Proposal Intan

8/3/2019 Proposal Intan

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1. SUMMARY

Metals have been associated with human activity for thousands of years. Although many

heavy metals serve as micronutrients for microorganisms, all are toxic in high concentrations due

to their ability to denature proteins. Resistance to heavy metals may develop in soil bacterial populations, but this may take several years. Nowadays, the heavy application of sewage sludge

has produce high concentration of cadmium and zinc that may lead to the soil pollution. The

contamination may disturb the biological activity in soil. The targets of this project are todetermine the species of bacteria that resistant to these heavy metals. Furthermore, to isolate and

observe the frequencies and characteristics of bacterial that do resistant to both cadmium and

zinc instead of determine the full range of bacterial resistance to that heavy metals and making

comparison between Gram-negative and Gram-positive bacteria for its resistance if possible. Themethod for this project is the sampling that will be done at the sewage sludge area between

20cm-50cm depth. Next are the isolation bacteria from the various samples in order to compare

the total number of bacterial isolates. Then is the testing of the bacteria isolates for their resistance against Cd2+ and Zn2+. Finally the screening of bacteria isolates recovered from the soil

sample for the presence of plasmids that may locate the gene of resistance against the Cd2+ and

Zn2+. Bacterial species that are expected to be obtain including Pseudomonas sp. and Alcaligene

sp. for Gram-negative bacteria and Bacillus sp. for Gram-positive bacteria.

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2. INTRODUCTION

Soil is defined in soil science as an independent body in nature with a unique morphology

from the surface down to the parent material as expressed by the soil profile. Soil is the product

of biochemical weathering of the parent material, and its formation is influenced by the soil

formation factors that including climate, organisms, parent material, relief and time (Jenny,

1941; Brady, 1990). Heavy metal contamination in soils is one of the world`s major

environmental problems. Heavy metal polluted soils become a major environmental problem

because many of the element will persist in the soil for hundred or even thousands of years, and

already polluted soils therefore require careful management. In particular, soils in such region

have been polluted from a wide range of sources with nickel (Ni), cadmium (Cd), Zinc (Zn),

mercury (Hg), cobalt (Co) and other heavy metals. Heavy metals occur naturally in soils at

relatively low concentration, as a result of the weathering and other pedogenic process acting on

the rock fragments on which the soils developed. Cadmium (Cd) for example, in soils it is

derived from both natural and anthropogenic sources. Natural sources include underlying

bedrock or transported parent material such as glacial till and alluvium. Anthropogenic input of

cadmium to soils occurs by aerial deposition and sewage sludge, manure and phosphate fertilizer

application. The determination of manganese (Mn) in soils is due to the present of it in small

quantities in many crystalline rocks. It is released into the soil by rock weathering and is

redeposited in various forms of Mn oxides such as pyrolusite (MnO2), braunite (Mn2O3) and

manganite (MnO(OH) or Mn2O3.H2O). For zinc (Zn), it seems to be distributed rather uniformly

in the magmatic rocks. The Zn concentration in argillaceous sediments and shales is higher than

in sandstones and carboniferous rocks. The Zn occurs chiefly as single sulfides (ZnS), but is also

known to substitute for Mg2+ in silicates (Alina, K.P. and Henryk, P., 1984). While copper (Cu),

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is classified as a native element, which means it can occur as a native element in the earth crust.

Copper belongs to the gold group, a noble element. However, the most common form of Cu in

soils is in the form of minerals such as sulfides, oxides, carbonates and silicates. Many of these

minerals are major constituents of copper deposits in copper mines (Hurlbut and Klein, 1977).

Some of heavy metals are essential and are required by the organisms as a micro nutrients

and are known as trace element (Bruins,M.R., et al , 2000). They are involved in redox processes,

in order to stabilize molecules through electrostatic interactions. Although some heavy metals

are essential trace elements, at high concentrations, it will be toxic to all branches of life

including microbes by forming complex with them. Because heavy metals are increasingly found

in microbial habitats due to natural and industrial processes, microbes have evolved several

mechanisms to tolerate the presence of heavy metals by few reactions. They are including efflux,

complexation, or reduction of metal ions. The objectives of these studies are actually to

determine type of bacterial species that resistance towards heavy metal in soils and to isolate and

characterize the full range of the resistance bacterial strain to heavy metals contaminated soils.

