Environmental biotechnology laboratory manual

Collection, disposal and treatment 36-37 Interaction of Plant Seeds with Diesel for Potential Use in the Remediation of Diesel fuel Contaminated Soils 38-46 Detection of Alkylbenzenesulf

Environmental Biotechnology

Laboratory Manual

ISLAMIC UNIVERSITY OF GAZA DEPARTMENT OF BIOTECHNOLOGY

ENVIRONMENTAL BIOTECHNOLOGY

LABORATORY MANUAL

Prof Dr Ismail Saadoun

Department of Applied Biological Sciences, Jordan University of Science and Technology, P.O Box 3030, Irbid- 22110, Jordan

Phone: +962-2-7201000-Ext 23460; Fax: +962-2-7201071 E-mail

address: isaadoun@just.edu.jo

Copyright 2008

All rights reserved No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without prior written permission of the author

Prof Dr Ismail Saadoun

Department of Applied Biological Sciences, Jordan University of Science and Technology, P.O Box 3030, Irbid- 22110, Jordan Phone: +962-2-7201000-Ext 23460; Fax: +962-2-

7201071 E-mail address: isaadoun@just.edu.jo

PREFACE

This manual has been designed for an undergraduate level laboratory sessions in environmental biotechnology The manual is divided into experiments that belong to a particular category An experiment will be carried out each week and some times may be continued in the week after

It should be noted that the first exercise in this manual require a repetition of basic techniques, and most results call for observations and tabulations

Prior to each lab session, careful orders and preparations are required which can be found in the procedure or the appendix sections Each experiment contains the following basic sections:

Results

The experimental analysis data are lay out as tables and figures Reports of the field visits are also included as instructed

References and further readings

A listing of useful articles and books is also provided

Appendix

Media, buffers and solutions used in each experiment are provided Their composition and companies which supply them are also included

Prof Dr Ismail Saadoun

Dept of Biotechnology and Genetic Engineering

Dept of Applied Biological Sciences

Jordan University of Science and Technology

ISLAMIC UNIVERSITY OF GAZA

Dept of Biotechnology Environmental Biotechnology Lab

Environmental Detection of Streptomycin-Producing Streptomyces spp

by Using strb1 and 16S rDNA-Targeted PCR

25-28 Field Trip (Main Wastewater Treatment Plant in Gaza) 29

Molecular Detection of Fecal Coliforms (E coli) in Water by PCR 30-35 Field Trip (Main Landfill Site in Gaza)

How the community deals with domestic solid waste?

(Collection, disposal and treatment)

36-37

Interaction of Plant Seeds with Diesel for Potential Use in the

Remediation of Diesel fuel Contaminated Soils 38-46 Detection of Alkylbenzenesulfonate-Degrading Microorganisms 47-49 Risks of Genetically Modified Organisms (GMOs) 50-55

ISLAMIC UNIVERSITY OF GAZA

Dept of Biotechnology Environmental Biotechnology Lab Lab Schedule

The aim of this lab course is to provide an understanding of the metabolic capability of

microorganisms to reverse and prevent environmental problems Topics will cover: Sewage

treatment, control of domestic, agricultural and industrial wastes, biocontrol of pests and

molecular detection of microorganisms in the environment Scientific visits are hopefully to be

worked on with proper arrangements

Week Exercise Pages

1 Introduction and Orientation/ Review of Microbial Techniques 6-12

2 Isolation and Characterization of Bacteria from Crude Petroleum Oil

Contaminated Soil

13-15

4 Growth Response of Bacteria on Petroleum Fuel (Diesel) 16-21

5 Enrichment for Uric Acid Utilizing Bacteria 22-24

6 Environmental Detection of Streptomycin-Producing Streptomyces spp

7 Mid Term Exam -

8 Field Trip (Main Wastewater Treatment Plant in Gaza) 29

9 Molecular Detection of Fecal Coliforms (E coli) in Water by PCR 30-35

Field Trip (Main Landfill Site in Gaza)

How the community deals with domestic solid waste?

(Collection, disposal and treatment)

36-37

13 Interaction of Plant Seeds with Diesel for Potential Use in the

Remediation of Diesel fuel Contaminated Soils

38-46

14 Detection of Alkylbenzenesulfonate-Degrading Microorganisms 47-49

15 Risks of Genetically Modified Organisms (GMOs) 50-55

16 Final Exam -

Review of Microbial Techniques CULTURAL TRANSFER

The procedure for transferring a microbial sample from a broth or solid medium or from a food sample is basically the same The sample is collected with a sterile utensil and transferred aseptically to a sterile vessel Two implements commonly used for collecting and transferring inoculum are the cotton swab and the platinum needle or loop The swab is used

in instances where its soft nature and its fibrous qualities are desired such as in taking a throat mucus sample or in sampling the skin of an apple A platinum needle or loop is used in those instances where a more concentrated microbial sample is available, such as in a contaminated water sample A typical culture transfer proceeds as follows:

