R. Ind Biot Manual Gaza

Industrial Biotechnology

Laboratory Manual

Prof. Ismail Saadoun

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ISLAMIC UNIVERSITY OF GAZA DEPARTMENT OF BIOTECHNOLOGY

INDUSTRIAL 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: [email protected]

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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: [email protected]

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PREFACE This manual has been designed for an undergraduate level laboratory sessions in industrial 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: [email protected]; [email protected]

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Islamic University of Gaza Faculty of Science/Department of Biotechnology

Industrial Biotechnology Lab

Contents Pages

Introduction, Orientation/Industrial Microbiology Power Point Presentation - Review of Microbial Techniques 6-12 Isolation of industrial microorganisms (streptomycetes) from soils of Gaza and their potential to produce antibiotics

13-16

Effect of Penicillin on Cell Wall Synthesis (Penicillin-Induced by Spheroplast Formation)

17-18

Upstream Processing (Microbiology of Milk & Dairy Products/ Testing of Penicillin in Milk)

19-21

Chymosin Demonstration 22 Production & Characterization of Citrinin 23-25 Fiber Hydrolytic Enzyme Activity of Streptomycetes Isolated from Gaza Soils

26-30

Degradation of Different Agricultural Waste Products by Soil Streptomycetes (Tomato, Apple and Orange Pomaces)

31-34

Pickling, Brewing and Cheese Preparation 35-36 Detection of Alcohol Production 37-38 Cellular and Enzyme Immobilization/ Immobilization of Yeasts 39-42 Open Lab (Ingredients of Different Food Products) Scientific Trip (Fermentation Units & Food Industries in Gaza) -Dairy Industry and Dairy Products Shops in Gaza -Dried Yeast and Starch Industry -Pharmaceutical Industry

43-44

Screening for Ethanol-Producing Yeasts 45 Appendices 46-49

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Islamic University of Gaza

Faculty of Science/Department of Biotechnology Industrial Biotechnology Lab

Lab Schedule The lab of this course focuses on the application of microorganisms in the industry with emphasis on metabolites of industrial microorganisms. Topics will include antibiotic production, action of antibiotics, microbiology of milk, chymosin, citrinin and wine production, and immobilization of microbial cells. Scientific visits are hopefully to be worked on with proper arrangements.

Week Exercise Pages

1 Introduction, Orientation/Industrial Microbiology Power Point Presentation - 2 Review of Microbial Techniques 6-12 3 Isolation of industrial microorganisms (streptomycetes) from soils of Gaza

and their potential to produce antibiotics 13-16

4 Effect of Penicillin on Cell Wall Synthesis (Penicillin-Induced by Spheroplast Formation)

17-18

5 Upstream Processing (Microbiology of Milk & Dairy Products/ Testing of Penicillin in Milk)

19-21

6 Chymosin Demonstration 22 7 Production & Characterization of Citrinin 23-25 8 Mid Term Exam - 9 Fiber Hydrolytic Enzyme Activity of Streptomycetes Isolated from Gaza

Soils 26-30

10 Degradation of Different Agricultural Waste Products by Soil Streptomycetes (Tomato, Apple and Orange Pomaces)

31-34

11 Pickling, Brewing and Cheese Preparation 35-36 12 Detection of Alcohol Production 37-38 13 Cellular and Enzyme Immobilization/ Immobilization of Yeasts 39-42 14 Open Lab (Ingredients of Different Food Products)

Scientific Trip (Fermentation Units & Food Industries in Gaza) -Dairy Industry and Dairy Products Shops in Gaza -Dried Yeast and Starch Industry -Pharmaceutical Industry

43-44

15 Screening for Ethanol-Producing Yeasts 45 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 pipet should be clean and sterile. It should be equipped with a pipet bulb or propipet so that oral contact and the potential danger of inhaling the microbial sample is avoided. Always place pipets 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

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transfer loop is reflamed, 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 reflamed, 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 reflamed 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

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

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

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

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

B. Microbial Motility 1. Obtain 3 tubes of semi-solid agar and inoculate each tube with one of the 3 culture types using

an inoculating needle. Omit the mixed culture sure to label each tube, incubate tubes at 37c for 48 hr.

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

1. Make gram stains of the E. coli, S. aureus, B. subtilis and the mixed culture according to the procedure described by your instructor. Observe these stains under the microscope using the oil immersion magnification.

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

Dilution Calculations Dilution factor = initial dilution x subsequent dilutions x amount plated Count per ml (or g) = reciprocal of dilution factor x colonies counted Example: A sample was diluted initially 1:100 (1 ml of in 99 ml sterile diluent). A subsequent 1:10 dilution (1 ml of the initial dilution into 9 ml sterile water) was prepared. Finally, 0.2 ml of the final dilution plated and 64 colonies were counted on the plate.

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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

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 of Industrial Microorganisms (Streptomycetes) from Soils of Gaza and their Potential to Produce Antibiotics

Introduction There are few hundred species of microorganisms that are used to make products useful to man. These are called industrial microorganisms, they include: 1- Bacteria 2- Actinomycetes (filamentous bacteria) 3- Molds (filamentous fungi): Penicillium chrysogenum, Penicillium notatum, Aspergillus niger,

Aspergillus oryzae 4- Yeasts : Saccharomyces cerevisiae , Candida utilis

This lab shows how basic science can lead to discovery. The group of actinomycetes that interests us most is the streptomycetes. None of the streptomycetes are pathogenic but many are medically significant because of the antibiotics that they produce. Streptomyces is the best recognized genus in the family Streptomycetaceae, because of its wide distribution in nature especially in soil. Further, this genus harbours a great number of the most important producer of antibiotic and other secondary metabolites. Therefore, various attempts were made to isolate these valuable organisms.

The purpose of this exercise is to try and discover actinomycetes from local soil samples that have antibiotic properties. Students attempt to identify streptomycetes with antibiotic properties in local soil samples. The use of known species of streptomycetes that produce antibiotics can be easily seen as they produce zones of inhibition on a lawn of bacteria; the antibiotic activity from local soil samples is variable and takes many samples to find a few that produce zones of inhibition. PROCEDURE

Sampling:

-Collect soil samples from cultivated or uncultivated soils from different locations in Gaza.

-Take these samples with an auger up to 10 cm depth, after removing approximately 3 cm of the soil surface.

-Place them in polyethylene bags, close tightly and store in a refrigerator.

Isolation:

-Mix the soil samples thoroughly and sieve them by 2 mm pore size mesh (Retsch, Germany).

-Suspend samples of 1g in 100 ml distilled water on a water-bath shaker (140 rpm, 30 min).