Besides we also are going to do the comparison between characteristic of bacterial presence for

heavy metals tolerance.

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3. LITERATURE FEVIEW

3.1 Heavy Metal

Heavy metal refers to any metallic chemical element that has a relatively high density and

is toxic or poisonous at high concentrations. Instead of a kind of metalloids which associated

with pollution and toxicity, it also includes some elements which are essential for living

organisms at low concentration. Heavy metals are natural components of the Earth's crust which

cannot be degraded or destroyed. Heavy metals are dangerous because they tend to

bioaccumulate, which means they tend to increase in the concentration of a chemical in a

biological organism over time, compared to the chemical's concentration in the environment.

Due to Niargu, J.O., 1988, we may be experiencing a ‘silent epidemic of environmental metal

poisoning’ from the ever increasing amounts of metals wasted into the biosphere.

3.2 Heavy Metals Pollution

Heavy metals give a high risk to the environment. For example, large quantities of Cu

and Zn salts used in pesticides, which been introduced in the environment during the spraying

the crops. Antimony is a metal used in the compound antimony trioxide, as a flame retardant. It

can also be found in batteries, pigments, and ceramics and glass.

Cadmium, Cd, is highly toxic to plants and animals. However, the concentrations of Cd

normally encountered in the environment do not cause acute toxicity (Alloway, B.J., 1993). The

predominant soluble Cd species in soils polluted from several different sources were fractionated

by Tills and Alloways, 1983, using combination of ion-exchange and reverse phase

chromatography. Cadmium tends to be more mobile in soils and therefore more available to

plants than many other heavy metals, including Pb and Cu (Alloway, B.J., 1993). The use of

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cadmium-containing fertilizers and sewage sludge is most often quoted as the primary reason for

the increase in the cadmium content of soils over the last 20 to 30 years in Europe (Jensen and

Bro-Rasmussen 1992). According to recent soil survey done in China, at least 13 330 ha of

farmland in 11 provinces were contaminated by varying degrees of Cd (Zhang and Huang 2000).

Followed by Ayten K. et al (2002), contamination has a large detrimental effect on nutrient

cycling and microbial activity and that the effects of Cd are reduced by fertilizer and sewage

additions. The sludge treatment also significantly decreased the bioaccumulation of cadmium,

copper, and zinc in the exposed species (Agnes Y.R. et al. 2006). The main pollutant sources for

Zn in soil are metalliferous mining activities, agricultural use of sewage sludge and composted

materials, and the use of agrochemicals such as fertilizers and pesticides (Alloway, B.J., 1993).

There is many more heavy metal compounds react in various ways in soil. The extensive used of

it may lead to contamination in soil which probably will effect the environment.

3.3 Heavy Metal Resistance in Bacteria

Over a year, various types of species or genera of bacterial strains resistant towards heavy

metals have been isolated and identified. They also have been further characterized for their

benefits in order to resistant to heavy metals. Microorganisms within species of the same genus

or within strains of the same species can differ in their sensitivity to metals (Dong, J. et al. 2007).

Giller et al. 1993 demonstrated that Rhizobium meliloti was less sensitive, in terms of growth, to

Cd than R. leguminosarum and R. loti. Pseudamonas fluorescens G10 and Microbacterium sp.

G16 have been isolated as a lead (Pb)- resistant bacteria from rape roots grown in heavy metal

contaminated soil and it has been characterized (Sheng, X.F., et al. 2008). For the same

experiment, Saravanan, et al., 2007, proposed that low molecular weight organic acids excreted

by bacteria might facilitate increased heavy metal availability. Instead of resistant to lead (Pb),

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they also resistant other kind of heavy metals including Zn, Cd, Cu and Ni. Pseudomonas

aeruginosa was said to be resistant to Cd, Pb, Mo and U instead of also resistant to antibiotics

(Marques, A.M., et al., 1979). Concurrently, two Gram-positive bacterial, Paenibacillus sp. and

Bacillus thurigeinsis were identified as both do tolerant to Cu2+, Zn2+ and Cd2+ (Rathnayake

