Environmental Biotechnology Laboratory Manual Prof Ismail Saadoun 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 i 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: +962-27201071 E-mail address: isaadoun@just.edu.jo ii 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 iii ISLAMIC UNIVERSITY OF GAZA Dept of Biotechnology Environmental Biotechnology Lab Contents Introduction and Orientation/ Review of Microbial Techniques Isolation and Characterization of Bacteria from Crude Petroleum Oil Contaminated Soil Growth Response of Bacteria on Petroleum Fuel (Diesel) Enrichment for Uric Acid Utilizing Bacteria Environmental Detection of Streptomycin-Producing Streptomyces spp by Using strb1 and 16S rDNA-Targeted PCR Field Trip (Main Wastewater Treatment Plant in Gaza) Molecular Detection of Fecal Coliforms (E coli) in Water by PCR Field Trip (Main Landfill Site in Gaza) How the community deals with domestic solid waste? (Collection, disposal and treatment) Interaction of Plant Seeds with Diesel for Potential Use in the Remediation of Diesel fuel Contaminated Soils Detection of Alkylbenzenesulfonate-Degrading Microorganisms Risks of Genetically Modified Organisms (GMOs) Appendices iv Pages 6-12 13-15 16-21 22-24 25-28 29 30-35 36-37 38-46 47-49 50-55 56-58 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 Introduction and Orientation/ Review of Microbial Techniques Isolation and Characterization of Bacteria from Crude Petroleum Oil Contaminated Soil Continue Growth Response of Bacteria on Petroleum Fuel (Diesel) Enrichment for Uric Acid Utilizing Bacteria Environmental Detection of Streptomycin-Producing Streptomyces spp by Using strb1 and 16S rDNA-Targeted PCR Mid Term Exam Field Trip (Main Wastewater Treatment Plant in Gaza) Molecular Detection of Fecal Coliforms (E coli) in Water by PCR Continue 10 Field Trip (Main Landfill Site in Gaza) How the community deals with domestic solid waste? (Collection, disposal and treatment) Interaction of Plant Seeds with Diesel for Potential Use in the 13 Remediation of Diesel fuel Contaminated Soils Detection of Alkylbenzenesulfonate-Degrading Microorganisms 14 Risks of Genetically Modified Organisms (GMOs) 15 16 Final Exam v Pages 6-12 13-15 16-21 22-24 25-28 29 30-35 36-37 38-46 47-49 50-55 - 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: In one hand hold the wire loop as you would a pencil; Heat the wire loop until red; Allow it to cool for a moment (this prevents burning or boiling of the medium when it contacts the loop); 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; Flame the container by passing the vessel top through the flame slowly (2 to sec) in order to sterilize the rim; Insert the wire loop and take the sample; Reflame the container top and replace the lid; Open and flame the top of the receiving vessel as you did with the sample vessel; 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 ml of a sample into 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: Pipette or 0.1 ml of a known dilution of a sample into the bottom section (smaller plate) of a sterile Petri dish; Within 20 add 12-15 ml of warm (46-48°C) fluid medium to this Petri dish; Cover the dish; 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); Allow the plate to stand, cool, and solidify; 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: Transfer cultures, streak plates and inoculate slants; Carry out dilution schemes to obtain microbial counts; Determine microbial motility by two methods; 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) ml pipettes Petri plates 99 ml dilution blanks Gram and spore stains Semi-solid agar tubes Procedure: A Microbial Isolation 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 The instructors have prepared different types of broth cultures You will dilute out each of these different cultures, by spread plating techniques and 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 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: 311316 U.S Army Corps of Engineers (USACOE), Waterways Experiment Station Phytoremediation: The Process (1997): pp Online Internet 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 43 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 redtris(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 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 44 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 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 to 10 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 45 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 Ambe, Y 1973 Alkylbenzenesulfonate (ABS) in the bottom muds of the inner part of Tokyo Bay J Oceanogr Soc Jap 29:1-7 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 Ichikawa, Y., and J Asaka 1966 Microbial decomposition of alkylbenzene sulfonate J Food Hyg Soc Jap 7:403-408 McKinney, R E., and J M Symons 1959 Bacterial degradation of ABS I Fundamental biochemistry Sewage Ind Wastes 31:549-556 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 Ohwada, K 1975 Agar plate method