Isolation Of Microorganisms

Environmental Biotechnology 3 Material and Methods

ISOLATION AND ENRICHMENT OF MICROORGANISMS CAPABLE
OF DETOXIFYING ENVIROMENTAL POLLUTANTS

Irfan, K., Zara, K. W., and Daud, M. K.

Environmental Biotechnology, Department Of Biotechnology and Genetic Engineering,
Kohat University Of Science and Technology, City Campus, Kohat Pakistan.

Author for correspondence: Email address china_zju@yahoo.com

Abstract:
Our environment is going to be polluted and polluted day by day due to human activities.
And pollutants are hazardous for life. Pollution in environment is of serious environmental
concern and interest in microorganisms capable of detoxifying environmental pollutant is of
practical significance. Scientists are using different techniques to remediate environmental
pollutants such as bioremediation. For this we need microorganisms capable of detoxifying
environmental pollutants. The objective of this study is to isolate and optimize the growth
conditions of microorganisms that showed high potential for degradation of environmental
pollutants. Different methods and techniques are used for the isolation of different
microorganisms.

Keywords: pollutants, microorganism, isolation, enrichment, culture, media.

Irfan, Zara and Daud, 2010 Isolation 3 Enrichment Of Microorganisms Capable Of
And
Detoxifying Environmental Pollutants


INTRODUCTION

Environmental biotechnology mainly deals with the “development and regulation of
biological systems for the remediation of contaminated environment and for environment
friendly processes. EB is mainly applied to study the natural environment. The main objective of
EB is the “stoppage of further pollution and cleaning up of accumulated pollution. Pollutant may
be various gases (CO, SO2, and NO) or solids (pesticides, herbicides, chlorinated hydrocarbons
etc.) or radiations, roadway noise, or industrial noise, or heavy metals. These pollutants cause
instability, disorders and harm or discomforts to the ecosystem. They are hazardous and toxic for
human health and should be removed or detoxified. There are a number of strategies to control
environmental pollution; bioremediation, biodegradation, phytoremediation etc. Usually
microorganisms are used to control environmental pollution i.e. bioremediation. So we have to
isolate and identity microorganisms capable of detoxifying environmental pollutants. For the
isolation of such microorganisms we use enrichment culture techniques. Enrichment culture
techniques rely on creating a condition, in which the survival and growth of bacterial cultures,
with whatever traits are desired, is favored. The nutritional composition of the microbial growth
media can be adjusted so that: an environmental contaminant serves as the only available source
of food and energy; or the growth conditions favor the growth of only those bacteria that can
grow at a certain temperature; or in the presence of other chemicals. In these ways, the
conditions can be controlled in the laboratory to allow for the selection of those bacteria that can
provide solutions to various problems.

In addition to selecting for naturally occurring microbial cultures that possess a desired
metabolic trait, it is also possible to use enrichment culture techniques to develop microbial
cultures with unique biochemical traits. The substrate range of enzymes catalyzing a certain
reaction can be expanded through the use of enrichment culture
techniques(www.gastechnology.org/webroot/app/xn/xd.aspx?it=enweb&xd=4reportspubs
%5C4_8focus%5Cenrichmentcultures.xml)

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MATERIAL AND METHODS

1) Isolation and enrichment of marine bacteria capable of detoxifying toxic
heavy metals:

Pollution in industrial areas is of serious environmental concern and interest in bacterial
resistance to heavy metals is of practical significance. Mercury (Hg), Cadmium (Cd) and lead
(Pb) are known to cause damage to living organisms including human beings. Several marine
bacteria highly resistant to mercury (BHRM) capable of growing at 25 ppm (mg L-1) or higher
concentrations of mercury have potential to detoxify Hg, Cd and Pb.

Metal-resistant microorganisms can be isolated from various polluted environments.
Samples were collected from contaminated areas. Grow these sample in seawater nutrient broth
(SWNB: SWNA without agar) amended with 25 ppm Hg. Then Seawater nutrient agar ( peptone
5.0 g, beef extract 1.5 g, yeast extract 1.5 g, sodium chloride 15 g, aged seawater 500 ml,
deionized water 500 ml and agar 15 g), amended with 10 ppm HgCl2 (ca. 50 mM Hg)(Merck,
Germany) is used for their isolation and enrichment. Bacterial colonies capable of growth when
streaked onto SWNA plates with 25 ppm mercury were termed bacteria highly resistant to
mercury (BHRM). Purified cultures of 13 BHRM isolates are capable of detoxifying
environmental pollutants (Cameron, R.E. 1992).