I.V.N., et al., 2009). However it seems to be Paenibacillus sp. has more potential in developing a

biosensor to detect Cu2+ in environment samples. Zinc is potentially toxic to sediment bacteria

including Bacillus sp., Salmonella sp. and Arthrobacter sp. and could pose serious threat to their

metabolism in natural environments (Nweke, C.O., et al, 2007). Cyanobacteria are

photosynthetic prokaryotes that require a variety of metal cations such as Cu

2+

, Ni

2+

, Fe

2+

and

Zn2+ to maintain their cellular metabolism (Cavet et al., 2003). Additionally, Pages, D., et al.,

2008, described that there was a presence of clusters of genes contain a cadmium efflux

determinant (cadA) from Gram-positive bacteria involved in both antibiotics and heavy metals

resistance in Stenotrophomonas maltophilia D457R. Huang et al., 2005 state that copper and zinc

accumulation in the roots and shoots of mycorrhizae infected plants were significantly lower

than those in the non-infected plants might also suggest that mycorrhizae efficiently restricted

excessive copper and zinc absorptions into the host plants. Finally, Yilmaz, E. and Ensari, N.,

2005 state that Bacillus circulans strain EB1 showed a high cadmium biosorption capacity

coupled with a high tolerance to this metal when grown in its presence.

3.4 Heavy Metal Resistance Mechanisms

To survive under metal-stressed conditions, bacteria have evolved several types of

mechanisms to tolerate the uptake of heavy metal ions. The mechanisms including efflux of

metal ions outside the cell, accumulation and complexation of the metal ions inside the cell, and

reduction of the heavy metal ions to a less toxic state (Nies, 1999). The ability of metal

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bioaccumulation by some Gram-negative bacterial species such as Escherichia coli,

Pseudomonas putida, Pseudomonas syringae, Pseudomonas aeruginosa (HASSEN et al., 1998)

was established on production of intracellular cadmium-binding proteins.

3.5 Genetic Studies in Bacterial Resistance

In order to identify the location of genes that enable bacterial to be resistance against heavy

metals, few genetic studies have been done. Nies, D. H., and S. Silver. 1989 found that the czc-

mediated resistance to Co2+, Zn2+, and Cd2+ encoded by plasmid pMOL30 is inducible in A.

eutrophus. Cd-resistance of soil bacteria is mediated by several genetic systems. Zhang, Y., et al

2008 had proposed that these had been well studied in the Cd efflux system coded by cadmium-

resistant gene cadA operon that widespread in Gram-positive bacteria such as Staphylococcus

aureus, Bacillus firmus and in Gram-negative bacterium Stenotrophomonas maltophilia.

Followed by Lee S. W., et al, 2001, a chromosomal fragment encoding Cd resistance was cloned

from Pseudomonas putida 06909, a rhizosphere bacterium, and sequence analysis revealed two

divergently transcribed genes, cadA and cadR. Mutational analysis indicated that cadA and cadR

are fully responsible for Cd resistance and partially for Zn resistance. The cadA promoter is

responsive to Cd(II), Pb(II), and Zn(II), while the cadR promoter was only induced by

Cd(II).Whereas the ability of P. aeruginosa resistant to nickel and ampicillin is plasmid

mediated and transferable to other strains whereas Cd, Cr and Pb could be chromosomal encoded

(Raja, C. E. and Selvam, G. S. 2009).

4. MATERIALS AND METHODS

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4.1 Sample Collection

Soils samples collection is taken from the sewage sludge, at a wetland area in Kuching,

Sarawak. The sample is taken in range 15-40cm below the soil surface. All samples are taken

using a sterile stainless steel spatula and put into the sterile glass bottles. The sample that have

been taken need to be stored at 4ºC until examination. The time interval between sampling and

bacterial isolation is within two weeks time.

4.2 Preparation for Heavy Metal Stock Solution

Prepare the heavy metal salt that need to be used in the experiment. Dissolve respectively

heavy metal salts in ultrapure water. Then calculate the concentration for the heavy metal stock

solution based on the solubility heavy metal salt in water. Sterilize the stock solutions by

filtration. Store the stock solution at 4ºC. Appropriate volumes of the heavy metal stock solutions

were added into Mueller Hilton agar (MHA) cooled to about 55 ºC to give desire final

concentrations.

