Environmental Biotechnology

Laboratory Manual

Prof. Ismail Saadoun

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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

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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

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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: Introduction Background and principles behind the assays performed. Procedure A detailed description of the materials, equipment needed to conduct the experiment and the method to be followed. Detailed listing of laboratory media, cultures, and special chemicals are also included. 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 Irbid-22110, JORDAN Tel: (Work) 962-2-7201000, Ext. 23460 Fax: 962-2-7201071 E-mail: isaadoun@just.edu.jo; isaadoun_1962@yahoo.com

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ISLAMIC UNIVERSITY OF GAZA Dept. of Biotechnology

Environmental Biotechnology Lab

Contents Pages Introduction and Orientation/ Review of Microbial Techniques 6-12 Isolation and Characterization of Bacteria from Crude Petroleum Oil Contaminated Soil

13-15

Growth Response of Bacteria on Petroleum Fuel (Diesel) 16-21 Enrichment for Uric Acid Utilizing Bacteria 22-24 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 Appendices 56-58

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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

3 Continue - 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.

by Using strb1 and 16S rDNA-Targeted PCR 25-28

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 10 Continue 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 -

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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.

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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

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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.

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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

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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- 2 x 1 0 - 1 x 2 x l 0 - 1 = 2 x 1 0 - 4

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reciprocal of dilution factor x colonies counted = count per ml 5000 (or 5 x 103) x 64 = 320,000 (or 3.2 x 105)

A. Plate count results should be reported to two significant figures only.

Example:

If the dilution factor used was 106 and 212 colonies were counted, the count/ml would be calculated thus, Reciprocal of dilution factor x colonies counted count/ml = count/ml 106 x 212 = 2 . 1 2 x 1 0 8

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.

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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.

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-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 x 105/gm

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

Growth at 42 ºC

Motility Species Identified

Sp. 1

Sp. 2 Sp. 3 Sp. 4 Sp. 5

Gram reaction for the isolates:

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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 Ecology 12: 155-172. BARTHA, R. and BOSSERT, I., 1984. The treatment and disposal of petroleum refinery wastes. In Petroleum Microbiology, ed. ATLAS, R.M. New York: Macmillan Publishing Co. pp. 1-61. BARTHA, R. and BOSSERT, I., 1984. The treatment and disposal of petroleum wastes. In Petroleum Microbiology, ed. Atlas, R.M.. New York: Macmillan Publishing Co. pp. 553-578. BOSSERT, I.D., KACHEL, W.M., and BARTHA, R., 1984. Fate of hydrocarbons during oily 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. 77-126. BOSSERT, I.D., and BARTHA, R., 1984. The fate of petroleum in soil ecosystems. In Petroleum Microbiology, ed. ATLAS, R.M. New York: Macmillan Publishing Co. pp. 435-474. Cappuccino, J. G. & Sherman, N., 1996. Microbiology: A Laboratory Manual. The Benjamin/Cummings Publishing Company, Inc. New York, pp. 129–182. ISBN 0-8053-6746-1

DAGLEY, S., 1975. A biochemical approach to some problems of environmental pollution. Essays Biochem., . 11: 81-138. MARGESIN, R. and SCHINNUR, F., 1997 Efficiency of indogenous and inoculated cold-adapted soil microorganism for biodegradation of diesel oil in Alpine Soils. Appl. Environ. Microbiol., 63, 2660-2664. Further Readings 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.

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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+) ↑↓ alcohol dehydrogenase acetaldehyde + NAD+ + H+ NAD+ + H+ + 2,6-dichlorophenolindophenol (DCPIP) [oxidized, blue] ↑↓ 5-methyl-phenazinium methylsulphate (MPMS) NAD+ + 2,6-DCPIP [reduced, yellow]

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PROCEDURE Microbial culture: The different bacterial species recovered from different soils contaminated with petroleum fuel spills (previous experiment) will be used to study their ability to degrade diesel fuel. Growth on diesel: -Incoculate colonies of different bacteria (previous experiment) into 50 ml mineral salts medium (MSM) of Leadbetter and Foster (1958) (Per liter: FeSO4 1 mg, MgSO4.7H2O 200 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:

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-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 2,6-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. -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 Dry Weight (mg/ml)

7 14 21

Readings at zero time = 0.0

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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 condition

Time Sp. 1 Sp. 2 Sp. 3 Sp. 4 Sp. 5 Sp. 5

+NAD+/29˚C 1h.

2h. 6h. 12h. Result -NAD+/29˚C 1h. 2h. 6h. 12h. Result +NAD+/90˚C 1h. 2h. 6h. 12h. Result

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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.

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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. Trace salt: FeSO4.7H2O 300 mg MnCl2.4H2O 180 mg Co(NO3)2.6H2O 130 mg ZnSO4.7H2O 40 mg H2MoO4 (Molybdic acid) or molybdenum trioxide

20 mg

CuSO4.5H2O 1 mgCaCl2 1000 mgHCl (0.1 N) 1000 ml2-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

19

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. 6-Incubate at 37 ºC /3 days. 7-Look for white rhizoid spreading colonies which adhere tenaciously to the agar surface. Prepare Gram stains of one of the colonies. 8-Transfere a single colony to a sterile test tube containing 0.5 ml of water. Mix thoroughly to make a turbid suspension. 9-Inoculate slants of UA agar, YE-NA, glucose-MS agar, glucose-casein hydrolysate agar and sodium malate-YE agar. Incubate at 37 ºC for 5-7 days. 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.

20

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

21

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).