1 In one hand hold the wire loop as you would a pencil;

2 Heat the wire loop until red;

3 Allow it to cool for a moment (this prevents burning or boiling of the medium when it contacts the loop);

4 Holding the culture container in the other hand, remove the cover by grasping it between the small finger and the palm of the loop holding hand;

5 Flame the container by passing the vessel top through the flame slowly (2 to 3 sec) in order

to sterilize the rim;

6 Insert the wire loop and take the sample;

7 Reflame the container top and replace the lid;

8 Open and flame the top of the receiving vessel as you did with the sample vessel;

9 Inoculate the sample into the vessel;

10.Reflame and cap the receiving vessel-;

11.Flame the loop to resterilize it

All vessels used need to be clearly labeled for identification The date and name of the person using the vessel should be included along with the other pertinent information, (e.g, medium type, control, concentration, etc.) All swabs, medium tubes, culture plates, and other items contaminated with microbes should be autoclaved before washing or disposal

PLATING

Isolation of individual microbial types may be obtained by dilution methods The dilution, a reduction of microbial cell concentration, may be achieved by spreading a small amount of culture across a wide medium surface This technique is called streaking Bacterial cell dilution may also be carried out using a series dilution scheme, a small amount of initially concentrated culture is introduced into a volume of medium or physiological saline and then homogeneously dispersed into that volume Physiological saline (0.85% NaCl) is used to protect cells from sudden osmotic shock thus preventing cell rupture, a sample of the new volume may be redispersed in yet another dilution volume to achieve further cell number reduction, by transferring known volumes of sample culture to known volumes of dilution media, one can calculate the reduction in cell concentration achieved, for example, if one introduced 1 ml of a sample into 9 ml of medium, one would have reduced the initial concentration by a factor of ten

Please refer to a dilution scheme for practice in making dilutions In dilution schemes one must maintain aseptic technique All transferring items must be microbe free All new media

or dilution media must be sterile

A pipet is used to transfer volumes of liquid The pipette should be clean and sterile It should

be equipped with a pipette bulb or pro-pipette so that oral contact and the potential danger of inhaling the microbial sample is avoided Always place pipettes in germicidal washing solution immediately after use

Dilution of cultures made by volume dilution may be plated out in Petri dishes and then incubated to allow the microbes time to grow A typical plating procedure would be as follows:

1 Pipette 1 or 0.1 ml of a known dilution of a sample into the bottom section (smaller plate) of a sterile Petri dish;

2 Within 20 min add 12-15 ml of warm (46-48°C) fluid medium to this Petri dish;

3 Cover the dish;

4 Swirl it gently to disperse the sample throughout the medium, (a figure eight pattern holding the dish flat on the table is the recommended swirl pattern: care should be taken to prevent splashing of the medium onto the lid of the dish);

5 Allow the plate to stand, cool, and solidify;

6 Invert the Petri dish (medium surface pointing down) and incubate in this position

Petri dishes are incubated upside down to prevent water from condensation from standing on the medium surface during incubation Pools of surface water would result in the loss of individual surface colonies since bacterial cells forming in the colonies could use the water pools as vehicles to reach the medium After a period of incubation microbial growth may be observed If sufficient dilution has been achieved, individual colonies of microbes may be clearly seen It is assumed that colonies arises from single microbial cells, thus an individual colony represents only one microbial type This assumes that the microbes in the original culture were not clustered and that a true homogenous dispersion was achieved (Shaking the solution with glass beads helps to break up cells clusters.) by picking out individual colonies and transferring them to a new sterile medium, microbial isolation can be achieved

Isolation is also achieved using the streaking technique This involves the aseptic transfer of a small quantity of culture to a sterile Petri dish containing medium The most common implement for streaking is the wire loop

Streaks should be performed by initially introducing an inoculum of the culture onto a small area of the medium plate surface This is called ‘the well’ After inoculating the well, the transfer loop is re-flamed, allowed to cool, and then touched on a remote corner of the plate to remove any heat remaining Beginning with the sterile loop in the well a streak is made across a corner of the medium surface (This spreads a bit of the culture out over the

medium—dispersing or diluting the culture.) the loop is re-flamed, cooled, and the streaking continued until all the available medium surface is utilized On a typical plate 3-5 streaks can

be made

Remember: the streaking loop must be re-flamed after each streak

Both processes, streaking and volume dilution, reduce and disperse the cell concentration onto the medium Upon incubation both dilution procedures should produce isolated colonies of a single strain The dilution technique has added use, in that upon sufficient dilution, all the colonies from the dilution can be seen as separate individual spots when plated By counting these spots and multiplying that number by the dilution factor for the plate, one can arrive at an estimate of the number of organisms in the original culture solution