for detection and enumeration of alkylbenzenesulfonate-degrading microorganisms App Microb 29: 40-43 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 Sakurai, Y 1967 Cultural conditions for plate count of heterotrophic bacteria in waters Jap J Water Treat Biol 2:21-27 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 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 46 12 ZoBell, C E 1946 Marine microbiology, p 240 Chronica Botanica Company, Waltham, Mas 47 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 1Direct 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 48 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 Methods used for the modification Methods used to construct and introduce the insert(s) into the organism or to delete a sequence Description of the insert and/or vector construction 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 Sequence, functional identity and location of the altered, inserted or deleted DNA with particular reference to any known harmful sequences Potential Environmental Impacts Potential for excessive population increase in the environment Competitive advantage of the GMO over the unmodified organism Identification and description of the target organisms Anticipated mechanisms and results of interactions between the released modified organism and the target organisms Identification and description of non-target organisms that may be affected by accident Probability of shifts in biological interactions or host range after release Known or predicted effects on non-target organisms in the environment, impact on population levels of competitors-prey, hosts, symbionts, predators, parasites and pathogens 49 Known or predicted involvement in biogeochemical processes Other potentially significant interactions with the environment 50 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 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 51 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, and 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 52 SURVEY RESULTS 1) Have you heard of GMF? 4% yes no 96% 3) Do u think we need GMFs? 44% 56% ye s no 2) Do you support labeling of GMFs? 28, 28% yes no 72, 72% 53 4) What you think GMFs might cause? 10% cancer 12% AIDS allergies 57% 21% Alzheimer birth defects 0% 5) Do you think eating GMFs is unethical? 26% yes no 74% 54 Appendices Appendix 1: Media Composition: 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 Oatmeal Agar Oatmeal Agar (pH = 7.2) Quaker, UK 20.0g Himedia, India 18.0g Trace salt solution: FeSO4.7H2O 1.0 ml Chemlab, England MnCl2.4H2O ZnSO4.7H2O 0.1g/100ml BDH, England BDH, England 0.1g/100ml 0.1g/100ml Distilled Water 100ml 55 CMC Agar (pH = 7.0-7.4) Yeast-extract CMC KH2PO4 Himedia, India 1.0g GCC, UK 10.0g 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 Pectin Agar (pH = 7.0-7.4) Yeast-extract Himedia, India 1.0g 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 Pectin Xylan Agar (pH = 7.0-7.2) Yeast-extract Xylan KH2PO4 NaCl Himedia, India 1.0g Sigma, USA 10.0g Laboratory Rasayan, India Merck, Germany 56 4.0g 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 Mueller-Hinton Agar Muller Hinton Agar Himedia, India D H2O Skim Milk Agar (pH 6.5-7.0) Skim Milk Agar D H2O 30.0 g 1L Himedia, India Himedia, India 30.0 g 18.0 g 1L 8- Mineral salts medium (MSM) of Leadbetter and Foster (1958) (Per liter) FeSO4 mg, MgSO4.7H2O 200 mg, Na2HPO4 210 mg, NaH2PO4 90 mg, CuSO4.5H2O 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 mg 9- T3 medium (Per liter) g tryptone, g tryptose, 1.5 g yeast extract, 0.05 M sodium phosphate [pH 6.8], and 0.005 g of MnCl2 57 [...]... Microbiology, ed ATLAS, R.M New York: Macmillan Publishing Co pp 435474 Cappuccino, J G & Sherman, N., 1996 Microbiology: A Laboratory Manual The Benjamin/Cummings Publishing Company, Inc New York, pp 129–182 ISBN 0-8053-67461 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... 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... Microbiology and Biotechnology DOI 10.1007/s 11274-0089729-z 17 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. .. Reduction Growth at 42 ºC Motility Species Identified 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,... swelling of the sporangium; have a stainable surface after release 19 Fig 1 Degradation of purines results in the formation of uric acid which can be metabolized further yo glyoxylate, urea and CO2 20 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... 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 23 Further Readings 1-Malkawi, H.I., I Saadoun, F Al-Momani and M.M Meqdam 1999 Use of RAPD-PCR fingerprinting... 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 24 Wastewater Treatment Plant (Field Visit) Wastewater background Treatment of wastewater Preliminary Treatment Screening Removal of oil... 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 10 Growth Response of Bacteria on Petroleum Fuel Introduction Information on hydrocarbon (HC) degradation is required to determine the feasibility to use microorganisms