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-Dilute serially up to 10-6 and spread (0.1 ml) over the surface of glycerol nitrate casein agar (Kuster & Williams, 1964) plates with sterile L-shaped glass rods. Triplicate plates will be used for total streptomycete count.

-Incubate the plates at 27 ºC and determine the number of colonies after 10 days. Dilutions that gave 20-200 colonies will be chosen for the isolation.

-Select some colonies then purify them by repeated streaking. Notes for teacher: 1. Fungi do not grow overnight; they may take a few days to a week to grow. 2. Known species of streptomycetes can be ordered from: American Type Culture 12301 Parklawn Drive Rockville, Maryland 20852 Fax 1-301-231-5826

Characterisation of the isolates:

Streptomyces colonies are characterised morphologically and physiologically following the directions given for the International Streptomyces project (ISP) (Shirling & Gottlieb, 1966).

-Determine the general aerial mass color on oatmeal agar plates, incubated in the dark at 27 ºC for 10 days. These colors are: grey, white, yellow, red, blue, green and violet. Note the different colors, colony surface shapes.

-Preserve these isolates under proper conditions for further experiments.

Antimicrobial activity:

-Perform this is test by plate diffusion method against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Candida albicans.

-Grow the above test organisms in 250 ml flasks containing 50 ml nutrient broth medium and incubate at 37 ºC with shaking at 100 rpm for overnight.

-Inoculate each culture by a sterile cotton swab on the surface of Mueller-Hinton agar plates.

-Cut discs (9 mm in diameter) from the oatmeal agar cultures with the Streptomyces isolates grown on for 10 days.

-By a sterile cotton swab inoculate the surface of Mueller-Hinton agar with the above test organisms.

-Place the oatmeal agar cultures on the surface of Mueller-Hinton agar cultures.

-Incubate at 27 ºC, and then check the inhibition zones after 24 hrs.

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Evaluation 1. Did you observe any unknowns with antibiotic properties? 2. How do you go from discovery of antibiotic properties to isolation of the active factor? 3. How do you get a new compound to market? Extensions 1. Do we have enough new antibiotics to fight the new resistant bacterial strains? Teachers

should discuss this problem of resistant strains of bacteria with students. Check newspapers and magazines for articles dealing with these new resistant strains and what antibiotics are needed.

2. Investigate how antibiotics work. Use sensitivity discs to demonstrate the effects of antibiotics on different lawns of bacteria. These discs are available from most supply companies.

RESULTS Table 1. Antibacterial and antifungal activity of the Streptomyces isolates. Colour Series Antibiosis White Grey Yellow Red Blue Green Violet TotalIsolates Active isolates

% S. aureus % E. coli % B. subtilis % C. albicans %

Table 2. Action of the most active Streptomyces strains.

Isolate No. Antibiosis S. aureus E. coli B. subtilis C. albicans

1 2 3 4 5

Record the results in Table 2 as + or -. You can measure also the diameter of the inhibition zone and compare between the isolates.

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REFERENCES

Kuster, E. & S.T. Williams (1964). Selection of media for isolation of streptomycetes, Nature, 202: 928-929.

Shirling, E.B. & D. Gottlieb (1966). Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol., 16: 313-340. Further Readings 1-Saadoun, I. and R. Gharaibeh. 2002. The Streptomyces flora of Jordan and its’ potential as a source of antibiotics active against antibiotic-resistant Gram-negative bacteria. World J. Microbiol. Biotech. 18: 465-470. 2-Saadoun, I., L. Wahiby, Q. Ababneh, Z. Jaradat, M. Massadeh and F. Al-Momani. 2008. Recovery of soil streptomycetes from arid habitats in Jordan and their potential to inhibit multi-drug resistant Pseudomonas aeruginosa pathogens. World J. Microbiol. Biotech. 24: 157-162

Screening of new metablites after Hamill (1977).

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Effect of Penicillin on Cell Wall Synthesis (Penicillin-Induced by Spheroplast Formation)

Introduction: Ehrlich defined chemotherapeutics as substances which are bound directly to pathogenic microbial cells, damaging the microbe without affecting the host. At first this concept was not generally accepted. The prevailing idea was that the action of chemotherapeutic agents consisted in stimulating the body defenses against the invading microbes. Later this idea appeared to be supported by the demonstration that prontosil was effective in vivo but not in vitro. Ehrlich's concept was fully substantiated only when it was established that the active substance in prontosil was sulfanilamide, which was split off in the body, and that this substance was active both in vitro and in vivo. The action of antibiotics upon pathogenic organisms served to support this idea further, thus contributing materially to the rapid progress in the utilization of antibiotics as chemotherapeutic agents.

Mechanism of Action: Being such an important medical compound the mechanisms by which antibiotics kill bacteria have been under scrutiny for decades, with such studies being instrumental in the design of new and improved compounds. There are a five mode of action that acts the cell of infection:

1. Inhibition of Cell Wall Synthesis (most common mechanism). 2. Inhibition of Nucleic Acid Synthesis. 3. Inhibition of Protein Synthesis (Translation) (second largest class). 4. Alteration of Cell Membranes. 5. Antimetabolite Activity.

Inhibition of Cell Wall Synthesis: Bacterial cell wall determines the shape and strength of the cells of which is provided by peptidoglycan (the major structural component of the cell wall). Gram-positive bacteria have thick cell walls composed primarily of this substance; where as Gram-negative bacteria have multi-layer cell walls that are thinner than those of the Gram-positive cells.

Peptidoglycan (PG) is synthesized in three stages in three different parts of the cell. Firstly, the N-acetylmuramic acid (NAM sugar) is linked to the alanine, glutamic acid, lysine and alanine precursor in the cytoplasm, forming the basic subunit of PG. Secondly, the NAM/peptide is linked to the N-acetylglucosamine (NAG sugar) at the cell membrane. And lastly, now at the cell wall, the newly synthesized PG subunit is transferred to the growing point of the cell wall’s PG by a bond between the old PG and the new NAM-NAG disaccharide. Here the lateral cross-linking by the pentaglycine peptide can occur.

Antibiotics are active at every step of PG synthesis. Among the most famous of the antibiotics to interfere with PG synthesis are the ß-lactams, of which penicillin is an example. Penicillins resemble the terminal amino acids of the NAM+tetrapeptide precursor synthesized in the cytoplasm and actually bind the enzyme that catalyzes the pentaglycine cross-linking reaction. This binding prevents the cross-linking reaction from occurring and thus weakens the cell wall. Eventually, the turgor pressure of the cell causes the cell to lose its shape and eventually burst if the surrounding solution is hypotonic.

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Generally antibiotics exert their selective toxicity against eubacteria because human cells lack cell walls.