As a rule of thumb only those incubated plates which have between 30 and 300 colonies are used to determine organism concentration in the original culture Thirty is taken

as the lower limit since statistically this many individual colonies are required for accuracy in calculation Three hundred is taken to be the upper limit] because difficulty is encountered in counting more than this number of colonies accurately

Motility Testing

Many microbes are motile Motility can be checked by inoculating a culture sample into a semisolid medium This is done with an inoculating needle which is stabbed straight down and pulled straight out of the tube Upon incubation, a non-motile colony will produce a single line of growth along the needle jab line, while a motile colony will give a wider band

of growth

The hanging drop mount is used to check motility It is prepared by placing a ring of lubricating grease around the rim of the recession in the hanging drop slide A drop of culture medium or a water suspension of a culture is then placed on a coverslip The coverslip is inverted so that the drop is clinging to the lower side, and the coverslip is laid to rest on the slide—being supported by the ring of grease This mount has the advantages that motility of live, motile microbes can be observed

Staining

A method of biochemical differentiation is staining Staining operates on the principal that different types of microbes have different chemical constituents making up their cellular components For example, the Gram stain operates on the principle that some cells retain a crystal violet-iodine complex after leaching with an alcohol solvent, these cells generally have complex membranes which result in retention of the blue complex and are thus called gram positive Other microbials with less complex membranes are not affected by the mordant, iodine The dye in these cells is washed out and replaced by a safranin counter-stain (red) These cells are said to be gram negative There are many other types of cellular dyes There are basic dyes specific for nuclear material, other cellular elements, and spores

Objectives:

This exercise will review the technical skills required to successfully function in an analytical microbiology laboratory This exercise will enable you to:

1 Transfer cultures, streak plates and inoculate slants;

2 Carry out dilution schemes to obtain microbial counts;

3 Determine microbial motility by two methods;

4 Carry out gram and spore stains;

Materials:

Broth and slant of: Escherichia coli, Bacillus subtilis, Staphylococcus aureus

Broth mix of: Staphylococcus aureus and Escherichia coli

Tryptone glucose extract agar (TGEA)

1 ml pipettes

Petri plates

99 ml dilution blanks

Gram and spore stains

Semi-solid agar tubes

Procedure:

A Microbial Isolation

1 Flask of agar medium are kept in a 48°c oven to maintain their fluidity, label _

plates of TGEA and pour 1—15 ml of the medium into these plates and allow them to cool and solidify for streaking and spread plating

2 The instructors have prepared 4 different types of broth cultures You will dilute out each

of these 4 different cultures, 2 by spread plating techniques and 2 by pour plating methods Your instructor will explain these procedures, as well as designate which of the cultures are

to be spread or pour plated and to what dilution Dilution schemes should be worked out

first on paper to avoid confusion (Note —examples of dilution schemes are given at the end of this exercise)

3 If the TGEA plates prepared in step 1 have solidified, proceed to streaking so that isolated colonies may be observed Streak out samples from all 4 broth cultures

Which of the cultures are to be spread or pour plated and to what dilution? Dilution schemes should be worked out first on paper to avoid confusion (Note —examples of dilution schemes are given at the end of this exercise)

4 When all plates have cooled and solidified, invert and incubate at 37 c for 48 hr Count the plates from the dilution(s) yielding between 25 and 250 colonies Calculate the bacterial cell concentration in the original culture Observe the streak plates Exchange class data

B Microbial Motility

1 Obtain 3 tubes of semi-solid agar and inoculate each tube with one of the 3 culture types using an inoculating needle Omit the mixed culture sure to label each tube, incubate tubes at 37c for 48 hr

C Staining

Use the broth cultures provided and the plates streaked for isolation as sources for microorganisms to stain

1 Make gram stains of the E coli, S aureus, B subtilis and the mixed culture according to

the procedure described by your instructor Observe these stains under the microscope using the oil immersion magnification

2 Make a spore stain of the cultures assigned to you Observe it under the microscope using the oil immersion objective Can you observe distinct spore bodies? If so, are they terminal, subterminal, or central? Are cells swollen at the spore location?

Dilution Calculations

Dilution factor = initial dilution x subsequent dilutions x amount plated Count per ml (or g) =

reciprocal of dilution factor x colonies counted

Example:

A sample was diluted initially 1:100 (1 ml of in 99 ml sterile diluent) A subsequent 1:10 dilution (1 ml of the initial dilution into 9 ml sterile water) was prepared Finally, 0.2 ml of the final dilution plated and 64 colonies were counted on the plate

Initial dilution x subsequent dilutions x amount plated = dilution factor

1/100 x 1/10x0.2 =0.0002 or 10- 2x 1 0- 1x 2 x l 0- 1 = 2 x 1 0- 4

reciprocal of dilution factor x colonies counted = count per ml

Then, the answer should be rounded off to 2.1 x 108 colony forming units (CFU) per ml