4.3 Isolation of Bacteria and Culture Conditions

The time interval between sampling and bacterial isolation can not exceed two weeks. A 10-

fold up to 10-7 of fresh soil (1g) is done in phosphate buffered saline and transfer the aliquots

samples (0.1ml) of the 10-5 to 10-7 dilutions is spread on nutrient agar (NA) plates. Prepare

three replicates for each dilution and incubate the plates at 30 C for 72 hours. Select the⁰

colonies randomly with different morphological appearance from that culture plates. Purify is

done by further sub-culturing in the same media.

4.4 Storage of Bacteria Isolates

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Keep the sub-culture bacteria on nutrient agar slants. Store all cultures at -80ºc in the 20%

glycerol broth. Prepare the stock cultures on slant agar, streaking single colony on the slant agar

surface and incubate at 30ºc for 48 hours.

4.5 Biochemical Identification of Bacteria Isolates

Incubate the bacteria isolates on nutrient agar for 24 hour before the identification

procedure at 30°C. Identify the isolates both morphological appearance and biochemical test

using analytical profile index (API) 20 NE test kit or API 20 E test kit (biomereux, france). API

20 NE test kit is standardizing system to identify non-fermentative bacteria while API 20 E test

kit system is used to determine Gram negative rods bacteria. Strip the API and inoculate with the

bacterial suspension of overnight culture on agar medium and incubate for 24 hours at 37°C.

Observe the colour changes (to distinguish the gram positive and gram negative).

4.6 Screening of Heavy Metal Resistance Bacterial Isolates

From the bacteria colonies recover on NA after overnight incubation at 30ºc, pick one

colony and spot it on MHA plates supplemented with different heavy metal salts. Incubate agar

plates selectively at 30ºc overnight.

4.7 Determination of Minimum Inhibitor Concentration (MIC)

MIC is to measure the level of heavy metal resistance. The method that been use for the

determination is refer to Filali, et al (2000). Inoculate single colony in LB media and grow it at

30ºc. Prepare a 10-2 dilution of the culture then amount of it need to be spread on MHA plates

containing different concentration of heavy metal salts solution. Incubate the plate at 30ºc 24-48

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hours. Lower the MIC values indicate more toxic metals and higher the MIC values indicate less

toxicity.

4.8 Isolation of DNA of bacteria and Electrophoresis

Grow the isolate bacteria on a plate containing nutrient agar. Inoculate a colony of

bacteria species into Luria Broth (LB) media. Incubate with shaker overnight at 37ºc. Keep it at

4ºc for further used in DNA analysis protocols.

4.8.1 Isolation and Preparation of Genomic DNA from Bacteria

Transfer amount of culture into eppendorf tube and centrifuge. Remove the supernatant and mix

the pellet with TE buffer by vortexing. Add sodium dedocyl sulphate (SDS), proteinase K and

amount of nacl and well mix them together. Incubate the mixture in water bath for 10 minutes at

65ºc. Add 25:24:1 of phenol / chloroform / isomyl and centrifuge again. Transfer a clear

supernatant into new eppendorf tube and ad 24:1 of chloroform / isomyl. Centrifuge and the

pellet then need to mix with isopropanol by vortexing until a white precipitate appear. Dry the

pellet. Dissolve it with TE buffer and store at 4ºc for next use.

4.8.2 Isolation of Plasmid DNA from Bacterial Culture

Same as the first procedure for genomic DNA preparation, transfer amount of culture into

Eppendorf tube and centrifuge. But after remove the supernatant, suspend it with Solution I and

straight away keep it on ice. Add Solution II and invert it few times and keep it in room

temperature for 5 minutes. Add Solution III and invert it until white precipitate appears.

Centrifuge and transfer the supernatant into a new tube. Add ethanol and invert it again.

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Centrifuge for 5 minutes at room temperature. Air dry the pellet then resuspend it in sterile ultra

pure water. Finally, run it on agarose gel electrophoresis.

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EXPECTED OUTCOME

Due on this project, I could expect that I will obtain few bacterial strain from both Gram

positive and Gram negative isolated from the samples that able to tolerate to high concentration

of cadmium or even zinc in the sewage sludge taken from wetland area. The result might be

varied in term of frequency within resistance bacterial species depends on how long the exposure

of bacteria to heavy metal. Besides, if genes able to be identify in the bacterial resistance, then it

might be help for the other microorganisms to detoxify heavy metals and reduce pollutant by

cloning and introduce into the microorganism resistance level.