22

-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

RI 7

GTGAAAGCCCGGG

GCTTAAC

Region located around

nucleotide position 576. Gharaibeh et al. 2003

RI 8

CACCGACCACAAG

GGGGGCA

Region located around

nucleotide position 995. Gharaibeh et al. 2003

AM 45

GTGAGTCCCCAGAT

CACCCCGAAG

Region located around

nucleotide position 1100.

Mehling et al. 1995b

AM 47

GTGGGCAATCTGCC

CTTGCACTCT

Region around position 120.

Mehling et al. 1995b

Forward

Primer

TGAGCCTTGTA

AGCGTCCAC

strb1 gene

Huddleston et al. 1997

Reverse

Primer

TTCATGCCGTGCTT

CTCCAG

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.

23

-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 16S-23S ribosomal DNA intergenic spacers. Inter. J. System. Bacteriol. 47: 202-206. 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.

24

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.

25

Wastewater Treatment Plant (Field Visit)

Wastewater background Treatment of wastewater Preliminary Treatment Screening Removal of oil and grease Grit removal Primary Treatment (settlement) Secondary Treatment Biological filtration Activated sludge Nitrification

Biological Denitrification Nutrient removal

Tertiary Treatment

Disinfection Treatment of Sludges

26

Molecular Detection of Fecal Coliforms (E. coli) in Water by PCR Introduction Gastrointestinal illnesses and enteric infections occur everywhere and nowhere at the same time. A large proportion of these illnesses are due to infections by a wide variety of enteric microorganisms, including bacteria, viruses, and intestinal parasites. It is estimated that, more than 100 different types of potentially pathogenic microorganisms that may cause an immense range of diseases and clinical symptoms can be present in polluted water (Ottoson, 2004). Diseases can be transmitted to humans via water through groundwater, estuarine water, seawater, rivers, aerosols emitted from sewage treatment plants, insufficiently treated water, drinking water, and private wells that receive treated or untreated wastewater either directly or indirectly (Fong and Lipp, 2005). In some places, crops are irrigated with wastewater and transmission of pathogens to humans (and animals) can occur directly or via crops, aerosols and potentially via groundwater.

Up to the present time, only microorganisms which can be grown in laboratory culture media could be directly related to water-borne outbreaks of disease. If microorganisms could not be grown or easily observed in the laboratory, they could not be detected in water. To overcome the drawback of these traditional laboratory culture techniques, scientists implemented new, rapid, sensitive and specific methods for detection of pathogens in the environment. Among these is the Polymerase Chain Reaction (PCR) (Josephson et al. 1991). In this experiment, indicator fecal coliform (E. coli) in water sample will be detected by using the PCR. Monitoring treated wastewater and sludge by the PCR would provide increased public health protection since the PCR is much more sensitive for detecting specific pathogenic microorganisms than traditional culture techniques are.

27

PROCEDURE Your instructor will provide you with a water sample that is contaminated with E. coli. The PCR will be used to amplify a fragment of the E. coli uidA gene that codes for ß-D-glucurinidase (Tsai et al. 1993). Although this gene may be found in some Shigella spp. (Bej et al. 1991), it is most commonly present in E. coli (Jochimsen et al. 1975). Primers UAL-754 (5’-AAAACGGCAAGAAAAAGCAG-3’) and UAR-900 (5’-ACGCGTGGTTACAGTCTTGCG-3’) (Bej et al. 1991). These primers will be used to amplify the 147-bp coding region of the uidA gene.

28

29

30

31

References Bej, A.K., J.L. DiCesare, L. Haff and R.M. Atlas. 1991. Detection of Escherichia coli and shigella spp. In water by using the polymerase chain reaction and gene probes for uid. Appl. Environ. Microbiol. 57: 1013-1017. Fong T-T., Lipp E. K. (2005) Enteric viruses of humans and animals in aquatic environments: health risks, detection, and potential water quality assessment tools. Microbiology & Molecular Biology Review., 69, 357–371. Jochiimsen, B., P. Nygaard and T. Vestergaard. 1975. Location on the chromosome of Escherichia coli of genes governing purine metabolism. Mol. Gen. Genet. 143: 85-91. Josephson, K.L., Pillai, S.D., Way, J., Gerba, C.P. and Pepper, I.L. (1991). Detection of fecal coliforms in soil by PCR and DNA:DNA hybridizations. Soil Science Society of America Journal 55: 1326-1332. Ottoson J. (2004) Comparative analysis of pathogen occurrence in wastewater – management strategies for barrier function and microbial control. PhD Thesis. Department of Land and Water Resources, Engineering Royal Institute of Technology (KTH), Stockholm, Sweden. Tsai, Yu-Li, C. J. Palmer and L. R. Sangermano. 1993. Detection of Escherichia coli in sweage and sludge by polymerase chain reaction. Appl. Environ. Microbiol. 59: 353-357.

32

Domestic Solid Waste Treatment and Landfill Sites in Gaza

-Field Trip (Main Landfill Site in Gaza) -How the community deals with domestic solid waste? (Collection, disposal and treatment) -Be familiar with the landfill sites in other countries as shown below in the table. Try to collect such information about the landfill sites in Gaza or West Bank and their actual operation and activities. -What are the products of these sites and how they can be modified to achieve the optimum operation and products.