B Only those plates with between 25 and 250 colonies should be used to calculate plate

counts

Counting Colonies on Plates and Recording Results

Refer to the prepared handout for details

References:

American Public Health Association 1985 Standard methods for the examination of dairy

products 15th edition (APHA: N.Y.) Chapter 5, standard plate count method

Isolation and Characterization of Bacteria from Crude Petroleum Oil

Contaminated Soil Introduction

Petroleum fuel spills as a result of pipeline raptures, tank failures and various other production storage and transportation accidents is considered as the most frequent organic

pollutants of soil and ground water (BOSSERT et al 1984; MARGESIN and SCHINNUR,

1997) and classified as hazardous waste (BARTHA and BOSSERT, 1984)

DAGLEY (1975) suggested that indigenous oil utilizing microorganisms, which have the ability to degrade organic compounds, have an important role in the disappearance of oil from soil This microbiological decontamination (bioremediation) of the oil-polluted soils is claimed to be an efficient, economic and versatile alternative to physiochemical treatments (ATLAS, 1991; BARTHA, 1986)

In this experiment, enumeration of bacteria and assessment of microbial diversity will be conducted for soils polluted by petroleum fuel spills Also, the ability of different bacterial cultures to transform diesel fuel using a simple and rapid test will be investigated

PROCEDURE

Collection of samples:

-Collect soil samples of 1 kg from different gas stations contaminated with petroleum fuel

spills They can collected down to 10 cm depth, after removing approximately 3 cm of the soil surface

Sample processing:

-Crush each soil sample, thoroughly mixe and sieved through a 2 mm pore size siever

(Retsch, Germany) to get rid of large debris The sieved soil will then be used for the isolation purposes

-Place the samples in polyethylene bags, close tightly and store at 4±1 °C

Isolation of bacteria:

-Suspend samples of 1g in 100 ml of sterile distilled water, agitate on a water-bath shaker

(100 rpm, 30 min), serially dilute up to 10-6

-Spread aliquots of 0.1 ml from each dilution over the surface of nutrient agar plates

Bacterial identification:

-The morphological characterization of each isolate will be first performed, noticing color,

size, and colony characteristics (form, margin, and elevation)

-Perform Gram stain test for each isolate

-Grow the isolates at 42 °C

Biochemical tests:

The following biochemical tests will be used in the identification studies: gelatin liquefaction;

citrate utilization; oxidase; catalase; growth at 6.5% sodium chloride; fluorescent pigment

production; indole formation; glucose fermentation and nitrate reduction (CAPPUCCINO and

SHERMAN 1996)

-Place the isolates in phenol red glucose broth to determine glucose fermentation as well as

gas production

Results

Table 1 Total bacterial count and diversity in soils (at 10 cm depth) polluted with petroleum

fuel

Sample

No Locality

Time of Exposure (Year) to Petroleum Oil Spill Colour CFU 10 5 /gm x Colony Types

1

2

3

Table 2 Morphological and physiological properties of the different bacterial isolates

Species

Biochemical and cultural criteria Oxidase Citrate Mr/VP Indole TSI

Gelatin

Nitrate Reduction at 42 ºC Growth Motility

Species Identified

Sp 1

Sp 2

Sp 3

Sp 4

Sp 5

Gram reaction for the isolates:

References

ATLAS, R.M., 1991 Microbial hydrocarbon degradation-bioremediation of oil spills J

Chem Technol Biotechnol 52, 149-156

BARTHA, R., 1986 Biotechnology of petroleum pollutant biodegradation Microbial

sludge disposal in soil Appl Environ Microbiol., 47, 763-767

BOSSERT, I.D., and COMPEAU, G.C 1995 Cleanup of petroleum hydrocarbon contamination in soil In Microbial Transformation and Degradation of Toxic Organic Chemicals, ed YOUNG, L.Y., and CERNIGLIA, C.E New York: Wiley-Liss, Inc., pp 77-

1-Saadoun, I 2002 Isolation and characterization of bacteria from crude petroleum oil contaminated soil and

their potential to degrade diesel J Basic Microbiol 42 (6): 420-428

2-Saadoun, I 2004 Recovery of Pseudomonas spp from chronocillay fuel-oil polluted soils in Jordan and the study of their capability to degrade short chain alkanes World J Microbiol Biotech 20 (1): 43-46

3-Saadoun, I 2005 Production of 2-methylisoborneol by Streptomyces violaceusniger and its transformation by selected species of Pseudomonas J Basic Microbiol 45 (3): 236-242

4-Ziad Al-Ghazawi, I Saadoun and A Al-Shak’ah 2005 Selection of bacteria and plant seeds to grow on diesel

fuel to be used in remediation of diesel contaminated soils J Basic Microbiol 45 (5): 251-256