In this exercise, the formation of defective cell walls to spheroplasts in E. coli will be observed.

Culture: Escherichia coli, 4-6 hr culture, grown in nutrient broth at 37 °C with aeration (Use heavy inoculum from overnight growth, grow 4-6 hours). Reagents: Sterile solutions: Autoclaved solutions - 0.5 M sucrose Nutrient Broth (double strength) Sucrose (2M, 684 mg/ ml) MgSO4. 7H2O (0.1 M, 246.5 mg/10 ml) D. H2O Filtered (or dilution of sterile ampule) Penicillin (25,000 units I ml)

Non -sterile solutions: Gram-stain reagents Equipment needed:

Erlemenyer flasks, sterile (125 ml) Sterile pipetts (10 ml and 5 ml) ~ Incubator shaker (37 °C) Microscope slides Microcentrifuge Eppendorf centrifuge tubes

Procedure: Set up the following protocol in 125-ml sterile flasks, using aseptic procedures in adding solutions and culture (measurements are in ml) Component Flask No. 1 2 3 4 Nutrient Broth 8 8 8 8 Sucrose (2 M) — -- 4 4 MgSO4.7H2O -- 1.6 -- 1.6 Penicillin — 0.4 -- 0.4 H2O 8 6 4 2 E. coli culture 5 5 5 5

Incubate the flasks at 3 7 ºC with gentle shaking (or stirred with stirring bar), for 90 min. At the end of the experiment, compare the turbidity in the four flasks, noting evidence for growth or evidence for lysis (clearing).

Record results. Remove 1.5 ml from each flask and centrifuge in Eppendorf tubes at 2200 g for 5 min. Pour off supernatants, and add 0.1 ml of 0.5M sucrose to re-suspend cells.

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Microbiology of Milk & Dairy Products Testing of Penicillin in Milk

Milk is a completed food. It is an aqueous solution of proteins, fats, carbohydrates, vitamins, and minerals and it represents an ideal growth medium for microorganisms. The latter may cause spoilage in the dairy product and yield distasteful end products, or the milk may be a source of disease as organisms inter the milk from the cow or in the processing. Curdling, stormy fermentation, ropiness, and discoloration are example of spoilage in milk. Salmonellosis and Campylobacteriosis typify milkborne disease of humans. A. Methylene Blue Reduction Test. The methylene blue reduction test is a rapid procedure for determining the relative amount of bacteria in a sample of milk. A solution of methylene blue is added to the milk and the time needed for the color to disappear is determined. The color loss is brought about by the methylene blue's uptake of electrons, which are released during the fermentation of lactose by bacteria. The principle of the test is that the number of bacteria present is proportional to the number of electrons released and thus, the amount of time necessary for the color loss. The enzyme which accomplishes the transfer is called dehydrogenase (reductase). Materials:

• Methylene blue thiocyanate solution (Dissolve 0.05 g of MB thiocyanate in 1 L D-H2O) • Sterile 1.0 ml and 10 ml pipetts. • Sterile test tubes with rubber stoppers Procedure : 1- Aseptically pipette 10 ml of the milk sample to a sterile test tube with a rubber stopper and

lable the tube with your name. 2- Pipette 1.0 ml of the MB solution to the milk, tighten the rubber stopper, and carefully invert

three times to mix Place the rack in the water bath at 37 °C and record the time. 3- Check the tube at 30-minute intervals for the loss of blue color. As this takes place, the milk

will return to its normal white. When four- fifth of the tube has become white, the test is completed.

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Refer to Table 1 and determine the quality of the milk according to reduction time for methylene blue. Table 1. Interpretation of Methylene blue (MB) reduction test Description of Reaction Incubation Time with MB Quality of Milk No reduction of blue color Up to 8 hr Excellent Reduction of blue to colorless 6.5 to 7.5 hr Good Reduction of blue to colorless 2.5 to 6 hr Fair Reduction of blue to colorless < 2 hr Poor B. Testing of Penicillin in Milk MATERIAL

- Bacteria broth sensitive to penicill in. - Nutrient agar plates - Penicillin, 0.05 unit/1 ml - Sterile filter paper discs - Sterile forceps - L- shape gloss rod - Beaker with 70% alcohol - Pipett, 1 ml, 5 ml - Water bath, 82 °C. - Incubator, 37 °C.

PROTOCOL 1. Prepare bacterial lawn (sensitive to penicillin); pipett 1 ml of bacterial broth

into a plate and use a glass spreader to achieve uniformity and carefully pipetting off the excess bacterial broth into disinfectant.

2. Using sterile forceps aseptically touch the edge of the disc into milk sample and allow to become completely wet by capillary action. 3. Place the wet disc on the surface of the bacterial lawn, touching disc gently with the tip of the forceps to insure proper contact. 4. Place a control disc containing 0.05 unit/ml of penicillin on the bacterial lawn, insure that the disc are at least 20 mm apart when measured from center to center to avoid overlapping zones. 5. Invert plates and incubate at 37°C, overnight.

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6. If the milk sample gave an inhibition zone, heat the sample to 82 °C for 2-5 mm. Heating inactivates naturally occurring substances. Retest heated milk sample as in step 1—4. Any clear zone around the discs of a heated sample is a positive test.

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Chymosin Demonstration Introduction The following demonstration provides an example of the use of a protein produced by biotechnology for making ice cream custard, and cheese. Chymosin is a protein that occurs in rennet, which has been used for decades by cheese-makers. Until 1990, the chymosin (rennet) was obtained from the stomach of animals, preferably milk-fed calves. In 1990, the U.S. Food and Drug Administration approved the use of chymosin produced by genetically engineered bacteria. The chymosin from the bacteria is identical to that obtained from calves. The teachers or the students can do the demonstration. Only kitchen equipment and utensils are needed. Equipment and materials needed:

• Chymosin - should be stored in a refrigerator, not a freezer, when not in use. • A medicine dropper or other appropriate utensil for adding a drop of chymosin to milk in a glass

measuring cup.

• A glass-measuring cup commonly used in a kitchen. The one-cup size works well; however, any available size is satisfactory.

• An oven or hot water bath that can be kept between 35oC and 43oC for about 30 minutes. An oven thermometer or a candy thermometer can be used to measure temperature.

• Milk at room temperature. Whole milk works well, but milk with less fat content may also be satisfactory Procedure: 1. Pour about 50 ml of warm milk in the measuring cup. Use the ml marks on the measuring cup

and pour the milk directly from the container into the cup. 2. Add only one drop of chymosin to the milk. It is unnecessary to use more than one drop in up

to 200 ml of milk. 3. Swirl the milk in the cup gently for 10 seconds, then place the cup in the oven or hot water.