2) Isolation and enrichment of hydrocarbon degrading bacteria:

Medium an d Cult u r e Con diti o n s :

The gravel samples from beach simulator tank were inoculated to enrichment cultures and
subcultures. The enrichment medium as inorganic medium was contained (1-1 filtered seawater):
NH4NO3 1g; K2HPO4 0.2g; FeC6H5O7.nH2O 0.02g. The pH was adjusted to 7.6-7.8. For
enrichment and for better growth of pure cultures, the growth medium was supplemented with
hydrocarbon compounds: Naphthalene, Phenanthrene, Trichlorodibenzofuran, Benzo[a]pyrene

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and crude oil. Pure cultures were obtained after isolation from agar plate dilution series and
incubated at 20 0C. Agar plate was contained (1-l distilled water) as ONR7a medium: Na2SO4
3.98g; NaCl 22.79g; NH4Cl 0.27g; KCl 0.72g; Tapso 1.3g; Gelrite; NaBr; NaF; NaHCO3;
H3BO3; Na2HPO4; MgCl2.6H2O 55.9g; CaCl2.2H2O 7.3g; SrCl2 0.5g; FeCl2.4H2O.

Isolation and enrichment: Gravel was collected from beach simulator tank and inoculated to
enrichment cultures containing hydrocarbon compounds. This culture was incubated at 200 C for
1 month with constant shaking to promote the growth of hydrocarbon degrading microorganisms
indigenous to the oil paste and/or seawater. Colonies number reached on medium which
supplemented with the hydrocarbon source. An increase in cell number was observed when the
sea water was supplemented with the fertilizers. Although the above mentioned techniques are
quite useful for studying functional and physiological traits of microbial populations in the
environment, pure culture experiments are indispensable for detailed analyses of function of each
population particularly for manifesting concealed physiological traits likely to be important for
the establishment of the consortium. To date, many pollutant-degrading bacteria have been
isolated from natural mixed populations after batch-culture enrichment in media containing
relatively high concentrations of the pollutant. However, this batch-culture enrichment is not
considered suitable for ecological studies, because such methods isolate a very limited number of
bacteria that always grow most rapidly in laboratory media. Pure cultures were stored in Marine
Broth agar and ONR7a agar (contain a single hydrocarbon compounds); pure cultures in liquid
medium were stored in 60 ml screw cap bottles as cultivation for next tests contained single
hydrocarbon compounds.

3) Isolation and enrichment of Bacteria degrading oil hydrocarbon:

Oil spills are an inevitable consequence of using petroleum. Spills can occur at the oil
refinery, during transportation, natural seeps or during routine maintenance of infrastructure. These
oil spills devastate the soil, surface and ground water and alter the microbial population at the
polluted sites. Biodegradation by microorganisms is more favorable than chemical treatment for
dealing with oil pollution since the microbes modify crude oils in beneficial ways and the end
products are environmentally safe to all living things (Toledo et al. 2006).

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Sampling of soils and media:

Soils contaminated with oil were collected from any oil refineries. Two types of medium
were used, mineral salt medium (MSM) (Zajic & Supplinson 1972) supplemented with 1% (v/v)
crude oil and vitamin mix (Bochaez et al. 1995) and MSM supplemented with 1% (v/v) oil but
lacking vitamin mix. The pH of the medium was adjusted to 6.5 with 1N HCl or 1M NaOH. Two
types of crude oil were used. The medium was sterilised at 121°C for 15 minutes then added with
1% (v/v) crude oil and 0.01% mycostatin as a fungal inhibitor.

Isolation and enrichment of bacteria:

Ten grams of soil were mixed with 50 ml 0.85% normal saline and incubated at 30°C for 30
minutes in an orbital shaker to homogenize the mixture, and the pH of the soil was measured
(Metter Toledo pH meter). The mixture was centrifuged at 10414 g, 4°C for 10 min. The pellet was
mixed and serially diluted with normal saline. The diluted suspension was plated onto MSM oil
agar with and without vitamin mix by the spread plate method for isolation of hydrocarbon
degrading bacteria. For the total count of bacteria, the diluted suspension was plated onto nutrient
agar.

In order to isolate the desired bacteria, enrichment culture was used. Briefly, 1 mL of soil
mixture was inoculated into 50 mL of MSM containing 1% (v/v) crude oil. The flasks were
incubated aerobically for 7 days. After incubation, an inoculation loop of culture was streaked onto
MSM agar containing 1% crude oil and incubated at 37°C for 24 h. Microbial colonies were picked
randomly and subculture by streaking onto MSM plates several times until pure colonies were
obtained (Anion, H et al. 2010).