5 Saadoun, I., M Alawawdeh, Z Jaradat and Q Ababneh 2008 Growth of hydrocarbon-polluted soil

Streptomyces spp on diesel and their analysis for the presence of alkane hydroxylase gene (alkB) by PCR World Journal of Microbiology and Biotechnology DOI 10.1007/s 11274-0089729-z

Growth Response of Bacteria on Petroleum Fuel Introduction

Information on hydrocarbon (HC) degradation is required to determine the feasibility

to use microorganisms such as bacteria in the removal of petroleum-based pollutants from the environment Degradation of these pollutants by microorganisms has been assessed by a variety of strategies Early efforts at petroleum prospecting were based on the detection and enumeration of HC-degrading bacteria that were associated with soils overlaying petroleum-bearing formation (BRISBANE and LADD, 1965; DAVIS, 1967) Others included the seeding of the environment with cocktails of oil-utilizing bacteria (Dave et al 1994)

The straight-chained alkanes are usually the easiest hydrocarbons to be degraded which are usually converted to alcohol via a mixed function oxygenase activity and through a chemical pathway resulting finally in the formation of fatty acids (Sanger and Finnarty 1984)

Since alkanes are one of the main components of diesel fuel, thus the detection of alcohol production as a result of alkane oxidation would be an applicable approach for detecting the activity of microorganisms on diesel

Simple Alkane

Monooxygenase ↓ O2/ NADH+H +

Alcohol + NAD + +H2O Alcohol Dehydrogenase ↓ NAD +

Aldehyde + NADH+H +

Aldehyde Dehydrogenase ↓ NAD +

Fatty Acid + NADH+H +

↓

β-Oxidation

Jacobs et al (1983) reported that the detection of alcohol formation is a simple, rapid

and suitable method for the primary, semiquantitative screening of organisms capable of ethanol production Saadoun and E-Magdadi (1998) adopted this method to screen organisms capable of degrading geosmin In this experiment, the method of Jacobs et al (1983) has been modified in order to determine the ability of different bacterial strains in degrading diesel fuel

by transforming the diesel fuel to alcohol The test is based on the following reactions:

Ethanol + nicotinamide adenine dinucleotide (NAD+)

mg, Na2HPO4 210 mg, NaH2PO4 90 mg, CuSO4.5H2O 5 µg, H3Bo3 10 µg, MnSO4.5H2O 10

µg, ZnSO4.7H2O 70 µg, MoO3 10 µg, CoSO4 10 µg, KCl 40 mg, CaCl2 15 mg, NH4Cl 500 mg and NaNO3 2 mg) supplemented with 0.05% (v/v) diesel sterilized by filtration through 0.45

unit membranes (Millipore Corp MA, USA)

-Incubate at 28°C and 200 rpm for 21 days

-The growth response of each of the above isolated bacteria on diesel can initially determined

at 7 days intervals by physical appearance (turbidity) and measuring the optical density (O.D.)

at 540 nm using Bausch and Lomb Spectronic colorimeter 20 (Bausch and Lomb Inc., Rochester, NY)

-Determine the dry weight of cells/ml of the cell suspension by placing 2ml volume of the final cell suspension in pre-weighed aluminum tares and dry at 65°C for over night before weighing

-Determine the growth on diesel by the ‘hole-plate diffusion method’ as follows:

-Pour 20 ml of mineral salts agar medium (MSM) into Petri dishes

-Inoculate plates with the above test organisms using a sterile swab

-Remove cores of 6 mm diameter from the agar Fill up the holes with 50 µl of filter sterilized diesel The control hole will be filled with sterile distilled water only

-Incubate the agar plates with the bacterial isolates overnight at 28 °C

-Record the results after 48 hrs by physical appearance of growth surrounding the holes

Growth conditions:

-Inoculate 6 slants of yeast extract-dextrose (YD) agar [per liter: 10 g dextrose, 10 g yeast

extract (YE), 0.5% (v/v) glycerin, pH 7.5] with the different bacteria then incubate at 28 ºC for 48h

Adaptation of bacteria on diesel:

-Inoculate cells into 100 ml broth of yeast extract (YE) (0.1%)-peptone (0.1%) plus 0.1%

(v/v) diesel, then incubate at 28 °C with shaking at 100 rev/min for 12 h

-Centrifuge the whole mixture of each flask for 5 min at 4000 rev/min then suspend the pellet

in the same medium and incubate under the same conditions The last step will be repeated three times, then wash the cells three times with 0.1 mol/L phosphate buffer, pH 7.5

-Suspend the pellets in a small volume (5 ml) of the same phosphate buffer

Assay for diesel degradation:

The test of JACOBS et al (1983) will be conducted to detect the biodegradation of diesel,

hoping that the bacterial strains used the monoxygenase pathway in the biodegradation process