Within 30 minutes, the milk protein and fat will have coagulated and a solid curd will be present. T he demonstration is complete.

Note: One drop of chymosin can be used to coagulate 200 ml of milk. The curd is the most firm when 50 ml of milk is used. Use of about 200 ml of milk does not provide as solid a curd as with only 50 ml, but does make the whey (water) more obvious, if this is important for the student to see.

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Production & Characterization of Citrinin Introduction Citrinin (4,6-Dihydro-8-hydroxy-3,4,5-trimethyl-6-oxo-3H-2-benzopyran-7-carboxylic acid, C13H14O5, M.Wt. 250.25) (Fig. 1) is a mycotoxin produced by Penicillium citrinum and Aspergillus candidus. It is a contaminant of wheat, barley, rye, and oats ( 2, 4, 6), thus this toxin is potentially important myxotoxin that may be ingested by man and animals.

Fig. 1. Structural formula of citrinin

Hetherington and Raistrick (3) obtained yields of 525 to 700 mg of citrinin per 350 ml of

medium with P. citrinum Ad. 23 in 12 to 16 days at 28 to 32 C. Timonin and Rouatt (7) obtained 500 to 700 mg of citrinin per 200 ml of mineral salts medium with Aspergillus candidus incubated for 20 days at 26 C. Highest yields of citrinin were produced by A. candidus on media containing honey (7) or maple syrup (8) rather than glucose as the carbon source. In 1966 Rodig (5) produced the equivalent of 2.6 g of citrinin per liter in 14 to 16 days at 40 C with Aspergillus niveus and 1.2 g of citrinin in 35 to 40 days at room temperature with P. citrinum using Timonin's medium containing twice as much glucose. This exercise demonstrates production of citrinin in 5-gallon (18.925 liter) carboys containing 4 liters of 4% sucrose and 2% yeast extract nutrient solution (1) CULTURES: 20 ml spore suspension of Penicillium citrinum MEDIA: PER CLASS 2 4 liter Y.E.S broth in 5 GAL. Glass carboy with 4% Sucrose and 2% YE. MATERIALS: PER CLASS 4 50 ml SYRINGES 4 STERILE CANNULA 4 25 ml DIST. WATER 8 Big BLACK RUBBER STOPPERS # 13 DUST MASK

19

A. Culture of the organism: 1. Medium: sucrose ------------------------------- 4% Yeast extract ----------------------- 2% 2. Place four liters of medium into a 5-gallon plug and autoclave 20 minutes at 121 oC. 3. Cool, inoculate with conidia of Penicilliurn citrinum, and incubate 28 days at room

temperature as a stationary culture. Place carboy on side to obtain the maximum surface area to volume ratio.

Note: After sporulation, P. citrinum cultures are stored at 4 to 5 C on slants of agar containing 2% dextrose, 0.7% yeast extract (Difco), 0.5% KH2PO4, and 2% agar, and also as lyophilized cultures stored at -30 C. Spore suspension of this fungus will be supplied as 20 ml by the instructor. To prepare that spore suspension, place a drop of Tween 20 on sterile cotton swab, swab a culture of P. citrinum growing on PDA plate, and suspend the swab contents in sterile dionized water. B. Recovery and purification of citrinin: 1. Filter the fermented medium through a double layer of cheesecloth to remove the mycelial mat

then suction filter through a 33 cm No. 1 filter paper. 2. Adjust the pH of the filtrate to 1.5 with HCl:H2O (l:l) (caution) in carboy, while stirring with

magnetic stirrer. A yellow precipitate should form. Continue to stir 15-20 minutes. 3. Collect the yellow precipitate (crude citrinin) using buchner funnel and reduced pressure.

Continue suction until precipitate appears relatively dry. 4. Dissolve precipitate in chloroform, filter through # 41 paper, and evaporate solution to dryness. 5. Dissolve residue in minimum necessary volume of hot absolute ethanol. 6. Reduce volume by about 1/3 on steam bath under air stream. 7. Place hot ethanol solution in Ice water bath. Needle-shaped yellow crystals should form. If crystals do

not form, evaporate additional solvent. 8. Filter crystals using reduced pressure, continue vacuum until crystals appear dry. Re-crystallize.

9. Dry crystals in vacuum desiccator over dry calcium chloride 48 hr.

10. Weigh.

Proof of Identity of Citrinin: Chemically characterize the product you believe to be citrinin according to: melting point, UV spectrum, IR spectrum, mass spectrum, NMR spectrum, TLC Rf values, optical rotation, and color reaction with FeCl3. The instructor will demonstrate the methods and equipment. Compare your results with the literature on citrinin.

20

References

1. Davis, N.D., D.K. Dalby, U.L. Diener and G.A. Sansing. 1975. Medium-scale production of citrinin by Penicillium citrinum in a semisynthetic medium. Appl. Microbiol. 29: 118-120. 2. Hald, B., and P. Krogh. 1973. Analysis and chemical confirmation of citrinin in barley. J. Ass. Offic. Anal. Chem. 56:1440-1443. 3. Hetherington, A. C., and H. Raistrick. 1931. Studies in the biochemistry of micro-organisms. XI. On the production and chemical constitution of a new yellow colouring matter, citrinin, produced from glucose by Penicillium citrinum Thom. Phil. Trans. Roy. Soc. Ser. B. 220:269-297. 4. Krogh, P., B. Hald, and E. J. Pedersen. 1973. Occurrence of ochratoxin A and citrinin in cereals associated with mycotoxin porcine nephropathy. Acta Pathol. Microbiol. Scand. Sect. B 81:689-695. 5. Rodig, D. R., L. C. Ellis, and I. T. Glover. 1966. The biosynthesis of citrinin in Pencillium citrinin. I. Production and degradation of citrinin. Biochemistry 5:2451-2458. 6. Scott, P. M., W. van Walbeek, B. Kennedy, and D. Anyeti. 1972. Mycotoxins (ochratoxin A, citrinin, and sterigmatocystin) and toxigenic fungi in grains and other agricultural products. J. Agr. Food Chem. 20:1103-1109. 7. Timonin, M. I., and J. W. Rouatt. 1944. Production of citrinin by Aspergillus sp. of the candidus group. Can. J. Pub. Health 35:80-88. 8. Wyllie, J. 1945. A method for the rapid production of citrinin. Can. J. Pub. Health 36:477-483.

21

Fiber Hydrolytic Enzyme Activity of Streptomycetes Isolated from Gaza Soils

INTRODUCTION Streptomyces species have been always a source of thousands of bioactive compounds that attracted the researchers many years ago. Enzymes are one of the important products of this unique group of bacteria and among these are the fiber hydrolytic enzymes that are essential for the turn over and recycling of the carbon locked in plant polymers. For this reason, actinomycetes and particularly Streptomyces species are considered as important decomposers of plant and plant components (Tsujibo et al., 1990).