4) Isolation and characterization of diesel oil degrading indigenous
microorganisms:

Petroleum continues to be used as the principle source of energy; however, despite its
important usage, petroleum hydrocarbons also pose as a globally environmental pollutant (Plohl
et al., 2002). Petroleum contamination results from leakage above ground and underground
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storage tanks, spillage during transport of petroleum products, abandoned manufactured gasoline
sites, other unplanned releases and current industrial processes (Mishra et al., 2001; Sarkar et al.,
2005). Uncontrolled releases of these compounds into soil and ground water are frequent as a
result of accidents or poor control practices and attract public interest (Mishra et al., 2001)
Petroleum compounds are considered to be recalcitrant to microbial degradation and persist in
ecosystems because of their hydrophobic nature and low volatility and thus they pose a
significant threat to the environment (Abed et al., 2002). The constituents of these contaminants
such as diesel oil, are carcinogenic, mutagenic and are potent immunotoxicants, thus posing a
serious threat to human and animal health. Oil spills, especially in soil contamination have
prompted research on cost-effective, environmentally benign cleanup strategies (Margesin and
Schinner, 2001).

Collections of diesel contaminated soil samples:

Eight diesel-contaminated soil samples were collected from different transport
companies. 100 g of each sample was placed into 500 ml Schott bottles and stored at 4°C until
further study. The diesel was purchased from a local garage and stored in the dark at ambient
temperature throughout the study.

Isolation and enrichment of bacterial diesel degraders:

Bushnell – Haas (BH) medium (Atlas, 1994) was used as the enrichment media with 10%
(v/v) diesel as the sole carbon source to isolate diesel–degrading bacteria. 10 g of the
contaminated soil was added and incubated at 30oC at 170 rpm. After 2 weeks, 1 ml of enriched
media was transferred into freshly prepared enrichment media and incubated at the same
conditions as described above.

Serial dilutions (1/10) from the third enrichment process were plated out onto BH agar
plates, which were covered with 100 μl of diesel oil and incubated at 30oC. The single colonies
were streaked onto nutrient agar plates, incubated at 30oC overnight, and stored at 4oC until
further use.

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5) Isolation and enrichment of malathion detoxifying strains of pseudomonas
aeruginosa from an insecticide impregnated soil habitat:

Malathion is one of general purpose organophosphate, household and agricultural
pesticide recommended by manufactures for control of most of insects including aphids, thrips,
mites and codling moth on vegetables, ornamental flowers and fruit trees (Wester and Cashman,
1989). This chemical is a “contact insecticide” that interferes with the action of important
enzymes, obilerating the insect’s nervous system within two hours of exposure. Blockage of
nervous system pathways causes rapid twitching and paralysis of muscles, which results in death.
Toxic effects of Malathion are similar to those observed with other organophosphates, except
that larger doses are required to produce them (U.S. Public Health Service, 1995). Its toxicity has
been observed in the mouse, rats, infants and small children (Gosselin et al., 1984) in adults who
are in direct or indirect contact with Malathion freshwater and marine fishes invertebrates and of
its fate after application on estuarine life. Apart from long list of health adverse effects of
malathion the chemical is a proven teratogen. Malathion tends to concentrate in peel and may not
readily remove by washing in water alone. Peeling, cooking and beat processing reduce the
residues. In one greenhouse study malathion applied at recommended rates was easily detected
on plant surfaces upto 9 weeks after spraying (Delmore and Appelhans, 1991).

Soil samples were collected in sterilized glass bottles from different sites along side
canal, receiving drained water of a factory involved in the formulation of pesticides. These soil
samples were processed on the day of collection by mixing 10 grams of each soil sample in 100
ml autoclaved water and keeping them in orbital shaker (DIGITEK INSTRUMENTS) at 100
rpm for 24 hours at room temperature 30°C. A selective medium (M-1) was prepared containing
0.5% malathion (commercial grade 50%) as sole source of carbon in addition to 0.1% K2HPO4,
0.5% NH4NO3, 0.02% MgSO4 and 1.5% agar in distilled water. To this solution 20μl of a
mineral solution containing FeSO4 10%, CaCl2 10%, copper nitrate 0.5%, zinc powder 0.5%
and MnCl2 was added. From each processed sample, 0.5 ml was spread over the surface of M-1
with the help of a glass spreader. The inoculated plates were subsequently incubated at 37°C.
The growth was streaked for pure culturing on nutrient agar and then on the selective medium

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M-1. The growth was preserved as nutrient agar slants and glycerol stocks (Shagufta A and J
Iqbal 2008).