-Perform the test in duplicate at 28 ºC in a small test tube containing the following: 20 µl dichlorophenolindophenol (DCPIP) (Acros organic, NJ, USA), 0.05 mol/L; 30 µl 5-methyl-phenazinium methylsulphate (5-MPMS) (Acros organic, NJ, USA), 0.05 mol/L; 25 µl of 0.1% (v/v) diesel, 5 µl of 0.15 M NAD solution and 25 µl of washed cells

2,6 Compare the change in the colour with four controls The first control contains no diesel (substrate), the second contains no NAD+ and the third contains no cells A fourth control consists of heating the cells for 10 min at 90 ºC

-Follow the reaction at 1 hour, 2 hour, 6 hr and 12 hr

Results

Table 1 Growth response of different bacterial isolates on diesel as measured by turbidity

and dry weight

Growth Measurements/Time (days) Bacteria O.D (540 nm)

7 14 21 7 14 21 Dry Weight (mg/ml)

Readings at zero time = 0.0

Table 2 Action of different bacterial species on diesel as indicated by colour change

Colour change from dark blue to other colours at different time intervals by each bacterial

species Reaction

References

BRISBANE, P.G., and LADD, J.N., 1965 The role of microorganisms in petroleum

exploration Annu Rev Microbiol., 19, 351-364

DAVIS, J.B., 1967 Petroleum Microbiology Elsevier Publishing Co., New York

JACOBS, C.J., PRIOR, B.A and DEKOCK, M.J 1983 A rapid screening method to detect

ethanol production by microorganisms J Microbiol Methods 1, 339-342

LEADBETTER, E.R and FOSTER, J.W 1958 Studies of some methane utilizing bacteria

Arch Microbiol 30, 91-118

SAADOUN, I and EL-MIGDADI, F., 1998 Degradation of geosmin-like compounds by

selected species of Gram–positive bacteria Lett Appl Microbiol, 26, 98-100

SANGER, M and FINNARTY, W 1984 Microbial metabolism of straight-chain and

branched alkanes In Petroleum Microbiology ed ATLAS, R.M.New York: Macmillan pp

1-61

Further Readings

1-Saadoun, I and F Al-Meqdadi 1998 Degradation of geosmin like compounds by selected species of

Gram-positive bacteria Lett Appl Mibrobiol 26: 98-100

2-Ziad Al-Ghazawi, I Saadoun and A Al-Shak’ah 2005 Selection of bacteria and plant seeds to grow on diesel

fuel to be used in remediation of diesel contaminated soils J Basic Microbiol 45 (5): 251-256

3 Saadoun, I., M Alawawdeh, Z Jaradat and Q Ababneh 2008 Growth of hydrocarbon-polluted soil

Streptomyces spp on diesel and their analysis for the presence of alkane hydroxylase gene (alkB) by PCR World Journal of Microbiology and Biotechnology DOI 10.1007/s 11274-0089729-z

Enrichment for Uric Acid Utilizing Bacteria

Introduction

Enrichment or selective culture techniques were first used by Winogradsky and Beijernick in their extensive studies of soil microorganisms It is based upon the diversity of microorganisms which exist in nature Microbiologists use this technique to create an in vitro environment in the laboratory which favors the isolation of a particular microorganism This

is achieved in two ways:

1-Optimal conditions for growth are selected

2-The most rapid growth rate for the desired organism is selected

Generally enrichment culture is done in liquid-batch culture where medium composition and physical parameters such as temperature can be controled and or varied, selective inhibitors can also be added to the medium to control or inhibit unwanted organisms For example, cyclohexamide added to the medium will inhibit the growth of fungi which might otherwise overgrow the desired bacterial species Of course, also important is the source of the inoculum for the enrichment

Enrichment for the bacterium Bacillus fastidiosus that is able to grow on uric acid or

allantoin was first described by den Doceren de Jung in 1929 Only uric acid or allantoin can

be metabolized by the bacterium Uric acid and allantoin are breakdown products of purine (Fig 1)

In this experiment, you will enrich for B fastidiosus which can grow on uric acid

Media

1-Uric acid broth tubes

0.5% uric acid + mineral salt (MS) base in tap water

MS base: NH4Cl 1.0 g; Na2HPO4.2H2O 2.14 g; KH2PO4 1.04 g; MgSO4.7H2O 0.2 g; Trace salt solution 10 ml; water 1000 ml, pH 7.0

2-Uric acid (UA) agar plates: MS base + 0.5% UA + 1.5% agar

3-UA-yeast extract (YE) broth and agar: same as 1 and 2 + 0.5% YE

4-Glucose MS agar: Glucose 0.5%, MS + 1.5% agar The glucose is sterilized separately and added to the sterile MS agar base

5-Glucose-Casein hydrolysate agar: glucose 0.2%, casein hydrolysate 0.5%, 1.5% agar

6-Malate-YE slants: 0.2% malate, 0.5% YE, 1.5% agar

7-YE-NA: 1.5% NA, 0.2% YE, 1% agar in tap water (water agar)

Procedure

1-Inoculate each of 2 tubes of uric acid broth with about 0.2 gm of soil It is preferable to get soil sample that is contaminated with chicken manure Why?

