
value of 4.16 × 108 nos of cells/mL with maximum
biosorption of lead at initial pH value of 4.5 (Figs 3 &
4). The bacterial growth is found as 3.72×108, 3.85 ×
108 and 3.65 × 108 nos of cell/mL for initial pH values
of 2.5, 3.5 and 5.5, respectively. Variation in pH of
the medium result in changes in the activity of the
bacteria and hence the bacterial growth as well as the
biosorption rate. Bacteria are very active over a
certain pH range. When pH differs from the optimal
value, the maintenance energy requirements
increase24-26.
Effect of temperature on lead biosorption
The effect of reactor medium temperature on
biosorption of lead(IV) ion with B. subtilis is shown
in Fig. 5. The optimum temperature is 40°C at which
the maximum lead ion biosorption occurs. The
maximum lead biosorption is 89.68% (w/w) at
optimum temperature of 40°C for 700 ppm of initial
lead ion loading. The biosorptions of lead ion are
observed as 80.34, 83.72 and 77.55% (w/w) for
temperatures of 30, 35 and 45°C, respectively with
700 ppm of initial lead ion loading. With increase in
temperature beyond 40°C, the biosorption of lead ion
decreases with bacteria B. subtilis. The bacterial
growths with different temperatures are shown in
Fig. 6. The bacterial growth is noticed maximum of
4.55 ×108 nos of cells/mL at 40°C after 48 h of
incubation. The bacterial growths are found to be
4.25×108, 4.30×108 and 4.10×108 nos of cell/mL at
temperatures of 30, 35 and 45°C with 700 ppm of
initial lead (IV) ion loading, respectively after 48 h of
incubation.
Every type of bacteria has an optimum, minimum
and maximum growth temperature. Temperatures
below the optimum for growth depress the rate of
metabolism of bacterial cells. Above the optimal
temperature, the growth rate decreases and thermal
death may occur24-26. At high temperature, death rate
exceeds the growth rate in the studies, which causes a
net decrease in the concentration of viable bacterial
cells.
Effect of shaking speed on lead biosorption
The effect of shaking speed on biosorption of
lead(IV) ion with B. subtilis is shown in Fig. 7. The
maximum lead ion biosorption is noticed as 97.68%
(w/w) at shaking speed of 60 rpm. The biosorptions of
lead(IV) ion are noticed as 89.68, 93.42 and 86.77%
(w/w) at shaking speed of 20, 40 and 80 rpm,
Fig. 5 — Effect of temperature on lead biosorption
Fig. 4 — Effect of pH on bacterial growth
595
Fig. 6 — Effect of temperature on bacterial growth
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INDIAN J. CHEM. TECHNOL., NOVEMBER 2006
as 40°C for maximum biosorption of lead ions in the
present studies. The optimum shaking speed is
noticed as 60 rpm. The maximum bacterial growth is
observed as 4.90×108 nos of cells/mL at optimum
bioprocess conditions.
References
Fig. 7 — Effect of shaking speed on lead biosorption
Fig. 8 — Effect of shaking speed on bacterial growth
respectively. The biosorption of lead ion increases
with increase in shaking speed up to 60 rpm, then it
declines. This is because of the binding of lead metal
ions to the bacterial surface is highest as well as
bacterial population is maximum at this optimum
shaking speed of 60 rpm. The bacterial growth is
maximum of 4.90 × 108 nos of cells/mL at 60 rpm at
optimum biological conditions (Fig. 8). The bacterial
growths are noticed as 4.36×108, 4.52×108 and
4.35×108 nos of cells/mL for shaking speed of 20, 40
and 80 rpm, respectively at optimum bioprocess
conditions (Fig. 8). Increase in mechanical forces
(increase in shaking speed) can disturb the elaborate
shape of enzyme molecule of the bacteria to such a
degree that denaturation of the protein occurs and
deactivates the bacterial growth24-26.
Conclusion
Biosorption of lead (IV) ion by Bacillus subtilis is
shown to be an effective bacterial bioremoval process.
The maximum biosorption to be obtained is up to
97.68% (w/w) for 700 ppm of initial lead ion loading
by 48 h incubation. The optimum initial pH is
observed as 4.5 and optimum temperature is noticed
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