33

Active Depo sites 2006 (Jordan)

34

مالحظاتComments

تاريخ

التشغيلDate

Started

المحافظةMunicipality

كمية

)يوم/طن(النفاياتDeposited

Garbage in Ton/Day

المساحة

دونمSize of the

area in !000 Sq m

اسم المكبName of the Depo

site

Akadirاالآيدر Irbid 700 806اربد 1980 Mafraqالمفرق 1986 Mafraqالمفرق 180 170

Mafraqالمفرق 2003 43 360 البادية

NorthernالشماليةBadia

Mafraq 5المفرق 2003 Rawishidالرويشد 179

Irbid 200 67اربد 1983 الشونة

NorthernالشماليةShouna

العاصمة 2003 Amman Ghabawiالغباوي 1947 2500

يستقبل نفايات ذيبان وجزء من عمان وشرآات

Receives garbageخاصهfrom Theiban some from

Amman and Industry

مادبا 1974Maadaba 500 87 مادباMaadaba

Notغير مناسب النحدار الموقعsuitable for its presence

on a slope location -Al)السلط(الحمرا Balqaa 450 275البلقاء 1990

Hamra (Sault)

الزرقاء 1991 Azarqaa Al-Thalielالضليل 270 295

DirAllaدير عال Balqaa 290 363البلقاء 1998 سيتم اغالقه . غير معتمد

Notوتشغيل البديل المعتمدcertified due to shut

down and use of alternative one

Zarqaaالزرقاء 1983 17 غير

حددةمUndtermi

ned

)الحالي(االزرقAzraq (Now)

Balqaa 55البلقاء 1988 غير محددة

Undetermined

الشونة الجنوبية

Southerns Shouna

Karak 22 205الكرك 1997 غير معتمدغور

-Ghour Alالمزرعةmazraa

Karak 190 485الكرك 1995 )اللجون(الكرك

Karak (Al-Lugoune)

Ghourغور الصافي Karak 25 153الكرك Not certified 1997غير معتمدAssafi

سيتم تحويله الى محطة Due to be تحويلية

changed to a conversion station

-Ashالشوبك Maan 45 26معان 1983shoubak

Tafilaالطفيلة Tafila 65 450الطفيلة 1990 Aielايل Maan 42 274معان 1984 Maanمعان Maan 90 502معان 1994 Al-Quiaraالقويرة Aqaba 25 270العقبة 2000

سيتم انشاء حارقة . غير مناسب Not apropiateمرآزية

Inator is due to irrected Aqabaالعقبة Aqaba 115 60العقبة 2000

35

Interaction of Plant Seeds with Diesel for Potential Use in the Remediation of Diesel Fuel Contaminated Soils

Introduction

The pollution with petroleum, heavy metals, xenobiotics, organic compounds and other contaminants is a growing environmental concern that harms both terrestrial and aquatic ecosystems. Bioremediation as a cleanup method and through the exploitation of the activities of microorganisms would degrade or attenuate such contaminants. Phytoremediation as one of the developed and implemented technologies of bioremediation is another option for cleaning up environmental pollution which, focuses on the use of living green plants (trees, grasses and aquatic plants) for the removal of contaminants and metals from soil, although some phytoremediation applications are believed to work through stimulation of rhizosphere bacteria by the growing plant root (Glass 2005). For hydrocarbon contamination, terrestrial, aquatic and wetland plants and algae can be used for the phytoremediation process under specific cases and conditions (Nedunuri et al. 2000, Radwan et al. 2000, Siciliano et al. 2000). The specific mechanisms involved in phytoremediation include: enhanced rhizosphere activity and subsequence biodegradation; phytodegradation; phytoextraction; phytovolatilization, and hydraulic pumping (USACOE, 1997).

An inventory of plant species in terrestrial and wetland environments in western Canada with a demonstrated potential to phytoremediate or tolerate petroleum hydrocarbons was developed by Farrell et al. (2000). One of the search results generated by this database is a list of 11 plant species capable of degrading (or assisting in the degradation of) a variety of petroleum hydrocarbons, and which may have potential for phytoremediation efforts in western Canada. Novak and AL-Ghazawi (1997) reported that both Fescue grass and Squash can enhance the bioremediation of hydrocarbon contaminated soils. Broad beans (Vicia faba) and lupine (Lupine albus) plants were tested by Radwan and his colleagues and the results showed that V. faba tolerated up to 10% crude oil (sand/crude oil, w/w) (Radwan et al. 2000). However, L. albus died after three weeks of exposure to a 5% oil concentration. Also, the leaflet areas of V. faba and L. albus, were respectively reduced by 40% and 13% at a concentration of 1% of oil. Other plants, such as Bermuda grass and Tall fescue were also investigated for their capabilities to remediate petroleum sludge under the influence of inorganic nitrogen and phosphorus fertilizers. About a 49% reduction of TPH occurred in the first six months, but there were no significant differences between the two species and the control (unvegetated). After one year, TPH was reduced by 68, 62 and 57% by Bermuda, fescue, and control, respectively. Radwan and his colleagues (2000) concluded that the optimal remediation was obtained by fertilization that produced a C:N:P ratio of 100:2:0.2. AL-Ghazawi et al. (2005) also tested seeds of two plants, Fescue grass (Cyndon dactylon) and Alfalfa (Medicago sativa) in their selection of plant seeds to resist diesel pollution as indicated by seed germination of these plants.

However, before starting a phytoremediation project, determining the toxicity of the contamination toward the plants is essential. Therefore, a successful approach requires the selection of plant seeds that have high resistance to the pollutant to be treated. For this reason, this experiment aimes to initiate an inventory process in Gaza for plant species in terrestrial environment with a potential to tolerate high levels of petroleum contamination and screen

36

these local plants or plant seeds for their resistance to high levels of diesel contamination to be recommended in future remediation of contaminated soils. PROCEDURE

Preparation of diesel:

-Purchase one liter of diesel from a Gas Station/Gaza.