2-Pasteurize one broth tube by heating in a water bath at 80-85 ºC/5 min

3-Incubate the tubes at 37 ºC / 3 days

4-Prepare wet mounts and Gram stains of your culture

5-Repasteurize the culture which you pasteurized in the first lab period and streak this culture and the unpasteurized culture on separate plates of uric acid agar Be sure to mark your plates

as pasteurized and unpasteurized

11-Record your results Did growth occur on any of the media other than the uric acid agar? If

so, prepare Gam stains and characterize the organism which grew on these media

Bacillus fastidiosus:

Large rods, 1.5-2.5 µm x 3-6 µm; stain uniformly; often in chains, motile, with lateral flagella Gram positive in the early stages of growth Endospores oval to cylindrical, 1.4 -1.7

µm x 1.8-3 µm; occupy most of the interior of the shorter sporangia; terminal or subterminal

in longer rods; may lie obliquely to the axis of closely septate filaments; produce little or no swelling of the sporangium; have a stainable surface after release

Fig 1 Degradation of purines results in the formation of uric acid which can be metabolized further yo glyoxylate, urea and CO2

Environmental Detection of Streptomycin-Producing Streptomyces spp by Using strb1

and 16S rDNA-Targeted PCR Introduction

Streptomyces species have always been a unique group of prokaryotes in respect to their

morphological diversity and their metabolic products, antibiotics and many enzymes of industrial interest (Tanaka and Omura, 1990) They are natural inhabitants of soil and they

together with the other genus Nocardia are the most abundant actinomycetes found in the soil (Williams et al., 1989)

Traditional detection and identification of Streptomyces spp specially

antibiotic-producers is time-consuming, requires multiple procedures and involves extensive

experimental work (Hain et al., 1997; Mehling et al., 1995; Williams et al., 1983)

The economic importance of the antibiotic-producing Streptomyces and the importance of Streptomyces in controling soil-born pathogens by antibiosis (Saadoun and Al-

Momani 1997; Tulemisova and Chormonova 1989; Weller and Thomashow, 1990) have promoted several workers to detect and characterize these organisms by simple and rapid procedures Detection and identification of these organisms in their natural habitats by rapid and sensitive tests such as PCR-based methods are needed to demonstrate their potential for antagonism against pathogens in soil

This experiment will attempt to rapidly detect strptomycin-producing Streptomyces spp in soils by PCR-based method using DNA isolated directly from soil Streptomycin was chosen to be screened because its coding gene strb1 is highly conserved between streptomycin producers (Retzlaff et al., 1993)

PROCEDURE

As isolates of Streptomyces are recovered Streptomyces-like colonies will be purified by repeated streaking then tested for streptomycin production (Gharaibeh et al., 2003) using streptomycin sensitive (Escherichia coli and Bacillus subtilis) and resistant (Klebsiella pneumoniae and Staphylococcus aureus) bacteria An isolate was considered to be active streptomycin-producer if it inhibited the growth of E coli with inhibition zone diameter of 18

mm or greater

Extraction of Bulk soil DNA

-Extract total DNA from the soil samples that have collected previously by a modification of

the direct lysis method (Ogram et al., 1987, Wellington et al., 1992) as follows:

-Suspend one g soil in 10 ml sterile distilled water, incubate for 1 hour at 28 ˚C with shaking

at 200 rpm

-After settling, centrifuge the supernatant at 3,000 rpm for 10 min

-Re-suspend the pellet in 50 mM EDTA and add SDS to a final concentration of 2%, then incubate in a water bath at 100 ˚C for 10 min

-Centrifuge the mixture at 10,000 rpm for 10 min, then transfer the supernatant to a clean sterilized Eppendorf tubes containing isopropanol (3:1 ratio)

-Invert the tubes several times, and centrifuge at 10,000 rpm for 1 min

-Discard the supernatant and add 100% ethanol

-Invert the tubes gently, then centrifuge at 13,000 rpm for 2 min

-Drain the tubes for 15 min to remove all of the ethanol then rehydrate the DNA pellet by adding 50 µl of sterile TE buffer [10 mM Tris-HCl pH = 7.4, 1 mM EDTA pH = 8.0] -Three sets of primers will be used in this experiment The first (RI7 and RI8) and second

(AM45 and AM47) sets of primers were taken from Gharaibeh et al., (2003) Both sets amplify 16S rDNA conserved regions found only in Streptomyces spp The third set represents the forward and reverse primers of strb1, a biosynthetic gene that codes for streptomycin amidinotransferase (Distler et al., 1992) according to Huddleston et al., (1997)

using the following primers: forward primer 5`-TG AGC CTT GTA AGC GTC CAC-3` and reverse primer 5`-TT CAT GCC GTG CTT CTC CAG-3` (OPERON Technologies, USA) to yield a 940 bp product