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WORK SCHEDULE

Project activities 2009 2010

Jul Aug Sep Oct Nov Dec Jan Feb Mac Apr May

Identify source

of data

► ► ●

Proposal writing

and presentation

► ●

Data collection ► ► ●

Benchwork andsample

processing

► ► ► ► ●

Progress report ► ► ●

Data analysis ► ► ► ► ●

Data validation :

statisticalanalysis

► ► ► ► ●

Report writing

and presentation

► ► ●

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REFERENCES:

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Alloway, B.J., 1993. Heavy Metals in Soils. Cadmium, 101-121. Blackie Academic &

Professional, Glasgow.

Agnes Y.R. et al. 2006. Environmental Pollution.Vol 145, Issue 1,pp 41-50

Ayten K. et al. 2002. Biology and Fertility of Soils. Volume 35, No. 6, pp 428-434. springer.

Bloomfield, C. and Pruden, G., 1980. The Behaviour of Cr(VI) In Soil Under Earobic and

Anaerobic Conditions, Environment Pollution, 23a,103.

Brady,N.C. 1990. The Nature and the Properties of Soils. Macmillan Publ. Co., New York.

Bruins M.R., Kapil S., Oehme F.W., 2000. Microbial Resistance to Metals in The

Environment. Ecotoxicology and Environmental Safety vol.45, pp.198-207

Cavet, J. S. et al., 2003. Zn, Cu and Co in cyanobacteria: selective control of metal availability.

FEMS Microbiol Rev 27: 165–181.

D.S. Holmes, M. Barreto, J. Valdez, C. Dominguez, C. Arriagada, S. Silver, S. Bueno and

E. Jedlicki. 2001. Whole Genome Sequence Of Acidithiobacillus Ferrooxidans:

Metabolic Reconstruction, Heavy Metal Resistance And Other Characteristics.

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A" (V.S.T. Ciminelli and O. Garcia, Jr., Eds.), Elsevier Publishers, Amsterdam, pp. 237-251

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G. Endo, M. Narita, C.-C. Huang, and S. Silver. 2002. Microbial Heavy Metal Resistance

Transposons and Plasmids: Potential Use for Environmental Biotechnology. J. Environ.

Biotech. 2, 71-82

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Biotech. 2, 71-82

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Than R. Leguminosarum Bv. Trifolii or R. Loti. Soil Biol. Biochem., 25: 273–278.

Hassen, A., Saidi, N., Cherif, M. & Boudabous, A. 1998. Effects of heavy metals on

Pseudomonas aeruginosa and Bacillus thuringiensis. Biores. Technol. 65: 73–82.

http://www.lenntech.com/heavy-metals.htm

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Appl. Microbiol. Biotechnol. 56, 736-741

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Deduced From Analysis of its Partial Genome Sequence. Hydrometallurgy 71, 97-105

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Porquet, F. Quentel and S. Silver. 2003. Antimony in Aquatic Systems. J. Phys. IV

France 107, 475-478

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Isolated From Sediment of Minamata Bay. FEMS Microbiol. Letters 223, 73-82

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sthenotrophomonas malthophilia. PLoS ONE 3(2) : e1539.doi:10.1371/journal

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IJEST.

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http://www.ssd.sarawak.gov.my/home_power.php

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Isolate the bacteria from the sewage sludge. Weight sludge contained metals (wet

weight). Mix the sludge with liquid mineral medium then incubate it for 2 hours at 30°C in the

shaker incubator. Withdrawn the subsample and serial diluted plated on mineral medium

amended with agar and CdCl2 or ZnCl2. Incubate aerobically or anaerobically for 24-48 hours at

30°C. sub-culture the recover bacterial on nutrient agar to obtain a pure cultures before screening

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for heavy metal isolates. Stain the pre-pured culture several times by Gram procedure and

observe the colour development as well as size and other characteristics using light microscopy.

Determine the MICs of the metals using the plate dilution method (Malik and Jaiswal,

2000). Prepare the stock of metal salts in distill water and use the filter membrane to sterilize

them. Store the stocks at 4 ºC. Add the heavy metal solution to the nutrient agar in various

concentrations range (3.5 – 3200 μg/mL). Then spot the inoculated with approximately 3 x 106

bacteria. Incubate the plates at 35-37 ºC for 48 hours and the MIC is defined as the lowest

concentration that completely inhibits growth.

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Proposal Intan

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