Streptomyces play an important role in soil as decomposers of plant and other materials,

thus participating in the turnover of relatively complex and recalcitrant polymers (Daffonchio et al., 1996). The production of wood hydrolytic enzymes, such as cellulases and hemicellulases (xylanases) by several Streptomyces species has been demonstrated by many workers (Beg et al., 2000; Elegir et al., 1994; Gilbert et al., 1995).

Cellulases, xylanases and other hemicellulolytic enzymes have received attention

worldwide due to their potential applications especially in the biodegradation of agricultural wastes as tomato pomace, olive mill wastes (Squires et al., 1991) in addition to their applications in the processing and clarification of fruit juices and wine (Coughlan and Hazlewood, 1993). Also, the digestibility of plant materials has been increased by applying these enzymes, thus the animal feed industry has developed (Blanco et al., 1995). Xylanases have an important industrial applications in processing and bleaching of pulp and paper (Elegir et al., 1994; Niehaus et al., 1999).

In this experiment, streptomycetes isolated breviously from different locations in Gaza

will be screened for their potential to produce fiber hydrolytic enzymes (cellulases, pectinases, xylanases, proteases, lipases and amylases). PROCEDURE Screening for xylanase –producing streptomycetes: -Culture each pure isolates on xylan agar and incubate for 4 days at 28°C.

-Flood plates with absolute ethanol (99%) and leave for 16 hours at room temperature.

-Colonies producing xylanase enzyme will show clear zones against the opacity of non hydrolyzed media (Priest 1985).

-Test the positive isolates again for confirmation.

-Store the confirmed isolates on xylan agar slants at 4°C (Priest et al., 1988)

Screening for cellulase–producing streptomycetes: -Culture each pure isolate on cellulose agar and incubate for 4 days at 28°C.

22

-Flood the plates with 0.1 % Congo Red and leave for 15-20 minutes.

-Wash plates with 1 ml NaCl and incubate at 5 C over night.

-Bacterial colonies producing cellulase will show clear zones against red color of non-hydrolyzed media (Sharma et al. 1990).

-Test the positive isolates again for confirmation (Carder 1986) and then store on cellulose agar slants at 4°C as a primary source (Priest et al., 1988).

. Screening for Pectinase –producing streptomycetes: -Culture pure isolates on pectin agar and incubate for 4 days at 28 °C. -Flood the plates with 0.1 % solution of polysaccharide precipitant cetavlon (acetyl trimethyl ammonium bromide dissolved in 15 % alcoholic solution) to detect pectinase production after 30-60 minutes. -Colonies producing pectinase showing clear zones against an opaque color of the non-hydrolyzed medium will be considered as positive (Priest 1985). -Test the positive again for confirmation and then store on pectin media slants at 4°C.

Screening for lipase-Producing streptomycetes: -Inoculate each pure isolate onto egg yolk agar base supplemented with egg yolk. The appearance of clear zones around the colonies will indicate the presence of lipase activity. Screening for amylase-producing streptomycetes: -Inoculate each pure isolate onto starch casein nitrate agar (SCNA) and incubate at 28° C for 4 days. -Flood the plates with iodine solution and leave for 30 minutes, then wash with distilled water. -Bacterial colonies producing amylase will show clear zone against black colour of stained starch (Santos and Martins, 2003). Screening for protease-producing streptomycetes: -Inoculate each pure isolate onto skimmed milk casein agar plates. -The appearance of clearing zones around colonies indicates the presence of proteolytic activity in these isolates by hydrolysis of casein (Sharmin et al. 2005).

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Results Table 1. Screening for hydrolytic enzymes produced by different active Streptomyces isolates.

Isolate Activity of the different Streptomyces isolates to produce fiber hydrolytic enzymes

CMCase Pectinase Xylanase Lipase Protease

*Diameter of clear zone, (+1); < 5 mm; (+2): 5-10 mm; (+3): > 10 mm

Plate diffusion method showing the lipase enzyme activity

24

REFERENCES Beg, Q. K., Bhushan, B., Kapoor, M. and Hoondal, G. S., 2000. Effect of amino acids on production of xylanase and pectinase from streptomyces sp. QG-11-3. World J. Microbiol. Biotechnol.16: 211-213. Blanco, A., Vidal, T., Colom, J. F. and Pastor, F. I. J., 1995. Purification and properties of xylanase A from alkali-tolerant Bacillus sp. strain Bp-23. Appl. Env. Microbiol. 61: 4468-4470. Carder, J. H., 1986. Detection and quantitation of cellulase by congo red staining of substrates in a cup-plate diffusion assay. Analyt. Biochem 153: 75-79. Crawford, D. L. and Sutherland, J. B., 1979. The role of actinomycetes in the decomposition of lignocellulose. Dev. Indus. Microbiol. 20: 143-151. Elegir, G., Szakaacs, G. and Jeffries, T. W., 1994. Purification, characterization and substrate specificities of multiple xylanases from Streptomyces sp. strain B-12-2. Appl. Env. Microbiol. 60: 2609-2615. Gilbert, M., Morosoli, R., Shareck, F., and Kluepfel, D., 1995. Production and secretion of proteins by streptomycetes. Crit. Rev. Biotech. 15: 13-39. Niehaus, F., Bertoldo, C., Kahler, M. and Antranikiar, G., 1999. Extremophiles as a source of novel enzymes for industrial application. Appl. Microbiol. Biotechnol. 5: 711-729. Santos EO. and L.M. Martins. 2003. Effect of the medium composition on formation of Amylase by Bacillus sp. 46: 129-134. Sharmin S., T. Hussein and M.N. Anwar. 2005. Isolation and Characterization of a Protease Producing Bacillus amovivorus and Optimization of Some Factors of Culture Conditions for Protease Production. J. Boil. Sci. 5: 358-362. Squires, M. W., Naber, E. C. and Toelle, V. D., 1991. The effects of heat, water, accid and alkali treatment of tomato cannery wastes on growth, metabolizable energy value and nitrogen utilization of broiler chicks. Poult. Sci. 71: 522-529.

Further Readings 1-Rawashdeh, R., I. Saadoun and A. Mahasneh. 2005. Effect of cultural conditions on xylanase production by Streptomyces sp. (strain Ib 24D) and its potential to utilize tomato pomace. African J. Biotechnol. 4 (3): 251-255 2- Saadoun, I., R. Rawashdeh, T. Dayeh, Q. Ababneh and A. Mahasneh. 2007. Isolation, characterization and screening for fiber hydrolytic enzymes-producing streptomycetes of Jordanian forest soils. Biotechnology 6(1); 121-129.

