PRACTICAL REPORT OF BIOREMEDIATION 2016

The Effect of Pb in The Growth of

Azotobacter sp.
1
Intan Prima Sari (1514100012), 2Agita Nur Indahsari (1514100071), 3Diana Yunita Sikaputri
(1514100064), 4Fila Oxi Wardhani (1514100060), 5Desy Lailatul Rachmah (1514100053), 6Arif
Fuad Saifulloh (1514100072)
Biology Department, Fakultas Matematika dan Ilmu Pengetahuan Alam, Institut Teknologi Sepuluh
Nopember (ITS)
Jl. Arief Rahman Hakim, Surabaya 60111 Indonesia
e-mail: hidayatulalami@bio.its.ac.id
Abstract Heavy metals are highly hazardous to the water treatment where microorganisms are directly involved
environment and organisms. It can be enriched through the food in the decomposition of organic matter in biological processes
chain. Once the soil suffers from heavy metal contamination, it for waste water treatment, because often the inhibitory effect
is difficult to be remediated. Lead is a chemical which is of heavy metals is a common phenomenon that occurs in the
included in the heavy metal groups that became one of the main biological treatment of waste water and sewage [4].
pollutants in the environment. This happens because the main
source of lead pollution is from vehicle exhaust emissions.
Mechanisms of metal resistance in microbes include
Azotobacter is a bacteria that fix nitrogen-free non-symbiotic precipitation of metals as phosphates, carbonates and/or
abundant in the rhizosphere of agricultural land and an EPS- sulfides; volatilization via methylation or ethylation; physical
producing bacteria that can function as chelating metals. The exclusion of electronegative components in membranes and
purpose of the practicum is to determine the potential of extra cellular polymeric substances (EPS); energy-dependent
Azotobacter in degrading Pb and to determine the effect of Pb metal efflux systems; and intra cellular sequestration with low
on the growth of Azotobacter. The method of the practicum is to molecular weight, cysteine-rich proteins[5,6]. There are
grow the bacteria in Pb with various concentrations are 0 ppm, several techniques to remove these heavy metals, but most of
50 ppm and 200 ppm, then the observed growth every 6 hours these techniques become ineffective when the concentrations
for 48 hours. After it made the growth curve of bacteria. The of heavy metals are less than 100 mg/L [7]. Lead (Pb), a
result is the highest growth of Azotobacter is at a concentration
major pollutant that is found in soil, water and air. This metal
of 200 ppm, based on such things Azotobacter able to survive the
Pb concentration of 200 ppm, so as Azotobacter able to degrade is also hazardous waste and highly toxic to human, animals,
Pb. plants and microbes [8]. Azotobacter is one of group
microorganism that can be agent of bioremediation of lead.
Key Word Azotobacter, Lead (Pb), Remediation Azotobacter was a non-symbiont genus of bacteria that were
abundant in the land and it able to bind free nitrogen, some of
I. INTRODUCTION its species were siderophore bacteria, phytohormones
producer and thus it potentially used as biofertilizer [9].

L iving system requires special transport and handling

mechanisms to keep them from toxic metals [1]. The
toxicity occurs in humans due to environmental pollution via
Diversity of Azotobacter in it habitat affected by the physical
and chemical properties of soil as well as interactions among
microorganisms in the soil [10]. Some members of the
soil or water contamination or due to occupational exposure. Azotobacter was high salinity tolerant and resistant to
Some of these metals are useful to us in low concentrations mercury so it also potentially used as a mercury
but are highly toxic in higher concentrations [2]. bioremediation on high salinity area [11,12].
Bioremediation processes are very attractive in comparison
with physicochemical methods such as electrochemical
treatment, ion exchange, precipitation, reverse osmosis,
evaporation, and sorption for heavy metal removal techniques
because they can have lower cost and higher efficiency at low
metal concentrations. Moreover, Bioremediation is an
innovative and promising technology available for removal of
heavy metals and recovery of the heavy metals in polluted
water and lands. Since microorganisms have developed
various strategies for their survival in heavy metal-polluted
habitats, these organisms are known to develop and adopt Azotobacter sp. [13]
different detoxifying mechanisms such as biosorption,
bioaccumulation, biotransformation and biomineralization,
which can be exploited for bioremediation either ex situ or in II.MATERIAL AND METHOD
situ [3]. There are a number of bio materials that can be use
to remove metal from waste water, such molds, yeasts, A.Time and Place
bacteria, and seaweeds. The ability of microbial stains to grow Practical effect of concentration on the growth of
in the presence of heavy metals would be helpful in the waste Azotobacter sp pbcl conducted on 22 November to 20
PRACTICAL REPORT OF BIOREMEDIATION 2016
December in the laboratory of microbiology and
biotechnology biology majors FMIPA ITS.