-Weigh 10 ml of disel then place them in a chemical hood for evaporation at room temperature for 24 hours.

-Measure the remaining amount of diesel in trem of volume (ml) and weight (gm). Diesel measurement is necessary to prepare the different concentrations of diesel.

-Prepare 200 ml of 5000 ppm diesel by disolving 1 ml of evaporated diesel in 199 ml of methylene chloride. This stock is used to prepare the other diesel concentartions; 100, 200, 500, 1000 and 2000 ppm. Use the following chart for calculations: 1 mg / L = 1 mg /kg = 1 mg/1000 g = 1/1000,000 = 1 ppm 0.5% = 0.5 g/100 ml = 5 g /1000 ml = 5000 mg /1000 g = 5000 ppm 10,000 mg/kg = 10 g/kg 2 g cotton →→→→→→→ 2 x 10-3 kg 1 kg soil →→→→→→→ 10g = 10,000 ppm 2 g →→→→→→→ ? 2 x 10 = 20 = 0.02 g = 20 mg 1000 1000 10,000 ppm →→→→→→ 1L 1 kg soil →→→→→→ 10,000 ppm 2 g cotton →→→→→→ ? 2 x 10 = 20 = 2% g to give us 10,000 ppm 1000 1000

37

-Use methylene chloride and distilled water as controls.

-Add 2 ml of each diesel concentartion, methylene chloride and water the glass Petri dishes.

-Uncover the plates that have been treated with diesel or methylene chloride for 10 min at room temperature.

38

Plant seeds:

-Different plant seeds may either provided by the National Center for Agricultural Research and Technology Transfer (NCARTT/Ministry of Agriculture-Jordan) or purchased from the local market.

-The seeds represent monocot or dicot plants and their viability is provided from the source they are obtained from.

-Give each seed type an alphabet letter (Table 1).

-Grow 2 replicates from each type of seed at different diesel concentrations (0.0, 100, 200, 500, 1000, 2000, and 5000 mg/kg).

-The germinated seeds under these conditions are expected to be tolerant to diesel and thus may be recommended for phytoremediation of diesel contaminated soils.

-Growth on cotton-based filter papers is not the same as growth in soil and results of this experiment are only first indication of diesel phytotoxicity of seeds under real soil conditions.

Table 1. Example of plant seeds that can be used in this experiment.

Plant Common Name Source Viability % Triticum durum Solid Horani

Wheat Local Market

Var. sacharata Zea Maize (Corn) NCARTT Hordeum vulgare Barley Local Market Cucuvita pepo Slasil (Squash) NCARTT Phaseolus vulgaris Romano NCARTT Triticum aestivum NCARTT Atriplex halimus NCARTT Hordeum spontaneum NCARTT

Phytotoxicity and indexing the samples:

-Formulate an index to assign the sample number and diesel concentration.

-Letters are assigned to each plant seed as such:

-Digits 00, 01, 02, 05, 10, 20, and 50 are assigned to diesel concentration of 0.00, 100, 200, 500, 1000, 2000, and 5000 mg/kg, respectively. For example, the A205 index refers to A plant, replicate number 2 with diesel concentration of 500 mg/kg. -Place 2 grams of cotton in glass Petri dishes of 11 cm diameter, and the required volume of diesel is added. -Dissolve diesel in methylene chloride to provide larger volume and thus make sure that the diesel is fully and homogeneously distributed all over the entire cotton. -After adding the diesel contamination, open the dishes inside a fume hood to evaporate the methylene chloride. -The zero diesel concentration is used as a control to determine the viability of the seeds. -Introduce seeds (10-15) of each species to each dish, and then add 2 ml of sterile distilled water. -Seal the dishes and incubate in the dark at room temperature to simulate below ground soil conditions.

39

-After 10 days, open the dishes and count the germinated seeds. Control Petri dishes includes seeds of each plant seeds and 2 ml of either sterile distilled water or methylene chloride. Incubation: -Incubate all plates at room temperature for 10 days. -At 2 days intervals, uncover plates and spray them 3 times with tap water. -Physically observe for seed germination or fungal contamination. -After incubation, count the number of germinated seeds and determine the length (mm) of plant sprout using a calliper.

40

Results

Table 2. viability of seeds of each plant type as compared to the viability being provided from the source.

Plant Common Name Source Viability %

Tested Viability %

Triticum durum Solid Horani Wheat

Local Market

Var. sacharata Zea Maize (Corn) NCARTT Hordeum vulgare Barley Local Market Cucuvita pepo Slasil (Squash) NCARTT Phaseolus vulgaris Romano NCARTT Triticum aestivum - NCARTT Atriplex halimus - NCARTT Hordeum spontaneum

- NCARTT

* NCARTT: National Center for Agricultural Research and Technology Transfer (Ministry of Agriculture-Jordan) **Number of plant seeds being used is ??. Number of other plant seeds is ??.

Table 3. Germination of different plant seeds at different diesel concentration1. Plant Seed Diesel Concentration (Mg/Kg) 0 100 200 500 1000 2000 5000 Triticum durum I II III Var. sacharata I II III

Hordeum vulgare I

II III Cucuvita pepo I II III Phaseolus vulgaris I II III Triticum aestivum I II III Atriplex halimus I II III Hordeum spontaneum I II III 0 diesel concentartion represent treatment of seeds with distilled water I: Average number of germinated seeds; II: Average % of germinated seeds, III: % decrease in seed germination as compared with control (0.0 diesel).