25

Fig. 2. Plate diffusion method to detect the enzymatic activity of Streptomyces isolates. (A) Amylase enzyme production on SCNA plate. (B) Protease enzyme production on skimmed milk casein agar plate.

26

Degradation of Different Agricultural Waste Products (Tomato, Apple and Orange Pomaces) by Soil Streptomycetes

Introduction Pectic substances are widely distributed in fruits and vegetables (10-30%). They are found in pulps of tomato, pineapple, orange, apple and lemon. In addition, they are found in turnips and in peels of oranges and other citrus fruits, hence, such substances form important natural substrates for the pectinase [1-3]. Therefore, pectinases are mainly used for enhancing filtration efficiency and clarification of certain fruit juices. Furthermore, they are widely used in maceration, liquefaction and extraction of vegetable tissues and have some uses in wood preservation [3-4]. Pectinase is a generic name for a family of enzymes that catalyze the hydrolysis of glycosidic bonds in pectic polymers. Pectinases are classified based on their mode of action into: polygalacturonase (EC 3.2.1.15), pectin esterase (EC 3.1.1.11), pectin lyase (EC 4.2.2.10) and pectate lyase (EC 4.2.2.2) [1, 5-6].

Pectinolytic microorganisms are widely spread in soil. Yeast and filamentous fungi are known to produce pectinases [7-8]. A wide variety of bacteria such as Pseudomonas, Xanthomonas, Erwinia, and Bacillus [1,9] are known as pectinase enzyme producers. Actinomycetes and particularly streptomycetes are being the best known enzyme producers [10]. Production and characterization of thermostable xylanase and pectinase from Streptomyces sp. QG-ll-3 was isolated by Beg et al. [11].

Few studies have been conducted on Streptomyces isolated from Jordan soil for their potential to produce enzymes of industrial importance. Rawashdeh et al. [12] isolated several Streptomyces isolates that were able to grow on tomato pomace. Upon further characterization, these isolates were able to produce cellulase, pectinase and relatively large amount of xylanase. Tahtamouni et al.[13] isolated indigenous Streptomyces isolates that were capable of producing chitinase.

This experiment is designed to isolate soil streptomycetes from different habitats in Gaza and screen them for their ability to utilize pectin as a sole source of carbon. The most active isolates will be tested for its potential to degrade apple, tomato and orange pomaces.

Materials and Methods

Location, sampling, treatment of soil samples and isolation technique: -Your instructor will describe to you the method of soil sampling and isolation of streptomycetes. Refer to the previous experiments on how these organisms are isolated. Your Instructor will be provide you with these organisms. Screening for fiber hydrolytic enzyme Streptomyces-producers -This is done previously. Pectinase production in tomatoes, apple and orange pomaces media

27

-The ability of the active Streptomyces sp. to grow in apple and orange broth media will be tested. -Prepare the media as follows: - Suspend 1g of dried fruit pomaces (tomatoes or apple or orange) in 100 ml distilled water, then supplement with 0.1 ml trace salt solution composed of 1 ml/L of FeSO4.7H2O, 0.1 g of MnCL2.4 H2O and 0.1 g of ZnSO4.7H2O. -Adjust the pH of the media to 7.5 prior to autoclaving. -Inoculate flasks containing the media with 1 ml spore suspension. -Incubate cultures at 28 °C for 8 days. -Perform pectinase, cellulase and xylanase enzymes activity daily using the standard DNS method and as follows: Pectinolytic activity assay -Pectinase assay will be performed following the procedure of Miller [19] with some modification. -Briefly, incubate a reaction mixture composed of 0.2 ml of crude enzyme solution, plus 1.8 ml of 1.0% (w/v) citrus pectin in 50 mM sodium phosphate buffer (pH 7.0) at 37 °C in a shaker water bath for 30 min. -Terminate the reaction by adding 3 ml of DNS reagent. The color is then developed by boiling the mixture for 5 min. -Measure the optical densities of samples at 575 nm against a blank containing the reaction mixture minus the crude enzyme. -Compare the results to controls inoculated with an inactive pectinolytic streptomycete isolate. -Interpret the results of enzyme activity in which one unit (U) of enzyme activity is defined as the mount of enzyme, which liberates 1 µmol of reducing groups (D – galacturonic acid) from the substrate [14]. Cellulase activity Assay. -Cellulase activity can be measured following the method of Miller (1959). -Briefly, incubate a reaction mixture composed of 0.2 ml of crude enzyme solution, plus 1.8 ml of 0.5% carboxymethyl cellulose (CMC) in 50 mM sodium phosphate buffer (pH 7.0) at 37 °C in a shaking water bath for 30 min. -Terminate the reaction by adding 3 ml of DNS reagent. The color is then developed by boiling the mixture for 5 min.

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-Measure the optical densities of samples at 575 nm against a blank containing the reaction mixture minus the crude enzyme. -Compare the results to controls inoculated with an inactive cellulytic streptomycete isolate. -Interpret the results of enzyme activity in which one unit (U) of enzyme activity was defined as the amount of enzyme, which liberates 1µmol of glucose per minute under the above assay conditions (Miller, 1959).

Xylanase activity assay Assays for crude xylanase will be performed using 0.5% soluble oat spelt xylan (Sigma) in 50mM sodium phosphate buffer, pH 7.0.

-The reaction mixture is composed of 1.8 ml substrate and 0.2 ml crude enzyme.

-Incubate the mixture in a water bath at 60 oC for 15 min.

-Terminate the reaction by adding 3 ml of 3,5-dinitrosalicylic acid (DNSA) (Miller, 1959). -The color (reddish brown) is then developed after placing the reaction tubes in a boiling water bath for 5 min. -After cooling the reaction tubes to room temperature, measure the O.D. at 575 nm with xylose as the standard.

-Compare the results to controls inoculated with an inactive xylanatic streptomycete isolate. -Interpret the results of enzyme activity in which one unit (U) of xylanase activity is defined as the amount of enzyme that releases 1µmol xylose/ min/ml under the above mentioned conditions. Results To evaluate the production of pectinase, cellulose and xylanase in media containing fruit pomaces, 3 different types of fruit pomaces were used as carbon sources. -Record the highest pectinase, xylanase and cellulase activity when apple pomace was used. -Record the highest pectinase, xylanase and cellulase activity when orange pomace was used. -Record the highest pectinase, xylanase and cellulase activity when tomato pomace was used. -Compare the substrate for the best enzyme activity. -Compare the best time for enzyme activity.