B.Tools and Material
Prepared flasks, test tubes, Nutrient Agar, Nutrient Broth,
PbCl, Azotobacter sp isolates, autoclave, LAF, and the
balance sheet
C. Method
2.1 Subculture Azotobacter sp
0.14 g Nutrients Agar mixed in 5 ml of distilled water and
dissolved in a hot plate magnetic stirrer. Subsequently
sterilized using an autoclave for 15 minutes at a temperature
of 121 degrees Celsius. Then tilted and left to solidify. Pure
isolates of Azotobacter sp was taken one loop and inoculated
into medium NA that has been made. Then incubated it for 24
hours.
2.2 Screening Leads Concentration
6 pieces flask prepared and labeled 0 ppm, 10 ppm, 25
ppm, 50 ppm, 100 ppm and 200 ppm. Each flask included
0.28 grams of Nutrients Agar are mixed with 10 ml of
distilled water. The next on each flask added pbcl accordance
with ppm concentrations. 0 ppm is not added pbcl, 10 ppm
was added 0.1 mg pbcl, 25 ppm pbcl added 0.25 mg, 0.5 mg
added 50 pbcl ppm, 100 ppm added 1 mg pbcl, and 200 ppm
added 2 mg pbcl. Then homogenized using a magnetic stirrer
hot plate. In another, 6 test tube is also prepared. After
homogeneous, NA + pbcl medium together with test tubes
were sterilized on autoclave.After sterile, respectively each
medium was transferred to a test tube for 5 ml. Then tilted
and left to solidify. Azotobacter sp isolates were inoculated
one loop on each medium that has been created and then
incubated it for 24 hours. After 24 hours, screening the
concentration to find the moderate and high concentration
that can make Azotobacter sp grow. In this practice make 3
concentration to bioremediation test that is 0 ppm, 50 ppm
(moderate) and 200 ppm (high).
2.3 Bioremediation Test
A. Making Nutrient Broth Medium
3 pieces flask prepared and labeled 0 ppm, 50 ppm
(moderate), 200 ppm (High). Each flask included 1,3 grams
of Nutrients Broth that are mixed with 100 ml of distilled
water. The next on each Flask added pbcl accordance with
ppm concentrations. 0 ppm is not added pbcl, 50 ppm added
5 mg pbcl, and 200 ppm added 20 mg pbcl. Then
homogenized using a magnetic stirrer hot plate. Subsequently
sterilized using an autoclave for 15 minutes at a temperature
of 121 degrees Celsius. Next prepare 100 ml of sterile
distilled water.
B. Inoculation
1 loop Azotobacter sp isolates that have been grown in the
medium NA + pbcl inoculated into 5 ml sterile distilled water
and poured into the appropriate concentration pbcl NB
medium. Subsequently incubated in a rotary shaker for 48
hours.
2.4 Growth Curve
After being transferred to the medium NB, subsequently
measured growth curve of Azotobacter sp. 2 ml inoculum was
taken and poured into a cuvette. Then measured absorbance at
600 nm wavelength using spektrofotometry. Measurements
were performed on the hour to 0 and was continued every 6
hours for 48 hours.
III. RESULT AND INVESTIGATION
A. Gambar dan Tabel
Agroekosistem pollution by heavy metals can harm the
food chain, food safety and human health. Nonessential heavy
metals lead (Pb) are naturally present on the farm, but the
concentration may increase as air pollution and the use of
animal manure, inorganic fertilizers, and pesticides
containing lead arsenate [14]. Practicum bioremediation of
what we do is intended to determine the effectiveness of the
use of bacteria Azotobacter sp. for remediation of Pb with
various concentrations. To prevent an increase in the content
of Pb in the farm needed a method to lower the concentration
of Pb in the media. One method of bioremediation of heavy
metal contaminated soil by phytoremediation is the use of
plants to extract, mensekuestrasi, and detoxify pollutants
[15].