-Primers can be synthesized by Operon Technologies (Operon, USA)

Primer Sequence (5` to 3`)* Corresponding region Reference

strb1 gene Huddleston et al 1997

*Operon Technologies, USA

Detection of streptomycin-producers in soil

-Detection of streptomycin-producers will be carried out by the amplification of strb1 gene

-PCR amplification will be carried out as mentioned above except that the annealing temperature is 55 ˚C

-Use nuclease free water (Promega, USA) as negative controls

Electrophoresis and Photography

PCR products are checked for DNA by standard electrophoresis procedures (Sambrook et al., 1989) Gels can be viewed using U.V illuminator and photographed using Polaroid MP4+

Instant Camera System (Polaroid corp., USA)

Results

The ability of the constructed (RI7/RI8) and the control primers (AM45/AM47) to detect

Streptomyces directly from soil can be shown by the presence of a single DNA band of 438

bp

Detection of the presence of streptomycin producers directly from soil using crude DNA extracted from soil samples that contained streptomycin producers as a template for PCR to

amplify the Strb1 gene; can be clearly indicated by a single band with 940 bp in size for the

soil samples tested

References

Gharaibeh, R., I Saadoun and A Mahasneh 2003 Evaluation of combined 16S rDNA and

strb1 gene targeted PCR to identify and detect streptomycin-producing Streptomyces J Basic Microbiol 4: 301-311

Hain, T., N Ward-Rainey, M Kroppenstedt, E Stackebrandt and F Rainey 1997

.Discrimination of Streptomyces albidoflavus strains based on the size and number of 23S ribosomal DNA intergenic spacers Inter J System Bacteriol 47: 202-206

16S-Mehling, A., U.F Wehmeier and W Piepersberg 1995 Application of random amplified polymorphic DNA (RAPD) assays in identifying conserved regions of actinomycete

genomes FEMS Microbiol.Lett 128:119-126

Retzlaff, L., G Mayer, S Beyer, J Ahlert, J., S Verseck, J Distler and W Piepersberg 1993 Streptomycin production in stretomycetes: A progressive report Pp 183-194 In: Blatz R.,

G.D Hegeman and P.L Skatrud (eds) Industrial Microorganisms: Basic and Molecular Genetics ASM Press, Washington, D.C

Saadoun, I and F AL-Momani 1997 Steptomycetes from Jordan soils active against Agrobacterium tumefaciens Actinomycetes 8: 29-36

Tanaka, Y and S Omura 1990 Metabolism and products of actinomycetes – An

introduction Actinomycetologica 4: 13-14

Tulemisova, E and T Nikitina 1989 Search for actinomycetes antagonists of fungi causing

sugar beet root rot Acta Biotechnol 9: 389-391

Weller, D.M and S.L Thomashow 1990 Antibiotics: evidence for their production and sites

where they are produced pp 703-711 In: , Barker R.R., P.E Dunn and R Alan (eds) New Directions in Biological Control: Alteration for Suppressing Agricultural Pests and Diseases

Liss Inc., New York

Williams, S T., M Goodfellow, G Alderson, E.M Wellington, P.H Sneath and M.J Sackin

1983 Numerical classification of Streptomyces and related genera J Gen Microbiol 129:

1743-1813

Williams, S T., M Goodfellow and G Alderson 1989 In: Bergey’s Manual of Systematic Bacteriology Williams, S.T., M.E Sharpe and J.G Holt, J G vol 4, pp 2452-2508

Further Readings

1-Malkawi, H.I., I Saadoun, F Al-Momani and M.M Meqdam 1999 Use of RAPD-PCR fingerprinting to

detect diversity of soil Streptomyces isolates New Microbiologica 22: 53-58

2-Saadoun, I., F Al-Momani and A Elbetieha 1999 Genetic Determinants of active antibiotic-producing soil

streptomycetes New Microbiologica 22: 233-239

3-Gharaibeh, R., I Saadoun and A Mahasneh 2003 Genotypic and phenotypic characteristics of

antibiotic-producing soil Streptomyces investigated by RAPD-PCR J Basic Microbiol 43 (1): 18-27

4-Gharaibeh, R., I Saadoun, and A Mahasneh 2003 Evaluation of combined 16s rDNA and strb1 gene targeted PCR to identify and detect streptomycin-producing Streptomyces J Basic Microbiol 43 (4): 301-311

5-Saadoun, I and R Gharaibeh 2008 Usefulness of strb1 and 16S rDNA-targeted PCR for detection of

Streptomyces in environmental samples Polish Journal of Microbiology 57 (1): 81-84

Wastewater Treatment Plant

Tertiary Treatment

Disinfection

Treatment of Sludges