41

Table 4. Length (mm) and weight (mg) of each tested plant sprout at different

diesel concentration1. Plant Seed L Diesel Concentration (Mg/Kg) 0 100 200 500 1000 2000 5000 Triticum durum Var. sacharata

Hordeum vulgare

Cucuvita pepo Phaseolus vulgaris Triticum aestivum Atriplex halimus Hordeum spontaneum 0 diesel concentartion represent treatment of seeds with distilled water L: Average length (mm) of plant sprout

42

Table 5. Coparison of the most sensitive and resistant plant seeds to different diesel concentration1. Plant Seed Diesel Concentration (Mg/Kg) 0 100 200 500 1000 2000 5000 I II III IV V I II III IV V I II III IV V I II III IV V 0 diesel concentartion represent treatment of seeds with distilled water I: Average number of germinated seeds; II: Average % of germinated seeds, III: % decrease in seed germination as compared with control (0.0 diesel), IV: Average length (mm) of plant sprout; V: % decrease in sprouts' length as compared with control (0.0 diesel);

43

References Al-Ghazawi, Z, Saadoun, I. and Al-Shak’ah, A. 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. Farrell, R.E., C.M. Frick and J.J. Germida. 2000. “PhytoPet©: A database of plants that play a role in the phytoremediation of petroleum hydrocarbons. Pages 29-40 in Proceedings of the Second Phytoremediation Technical Seminar, Environment Canada, Ottawa. Glass, D. J. 2005. Commercial use of genetically modified organisms (GMOs) in bioremediation and phytoremediation. In: Bioremediation of Aquatic and Terrestrial Ecosystems. Eds. Fingerman, M. and Nagabhushanam, R. pp. 41-96. Science Publishers, Enfield (NH), USA. Nedunuri, K.V., R.S. Govundaraju, M.K. Banks, A.P. Schwab, and Z. Chen. 2000. Evaluation of phytoremediation for field scale degradation of total petroleum hydrocarbons. J. Environ. Eng. 126: 483-490 Novak, J. and AL-Ghazawi, Z., 1997. Plants-assisted Bioremediation of hydrocarbon contaminated soils, Proceedings of the mid-Atlantic hazardous waste conference, Blacksburg, Virginia, July 1997. Radwan, S.S., H. Awadhu and I.M. El-Nemr. 2000. Cropping as a phytoremediation practice for oily desert soil with reference to crop safety as food. Int. J. Phytoremed. 2: 383-396 Siciliano, S.D. and C. Greer. 2000. Plant-bacterial combinations to phytoremediate soil contaminated with high concentrations of 2,4,6-Trinitrotolene. J. Environ. Quality 29: 311-316

U.S. Army Corps of Engineers (USACOE), Waterways Experiment Station. Phytoremediation: The Process. (1997): 5 pp. Online. Internet. 1 July 1998. Available: http://www.wes.army.mil/ el/phyto/backgrnd.html.

Further Readings 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.

44

Detection of Alkylbenzenesulfonate-Degrading Microorganisms

Introduction

The surfactant alkylbenzenesulfonate (ABS) is a component of domestic and industrial detergents and considered as a recalcitrant molecule. A steady increase in detergent use has been accompanied by pollution problems such as foaming in river waters and sewage systems (4). Considerable amounts of ABS accumulate in the bottom sediment of coastal marine areas as well as inflowing rivers (1, 5). Many microorganisms are known to degrade these surfactants (2, 9, 10) as the ABS acts as the sole sulfur source. Benarde et al. (2) were able to isolate ABS metabolizing bacteria from various sources, using ABS-glucose-iron agar. A discoloration of the medium around colonies of ABS-degrading bacteria, as a result of iron sulfide formation, was noted. Ichikawa and Asaka (3) observed the formation of a halo around the colonies of ABS decomposing bacteria when methylene blue solution was poured over the surface of ABS nutrient agar plates. However, the halo was not very clear.

In this exercise, a simple agar plate method for isolation and clear demonstration of ABS degrading microorganisms in both seawater and freshwater will be explored by modifying a method for the volumetric determination of anionic surfactant. Neutral red (NR) is used as an indicator (11). The method depends upon the color responses of neutral red in alkaline medium. Neutral red changes from pink, when it enters into ABS micelles, to yellow, when the ABS is degraded, and does not form micelles. When neutral red-tris(hydroxymethyl)-aminomethane buffer solution and then cationic surfactant solution are sprayed onto the agar surface of ABS-nutrient agar cultures, transparent haloes appear around the colonies of ABS-degrading microorganisms against a pink background.

Procedure Culture Media 1-Estimation the number and presence of viable aerobic heterotrophic bacteria Use the following culture media: aerobic heterotrophic bacteria For Seawaters: ZoBell medium 2216E (12). For freshwater: Sakurai medium (8) [polypeptone, 2.0 g; Bacto-yeast extract, 1.0 g; glucose, 0.5 g; agar, 15.0 g; and tap water to 1,000 ml (pH 7.2)]. 2-Enumeration of ABS-resistant bacteria Add ABS to seawater or freshwater medium at a concentration of 1 g/liter. Bacteria capable of producing colonies on the ABS-nutrient medium are considered to be ABS resistant. 3-ABS-degrading bacteria/bacteria using ABS as sole source of carbon Examine further the agar plates upon which ABS-resistant bacterial colonies developed to enumerate the ABS-degrading bacteria (utilizing ABS as sole carbon source) following the procedure described below.