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References 1. Gummadi, S. N., Panda, T.: Purification and biochemical properties of microbial pectinases. Proc Biochem 38: 987-996 (2003) 2. Rai, P., Majumdar, G.C., DasGupta, S., De, S.: Optimizing pectinase usage in pretreatment of mosambi juice for clarification by response surface methodology. J Food Eng 64: 397-403 (2004) 3. Sreenath, H.K., Kogel, F., Radola, B.J.: Macerating Properties of a Commercial Pectinase on Carrot and Celery. Ferment Technol 64: 37-44 (1986) 4. Hours, R.A., Vogrt, C.E. and Ertola, R.J.: Apple pomace as raw material for pectinase production in solid state culture. Biological Wastes 23: 221-228 (1988) 5. Beg, Q.K., Bhushan, B., Kapoor, M. and Hoondal, G.S. 2000. Effect of amino acids on production of xylanase and pectinase from Streptomyces sp. QG-11-3. World J Microbiol Biotechnol 16: 211-213 (2000) 6. Aguilar, G., Huitorn, C.: Stimulation of the production of extracellular pectinolytic activities of Aspergillus species by galacturonic acid and glucose addition. Enzyme Microb Technol 9: 690-696 (1987) 7. Manachini, P.L., Fortina, M.G., Parini, C.: Purification and properties of endopolygalacturonase produced by Rhizopus stolonifer. Biotechnol Lett 9: 21-/224 (1987) 8. Liu, Y.K., Luh, B.S.: Purification and characterization of endopolygalacturonase from Rhizopus arrhizus. J Food Sci 43: 721-726 (1999) 9. Dosanjh, N.S., Hoondal, G.S.: Production of constitutive, thermostable, hyper active exo-pectinase from Bacillus GK-8. Biotechnol Lett 12: 1435–1438 (1996) 10. Vinogradova, S.P., Kushnir, S.N.: Biosynthesis of hydrolytic enzymes durig cocultivation of macro- and micromycetes. Appl Biochem Microbiol 39: 573-575 (2003) 11. Beg, Q.K., Bhushan, B., Kapoor, M., Hoondal, G.S.: Production and characterization of thermostable xylanase and pectinase from Streptomyces sp. QG-ll-3. J Indust Microbiol Biotechnol 24: 396-402 (2004) 12. Rawashdeh, R., Saadoun, I., Mahasneh, A.: Effect of cultural conditions on xylanase production by Streptomyces sp. (strain lb 24D) and its potential to utilize tomato pomace. African J Biotechnol 4: 251-255 (2005) 13. Tahtamouni, M.E.W., Hameed, K.M., Saadoun, I.M.: Biological control of Sclerotinia sclerotiorum using indigenous chitinolytic actinomycetes in Jordan. Plant Pathol. J. 22: 107-114 (2006) 14. Miller, G.L.: Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chem 31: 426-428 (1959).

30

Pickling, Brewing and Cheese Preparation

A- Fermentation of Sauerkraut The word sauerkraut is derived from German roots ‘sour-cabbage’. This food product is formed by species of Lactobacillus (Gram positive bacilli) and Leuconostoc (Gram positive cocci) which normally occur in cabbage. The acid they produce inhibits the growth of other organisms and provides a natural preservative in the product. The high salt content enhances preservation. Procedure 1. Remove the core and outer leaves from a quarter of a head of cabbage and weigh it on a balance. Weigh out 3% of this figure as noniodized salt. Noniodized salt is recommended to avoid the iodine which may interfere with bacterial growth. 2. Shred the cabbage and mix it thoroughly with the salt. Pack the cabbage into a sterile 1L beaker. Cover the cabbage with a sterile Petri dish lid or a watch glass, and add a foil-wrapped stone or brick to weigh down the cover. This will encourage the anaerobic conditions necessary for fermentation of the cabbage. Replace the foil over the beaker and incubate the cabbage aside at approximately 30 ºC for 1 to 2 weeks. 3. Observe the cabbage at regular intervals and note the accumulation of fluid as water is drawn out of the plant cells by the salt. At intervals, take fluid samples and do Gram staining and observe the change in the flora. 4. At the end of incubation, taste the sauerkraut and enter your observations. B- Fermentation of Wine and Beer The production processes for wine and beer are highly specialized and sophisticated industries throughout the world. Procedure 1. Select tubes of malt extract broth and sterile grape juice supplemented with 5% sucrose containing cotton plugs. The cotton plugs will permit evolved CO2 to escape. Inoculate each tube with a heavy loopful of Saccharomyces cerevisiae or commercial yeast and incubate the tubes at approximately 30 ºC for 1 week or longer at the direction of the instructor. 2. Shake the tubes carefully and note the foaming at the surface which indicates that CO2 has been produced. Note the winelike aroma in the grape juice and the beerlike odor of the malt extract broth. Taste the product to determine its quality and prepare stains of the sediment at the bottom of the tubes to observe the yeast cells. 3. Production of alcohol by organisms as a result of their growth on a certain substrate can be detected enzymatically. This test will be done next week.

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C- Preparation of Cheese Cheese is prepared from the casein portion of the milk. The casein may be precipitated as a curd by the enzyme rennin as well as by the acid produced during bacterial growth. The curd is an unripened cheese which may be sold as cottage cheese or pot cheese. Procedure 1. Milk curds may be prepared as follows: Place a quart of warm milk in a large container and add a rennin tablet as directed by the instructor. Curds will begin to form in a few minutes and, when curdling is complete, the curds may be emptied into a colander lined with cheesecloth. 2. Squeeze as much liquid from the curds as possible and thoroughly mix a small piece of blue cheese (source of Penicillium roqueforti) into the curds with a spoon. 3. Shape the curds into a compressed mass and salt the outside layer lightly to add flavour and inhibit bacterial growth. Replace the cheesecloth covering. 4. Place the cheese into a plastic container, cover tightly, and incubate at a cool temperature. If cottage cheese has been used, the original package may be used for storage. Cheesecloth may be used for wrapping and the excess water may be removed as it appears in the container. 5. Taste the cheese at weekly intervals and note the development of blue veins on Penicillium mold and the characteristic blue cheese flavour and aroma. References I. Edward Alcamo. 1991. Laboratory Fundamentals of Microbiology. The Benjamin/Cummings Publishing Company, Inc. Redwood City, California, USA. pp. 176-188.

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Detection of Alcohol Production

Introduction 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. In this experiment, the method of Jacobs et al. (1983) will be applied in order to determine the ability of the yeasts from the previous lab to produce 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] PROCEDURE 1-Obtain different types of broth media from your instructor. Tubes containing the broth media from the last experiment (malt extract and grape juice) can be used. Assay for alcohol production: The test of JACOBS et al. (1983) will be conducted to detect alcohol production. -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 filtered broth (malt extract, grape juice, ……..etc.), 5 µl of 0.15 M NAD solution and 25 µl of washed cells. -Compare the change in the colour with four controls: 1st control contains no (substrate) 2nd control contains no NAD+ 3rd control contains no cells 4th 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.