The results of bacterial growth charts Azotobacter sp.
grown on NA media with Pb added as much as 0 ppm (as a
control), 50 ppm, and 200 ppm are as follows:
Figure 1. Graph of growth of Azotobacter sp. with the
addition of 0, 50, and 200 ppm Pb
We know the growth phase of bacteria contained adaptation
phase (log phase), the growth phase (lag phase), the
stationary phase, and the phase of death. Likewise, the results
of this bioremediation lab, media supplemented with heavy
metals Pb with such concentration causing bacterial growth
becomes unbalanced. Adaptation phase that is characterized
by the growth of bacteria's silence for adjusting to the new
media environment and become invisible because of the early
hour measurements of bacterial growth seen that Azotobacter
sp. with a concentration of 0 ppm and 200 ppm increased
while Azotobacter sp. with a concentration of 50 ppm Pb
decreased. Then the growth of bacteria on the medium with a
concentration of 50 ppm increase until the 24th hour
measurement, in this case we conclude the bacteria are
experiencing a growth phase. While at the concentration of 0
ppm is only experiencing a growth phase until the 18th hour
and then growth slowed at the 24th hour. For media with
PRACTICAL REPORT OF BIOREMEDIATION 2016
highest metal concentrations (200 ppm) only increased
(growth stage) only until the 12th and then decreased until
the 30th. Then, for all the bacteria with concentrations
increase until the 42 th hour, then decreased. This we
conclude bacterium is undergoing a phase of death.
From the results of the graph, we conclude that the growth
of bacteria that the most effective way is to increase the
concentration of Pb 50 ppm because at these concentrations of
bacteria through a phase of growth of the longest ie until the
24th hour, while in addition to the concentration of 0 and 200
ppm already decreasing the concentration of bacterial cells in
advance. Although at the beginning of the measurement
decreased the concentration of 50 ppm this may occur due to
the adaptation of bacterial cells that adapt to the new
environment, so it can still happen. Then, the average
stationary phase at all concentrations up on all 42 hours of
measurement, which in turn is a phase of death, namely the
48th hour. From the shape of the graph of the observed fact is
still not too resembles the chart the growth of bacteria that is
known from the literature [16]. because the chart is still up
and down, and no significant changes have occurred. This
may occur because of a spectrophotometer is used to measure
the density of bacterial cells are often damaged, and because
of lack of sterility of the practitioner in performing the
treatment in the lab.
One of the mechanisms that micro-organisms use to avoid
the toxicity of metals of no biological function is to limit their
movement across the cell envelope. The cell wall is a natural
barrier for Pb(II), since the functional groups of several
macromolecules are involved in binding this metal [17].
Azotobacter sp. is Gram-Negative bacteria [18] . In Gram-
negative bacteria, this role is played mainly by
lipopolysaccharide, a significant component of the outer
membrane. Then , Azotobacter sp can synthetize Extracellular
polymer (EPs) that bind cations of toxic metals, thus
protecting metal-sensitive and essential cellular components
[18][14] . The composition of EPs is very complex, including
proteins, humic acids, polysaccharides and nucleic acids,
which chelate metals with different specificity and affinity
[17]. Azotobacter sp produce Eps for minimalize
concentration of intracelluler O2 so the activity of
Nitrogenase keep going [14]. The Extracellular polymer have
potential to mobilize heavy metal including Pb and can bind
cations of toxic metals, thus protecting metal-sensitive and
essential cellular components. Azotobacter sp can develop the
resistant system to heavy metal hrough Fitokleatin which
celates metal and keep them in their vacuola [14].
Azotobacter sp usually treating heavy metal contaminated
soil as a consortium with phyoremediation because it can
enhance the plant growth [19] .
IV. CONCLUSION
From the Practical work we can conclude that the growth
of bacteria that the most effective way is to increase the
concentration of Pb 50 ppm because at these concentrations of
bacteria through a phase of growth of the longest ie until the
24th hour, while in addition to the concentration of 0 and 200
ppm already decreasing the concentration of bacterial cells in
advance.
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