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Seawater ABS inorganic seawater medium: [(NH4)2S04, 1.0 g; K2HPO4, 0.01 g; ABS, 1.0 g; washed agar, 15.0 g; and charcoal-treated seawater (7) to 1,000 ml (pH 7.6)] Freshwater ABS inorganic freshwater medium (6) [(NH4)2S04, 2.0 g; KH2PO4, 2.0 g; Na2HPO4, 3.0 g; MgSO4 H20, 0.01 g; ABS, 1.0 g; washed agar, 15.0 g; and charcoal-treated tap water to 1,000 ml (pH 7.2) ] -Serially dilute water samples as described in the first experiment. -Place portions (0.1 ml) of serially diluted water sample on a nutrient medium and spread evenly over the agar surface with a glass rod. -Incubate the plates aerobically at 20 ºC for 2 weeks. Detection and enumeration of ABS-degrading microorganisms. 1-Reagents: For ABS, use tetrapropylene-derived sodium dodecylbenzenesulfonate (TBS). For NR solution, dissolve 0.3 g of NR in distilled water to 100 ml. For tris (hydroxymethyl) aminomethane (Tris) buffer solution, dissolve 2.42 g of Tris in 100 ml of distilled water. To this solution add 24.4 ml of 0.2 M HCl and increase the volume to 400 ml with distilled water. Adjust the pH to 8.6. For NR-Tris solution, prepare on the day of use by mixing the NR and Tris buffer solutions (1:9, vol/vol). For cationic surface active agent (CSAA) solution, dissolve 0.64 g of cetyl trimetyl ammonium chloride in distilled water to 1,000 ml. 2-Assay procedure: -Spray evenly the NR-Tris solution onto the surface of ABS-nutrient agar plates upon which colonies of ABS-resistant microorganisms had developed. -Leave the plates to stand at room temperature for 5 to 10 min to dry the surface, and then spray evenly a small amount of CSAA solution onto the agar. -Observe the appearance of transparent haloes around certain of the colonies. RESULTS The typical appearance of ABS-degrading bacterial colonies on a plate inoculated with a seawater sample. Transparent rings that appeared around colonies of presumed ABS-degrading bacteria are seen against the pink background of the ABS agar.

46

Typical halo formation of ABS-degrading bacteria after treatment as described in Materials and Methods. Transparent rings appeared around the colonies of ABS-degrading bacteria, and these can be seen against the pink background of the agar surface. One ABS-nondegrading bacterium can be seen against the edge of the plate (6). Rrefernces 1. Ambe, Y. 1973. Alkylbenzenesulfonate (ABS) in the bottom muds of the inner part of Tokyo Bay. J. Oceanogr. Soc. Jap. 29:1-7. 2. Benarde, M. A., B. W. Koft. R. Horvath, and L. Shaulis. 1965. Microbial degradation of the sulfonate of dodecyl benzene sulfonate. Appl. Microbiol. 13:103-105. 3. Ichikawa, Y., and J. Asaka. 1966. Microbial decomposition of alkylbenzene sulfonate. J. Food Hyg. Soc. Jap. 7:403-408. 4. McKinney, R. E., and J. M. Symons. 1959. Bacterial degradation of ABS. I. Fundamental biochemistry. Sewage Ind. Wastes 31:549-556. 5. Okubo, K. 1972. Methylene blue active substances (MBAS) as an indicator of anionic surfactants detected in Tokyo Bay and its adjacent sea areas. Bull. Tokai Reg. Fish. Res. Lab. 70:45-51. 6. Ohwada, K. 1975. Agar plate method for detection and enumeration of alkylbenzenesulfonate-degrading microorganisms. App. Microb. 29: 40-43. 7. Ryther, J. H., and R. R. L. Guillard. 1962. Studies of marine planktonic diatom. II. Use of Cyclotella nana Hustedt for assay of vitamin B,2 in seawater. Can. J. Microbiol. 8:437-445. 8. Sakurai, Y. 1967. Cultural conditions for plate count of heterotrophic bacteria in waters. Jap. J. Water Treat. Biol. 2:21-27. 9. Standard, P. G., and D. G. Ahearn. 1970. Effects of alkylbenzene sulfonates on yeasts. Appl. Microbiol. 20:646-648. 10. Swisher, R. D. 1970. Surfactant biodegradation, p. 496. Surfactant science series, vol. 3. Marcel Dekker, Inc., New York. 11. Uno, T., and K. Miyajima. 1962. Determination of surface-active agent. III. Volumetric determination of anionic surface-active agent using neutral red as an indicator. Chem. Pharm. Bull. 10:467-470.

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12. ZoBell, C. E. 1946. Marine microbiology, p. 240. Chronica Botanica Company, Waltham, Mas.

48

RISKS of GMOs

The organisms resulting from the use of modern biotechnological techniques are commonly referred to as genetically modified organisms (GMOs). The use of GMOs in different human enterprises has been an object of discussion for some time. To minimize risks to human health or the environment, the risk assessment of GMOs and their use has been regarded as an essential precaution in various countries. Earlier studies concerning risk assessment of GMOs have generally concentrated on the transgenes, their traits and the methods of their detection. Therefore, directive regulations or guidance documents have been proposed or published to supply necessary information for the risk evaluation of GMOs and to support the authorities. These directive regulations include the following: 1-The safety of the use of GMOs 2-Risk assessment of novel foods 3-The occupational safety of workers dealing with GMOs 4-The contained use of GMMOs 5-The deliberate release of GMOs

Risk assessment is a valuable principle that has become incorporated into modern

safety legislation. "Risk" is defined as an estimate of the probability that an event (harm, injury, or disease) will have an adverse effect and an estimate of the magnitude of that effect. I other words, what is the probability that something bad will happen, and, if it does happen, how serious are the consequences.