33

Results Table 1. Action of different bacterial and yeast species on ……..as indicated by colour change

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

Reaction condition

Time 1 2 3 4 5 6

+NAD+/29˚C 1h.

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

References

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.

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CELL AND ENZYME IMMOBILIZATION Immobilization of Yeasts

Immobilization: The conversion of enzymes from a water soluble, mobile state to a water-insoluble immobile state

Advantage of immobilizing enzymes compared to free enzymes: 1- Extending the purified enzyme, preparations effectiveness for long time

النه يصعب تخزين أنزيمات فعالة لمدة طويلة2- Stabilizing enzymes to be used over and over again so that conversion of substrate

to product can be carried out continuously .ستعمال أو الخزن ألن األنزيمات تتعرض لعوامل آيماوية ، فيزيائية ، بيولوجية تثبط من فعالية األنزيمات خالل اإل

Advantages of immobilizing cells compared to free cells:

1- The capacity to use them repeatedly 2- The easy separability (sieve) 3- Their application to continuous reactions -Early used 1- 18th century for vinegar production

- 20th century for disposal of sewage effluent

35

36

Immobilization of Saccharomyces cerevisiae Materails: Per Class 2 Balances Weight Boots Spatulas 12 400 ml sterile Beakers 12 Glass Sterile Stirring rods 2 Blenders

4 50-60 cc syringes 10 5 ml or 10 ml wide pore pipettes 12 100 ml Distilled water Pipett Bulbs 1 Bottle Alginic acid (Sigma, Type 4, Sodium, Salt) Refrigerator Space. 0.09 M Calcium chloride

0.009 mM phosphate buffer (20 gm CaCl2 in 2L water. 0.15 gm KH2PO4 in 250 ml H2O, mix 4 ml phosphate buffer in CaCl2 solution).

37

Procedure Sodium alginate (50 ml; 40% wt/vol) is mixed with 50 ml of cell suspension (containing 25 g [wet weight] of cells) until homogeneous. The mixture is passed through a narrow tube and into 0.05 M CaCl2 solution (pH 6 to 8) at 37°C forming beads 2.8 to 3.0 -mm in diameter. The beads are allowed to "cure" at 20 to 22 °C for 1 hr, rinsed with H2O, and equilibrated overnight in a 0.05 M CaC12 solution at 4 °C. The immobilized S. cerevisiae cells can produce ethanol from glucose. That can be compared to free yeast cells.

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Islamic University of Gaza Dept. of Biotechnology

Industrial Biotechnology -------------------------------------------------------------------------------------------------------------------- Name : -------------------------------------------------------------------------------------------------------------------- Ingredient Commercial Product High Fructose Corn Syrup ( HFCS )

1

2 3 4 5 Xanthan 1 2 3 4 5 Dextran 1 2 3 4

5 Other

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ISLAMIC UNIVERSTITY OF GAZA

Fermentation Request

Date: Name:

Department:

Phone:

Requested Date of Fermentation Run: The following information will greatly facilitate your request for use of the fermentation facility. This information and a brief meeting between yourself and the manager of the fermentation unit will be used to determine initial operating parameters for your run. On a separate sheet please give the following information with as much detail as possible, and attach to this form. Include any known data that is pertinent to the task, including detailed media recipes, shake flask studies, etc. I. Name and cellular morphology of organism.

II. State objective (biomass or microbial metabolite production, bioprocessing such as cell disruption, etc).

III. How much material do you require? Iv. Describe known characteristics of the organism (i.e. growth rate; temperature and pH optimum; preferred growth medium, etc.). V. If production of a microbial metabolite is the objective, state known details of production, concentration and storage.

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Screening for Ethanol- Producing Yeasts Introduction Ethanol is a clear, colorless, flammable, oxygenated hydrocarbon with the chemical formula C2H5OH. Ethanol can be used as transport fuel in at least four forms: anhydrous ethanol (100 % ethanol), hydrous ethanol (95 % ethanol and 5 % water), anhydrous ethanol-gasoline blends (10-20 % ethanol in gasoline) and raw materials for ethyl tert-butyl ether (ETBE) (1). The ability of microorganisms to produce alcohol from sugars has been known since ancient Egyptian times and could be regarded as one of the first uses of biotechnology, parts from its use in the brewing, wine and spirits industries. Ethanol is widely used in the chemical industry and is used as a motor fuel (1). The use of ethanol as a fuel started in the 1930 in the USA where ethanol produced from maize was used at a concentration of 20 % to produce gasohol called Agrol, also Canada and Brazil are major producers and users of this energy source, much of it derived from grains such as corn and wheat. The main organism used in fermentation of carbohydrates to ethanol is Saccharomyces cerevisiae, other bacterial species can also produce ethanol such as; Zymomonas mobilis, Clostridium thermocellum (1, 2). Procedure -Obtain different yeasts from your instructor. These organisms should be grown on Edinburgh Minimal Medium agar (Per 1L: Xylose 20 g, Agar 15 g) plates for 3-5 days at 28° C. -The ability of these organisms to produce ethanol will be tested according to the method of Jacob et al (2). -Add 5 ml of molten agar (50º C) which consisted of agar (10 g/L) made up in a sodium phosphate buffer (0.1 M; pH 8). -Add then the reaction mixture which consisted of 50 µl 0.05 M 2,6-dichlorophenolindophenol (DCP; Merk), 100 µl 0.15 M NAD solution (Boehringer Mannheim; product no. 223468). -Incubate the plates at room temperature for 30 min. -Spread 3 ml of 0.005 M 5- methyl-phenzinium methyl sulfate (Merk; product no. 10819) over each plate. -Incubate at 30º C for 30min. -The presence of ethanol is indicated by observing the yellow color (reaction zone) on a blue background.

References

1- SCRAGG A. 1999. Environmental Biotechnology. Addison Wesly Longman. pp. 105-174. 2- 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.

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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


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



MnSO4 BDH, England 0.05g


CaCl2.2H2O H&W, England 2.0g

NH4Cl Merck, Germany 2.0g


4. Pectin Agar (pH = 7.0-7.4)


Pectin Sigma, USA 5.0g









5. Xylan Agar (pH = 7.0-7.2)


Xylan Sigma, USA 10.0g


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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

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Appendix 2: Glucose and Xylose Standard Curves.

A. Glucose Standard Curve.

B. Xylose standard curve.

Documents

R. Ind Biot Manual Gaza