6) Isolation of Surfactant-Resistant Bacteria from the Surface Microlayer:

The surface microlayer (SML) is the uppermost millimetre of the water column and
bacteria living in this compartment (bacterioneuston) face a challenging environment in terms of
exposure to solar irradiation, accumulation of pollutants and surface tension (Agogué et al.,
2005).

SML samples were collected using the glass-plate method as described by Agogué et al.
2004. The glass plate was dipped in the water and left to drip for approximately five seconds.
Immediately after, the layer of SML was collected by passing the glass plate through a grid.

For the preparation of enrichment cultures, samples of SML water were spread on triplicate
plates of PY (peptone-0.1% and yeast extract-0.01%, diluted in brackish water salinity 17 gL–1)
containing 70 mM SDS (Sodium dodecyl sulphate, Bio-Rad), 1 mM CTAB (cetyl
trimethylammonium bromide, Sigma) or without surfactant. These samples were considered to
represent the initial inoculum used in the enrichment cultures. In parallel, two microcosms were
prepared using 200 mL of SML sample and 800 mL of basal saline medium (BSM) (adapted
from Amirmozafari et al., 2007). The anionic surfactant SDS or the cationic surfactant CTAB
were added to each of the microcosms in approximate critical micelle concentrations (CMC) of 8
mM and 1 mM, respectively. The cultures were incubated at room temperature for two weeks on
a rotary shaker operating at 90 rpm. Aliquots were collected weekly and spread on PY plates
amended with the approximate CMC concentrations of SDS or CTAB (3 replicates Fig. 2.
Variation of the mean CFU counts in enrichment cultures of bacterioneuston in microcosm
amended with 1 mM CTAB or 8 mM SDS. 92 A. LOUVADO et al. of each). After 3 days of
incubation at 25i C, bacterial abundance was estimated by colony counting.

Irfan, Zara and Daud, 2010 Isolation10 Enrichment Of Microorganisms Capable Of
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7) Enrichment and Isolation of a Mixed Bacterial Culture for Complete
Mineralization of Endosulfan:
Endosulfan (6, 7, 8, 9, 9a-hexahydro-6, 9-methano, 3, 4-benzo (e. dioxathiepin-3-oxide) is a
chlorinated cyclodiene insecticide, acaricide widely used throughout the world to control
numerous insects and mites in many food and nonfood crops.
Technical-grade endosulfan is a mixture of two stereo isomers, alpha and beta Endosulfan, in a
ratio of 70:30. Both the isomers are extremely toxic to aqueous organisms. Because of its
widespread usage and potential transport, endosulfan contamination is frequently found in the
environment at considerable distances from the point of its original applications.
Sample Collection for Enrichment Studies:

Soil samples used in this study were collected from the premises of an endosulfan
processing industry. Topsoil was collected from the first 15 cm and preserved at 4◦C (at the
site).The preserved soil samples were brought to the laboratory on the same day, and the
microbial enrichment was started immediately.

Enrichment of Mixed Bacterial Culture:

A soil suspension (10 g /50 ml) was made in a nutrient broth (NB). The composition of NB is as
follows: KH2PO41.0 g, K2HPO4; 1.0 g, NH4NO3; 1.0 g, NaCl; 1.0 g, MgSO4·7H2O; 0.2 g,
CaCl2; 0.02 g, Fe (SO4)3; 0.02 g, trace metal solution [12] 1 ml dissolved in 1 liter of distilled
water; final pH of the minimal medium was adjusted to 7 using HCl and/or NaOH. The soil
suspension was kept in an orbital shaker (Remi Instruments Ltd., India) set for 28 ± 2◦C for 24 h
at 150 rpm. The solid particles were allowed to settle for 1 h, and 1 ml of 84 the supernatant was
inoculated into a fresh 100 ml Erlenmeyer flask containing 10 ml of fresh NB spiked with 50 mg
l−1 of endosulfan. The contents were incubated at 28 ± 2◦C and 150 rpm for one week.
Thereafter, 1 ml of the suspension was transferred into a fresh Erlenmeyer flask and cultured as
above (at each culturing step, the concentration of endosulfan was increased, and at the end, the
concentration of Endosulfan was around 500 mg l−1 in the NB). After six transfers, a loop full of
this inoculum was streaked onto agar plates and incubated for 24 h at 28 ± 2◦C. Dilution and
streaking of colonies with same morphology was carried out three times. The colonies with
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distinct morphology were streaked on agar slants for 24 h at 28 ± 2◦C and refridgerated 4◦C for
further use (Mathava, K. and L, Philip 2006).