Systematic approach is expected to benefit the risk assessment of GMOs cultivation. The advantages of applying this approach are: limited cultivation area, controlled agricultural procedures and short time scale. However, the problems of such approach are: limited availability of ecological and biological data and lack of reliable data for risk analysis. Therefore, risk assessment of GMOs requires good knowledge in the field of sciences involved (gene technology, biology, ecology, agriculture, etc.).

THE POSSIBLE EFFECTS OF RELEASING GMOS INTO THE ENVIRONMENT

1- Direct Effects: - Related to the behavior of the GMOS in the environment and the particular genes which have been transferred Ex: -If a wild plant that is related to GM crop is growing nearby, there could be cross pollination and transfer of the foreign genes into native flora. -Antibiotic resistance The first GM crop in Europe is a maize variety containing and Ampr gene as well as insect and herbicide resistance. This has raised considerable controversy because of the clinical importance of ampicillin and the risk of the antibiotic resistance gene being transferred to the bacterial flora in the intestine of animals eating the maize (which is intended for animal feed production) or in the soil. This resistance could eventually be transferred to human or animal pathogens, increasing clinical problems with antibiotic resistance.

49

2- Secondary Effects

Ex: a- The use of crops resistance to broad-spectrum herbicides could alter weed flora and remove important food sources for birds already under pressure from conventional agriculture systems b- Insect resistant crops, could harm non-targeted beneficial insects ingesting pests which have fed on the crop. 3-Socio-economic impacts The presumption behind policy is that GMOS are good for competitiveness, jobs and agriculture. * Efficient agriculture// job losses, not gains * Biotechnology industry will also replacing traditional crop breeders.

Therefore, when considering the release of transgenic plants the risks are considered on a case-by-case and the following should not be forgotten: � Of the antibiotic resistance genes only Kanamycin has been tested for its effect on humans and this type of marker should be replaced by non-antibiotic markers if possible. � Field trials are needed to determine the best strategies to avoid the transference of genes on a case-by-case basis. Field evaluation is needed to determine the level of expression which can vary greatly

Information Requirements for the Release of GMOs Characteristics of the Modified Organism 1. Methods used for the modification. 2. Methods used to construct and introduce the insert(s) into the organism or to delete a

sequence. 3. Description of the insert and/or vector construction. 4. Purity of the insert from any unknown sequence and information on the degree to which

the inserted sequence is limited to the DNA required to perform the intended function. 5. Sequence, functional identity and location of the altered, inserted or deleted DNA with

particular reference to any known harmful sequences. Potential Environmental Impacts 1. Potential for excessive population increase in the environment. 2. Competitive advantage of the GMO over the unmodified organism. 3. Identification and description of the target organisms. 4. Anticipated mechanisms and results of interactions between the released modified

organism and the target organisms. 5. Identification and description of non-target organisms that may be affected by accident. 6. Probability of shifts in biological interactions or host range after release. 7. Known or predicted effects on non-target organisms in the environment, impact on

population levels of competitors-prey, hosts, symbionts, predators, parasites and pathogens.

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8. Known or predicted involvement in biogeochemical processes. 9. Other potentially significant interactions with the environment.

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Release of GE plants and Microorganisms needs approval by relevant bodies, such as the Deliberate Release Directive (90/220/EEC)

- The possible problems which may apply to the release of transgenic plants * Introduction of marker genes (Antibioticr) along with the target gene ntp11 gene (neomycin phasphotransferase 11) → Kanamycin r The concern is that the ntp11 gene product 1- will be toxic 2- may be transferred to other M.OS in the environment * Production of allergenic pollen from transgenic plants * Transfer of the genes from GMOS TO M.OS in the environment - Bacteria can transfer DNA by transformation, conjugation, transduction - Plants can transfer DNA by cross-pollination - The extent of gene transfer from crop plants to wild populations depends on: 1- crop plants and wild species must be compatible 2- growing at the same location 3- flower at the same time 4- have a means of pollen transference - The genetic strategies to avoid the transference 1- Introduction of male sterility so that the transgenic plant produces no pollen 2- Linking the gene with a gene that is lethal in pollen 3- Removal of flowers from the transgenic plant 4- Removal of compatible species 5- Planting of buffer plants 6- Direct gene introduction to the chloroplast which is not transferred to the pollen and

therefore cannot be transferred in cross-breeding * The new gene may give the plant a selective advantage, causing it to become a new pest or weed. * The transgenic plant may compete with local beneficial plants and upset the plant communities.

References:

1- Herbert M. What Is Genetically Modified Food (And Why Should You Care)? http://www.earthsave.org/newsletters/genfood2.htm

2- Krebs J. (2004). What's on the label? Science. Nov 12;306(5699):1101. 3- Burke D. (2004). GM food and crops: what went wrong in the UK? Many of the

public's concerns have little to do with science. EMBO Rep. May;5(5):432-6. 4- LeBlanc JG, Silvestroni A, Connes C. (2004). Reduction of non-digestible

oligosaccharides in soymilk: application of engineered lactic acid bacteria that produce alpha-galactosidase. Genet Mol Res. (3):432-40.

Links: 1- http://ohioline.osu.edu/gmo/a1.html. 2- http://wheat.colostate.edu/above.html 3- http://www-infocris.iaea.org/MVD/ 4- http://www.jmu.edu/biology/biofac/facfro/cloning/cloning.html 5- http://croptechnology.unl.edu/viewLesson.cgi?LessonID=957885612 6- http://barleyworld.org/NABGMP/QTLFIG.HTM 7- http://wheat.pw.usda.gov/ggpages/1rscom.html 8- http://www.wintv.com.au/science/barley.shtml 9- http://www.regional.org.au/au/abts/2001/t4/broughton.htm

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Survey -Provided below a survey results of a questionnaire done by biotechnology major students

Students Opinion about Genetically Modified Organisms (GMOs) and their Possible Risks

Ismail Saadoun

Department of Biotechnology and Genetic Engineering, Jordan University of Science and Technology, P.O. Box 3030, Irbid- 22110, Jordan. (E-mail: isaadoun@just.edu.jo)

ABSTRACT

Genetically modified organisms (GMOs) are those that resulted from the use of modern biotechnological techniques. The use of such organisms in different human enterprises has been an object of discussion for some time. To extend this discussion and to increase the awareness of people about GMOs and their possible risks, a questionnaire of 22 questions was conducted on a group of 280 undergraduate students of biotechnology and genetic engineering major at Jordan University of Science and Technology (JUST). The study focused on three main topics: molecular biology information (9 questions), risks of GMOs on human health and environment (7 questions) and the legislation that regulate the process of genetic engineering (6 questions). The answer for each question was either yes or no or not sure with a scale of 3, 1 and 2 points for these answers, respectively. Results revealed an average of 2.21, 1.98 and 2.34 points for the questions of the first, second and third topic, respectively, which indicate that the students have considerable information about molecular biology and the legislation of genetic engineering. However, the students’ background about the possible risks of GMOs needs to be re-evaluated.

-Based on the above survey, a questionnaire can be formulated by the students with the help of the instructor.

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SSUURRVVEEYY RREESSUULLTTSS

1) Have you heard of GMF?4%

96%

yesno

3) Do u think we need GMFs?

44%

56%

yesno

2) Do you support labeling of GMFs?

72, 72%

28, 28%

yesno

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4) What do you think GMFs might cause?

57%

0%

21%

12%

10%

cancer

AIDS

allergies

Alzheimer

birthdefects

5) Do you think eating GMFs is unethical?

74%

26%

yesno

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Appendices

Appendix 1: Media Composition:

1. Starch Casein Nitrate Agar (pH = 7.2)Components Company Per Liter

Casein Difco, USA 0.3g

Starch Riedel-de Haen, Germany 10.0g

KNO3 GCC, UK 2.0g

MgSO4.7H2O Chemlab, England 0.05g

FeSO4.7H2O Chemlab, England 0.01g

CaCO3 Chemlab, England 0.02g

NaCl Merck, Germany 2.0g

K2HPO4 Laboratory Rasayan, India 2.0g

Agar Himedia, India 18.0g

2. Oatmeal Agar (pH = 7.2)

Oatmeal Quaker, UK 20.0g

Agar Himedia, India 18.0g

Trace salt solution: 1.0 ml

FeSO4.7H2O Chemlab, England 0.1g/100ml

MnCl2.4H2O BDH, England

0.1g/100ml

ZnSO4.7H2O BDH, England 0.1g/100ml

Distilled Water 100ml

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3. CMC Agar (pH = 7.0-7.4)

Yeast-extract Himedia, India 1.0g

CMC GCC, UK 10.0g

KH2PO4 Laboratory Rasayan, India 4.0g

NaCl Merck, Germany 2.0g

MgSO4.7H2O Chemlab, England 1.0g

MnSO4 BDH, England 0.05g

FeSO4.7H2O Chemlab, England 0.05g

CaCl2.2H2O H&W, England 2.0g

NH4Cl Merck, Germany 2.0g

Agar Himedia, India 15.0g

4. Pectin Agar (pH = 7.0-7.4)

Yeast-extract Himedia, India 1.0g

Pectin Sigma, USA 5.0g

KH2PO4 Laboratory Rasayan, India 4.0g

NaCl Merck, Germany 2.0g

MgSO4.7H2O Chemlab, England 1.0g

MnSO4 BDH, England 0.05g

FeSO4.7H2O Chemlab, England 0.05g

CaCl2.2H2O H&W, England 2.0g

NH4Cl Merck, Germany 2.0g

Agar Himedia, India 15.0g

5. Xylan Agar (pH = 7.0-7.2)

Yeast-extract Himedia, India 1.0g

Xylan Sigma, USA 10.0g

KH2PO4 Laboratory Rasayan, India 4.0g

NaCl Merck, Germany 2.0g

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MgSO4.7H2O Chemlab, England 1.0g

MnSO4 BDH, England 0.05g

FeSO4.7H2O Chemlab, England 0.05g

CaCl2.2H2O H&W, England 2.0g

NH4Cl Merck, Germany 2.0g

Agar Himedia, India 15.0g 6. Mueller-Hinton Agar Muller Hinton Agar Himedia, India 30.0 g

D. H2O 1 L 7. Skim Milk Agar (pH 6.5-7.0)

Skim Milk Himedia, India 30.0 g Agar Himedia, India 18.0 g D. H2O 1 L

8- Mineral salts medium (MSM) of Leadbetter and Foster (1958)(Per liter) FeSO4 1 mg, MgSO4.7H2O 200 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. 9- T3 medium (Per liter)

3 g tryptone, 2 g tryptose, 1.5 g yeast extract, 0.05 M sodium phosphate [pH 6.8], and 0.005 g of MnCl2.