8) Enrichment, Isolation, and Phylogenetic Identification of Polycyclic
Aromatic Hydrocarbon-Degrading Bacteria:

Polycyclic aromatic hydrocarbons (PAHs) are common ubiquitous compounds found
worldwide in soils and sediments as a result of both natural and anthropogenic production (7
Chung, W. K., and G. M. King. 2001). High-molecular-weight PAHs from anthropogenic
sources can reach toxic concentrations that are detrimental to the environment and human health.
These compounds persist in the environment and, due to their hydrophobicity, become associated
with particulate matter, such as clays and humics that are deposited in soils and sediment. PAHs
are lipophilic and have the potential to biomagnify through the food chain. Recently, the U.S.
Department of Health and Human Services listed 15 PAHs as being carcinogenic.

Four ~1-g sediment samples were inoculated into separate Erlenmeyer flasks containing
100 ml BH minimal medium and 1.5% (wt/vol) NaCl. One of the following PAHs (5 mg) served
as the sole carbon and energy source for growth: naphthalene, phenanthrene, fluoranthene, or
pyrene. The PAHs were added to empty flasks, the solvent was allowed to evaporate, and liquid
medium was added, followed by an inoculum. An additional culture flask was set up for each
sample site with no added carbon source to serve as a negative control. Two successive
enrichments were prepared, with 1% (vol/vol) culture fluid used as the inoculum for each
subculture at 3-week intervals. The cultures were incubated aerobically at room temperature with
shaking at 175 rpm. The initial enrichment culture was designated A and the second and third
subcultures were designated B and C. To obtain PAH-degrading isolates, the B and C
enrichments were diluted in BH, plated on BH agar plates, and sprayed with the same PAH used
in the enrichment. Colonies surrounded by zones of clearing were subcultured to BH agar plates
and sprayed with the same PAH. Replicate plating was done using LB agar plates, BH agar
plates sprayed with PAH, and BH agar plates without PAH (control). The criterion for selection
of naphthalene-degrading strains was enhanced growth on plates with added substrate compared
to the control plates without added substrate. Selected pyrene-degrading isolates from pyrene

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spray plates were subcultured to BH agar plates and oversprayed with either fluoranthene,
chrysene, benzo[a]pyrene, dibenzo[a,i]pyrene, or a mixture of phenanthrene/pyrene for
cometabolism studies.

Twelve isolates representing each genus were grown in liquid culture tubes containing 4
ml (BH) minimal medium and 0.5 mg of one of the following PAHs: naphthalene, phenanthrene,
fluoranthene, pyrene, chrysene, or benzo[a]pyrene. Percent transmittance was measured at 540
nm on a Spectronic 20 (Milton Roy Company) to monitor growth. Overnight cultures grown in
LB broth (Difco Laboratories) were diluted 1:20 in BH minimal medium for use as inocula in the

mineralization experiment. PAH mineralization was determined with 14C-labeled substrates by a
method modified from previous studies (Boyd et al., 2005). One milliliter of the diluted culture

fluid was injected into a 40-ml prestoppered serum vial charged with one of the following 14C-

labeled substrates: 1.76 μg [14C]naphthalene, 18.6 mCi/mmol; 1.33 μg [14C]phenanthrene, 9

mCi/mmol; 0.91 μg [14C]fluoranthene, 45 mCi/mmol; or 0.34 μg [14C]pyrene, 55 mCi/mmol
(Sigma-Aldrich Co.). Vials were prepared in quadruplicate, with one vial serving as an acidified
control. Each stopper also contained NaOH-soaked filter paper suspended in the headspace.
After incubation for 46 h, samples were acidified with 1 ml 1 M H2SO4 and incubated

overnight. The filter papers were removed and placed into vials containing 5 ml scintillation
cocktail. Disintegrations per minute were measured on a Beckman LS6500 scintillation counter,
and the values were used to calculate mineralization. Mineralization rates were calculated from

the 14CO2 recovered and the specific activity of the initial 14C-labeled substrate.

Enrichment and isolation

Enrichment cultures were harvested 3 weeks after inoculation. Positive growth was
determined by an increase in the turbidity of the flasks containing PAH as a sole carbon and
energy source compared to the negative control flasks. Serial dilutions of enrichment cultures
were plated on solid media and oversprayed with PAHs, enabling selection of colonies

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surrounded by zones of clearing. Representative colonies of different morphotypes were from
each final enrichment were selected for further characterization.

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Review 15 Environmental Biotechnology

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Environmental Pollutants

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